Louisiana State University LSU Digital Commons

LSU Historical Dissertations and Theses Graduate School

1972 Penetration and the Host-Parasite Interface of Phytophthora Infestans on Tomato Leaf Tissue. Alice Sheppard badgett eT mplet Louisiana State University and Agricultural & Mechanical College

Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses

Recommended Citation Templet, Alice Sheppard badgett, "Penetration and the Host-Parasite Interface of Phytophthora Infestans on Tomato Leaf Tissue." (1972). LSU Historical Dissertations and Theses. 2364. https://digitalcommons.lsu.edu/gradschool_disstheses/2364

This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS

This dissertation was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted.

The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction.

1. The sign or "target" for pages apparently lacking from the document photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you complete continuity.

2. When an image on the film is obliterated with a large round black mark, it is an indication that the photographer suspected that the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame.

3. When a map, drawing or chart, etc., was part of the material being photographed the photographer followed a definite method in "sectioning" the material. It is customary to begin photoing at the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections with a small overlap. If necessary, sectioning is continued again — beginning below the first row and continuing on until complete.

4. The majority of users indicate that the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to the understanding of the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the Order Department, giving the catalog number, title, author and specific pages you wish reproduced.

University Microfilms

300 North Zeeb Road Ann Arbor, Michigan 48106

A Xerox Education Company I 73-13,688

TEMPLET, Alice Sheppard Badgett, 1943- PENETRATION AND THE HOST-PARASITE INTERFACE OF PHYTOPHTHORA INFESTANS ON TOMATO LEAF TISSUE.

The Louisiana State University and Agricultural and Mechanical College, Ph.D., 1972 Botany

University Microfilms,A XEROX Company, Ann Arbor, Michigan

© 1973

ALICE SHEPPARD BADGETT TEMPLET

ALL RIGHTS RESERVED

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. PENETRATION AND THE HOST-PARASITE INTERFACE

OF PHYTOPHTHORA INFESTANS ON

TOMATO LEAF TISSUE

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

in

The Department of Plant Pathology

by Alice Sheppard Badgett Templet B.A., Duke U n iv e rsity , 1965 M.S., Louisiana S ta te U n iv e rsity , 1968 December 1972 PLEASE NOTE:

Some pages may have

indistinct print.

Filmed as received.

University Microfilms, A Xerox Education Company ACKNOWLEDGEMENTS

Sincere appreciation is extended to Dr. L. L. Black for his guidance and advice during the course of this study and the final preparation of this manuscript.

S pecial thanks are expressed to Mr. H. W hitecross and Dr. A.

B. Merkle for instruction in the preparation and examination of material for the scanning electron microscope in the School of

Geoscience.

Appreciation is extended to Dr. S. J. P. Chilton of the

Department of Plant Pathology and to Dr. C. A. Schexnayder of the

Department of Botany for making electron microscope materials and facilities available.

In addition, gratitude is expressed to Drs. G. E. Holcomb,

K. S. Derrick, N. L. Horn, C. A. Schexnayder, and M. D. Socolofsky for helpful criticism in the preparation of this manuscript. TABLE OF CONTENTS

Page

Acknowledgement...... i

Table of Contents...... i i

List of Illustrations...... i i i

A b s tra c t...... iv

I. Introduction...... 1

II. Materials and Methods...... 3

III. Results...... 8

A. Scanning ElectronMicroscopy...... 8

B. Leaf C le a rin g s...... 14

C. H isto lo g y ...... 19

IV. Discussion...... 39

A. Scanning Electron Microscopy...... 39

B. Leaf C le a rin g s ...... 41

C. H is to lo g y ...... 43

V. Summary...... 30

VI. Literature C ited...... ^2

VII. V ita ...... 56

i i LIST OF ILLUSTRATIONS

Page

I. Scanning Electron Microscopy

Fig. 1-11...... 11-13

II. Leaf Clearings

Fig. 12-21...... 16-18

III. Histology Fig. 22-67 ...... 27-38

i i i ABSTRACT

Tomato leaf tissue inoculated with race 1 (compatible) and race 0 (incompatible) of Phytophthora infestans (Mont.) de Bary was examined with light, scanning electron and transmission electron microscopes. Germinated zoospores and sporangia were observed on the inoculated leaf surface. Germ tubes of sporangia had penetrated epidermal cells directly; others had entered through stomatal open­ ings. Hyphae from surface mycelium were observed to pass through stomatal openings. Intercellular mycelium ramified between the palisade mesophyll cells, penetrating host cells with one to several long, curved haustoria. Mycelium was found intracellularly in xylem vessels of tomato leaflets.

A matrix which stained differently than the fungal or host cell wall was found between the cell walls of the host and parasite. Similar material was present between adjacent mycelial s tra n d s .

The initial stage in the development of a haustorium is the initiation of a papilla-like structure of modified host cell wall material at the point where the fungus and the host cell come in contact. This is followed by the development of a young capitate haustorium which is surrounded by a sheath of modified cell wall material often containing particulate material. A mature haustorium has a constricted neck where it passes through the host wall. A collar of sheath material encircles the basal portion of the haustorium. The distal portion of the haustorium is not covered

iv by a sheath, thus the haustorial cell wall appears to lie directly against the host plasma membrane.

Swollen endoplasmic reticulum, increased golgi activity, distorted mitochondria, chloroplast destruction and wall lesions were observed in cells of infected leaves. Most of these effects on host cells were observed in the leaf lesion area whether or not the cell was penetrated by haustoria.

v INTRODUCTION

The .infection process and the anatomical relationship between

a plant pathogen and its host have been the subject of numerous

morphological studies. Light, scanning electron and transmission

electron microscopes have been used to study the host-parasite

interface of certain diseases. The results of these studies have

contributed to an understanding of the process of pathogenesis.

Review articles of the infection process of plant cells and/or

surfaces by fungal pathogens have been written by Wood (30)

and Ehrlich and Ehrlich (11).

Phytophthora infestans (Mont.) de Bary, the causal agent of

the late blight disease of potato and tomato plants, is a very

destructive pathogen. Under extreme conditions, it causes total

crop failure. Most of the research on the late blight disease

has been directed toward a study of the physiology, etiology and

anatomy of the disease on the economically important potato plant.

Blackwell (5) made an extensive light microscopic study of

the haustoria of P. infestans in potato tuber and leaf tissue.

Pristou and Gallegly (21), Tomiyama (27), Takakuwa and Tomiyama

(26) and Sakuma and Tomiyama (25) presented work related to the

infection process of the late blight pathogen on potato tissue.

Ehrlich and Ehrlich (12) conducted an electron microscopic study of the hyphae and haustoria of this fungus in potato tissue.

In 1962, Kishi (17) presented a light microscopic study of

1 2

the cytology of the host-parasite relationship of the tomato-

P. infestans disease complex. He studied specifically the

infection process and the host reaction to fungal invasion of tomato midrib leaf tissue. To date no further cytological investigations of the tomato- P. infestans disease complex have been reported.

The purpose of the present investigation was to study microscopically tomato leaf tissues infected with P. infestans in relation to the i) primary infection process, ii) physical location of the fungal thallus, and iii) the host-parasite inter­ face . MATERIALS AND METHODS

The tomato plants, Lycopersicon eseulentum L., used in this study were derived by selfing individual plants from the breeding line 386-1-5 supplied by Dr. M. E. Gallegly at West Virginia

University, Morgantown, West Virginia. Progeny from plant Selection

33 used in this study were homozygous resistant to race 0 of

P. infestans and susceptible to race 1 of P. infestans.

The plants were grown in vermiculite in one quart plastic containers, four plants per container. Full strength HoaglandTs solution (16) was used as the nutrient source at the time of planting. After planting, one half strength Hoagland’s solution was used to water the plants at one day intervals. The plants were grown in an environmental chamber at 20-23 C and illuminated with 1400 ft-c fluorescent and incandescent light for 14 hr daily.

Race 0 and race 1 of P. infestans, were obtained from Dr. M.

E. Gallegly, West Virginia University, Morgantown, West Virginia.

The cultures were maintained in an incubator at 20 C on lima bean agar (.10 oz package frozen baby lima beans blended for 2 min, 14 g Fisher agar, and 1 liter distilled water) slants in test tubes. Zoospore-sporangia! suspension inoculum was obtained from flood plate cultures of the fungus grown on lima bean agar in petri dishes. Seven day old cultures were used in each study.

Twenty-five ml of distilled water was added to flood each plate and the growth loosened from the agar surface with a sterilized

3 glass rod. The suspension was filtered through two layers of

cheesecloth into a 200 ml flask and the concentration adjusted

to 40,000 sporangia/ ml distilled water using a Neubauer hemocyto

meter. The fungal suspension was incubated at 12 C for 2 hr to

induce zoospore liberation. The resulting zoospore-sporangial

suspension was used as the inoculum for this study.

When the tomato plants were 3 weeks old, each container was placed in a holder equipped with flat supportive stages to hold

the leaves level for inoculation. The leaflets were attached to each stage with thin strips of masking tape. Plastic rings,

5 mm in diameter with a 3 mm rim, were attached to the leaflet surface by a thin coat of Petrolatum. One tenth ml of inoculum was pipetted onto the leaf surface within the area of the plastic

ring. After inoculation, the plants were placed in a dark box at

100% relative humidity and approximately 20 C for 24 hr. After

24 hr, the containers were removed from the holders, the plastic

rings removed from the leaflets, and the plants returned to the environmental chamber. Leaflet material was removed from the plants for processing at various time intervals after inoculation

Scanning Electron Microscopy

The zoospore-sporangial suspension used in th is study was washed three times in distilled water by repeated low speed centrifugation before being used as the inoculum. Leaf discs approximately 5 mm in diameter were cut with a cork borer from

inoculated leaflet material. The leaf discs were cut at 1, 2, 3, 5

6 and 12 hr intervals after inoculation. The discs were quick frozen in liquid nitrogen and placed on coverslips pre-cooled on a chilled lypholizer tray in a freezer for 30 min. The tray was then transferred to the lypholizer for 1 hr.

The lypholized leaf discs were attached to copper stubs with double s tic k y tap e. The stu b s were placed in a vacuum evaporator and a layer of carbon and a layer of gold-palladium (4U:60 Ladd

Research Ind.) metal was evaporated on the leaf surface. The coated leaf discs were viewed with a Jeolco MT2 scanning electron microscope at 25KV.

Cleared Leaf Discs

Leaf discs 5 mm in diameter were obtained as outlined previously. Disc samples were removed at 1, 2, 3, 4, 6, 8, 12, 24,

48, and 72 hr after inoculation. The material was placed in a

0.5% NaCl solution for 12 hr, washed in distilled water, and transferred to a saturated chloral hydrate solution for several days until the discs were decolorized. The discs were washed in distilled water and placed in a staining solution of 1% aniline blue in lactophenol (6) for 2-3 days. The discs were then heated in the staining solution until the mixture fumed. They were then cooled and rinsed in lactophenol to remove excess stain. The discs were transferred to a microscope slide in a drop of Permount and covered with a coverslip. The inoculated leaf discs were examined with a Leitz Ultraphot light microscope using bright field illumination to locate the fungal mycelium in the host tissue. 6

Photographs were taken on Kodak Panatomic X black and white film.

H istology

Leaf discs 1 mm in diameter were obtained from leaflet material previously inoculated with a zoospore-sporangial suspension of P. infestans at the time intervals previously mentioned. The plant material was fixed in either (i) 3% glutaraldehyde-3% acrolein in

0.02 M sodium cacodylate buffer at pH 7.2 for 1 hr, washed in b u ffer fo r 1 h r and p o stfix ed in 2% osmium te tro x id e fo r 1 h r (2); o r ( ii) 2% potassium permanganate in a 0.02 M sodium cacodylate buffer at pH 7.2 for 1 hr. Tissue fixation and dehydration were carried out in an ice bath at approximately 2 C. Dehydration of material fixed by either method was with a graded ethanol series followed by embedding in either Maraglas (13) or Epon-Araldite epoxy resin (18).

Sections used for examination with the light microscope were cut with a glass knife on a Porter-Blum ultramicrotome to a thickness of 1 u. The sections were placed in a drop of water on a clean glass microscope slide and heated until the water evaporated from the preparation. A drop of stain (1% aniline blue in 10% boric acid) was placed on the section and heated to fuming (23).

Excess stain was removed with water and the section air dried.

The slides were examined with a Leitz Ultraphot light microscope using bright field illumination. Photographs were taken on Kodak

Panatomic X black and white film.

Sections used for viewing with the electron microscope were 7 cut with a Dupont diamond knife on a Porter-Blum ultramicrotome, placed on collodion coated copper grids, post stained with uranyl acetate (19) and lead citrate (22) and examined in an

Hitachi HU11A electron microscope at 50KV. RESULTS

Because of the large number of spores per ml of inoculum, disease symptom expression was very rapid in inoculated leaflet tissue. In tomato tissue inoculated with race 0 (incompatible) of P. infestans. hypersensitive flecks were observed on the leaflet surface as early as 18 hr after inoculation. By 72 hr, a maximum number of flecks was observed.

As early as 21 hr following inoculation, brown, water-soaked spots appeared on the leaflets inoculated with race 1 (compatible).

The water-soaked areas had begun to coalesce, rendering the leaflet almost completely brown and flaccid 18 hr after inoculation.

By 72 hr, the leaflet tissue became dry and brittle, the petiole had lost its turgor, and brownish discoloration was noted in the stem portion of the plant adjacent to the infected leaf.

Scanning Electron Microscopy

Healthy leaf tissue (Fig. 1) appeared to be devoid of much surface debris and contamination. Because of the great number of spores in the inoculum, the inoculated leaf tissue showed a large number of sporangia and/or zoospores per leaf disc.

Frequently an accumulation of sporangia was found around cells at the base of a large leaf hair (Fig. 2). Mycelial strands of the fungus were also observed to be present on the leaf surface as early as 2 hr after inoculation. Sporangia were seen to accumulate in the crevices between rows of glandular hairs on the disc surface (Fig. 3). At higher magnification, individual sporangia 9 and zoospore material were observed (Fig. 4 and 6). Race 1 zoospores and sporangia were evident 2 hr after inoculation

(Fig. h). A zoospore of race 0 was observed which had germinated in close proximity to a sporangium 3 hr after inoculation (Fig.

6). Another germinated zoospore with what appears to be an appressorium is shown in F ig . 5.

Sporangia of P. infestans may germinate directly and penetrate host tissue causing infection or produce zoospores which are agents of infection. A sporangium shown in Fig. 7 has germinated and penetrated an epidermal cell directly. An open stomate is evident above the sporangium. A high magnification micrograph of the zone of penetration revealed a collar of material surrounding the penetration peg (Fig. 8).

Germinating sporangia with long germ tubes were also found on the disc surface. In a preparation 12 hr after inoculation, the germ tube of a sporangium had bifurcated, one side terminating in an enlarged bulb (Fig. 9).

Two sporangia are shown in Fig. 10. One of the sporangia has germinated and penetrated a stomatal opening. Mycelium was generally found to be present on the leaf surface. Some hyphal strands were observed which had passed through a stomatal opening on the epidermal surface (Fig. 11). 10

KEY TO LABELING OF THE FIGURES

A = appressorium

B = bulb

C = collar

GH = glandular hair

LH = leaf hair

M = mycelium

S - sporangium

ST = stornate

Z = zoospore F ig. Healthy tomato leaf epidermal cells x 600.

F ig . Survey view of tomato leaf tissue inoculated with race 1. Sporangia (S) and mycelium (M) are shown around the base of a large leaf hair (LH) x 160.

F ig. Survey view of tomato leaf tissue inoculated with race 1. Sporangia (S) are shown aggregating in the crev ices between rows of glandular hairs (GH) x 160.

F ig. Race 1 sporangia (S) and zoospores (Z) on the leaf surface 2 hr after inoculation x 600.

F i g . A race 0 sporangium (S) and a germinated zoospore (Z) on the leaf surface 3 hr after inoculation. What appears to be an appressorium (A) has been produced at the tip of the zoospore germ tube x 1S00.

F ig. A race 1 sporangium (S) and a germinated zoospore (Z) on the leaf surface 2 hr after inoculation x 1MU0.

F ig. A race 1 sporangium (S) has germinated, penetrating an epidermal cell directly 2 hr after inoculation. A stomatal opening (ST) is to the upper right of the sporangium (S) x 1200.

F ig. The sporangium (S) of race 1 has a collar (C) of material surrounding the penetration peg, 2 hr after inoculation x 3500. '(&&&?> > 'v 1 * * * ri Fig. 9 A germinated sporangium (S) of race 1 12 hr after inoculation. The germ tube has bifurcated, one tube terminating in a bulb (B) which appears similar to an appressorium x 1H00.

Fig. 10 A germinated sporangium (S) of race 0 with its germ tube penetrating a stomatal opening (ST) 2 hr after inoculation x 1500.

Fig. 11 A mycelial strand (M) of race 0 penetrating a stomatal opening (ST) 2 hr after inoculation x 1500.

1 4

Leaf Clearings

Leaf cells from healthy tissue were clear after aniline blue staining (Fig. 12). Germinated sporangia and zoospores and an abundance of mycelium were found on the cleared and stained inoculated leaf discs when observed between 12 and 72 hr after inoculation (Fig. 13, 15, 16). Empty sporangial cases, ungerminated sporangia and sporangia containing unliberated zoospores from the original inoculum were seen on the surface of all leaf discs following inoculation (Fig. 14).

Intercellular mycelium with haustoria protruding into host cells was found in abundance 48 hr after inoculation throughout the tomato leaf tissue (Fig. 17). A number of the palisade mesophyll cells contain haustoria 48 hr after inoculation (Fig.

18, 19, 20 and 21). The mycelium appears to grow between the cells of the palisade mesophyll, branching in several directions. There are one to several haustoria per cell. The haustoria are finger­ like, slender and curved (Fig. 19, 20, 21) to curled (Fig. 18) projections into the host cells. Host cell walls are outlined and nuclei are observable in some cells. Two or more haustoria may arise in close proximity to one another and penetrate the same cell (Fig. 21). Numerous haustoria were observed to penetrate single cells when intercellular mycelium partially encircled the cell (Fig. 18). 15

KEY TO LABELING OF THE FIGURES

ES = empty sporangium

GT = germ tube

H = haustorium

HC = host cell

M = mycelium

N = nucleus

S = sporangium

ST = stomate

UZ = unliberated zoospores

Z - zoospore F i g . 12 Healthy tomato leaf epidermal cells x 650.

F ig . 13 Sporangia (S) and mycelium (M) of race 1 on the leaf surface 48 hr after inoculation x 500.

F ig . 14 Empty sporangial cases (ES), u n d iffe re n tia te d sporangia (S) and sporangia with unliberated zoospores (UZ) on the leaf surface 48 hr after inoculation x 800.

Fig. 15 A germinated sporangium (S) is lying on the guard cell of a stomate (ST) on the leaf surface 48 hr after inoculation x 1500. JLO Fig. 16 The germ tube (GT) of a germinated zoospore (Z) is lying at the junction between two epidermal cells, <18 hr after inoculation. Mycelial strands (M) are darkly stained x 1700.

F ig. 17 Ramifying mycelium (M) of race 1 with numerous haustoria (H) easily visible within the leaf tissue 48 hr after inoculation x 400.

F ig. 18 Mycelium (M) of race 1 ramifying between host cells (HC). Numerous haustoria (H) have invaded the host cells (HC). The cell nuclei stain darkly x 1300.

F ig. 19 Mycelium (M) of race 1 has bifurcated around a host cell (HC). A haustorium (H) has penetrated the cell at this point. The host cell nucleus (N) is in close proximity to the haustorium (H) 72 hr after inoculation x 1400.

Fig. 20 Mycelium (M) of race 1 with several haustoria protruding into host cells (HC) 48 hr after inoculation x 1300.

Fig. 21 Higher magnification of Fig. 20 x 1900.

19

Histology

Samples taken prior to 98 hr after inoculation and fixed for processing for light and electron microscopy did not have sufficient fungal mycelium in the host tissue to be located with ease. Thus the material reported in this study was obtained from tissue 98 and 72 hr after inoculation when disease expression was advanced. Cells in these tissue pieces were found to be in various stages of disruption as a result of the fungus infection.

A cross section of uninoculated, healthy tomato tissue shows the epidermis, palisade and spongy mesophyll and a minor vein area of the leaf (Fig. 22). A montage’ electron micrograph (Fig.

23) was prepared from the same block o f tis s u e . The epiderm al and guard cells are to the left. Palisade cells are the chloro- plast containing cells to the right of the photograph. Spaces between the palisade cells are intercellular spaces.

Light micrographs of tissue infected with P. infestans showed portions of fungal mycelium intercellularly with numerous haustoria protruding into cells (Fig. 29, 99, 60, 69). Cell destruction was almost complete in the lesion area after 72 hr (Fig. 99).

Some of the cells have darkened cellular contents. Cell outlines are very irregular. In some tissue segments, a number of cells were not totally destroyed, but were in various stages of cell disruption.

A matrix which stained differently than the host cell wall or the fungal cell wall was found between the two frequently (Fig. 20

25, 29, 30, 91, 62, 63). What appears to be the same material also was found to be present between adjacent pieces of mycelium

(Fig. 36, 93).

Prior to the development of a haustorium in a host cell, cell wall material accumulated or was modified to form a papilla-like structure in the area of contact of the mycelium and the cell wall

(F ig. 25) . The h o st plasma membrane rem ains continuous around the papilla (Fig. 25, 95). During or after papilla formation, the fungal cell wall begins to ingress into the papilla area (Fig.

95). The fungal cell wall is distinguished from the host cell wall by the differential staining properties of each. At the point where the haustorium passes through the cell wall, it is constricted. Beyond the point of constriction, the haustorium begins to increase in diameter. The haustorium is bulbous in the initial stages (Fig. 26, 27, 28), but develops into a long straight to curved finger-like projection into the cell as it matures

(Fig. 32, 33, 39). Occasionally the haustoria are coiled or branched.

As the young haustorium pushes through the host cell wall at the site of the papilla (cell wall lesion), the papilla material encapsulates the haustorium (Fig. 27, 28). The papilla material which forms the layer around the young haustorium has staining properties similar to the host wall. Wedges of this papilla material at the constricted neck of the haustorium contain p a r tic le s th a t s ta in d ark ly . The h o st plasma membrane invaginates 21 as the haustorium pushes in and remains continuous between the host cytoplasm and the encapsulated haustorium.

When the haustorium increases in size inside the host cell,

the host plasma membrane remains intact around the haustorium

(Fig. 34, 35). As the haustorium elongates, the papilla material which covered the young bulbous haustorium is left as a collar

surrounding the neck and base of the mature haustorium (Fig. 32,

33, 51). Thus, the major part of the mature haustorium appears to

be separated from the host cytoplasm only by the host plasma

membrane (F ig. 34). Sections through the base of curved haustoria (Fig. 29, 30)

show that they are separated from the host cytoplasm by the host

plasma membrane and a la y e r o f c o lla r m a te ria l sim ila r in

staining properties to the host wall. Host cell wall lesions

containing darkly stained particulate matter were found near the

neck of some haustoria (Fig. 30, 31). Some of the host cell wall

lesions located near the constricted neck of haustoria were found

to be continuous with the collar material between the haustorium

and the ho st plasma membrane (F ig. 33).

A prominent feature of young haustoria is the presence of

numerous mitochondria and endoplasmic reticulum (Fig. 31, 32,

33, 34, 35, 50, 51). Mitochondria and endoplasmic reticulum pass

freely through the haustorial neck into the developing haustorium.

No nuclei were found in the haustoria observed.

Frequently particulate matter was observed in host walls in 22 close proximity to fungal mycelium. Vesicles similar to golgi vesicles were observed near the host plasma membrane (Fig. 27,

36, 42). Vesicle or membrane remnants were discernible beneath the plasma membrane in the host w all (Fig. 36, 42). G enerally, the host plasma membrane became convoluted around the areas adja­ cent to the regions of haustorial penetration (Fig. 48, 61,

63) .

Oblique sections through some haustoria revealed an irregularly shaped accumulation of material between the host plasma membrane and the fungal wall (Fig. 46, 47, 48, 53). This material was present irrespective of the fixing procedure used. The plasma membrane was continuous around these accumulations.

The penetration of a haustorium into a host cell brings about certain changes in the host cytoplasm. The chloroplast shows sig n s o f d is ru p tio n . The o u te r membrane of the c h lo ro p la st begins to breakdown (Fig. 27, 29, 42) and the grana and intergranal lamellae separate, becoming disorganized. Even though the grana become disorganized, they do not completely breakdown and separate but remain in a loose association (Fig. 46, 48, 50, 52,

54) . Host cell nuclei and swollen endoplasmic reticulum were often closely associated with some of the haustoria (Fig. 35, 61).

Certain of the cells in the leaf were in late stages of destruction (Fig. 49, 54, 55, 65). The host cell contents stained darkly with few organelles discernible (Fig. 49, 55). Haustoria from intercellular mycelium (Fig. 55) invaded two adjacent cells, 23 the contents of both cells are dark. Certain leaf cells were invaded by several haustoria (Fig. 54) . Portions of some of the chloroplast grana could be recognized. Even in the late stages of infection, the host cell walls retain their integrity (Fig. 54,

55) .

In some cells of leaf tissue inoculated with race 0 (incompatible), the cellular contents have accumulated around the haustorium

(Fig. 65). In addition to the host cytoplasm, the cytoplasm in the mycelium and haustorium of the fungus stained darkly.

A number of the cells in diseased tissue showed cellular disorders attributable to the fungal invasion even though they were not penetrated by haustoria. Disruption of the chloroplast g ran al o rg an izatio n and the breakdown o f the membrane around the chloroplast are some of the first observable effects on cells that lie in close proximity to the intercellular mycelium of the invading fungus (Fig. 39, 40, 43). In some cells the chloroplast membrane was com pletely destroyed (Fig. 57).

A common feature of leaflet infection was the observance of wall lesions (Fig. 30, 31, 33, 36, 37, 38, 40, 41, 58, 59, 66, 67)

In some cells darkly stained particulate matter was found in the lesion area (Fig. 30, 31, 33, 36, 37, 38, 41, 66). Several uninfected cells showed extensive wall lesions (Fig. 59, 66, 67).

An accumulation of vesicular material in the cell wall exterior

to the plasma membrane was observed (Fig. 66). The le sio n s appeared to be similar in adjacent cells. The plasma membrane 24 was convoluted along the lesion surface but appeared to remain

intact. Lesion formation was present irrespective of the race of

P. infestans used as the inoculum.

Sections through the minor vein areas of the leaf revealed

the presence of fungal mycelium intracellularly in xylem vessels

(Fig. 56) when the mycelium of the fungus invaded mesophyll cells

in the vicinity of the vein area. 25

KEY TO LABELING OF THE FIGURES

C = chloroplast

CO = collar

CY = cytoplasm

EP = epidermal cell

ER = endoplasmic reticulum

FC = fungal cell

FN - fungal nucleus

FW = fungal wall

G = golg i

H = haustorium

HC = host cell

HN = host cell nucleus

HW = host cell wall

IN = intercellular space

M = mitochondrion

MA = matrix

MV = minor vein

MY = fungal mycelium

NE = haustorial neck

P = papilla

PAL = palisade mesophyll cell

PM = plasma membrane

S = sheath

SPO = spongy mesophyll cell ST = stomate

V = vacuole

VE = vesicles

WL = wall lesion

X = xylem vessel Fig. 22 Light micrograph of a one micron cross section through a healthy tomato leaf. The epidermis (EP), palisade mesophyll (PAL), spongy mesophyll (SPO) and minor vein (MV) are shown in the cross section x 900.

Fig. 23 A montage' electron micrograph of healthy tomato leaf tissue. The epidermis (EP), a stomate (ST), palisade mesophyll cells (PAL) and intercellular spaces (IN) are shown in the montage', (OsO^ x 5200). PAL F ig . 24 Light micrograph of a one micron section of tomato leaf tissue. The fungal mycelium (MY) is shown ramifying between the host cells (HC). The upper epidermis (EP) is to the left of the micrograph (72 hr, race 1, KMnO^ x 800).

F ig . 25 The initial stage in the development of a haustorium. A papilla-like structure (P) of modified host cell wall (HW) is ingressing into the cell cytoplasm. Matrix material (MA) is observed between the fungal cell wall (FW) and the host cell wall (HW) (72 hr, race 1, KMn04 x 10,400).

F ig . 26 Survey view of leaf tissue showing haustoria (H) in host cells (HC) and intercellular mycelium (MY) (72 hr, race 1, KMnOq. x 11,000).

F ig. 27 Higher magnification of Fig. 26. This is a section through a young haustorium (H) which is surrounded by a sheath (S) of cell wall material. The chloroplast (C) shows some grana1 disorganization. Golgi (G) and vesicles, presumbly golgi vesicles (VE), are observed in the vicinity of the haustorium (H) (72 hr, race 1, KMnOij. x 10,000) . c. u

EP1

>jf-v)r$

A k c ^ i t v ^ 14 tr!^-sr:j|#^

F W ^ )„ v ¥ V (; ^ - PM Px

% § P f c

Itoi G

r &£>;. ., jZ\f

; ■*‘A v t% ^ - :^-. - '"'V V v \< f> ' ' '.;, V*i>E)f

i o SI K''c J « # i 27 Fig. 28 Serial section through the haustorium (H) in Fig. 27. The haustorium (H) is surrounded by a sheath (S) of modified host cell wall which has particulate matter embedded in it. The plasma membrane (PM) is continuous around the sheath (S) (72 hr, race 1, KMnO^ x 22,000).

F ig . 29 Section through the base of a curved haustorium (H). The collar (CO) of sheath material lies between the host plasma membrane (PM) and the fungal cell wall (FW). Matrix material (MA) is present between the fungal cell wall (FW) and the host cell wall (HW) (72 hr, race 1, KMnOq. x 20,000).

F ig . 30 Section through a haustorium (H) showing the constricted neck (NE). The collar (CO) of sheath material surrounds the haustorium (H). Matrix material (MA) is present between the fungal cell wall (FW) and the host cell wall. Wall lesions (WL) are observed on either side of the haustorium (72 hr, race 1, KMnOq. x 20,000) .

F ig . 31 A section through a haustorium (H) with a constricted neck (NE) . A wall lesion (WL) is observed in close proximity to the haustorial neck (NE) (72 hr, race 1, KMnO^ x 21,000). M V L * W i

■%4 ' H FW

1 29 sf?*: •• ;• , ■• ♦

, <■ ^7 • • s*

-n & M , ■'LijV ••’

WWi/v* 4cul A| . '-■■ >-'■*- /f \ i '&'''■■■'$ |4j •: ’* k

-V W, o Fig. 32 Longitudinal section through a mature haustorium (H). A collar (CO) of sheath material is present around the constricted neck of the haustorium. Mitochondria (M) and endoplasmic reticulum (ER) are present in the haustorium (H) (72 hr, race 1, KMnOij. x 11,200).

F ig. 33 Higher magnification of Fig. 32. Wall lesions (WL) are observed on either side of the haustorium (H). The wall lesion (WL) to the right is continuous with the collar (CO) of sheath material (72 hr, race 1, KMnOij. x 22,400).

F ig. 34 Longitudinal section through a haustorium (H). The distal end of the haustorium (arrow) is separated from the host cytoplasm only by the host plasma membrane. Numerous vesicles are observed in the host cytoplasm around the haustorium (H) (72 hr, race 1, KMnOi* x 7200).

F ig. 35 Higher magnification of Fig. 34. The haustorium is separated from the h o st plasma membrane (PM) by a c o lla r (CO) of sheath material around the constricted neck (72 hr, race 1, KMnOq. x 22,500). ^ - 4 1 1 Fig. 36 Cross section through a haustorium (H) and intercellular mycelium (MY) of the fungus. Vesicles (VE) are observed in abundance near the host plasma membrane (PM). Some of the v e sic le s are shown to be e x te rio r to the h o st plasma membrane (PM). Matrix material (MA) is found between adjacent mycelial strands (72 hr, race 1, KMnOq. x 10,000).

F ig. 37 Particulate material (arrows) is found in the host cell wall (HW) in close association to the fungal cell wall (FW) (72 hr, race 1, KMnOi^ x 26,000).

F ig. 38 Wall lesions (WL) are observed in cells with intercellular mycelium (MY) in close association to the host cell wall (72 hr, race 1, KMnOq. x 10,000) .

Fig. 39 Chloroplast (C) disorganization in uninfected host cell with intercellular mycelium in close association (72 hr, race 1, KMn04 x 9000).

Fig. 40 A survey view of host cells (HC) in close association to intercellular mycelium (MY) (72 hr, race 1, KMnOij. x 11,000) .

Fig. 41 Higher magnification of Fig. 40. A wall lesion (WL) is found in close association to intercellular mycelium (MY). A cross section through a haustorium (H) is observed. Matrix material (MA) is found between the fungal and the host cell walls (72 hr, race 1, KMnOq. x 15,000).

Fig. 42 Section through a cell with cross sections through several haustoria (H). Endoplasmic reticulum (ER) is shown surrounding a haustorium (H). Numerous vesicles (VE) were observed close to the host cell wall (72 hr, race 1, KMnO^ x 8400).

F ig. 43 Matrix material (MA) is shown between adjacent mycelial strands (MY). Golgi (G) is observed and chloroplasts (C) with granal disorganization (72 hr, race 1, KMnOq^ x 9000 ) .

Fig. 44 Light micrograph of a one micron section through tomato leaf tissue with fungal mycelium (MY) among the spongy mesophyll cells (SPO) (72 hr, race 1, OsOq x 800).

Fig. 45 An initial stage in haustorial penetration is the development of a papilla-like structure (P) of cell wall material. The fibrillar nature of the cell wall can be discerned. The fungal cell begins to ingress (arrow) in the region of the p a p illa (P ). The plasma membrane (PM) is continuous around the papilla area (72 hr, race 1, OsOq x 18,500).

Fig. 46 Section through a haustorium (H). The haustorial neck is surrounded by a sheath (S) (72 hr, race 1, OsOq x 12,000).

Fig. 47 Higher magnification of Fig. 46. The haustorium (H) is surrounded by a sheath (S) of ir re g u la r o u tlin e (72 h r, race 1, OsOq. x 14,000) .

Fig. 48 Section through a haustorium (H) surrounded by a collar (CO) o f sheath m ateria l of irre g u la r o u tlin e . The plasma membrane (PM) remains continuous around the collar (CO) (72 hr, race 1, 0s0(| x 13,500) .

F ig. 49 Section through a haustorium (H) surrounded by a collar (CO) of sheath material. The host cell (HC) contents have darkened (72 hr, race 1, OsOq. x 13,500).

F ig. 50 Section through a haustorium (H). The host cell contents are in a late stage of destruction (72 hr, race 1, OsOij. x 14,500) „

F ig. 51 Higher magnification of Pig. 50. A collar (CO) of sheath material surrounds the constricted haustorial neck. The distal portion of the haustorium (arrows) is not surrounded by a sheath (72 hr, race 1, OsO^ x 21,000).

Fig. 52 Section through haustoria (H) in a host cell. The mitochondria (M) are distorted (72 hr, race 1, OsOij. x 4500) .

Fig. 53 Higher magnification of Fig. 52. The haustorium (H) is surrounded by a collar (CO) of modified host cell wall material (72 hr, race 1, OsO^ x 13,000).

Fig. 54 Section through haustoria (H) in a host cell. The host cell is in a late stage of destruction (72 hr, race 1, OsO^ x 10,000).

Fig. 55 Section through haustoria (H) in host cells which are in late stages of destruction. The host cell contents are darkened (72 h r, race 1, OsOij. x 11,000) . J* - *fc a p-j,™->-H '"1 •'7TO -. ( !- .» SS -iS^l ’W U-,

m r "

fc ;™ / ' {r«« ^ A -s&.t m im aim - : r 4 , ^ , ? >

SjEfJ ©’.' S hew p*** t 1 «f -f

^S&8PnSjffA,A .4^% ».fci

K

4 ' ’ W H

o' , -• •> r * r m

AIM “s F ig . 56 Intracellular mycelium (MY) in xylem vessel elements (X) (72 hr, race 1, OsO^ x 13,500).

F ig. 57 Chloroplast (C) destruction in uninfected leaf cells in close proximity to intercellular mycelium (72 hr, race 1, OsOij. x 10,000).

F ig. 58 Wall lesions (arrows) in uninfected leaf cells adjacent to infected cells (72 hr, race 1, OsOq. x 1600) .

F ig. 59 Wall lesions (arrows) in uninfected leaf cells adjacent to infected cells (72 hr, race 1, OsOij. x 1700) . 36 F ig . 60 Light micrograph of a one micron section of tomato leaf tissue infected with fungal cells (FC) of JP. infestans in the spongy mesophyll cells (SPO) of the leaf (48 hr, race 1, OsOij. x 700) .

Fig. 61 Section through a portion of a haustorium. The host cell nucleus (HN) is in close proximity to the fungal haustorium and the h o st plasma membrane (arrow) is convoluted in the vicinity of the haustorium (48 hr, race 1, 0s0i| x 13,800).

F ig. 62 Section through a haustorium. Matrix material is found between the fungal and the host cell walls (arrow) (48 hr, race 1, OsO^ x 12,500) .

Fig. 63 Higher magnification of Fig. 62. The fungal cell wall (FW) i s separated from the h o st plasma membrane by a sheath. Matrix material is found between the fungal and the host cell w alls (arrow ). The h o st plasma membrane (PM) is convoluted (48 hr, race 1, OsOq. x 18,600). k t S i w F ig . 64 Light micrograph of a one micron section through tomato leaf tissue infected with fungal cells (FC) of race 0 in the spongy mesophyll (SPO) cells (72 hr, race 0, OsOq x 800) .

F ig . 65 Section through a haustorium (H) in a spongy mesophyll cell (SPO). A portion of the host cell contents appear to have gelatinized around the haustorium (H) (72 hr, race 0, OsOq. x 10,800).

Fig. 66 Wall lesions (WL) in uninfected leaf cells. Numerous vesicle material is noted in the cell wall lesion (WL) area (72 hr, race 0, OsOq. x 30,000) .

F ig. 67 Wall lesion (WL), darkly stained, in an uninfected leaf cell (72 hr, race 0, OsOq. x 13,500). 38 DISCUSSION

Scanning Electron Microscopy

Use of the scanning electron microscope in the study of surface infections has become an increasingly valuable tool for researchers. Several studies have been done which have utilized the scanning electron microscope to study early stages of infection on plant surfaces by pathogens (3, 29) . The purpose of the present investigation was to study the primary infection process of P. infestans on tomato leaf tissue.

As is shown in the two survey views of the leaf surface (Fig.

2 and 3), the inoculum was randomly distributed on the surface.

However, in the areas where leaf hairs were present, the sporangia had a tendency to accumulate around these structures or in the crevices between rows of glandular hairs. In areas absent of leaf hairs, the sporangia showed no definite distribution pattern.

Kishi (17) observed that zoosp@i?es of certain isolates of P. infestans showed a taxis for the basal cells of leaf hairs on tomato. This was especially true if the hairs had been injured.

He also showed that in cases where the basal cells became infected, a hypersensitive reaction was observed on both susceptible and resistant hosts.

In this study fewer zoospores were observed than was anticipated. Although some zoospores were observed which had germinated (Fig. 5 and 6), Pristou and Gallegly (21) observed

39 40

from 1ight microscopic observations of sectioned potato leaf

tissue that the P. infestans zoospores were the primary agents of infection, usually entering the host directly through the

epidermal layer. Occasionally, the germ tubes of zoospores

entered through stomata. The fixing methods used in the present

study may have destroyed or dislodged many of the zoospores which would account for the difficulties experienced in finding and

recognizing them.

When a sporangium germinated and penetrated an epidermal cell

directly, a collar zone was evident around the penetrating hypha

(Fig. 8). Its appearance would suggest that the collar is probably

composed of cuticular leaf material instead of being of fungal

origin. It is likely that enzymes secreted by the fungus around

the point of penetration could have brought about this collar of

leaf cell exudate.

Penetration hyphae of sporangia were shown to pass through

open stomates on the leaf surface. When this occurred, it

appeared that the sporangium had germinated on or near a guard

cell and passed through an open stomate (Fig. 10).

A number of the sporangia which germinated on the leaf surface,

not in close proximity to guard cells or stomates, produced

hyphae which ramified over the leaf surface. One such sporangium

is shown in Fig. 9. A short distance from the sporangium the

hyphal strand bifurcated, one side terminating in a bulbous

structure. This bulbous structure is probably similar to that 41 produced by germinated zoospores. This structure may be an appressorium.

Mycelial strands were observed on most leaf discs several hours following inoculation. Some strands were observed to pass through opened stomates on the leaf surface (Fig. 11).

Most observations dealt with sporangia of P. infestans penetrating the leaf surface in one to several ways. Improved fixation procedures may enable the zoospores of this fungus to be studied more successfully during the early infection process.

Leaf Clearings

Tissue clearing methods have been useful in helping to elucidate information about host-pathogen relationships. Blackwell

(5) presented an extensive light microscopic study of the haustoria of P. infestans in both potato tuber and leaflet tissue. Boothroyd

(6) has outlined a generalized leaf clearing procedure for observing zoospore germination and infection of P. infestans on potato tissue.

A study of the process of infection and the reaction of host tomato leaf tissue to P. infestans invasion was reported by Kishi (17).

He used epidermal tissue strips, treated with a vital stain, to observe the germination and infection of zoospores in tomato cells.

The purpose of the present study was to observe the location of the late blight fungus, P. infestans. in tomato leaf cells.

For this study, leaf clearings were made of tomato tissue infected with race 1 (compatible).

Material taken from healthy leaf tissue showed the epidermal ‘12 cells and stomates clearly on the leaf surface. By focusing downward, the palisade and spongy mesophyll cells were easily

recognized (Fig. 12).

On inoculated leaf discs, abundant mycelium was observed as

early as 12 hr after inoculation (Fig. 13). The mycelium ramified

over the surface and was very abundant. Sporangia were recognized

easily on the surface by the intense staining of the sporangial

contents. Empty sporangial cases and sporangia, some with differentiated zoospores were observed in small groups on the

leaf surface (Fig. I1!) . When the internal contents of the sporangium had differentiated into zoospores, six to seven zoospores were observed.

Sporangia may germinate directly on the leaf surface instead of producing zoospores. One such sporangium is shown in Fig. 15

The sporangium is in close proximity to a stomatal opening and

the germ tube of the sporangium is lying on the edge of a guard

cell. As was shown in Section I, Scanning Electron Microscopy,

germ tubes of sporangia were observed to pass through stomatal

openings, making it possible for sporangia to bring about

in fe c tio n .

Zoospores are considered to be the primary agents of infection

(28). In Fig. 16, a germinated zoospore is shown. The tip of the

germ tube is at the junction between two epidermal cells.

Observations of germinated zoospores on the leaf surface were

rare. It may be assumed that the procedure used in clearing the *43 leaf t-issue could have destroyed such delicate spore material.

The fungal mycelium stained intensely with the aniline blue, permitting its location in the host tissue to be determined with ease (Fig. 17). The mycelium was very abundant, ramifying intercellularly throughout the leaf disc in all directions.

Haustoria are long, slender, curved structures protruding into the host cells from the hyphal surface (Fig. 18, 19, 20, 21).

One to many haustoria were observed in a number of mesophyll cells.

Host nuclei stained intensely with the aniline blue and the cell walls were prominent. At times the mycelium bifurcated around a cell generally producing a haustorium at the junction of the bifurcation (Fig. 19). This pattern of movement through the mesophyll is similar to that reported by Blackwell (5) in potato leaf tissue.

Histology

Intercellular mycelium of P. infestans was found in abundance ramifying between the cells on the interior of the tomato leaf as early as 98 hr after inoculation with race 1 (Fig. 29, 99, 60).

Observations of mycelium were rare in leaf tissue inoculated with race 0 (Fig. 69). By 72 hr after inoculation, portions of the fungal mycelium were found intracellularly in the xylem vessels

(Fig. 56). The presence of mycelium in the vascular elements has not been reported previously for this disease. This, in part, could account fo r the rap id c o llap se o f tis s u e , a symptom of the late blight disease in tomato plants. 44

A matrix material with staining properties different from either the fungal or host cell walls was found between adjacent fungal and host cell walls (Fig. 25, 27, 29, 30, 36, 38, 62, 63).

The matrix was also present between adjacent segments of mycelium

(Fig. 35, 43). It is possible that this matrix is an adhesive coating secreted by the fungus in the vicinity of the host cell wall. Bracker (7) observed a similar coating on the surface of

Erysiphe graminis. However, no mention was made of the presence of this matrix between adjacent mycelial strands of the pathogen.

Penetration of a tomato leaf cell by a haustorium of P. infestans begins with the development of a small papilla of presumably host wall material in the vicinity of the future haustorium (Fig. 25, 45). In some cases, penetration was preceeded by the development of cell wall structures in which the fibrillar composition of the cell wall was evident (Fig. 45).

A portion of the fungal cell wall begins to ingress into the papilla area of the host cell wall. Ehrlich and Ehrlich (11) citing unpublished data claim to have observed papilla formation by P. infestans in potato tissue. Hanchey and Wheeler (14) observed similar structures in cells of tobacco roots infected with P. parasitica. They suggest that the irregular wall border may have a gel-like consistency due to minor cell wall dissolution and imbibition of water.

Young capitate haustoria were individually surrounded by a 4 5 layer of sheath material similar in staining properties to the host cell wall. This layer is presumably modified cell wall

(Fig. 27, 28). Particulate matter (Fig. 28) is present in the

sheath matrix proximal to the constricted neck of the haustorium.

Serial sections through the haustorium indicated that the entire haustorium was enclosed by the sheath of modified host wall material. Particulate matter in the sheath and the irregularity of the border of wall material indicate that the host cell wall may have softened in this region. The plasma membrane remains

continuous around the border.

Berlin and Bowen (4) suggest that the term sheath be used to

connote those areas around the parasite which are of cell wall

origin, whether the structure forms a collar around the

haustorial neck or covers the entire haustorium. The term sheath

has been used in this study to designate a layer of modified host

cell wall material between the parasite cell wall and the host

plasma membrane.

Ehrlich and Ehrlich (12) have used the term encapsulation to

designate the layer of material around the haustoria of P.

infestans on potato leaf tissue. They describe this area as a

region o f unknown m a te ria l e x is tin g between the ho st plasma

membrane and the p a ra s ite c e ll w a ll, n e ith e r ty p ic a lly o f ho st

nor of fungal origin. However, their micrographs indicate that

this area of the encapsulation has a similarity in staining 46 p ro p e rtie s to the host c e ll w a ll. The term sheath would seem more appropriate to their description than the term encapsulation.

Older haustoria are surrounded by a collar of sheath material between the fungal c e ll w all and the ho st plasma membrane (Fig.

29, 30, 32, 33, 34, 35, 48, 49, 50, 51, 53). The host plasma membrane is continuous around the haustorium . L ongitudinal se c tio n s through mature haustoria suggest that the host cell wall is modified in the vicinity of the haustorial neck (Fig. 33, 35) forming a collar around the basal portion of haustoria. However, the distal portion of the haustorium is not surrounded by a sheath

(Fig. 34) suggesting that as the haustorium elongated, it grew through the sheath thus forming the collar. No wall material or fungal secretory material can be discerned in this region. Numerous vesicles appear in the cytoplasm around the haustorium (Fig. 34) .

However, no vesicles were observed which merged with the plasma membrane.

Calonge (10) found that the entire haustorial complex of P. palmivora was not enclosed by a typical sheath or an encapsulation.

The distal portion of the haustorium appeared to lie naked against the host cellular debris.

In oblique sections through mature haustoria, the sheath material is very irregular in outline (Fig. 46, 47, 48, 52, 53).

It is possible that the irregular outline of the sheath in these * cells has a gel-like consistency as suggested by Hanchey and

Wheeler (14) who observed similar host wall modifications around 47

the haustoria of P. parasitica in tobacco root cells.

Ehrlich and Ehrlich (12) refer to what is called sheath in

this study as an encapsulation. They suggest that the entire

haustorium is "encapsulated" by material which is neither

distinctly of host nor of fungal origin. As mentioned previously,

this study suggests that the sheath becomes only a collar around

the base of the haustorium. The Ehrlichs* interpretation of the

encapsulation region may have been made from sections of young

haustoria or oblique sections through older haustoria, either

of which would lead one to conclude that the entire haustorium was "encapsulated". None of their micrographs show longitudinal

sections through mature haustoria. They describe haustoria of

P. infestans as "small globes to short, straight or curved pegs".

This description does not fit well with haustoria of P. infestans

in potato tissue described by Blackwell (5) as straight to curved

finger-like projections occasionally being curled or the

haustoria in tomato leaf cells observed in this study.

In a number of cells, the host cell nucleus was in close

proximity to the haustorium (Fig. 61). Kishi (17) observed that

the nucleus of the tomato leaf cells moved to the haustorial locus

in infected cells.

Swollen endoplasmic reticulum, increased golgi activity,

distorted mitochondria and chloroplast destruction were disease

symptoms observed in h o st c e l l s . These o b serv atio n s are

consistent with the findings of other workers (4, 8, 10, 11, 12, 1+8

1*+, 15, 20, 29) for host cell symptoms of disease.

Vesicle material similar in appearance to golgi vesicles were observed in the vicinity of the pathogen (Fig. 36, *+2). Some o f the v e sic le s are p o sitio n ed e x te r io r to the plasma membrane

(Fig. 36). Peyton and Bowen (20) observed secretory vesicles in the cytoplasm around the haustoria of Peronospora manshurica in

Glycine max host cells. These vesicles were not found in control cells. They proposed that these vesicles fused with the plasma membrane, discharging their contents into the zone between the host plasma membrane and the parasite cell wall. Small segments of membrane were observed in this zone. A similar mechanism could be postulated for P. infestans in tomato cells based on the particulate matter found in the sheath material at the base of the haustoria. However, no vesicles were seen to fuse with the h o st plasma membrane in the c e lls stu d ie d .

Kishi (17) observed cellular discoloration in infected cells

15 to 29 hr after penetration of susceptible tomato epidermal cells by race 1 of P. infestans. The discoloration proceeded from brown to black. Some cells observed in the present study were in late stages of cell destruction (Fig. 99, 59, 55, 65).

The cell contents are darkened. Since cell darkening is associated with phenolic compound synthesis (1, 9, 25), it is likely that these cells contain an accumulation of phenolic compounds.

A haustorium of race 0 is shown in Fig. 65. The cell contents 49 appear to have accumulated around the haustorium. Whether this is characteristic of the resistant host reaction was not determined because observation of race 0 haustoria were rare.

The dark staining haustorium and portion of the intercellular mycelium suggest that this haustorium may be a necrotic portion of the fungus, similar to the necrotic haustoria of Albugo Candida observed by Berlin and Bowen (4). Kishi (17) observed with race 0 of P. infestans that cellular discoloration occurred within 2 hr after penetration. All cellular movement ceased 3 hr after penetration followed by the cell contents becoming gelatinized around the fungal haustorium.

Wall lesions were observed in both infected and uninfected cells in infected tissue (Fig. 30, 31, 33, 36, 37, 38, 40, 41,

58, 59, 66, 67). In some cells particulate matter was found in the lesion area (Fig. 30, 31, 33, 36, 37, 38, 41, 66). This material could represent secretory material and/or vesicle remnants. Some cells have lesions which are darkly stained as are the cell walls in those areas (Fig. 58, 59, 67). It is likely that these cells have had increased phenolic compound synthesis

(25) resulting in the stained walls. More extensive wall lesions were observed in uninfected cells than in infected cells which suggests that a fungal toxin is involved in pathogenesis. Host cell wall lesions were frequently associated with extensive cellular damage. SUMMARY

Scanning Electron Microscopy

1. Germinated zoospores of P. infestans were observed on the

surface of inoculated tomato leaf tissue.

2. Some sporangia of P. infestans germinated and penetrated the

host tissue either directly through the epidermal cells or

through stomatal openings on the leaf surface.

3. Mycelial strands of the fungus were observed to pass through

stomatal openings.

Leaf Clearings

1. Germinated and ungerminated sporangia, zoospores and mycelium

of P. infestans were found on the surface of stained tomato

leaf discs.

2. Intercellular mycelium with slender, finger-like, curved

haustoria were found in abundance throughout tomato leaf

tissue 48 hr after inoculation.

H istology

1. Matrix material which stained differently than the fungal

cell wall or the host cell wall was found between the two

cell walls. Similar material was present between adjacent

mycelial strands.

2. A papilla-like structure of modified host cell wall material

preceeds penetration and development of a haustorium.

3. Young c a p ita te h a u sto ria of P. in fe sta n s are surrounded by a

sheath of modified cell wall material which often contains particulate material embedded in it.

Mature haustoria have a collar of sheath material around

the basal portion of the haustorium. The distal portion of

the haustorium is not covered by a sheath, but appears to

lie directly against the host plasma membrane.

Swollen endoplasmic reticulum, increased golgi activity, distorted mitochondria and chloroplast destruction were

effects of the disease observed in host cells.

Wall lesions were observed in both infected and uninfected cells. More extensive wall lesions were found in uninfected

than infected cells.

Mycelium was found intracellularly in xylem vessels of tomato

l e a f l e t s . LITERATURE CITED

Agrios, G. N. 1969. Plant pathology, p. 120-133. Academic

Press. New York.

Arnott, N. J., S. W. Russo, and K. M. Smith. 1969. Modification

of plastid ultrastructure in tomato leaf cells infected

with tobacco mosaic virus. J. Ultrastructural Res. 27:

149-167.

Barnes, G. and Neve, N. F. B. 1968. Examination of plant

surface microflora by the scanning electron microscope.

Trans. Brit. Mycol. Soc. 51: 811-812.

B e rlin , J . D. and C. C. Bowen. 1964. The h o s t-p a ra s ite

interface of Albugo Candida on Raphanus sativus. Amer.

J. Bot. 51: 445-452.

Blackwell, E. M. 1953. Haustoria of Phytophthora infestans

and some other species. Trans. Brit. Mycol. Soc. 36:

138-158.

Boothroyd, C. W. 1967. Direct penetration of potato leaves

by germ tubes from sporangia of the potato late blight

fungus, p. 63-64. Sourcebook of laboratory experiments

in plant pathology. W. H. Freeman and Company.

Bracker, C. E. 1967. Ultrastructure of fungi. Annu. Rev.

Phytopathol. 5: 343-374.

Bracker, C. E. 1968. Ultrastructure of the haustorial apparatus

of Erysiphe graminis and its relationship to the epidermal

cell of barley. Phytopathology 58: 12-30. S3

9 . Brenneman, J. A. 1970. Respiration and terminal oxidases in

tomato leaves infected by Phytophthora infestans. Ph.D.

Thesis. Louisiana State Univ., 35 p.

10. Calonge, F. D. 1969. Ultrastructure of the haustoria or

intracellular hyphae in four different fungi. Arch.

Mikrobiol. 67: 209-225.

1 1. Ehrlich, M. A. and H. G. Ehrlich. 1971. Fine structure of

the host-parasite interfaces in mycoparasitism. Annu.

Rev. Phytopathol. 9: 155-189.

1 2. Ehrlich, M. A. and H. G. Ehrlich. 1966. Ultrastructure of

the hyphae and haustoria of Phytophthora infestans and

hyphae of P. parasitica. Can. J. Bot. 99: 1995-1503.

13. Freeman, J. and Spurlock, B. 1962. A new epoxy embedment for

electron microscopy. J. Cell Biol. 13: 937.

19. Hanchey, P. and H. Wheeler. 1971. Pathological changes in

ultrastructure: tobacco roots infected with Phytophthora

parasitica var. nicotianae. Phytopathology 61: 33-39.

15. Hanchey, P., H. Wheeler, and H. H. Luke. 1968. Pathological

changes in ultrastructure: Effects of victorin on oat

roots. Amer. J. Bot. 55: 53-61.

16. Johnson, C. M., P. R. Stout, T. C. Broyer and A. B. Carlton.

1957. Comparative chlorine requirements of different

plant species. Plant and Soil 8: 337-353. 54

17 . Kishi, K. 1962. Studies on the physiological specialization

of Phytophthora infestans on tomatoes III. On the process

of infection and reactions of host tissues. Bull. Hort.

Res. Sta., Okitsu. 1: 142-162.

18. Mollenhauer, H. H. 1964. Plastic embedding mixtures for use

in electron microscopy. Stain Technol. 39: 111-114.

19. Pease, D. C. 1964. Histological techniques for electron

microscopy, p. 234-235. 2nd Ed. Academic Press, New York.

20. Peyton, G. A. and C. C. Bowen. 1963. The h o st-p a ra site

in te rfa c e of Peronospora manshurica on Glycine max. Amer.

J. Bot. 50: 787-797.

21. Pristou, R. and M. E. Gallegly. 1954. Leaf penetration by Phytophthora infestans. Phytopathology 44: 81-86.

22. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J.

Biophys. Biochem. Cytol. 17: 208-212.

23. Richardson, K. C., L. Jarett and E. H. Finke. 1960. Embedding

in epoxy resins for ultrathin sectioning in electron

microscopy. Stain Technol. 35: 313.

29. Royle, D. J. and G. G. Thomas. 1971. Observations with the

scanning electron microscope on the early stages of hop

leaf infection by Pseudoperonospora humuli. Physiol.

Plant Pathol. 1: 345-349. 55

25. Sakuma, T. and K. Tomiyama. 1967. The ro le o f phenolic

compounds in the resistance of potato tuber tissue to

infection by Phytophthora infestans. Ann. Phytopathol.

Soc. of Japan. 33: 48-59.

26. Takakuwa, M. and K. Tomiyama. 1957. Cell-physiological studies

on resistance of potato plants to Phvtoohthora infestans.

VI. The time required for the browning process of midrib

cells induced by the infection with two different

pathogenic strains of Phytophthora infestans in potato.

N at'l Agri. Exper. Stat. Res. Bull. Hokkaido No. 73:

93-99.

27. Tomiyama, K. 1956. Cell physiological studies on the resistance

of potato plant to Phytophthora infestans. IV. On the

movement of cytoplasm of the host cell induced by the

invasion of Phytophthora infestans. Ann. Phytopathol.

Soc. of Japan 21: 54-62.

28. Walker, J. C. 1969. Plant pathology. 3rd Edition, p. 574-

589. McGraw-Hill Book Company, New York, New York.

29. Wheeler, H. and P. Hanchey. 1968. Permeability phenomena in

plant disease. Annu. Rev. Phytopathol. 6: 331-350.

30. Wood, R. K. S. 1967. Physiological plant pathology, p. 15-54.

Blackwell Scientific Publications, Oxford, England. VITA

Alice Sheppard Badgett Templet was born on March 16, 1943 in Winston Salem, North Carolina. She received her elementary education in the Forsyth County school system. In 1960, she moved to Atlanta, Georgia where she completed her secondary education in the Atlanta City school system, graduating from

Southwest High School in June, 1961.

She attended Duke University in Durham, North Carolina and received her B. A. degree in Science Education from Duke

University in June, 1965. In September, 1965, she began study in the Department of Botany and Plant Pathology at Louisiana State

University and received her M. S. degree from that institution in January, 1968. She began her doctoral work in the Department of Plant Pathology under the direction of Dr. L. L. Black. She is presently a candidate for the degree of Doctor of Philosophy.

56 EXAMINATION AND THESIS REPORT

Candidate: Alice Sheppard Badgett Templet

Major Field: Plant Pathology

Title of Thesis: Penetration and the Host-Parasite Interface of Phytophthora Infestans on Tomato Leaf Tissue Approved:

Major Professor and Chairman

' Deap of the Graduate School

EXAMINING COMMITTEE:

Date of Examination:

July 31, 1972