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This is to certify that the

dissertation entitled

THE ULTRASTRUCTURE AND HISTOCHEMISTRY OF
INFECTION BY COLLE'IUI‘RICHUM LAGENARIUM IN
CUCUMBERS INDUCED FOR RESISTANCE

presented by
Barry David Stein
has been accepted towards fulfillment

of the requirements for

Ph.D degree in Botany; and Plant Pathology l

Major professor

Date Febmag 7, 1991

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

F——_————.—————_—_—.—__—-——W
DATE DUE DATE DUE DATE DUE

JUL 0 5 1995‘]
____l| '

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

if?

usu Is An Affirmative Action/Equal Opportunity Inaitution

 

 

 

I!

 

THE ULTRASTRUCTURE AND HISTOCHEMISTRY OF
INFECTION BY COLLETOTRICHUM LAGENARIUM IN

CUCUMBERS INDUCED FOR RESISTANCE

BY

Barry David Stein

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Botany and Plant Pathology

1991

153363113:

ABSTRACT
THE ULTRASTRUCTURE AND HISTOCHEMISTRY OF

INFECTION BY COLLETOTRICHUM LAGENARIUM IN
CUCUHBERS INDUCED FOR RESISTANCE

BY

Barry David Stein

Induced resistance in cucumbers seems to be a function of
the epidermis. Since induced resistance is nonspecific, it
has been hypothesized that the inhibition of penetration
into cells is one of the primary mechanisms. Whether
inhibition is a function of cell walls or cytoplasm is not
known. To determine the possible location of induced
resistance, cucumber plants induced for resistance by
injection with gsggggmgngg gyringag pv syringe; were
compared to control plants by electron microscopy,
ultrastructural histochemistry, and energy dispersive X-ray
microanalysis. Plants were also assayed for peroxidase,
phenolics, and extensin in the petiolar epidermis and bulk
petiole of induced and control cucumber plants. Leaves of
induced plants, which were infected by leletotrichum
lagenarium, were observed to have electron-dense
modifications of the walls of infected cells. Energy
dispersive X-ray microanalysis (EDS) of these cell walls
showed that silicon accumulation accounted for much of the
electron-density. Silicon was found in cells prepared by

standard glutaraldehyde/osmium tetroride fixation and by the

bromine procedure for lignin localization. These cell wall
areas also stained with potassium permanganate indicating
the presence of lignin. The binding of potassium
permanganate to the electron-dense areas was corroborated by
EDS for Mn. Papillae developed where the fungus started to
penetrate into epidermal cells. Papillae also stained with
potassium permanganate; this was corroborated by EDS.
Hydroxyproline, a component of the cell wall protein
extensin, was not found to vary in concentration as a result
of cucumber plants being induced for resistance.

Peroxidase, which is thought to be responsible for
polymerizing extensin and lignin, was found to occur in
increased concentrations in the epidermis and bulk tissue of
the petioles of induced cucumber plants. The epidermis of
induced plants appeared to have higher concentrations of
lignin than the petiole, a further indication that the
epidermis is important in induced resistance. Carbazole
staining of peroxidase for light microscopy also showed
higher concentrations of peroxidase in induced cucumber
leaves, though localization to a cell type was not possible.
Diaminobenzidine staining for the electron microscopic
localization of peroxidase suggested that peroxidase was
concentrated between the electron-dense cell walls and the

plasmalemma.

ACKNOWLEGEMENTS

I would like to thank the members of my Dissertation
Committee Ray Hammerschmidt, Frank Ewers, Derek Lamport and,
most especially, Karen Klomparens for their help during the

the time that I have been at Michigan State University.

iv

TABLE OF CONTENTS

Page
LIST OF TABLES...........................................vii
LIST OF FIGURES................................. ........ viii
CHAPTER I LITERATURE REVIEW.............................l
Historic review of induced resistance..................2
Ultrastructure of fungal infection.....................9
Role of lignin and peroxidase in resistance...........10
Ultrastructural localization of lignin................12
Ultrastructural localization of silicon...............13
Ultrastructural localization of peroxidase............14
Resistance induced by heatshock of seedling cucumbers.14
Objectives............................................15
CHAPTER II ULTRASTRUCTURE AND HISTOCHEMISTRY OF THE
INFECTION OF CUCUMBERS, INDUCED TO BE
RESISTANT: BY QQLLETQIBIQHHM LAQENABIHMo-o-ool7
ABSTRACT..............................................18
INTRODUCTION..........................................20
METHODS AND MATERIALS.................................20
Plant Material....................................20
Electron Microscopy...............................21
Potassium Permanganate Staining...................22
Bromine Staining Procedure........................23
Energy Dispersive X-ray Microanalysis.............24
Diaminobenzidine Staining.........................24
RESULTS...............................................25
Inoculation of Plants.............................25
Ultrastructure of Appressoria.....................25
Effects of Fungus on Plant Cell Wall..............34
Necrotic Appressoria...................... ........ 41
Papillae..........................................41
Peroxidase Localization...........................44
Bromination of Pumpkin Hypocotyls.................49
DISCUSSION............................................50
Appressoria Ultrastructure........................50
Matrix Layer......................................51
Effects of Fungus on Plant Cell Wall..............51
Plant Cell Reactions to Fungus....................53
Papillae..........................................57
Peroxidase........................................58
Cell Wall Barriers to Infection...................58
CHAPTER III EFFECTS OF HEATSHOCK UPON THE CELL WALL
THICKNESSES OF EPIDERMAL CELLS OF THE
HYPOCOTYL OF CUCUMBER SEEDLINGS..............60
ABSTRACT.....................................61
INTRODUCTION..........................................62
METHODS AND TECHNIQUES................................62
Experiment 1......................................62

vi

Experiment 2......................................63
Morphometry.......................................63
RESULTS...............................................64
Morphometry.......................................64
Electron Microscopy...............................70
DISCUSSION............................................73
CHAPTER IV EFFECTS OF INDUCED RESISTANCE ON
PEROXIDASE, PHENOLICS AND EXTENSIN
CONCENTRATIONS IN CUCUMBER LEAVES...........7S
ABSTRACT..............................................76
INTRODUCTION..........................................77
METHODS AND TECHNIQUES................................78
Preparation of plant samples......................78
Bacteria Culture..................................79
Histochemistry and microscopy.....................79
Acetone Powders...................................79
Assay for Cell Wall Bound Phenolics...............80
Assay for Peroxidase..............................80
Assay for Hydroxyproline..........................81
RESULTS...............................................81
Microscopy........................................81
Cell Wall Bound Phenolics.........................84
Peroxidase........................................84
Hydroxyproline....................................85
DISCUSSION............................................87
CHAPTER V TECHNIQUES FOR THE ULTRASTRUCTURAL
LOCALIZATION OF LIGNIN USING POTASSIUM
PERMANGANATE AND BROMINE STAINING AND
EDS.........................................89
ABSTRACT..............................................90
INTRODUCTION..........................................91
MATERIALS AND METHODS.................................91
RESULTS...............................................93
DISCUSSION............................................98
CHAPTER VI WORK ON THE IMMUNOCYTOCHEMICAL LOCALIZATION
OF EXTENSIN AND PEROXIDASE IN THE LEAF OF
CUCUMBERS INDUCED FOR RESISTANCE...........102
ABSTRACT.............................................103
INTRODUCTION................................... ...... 104
METHODS AND TECHNIQUES...............................105
Cucumber Seedlings...............................105
Cucumber Plants..................................105
Immunocytochemistry..............................106
PBS Formula......................................107
RESULTS AND DISCUSSION...............................107
SUMMARY..................................................112
LIST OF REFERENCES.......................................115

vii

LIST OF TABLES

TABLE . Page
2.1. Occurrence and appearance of internal flanges
observed in sections of appressoria................. ...... 31

2.2. The presence of electron-dense cell walls at the

sites of infection caused by ggllgtgtgignum lagenarigm....35

3.1. Comparison of epidermal outer cell wall thickness of
control and heat shocked hypocotyl 0, 24 and 48 hours
after experimental hypocotyls were treated................65

3.2. The second comparison of epidermal outer cell wall
thickness of control and heat shocked hypocotyl 0, 24 and
48 hours after experimental hypocotyls were treated.......66

3.3. A comparison of the epidermal outer cell wall
thickness of hypocotyls of the cucumber cultivar Marketer
which had been placed into water at various different
temperatures..............................................67

4.1. Measurements of the concentrations of hydroxyproline
in the tissues of bulked samples from the cucumber
epidermises and petioles used in the assay for peroxidase.86

5.1. Comparison of techniques used in the fixation and
staining of plant samples for the presence of lignin. ..... 99

viii

LIST OF FIGURES

Figure Page
2.1. First true leaf of Qgggmig gatiyg cv SMR 58 plant
induced for resistance one week earlier by infection into
the abaxial surface with Efiggggmgngs syringag pv syringgg.
Necrotic lesions (N) are the result of bacteria
injection.................................................27

2.2. Second leaf of injected cucumber plant, such as in
Fig. 1, where the injection has resulted in a type of
ChlorOSisO00.0.00...OOOOOOOOOOOOOOOOOOOO... ..... O ......... 27

2.3. Injected SMR 58 cucumber plants are located on the

left of rear bench next to uninjected controls on the right
of rear bench. The controls (arrowhead) are larger in size
than the injected plants..................................27

2.4. Scanning electron micrograph of germinated conidia (C)
of Colletotrichum laggnarium with germination tube (GT) of
variable length and appressoria (A) 24 hrs. after
inoculation upon control leaves. Bar = 10 um ............. 29

2.5. Scanning electron micrograph of conidium (C) with
appressorium (A) on control leaf 24 hours after inoculation
with Q. lagenariun showing matrix layer (ML) around
appressorium. Bar = 1 um..........................;......29

2.6. Scanning electron micrograph of germinated spore on
induced leaf 24 hrs. after inoculation with Q. lagegagigmy
showing matrix layer (ML) (arrows) around appressorium (A).
Conidium (C) and Bacteria (B). Bar = 1 um................29

2.7. Transmission electron micrograph of ultrathin section
of appressorium (A), portion of conidium (C), and plant cell
wall (CW) from control plant 48 hrs. after inoculation with
Q. laggngri_m. Section was uranyl acetate and lead citrate
stained. Conidium wall appears less electron-dense than
appressorium wall. The plant cell wall under the
appressorium is indented (arrow). Internal flange (F),
extracellular matrix layer (ML), and bacterium (B). Bar = 1

“m0...O..0.0..0...0..00...OOOOOOOOOOOOOOOOOOO0.00.00.00.0029

2.8. Appressorium on control plant, transmission electron
micrograph of ultrathin section, 48 hrs. after inoculation
with Q. la enar' , the infection peg (IF) has broken
through plant epidermal cell wall (CW). Section stained

ix

with uranyl acetate and lead citrate. Cell wall next to
infection peg is less electron-dense than surrounding wall.
Wall under appressorium (A) appears indented towards the
plant cell cytoplasm but rest of cell wall layers do not
appear to be disturbed. Bar = 1 um.......................33

2.9. Appressorium from control plant. Transmission
electron micrograph of the appressorial cell wall shows
outer (O), and inner (I) layers. The internal flange (F)
and the extension (E) differ in electron density from each
other. Bar = 0.2 um......................................33

2.10. Transmission electron micrograph of ultrathin section
of appressorium (A) and plant epidermal cell wall (CW) from
control plant 48 hrs. after inoculation with Q. laggnari_m.
Section was not stained with uranyl acetate and lead
citrate. Bar = 1 pm......................................33

2.11. Transmission electron micrograph of ultrathin
section, stained with uranyl acetate and lead citrate, of
appressorium (A) on protected tissue at 48 hrs after
infection with Q. lagenarium. Cell wall (CW) of plant under
appressorium is electron-dense (D) with electron-dense
granules (GR) between host cell wall and plasmalemma (PL).

Bar:1“m0.000000000000000000000.0.0.0000...0.0.0.0....0.33

2.12. Transmission electron microscopy ultrathin section,
stained with KMnOU of appressorium (A) on induced plant
inoculated 48 hrs previously with g. laggnazium.
Appressorial walls (AW) are heavily stained by 10an4 due to
the presence of melanin. The region of the plant cell wall
between the arrowheads and proximal to the appressorium is
electron-dense. Bar = 1 um...............................38

2.13. Unstained serial section of specimen in Fig. 12.
Region of plant cell wall between the arrowheads corresponds
with that of Fig. 12. Appressorium (A). Bar = 1 um......38

2.14. EDS dot map from a serial section of specimen in Fig.
12 indicating the localization of manganese. The electron-
dense cell wall (marked by arrowheads in Fig. 12) of the
plant under the appressorium is more heavily stained than
surrounding areas of the plant cell wall. Appressorium

(A).......................................................38

2.15. EDS dot map of Si in Fig. 13. The arrowheads
correspond in position to those found in Figs. 12, 13, and

14......OOOOOOOOOOOOOCOOOOOOOOOIOOOOOCOOOOOOO 0000000000000 38

2.16. Transmission electron microscopy ultrathin section of
brominated mature cucumber leaf that was fixed 48 hours
after inoculation with Q. lagenazium. Plant cell wall (CW)

X

proximal to appressorium (A) is electron-dense (marked by
arrowneadS). Bar:1“mOOOOOOCOOIOOOOOOOOOOO0.0.00.00000040

2.17. Br EDS dot map of Fig. 16. arrowheads below
appressorium (A) correspond to the plant cell wall (CW)
similarly marked in Fig. 160.000.00.0000000000.00.00.00.0040

2.18. Si EDS dot map of Fig. 16 with arrowheads in the same
position as those in the thin section.....................40

2.19. Transmission electron micrograph of ultrathin section
of tissue, from protected plant at 48 hrs after inoculation
with Q. lagenarium. Section was stained with KMnO,. ZPlant
cell wall under appressorium (A) is more electron—dense than
surrounding wall. Appressorium is misshapened and appears
necrotic. Bar = 1 um.....................................40

2.20. Transmission electron micrograph of ultrathin section
of necrotic appressoria on induced tissue 48 hrs. after
inoculation with Q. lagenarium. Pockets of unsuccessful
penetrations, as well as successful ones, often occurred.
Papilla (P). Bar = 10 um.................................43

2.21. Transmission electron micrograph of ultrathin section,
stained with uranyl acetate and lead citrate, of papilla of
protected plant at 48 hrs after inoculation with Q.
lagenarium. Papilla (P) with granular material.
Plasmalemma of plant cell (arrowhead) surrounds papilla.

Bar:1“m0.0000000COOOCOOCOOOOOOOOOOOOOOC0.00.00.00.0000043

2.22. Transmission electron micrograph of ultrathin section
of primary hypha (PH), 72 hrs., showing successful
penetration of Q. laggnarium into protected plant. Section
was stained with KMnO4. Material between hypha and
plasmalemma appears more fine grained than at 48 hrs. (Fig.
21). The material surrounding the hypha, adjacent to the
plant cell wall (arrow), is stained with KMnO4 indicating
the presence of lignin. Bar = 1 um.......................43

2.23. Transmission electron micrograph at 48 hours after
inoculation of induced leaf with Q. lagenazium. Section was
stained with KMnO,. Primary hypha (PH) has elongated into
plant cell and is surrounded by papilla (P). Bar = 1 um..43

2.24. Transmission electron micrograph of ultrathin section
of hypha (H) with papilla (P), on a protected plant
inoculated 48 hrs. previously with Q. lagepazium. Section
was stained with I<Mn0,. Material in papilla around hypha is
electron-dense, indicating the presence of lignin. Bar = 1

“m0.... ......... 00.000.000.000...00.0.00...O ......... 0.00.46

2.25. Unstained transmission electron microscopy ultrathin

xi

serial section of same material as Fig. 24. Papilla (P),
hypha (H). Bar:1um....................................46

2.26. Scanning transmission electron microscopy x-ray
microanalysis dot map demonstrating the presence of
manganese (from KMnO) in a serial section of hypha shown in
Fig. 24. Papilla (P), hypha (H).......................... 46

2. 27. Transmission electron microscopy ultrathin section of
Diaminobenzidine-030, stained protected tissue at 48 hrs.
after inoculation with Q. laggnarigm showing increased
staining (arrowhead) around outside of appressorium and also
staining between electron-dense plant cell area and plant
plasmalemma, demonstrating the localization of peroxidase.

Bar:1"m................................................46

2.28. Transmission electron micrograph of section of
control plant, inoculated 48 hours before fixation with Q.
lagenarium, and stained for peroxidase using
Diaminobenzidine-0804 (DAB). The interface between the
electron-dense area of the plant cell wall and the
plasmalemma as well as the outer surface of the appressorium
are stained by the DAB. Bar = 1 um.......................48

2. 29. Transmission electron micrograph of material from
induced plant which had been inoculated with g. lagenagigm
48 hours before and stained for peroxidase using DAB. The
plant cell wall around area of fungal penetration lacks
staining (arrowhead). Bar = 1 um.........................48

2.30. Transmission electron micrograph of the epidermis of

the hypocotyl of seedling of the Spookie cultivar of pumpkin
(resistant) which had been inoculated 48 hours earlier with
Q. laggnarium and fixed with paraformaldehyde and incubated
in bromine/chloroform solution. Appressorium (A) and plant
cell wall (CW). Bar = 1 um...............................48

2.31. Scanning transmission electron microscopy X-ray
microanalysis dot map from a serial section of Fig. 29
indicating the localization of manganese. The electron-
dense cell wall of the plant under the appressoria (A) is
more heavily stained than surrounding areas of the plant
cell wall (CW) indicating the presence of lignin. Cell
walls of adjacent cells are not stained...................48

3.1. Scanning electron micrograph of cucumber hypocotyl
cross section. Bar=1oo um..............................69

3.2. Transmission electron micrograph of heat shocked
cucumber seedling hypocotyl epidermal cells. The
endoplasmic reticulum (ER) are swollen, the nucleoli (NU)
are electron-dense and the epidermal outer cell walls (C)

xii
are layered. Bar:1“m..................... ............. 69

3.3. TEM micrograph of control cucumber hypocotyl epidermal
cells. Endoplastic reticulum (ER), nucleoli (NU) and
epidermal outer cell wall (C). Bar = 1 um................69

3.4. TEM of epidermal cells of cucumber hypocotyl from
seedling which had been placed into 22.5°C water.
Endoplasmic reticulum (ER). Bar = 1 um...................69

3.5. TEM of epidermal cells of cucumber hypocotyl 24 hours
after heat shock. Bar - 1 um...................... ..... ..72

3.6. TEM of epidermal cells of control cucumber hypocotyl
24 hours after experimental seedlings were heat shocked.

Bar:1“moose...ooeooeooooooooooooeooooooooooooo00000000072

3.7. TEM of epidermal cells of cucumber hypocotyl 48 hours
after seedlings were heat shocked. Bar = 1 um............72

3.8. TEM of epidermal cells of control cucumber hypocotyl
48 hours after experimental seedlings were heat shocked.

Bar:1”m0..0...0.0..00........O.........................72

4.1. Light micrograph of cucumber leaf, induced by
injection with 2mm: sittings: pv utilises. sectioned
with a cryostat and stained with carbazole for peroxidase.
Bar = 100 um..............................................83

4.2. Light micrograph of control cucumber leaf, not
injected, sectioned with a cryostat and stained with
carbazole for peroxidase. Bar = 100 um...................83

5.1. Ultrathin section of bromine-incubated,
paraformaldehyde-fixed material showed poor ultrastructural
preservation. Cytoplasm (CY) was present, though organelles
and other constituents of the cytoplasm could not be
differentiated. Walls directly under appressorium (A) were
more electron dense than walls of neighboring cell (arrow).

Bars1“moosoooooooooeoooooeooooooooooooooooooeo00000000095

5.2. Energy dispersive x-ray microanalysis (EDS) dot map
showing localization of Br in Fig. 1. The wall of the cell
under the appressorium (A) was more heavily stained than the
neighboring cell (arrow)..................................95

5.3. Ultrathin section of material fixed with
glutaraldehyde and OsO,; sections from epoxy blocks were
stained with KMn0,. IPreservation of sample was comparable
to that seen in standard UA/ Pb citrate stained samples.
Cell walls (CW) were heavily stained with KMnO . Cytoplasm
(CY) was not as heavily stained. Fungal conid1a (C),

xiii
appressoria (A) and hyphae (H) are present. Bar = 1 um...95

5.4. Photomicrograph of a serial section of Fig. 3 which
was not stained with KMnO,. ‘The cell walls of plant (CW)
and fungal hyphae (H) were electron-lucent, compared to the
electron-dense walls of the stained section in Fig. 3. The
wall of the appressoria (A) was electron-dense. Bar = 1

um...O..............O.......................O........ ..... 95

5.5. EDS dot map for Mn in Fig. 3. The heaviest
concentrations of Mn were found in hyphae and plant cell
walls which were the areas which became more electron-dense
in Fig. 3 as well as the cells walls of the appressoria (A)
which were electron-dense in both Figs. 3 and 4. The
cytoplasm (CY, same area as in Fig. 3) is only lightly
stained...................................................95

5.6. Ultrathin section of sample fixed with glutaraldeyde
followed by KMn04. The plant cell wall (CW) was less
electron-dense than the plant cytoplasm (CY), though there
were layers in the cell wall which were more heavily stained
by the KMnO4. 'The outer matrix layer (M) and cytoplasm of
the appressorium (A) were also well stained. The
appressorium cell wall was very electron dense. Bar = 1

“m....... ........ ........ ..... ............................95

5.7. EDS of Fig. 6. The greater concentration of Mn was
found in the cytoplasm (CY) of the plant cell and in the
appressorium (A) and its matrix layer (A). The plant cell
wall (CW) had little Mn associated with it ................ 95

6.1. TEM of epidermal outer cell wall of SMR 58 cucumber
plants. Tissues were embedded in Spurr's epoxy. Sections
were incubated with anti-peroxidase and Protein A/ colloidal
gold. Cell wall (C) has a heavy concentration of gold
particles but extra- (E) and intracellular (I) spaces have
only a scattering of particles. A greater number of
particles are found in a deposit layer which covered much of
the epidermis (D). Bar = 1 um...........................109

6.2. TEM of mesophyll cell from control, uninjected SMR 58
cucumber plant, the samples of which were embedded in
Spurr's epoxy. Sections were incubated in anti-peroxidase
and Protein A/ colloidal gold. Little binding of particles
occurs in the intercellular space (IS) but there is binding
over much of the cytoplasm (CY), chloroplasts (CH), starch
granules (G) and cell wall (C). Bar = 1 um..............109

6.3. TEM of induced resistant SMR 58 cucumber plant. Thin
sections were incubated with anti-peroxidase and Protein A/
colloidal gold. Most of binding occurs on chloroplasts
(CH), starch grains (G), ground cytoplasm (CY) and cell wall

xiv
(C). Bar:1um.........................................109

6.4. TEM of the hypocotyl of 48 hour heat shocked and 24
hours after inoculation of gladgspgrium cucumerigum onto

seedlings. Sections were incubated with anti—peroxidase and
colloidal gold. Particles are found on the nucleus (N),
nucleolus (NU), ground cytoplasm (CY) and cell wall (C) but
not as heavily on the intercellular space (I) and vacuole

(V). Bar:1“m.................................. ....... 109

CHAPTER I

LITERATURE REVIEW

LITERATURE REVIEW

ammawm

Acquired, systemic resistance in plants, which is induced
by prior infection, occurs in a number of plant/pathogen
interactions. Early studies were extensively reviewed by
Chester (1933), who gave abundant evidence for the existence
of acquired resistance but attributed the cause to a plant
immune system based on antibodies. Much of the recent
progress in determining the nature of this phenomenon dates
from the studies of Gilpatrik and Weintraub (1952). They
reported that viral infection of Qignthus barbatus with
carnation mosaic virus caused the development of systemic
protection in uninfected and apparently virus-free leaves
against further virus infection. If this effect had been
due to cross protection of one virus strain against another,
the protected leaves would also have been systemically
infected with the original virus.

Further studies by Ross (1961) showed that infection of
the three lower leaves, of trimmed five leaf tobacco plants
with tobacco mosaic virus (TMV), induced resistance against
challenge inoculation with TMV in the upper two leaves.
Lesion size on the induced leaves was reduced and generally

there were fewer lesions. No TMV could be recovered from

3
the leaves to be challenged before the challenge
inoculation. Resistance could not be induced by wounding or
by chemical injury. Ross (1961) further investigated the
time course of the development of resistance and reported
that resistance to TMV developed after a lag of two days,
reached a maximum at 7-10 days and persisted for at least 20
days. Either potato virus X (PVX) or tobacco ringspot virus
was also effective in inducing resistance against TMV
challenge. Plants induced by TMV were resistant against
turnip mosaic virus, another lesion producing virus.
Viruses that did not produce lesions did not induce
resistance. Ross (1961) concluded that any leaf necrosis
producing virus was as equally effective as TMV in inducing
resistance against further virus infection.

Induced resistance was also reported in plants that were
infected by fungi (Chester, 1933). Hecht and Bateman (1964)
reported that tobacco, inoculated with the fungus
Thielaviopsis basicola, developed necrotic lesions and
afterwards were resistant to TMV and TNV infection. Yarwood
(1954) reported that pinto bean leaves, previously infected
with bean rust, grgmyggg phgsggli, were comparatively
resistant to further infection by bean rust. He also noted
that germination of rust uredospores on bean leaves could be
inhibited by the presence of either rust spores or by the
close proximity of rust-infected leaves. He concluded that

the inhibitor was gaseous in nature and was given off by the

4
fungus. Yarwood (1956) duplicated this experiment using g.
phasggli to induce sunflowers which were challenged with
Puccini; helianthi and also used 2. helianthi to induce
beans for resistance against H. phggggli. Both fungi
therefore induced resistance against the other fungus.

The systemic nature of induced resistance in plants
infected by fungi was demonstrated by Cruikshank and Mandryk
(1960). Their research used tobacco plants that were
inoculated with Egrgngspgxa tabggina, which causes a stem
infection. The plants became more resistant to subsequent
infection by P. tabagina than plants which were not
previously infected. The effect was small on the mature
portions of the plant and was greatest on those parts which
developed after the infection.

Bell and Presley (1969) reported that attenuated or heat
killed conidia of yertigillgm albgzatzum were able to induce
resistance in cotton, demonstrating that live pathogens were
not necessary to induce resistance in certain cases.

Kuc et al. (1975) determined that gladgspgzium
cucumerinum or Colletotrichnm lagsnarium could be used to
systemically protect against further infection by g.
lagenazium in eight cultivars of cucumbers susceptible to
infection by Q. lagenarium. The size and number of lesions
on protected plants were smaller than those on the control
plants. Protection could be achieved by low levels of Q.

laggnarium such that only a single lesion of the protective

5
organism on a leaf could confer resistance. Damage of
leaves by dry ice did not produce protection. Cucumber
plants susceptible to infection by Q. cucumgninum were also
protected by infection with ggllgtgtgignum lindemuthianum, a
bean pathogen (Hammerschmidt et al., 1976).

Richmond et al. (1979) determined that induced resistance
to Q. lagenarium in cucumbers induced by Q. lagenagium was
caused by reduced penetration of appressoria on induced
resistant plants compared to the number of appressoria that
penetrated into uninduced plants. The number of conidia
that germinated to form appressoria on induced and uninduced
plants did not differ (Richmond et al., 1979; Hammerschmidt
and Kuc, 1982). In an earlier study, Jenns and End (1977)
reported that the size of the lesions produced by Q.
laggnarium on cucumber plants, induced by tobacco necrosis
virus, was also smaller than those on uninduced plants.

When the epidermises of induced resistant and genetically
resistant leaves were removed and the remainder of the leaf
challenged by Q. lgggngxigm, the induced resistant leaves
were found to be susceptible to infection, but the leaves of
plants with constitutive resistance remained resistant to
infection (Richmond et al., 1979). This indicated that
induced resistance in cucumbers was mediated by the
epidermis.

Hammerschmidt and Kué (1982) noted the lack of

penetration by appressoria of Q. laggggrigm into protected

6
epidermis and found that lignin formed in petiolar epidermal
cells, directly under appressoria. Lignin, though, was
associated only with some of the appressoria which failed to
penetrate. In other cases, penetration or failure of
penetration occurred with no signs of lignification and, in
a few of the cases lignification and penetration occurred
together. A large percentage (91.7%) of the penetrations by
gladgspgrium gugnmeninum into protected tissue were
lignified, compared to the small percentage in unprotected
tissues (6.2%) (Hammerschmidt and Kué, 1982). Increases of
lignin content in cucumber apical tissue occurred in
protected plants challenged by Q. cucumgzinum. When lignin
precursors were introduced into cucumber leaf disks from
plants which had been protected by prior infection with Q.
laggnarium, the precursors were incorporated into lignins
faster in the protected, compared with unprotected, tissues
(Dean and Kué, 1987). Lignin deposition was postulated,
therefore, to have a role in the resistance found in
cucumber plants.

A systemic increase in peroxidase activity was found in
the second true leaf of cucumber plants that were protected
by Q. lagenarium injection in the first true leaf
(Hammerschmidt et al., 1982). As in earlier reports, injury
with dry ice did not produce resistance and did not cause
the systemic increase of peroxidase levels above that of

uninjured plants. Smith and Hammerschmidt (1988) compared

7
the total activity of the soluble acidic peroxidases of
cucumber, muskmelon and watermelon and reported that
systemically induced leaves had a two-fold or greater
activity compared to control leaves. Peroxidases in these
species were found to migrate as a cluster of three bands on
high pH native polyacrylamide gels (Smith and Hammerschmidt,
1988).

Research on the source and movement of the signal that
causes systemic protection was done in cucumber plants by
grafting and removing the first leaves, which were infected
by Q. lagenarium (Dean and Kuc, 1986). Leaves grafted on an
infected plant became resistant, as long as the induced leaf
remained on the plant. Induced plants, where the infected
leaf was removed seven days after the initial infection and
challenged ten days later, were characterized by a
progressive lessening of the level of resistance, in newer
leaves, to challenge by Q. laggnazigm. This demonstrated
that the inducing signal comes from only the infected leaf
(Dean and Mud, 1986). Salicylic acid was shown to increase
in concentration in cucumber plants induced to become
resistant with both Q. lagenazium and tobacco necrosis virus
and to also induce resistance (Métraux et al., 1990). In
work with tobacco cultivars infected with tobacco mosaic
virus, it was found that salicylic acid levels increased in
resistant but not in susceptible cultivars and that

induction of proteins related to resistance rose in parallel

8
to salicylic acid (Malamy et al., 1990). These two studies
suggested that the inducing signal for induced resistance
may be salicylic acid.

Xuei et al. (1988) examined the ultrastructural changes
that occurred in the leaves of control and induced resistant
cucumber plants during infection. Plants were induced by
inoculation on the first leaf with Q. laggngzigm and
challenged with Q. lgggnazium on the second leaf. Few
appressorial penetrations into induced leaves at 48 hours
were observed to occur whereas approximately 70% of the
appressoria on control leaves had penetrated host epidermal
cells. They found no ultrastructural differences between
induced and control leaves. Conidia on control leaves
germinated to produce appressoria by 24 hours after
challenge. Appressoria had electron-opaque inner walls,
electron-lucent outer walls, and "associated cone-like
structures". In Q. lindemuthianum a similar structure was
termed an 'internal flange', attached to which were 'cone-
like structures' (Mercer et al., 1975). Xuei et al. (1988)
'did not differentiate between internal flanges and cone-like
structures. Penetration into epidermal cells by penetration
pegs occurred after 48 hours. Papillae occurred around the
sites of penetration between the plasmalemma and plant cell
wall. By 120 hours infected cells were dead. In some cases
infection by fungi into induced leaves were compatible as

happened in control leaves. The epidermal cell walls under

9

some appressoria were more electron-dense than was found in‘
control leaves. Fewer appressoria were seen to have
penetrated epidermal cells by 48 hours than were seen in the
control.
Ultrastructure of fungal infection

The infection of plants by other members of the genus
lelgtgtrighum has been studied ultrastructurally. Politis
and Wheeler (1973) examined the infection of maize leaves by
Q. graminiggla. Compatible (Mercer et al., 1975) and
incompatible interactions were studied in Bhaseglgs yulgaris
infected by Q. linggmutniannm (O'Connell et al., 1985).

Both :- graminicela and Q. lindemuthianum infected in the
same manner as Q. laggnazinn by directly penetrating through

the epidermal wall by means of melanized appressoria from
which penetration pegs develop. Q. graminiggla only took 9
hours to begin penetration after inoculation with conidia
(Politis and Wheeler, 1973). Penetration by Q.
lindemuthianum into Phaseglus yulgaris was reported to begin
40 to 50 hours after inoculation (Mercer et al., 1975).
Papillae formed in maize infected by Q. graminiggla (Politis
and Wheeler, 1973). The localization of hydroxyproline-rich
glycoproteins (HRGPs) in papillae, and of chitin by
immunocytochemistry were described for Eggseglus yulgaris
cultivars infected by races of Q. lindemuthianum (O'Connell
et al., 1990; O'Connell and Ride, 1990). HRGPs were found

in the cell walls of living cells next to dead

10
hypersensitive cells, which resulted from the incompatible
reaction to infection of the hypocotyl of Ehafigglus yulgaris
by Colletotrighum linggmntniangm (O'Connell et al., 1990).
HRGPs were suggested to act as a mechanical barrier to
further fungal penetration and as a place for deposition of
lignin (O'Connell et al., 1990).

Wolkow et al. (1983) examined the effects of melanin
biosynthesis inhibitors on the ultrastructure and
pathogenicity of Q. lindgmuthiangm. Comparisons of melanin
minus mutants and melanin biosynthesis inhibitor treated
conidia with normally melanized controls showed that melanin
is necessary for penetration of epidermal cell walls. The
ability of Maggapgzthg griggg, the rice blast pathogen, to
penetrate through the epidermal wall was also found to
depend on the melanization of the appressoria (Howard and
Ferrari, 1989). Melanin was found to allow appressoria to
resist plasmolysis when placed in higher solute
concentrations than could unmelanized appressoria. It was
hypothesized that melaninization creates a permeability
barrier allowing for a high internal hydrostatic pressure in
the appressoria which enabled the penetration peg to pierce
the plant cell wall (Howard and Ferrari, 1989).
mammwmm

Hammerschmidt et al. (1985) examined the nature of
constitutive resistance in cucurbits. They found that

epidermal peels from cucurbit hypocotyls inoculated with Q.

11
W. 5;- mm and WM arbo um.
which were stained by phloroglucinol-HCl, were also stained
by KMnO,. Treatment of epidermal peels with hot (80°C)
aminoethanol, which is a delignifying agent, removed the
material which reacted with phloroglucinol-HCI and KMnO,.
Hammerschmidt et al. (1984) determined that increases in
hydroxyproline, a constituent of extensin, and lignin
occurred in cucumber cultivars resistant to ngggspgzium
ngumeginum but not in susceptible cultivars. Earlier,
Esquerré-Tugayé et al. (1979) found that the resistance of
muskmelon (Cucumis mglg L.) to Q. lagenarium could be
enhanced by treating seedlings with hydroxyproline or by
treatment with ethylene which increased levels of extensin
in plants.

Lignin has been localized, using phloroglucinol staining
for light microscopy, around the sites of appressoria on the
petiolar epidermis of cucumbers with induced and cucurbits
with non-host resistance, where it appeared to cause the
inhibition of fungal penetration (Hammerschmidt and Kuc,
1982; Hammerschmidt et al., 1985). Lignins were shown to be
polymerized by peroxidase (Negrel and Lherminier, 1987).
Increased peroxidase activity occurs in induced plants, and
is the result of the enhancement of the levels of acidic
isozymes, which are thought to be the type of peroxidase
involved in the polymerization of lignin (Negrel and

Lherminier, 1987). However, peroxidase in induced plants

12

has not been localized using any form of microscopy (Smith
and Hammerschmidt, 1988). Ultrastructural localization of
peroxidase and lignin around the sites of infection could
provide additional evidence supporting the role of both
compounds in induced resistance.
glcgaccructural lccalizagicn cf lignin

Lignin has been localized at the ultrastructural level
with the KMnO4 staining procedure (Hepler et al., 1970;
Hayat, 1986) . Phloroglucinol, like KMnOU will combine with
many cellular components. Both, however, stain lignin and
not the other cell wall constituents, such as cellulose,
hemicellulose, pectins (Hepler et al., 1970). Tests on the
reactions of 10an4 with a range of substances further
substantiated the lack of reactivity of I<Mn04 with most cell
wall substances, except for polyphenolics such as lignin
‘(Bland et al., 1971). Both phloroglucinol and KMnO4
staining for light microscopy gave equivalent results on
the lignification in infected cucumbers (Hammerschmidt et
al., 1985) . The usefulness of 1041104 staining might
therefore be limited to structures, such as the cell wall,
where the constituents are limited in number and the
material of interest is the only one which reacts with
I<Mn0,.

Lignin has also been localized ultrastructurally in woody
plants by the use of bromine as a stain, which was then

detectable by energy dispersive x-ray microanalysis (EDS)

13
(Saka et al., 1978). This technique has not been applied to
research on herbaceous plants.

Energy dispersive x-ray microanalysis (EDS) is a
technique by which the x-rays produced by the interaction of
the electron beam and the sample, are gathered and converted
to a spectrum. Different elements have characteristic X-ray
peaks (Sumner, 1983). The use of EDS in histochemical
studies has occurred since the development of EDS (Hale,
1962). EDS, though, has not been extensively used for this
purpose (Sumner, 1983). In general, the greatest limiting
factor in the use of EDS with biological specimens has been
the mobility of the ions of interest during processing of
samples. Where this has been a problem in samples fast
freezing techniques have been used (Sumner, 1983).
Histochemical staining results in the deposition of
materials which are usually not mobile. In certain stains,
it is possible to determine the location of the stain due to
a characteristic element which is not ordinarily found in
the sample (Sumner, 1983). Then it is possible to determine
the localization of the stain with an electron microscope,
even if the stain is difficult to distinguish in the
electron microscope, as in the case of bromine and potassium
permanganate.

Ultrastructural lccclicccicc cf cilicon
Another use of EDS is to determine the number and

location of elements (Sumner, 1983). Kunoh and Ishizake

14
(1976) used EDS to show that silicon accumulated around the
penetration sites on barley of powdery mildew, Eryciphc
gzcminic hcrdei and in the papillae. Zeyen et al. (1983)
also used EDS with specimens of barley to localize silicon
in papillae and in epidermal cells that died after infection
by powdery mildew.

Kunoh and Ishizaki (1975) used microincineration to
demonstrate the presence of silicon around infection sites
of E. gccminic hcccci on barley but did not find silicon in
cucumber cotyledons infected by chlccccrichgm lcgcnccicm.
They suggested that silicon accumulation was a generalized
defensive response of barley.
ultrastrnc.tu__lra localization of 9219119251

Behnke (1969) adapted the use of the ultrastructural
technique for peroxidase staining of Graham and Karnovsky
(1966) to the staining of endogenous peroxidase in animal
tissue after fixation with glutaraldehyde fixation. Goff
(1975) used this technique to localize peroxidase in onion
root tips and determined that peroxidase was associated with
the cell wall, cell membrane, as well as other membranous
systems in root cells.

Resistance 1.1M bx the boat shook of seedling onounbota

Resistance can also be induced for a shorter period of
time in seedling cucumbers by heat shock (Stermer and
Hammerschmidt, 1984). For the first 12 hours after the heat

shock, seedlings are more susceptible to infection than are

15

control. After this period, resistance to infection
develops and this resistance can last for over two days
(Stermer and Hammerschmidt, 1984). Resistance induced by
both heat shock and prior infection are nonspecific and
effective against a wide range of pathogens (Stermer and
Hammerschmidt, 1984; Hammerschmidt et al., 1982). Also, in
both systems, resistance is correlated with the enhancement
of peroxidase concentrations of induced plants (Stermer and
Hammerschmidt, 1984; Hammerschmidt et al., 1982). It has
been postulated that the enhanced levels of extensin and
peroxidase are indications of the increased deposition of
cell wall material and that the increase in cell wall
thickness is responsible for the nonspecifity of resistance
due to the greater difficulty encountered by pathogens in
penetrating host cells (Esquerré-Tugayé et al., 1979;
Hammerschmidt et al., 1982).
9mm

The objectives of this research were to determine what
the role of the epidermal cell wall compared to the
cytoplasm was in the defense of cucumber plants against
infection by chlcccczichgm lcgcncgicm. This was to be
accomplished by studying the fine structure of the initial
events of infection to assess the relative importance of
different structures in the cell wall and cytoplasm to the
course of infection. Since lignin, extensin, and silicon

are known to be important in disease resistance in plants,

16
the localization of these materials was compared with the
various resistance structures in cells to see if there was a
correspondence between these materials and the cell
structures. Peroxidase is responsible for polymerization of
lignin and extensin so the localization of peroxidase was

determined and compared to that of lignin.

CHAPTER II

ULTRASTRUCTURE AND HISTOCHEMISTRY OF THE INFECTION OF

CUCUMBERS, INDUCED TO BE RESISTANT, BY QQLLETOTRICHUM

LAGENARIQM

17

18

ABSTRACT

Lignin is hypothesized to be a factor in the ability of
plants to resist infection. To determine if lignin could be
localized around sites of infection, control (susceptible)
cucumber plants and plants with systemic resistance induced
by injections of Ecccgcmcncc cycingcc pv. cygingac were
challenge inoculated with QQLIBLQLLiQhEE lagenarium.
Genetically resistant seedlings of pumpkin were also
inoculated and examined. The ultrastructure of plant and
fungus was examined 24 to 72 hours after inoculation.
Conidia produced germ tubes with appressoria by 24 hours.
Appressoria developed internal elaborations of cell wall
around the origin of penetration pegs. In cucumber,
penetration of plant epidermal cell walls occurred by 48
hours with papillae formed at the site of penetration. Both
elemental bromine and potassium permanganate were used to
histochemically stain for lignin, which has been associated
with resistance in cucumbers. Potassium permanganate, used
in concert with energy dispersive X-ray microanalysis (EDS),
confirmed the presence of lignin in papillae and in
electron-dense areas of plant cell walls directly underneath
appressoria. Using EDS, silicon was localized in the

electron-dense areas of plant cells in mature cucumber

19
leaves, but did not occur in seedling pumpkin hypocotyls.
Cell walls under appressoria in seedling pumpkin hypocotyls
stained with bromine, indicating lignin deposition. The
staining of hypocotyl cell walls by bromine was confirmed by
EDS. This is the first report of silicon in induced
cucumber tissue. There seemed to be no qualitative
difference in the resistance reactions that occurred in

induced and control plants.

20

INTRODUCTION

Inoculation of cucumber plants with a variety of
different pathogens can induce systemic resistance against
subsequent infection. In this study, the process of
infection by Q. lcgcnccicm race 1 into cucumber with induced
resistance or uninduced, was examined using electron
microscopy and histochemistry to localize lignin and
peroxidase and to determine if the enhancement of these
putative resistance factors could be further associated with

resistance.

METHODS AND MATERIALS

21cc; mcterial

Seeds of cucumber plants (Qccgmic sccivus L., cv SMR 58)
were placed in 1% sodium hypochlorite, washed in deo, and
left to germinate on moistened germination paper for 6 days.
Seedlings were planted in clay pots and grown in greenhouses
under natural light. Resistance was induced by ESBQQQEQDQS
cyzingcc pv. cyxingcc, cultures of which were inoculated
into nutrient broth 24 hours earlier, diluted to ca. 108
cfu/ml, and then injected into the first true leaf.

Injections were done with a 5 ml hypodermic syringe, without

21
a needle, through abaxial stomata. Plants received a second
inducing inoculation, in the second true leaf, 7 days later.
Control plants were not injected, since injury does not
produce the changes associated with induced resistance
(Hammerschmidt et al., 1982).

Seven days after the second inducing inoculation, the
third true leaf of both induced and control plants, were
removed and placed into a humid chamber at 23%:. Six 30 pl
droplets with 3 x 10° conidia/ml of W
lcgcnczicm (Pass.) Ell. and Halst. race 1 were placed on the
adaxial surface of the leaf. These conidia were harvested
into deo from 8 day old cultures which were grown on potato
dextrose agar (PDA) in the dark at room temperature. The
conidia were filtered through four layers of cheesecloth
before being used to inoculate leaves.

Eight day old pumpkin seedlings (9212212133 pcpc cv
Spookie) were also inoculated on the hypocotyl with Q.
lagenggium using an atomizer. Seedlings were rolled into
moist paper towels and kept moist and in the dark for 48
hours. Pumpkin is genetically resistant to infection by Q.
lcgcncgicm and served as an example of constitutive
resistance.

Electron nicrocconx

At 24, 48, and 72 hours, 1 mm square samples of induced

and uninduced cucumber leaves were fixed overnight in 2.7%

glutaraldehyde in 0.05 M, pH 6.8 phosphate buffer at 4%:

22
using vacuum infiltration, washed with buffer (4x),
postfixed in 1% OsO4 in buffer at 4°C for 90 minutes, and
dehydrated in a graded ethanol series. After dehydration
samples were prepared for scanning electron microscopy (SEM)
by critical point drying in a Balzers CPD 10, gold sputter
coated with an Emscope SC 500, and were examined with a JEOL
35CF SEM. Samples prepared for transmission electron
microscopy (TEM) were embedded in ERL epoxy resin (Spurrs,
1969), after the ethanol dehydration, and sectioned on a
Reichert Ultracut E ultramicrotome. Silver-gold sections
L90nm) were stained with saturated uranyl acetate in water
followed by lead citrate (Reynolds, 1963), and examined
using a Philips 201 TEM. During the course of this study
299 appressoria were examined and photographed.

Most comparisons were made at 48 hrs after infection,
since at 24 hours no examples of penetration of the
epidermal cell wall were seen, and at 72 hours, primary
hyphae had ramified well into host cells. Penetration was
found to occur at roughly 48 hours. 283 appressoria were

examined from samples of cucumber leaves at 48 hours after

infection by Q. lcgcnczicm.
1911194 Staining:

Ultrathin sections were collected on nickel grids, placed
into an 1.5 ml Eppendorf microcentrifuge tube filled with 1%
KMnO4 for 30 minutes, and washed in distilled water (Hayat,

1970) . 10an4 was used because it has been found to localize

23
lignin (Hayat, 1970; Hayat, 1986). Sections stained with
KMnO4 were examined for the presence and localization of
manganese with a JEOL 100CX TEM equipped with a scanning
attachment and a Link Systems AN 10000 energy dispersive X-
ray analyzer.
Bromine staining procedure:

At 48 hours after inoculation, 1 mm square samples of the
third leaf of induced and control plants and 1 mm long
portions of inoculated and uninoculated pumpkin seedlings
were fixed in 2% paraformaldehyde in 0.05M phosphate buffer,
pH 6.8 (Hayat, 1972). Pumpkin seedlings served as an
example of constitutive resistance that were compared with
the induced resistance of cucumber plants. Samples were
washed in buffer (4x) and brominated by the technique of
Saka et al. (1978), where specimens dehydrated in a graded
ethanol series were transferred to chloroform through
intermediate ethanol/chloroform steps, placed into a
solution of 0.3 ml bromine in 20 m1 chloroform for 3 hours
and washed in chloroform (4x) to remove bromine from the
solution. Samples were left in chloroform for 10 days with
the chloroform being changed daily to extract out unbound
bromine. Samples were taken through a chloroform/ethanol
transition into ethanol and through an ethanol/ERL epoxy
resin transition before being placed into 100% ERL resin
(Spurr, 1969). Samples were then embedded and sectioned as

described in the section on transmission electron

24
microscopy. Sections were not stained with uranyl acetate
or lead citrate and were examined for Br and Si using a JEOL
100 CX TEM equipped with a scanning attachment and a Link
Systems AN 10000 energy dispersive X-ray microanalyzer. In
this procedure 21 appressoria from seedling pumpkin and 16
appressoria from mature cucumber leaves were examined.
Enercx dispersing x2131 nicroanalxcie

x-ray spectra from 0.840 ev to 20.220 ev were collected
for each sample examined.

Energy dispersive X-ray microanalysis (EDS) dot maps of
KMnO4 stained sections and brominated material were
constructed by x-ray collection of a specified ion over a 1
hour time period. Black pixels in the computer image
indicated the presence of the ion.
121mm mining

Q. lcgcnccicm infected leaves of control susceptible and
induced resistant cucumber were fixed in glutaraldehyde, as
above, followed by treatment with a solution of 0.2 mg
diaminobenzidine (DAB) in 1.0 ml of 0.1 M Tris-HCl, pH 7.6,
with 0.2% HRH for one hour (Goff, 1975). Peroxidase
catalyzes the oxidation of DAB, producing an osmiophilic
precipitant. Samples were then washed twice with buffer,
postfixed (stained) with OsO, and prepared for TEM as above.
Samples of control and induced cucumber were not treated
with HRH to act as a check on treated samples. Ultrathin

sections were not stained with uranyl acetate or lead

25
citrate. The presence of peroxidase was indicated by the

formation of osmiophilic deposits (Goff, 1975).

RESULTS

Inocnlationofnlonto

Cucumber plants which were injected with Ecccccmcncc
cycingcc pv cyzingcc developed necrotic lesions at the sites
of injection (Fig. 1). The second true leaf, which
developed after the injection, frequently developed a type
of veinal chlorosis (Fig. 2). This was especially true if
the first true leaf was injected when the second true leaf
was less than 1.0 cm long. If the first true leaf was
injected after the second true leaf was over 30% of its full
size then the vascular chlorosis did not appear. Chlorotic
leaves appeared to possess less laminar than injected plants
without chlorosis and, in general, were smaller in size.
Also in general, injected cucumber plants were smaller than

the uninjected control plants (Fig. 3).

glczastzccture of QDPIQEEQLLQ

At 24 hours after inoculation, QQllQLQLLiQth lcgcncgicm
conidia had germinated and produced a germ tube (Fig. 4).

The germ tube was variable in length. Appressoria formed
from the end of the germ tube (Fig. 4). No ultrastructural
differences were seen in the appearance of the fungus or
timing of germination on induced or uninduced tissue (Figs.

5 and 6). An extracellular matrix produced by appressoria

26

Fig. 1. First true leaf of Qccumic cativc cv SMR 58 plant
induced for resistance one week earlier by infection into
the abaxial surface with Pseudomonac syringae pv syringac.
Necrotic lesions (N) are the result of bacteria injection.

Fig. 2. Second leaf of injected cucumber plant, such as in
Fig. 1, where the injection has resulted in veinal
chlorosis.

Fig. 3. Injected SMR 58 cucumber plants are located on the
left of rear bench next to uninjected controls on the right
of rear bench. The controls (arrowhead) are larger in size
than the injected plants.

 

 

28

Fig. 4. Scanning electron micrograph of germinated conidia
(C) of Colletotrichum lagenarium with germination tube (GT)
of variable length and appressoria (A) 24 hrs. after
inoculation upon control leaves. Bar = 10 um.

Fig. 5. Scanning electron micrograph of conidium (C) with
appressorium (A) on control leaf 24 hours after inoculation
with Q. lagenarium showing matrix layer (ML) around
appressorium. Bar = 1 pm.

Fig. 6. Scanning electron micrograph of germinated spore on
induced leaf 24 hrs. after inoculation with Q. lagenarium,
showing matrix layer (ML) (arrows) around appressorium (A).
.Conidium (C) and Bacteria (B). Bar = 1 pm.

Fig. 7. Transmission electron micrograph of ultrathin
section of appressorium (A), portion of conidium (C), and
plant cell wall (CW) from control plant 48 hrs. after
inoculation with Q. lagenacium. Section was uranyl acetate
and lead citrate stained. Conidium wall appears less
electron-dense than appressorium wall. The plant cell wall
under the appressorium is indented (arrow). Internal flange
(F), extracellular matrix layer (ML), and bacterium (B).

Bar = 1 pm.

 

30

was located at the area of contact between appressoria and
host plant and appeared to bind appressoria to the plant
surface (Figs. 5, 6 and 7). By 48 hours after inoculation,
penetration pegs formed in appressoria on control and
induced cucumbers and had broken through the plant epidermal
outer cell walls (Fig. 8). Appressorial walls appeared more
electron-dense than spore walls due to melanization (Fig.
7). The appressorial wall also possessed two wall layers:
the electron-dense, thick outer and an inner, more electron—
lucent layer (Fig. 9). The inner layer of the appressorial
wall turned inwards into the cytoplasm of the appressorium
(Figs. 7 and 9) and, based on examination of serial
sections, formed a funnel-like structure (Fig. 9). Cone-
like structures arose from the ends the ends of the internal
flange and were apparently continuous with the innermost
layer of the appressoral internal flange (Fig. 9). The
innermost layer of the internal flange appeared to be
continuous with the wall of the penetration peg (Fig. 8).
Sections prepared without post-staining with uranyl acetate
or lead citrate showed that the cone-like structures stained
poorly with OsO4 compared with the internal flange itself
(Figs. 9 and 10). The cone-like structures, towards the
top, often branched into different layers (Fig. 6).

Portions of the internal flange and the cone-like structures
were seen in most appressorial sections and appeared to
occupy much of the basal portion of the appressorium (Table

1).

31

TABLE 1
Occurrence and appearance of internal flanges observed in
ultrathin sections of appressoria

 

Whole cross Partial cross No internal flange
sections of section of
internal flange internal flange

 

Control 11 10 8

Induced 18 27 22

 

Numbers are taken from the inspection of micrographs of
samples, 48 hours after inoculation with chlccccgichgm
lcgcnccicm, prepared by glutaraldehyde/osmium tetroxide
fixation.

32

Fig. 8. Appressorium on control plant, transmission
electron micrograph of ultrathin section, 48 hrs. after
inoculation with Q. lagenaricm, the infection peg (IF) has
broken through plant epidermal cell wall (CW). Section
stained with uranyl acetate and lead citrate. Cell wall
next to infection peg is less electron-dense than
surrounding wall. Wall under appressorium (A) appears
indented towards the plant cell cytoplasm but rest of cell
wall layers do not appear to be disturbed. Bar = 1 pm.

Fig. 9. Appressorium from control plant. Transmission
electron micrograph of the appressorial cell wall shows
outer (O), and inner (I) layers. The internal flange (F)
and the extension (E) differ in electron density from each
other. Bar = 0.2 pm. .

Fig. 10. Transmission electron micrograph of ultrathin
section of appressorium (A) and plant epidermal cell wall
(CW) from control plant 48 hrs. after inoculation with Q.
lagenazium. Section was not stained with uranyl acetate and
lead citrate. Bar = 1 pm.

Fig. 11. Transmission electron micrograph of ultrathin
section, stained with uranyl acetate and lead citrate, of
appressorium (A) on protected tissue at 48 hrs after
infection with Q. lcgcncgicm. Cell wall (CW) of plant under
appressorium is electron-dense (D) with electron-dense
granules (GR) between host cell wall and plasmalemma (PL).
Bar = 1 pm.

     
      
      

  

.3'

j

. f!

  

’II'
t:

(II A}.

. .‘..‘.

34
Effecteofmncnconolontcellnall

The areas beneath the appressoria, the cuticle and upper
portion of the plant cell wall of both control and induced
plants were indented, suggesting that penetration by
penetration pegs through the cuticle was achieved by
mechanical force (Fig. 7). Control and induced plant cell
walls immediately next to successful penetrations were less
electron-dense than the wall areas 1pm further away from the
point of penetration (Fig. 8). The cell wall layers nearer
the cytoplasm were cleanly cut and not compressed as was the
cuticle and outer layers of the cell wall (Fig. 8). This
could indicate the breakdown of cell walls by enzymatic
degradation (Fig. 8).

Certain plant cells in contact with appressoria, in both
control and induced plants, had cell walls which were more
electron-dense than surrounding plant cells (Fig. 11). This
was noted in 4 out of 34 appressoria in the control
treatment at 48 hours and 8 out 85 appressoria in the
induced plants at 48 hours (Table 2). No cases were seen in
which these electron-dense cell areas were penetrated by the
fungi.

In appearance the electron-dense plant cell areas were
not much wider than the appressorium actually in contact
with this area (Fig. 11). In addition, a number of electron-
dense granules occurred between the electron-dense plant
cell wall and the plasmalemma (Fig. 11).

When 10an4 was used to stain sections in which the

35

TABLE 2
The presence of electron-dense cell walls at the sites of

infection caused by colletotricnnn lacenarinn

 

 

 

48 hours after 72 hours after

inoculation inoculation
Control 4/34 0/4
Induced 8/85 1/6

 

Samples were prepared from the third leaf of cucumber plants
which were induced for resistance by injections of
Eccudomonas syringac pv cycingcc into the first and second

true leaves. Qolletoccichum lggenagium was inoculated onto
the third leaf and the sites of inoculation were fixed 48

hours and 72 hours later with glutaraldehyde and osmium
tetroxide. The numerator expresses the number of
appressoria in contact with electron-dense plant cell walls.
The denominator is the total number of appressoria.

36
electron-dense epidermal cell areas occurred under
appressoria, the electron-density of these sites did not
appear to increase (Figs. 12 and 13). EDS indicated that Mn
from the KMnO4 stain was localized in this portion of the
host cell (Fig. 14). The staining of this area by KMnO4
indicated the presence of lignin. In addition, silicon was
found to be localized in the electron-dense plant cell areas
(Fig. 15).

To determine if silicon was actually present in the
electron-dense plant cell area before the tissue was
prepared, to confirm the localization of lignin that was
obtained by KMnO4 staining, and to find whether the
electron-dense plant cell areas were electron-dense without
OsO4 post-fixation, control and induced cucumber leaves were
inoculated with Q. lcgccccicm and sampled 48 hours later and
then prepared by the bromine technique.

The brominination and further preparation of mature
cucumber leaves, inoculated 48 hours previously with
Q.1cgcnccicm, extracted much of the cytoplasmic content of
the cells (Fig. 16). Electron-dense plant cell areas were
also found proximal to appressoria in this treatment (Fig.
16). This occurred in induced plants, in 1 out of 4 cases
and in uninduced plants in 2 out of 12 cases all of which
were analyzed for bromine. In appearance, the electron-
dense areas appeared to be very similar to those seen in
.glutaraldehyde/ 0804 fixed cucumber leaves.

All of brominated cucumber leaf samples with electron-

37

Fig. 12. Transmission electron microscopy ultrathin
section, stained with KMnO,,<of appressorium (A) on induced
plant inoculated 48 hrs previously with C. 1cgenaziu .
Appressorial walls (AW) are heavily stained by KMnO, due to
the presence of melanin. The region of the plant cell wall
between the arrowheads and proximal to the appressorium is
electron-dense. Bar = 1 pm.

Fig. 13. Unstained serial section of specimen in Fig. 12.
Region of plant cell wall between the arrowheads corresponds
with that of Fig. 12. Appressorium (A). Bar = 1 um.

Fig. 14. EDS dot map from a serial section of specimen in
Fig. 12 indicating the localization of manganese. The
electron-dense cell wall (marked by arrowheads in Fig. 12)
of the plant under the appressorium is more heavily stained
than surrounding areas of the plant cell wall. Appressorium
(A).

Fig. 15. EDS dot map of Si in Fig. 13. The arrowheads
correspond in position to those found in Figs. 12, 13, and
14.

 

39

Fig. 16. Transmission electron microscopy ultrathin section
of brominated mature cucumber leaf that was fixed 48 hours
after inoculation with Q. lagenarium. Plant cell wall (CW)
proximal to appressorium (A) is electron-dense (marked by
arrowheads). Bar = 1 pm.

Fig. 17. Br EDS dot map of Fig. 16. arrowheads below
appressorium (A) correspond to the plant cell wall (CW)
similarly marked in Fig. 16.

Fig. 18. Si EDS dot map of Fig. 16 with arrowheads in the
same position as those in the thin section.

Fig. 19. Transmission electron micrograph of ultrathin
section of tissue, from protected plant at 48 hrs after
inoculation with Q. lagenacium. Section was stained with
KMnO,. Plant cell wall under appressorium (A) is more
electron-dense than surrounding wall. Appressorium is
misshapened and appears necrotic. Bar = 1 pm.

 

41

dense cell areas were then analyzed for bromine and silicon.
It was found that in brominated samples, no examples of
bromine concentration in the electron-dense areas could be
found by EDS (Fig. 17), although silicon was readily found
(Fig. 18).
ficccccic appressczic

Induced and control plants inoculated with Q. lagenacium
showed many of the same features including, necrotic
appearing appressoria (Fig. 19). In many cases necrotic
appressoria were associated with deposition of cell wall
material (Fig. 19). Numbers of necrotic appressorium were
often found together on both induced and control plants
(Fig. 20).
aniline

Papillae usually formed where penetration hyphae had
pierced through the cell wall (Figs. 21 and 22). The
cytoplasm around papillae had large numbers of mitochondria,
golgi, abundant vesicles and rough endoplasmic reticulum.
In both induced and control plants, papillae consisted of a
layer of granular material (Fig. 21). The plant cell
membrane around this layer was highly invaginated. The
extraplasmalemmal material continued to surround hyphae as
the hyphae elongated through cells (Fig. 23).

At 72 hours, this layering of papillae and cytoplasm was
observed often to continue as primary hyphae ramified
throughout the plant cells (Fig. 22). Both the papillae and

the host plant cytoplasm surrounding the hyphae were thin.

42

Fig. 20. Transmission electron micrograph of ultrathin
section of necrotic appressoria on induced tissue 48 hrs.
after inoculation with Q. lagenacium. Pockets of
unsuccessful penetrations, as well as successful ones, often
occurred. Papilla (P). Bar = 10 um.

Fig. 21. Transmission electron micrograph of ultrathin
section, stained with uranyl acetate and lead citrate, of
papilla of protected plant at 48 hrs after inoculation with
Q. lcgcnarium. Papilla (P) with granular material.
Plasmalemma of plant cell (arrowhead) surrounds papilla.
Bar = 1 pm.

Fig. 22. Transmission electron micrograph of ultrathin
section of primary hypha (PH), 72 hrs., showing successful
penetration of Q. lagenarium into protected plant. Section
was stained with KMn04. Material between hypha and
plasmalemma appears to have smaller particles than at 48
hrs. (Fig. 21). The material surrounding the hypha,
adjacent to the plant cell wall (arrow), is stained with
KMnO, indicating the presence of lignin. Bar = 1 pm.

Fig. 23. Transmission electron micrograph at 48 hours after
inoculation of induced leaf with Q. lagenagium. Section was
stained with KMn04. Primary hypha (PH) has elongated into
plant cell and is surrounded by papilla (P). Bar = 1 pm.

 

44
The plant cell cytoplasm around the papillae possessed few

organelles at 72 hours after inoculation.

Papillae in sections of induced and uninduced tissue
prepared by glutaraldehyde/osmium tetroxide were often
stained by KMn04‘treatment (Figs. 24 and 25). The amount of
staining could be seen by comparing unstained and KMnO4
stained serial sections. Transmission EM energy-dispersive
X-ray microanalysis (EDS) of KMnO4 stained sections
confirmed the localized accumulation of manganese from the
KMn04‘treatment in papillae (Fig. 26). Appressorial walls
were also heavily stained. This was due to the presence of
melanin, a polyphenolic compound similar in structure to
lignin which also reacted with KM'nO4 (Figs. 12 and 14).
Pemidcce lmlizetion

Staining for peroxidase in infected control and induced
cucumbers using DAB revealed a layer of material around the
outside of appressoria (Figs. 27 and 28). This layer was
only faintly stained with uranyl acetate and lead citrate
staining (Fig. 21) and somewhat more densely stained with
mm, (Fig. 12).

In addition, DAB-OsO4 stained electron-dense plant cell
walls that were under appressoria in both control and
induced plants (Figs. 27 and 28). In some cases the plant
cell wall directly under the appressoria did not stain while

the areas surrounding this did stain with DAB (Fig. 29).

45

Fig. 24. Transmission electron micrograph of ultrathin
section of hypha (H) with papilla (P), on a protected plant
inoculated 48 hrs. previously with Q. lcgccczicm. Section
was stained with KMnOv Material in papilla around hypha is
electron-dense, indicating the presence of lignin. Bar = 1

pm.

Fig. 25. Unstained transmission electron microscopy
ultrathin serial section of same material as Fig. 24.
Papilla (P), hypha (H). Bar = 1 pm.

Fig. 26. Scanning transmission electron microscopy X-ray
microanalysis dot map demonstrating the presence of
manganese (from KMan) in a serial section of hypha shown in
Fig. 24. Papilla (P), hypha (H).

Fig. 27. Transmission electron microscopy ultrathin section
of diaminobenzidine-0504 stained protected tissue at 48 hrs.
after inoculation with Q. lcgcnaricm showing increased
staining (arrowhead) around outside of appressorium and also
staining between electron-dense plant cell area and plant
plasmalemma, demonstrating the localization of peroxidase.
Bar = 1 pm.

 

47

Fig. 28. Transmission electron micrograph of section of
control plant, inoculated 48 hours before fixation with Q.
lcgenacium, and stained for peroxidase using
diaminobenzidine-Oso4 (DAB). The interface between the
electron-dense area of the plant cell wall and the
plasmalemma as well as the outer surface of the appressorium
are stained by the DAB. Bar = 1 pm.

Fig. 29. Transmission electron micrograph of material from
induced plant which had been inoculated with Q. lcgenazium
48 hours before and stained for peroxidase using DAB. The
plant cell wall around area of fungal penetration lacks
staining (arrowhead). Bar = 1pm.

Fig. 30. Transmission electron micrograph of the epidermis
of the hypocotyl of seedling of the Spookie cultivar of
pumpkin (resistant) which had been inoculated 48 hours
earlier with Q. lagenarium and fixed with paraformaldehyde
and incubated in bromine/chloroform solution. Appressorium
(A) and plant cell wall (CW). Bar = 1 pm.

Fig. 31. Scanning transmission electron microscopy X-ray
microanalysis dot map from a serial section of Fig. 30
indicating the localization of bromine. The electron-dense
cell wall of the plant under the appressoria (A) is more
heavily stained than surrounding areas of the plant cell
wall (CW) indicating the presence of lignin. Cell walls of
adjacent cells are not stained.

 

31

49
fizcminaticn of RBEEKLD DYRQQQEYlE

Pumpkin hypocotyls were as heavily extracted by bromine
treatment as were mature leaves (Fig. 30). The EDS for
bromine in seedling pumpkin cells showed that areas of
greater bromine concentration were found in the cell walls
of the cells proximal to appressoria and not in neighboring
cells (Fig. 31). The portion of the pumpkin cell wall that
contained bromine was greater relative to the size of the
cell than the electron-dense area of the cucumber leaf (Fig.

12 and 13).

50

DISCUSSION

Appresscria ultrasczcctgce

Appressorial walls were found to be made up of two
layers, an electron opaque outer wall and an electron lucent
inner wall. Xuei et al. (1988) also described appressoria
on control leaves as having an electron-opaque inner wall
and electron-lucent outer wall. In addition a loose
fibrillar material surrounded the outer appressorial wall.
It was poorly seen in uranyl acetate/lead citrate stained
glutaraldehyde/osmium tetroxide fixed sections but 1(1an4 and
especially, DAB-OsO4 staining increased its visibility. A
similar layer was also found in Q. lindemuthianum (Wolkow et
al., 1983).

The internal flange existed in the appressoria as an
inturning of the cell wall surrounding the penetration peg.
Xuei et al. (1988) also noted the presence of these
elaborations of the wall structure in Q. lagenarium
appressoria, an identical structure in Q. lindemuthianum was
termed a internal flange (Mercer et al., 1975). Prominent
continuations to the internal flange were seen in this study
and were also reported to occur in Q. lingemuthianum where
the structure was called the associated cone-like structure
(Mercer et al., 1975).

In this study, appressorial walls of Q. lagenaricm were

51
stained by KMnO4. Melanin is known to a constituent of the

walls of appressoria in Q. lindcmcchicncm (Wolkow et al.,
1983) as well as other fungi (Howard and Ferrari, 1989).
10an4 is known to stain appressorial walls (Hayat, 1986) and
melanin, with a polyphenolic structure like that of lignin
would therefore account for the observed appressorial wall
staining by KMnO,.
Matrix leyet

In the area of contact between appressoria and plant
surface there occurred an extracellular matrix between
appressoria and plant cell walls. The matrix appeared in
both SEM and TEM. There is evidence to believe that the
threadlike appearance of the matrix seen in Fig. 2 is an
artifact since SEM of frozen hydrated specimens do not show
this feature (Ralph Nicholson, personal communication). The
matrix might be related in some way to the fibrillar
material that was found to surround appressoria. This
matrix appeared to bind appressoria to the plant surface
and, perhaps, allowed for the application of pressure by
appressoria against the plant surface, thereby causing the
plant cell wall indentations observed under appressoria.
Effecteo_f_c__fun usonnlenccellnoll

In the successful penetrations through the epidermis of
control and induced plants by appressoria, evidence was seen
in micrographs for the compression of the cuticle and
outermost layers of the host cell wall beneath the

appressorium. The inner wall layers of the epidermal cell

52
did not appear to be compressed by the penetration pegs but

the cell wall next to the penetration peg did not stain with
uranyl acetate and diaminobenzidine-0304 as intensely as the
surrounding cell wall. Q. lcgcnczigm was found to form an
indentation on the epidermal outer cell wall and penetrated
through plant cell walls without causing further disruption
of the wall layering. Xuei et al. though, reported little
or no compression. Wolkow et al. (1983) found that the base
of the appressorium of Q. linccmcchicngm also often fit into
a indentation in the plant cell wall and only the upper part
of the plant cell wall was affected, the rest appeared
undisturbed. In contrast, Q. gccmicicclc (Politis and
Wheeler, 1973), was found to break through plant cell walls
without compressing the walls.

An inhibitor of the melanin pathway was used to eliminate
the electron-dense portion of the appressorial wall in Q;
linccmcchiancm (Wolkow et al., 1983). This decreased the
ability of appressoria to penetrate through normal
Brycphyllcm epidermis, but not dewaxed epidermis (Wolkow et
al., 1983). This suggested that the pressure of the rigid
melanized appressorial walls against the plant surface
accounted for the ability of appressoria to penetrate
through plant cuticles. Wolkow et al. (1983) also
determined that a fungal cutinase inhibitor delayed, but did
not stop, penetration and that melanization was greatest on
the thickest film of formvar plastic upon which the

conidiospores were germinated. Bonnen and Hammerschmidt

53
(1989a) found that, while Q. lcgcnczicm did produce

cutinase, cutinase was not necessary for the penetration of
plant hosts by appressoria (Bonnen and Hammerschmidt,
1989b).

These studies are in accord with the observation that
only the outer layers of the cucumber plant cell appeared to
be pushed by the appressoria of Q. lcgccccicm. These
findings indicated that mechanical pressure of appressoria
allowed for the further penetration by means of enzymatic
degradation of plant cells by the fungus.

Eleni: cell all reactions to fnncns

Certain plant cell walls in contact with appressoria were
more electron-dense than adjacent areas of the plant cell
wall. These electron-dense cell areas were found in both
control and induced plants. Electron-dense cell areas were
found in samples prepared by the bromine technique and by
glutaraldehyde] osmium tetroxide fixation. In no case was
it seen that penetration pegs broke through electron-dense
areas. For this reason electron-dense cell areas appeared
to be an effective defense against infection. Electron-
dense areas of plant cells were intensely stained without
uranyl acetate/lead citrate or KMn04. 'The electron-density
of the staining did not seem to increase greatly when KMnO4
was used to stain the sections. EDS localized Mn from the
101an4 staining, which indicated that lignin occurred there.
Bromine stained material did not have Br concentrated in the

electron-dense cell walls.

54
Why KMnO4 staining indicated lignin in mature leaves but

Brz did not is not known, though it might have to do with
the chemistry of lignin in the cell walls. It was found
earlier that the composition of lignin deposited as result
of infection and wounding is different from lignin that is
normally deposited (Hammerschmidt et al., 1985;
Hammerschmidt, 1984). Saka et al. (1978) found that bromine
reacts more with syringyl units than with the guaiacyl
units.

Silicon was also found in the electron-dense cell walls.
It is possible that the Si was introduced during tissue
preparation but the fact that Si was also found in electron-
dense cell areas in the tissues prepared by bromination
shows this is unlikely since brominated mature leaves were
heavily extracted during preparation and the only solutions
used in common for the two treatment were phosphate buffer,
ethanol, and Spurr's embedding media.

Silicon was likely bound into the plant cell wall. For
this reason it was relatively immobile despite the
processing procedures used. Because of this bound nature,
the EDS dot maps for silicon most likely reflect the
situation in the living plant.

Sakai and Thom (1979) reported that silicon was
preferentially found in the middle lamella of stomatal cells
of sugar cane and suggested that silicon interacted with
lignin and other phenolics of the middle lamella as well as

other cell wall constituents. Raven (1983) postulated that

55
energetically it is more favorable for plants to deposit

silicon rather than deposit lignin.

Lignin and silicon in the electron-dense areas of the
cucumber cell wall could act as a barrier to further
penetration by the fungus and this would then account for
the fact that no penetration was seen through electron-dense
areas. Penetration through lignifications of the epidermis
of cucumber petiole has been reported (Hammerschmidt and
Rue, 1982). Whether petiole epidermal cells are siliconized
is not known.

Pumpkin seedlings were inoculated to serve as an example
of constitutive resistance, as opposed to the induced
resistance of cucumbers. No electron-dense deposits were
found in seedling pumpkin hypocotyls. Bromine staining gave
evidence that lignification occurred in the epidermal cells
of the hypocotyl of seedling pumpkin. The total area of
lignification in the pumpkin hypocotyl epidermal cell wall
as seen in EDS bromine dot maps was greater than the
electron-dense areas of cucumber epidermal cells. Staining
with phloroglucinol/HCl showed that epidermal cells of the
petiole of cucumber plants did lignify when infected with Q.
lcgcnccicm (Hammerschmidt et al., 1982). The outer
epidermal wall of the infected, as well the anticlinal wall
between the infected and as adjacent cells, were lignified
as seen by phloroglucinol staining and light microscopy.
Lignifications of the epidermal cells of the leaf blade,

when viewed by light microscopy, were confined to just

56
around the area pierced by the penetration peg and did not

extend to the anticlinal walls (personal communication, R.
Hammerschmidt). This corresponds in size to the electron-
dense plant cell walls seen in electron microscopy and
indicates that, in cucumber leaf epidermal cells, the
electron-dense cell walls and phloroglucinol-HCl stained
lignifications are the same. The pumpkin hypocotyl
lignifications were more similar to those seen in cucumber
petiole in terms of the size and the lack of silicon.

Silicon was not found in seedling pumpkin. Kunoh and
Ishizaki (1975) also did not report silicon in cucumber
cotyledons infected by Q. lcgcnczicm. Although the
localization of silicon may be artifactual, the fact that
silicon was found in tissues prepared in such divergent ways
as the bromine and the glutaraldehyde/030,‘bechniques would
argue that silicon was actually present in the electron-
dense regions of the infected plant cell wall. It would,
therefore, seem likely that petiolar epidermis of cucumbers
and hypocotyl of seedling lignify upon infection with Q.
lcgcpcgicm, but that mature leaves of cucumber plants
accumulate silicon. In addition, silicon has been shown to
be important in the defense of cucumber plants against
infection by Eccccicm wilt and by powdery mildew,

Sphacrctheca fuligincg (Miyake and Takahashi, 1983; Adatia
and Besford, 1986).

57
Recluse

Electron-dense deposits not only occurred in the cell
wall but also as granules between the plant cell wall and
the plant plasmalemma. These granules were previously
described as papillae (Xuei et al., 1988). In this
research, the location of the granules between the
plasmalemma and cell wall is also the same as described in
papillae (Xuei et al., 1988). In addition, granules were
more electron-dense and larger than the material that
composed papillae. Granulation in the area of the infection
site has been reported to precede papillae formation in Q.
gccmicicclc on susceptible maize (Cadena-Gomez and
Nicholson, 1987). The study, though, was done with light
microscopy so the appearance of granulation in maize and its
similarities to granules in cucumbers can not be determined.

We also noted that papillae possessed a layer of granular
material, which was between the plasmalemma and the outside
of the hyphae, surrounding the penetration hyphae 48 hours
after inoculation. At 72 hours, as the primary hypha
elongated, this layer of material was still present between
the hypha and plant host plasmalemma, although the particles
of the material in the papillae at 72 hours were smaller
than at 48 hours. This layer stained with KMnO4 indicating
the presence of lignin. A layer that was similarly granular
was also reported in papillae of Q. linccmcchicccm infecting
beans (O'Connell et al., 1985). Hammerschmidt and Kué

(1982) found that mycelia of Q. lcgcnccicm, incubated with

58
coniferyl alcohol, hydrogen peroxide, and a crude cucumber

peroxidase preparation, had a lignin-like substance
deposited on it. Lignification was also seen in papillae
around Q. lcgcnczicm and flclminchccpccicm carbonum in squash
and pumpkin (Hammerschmidt et al., 1985).

Numbers of necrotic appearing appressorium were seen on
control and induced plants, often in association with
papillae. Whether the papillae blocked penetration by the
appressoria and caused the cells to die or whether the
papillae formed after the appressoria became necrotic is not
known.

d se

DAB--OsO4 staining occurred in both induced and uninduced
plants at 48 hours after inoculation with C. lcgcnacicm,
demonstrating that increased amounts of peroxidase were
present in the electron-dense plant cell walls. Peroxidase
is thought to cause the polymerization of lignin (Negrel and
Lherminier, 1987).
cell cell terriers to infection

Barriers to infection in the form of lignification of
cell walls and papillae, as well as the development of
electron-dense deposits between plasmalemma and cell wall of
the host plant, occurred in both control and induced
cucumbers. These mechanisms of defense are, therefore,
common both to induced and control cucumbers, and the
differences in extent of symptom development are probably

related to the number of successful vs. blocked infection

attempts.

59

CHAPTER III

EFFECTS OF HEATSHOCK UPON THE CELL WALL THICKNESSES

AND APPEARANCES OF HYPOCOTYL OF CUCUMBER SEEDLINGS

60

61

ABSTRACT

There were distinct differences between the
ultrastructure of heat shocked and control seedlings, most
notably the dilation of the endoplasmic reticulum and the
increased electron-density of nucleoli of heat shocked
seedIings. Morphometry of the thickness of epidermal cell
walls of the hypocotyls ofheat shocked cucumber seedlings
gave some evidence that heat shock caused an increase in

thickness.

62

INTRODUCTION

Resistance to infection by pathogens can be induced by
the prior localized infection of the host plant by fungal,
bacterial or viral pathogens (Jenns and Kuc, 1980). This
type of induced resistance can last for several weeks
(Hammerschmidt et al., 1976).

If the deposition of new cell wall material is
responsible for resistance, it should be possible with
morphometric analysis to show a difference in cell wall
thickness between cells of induced plants and controls. The
aim, therefore, of this portion of the research was to: 1)
determine the differences between the epidermal cell wall
thicknesses of heatshocked and control seedling cucumbers
and: 2) determine if any difference in thickness could be

correlated with disease resistance.

METHODS AND TECHNIQUES

Experinentl

Seeds of cucumbers, Qcccmic ccciyc cv Marketer were
placed in 1% sodium hypochlorite for 10 minutes and then
washed in ng3. Seeds were then placed on moist paper
toweling. After 6 days, 10 seedlings were placed in 50°

Water for 40 seconds and fixed in 2.7% glutaraldehyde in

63
0.1M HEPES, pH 7.2. Ten control seedlings were also fixed.

Additional control and heat shocked seedlings were placed on
germination paper and rolled into 4 tubes of 10 seedlings
each which were put into 250 ml beakers which had 50 ml of
(EEO added into them to keep the tubes moist. Beakers and
tubes were wrapped in aluminum foil so as to prevent the
exposure of seedlings to light. At both 24 and 48 hours 10
seedlings each of control and heat treated were fixed in
2.7% glutaraldehyde. Samples were washed in buffer (4x) 24
hours after fixation, postfixed in 1% 050, in 0.1 M HEPES pH
7.2 for 90 minutes, washed in buffer (2x), dehydrated in a
graded ethanol series, infiltrated and embedded in ERL epoxy
resin (Spurr, 1969). Silver-gold sections were also taken
on a Sorvall Porter-Blum MT 28, placed on copper grids and
viewed with a Philips 201 transmission electron microscope
(TEM).
Exocrinenc 2

Seedlings germinated as in Exp. 1 were placed into
22.5°C, 35°C, 40°C, 45°, 50° and along with a control which
was not placed into water were fixed in 2.7% glutaraldehyde
in 0.1M HEPES, pH 7.2. Control and 50°C heatshock seedlings
were also fixed at 24 and 48 hours. Samples were prepared
for TEM as in Exp. 1.
Hornnonete'y

1 pm sections of specimen blocks were placed onto glass
slides and stained with toluidine blue. The sections were

covered with permount and coverslips.

64
Slides were analyzed by a Zeiss Photometric Digitizer,

courtesy of Dr. Ann Cornell-Bell of the Eye Research
Institution on Staniford St., Boston, Massachusetts. A
total of sixty measurements were taken from each hypocotyl
cross section, the measurements being made in three places
of twenty cells. Since the cross section of the cucumber
hypocotyl resembles the shape of a clover leaf the twenty
cells counted from each section were picked from each of the
four corners, five cells at a time.

The mean, variance, F-test for variance ratio and T-test
to compare means of a sample were determined for the

samples.

RESULTS

Mczphometry

The epidermal cells of the corners of the hypocotyl were
measured (Fig. 1). The epidermal outer cell wall of the
heat treated samples were significantly thicker than the
controls at 0, 24 and 48 hours (Table 1). When this
experiment was repeated and additional temperature steps
were run it was found that the results did not replicate
those of the first experiment (Table 2) and cell wall
thicknesses decreased, as the temperature increased (Table

3).

65

TABLE 1
Comparison of epidermal outer cell wall thickness of control
and heat shocked hypocotyl 0, 24 and 48 hours after
experimental hypocotyls were treated.

 

 

 

0 Hour 24 Hour 48 Hour
Control 1.7210.55 1.5210.39 1.6610.42
N=600 N=600 N=600
A C E
Heat shock 2.0610.58 1.8010.46 1.8410.52
N8600 N-600 Na600
B D F

 

The means of the control and heat shocked cell wall
thicknesses at 0, 24 and 48 hours were compared using
Student's t-test done at a significance level of 0.05.

66

TABLE 2
The second comparison of epidermal outer cell wall thickness
of control and heat shocked hypocotyl 0, 24 and 48 hours
after experimental hypocotyls were treated.

 

 

 

0 Hour 24 Hour 48 Hour
Control 1.7610.47 1.5410.35 1.60:0.34
N=300 N=300 N=300
A C D
Heat shock 1.5710.37 1.5410.31 1.57:0.41
N=300 N-300 N=300
B C D

 

The means of the control and heat shocked cell wall
thicknesses at 0, 24 and 48 hours were compared using
Student's t-test done at a significance level of 0.05.

67

TABLE 3
A comparison of the epidermal outer cell wall thickness of
hypocotyls of the cucumber cultivar Marketer which had been
placed into water at different temperatures

 

Undunked 1.76:0.47
22°C 1.84:0.38
35°C 1.671038
40°C ‘ 1.62:0.39
45°C 1.40:0.46
50°C 1. 57:0. 37

 

The means and standard deviations are from 300 measurements.

68

Fig. 1. Scanning electron micrograph of cucumber hypocotyl
cross section. Bar = 100 um.

Fig. 2. Transmission electron micrograph of heat shocked
cucumber seedling hypocotyl epidermal cells. The
endoplasmic reticulum (ER) are swollen, the nucleoli (NU)
are electron-dense and the epidermal outer cell walls are
layered (arrow). Bar = 1 pm.

Fig. 3. TEM micrograph of control cucumber hypocotyl
epidermal cells. Endoplasmic reticulum (ER), nucleoli (NU)
and epidermal outer cell wall (C). Bar = 1 pm.

Fig. 4. TEM of epidermal cells of cucumber hypocotyl from
seedling which had been placed into 35°C water. Endoplasmic
reticulum (ER). Bar = 1 pm.

 

70
Electronnicrosconz

The epidermal cells of the heat shocked seedlings
differed in a number of features from the control seedlings.
Immediately after heatshock the endoplasmic reticulum was
dilated, compared to controls (Figs. 2 and 3). Dilation of
the endoplasmic reticulum also occurred at 35°C (Fig. 4) .
The nucleoli of heat shocked cells were more electron-dense
than controls (Figs. 2 and 3). The outer epidermal cell
wall became demarcated into layers in heat shocked cells but
remained a single layer in controls (Figs. 2 and 3). At 24
hours the epidermal cells no longer exhibited these
differences (Figs. 5 and 6). This was also true at 48 hours

(Figs. 7 and 8).

71

Fig. 5. TEM of epidermal cells of cucumber hypocotyl 24
hours after heat shock. Bar = 1 pm.

Fig. 6. TEM of epidermal cells of control cucumber
hypocotyl 24 hours after experimental seedlings were heat
shocked. Bar = 1 pm.

Fig. 7. TEM of epidermal cells of cucumber hypocotyl 48
hours after seedlings were heat shocked. Bar = 1 pm.

Fig. 8. TEM of epidermal cells of control cucumber
hypocotyl 48 hours after experimental seedlings were heat
shocked. Bar = 1 pm.

 

73

DISCUSSION

The hypothesis that the basis for induced resistance in
heat shocked cucumber seedlings was due to increased
deposition of cell wall material was tested by comparing
heatshocked with control seedlings. Previously it was found
that the concentration of cell wall associated materials
extensin and peroxidase increased in induced seedlings
(Stermer and Hammerschmidt, 1984). In the first experiment
the cell wall thicknesses of control and heat shocked
cucumber seedlings were compared at 0, 24 and 48 hours. At
24 hours the plants became disease resistant as a result of
the heat shock (Stermer and Hammerschmidt, 1984). The first
experiment seems to support the hypothesis by showing that
the epidermal outer cell walls increased in thickness as a
result of heatshock at 24 and 48 hours. A statistical
significant increase in cell wall thickness was also seen at
0 hour when it would be thought that deposition of new cell
wall material would not have time to occur.

To determine whether the increase in cell wall thickness
at 0 hour in heat shocked seedlings was a result of water
imbibition seedlings, in the second experiment, were treated
in water at 22.5°C, 35°C, 40°, 45°C, and 50°C. If the cell
wall thickness increased as a result of the heat shock the

increases would be most evident at the greater temperatures.

74
If seedlings were imbibing water the increases would start

at 22.5°C and increase gradually as the temperatures went up
to 50°C. The measurements of cell wall thickness followed
neither course and actually diminished at the higher
temperatures. Included in the samples were control and heat
shocked seedlings at 24 and 48 hours. No difference was
found in the cell wall thickness of the control and heat
shocked seedlings at either 24 or 48 hours.

Some differences were seen in the fine structure of the
cells of heat treated plants. The cells from heat shocked
plants at 0 hour had dilated endoplasmic reticulum, darkly
staining nucleoli, and an outer cell wall demarcated into
two layers.

The present evidence gives no indications of what the
mechanism of disease resistance in heat shocked cucumber
seedlings is. Observation of the infection process of heat
shocked seedling cucumbers by QQllELQLEiEDEE lcgccccicm
using histochemistry, such as was done in Chapter II might
provide more information. Morphometric analysis proved to
be unsuitable for a study of this type, perhaps because of
the large error in the measurement of such small structures
or to other factors involving random differences between
different batches of seedlings which resulted in the
apparent differences in the first experiment between the

control and heat shocked seedlings.

CHAPTER IV

EFFECTS OF INDUCED RESISTANCE ON PEROXIDASE, PHENOLICS AND

EXTENSIN CONCENTRATIONS IN CUCUHBER LEAVES

75

76

ABSTRACT

Lignin, extensin and peroxidase are involved in disease
resistance in cucumber plants. Both lignin and extensin are
structural components of the cell wall. Peroxidase is
thought to polymerize the precursors of lignin and extensin.
Carbazole staining showed that cucumber leaf peroxidase
occurred in greater concentration in induced cucumber leaves
compared to control leaves. The staining did not seem to be
localized to any particular cell type. The concentrations
of phenolics, peroxidase and extensin were determined for
epidermal peels of petioles and for the whole petiole. The
difference between the epidermal layer of the petiole and
the entire petiole and between induced and control tissues
could therefore be determined. Assays for phenolics in cell
walls showed no difference in the concentration of phenolics
in control and induced epidermis and petiole. Peroxidase
isozymes were more concentrated in protected than control
epidermis and petiole and in induced epidermis than in
petiole. Hydroxyproline levels were higher in the epidermis

of the petiole compared to the entire petiole.

77

INTRODUCTION

Peroxidase occurs at an increased concentration in
cucumber plants induced to become resistant by prior
infection (Hammerschmidt et al., 1982). Lignin occurs at a
higher concentration in induced plants, compared to control
plants, after induced and control plants were inoculated
with Q. lagenagicm (Hammerschmidt and Kué, 1982). Extensin
was shown to be involved in resistance (Hammerschmidt et
al., 1984). Peroxidase is part of the pathway in which
polyphenolic precursors are polymerized to form lignins
(Negrel and Lherminier, 1987). Peroxidase, lignin and
extensin have not been localized in induced plants so that
the particular tissues in which these materials are enhanced
is not known.

Lignins and extensins are thought to be responsible for
resistance in cucumber plants by the stiffening of cell
walls, thereby inhibiting penetration of fungi through the
epidermis of plants (Hammerschmidt and Kué, 1982;
Hammerschmidt et al., 1984). Induced resistance was
eliminated once the epidermis was removed, but this was not
true for genetic resistance (Richmond et al., 1979). For
these reasons, most of the enhancement of peroxidase in
induced resistant cucumbers should occur in the epidermis.

To determine the location of peroxidase, lignin and

78
extensin enhancement and to confirm that enhancement does

indeed occur, cucumber plants were induced to be resistant.
The amounts of peroxidase, lignin and extensin were compared
in controls versus induced plants by measuring the amounts
in epidermis peeled from petioles. Amounts in entire
petioles were also determined so that the degree of
enhancement in the epidermis could be determined. Since
peroxidase can be stained using histochemical techniques, it
was used to examine the leaves of control and induced plants

for sites of enhancement.

METHODS AND TECHNIQUES

generation of plant sonoles

Cucumber seeds were placed into 1% sodium hypochlorite
for 10 minutes, rinsed in dH,O, placed onto moist paper
toweling in a pan covered by aluminum foil, and kept at room
temperature for 6 days. When seedlings were about 1 inch
high, they were planted into pots and placed in a
greenhouse. Once the first leaves of plants were
approximately 2/3 full size, Escgdcmonas syringae pv
cycicgcc (see below) was injected into leaves through
abaxial stomata using a 5 ml syringe without a needle. One
week later, similar injections were made into second leaves.
Leaves were harvested one week after the second injections.
Epidermal peels from petioles and whole petioles were

frozen. The procedures for preparation of epidermal peels

79
and petioles are described in the section on acetone powders

below. Samples of three leaves of each treatment were fixed

for histochemistry.

Bacterie culture

Psenconones syringes pv syringes were inoculated into
nutrient broth media (NB media) and placed on rotary shader

for 24 hours. 5 ml of culture was diluted with 100 ml of
dH,O to ca. 108 cfu/ml and used for injections.
Histocnenistrx one microscopy

Samples from three of the third true leaves from each
treatment were fixed overnight in 0.25% glutaraldehyde, 1%
paraformaldehyde in 0.05M, pH 6.8, phosphate buffer.
Samples were washed in buffer with 0.5M sucrose, then buffer
with 1.15M sucrose. Samples were placed into Tissue Tek and
sectioned in a cryostat at -20°. Cryostat sections were
placed into wells of a spot plate. Peroxidase staining
solution (20 pl of 40 mg/ml of 3-amino-9-ethyl carbazole in
n,n dimethyl formamide was added to 0.01% H55 in 0.05M
sodium acetate buffer, pH 5.6.) was added to sections for
one minute. Sections were then taken out of wells and
placed into 0.05M sodium acetate, pH 5.6 and finally into
drops of sodium acetate on slides. Cover slips were placed
on slides and sealed with nail polish.
Acccccc powdecc

Wet weight for cucumber plant epidermal peels and whole
petioles were determined. The samples were then placed into

mortars to which LN2 was added. Samples were ground into

80
fine powder. 10 ml of acetone at -20°C was twice added to

sample. Slurry was filtered and resultant powder stored at
-20°C.
A833! to: cell 2111 12931851 phenolics

100 mg of acetone powders from uninjected control and
Eccndcmcncc cycingcc pv cyzingcc injected, induced plants
were placed into 5 ml of 1.0N NaOH for 20 hours at room
temperature. Samples were centrifuged in a Precision Vari-
Hi-Speed Centricone at a setting of 70. The supernatant was
pipetted off from the cell walls pellet. The pellet was
washed with 3 ml of deo, 3 times and the washes were
combined with the supernatant. The pellet was discarded.
The combined supernatant and dH,0 wash was acidified to pH
2.5 with concentrated HCl. 10 ml of ethyl acetate was added
to the acidified solution (3x), vortexed, and pipetted off.
The ethyl acetate extract was dried in a Rotavapor at 35%:
and redissolved with 0.25 ml methanol. The methanol extract
was spotted onto a TLC plate and run with CHCl,:.Acetic
Acid: H20 (4:1:1) which was mixed together and the lower
phase drawn off and used. A total of three TLC plates were
run.
Asses for new—noose

20 mg of acetone powders were extracted with 1 ml of 0.5M
sucrose in 0.01M phosphate buffer pH 6.0. 100 pl of extract
was mixed with 10 pl of 50% dextrose and layered onto PAGE.
The voltage was set at 100V until the bromophenol blue

tracking dye reached the stacking gel at which point current

81
was turned up to 200V. Gels were stained for peroxidase for

1 hour in a solution of 3-amino-9-ethyl carbazole added to
0.01% HRH in 199 ml of 0.05M sodium acetate, pH 5.6. 8
gels were run, in total.
Asses for W

Acetone powders (0.019 of bulked cell wall material from
the assay for peroxidase) were prepared for hydroxyproline
assay by the method of Hammerschmidt et al. (1984) by being
placed into 5 ml of 0.015 M Sfirensen's phosphate buffer, pH
7.2 (Hayat, 1972). The material was centrifuged and the
pellet was washed in 10 ml of buffer (5x). The material was
washed in ng3 (4x), centrifuged, and the pellet was frozen
at -20°C. The frozen material was lyophilized at 60
millitorr and -20W3. 0.0059 of material was hydrolyzed with
400 pl of 5.5 N HCl at 110°C overnight. Hydrolyzed material
was blown dry in a stream of air. The dried material was
resuspended in 400 pl of 0.001 N HCl. 200 pl of the
solution was assayed for hydroxyproline (Lamport, personal

communication; Lamport and Miller, 1971).

RESULTS

Hicnescons

Carbazole treated induced cucumber leaf sections were
deeply stained by the treatment (Fig. 1). Control leaf
sections were green, though some carbazole staining occurred

on the epidermis (Fig. 2). The induced leaves appeared to

82

Fig. 1. Light micrograph of cucumber leaf, induced by
injection with Pseudomonas syringae pv cy;i_gcc, sectioned
with a cryostat and stained with carbazole for peroxidase.
Bar = 100pm.

Fig. 2. Light micrograph of control cucumber leaf, not
injected, sectioned with a cryostat and stained with
carbazole for peroxidase. Bar = 100 pm.

 

84 -
be stained more intensely in the palisade mesophyll. During

the staining of the sections, the spot plate wells
containing induced leaf sections could readily be
differentiated from the control since the staining solution
in the well turned red.
Cell well 22229 phenolics

No differences in intensity or compound occurred between
the comparisons of cell wall bound phenolics in control and
induced epidermis and petiole.
Eeroxioese

The isozymes that were most noticeable in the
polyacrylamide agarose gel electrophoresis of the anionic
isozymes of peroxidase in unprotected and protected
epidermis and whole petioles of cucumber were the 3 bands
which were the most anodic. The bands of isozymes from
protected petiole were darker than those of unprotected
petiole 6 times out of 8 whereas unprotected petiole was
darker 2 out of 8 times. Protected epidermis was darker
than unprotected epidermis 7 out of 8 times while both
appeared the same 1 out 8 times. Protected epidermis
appeared darker than protected petiole 6 of 8 times,
protected petiole was darker than protected epidermis 1 of 8
times, and both appeared the same 1 of 8 times. The ranking
of the comparisons between the different tissues on the
intensity of the three most anodic peroxidase bands was
Protected epidermis < Protected petiole < Unprotected

petiole s Unprotected epidermis.

85
W
The concentration of hydroxyproline in cucumber petiole
did not appear to be affected by the use of Eceuccmonas
cycingcc pv cycingcc as an inducing agent (Table 1). The
concentration of hydroxyproline in the epidermis of petioles
was higher than the concentration in the entire petiole

(Table 1).

86

TABLE 1
Measurements of the concentrations of hydroxyproline in the
tissues of bulked samples from the cucumber epidermises and
petioles used in the PAGE assay for peroxidase.

 

 

 

 

pg 1'9
0.005 g 1.0 9
Dry weight Fresh weight
Unprotected petiole 1.90 pg 9.45 pg
Unprotected epidermis 1.22 pg 18.89 pg
Protected petiole 2.40 pg 9.90 pg
Protected epidermis 0.90 pg 15.83 pg

 

Cucumber plants were induced by injection of Pseudcmonac
cyringce pv cyzingge into the first and second leaves. The
petiole of the third leaf was used for hydroxyproline
determinations. Petioles were either stripped of epidermis,
for the measurement of hydroxyproline in epidermis or used
whole for determination of hydroxyproline in the bulk
petiole.

87

DISCUSSION

The finding of increased peroxidase in induced leaf
tissue corroborates the previous findings of Hammerschmidt
et al. (1982) of increased peroxidase levels in induced
leaves. Reaction product appeared somewhat more
concentrated in the palisade mesophyll than in the
epidermis, spongy mesophyll or vascular tissue. Comparisons
of induced and control petiole and epidermis using PAGE
indicated that soluble peroxidases were more concentrated in
induced petioles than control petioles and that induced
petiole epidermis contained a higher concentration of
peroxidase than the bulk petiole. This indicates that in
the petiole of cucumber, induced plants have a higher
concentration of peroxidase than the uninduced control
plants and that there is a higher concentration of
peroxidase in the epidermis than in the bulk petiole tissue.
This supports the work of Richmond et al. (1979) which
indicated that the epidermis is the site at which induced
resistance operates. The peroxidases were also found as a
set of three bands concentrated to the aniodic end of the
gel as was reported by Smith and Hammerschmidt (1988).

The lack of a difference in the concentration of
phenolics between epidermal strips and bulk petiole and

protected and unprotected plants agrees with the observation

88
that lignification followed challenge inoculation by a

pathogen in induced plants (Hammerschmidt and Kué, 1982).
The lack of difference in hydroxyproline concentration

between the induced and control petioles indicates that

extensin levels do not change after cucumber plants are

induced for resistance.

CHAPTER V

TECHNIQUES FOR THE ULTRASTRUCTURAL LOCALIZATION OF LIGNIN

USING POTASSIUM PERMANGANATE AND BROMINE STAINING AND EDS

89

ABSTRACT

Bromine and potassium permanganate were used to localize
lignin in a herbaceous plant using energy dispersive X-ray
microanalysis (EDS) and transmission electron microscopy
(TEM). Bromine was used as part of the preparation
procedure, and in separate experiments, KMnO4 was used as
either a fixative or a post-section stain. Lignin
deposition was associated with sites of infection caused by
the fungus, QQIlfiLQSIiQth lcgcnczicn in pumpkin seedlings.
The procedure for using Br2 as an ultrastructural stain for
lignin in herbaceous plants included a modification of the
procedure originally developed for woody plant by adding 8
paraformaldehyde prefixation step. These stains provided
data on the ultrastructural localization of lignin which
contributed to the elucidation of its role in the
interactions between pathogenic fungi in resistant plant

hosts.

91

INTRODUCTION

Of the techniques available for the ultrastructural
localization of lignin, it was necessary to decide which was
the most useful for the cucumber model system. To make this
choice, it was important to compare the different techniques
in a similar situation to determine the relative merits of
each technique. For this reason the KMnO4 and Br,
procedures were modified for use with herbaceous plants and
compared ultrastructurally. Energy dispersive X-ray
microanalysis was used to localize Mn or Br, in the treated

samples.

MATERIALS AND METHODS

The upper 1 cm of the hypocotyl of the Spookie cultivar of
pumpkin, 6 days old, grown at 23°C in the dark was
inoculated with 3x10° conidia/ml of W
lcgcnccicm which was grown on PDA for 10 days. Hypocotyl
segments were fixed 48 hours after inoculation by one of the
following protocols:

(1) 2% paraformaldehyde in 0.05 M sodium phosphate buffer,
pH 6.8 (Hayat, 1972) for 4 hours, 3 buffer washes,
dehydrated in a graded ethanol series, placed into 1:1

ethanolzchloroform, 100% ethanol, then a solution of 0.5%

92
bromine in chloroform for 3 hours with agitation (Saka et

al., 1978), placed in CHCl, for 10 days during which the
CHCl, was replaced 8 times, followed by 1:1 propylene
oxide:CHCl,, 100% propylene oxide, and four changes of ERL
epoxy resin (Spurr, 1969), and then embedded (Spurr's resin)
and polymerized at 65°C for 8 hr.

(2) 1% I<Mn04 at 23°C for 15 minutes, four buffer washes.

(3) 1% KMnO, at 23°C for 15 minutes, four buffer washes,
2.67% glutaraldehyde in buffer, overnight at 4°, followed by
four buffer washes.

(4) 2.67% glutaraldehyde in buffer for overnight at 4W2,
four buffer washes, 1% KMnO, at 23°C for 15 minutes, and
again four buffer washes.

(5) 2.67% glutaraldehyde in buffer for overnight at.4PC, and
four buffer washes.

(6) 2.67% glutaraldehyde in buffer for overnight at 44:,
four buffer washes, 1% 0504 at 4°C for 90 minutes, and two
buffer washes.

The samples from treatments #2-5 were dehydrated in a
graded ethanol series and embedded in ERL epoxy resin
(Spurr, 1969). At least three blocks from all the
treatments were sectioned with a Reichert Ultracut E
microtome, although, in some of the treatments only one
block was found to have instances of fungal infection.
Sections from treatments #5 and #6 were placed on nickel
grids and stained with 1% KMnO4 for 30 minutes in a filled

and sealed 1.5 ml Eppendorf microcentrifuge tube and then

93
washed well with dH,0 (Hayat, 1970) . Sections were viewed

with either a Philips 201 transmission electron microscope
(TEM) or a JEOL 100CX II TEM. Energy dispersive X-ray
microanalysis (EDS) spectra were collected and dot maps for
bromine and manganese were made using the JEOL 100CX II in
the scanning transmission mode (STEM) at 100 kV with a Link
Systems AN 10000 energy dispersive X-ray analyzer. Each dot
map was generated by 20 scans which took approximately one

hour.

RESULTS

Conidia of chlcccczichcm lcgcnczicm germinated to
produce appressoria by 24 hours after inoculation. By 48
hours, appressoria had formed penetration pegs that had
broken through the outer walls of the plant epidermal cells.
It was at this stage that reaction by the plant to infection
by the fungus could be studied.

As expected the method for Br staining resulted in poor
ultrastructural preservation of the plant cells. Even with
the addition of a paraformaldehyde prefixation step, the
treatment with chloroform extracted the cytoplasm. Cell
walls were the primary visible elements of structure (Fig.
1). Cytoplasm was present, though organelles and other
constituents of the cytoplasm could not be distinquished.

The walls of the cells proximal to appressoria were often

more electron-dense than walls of those cells that were not

94

Fig. 1. Ultrathin section of bromine-incubated,
paraformaldehyde-fixed material showed poor ultrastructural
preservation. Cytoplasm (CY) was present, though organelles
and other constituents of the cytoplasm could not be
differentiated. Walls directly under appressorium (A) were
more electron dense than walls of neighboring cell (arrow).
Bar = 1 pm.

Fig. 2. Energy dispersive X-ray microanalysis (EDS) dot map
showing localization of Br in Fig. 1. The wall of the cell
under the appressorium (A) was more heavily stained than the
neighboring cell (arrow).

Fig. 3. Ultrathin section of material fixed with
glutaraldehyde and 050;; sections from epoxy blocks were
stained with KMn04. Preservation of sample was comparable
to that seen in standard UA/ Pb citrate stained samples.
Cell walls (CW) were heavily stained with KMnO . Cytoplasm
(CY) was not as heavily stained. Fungal conid1a (C),
appressoria (A) and hyphae (H) are present. Bar = 1 pm.

Fig. 4. Photomicrograph of a serial section of Fig. 3 which
'was not stained with KMn04. 'The cell walls of plant (CW)

and fungal hyphae (H) were electron-lucent, compared to the
electron-dense walls of the stained section in Fig. 3. The
wall of the appressoria (A) was electron-dense. Bar = 1 pm.

Fig. 5. EDS dot map for Mn in Fig. 3. The heaviest
concentrations of Mn were found in hyphae and plant cell
walls which correspond to the areas which were more
electron-dense (Fig. 3). High concentration in cell walls
of the appressoria (A) which were electron-dense in both
Figs. 3 and 4. The cytoplasm (CY, same area as in Fig. 3)
is only lightly stained.

Fig. 6. Ultrathin section of sample fixed with glutaraldeyde
followed by KMnO4. 'The plant cell wall (CW) was less
electron-dense than the plant cytoplasm (CY), though there
were layers in the cell wall which were more heavily stained
by the KMn04. The outer matrix layer (M) and cytoplasm of
the appressorium (A) were also well stained. The
appressorium cell wall was very electron dense. Bar = 1 pm.

Fig. 7. EDS of Fig. 6. The greater concentration of Mn was
found in the cytoplasm (CY) of the plant cell and in the
appressorium (A) and its matrix layer (A). The plant cell
wall (CW) had little Mn associated with it.
electron-density of the cell walls of infected cells, as
compared to non-infected cells, was caused by increased
amounts of bromine, as determined by EDS (Fig. 2). This
indicated that lignin was present in the walls of infected
cells.

 

96

in contact with appressoria (Fig. 1). The increased
Specimens fixed with glutaraldehyde and osmium tetroxide,
followed by 10an4 staining of ultrathin sections showed
preservation equal to that found in standard section
staining treatments with uranyl acetate and lead citrate,
though there was a greater number of staining artifacts from
the KMnO4 (Fig. 3). Comparison of stained and unstained
serial sections indicated that increases in electron-density
occurred in the plant cell wall and in the hyphae.
Appressorial cell walls and certain portions of plant cells
were electron-dense in the sections unstained by KMnO4
(Figs. 3 and 4). In order to determine whether increases in
electron-density were due to KMnO4 or to another cause, such
as OsO4 post-fixation, EDS was used to determine the
distribution of manganese (Fig. 5). The EDS Mn dot maps
showed that Mn occurred in the areas where KMnO, staining
resulted in increased electron-density, such as the plant
cell wall and in fungal hyphae, but that Mn also was
localized in the appressorial wall, which was electron-dense
in both KMnO4 stained and unstained sections.

Material fixed with KMnOU alone (method #2) , as well as
before or after glutaraldehyde (methods #3-4), were similar
in that the cell walls were not heavily stained and the
cytoplasm was poorly preserved, yet heavily stained (Fig.
6). The EDS of these samples showed that little of the
manganese was localized in the cell walls of the host plant,

but rather most of the Mn X-ray signal originated from

97
cytoplasm and appressoria (Fig. 7).

98

DISCUSSION

A lignin staining technique utilizing EDS to localize
bromine, which had previously been used on wood cells (Saka
et al., 1978), was applied here to an herbaceous specimen,
to study the localization of lignin in pumpkin seedlings
infected with the fungus, chlcccczichcm lcgcnccicm.

Lignin in infected epidermal cell walls in herbaceous
plants could be determined by the localization of bromine
with EDS (Table 1). The level of staining in both the
cytoplasm and in the rest of the background of epoxy
sections was very low. Cytoplasm, however, was heavily
extracted by the treatment, leaving few recognizable
cytoplasmic constituents. From the perspective of overall
specimen image quality, the bromine method appears more
suitable for specimens where the quality of the preservation
of cytoplasm is unimportant.

Lignification in herbaceous plants has been previously
studied by means of KMnOU which when used as a fixative,
acts also well as a stain for lignin (Hepler et al., 1970).
In this study, plant and fungal cell cytoplasm became
heavily stained during KMn042fixation, but plant cell walls
were not (Table 1). Energy dispersive x-ray microanalysis
(EDS) showed that there was little Mn in plant cell walls in

samples from the 10an4 fixation treatments (alone or before

99

Table 1.
Comparison of techniques used in the fixation and staining
of plant samples for the presence of lignin.

 

 

Treatment Quality of Ease of
Ultrastructural Localization
Preservation* of Lignin*

1. FA/Br +/- ++++

2 . m0, + +/—

3 . I<MnO,/Glut + +/-

4. Glut/KMnO4 ++ +/—

5. Glut +++ ++

KMnO4 on section

6. Glut/OsO4 ++++ +++
10an4 on section

 

The order of the treatments is the same as that
found in the Materials and Methods. In treatments

5 and 6 ultrathin sections were stained with KMnO,.
Paraformaldehyde (FA), Glutaraldehyde (Glut).
*Quality of ultrastructural preservation refers to
the integrity of membrane, cytoplasm, and organelles.
Ease of localization of lignin refers to the amount
of signal compared to background in the dot maps.

The meaning of the symbols are: +/- poor, + fair,

++ good, +++ very good, ++++ excellent.

100
glutaraldehyde). Since the overall staining of the

cytoplasm by KMnO, is very heavy, KMnO4 fixation would not
be useful in determining whether lignin is occurring in
papillae. Doubtlessly the wide variety of materials present
in cytoplasm include materials such as the polyphenolics
which were found to be a primary material that reacts with
KMnO, (Bland et al., 1971). Fixation with glutaraldehyde
alone did not improve Mn localization in sections stained
with KMnO,.

As expected, glutaraldehyde/osmium tetroxide fixed
material had better preservation of fine structure than did
the other techniques (Table 1). Ultrathin sections from
specimens fixed in this way and subsequently stained with
10an4 resulted in depositions of amorphous, electron-dense
puddles. Fungal walls and cytoplasm, cell walls of the
plant, and to a lesser degree plant cytoplasm, were stained
by KMnO,,¢as demonstrated by both EDS and bright field TEM.
Staining of appressorial walls was due to the presence of
melanin, a polyphenolic which also reacts with KMnO4 (Hayat,
1986).

Cytoplasmic and general background staining of sections
of glutaraldehyde/osmium tetroxide fixed material was higher
than that of the bromine technique but much lower than that
of the KMn042fixation techniques. Cell walls of both plant
and fungus from thin sections treated with KMnO, were
heavily stained with permanganate but that differences in

the cell wall structure were obscured by the high

101

concentration of Mn. As was done in this study, unstained
serial sections should be considered as an important control
for this procedure.

In summary, the bromine staining technique corroborated
with EDS resulted in excellent localization of lignin and
acceptable fixation of plant cell walls, but had the
distinct disadvantage of extraction and poor preservation of
cytoplasm. The KMnO4 thin section staining technique
resulted in good preservation of cytoplasm, but poorer
elucidation of cell wall structure due to an increase of
staining over the bromine technique. Both procedures
allowed the localization of specific stain (Br or Mn) with
EDS.

The KMn042fixation procedure resulted in poor fixation
and inferior localization of KMnO4 stained lignin. One
advantage of this technique, however, was that the
substructure of plant cell walls could be distinguished
under bright field TEM more readily, due to the lower levels
of staining that occurred, compared to the KMnO4 section
staining method. For these reasons both the 10an4 staining
technique and the bromine procedure were used for further

study of lignification during resistance against infection.

CHAPTER VI

PRELIMINARY OBSERVATIONS ON THE IMMUNOCYTOCHEMICAL

LOCALIZATION OF EXTENSIN AND PEROXIDASE IN THE

LEAF OF CUCUMBERS INDUCED FOR RESISTANCE

102

103

ABSTRACT

In preliminary experiments an antibody to peroxidase
bound to much of the cytoplasm and cell wall of the leaves
of induced and control cucumber plants. An antibody to

extensin did not exhibit any specificity.

104

INTRODUCTION

Both extensin and peroxidase are thought to be involved
in disease resistance in cucumber plants (Hammerschmidt et
al., 1984; Hammerschmidt et al., 1982). Extensin is a
component of plant cell walls and presumably the
nonspecificity of induced resistance in cucumbers is due to
the increase in the impenetrability of cell walls as a
result of either increased cell wall deposition or increased
crosslinkage of cell walls or possibly both. Extensin has
been found to increase after resistance was induced by both
prior infection and heat shock (Stermer and Hammerschmidt,
1984).

Peroxidase is thought to cause the polymerization of both
lignin and extensin in plant cell walls (Negrel and
Lherminier, 1987). The concentration of peroxidase, in
plants, increases after localized infection of mature plants
and heatshock of seedlings (Hammerschmidt et al., 1982;
Stermer and Hammerschmidt, 1984).

The localization of extensin and peroxidase to the
epidermal cells, the site of induced resistance in cucumbers
(Richardson et al., 1979) would corroborate the ideas that
both extensin and peroxidase are involved in the response by

cucumbers to infection.

105

METHODS AND TECHNIQUES

Manner seedlings

6 day old cucumber seedlings (Qcccmic ccciyc cv Marketer)
were heat shocked by plunging into 50°C water for 40
seconds. Control and heat treated seedlings were sampled at
0, 24 and 48 hours after treatment. In addition, both
control and heat treated seedlings were infected with
Qlccccpccicm ccccmccincm at 24 hours after the heat
treatment and were sampled at 48 hours after the heat
treatment. The uppermost 1 mm of the seedlings from the
different treatments were fixed in 0.25% glutaraldehyde, 2%
paraformaldehyde in 0.1 M HEPES, pH 7.2 for 3 hours.
Samples were dehydrated in 25% ethanol at 0°C and 50%, 75%,
95%, and 100% at -30°C, then placed in Lowicryl and
polymerized with U.V light at -30°C for 3 days. Samples
were also fixed in 2.7% glutaraldehyde in 0.1 M HEPES, pH
7.2 for 24 hours, post-fixed in 1% 0804 in buffer for 2
hours, dehydrated in ethanol and embedded in ERL media
(Spurr, 1969).
Qcccmcc; plangs

Cucumber plants (Qpccmic ccciyc cv Marketer) were
injected, with Ecccccmcncc cygingcc pv sygingae, into the
first true leaf when that leaf was at 2/3 of its mature
size. 10 injections were made using a syringe without a

needle into the abaxial surface. Portions of the second

106
leaf were fixed for 12 hours in 2.7% glutaraldehyde in 0.1 M

HEPES, pH 7.2, 6 days after injections. Samples were post-
fixed in 1% 0304 in buffer for 2 hours, dehydrated in
ethanol and embedded in ERL media (Spurr, 1969).
Immecxtocnenistrx

Sections were cut with a diamond knife, placed on 300
mesh nickel grids and stored in a vacuum desiccator.

The methods of Bendayan (1984) were used to stain
sections:
1). Grids with Spurr's media embedded sections were etched
with saturated sodium metaperiodate, tissue side down, for
one hour.
2). Spurr's media and Lowicryl embedded sections were rinsed
by placing section side down on surface of dH,O for 2-3
minutes in each spot (4x), grids were shaken off between
each wash.
3). Grids were placed on drops of PBS with 1% ovalbumin for
5 minutes.
4). Transfer without shaking or rinsing to 1:100 antibody
(anti-extensin, Ray Hammerschmidt, or anti-peroxidase,
Jennifer Smith) for 1 hour.
5). Rinsed on PBS in spot plates for 5 minutes (4x).
6). Transfered to drops of PBS with 1% ovalbumin for 5
minutes.
7). Incubated on colloidal gold/Staphylococcus Protein A
,(Bendayan, 1983) diluted 5 to 10 fold with PBS for 30

minutes.

107
8). Thoroughly jet washed with PBS and ngD.

9). Dried and stained with 2% aqueous uranyl acetate for 10
minutes.
BBS formula

pH 7.4 PBS is composed of 89 NaCl, 1.449 Na,HPO,x2H,O,

0.29 KH,PO,, 0.2g KCl, and brought up to 1 liter with dH,O.

RESULTS AND DISCUSSION

Spurr's media embedded mature cucumber leaves, when
stained with anti—peroxidase and 12 pn colloidal gold had
labeling on most of the cell wall (Fig. 1) and cytoplasm in
both control and induced sections (Fig. 2 and 3). Hypocotyl
sections were also labeled on both cell wall and cytoplasm
(Fig. 4).

In all these cases, there was varying amounts of
background, due to nonspecific binding, which can be seen
where colloidal gold particles occurred in extracellular
areas, such as outside the epidermal outer cell wall (Fig.
1) and in the gaps created by the shrinkage of starch
granules in chloroplasts (Fig. 2).

The lack of specificity of the anti-peroxidase can be
seen by the large amounts of staining occurring in areas
where peroxidase would not be expected, such as the starch
grains of chloroplasts (Figs. 2, 3 and 4). The nonspecific
binding of the antibody to such locations may be due to the

use of Freund's adjuvant which has been found to cause cross

108

Fig. 1. TEM of ultrathin section of epidermal outer cell
wall of SMR 58 cucumber plants. Tissues were embedded in
Spurr's epoxy. Sections were incubated with anti-peroxidase
and Protein A/ colloidal gold. Cell wall (C) has a heavy
concentration of gold particles but extra- (E) and
intracellular (I) spaces have only a scattering of
particles. A greater number of particles were found in a
layer which covered much of the epidermis (D). Bar = 1 pm.

Fig. 2. TEM of mesophyll cell from control, uninjected SMR
58 cucumber plant, the samples of which were embedded in
Spurr's epoxy. Sections were incubated in anti-peroxidase
and Protein A/ colloidal gold. Little binding of particles
occurs in the intercellular space (IS) but there was binding
over much of the cytoplasm (CY), chloroplasts (CH), starch
granules (G) and cell wall (C). Bar = 1 pm.

Fig. 3. TEM of induced resistant SMR 58 cucumber plant.
Thin sections were incubated with anti-peroxidase and
Protein A/ colloidal gold. Most of binding occurred on
chloroplasts (CH), starch grains (G), ground cytoplasm (CY)
and cell wall (C). Bar = 1 pm.

Fig. 4. TEM of the hypocotyl of 48 hour heat shocked and 24
hours after inoculation of Qladospocium ccccmcgiccm onto
seedlings. Sections were incubated with anti-peroxidase and
colloidal gold. Particles were found on the nucleus (N),
nucleolus (NU), ground cytoplasm (CY) and cell wall (C) but
not as heavily on the intercellular space (I) and vacuole
(V). Bar = 1 pm.

 

110
reactivity to components of plants due to the similarities

of the cell walls of bacteria which make up the adjuvant and
the cell walls and other polysaccharides of plants (G. de
Zoeten, personnel communication).

Lowicryl embedded material, besides being more difficult
to section and having poorer contrast preservation of fine
detail than epoxy embedded material, did not yield a greater
specificity of anti-peroxidase localization.

Anti-extensin, obtained from R. Hammerschmidt, did not
bind to sections to any greater degree than normal rabbit
serum.

To continue this research new antibodies to extensin and
peroxidase need to be produced. One method to gain greater
specificity of antibody to antigen would be exposing the
antigens to the fixative used in sample preparation. This
would cause the antibody to be produced to the conformation
of the antigen as it would occur in the fixed samples. As
mentioned above Freund's adjuvant could not be used.

Both extensin and peroxidase are glycosylated.
Antibodies to the glycosylated portion of the protein would
probably cross react with other glycosylated molecules
resulting in non-specific binding. Deglycosylation of the
proteins would yield antibodies which would likely be
blocked from the appropriate epitope by the glycosylation.
One solution might be to make antibodies to the fixed
deglycosylated form of the molecules and affinity purify

against the fixed glycosylated forms which should result in

111
antibodies which would bind to the antigen in fixed samples.

The problem is whether fixation of the deglycosylated form
of the protein would cause the protein to take up a
different conformation than the fixed glycosylated form.
The possibility of the production of an antibody to
lignin should be examined. The precursors of lignin are
comparatively low weight molecules but antibodies have been
produced to other low weight molecules in plants such as
abscisic acid (Sossountzov et al., 1986). Localization of
lignins would corroborate the histochemistry of lignin in
Chapter II. Since lignin is well bound into the cell walls
normal preparation procedures should suffice. If possible,
antibodies to the precursors of lignin as well as the
polymer should be made to see if the relative abundances of
the precursors in the cytoplasm of induced and infected
cucumbers could be determined and compared to the amounts of

lignin polymerized in the cell wall.

112

SUMMARY

Cucumber plants exhibit a modification of epidermal cells
where the outer wall, that is in contact with fungal
appressoria of QQllfiLQIIlQhHI lcgcncriun, becomes electron-
dense. Electron-dense cell walls were never seen without
appressoria. Appressoria were never seen to penetrate
through these walls. Energy dispersive X-ray microanalysis
(EDS) showed that one component of these walls, and probably
the one that accounted for the electron-density, was
silicon. Samples prepared by two different techniques still
were found to have silicon in the electron-dense cell walls
so the silicon is not likely an artifact of sample
preparation. Electron-dense cell walls were histochemically
stained for lignin using potassium permanganate and bromine.
EDS showed that Mn was bound into the electron-dense cell
wall, indicating lignin, but that Br was not. The reason
for the failure of this histochemical test to indicated
lignin while the potassium permanganate test did is not
known but might be caused by the special properties of
lignin deposited as a response to infection.

Papillae developed around the sites of fungal
penetration. Staining with potassium permanganate indicated

that lignin localized into papillae. The localization of Mn

113
form the stain to papillae was corroborated by EDS.

The ultrastructural reactions to infection were common to
both induced and control plants infected by Q. lcgcnccicm.
The differences were likely quantitative rather than
qualitative.

Heat shock of cucumber seedlings was shown to cause
induced resistance. To determine whether an increase in
epidermal cell wall thickness caused induced resistance,
measurements were made of heat shock and control seedlings.
Heat shock of seedlings caused several ultrastructural
effects, most notably an increase in the distance between
the layers of the endoplastic reticulum and an increase in
the electron-density of nuclei. Measurements of the
thickness of the outer wall of epidermal cells indicated
that heat shock caused the cell walls to increase in
thickness but this increase did not occur consistently.

Measurements of peroxidase in the epidermis of the
petiole and the bulk petiole of induced and control mature
plants showed that the induced tissue had a greater
concentration of peroxidase than control and that induced
petiolar epidermis a greater concentration than the bulk
petiole. This is in accord with the hypothesis that the
epidermis is responsible for the effect of induced
resistance since peroxidase is thought to be responsible for
the polymerization of lignin and extensin.

Measurements of the concentration of phenolics in induced

and control petiolar epidermis and bulk petiole did not

114
demonstrate any differences but the concentrations of

hydroxyprolines, a constituent of extensin, were found to be
higher in epidermal cells but no difference was seen between

control and induced tissue.

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115

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