Ultrastructure changes induced by brachyurum in roots of potato

Item Type text; Thesis-Reproduction (electronic)

Authors Schuerger, Andrew Conrad

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

Download date 26/09/2021 13:49:31

Link to Item http://hdl.handle.net/10150/557736 ULTRASTRUCTURE CHANGES INDUCED

BY IN ROOTS OF POTATO

by

Andrew Conrad Schuerger

A Thesis Submitted to the Faculty of the

DEPARTMENT OF PLANT PATHOLOGY

In Partial Fulfillment of the Requirements For the Degree of

MASTER OF SCIENCE

In the Graduate College

. THE UNIVERSITY OF ARIZONA

19 8 1 STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfillment of re­ quirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library,

Brief quotations from this thesis are allowable without special permission9 provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judg­ ment the proposed use of the material is in the interests of scholar­ ship, In all other instances9 however, permission must be obtained from the author,

SIGNED:

APPROVAL BY THESIS DIRECTOR

This thesis has been approved on the date shown below:

" 9 7 7 . f l . r7 7 l . . is. A / W m / M. A. McCLURE Date Professor of Plant Pathology ACKNOWLEDGMENTS

I would like to express my deepest appreciation to Dr„ Michael

A. McClure9 chairman of my advising committee5 for his guidance and support during my studies as an undergraduate and graduate student.

The work enclosed within this thesis is a direct result of his knowledge and expertise in electron microscopy•and his willingness to assist students in accomplishing their chosen goals.

Special thanks must also be extended to Mr. Raymond E. Wheeler for his technical assistance throughout my studies.

iii TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS...... v

ABSTRACT ...... vii

INTRODUCTION...... 1

MATERIALS AND METHODS...... 3

Feeding Behavior Observation Chambers...... 3 Preparation of Parasitized Roots for Transmission Electron Microscopy...... 5 Preparation of Control and Healthy Roots for Transmission Electron Microscopy ...... 6 Thick Sections for Light Microscopy...... 6 Bacterial Isolation ...... 7 Preparation of Parasitized Roots for Scanning Electron Microscopy ...... 8

RESULTS ...... 10

DISCUSSION ...... 40

■REFERENCES ...... 46

iv LIST OF ILLUSTRATIONS

Figure Page

1. Plexiglass chambers used for rearing potato seedlings for scanning electron microscopy...... 9

2„ Light micrographs of actively feeding photo­ graphed from petri dish observation chambers...... 11

3. Scanning electron micrographs illustrating early pene­ tration of potato roots by _S. brachyurum individuals. . . 12

4. Adult and larval nematodes penetrate potato root via the same entry hole ...... 13

5. Thick sections (1.5-2.0 pm) show feeding areas of parasitized potato root tissue, ...... 15

6. Scanning electron micrograph illustrating nearly complete penetration of root tissue by a S_. brachyurum individual. 16

7 o Transmission electron micrographs of control root tissue. 18

8. Gross-section through penetrated root tissue with stylet inserted into a cortical cell ...... 19

9. Serial sections through the membrane proliferation net­ work observed around the stylet in penetrated cells . . . 20

10. . Thin section above stylet entry illustrating overall ultrastruetural modifications and amphidial duct. .... 21

11. Longitudinal thin section near stylet entry of penetrated cells showing spherical inclusion bodies (i), stylet (st), and membrane proliferation (mp) ...... 22

12. Longitudinal thin section through a cortical cell of a root parasitized for 36 h ...... 24

13. Thin sections through the long axis of stylet entry of young parasitized (48 h) potato roots ...... 25

14. Feeding plug (fp) material had a close association with the buccol cavity (be) acting as an occulsive seal with the plantT s cell wall (cw)...... 26

v vi

LIST OF ILLUSTRATIONS— Continued

Figure Page

15. Longitudinal thin section showing macrofilaments (ma). . 27

16. Cross-sectional view of macrofilaments (ma)...... 28

17. Thin cross-section illustrating a membrane network dif­ ferent than the macrofilaments or membrane proliferations at stylet entry...... 30

18. Golgi and mitochondria breakdown was observed in cells penetrated by nematodes...... 31

19. Thin section, through proximal end of penetrated root cell where stylet penetration occurred approximately 40 - 50 pm distal to the section ...... 33

20. Section through root tissue parasitized for 72 to 96 h and vacated by nematodes for 20 to 48 h...... 34

21. Thin section of a cortical cavity (c) formed by the breaching of cell walls (cw) by the inward migration of the nematode ...... 35

22. Young (48 h) parasitized tissue illustrating cyto­ plasmic matrix in Cortical cavity but not in active feeding cell (fc) ...... e. 36

23. Scanning electron micrograph of a vacated penetration hole (h) in a potato root...... 37

24. Polar flagellated, gram negative bacteria isolated from potato root culture...... 39 ABSTRACT

Ultrastructural alterations of potato root tissue occurred only in cells directly penetrated by Scutellonema brachyururn. Membrane proliferations were consistently observed surrounding the stylet in penetrated cells and appeared to originate from the plasmalemma9 which invaginated around the stylet during penetration. Dense spherical inclusion bodies were associated with the membrane proliferations and bore striking resemblance in ultrastructure and density to material found in the stylet orifice and buccal cavity. Where the stylet pene­ trated the cell wall, a feeding plug was formed which appeared to be continuous with material emanating from the buccal cavity. A residual matrix containing dense spherical bodies was observed in penetrated root tissue parasitized for longer than 72 h. It is believed that the residual matrix was the final stage in cytoplasmic degeneration. Longi­ tudinally arranged "macrofilaments," variable in diameter (20 nm to

40 nm averaging. 30 to 35 nm), occurred in groups of up to 30 in pene­ trated potato root cells. A dark brown lesion began to form within 12 h after feeding began and eventually extended several hundred micrometers axially in the root. Lesions involved cells not directly penetrated by

brachyurum suggesting a chemical as well as a mechanical injury to tissue. .Feeding was always interacellular and generally restricted to epidermal and cortical cells. INTRODUCTION

Scutellonema brachyurum(Steiner, 1938)Andrassy.s 1958 is commonly found in soils of tropical crops and ornamentals. S_. brachyurum attacks Guinea Yam(Dioscorea rotundata) in Puerto Rico(2) and in Nigeria it. is considered a major problem on yams (DioJscorea spp.) (3s4). Other species of Scutellonema are associated with cotton in the southeastern United States (20,21,37) and in many other areas of the world (13). While injury to crops and gross symptomology have been described (1,2,20,21) histopathological studies have emphasized general feeding behavior (26) and the resulting heavily diseased or moribund tissue (3). Feeding of Scutellonema has been characterized as both truly ectoparasitic and superifIcially endo- parasitic (21), the latter description referring to temporary pene­ tration of epidermal and cortical cells of the root by the anterior portion of the nematode. _S. brachyurum also is reported to mature and reproduce within root tissue (21).

Several investigations have dealt with the histopathology of other members of the including Hoplolaimus (5,23,24,

25,34) and Helicotylenchus (27,29). These papers have examined more critically the morphological changes which occur in parasitized roots.

Lesions appear red or brown and often extend axially several hundred micrometers from the site of penetration(5,23,34). Surface depres­ sions and flecked cell contents are common around feeding sites which

1 2 usually support only a single nematode (23,24,27). These ectoparasites generally move through cells, not between them, creating large cavities within cortical tissue. Feeding has rarely been observed to extend into the endodermis or stele of the root.

Ultrastructural pathology of nematodes generally has been restricted to sedentary endoparasites (10,11,30) and ectoparasites in the order Dorylaimida (39). At the time of this writing, only two investigations have been concerned with the ultrastructural pathology of Hoplolaim feeding sites (18,25). Nothing is known regarding fine structure of cellular modifications induced by Scutellonema.

It was the purpose of this study to examine, ultrastructurally, host responses during initial penetration of Scutellonema brachyurum, and to establish, sequentially, cellular events occurring in para­ sitized potato roots. MATERIAL AND METHODS

Potatoe seeds(Solanum tuberosum) were obtained in 1980 from

Dre Ro Eo Webb9 Chiefs Vegetable Laboratory, Beltsville Agricultural

Research Centers Beltsville,-MarylandTomato seeds (Lycopersicon '> — ------esculenturn, CV Globemaster Hybrid 65) were obtained, in 1979 from

Burpee Seed Co. and used for all greenhouse cultivation.

Feeding Behavior Observation Chambers

A water agar system was used to observe directly, all aspects of feeding behavior and lesion development. Observation chambers were constructed by cutting a 4- x 5 cm rectangular hole in the bottom of plastic 9 cm petri dishes and securing a 4.5 x 6 cm glass coversiip over the hole with epoxy cement. Growth medium consisted of 86 ml water, 14 ml full strength Hoagland7s solution (15) and 1.25 g bacto agar. Full strength Hoagland? s solution was filter sterilized through a 0.22 pm millipore filter and added to the liquid water agar cooled to 50 C. Prior to solidification, 15 ml of this medium was poured into surface sterilized observation chambers. Observation chambers were surface sterilized by treatment in 20% bleach for 30 min at 23 C and rinsed twice in sterile distilled water.

Potato seeds, soaked for 24 h in sterile distilled water (38) were surface sterilized for 30 min in 1% aqueous chlorhexidine gluconate and washed three times in sterile distilled water. Seeds were germi­ nated on the growing medium, at 21 C, with a 12 h light cycle of

3 4,

20,000 lux* After.six days one-half dozen seedlings were individually transferred to observation chambers containing fresh growing medium.

Each seedling was then inserted into the growing medium, about 2-3 mm, and allowed to grow for 6-10 days prior to inoculation. During this time rootlets grew to the bottom of the chambers and laterally along the glass coverslip for several centimeters.

•' S/ brachyurum adult females and imature juveniles were obtained initially from an oleander hedge(Nerium oleander) on the campus of the University of Arizona. Greenhouse-reared tomato plants(seed­ lings grown in two parts 20 mesh silica sand and one part sandy loam soil) were inoculated three weeks after germination with mixed popula­ tions from oleander? which contained several types of bacterial feeding nematodes in addition to S_. brachyurumo Observation chambers were inoculated with either adult females and juveniles from oleander, or exclusively juveniles reared for two months on tomato. In both cases . the nematodes were extracted by sugar floatation (17) and treated with

0.5% aqueous chlorhexidine gluconate for 30 min at room temperature.

After washing three times in sterile distilled water they were placed for 24 h on a double layer of facial tissue, suspended over one centimeter of distilled water by a metal screen, all of which was sealed in a plastic refrigerator container. Nematodes were collected and 50 _S. brachyurum, either a random mixture of adult females and juveniles, or juveniles alone, were hand-picked to a dish of sterile distilled water, from which they were pipetted in a volume of one ml to observation chambers with 12-16 day old potato seedlings. After 5 inoculation, all chambers were kept in total darkness, except for observation periods, for the duration of the experiment. Observations on feeding behavior were made through stereo and compound light micro­ scopes over a 120 h period.

Preparation of Parasitized Roots for Transmission Electron Microscopy

All fixation and buffer washes were conducted at 4 C. Observa­ tion chambers, containing infected seedlings were chilled for 2 h at

4 C and then flooded with 3% glutaraldehyde in 0.20 M phosphate buffer, pH 6.8. This fixative was allowed to permeate the observation chambers for 3 h before removing cubes of agar, 4 mm on a side, which contained segments of infected roots. Agar encapsulated root segments were fixed an additional 15 h in fresh glutaraldehyde, washed six times in 0.20 M phosphate buffer, pH 6.8, over a period of 4 h, then postfixed for 16-

18 h in 1% osmium tetraoxide in 0.20 M phosphate buffer, pH 6.8. After washing an additional three times, over one hour, with chilled sterile distilled water, root sections were slowly allowed to come to room temperature (approximately 23C). At this point some of the roots were stained en block with 2% aqueous uranyl acetate for one hour at 55 C followed, by three post stain washes in sterile distilled water. Root tissue was dehydrated in an acetone series (10% increments) with three changes in 100% anhydrous acetone proceeding infiltration, overnight, with SpurrTs MBn medium (35). Blocks were heat-cured for 18-24 h at 70 C.

Silver-gold sections were cut using a Dupont diamond knife on a

LKB Ultratome III or Sorvall MT2-B ultramicfotome. Sections were placed 6 on naked 200 mesh copper grids and stained. En block stained root sections were stained additionally with Reynoldfs lead citrate (28) for.

10 min at room temperature. Sections not stained en block were treated with 2% aqueous uranyl acetate for 20 min at 70 C, then post-stained with

Reynold? s lead citrate for 10 min at room temperature. Sections were viewed and photographed in aHitachi H-500 or JEOL 100 CX transmission electron microscope.

Preparation of Control and Healthy Roots for Transmission Electron Microscopy

Two types of controls were prepared for transmission electron microscopy. One set consisted of uninoculated roots germinated and cultured as described above. The second set was mechanically injured with fine (0.15 mm diameter) stainless steel probes by piercing the root tip and cortical cells of different roots. Injured roots were allowed to continue growth for an additional 72 h before fixation. Uninjured control roots were 16 days old at the time of fixation. All controls were prepared for electron microscopy in the same manner as were diseased tissue.

Thick Sections' f or /Light Microscopy

Resin embedded roots were trimmed on a Sorvall MT2-B and 1.5 to

2.0 jim sections were cut using glass knives. Sections were placed in a drop of double distilled water, on a glass microscope slide, and dried on a hot plate for 70 C for 10 min. They were then flooded with

0.5% Toludine Blue in phosphate buffer, pH 7.4, heated to 70 C and 7 allowed to stand for no longer than five minutes. The Toludine Blue stain was not allowed to dry. Slides were then flushed gently with double distilled water at approximately 23 C until the water was clear and all the excess stain was removed.

After air-dryingj sections were covered with immersion oil and a #1 glass coverslip was secured in place using clear fingernail polish.

Selected sections were photographed on Kodak Panatomic X film.

Bacterial Isolation .

Bacterial contaminants of potato seed were cultured at 30 C by placing root sections, excised from observation chambers, on 4% potato dextrose agar with 10 g/1 peptone added (pH was adjusted to 8,2 with

3 N NaOH). Single colonies were visually examined and three separate colony-forming units were isolated on potato dextrose peptone agar.

Each of these isolates was tested with Gram stain. Kingf s B medium,

Pectate medium, and examined for flagellation.

Whole mounts of flagella were prepared for transmission electron microscopy by mixing one drop of 2% aqueous phosphotungstic acid, adjusted to pH 6.5 with 1 N NaOH, with one drop of bacterial suspension.

The bacterial suspension was prepared from each isolate, by suspending

3 x 10^ bacteria/ml, grown 2.4 h on potato dextrose-peptone agar, in sterile distilled water. After allowing this mixture to stand for 10 sec, a 200 mesh grid, with a carbon-formvar substrate layer, was placed on top of the drop of mixture, substrate down, for an additional

5 sec. Grids were air dried and viewed in a Hitachi H-500 transmission electron microscope. 8

Preparation of Parasitized Roots for Scanning Electron Microscopy

Six one-week old seedlings, germinated on the growing medium

used throughout these experiments, were transferred to plexiglass

chambers (Fig. 1) containing steam-sterilized 20 mesh silica sand.

Seedlings were allowed to grow to a height of 6 cm, approximately two additional weeks, before inoculation. Plexiglass chambers were kept on a 12 h light cycle of 20,000 Lux at 21 C. These chambers were kept

.underneath a 1000 ml glass beaker at all times. _S. brachyurum juveniles, raised on tomato were surface sterilized using 0.5% aqueous chlorhexidine gluconate for 30 min at room temperature. Approximately

400 juveniles, in three milliters of sterile distilled water, were added to each of three chambers. An equal volume of sterile distilled water was added to a fourth tank as a control. Quarter strength

Hoagland's solution, the sole water and nutritive source, was applied at the rate of 2 ml every 48 h until harvest.

Fixation, buffer washes, and acetone dehydration was the same as for roots prepared for transmission electron microscopy. Staining en block was not done on this material. Tanks were chilled for .2 h at

4 C and sand particles adhering to the roots were removed with a gentle stream of chilled distilled water. Washed roots were immediately placed in a chilled water bath and 5 to 10 mm segments of root tissue were cut and immersed in fixative at 4 C.

After fixation and dehydration, root segments were dried by the critical point method, mounted on double-sided sticky tape, coated with

15 nm of gold-palladium (60:40) in a Polaron E5100 sputter coater, 9

Fig. 1. Plexiglass chambers used for rearing potato seedlings for scanning electron microscopy.

Three-week old potato seedlings were inoculated with _S. brachyurum juvenile nematodes and kept under a 1000 ml beaker for the duration of the experiment. RESULTS

Juvenile _Sc brachyurumv raised on tomato, were more active than adult females from oleander. Juveniles began feeding as early as

12 h after introduction to.the agar medium, while adult females were never observed to initiate feeding before 24 h. Juveniles, from tomato, fed throughout the agar system on individual roots and root tipso Adult females preferred roots lying adjacent to other roots, and rarely fed on individual roots or exposed root tips. When more

- 'VA't „ : -i ?. i. V . . . - . S .'t < A. I s :• - - ' ’ than one nematude'"attacked two adjacent roots, all nematodes initially preferred only one of the roots. Migration to the adjacent root followed after considerable cell damage was sustained by the first root.

Adult females and juveniles assumed coiled positions during initial feeding and penetration (Fig. 2A). After 24 to 36 h of feeding, and after penetration to a depth of one or two cells, nematodes uncoiled and took on a variety of configurations (Fig. 2B). Typically, nematodes entered roots individually by puncturing one or two epidermal cells, usually in or near areas of dense root hairs, and expanded the penetration hole by physical pressure as they moved inward (Fig. 3).

Only rarely did more than one nematode use the same penetration hole

(Figs. 2B,4). Feeding generally was restricted to epidermal and cortical cells, with penetration always occurring intracellularly.

Feeding on endodermal and pericycle cells was observed periodically

10 11

Fig. 2. Light micrographs of actively feeding nematodes photographed from petri dish observation chambers.

Discoloration of cells began near the head of the nematode (A) eventually extending axially along the root for several millimeters (B). Lesion development stopped when nematodes vacated root tissue. 12

Fig. 3. Scanning electron micrographs illustrating early penetration of potato roots by S^. brachyurum individuals.

(AX 448, BX 579). 13

(X 962). 14

(Fig. 5A). Nematode orientation, within the cortex, appeared random with

cortical cavities being formed either centripetally (Fig. 5B) or by

disruption of several cells within a local area (Fig. 5A). Stylet

thrusts and pumping of the metacorpus were intermittent. A rest period ' • / of several hours, following stylet thrusts, preceeded metacorpal activity which lasted, no longer than one hour. This sequence was repeated several times before further penetration was observed. Up to

four cells were penetrated within 48 h, and as"many as six to eight cells were penetrated within 96 h. Feeding ceased and nematodes migrated from

• the roots between 96 and 120 h after feeding began.

Nematodes were never observed entirely within roots in the agar

system, but in the sand system, nematodes were observed coiled entirely within a root or with only their tails exposed (Fig. 6). No eggs were

observed within root tissue in either system, but were commonly seen in

the agar system near roots which supported actively feeding adult females.

Light tan to dark brown lesions developed within 24 to 48 h after feeding began at that site. Discoloration of cells initially

appeared near the head of the nematode (Fig. 2A) within 12. to 24 h and

spread to adjacent cells within 48 h, even if those cells were not penetrated by the nematode or its stylet. Lesions extended several mil­

limeters up and down the root away from the site of penetration. Lesion

development and extension ceased once nematode feeding stopped and

roots were vacated. During early stages (12 h) of feeding single

cortical cells became filled with dense granular contents. These dark­

ened cells developed into large extended lesions as the nematode

expanded its feeding zone (Fig„ 2B). 15

Fig. 5. Thick sections (1.5-2.0 ym) show feeding areas of parasitized potato root tissue.

Cortical cavities (c) were formed in one of two ways: either by disruption of several cells in a local area (A) or centripetually (B). Feeding was generally restricted to the cortex with intracellular penetration. Feeding cells (fc) were periodically observed in pericycle and endodermal tissue (B). 16

Fig. 6. Scanning electron micrograph illustrating nearly complete penetration of root tissue by a brachyurum individual.

(X 1032). 17

Thin sections cut through cells in lesions9 48 to 72 h after feeding began5 cells which were not penetrated directly but showed visual discoloration, were similar to those of healthy cells (Fig. 7

A-D) and showed no obvious ultrastructural alterations typically observed in penetrated cells. Ultrastructural alterations were not observed in mechanically injured potato root tissue. Cell organelles, in this part of the lesion appeared normal, with intact membranes and . no changes in shape or size. Cortical cells immediately adjacent to penetrated and parasitized cells, likewise failed to show obvious ultrastructural alterations. Only cells that were breached by the stylet were demonstrably modified. Most striking of these modifications was a network of membranous material that formed around, and was associated only with the stylet?s entry into cortical cell cytoplasm (Fig. 8).

The network was oriented in the plane of the stylet in sections directly through the long axis of stylet entry (Fig. 8,9A), but cross- sections of roots cut above or below the stylet entry hole revealed the network in cross-sectional view (Fig. 9B). ■ The membranes were composed of a double layer of material which was highly variable in shape and width, and had the appearance of an interwoven lattice system.

Associated with the stylet entry hole, and usually contained within the membranous network were dense inclusion bodies (Figs. 8,9,10).

These bodies appeared roughly circular in both longitudinal (Fig. 11) and corss-sections (Figs. 9B,10), and showed little internal sub­ structure except for a densely packed, grainy»matrix, They differed 18

Fig. 7. Transmission electron micrographs of control root tissue.

Cell organelles were regular in shape and contained intact membranes with easily recognizable substructure. A, the plasmalemma (p) was in close association with cell wall material (cw) forming excretory vesicles (e). Mitochon­ dria (mi) were oval in shape with a high integrity of membranes and cristae. The nucleus (n) was surrounded by an intact nuclear membrane (nm). B, rarely observed micro­ filaments (mf) were always straight and uniform in diameter (approximately 16-18). C, rough endoplasmic reticulum (r) were common ghroughout control sections. D, golgi (g) exhibited a well-formed stack with numerous transitory vesticles (tv).

(AX 39,117, BX 54,050, CX 35,305, DX 40,000). 19

d*k?

Fig. 8. Cross-section through penetrated root tissue with nematode stylet inserted into a cortical cell.

A feeding plug (pf) formed where the nematodes (ne) stylet (st) penetrated the cell wall. Associated with the stylet was a membrane proliferation (mp) network containing many spherical inclusion bodies (i) which had the same ultra­ structure and density of material found in the stylet’s orifice (sto). Macrofilaments (ma) were observed throughout the penetrated cell. Also characteristic of penetrated cells was a breakdown of the tonoplast (to), plasmalemma (p), and golgi (g).

(X 9,218). 20

Fig. 9. Serial sections through the membrane proliferation network observed around the stylet in penetrated cells.

Sections cut in sequence of one penetrated cell reveal a possible orientation of the membrane proliferation network. The network appeared to be oriented in the same plane as the stylet in sections cut directly through the long axis of stylet entry (A). Above or below stylet entry, membrane proliferations appeared to be oriented longitudinally in the cell (B). (i = inclusion bodies, mp = membrane pro­ liferation, p = plasmalemma).

(AX 20,666, BX 71,052). 21

Fig. 10. Thin section above stylet entry illustrating overall ultrastructural modifications and amphidial duct.

The material found within the amphidial duct (ad) was not continuous with material of the feeding plug, changing density and ultrastructure several times along the length of the amphidial duct. (to = tonoplast, g = golgi, mi = mitochondria, c = cortical cavity, ne = nematode, mp = membrane proliferation, pd = plasmodesmata, n = nucleus, ma = macrofilaments).

(X 11,538). 22

Fig. 11. Longitudinal thin section near stylet entry of penetrated cells showing spherical inclusion bodies (i), stylet (st), and membrane proliferation (mp).

(X 17,362). 23 from elongate vesicles5 found throughout altered cells (Fig. 12) in shape, internal substructure, and lac, of an external membrane.

Material with an appearance somewhat similar to those inclusion bodies was present in the stylet, orifice (Fig. 13A) and cell cytoplasm directly adjacent to an area where a stylet was previously inserted (Fig. 13B).

As the stylet penetrated the cell wall the plasmalemma invagi- nated around the stylet (Fig. 8) forming a channel that maintained its integrity even if the stylet was withdrawn (Fig. 9 ,13B). The plasmalemma was disrupted throughout the cell, and was observed to separate from the cell wall. Evidence of endo- and exocytosis, seen in controls and unaffected cortical cells (Fig, 7A), was not observed in penetrated i cells,

Material resembling a feeding plug formed during penetration of plant cell walls by the nematode? s stylet (Figsv 8,14), Plug material surrounded the stylet and lined the buccal cavity of the nematode

(Fig. 8). Its origin was not determined. Plug material appeared to

seal the gap between the stylet and the cell wall, and often filled

invaginations of the buccal cavity (Fig. 14).. Similar material was

found deep within the amphidial duct (Fig. 10), but no material

directly traceable from the amphids to the feeding plug was observed.

Common to all cells, recently penetrated (24 to 48 h ) , were

complexes of tubular structures (Figs. 8,12,15,16). These tubes • varied greatly in diameter (20 nm to 40 nm) (Fig. 16) and were almost

exclusively oriented longitudinally in the. cell (Figs. 12,15). Groups

of tubes were not only near the penetration hole but were found through­

out the parasitized cell. In cross and longitudinal sections their 24

Fig. 12. Longitudinal thin section through a cortical cell of a root parasitized for 36 h.

Elongate inclusion bodies (ei) differed greatly from the inclusion bodies observed in the membrane prolif­ eration network near stylet entry (mi = mitochondria, n = nucleus).

(X 10,671). 25

Fig. 13. Thin sections through the long axis of stylet entry of young parasitized (48 h) potato roots.

Material found in the stylet orifice (A) and in the penetrated cell's cytoplasm, near where the stylet orifice rested prior to nematode retraction (B) was similar in electron density and ultrastructure to the inclusion bodies found in the membrane proliferation network. (i = inclusion bodies, st = stylet, sto = stylet orifice, p = plasmalemma).

(AX 45,000, BX 50,000). 26

Fig. 14. Feeding plug (fp) material had a close association with the buccal cavity (be) acting as an occulsive seal with the plant’s cell wall (cw).

(X 51,875). 27

Fig.- 15. Longitudinal thin section showing macrofilaments (ma).

Macrofilaments were observed to be generally uniform along their lengths but variable in diameter (20 to 40 nm, averaging 30 to 35 nm). A unit membrane (u) made up the walls of the macrofilaments.

(X 52,500). 28

Fig. 16. Cross-sectional view of macrofilaments (ma).

A unit membrane wall (u) was observed in several macro­ filaments. Additionally, the macrofilaments varied in diameter (20 to 40 nm, averaging 30 to 35 nm) but the range clearly separated the macrofilaments from smaller microfilaments.

(X 187,687). 29 walls appeared to be a unit membrane (Figs. 15,16) of regular thickness

Each tube was. very uniform in diameter throughout its length. Groups consisted of up to 25 or 30 tubes, closely packed but lacking inter­ connections. Distinct from these tubular arrangements and from the membranous network surrounding the site of stylet entry, was a third system of tubes or vesicles which appeared randomly throughout para­ sitized cells (Fig. 17). These consisted of vesicle-like pouches or sacs bounded by a unit membrande. The diameter of the sacs was variable.

Other cell organelles, observed in controls, were altered to various degrees in penetrated cells. Golgi gradually were transformed into large convoluted vesicles which separated from the golgi stack and formed a loose group of variably shaped and sized vesicles (Figs.

10,18). Mitochondria became very distorted (Fig. 18A.) and often extremely elongate (Fig. 12). Their outer membrane sometimes separated and formed pouch-like vesicles (Fig. 18B) or the regularity of the intermembrane space was lost. The central vacuole, of mature cortical cells was lost after penetration as cytoplasmic material proliferated and the tonoplast degenerated (Fig. 10). The tonoplast, barely visible in control and unaffected cells,became swollen and disintegra­ ted allowing cytoplasm to mix with vacuole material (Figs. 8,10).

Nuclear membranes were poorly defined in penetrated cells (Fig.

12) and nucleoli were not observed in nuclei of penetrated cells.

Associated with the loss of the nucleolus was a reduction in identi­ fiable rough endoplasmic reticulum and of cellular or membrane-bound ribosomes. 30

Fig. 17. Thin cross-section illustrating a membrane network different than the macrofilaments or membrane pro­ liferations at stylet entry.

Vesicle-like pouches or sacs (vp) had a unit membrane wall but showed a large amount of irregularity in shape and diameter.

(X 50,757). 31

Fig. 18. Golgi and mitochondria breakdown was observed in cells penetrated by nematodes.

Mitochondria (mi) were altered, becoming extremely distorted in shape and often having degenerate membrane systems (d) (A). Golgi stacks (g) degenerated into large convoluted transitory vesicles (tv) with altered membranes (arrows) (B).

(AX 30,789, BX 70,010). 32

At a distance of about 40-50 micrometers from the stylet entry

hole9 the tonoplat appeared to be less affected and the central vacuole had a higher degree of integrity (Fig. 19). Furthermore9

tubular structures were common, mitochondria appeared less distorted and

less elongate, but golgi were observed to degenerate in a similar manner

as golgi were observed to breakdown near stylet entry.

Roots fed on for 72 to 96 h and vacated by the nematodes for

24 to 48 h showed a high number of bacteria present in cortical cavities but little evidence of cell alterations, noted earlier (Figs. 20,21).

Parasitized cells contained a dense matrix composed of numerous electron

dense spherical bodies (Fig. 20). The matrix was bound on the inner and

outer side by a unit membrane (Fig. 2033).. The matrix was also observed

in cortical cavities of old (72 to 96 h) parasitized tissue (Fig. 20 and

21) and in young (48h) parasitized tissue (Fig. 22). The matrix was most intact in feeding cells of old tissue (72 to 96 h) (Figs. 20,21)

but was never observed in cells actively supporting nematode feeding

activity (Fig. 22).

Bacteria were found in roots penetrated by S_. brachyufurn.

Present in the rhizosphere, these bacteria moved into rooots via open­

ings created by the nematode (Fig. 23). Thin sections revealed the

presence of the bacteria only in cells that were breached and/or dis­

rupted (Figs. 20,21,22). Enzymatic breakdown of cell wall material

was not observed in parasitized and ruptured tissue. Single colonies,

cultured from several agar observation chambers, were all amber in

color, smooth and glossy in texture, and had even margins. Several Fig. 19. Thin section through proximal end of penetrated root cell where stylet penetration occurred approximately 40 - 50 pm distal to the section.

Golgi (g), rough endoplasmic reticulum (r), and macro­ filaments (ma) at a distance of 40 - 50 pm from stylet penetration showed similar cytoplasmic alterations observed near stylet entry, but mitochondria (mi) and tonoplast (to) integrity were close to that observed in control cells. The intact nature of the tonoplast resulted in a central vacuole (v) similar to control cells.

(X 7,428). 34

Fig. 20. Section through root tissue parasitized for 72 to 96 h and vacated by nematodes for 20 to 48 h.

Cortical cavities (c) contained numerous bacteria (b) and a matrix (m) interpreted as coagulated cytoplasm (A). The matrix was also found in cells that had traceable disruption of cell walls and membranes, presumably these cells represented feeding cells (fc). Under high magnification the matrix was observed to be composed of dense spherical bodies and bound by a unit membrane on the inner and outer side (arrows) (B).

(AX 6,284, BX 74,117). 35

Fig. 21. Thin section of a cortical cavity (c) formed by the breaching of cell walls (cw) by the inward migration of the nematode.

Bacteria (b) found in vacated feeding cells (fc) entered through traceable openings. (m = matrix, cw = cell wall).

(X 3,214). 36

Fig. 22. Young (48 h) parasitized tissue illustrating cyto­ plasmic matrix in cortical cavity but not in active feeding cell (fc).

The matrix (m) observed in cortical cavities (c) of young parasitized tissue was intermixed with bacteria (b) and other debris similar to that observed in old (72 to 96 h) parasitized tissue but the matrix was not observed in the active feeding cell (A). The spherical bodies of the matrix were similar to the matrix observed in older, parasitized tissue (B). (u = unit membrane)

(AX 3,300, BX 38,182). 37 9WZ,«*$

Fig. 23. Scanning electron micrograph of a vacated penetration hole (h) in a potato root.

Note bacteria (b) on root surface. The penetration hole offers an obvious doorway for bacteria and other microorganisms for entry into root tissue.

(X 2610). isolates were tested and all were found to be rod-shaped, polar- flagellated bacteria (Fig. 24) which were pectate negative at 72 h,

King’s B positive flourescent in 24 h, and Gram positive in 24 h cultures. Flagella were grouped in polar tufts. Bacteria were cultured from control and parasitized roots with the latter having higher concentration of bacteria. 39

Fig. 24. Polar flagellated, gram negative bacterium isolated from potato root culture.

Initial tests indicate only one population of bacteria present in the potato root-nematode system and that most likely this bacteria was a flourescing Pseudomanad.

(X 29,750). DISCUSSION . •

Feeding behavior and extension of lesions axially along the

root 5 beyond cells directly fed upon by S /'~bfa'chyurum, was consistent with reports of feeding by related species (5,23,24,25,29) arid suggested

a chemical as well as a mechanical mechanism of injury to root tissue.

Obvious ultrastructural alterations were never observed in cells not

penetrated by the nematodeTs stylet including those cells directly

adjacent to feeding sites. Obvious darkening of these areas, therefore, may result from physiological and/or chemical alterations which may

either be lost in preparation of tissue for electron microscopy or not revealed by standard stains for election microscopy. If ultra-

structural alterations of parasitized cells are mediated- by specific

substances the inability of these substances to affect adjacent cells

could indicate that they are either bound to cell organelles, or are

not able to diffuse across membranes.

The membranous network surrounding the stylet of £[.. brachyurum .

was interpreted as a proliferation of the. plasmalemma induced by

nematode secretions or a result of host reaction to penetration by the

nematodeTs stylet. Similar membrane proliferations, termed paramural

bodies or plasmalemmasomes (14,32), have not been reported for nematode-

host interactions. Membrane proliferations of this type have been

observed for viroid infected plant tissue (14,32) and virus infected

40 41

tissue of mice (22). Such alterations and proliferations have been

implicated in nucleic acid metabolism (14,16,22,31,32,36) in which.

they act as possible sites of RNA and DNA incorporation and/or

synthesiso Scrapie viral DNA is thought to be membrane bound (16,22)

and its replication possibly involved in membrane proliferation.

Evidence also indicates that a large quantity of citrus exocortis viroid

RNA is associated with a component of the endomembrane system (31) but whether its presence is due to viroid RNA synthesis or an accumu­

lation of infective RNA is not known (32,33,37).

If we assume that there is a direct relationship between nucleic acids and membrane proliferations in viroid and viral-host systems, then

it seems reasonable to expect that a similar mechanism might account

for the proliferation of the plasmalemma observed in potato root cells parasitized by _S. brachyurum. It has been suggested that nematode

saliva, and presumably nematode esophageal secreations, contain

ribosomal RNA (7,12,18,30). In this study it was noted that electron

dense inclusion bodies, with, a homogeneous grainy matrix (Figs. 8,10) were associated with membrane proliferations induced by brachyurum.

These bodies bore striking resemblance to material associated with the

stylet orifice (Fig. 13) suggesting that the inclusion bodies could be

of nematode origin. Dense spherical inclusion bodies were also found

in cells of susceptible soybean parasitized by Rotylechulus reniformis

and Rebois, et al. (30) surmised that they were of nematode origin.

A membranous network similar to the one observed in potato cells 42

parasitized b y S_* brachyurum was not reported by Rebois, et al. although

they did record increased amounts of rough endoplasmic reticulum

(30)c That these inclusions are of nematode origin, and that they

contain RNA, has not been conclusively proven. If an interaction

between nematode secreted RNA and membrane alterations can be demon­

strated, a possible model is at hand to help explain other host

responses to pathogenisis like the tubular structures observed in potato root cells fed on by S_. brachyurum. It has been established

that stylet secretions of endoparasitic nematodes'play a significant

role in the formation of synctia and uninucleate giant cells (6,7)

but a similar role for nematode secretions in host cell alterations has not been well established for many ectoparasitic nematodes.

Other cell alterations induced by S. brachyurum indicated a

general senescence of cell cytoplasm. Degeneration of mitochondria,

rough endoplasmic reticulum, golgi, tonoplast, and central vacuole is

consistent with the effects of aging in senescing wheat and barley

leaves (13,33). The separation and contraction of the plasmalemma,

observed in this study also would be expected in senescing tissue.

Organelle breakdown is possibly enhanced and accelerated by nematode

penetration but these alterations are not unique to the _S. brachyurum-

potato interaction.

Formation of tubular groups, henceforth termed "macrofilaments",

in potato cells penetrated by S_. brachyurum, is one alteration not

generally anticipated in senescing cells. The size and construction *

of these macrofilaments differed markedly from either microtubules 43 or microfilaments typically encountered in cells. The size (20-40 nm) of the macrofilaments in this study was larger and more variable than microtubules (25 nm), microfilaments (4-6 nm) or 10 nm filaments typically found in cells (9). Cross-sectional view of true micro- tubles reveals a substructure of 11 to 13 protofilaments arranged helically (9)5 whereas macrofilaments were bounded by a unit membrane and lacked substructure! protofilaments. A comparison of the macro­ filaments found in parasitized potato cells and microfilaments found in control cells (Fig, 71) showed few similarities. Although both were linear and regular in diameter9 microfilaments were not bound by a unit membrane and were smaller in diameter (17 nm). Large tubular structures (46 nm in diameter) have been induced to form under low temperature conditions in the protozoan Sticholonche zanclia (8).

These "macro-tubules", as they were termed, derived from the disassembly of tubulin in microtubules but were radically different ultra- structurally from the macrofilaments, found in potato root cells parasitized by; S_. brachyurunu Additionally, the lack of macrofilaments in adjacent cells and in controls treated to the same low temperature fixation procedure indicates that another mechanism must be involved in large tubular formation,in potato cells parasitized by _S. brachyurum, than purely low temperature. The origin and function of macrofilaments in cells parasitized by _S. brachyurum are unknown. If these structures are related in any way to microfilaments or microtubules, then possibly they function in cytoplasmic movement. 44

The feeding plug formed by S. brachyurum was not as extensive as those formed by other nematodes (10,19,39). While the material forming the plug appeared to originate from the buccal cavity, an origin in the amphidial duct or gland could not be eliminated as a possibilityo The origin of feeding plug material in other nematodes has been suggested to flow from either the stylet orifice, the inner labial receptors, and/or the amphidial canals (10,39)»

In summary, the phase of this study to establish sequential events in early penetration and pathogenesis of root cells (24 to 48 h) demonstrated that initial penetration resulted in ultrastructural modifications which decreased in intensity away from point of entry«

Golgi breakdown progressed from increased production of transitory vesicles to a total breakdown of the stack which resulted in loose collections of vesicles with distorted and irregular unit membranes.

Mitochondria elongated and slowly lost the integrity of intermembranal space. Eventually internal cristae structure was lost and the outer membrane burst. Endoplasmic reticulum and ribosomal complexes were reduced in most cells penetrated by _S, brachyurum. Macrofilaments were formed early and occurred throughout the parasitized cell.

In contrast, older parasitized tissue (72 to 96 h) which had been vacated by _S. brachyurum (24 to 48 h) contained cells with a residue of material believed to be coagulated cytoplasm. This material was also observed in cells nearly destroyed by nematode passage, and in pericycle and endodermal cells which had served as feeding sites 45

24-48 h earlier. Based upon these observations, it was concluded

that those cells which contained the coagulated matrix represented

the final stages of cellular alterations. However, a sufficient

number of cells were not examined to reconstruct the sequence of events

leading to the death of the parasitized cell,

‘ The parasitized cell in Figures 8 and 10 was the third and

innermost cell penetrated by a juvenile S_. brachyurum within 48 h after

feeding was observed to begin. Assuming that the same sequence of

ultrastructrual alterations occurred in all three cells parasitized,

a very rapid response by host cells to penetration by S , brachyurum would be necessary, with modifications and ingestion of cytoplasm

requiring 12 to 14 h per cell. Further sectioning, of infected material and. a more precise method of determining the initiation of

feeding on individual cells, would be necessary to firmly establish

this assumption. REFERENCES

1. Acosta, N.,-and Ac Ayala. 1975. Pathogenicity of Pratylenchus ooffeae, , Meloidogyne incognita, and Rotylenchulus reniformis on Dioscorea•rotundata. I. Nematol. 7 :l-6 o

2. Acosta, No, and A. Ayala. 1976. Effects of Pratylenchus coffeae and Scutellonema bradys alone and in combination on Guinea Yam (Dioscorea rotundata) J. Nematol. 8:315-317.

3. Adesiyan, S. 0., R. A. Odihirin, and M. 0. Adenji. 1975a. Histopathology studies of the yam tuber (Dioscorea rotundata) Poir infected with Scutellonema bradys (Steimer and LeHew). Int. Biodetn. Bull. 11:48-55.

4. Adesiyan, S, 0., R. A. Odihirin, and M. 0. Adenjo. 1975b. Change in carbohydrate constituents induced in the yam tuber (Dioscorea rotundata Poir) by a. plant parasitic nematode Scutellonema bradys. Int. Biodetn. Bull. 11:124-226.

5. Ahmad, M. 1978. Studies on the biology, life cycle and patho­ genicity of Hoplolaimus geleatus ( C o f f 1913) Thorme, 1935 (Nematoda: Hoplolaiminae), Ph.D., Rutgers University, The State University of New Jersey (New Brunswick), Dept, of Plant Pathology. 98 pp. (Abstr.).

6. Bird, A. F. 1968. Changes associated with parasitism in nematodes. IV. Cytochemical studies on the ampulla of the dorsal esophangeal gland of Meloidogyne j avanica and on exudations from the buccal stylet. J. Parasit. 54:879-890,

7. Bird, A. F. 1969. Changes associated with parasitism in nematodes. V. Ultrastructure of the stylet exudation and dorsal esophageal gland contents of female Meloidogyne javanica. J. Parasit. 55:337-345.

8 . Cachon, J., and M. Cachon. 1980. Polymorphism of tubulin reassembly: I. Different aspects of allotropic transformations induced by low temperature. Biologic Cellulaire 37:23-34.

9. Dyson, R. D. 1978. Cell Biology: A molecular approach. 2nd edition, Allyn and Bacon, Inc., pp. 22-26.

10. Endo, B. Y. 1978. Feeding plug formation in soybean roots infected with soybean cyst nematode. Phytopathology 68:1022-1031.

46 47

11c Endos B, Y., and W. P, Wergin. 1973. Ultrastractural investigation of clover roots during early stages of infection by the root-knot nematode, Meloidogyne incognita. Protoplasma 78:365-379.

12c Epstein, E., and E. Cohn. 1973. Biochemical changes in roots infected by the nematode Longidorus africanus. Phytopara- sitica 1:58-59. (Ahstr.).

13. Parkas, G. L., L. Dezi, M. Horvath, K. Kisban, and J. Udvardy, 1964. Common pattern of enzymatic changes in detached leaves and tissues attacked by parasites. Phytopathol. Z. 49:343-354.

14. Hari, V. 1980. Ultrastructure of potato spindle tuber viroid- infected tomato leaf tissue. Phytopathology 70:385-386.

15. Hoagland, D. R., and D. I. Arnon. 1950. The water culture method for growing plants without soil.. Calif. Exp. Stn. Circ. 347, 32 p.

16.. Hunter, G. D. , R. H. Kimberlin, S. Collis, and G. C. Mil Is on, 1973. Viral and non-viral properties of the scrapie agent. Annals of Clinical Research 5:262-267.

17. Jenkins, W. R. 1964. A rapid centrifugal-floatation technique for separating nematodes from soil. Plant Disease Reporter 48:692..

18. Jones, R.. K. 1978. Histological and ultras tract ural changes in, cereal roots caused by feeding of Helicotylenchus spp. Nematologica 24:393-397.

19. Kisel, Me, J. Catillo, and B. M. Zukerman. 1971. An adhesive plug associated with the feeding of Hemicycliophora similis on cranberry. J. Nematol. 3:296-298.

20. Kraus-Schmidt, H., and S. A. Lewis. 1979. Scutellonema brachyurum: Host plants and pathogenicity on cotton. Plant Disease Reporter 63:688-691.

21. Kraus-Schmidt, H.; and S. A. Lewis. 1981. Dynamics of concomitant populations of Hoplolaimus columbus, Scutellonema brachyurum, and Meloidogyne incognita on cotton. J. Nematol. 13:41-48.

22. Lambert, P. J., J. Hooks, C. J. Gibbs, Jr., and D. C. Gajdusek. 1971. Altered plasma membranes in experimental scrapie. Acta Neuropath. 19:81-93. 48

23c Lansing5 E. 1979. Histological responses by wheat plants to infection by Hopolaimus (Hoplolaimus) sp. Helminthological Abstracts— Series B 48:118 (Abstr.).

24. Lewis9 S . A. 9 F. H,; Smiths and W. M. Powell. 1974. Histopathology of infection by Hoplolaimus columbus on cotton and soybean and aspects of its pathogenicity. J, Nematol, ~6:145 (Abstr.).

25. NG, 0. C .5 and T. A. Chen. 1980. Histopathological study of alfalfa root infected by Hoplolaimus galeatus. Phytopathology 70:466-467 (Abstr.).

26. Nong, L .9 and G. F. Weber. 1965. Pathological effects of Pratylenchus:scribneri and Scutellonema brachyurum on Amaryllis.

27. Orbin, D. P.:s and E. J. Cairns. 1971. Histopathology of' soybean roots infected with Helicotylenchus dihystera (Cobb) Sher. J. Nematol. 3:322-323 (Abstr.).

28. Pease, D. C. 1964. Histological techniques for electron microscopy. Academic Press, New York and London, pp. 223.

29. Rao, V. R., and G.. Swarup. 1975. Studies on the life history of Helicotylenchus dihystera and on the histopathology of infested sugarcane roots. Indian J. Nematol. 5:56-61.

30. Rebois, R. V., P. A. Madden, and B. J. Eldridge. 1975. Some ultrastructural changes induced in resistent and susceptible soybean roots following infection by Rotylenchulus reniformis. J. Nematol. 7:122-139.

31. Semanick, J. S ., D. Tsuruda, L. Zaner, J. L.M. C. Geelen, and J. G. Weathers. 1976. Exocortis disease: subcellular • distribution of pathogenic (Viroid) ENA. Virology 69:669-676.

32. Semahcik, J. S., and W. J. Vanderwoude. 1976. Exocortis viroid: cytopathic effects at the plasma membrane in association with pathogenic ENA. Virology 69:719-726.

33. Shaw, M . , and M. S. Manocha. 1965. Fine structure in detached, senescing wheat leaves. Canadian J. Botany 43:747-755.

34. Singh, K . , and S. R. Misra. 1976. Pathogenicity and histo­ pathology of Hoplolaimus indicus on sugarcane. Nematologica 22:433-436. 49

35. Spurr, A. R., 1969. A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26:31-34.

36. Wheeler, H. 1974. Cell wall and plasmalemma modifications in diseases and injured plant tissues. Canddian J. Botany 52:1005-1009.

37. Whitehead, A. G. 1959. Scutellonema clathricaudatum n. sp. (Hoplolaiminae: ), a suspected ectoparasite of the roots of the cotton plant (Gossypium hirsutum L. var. UK 51). Nematologica 4:56-59.

38. Wyss, U. 1978. Root and cell response to feeding by Xiphinema index. Nematologica 24:159-166.

39. Wyss, U . , R. Jank-Ladwig, and H. Lehmann, 1979. On the formations and ultrastructure of feeding tubes produced by Trichodorid nematodes. Nematologica 25:385-390.