CYTOPATHOLDGY 0F INCLUSIONS
CAUSED BY SIX STRAINS OF TOBACCO
MOSAIC VIRUS IN LEAF CELLS 0F
RESISTANT AND SUSCEPTIBLE
LYCOPERSICON ESCULENTUM MILL

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
ARTHUR S. ALLEN
1968

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Univ ersity

This is to certify that the

thesis entitled
CVTOPATHOLOGV 0F INCLUSIONS CAUSED BY SIX STRAINS
OE TOBACCO MOSAIC VIRUS IN LEAF CELLS OF RESISTANT AND
SUSCEPTIBLE LVCOPERSICON ESCULENTUM MILL.

 

presented by

ARTHUR S. ALLEN

has been accepted towards fulfillment
of the requirements for

_Eb..ll._ degree ian
Pfiamt Pathozogy

25y Sew/e/

Major professor

Date AUQUAJE 3, 7968 v

 

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ABSTRACT
CYTOPATHOLOGY OF INCLUSIONS CAUSED BY
SIX STRAINS OF TOBACCO MOSAIC VIRUS
IN LEAF CELLS OF RESISTANT AND
SUSCEPTIBLE LYCOPERSICON
ESCULENTUM MILL.

by Arthur S. Allen

Gross foliar symptoms produced in a susceptible
tomato, Bonny Best, and a resistant tomato, N32, caused
by infection with the following strains of TMV: Holmes
Rib-Grass Strain (ERG), John S. Boyle Tomato Internal
Browning Strain (JSB), Holmes Distorting Strain (HDS),
Alexander's Strain (SA), Yellow Aucuba Strain (YA) and
Bean Strain (5P) were studied in conjunction with cyto-
logical observations of inclusions. All plants were
grown under greenhouse controlled conditions both with
and without supplemental lighting. At intervals after
inoculation, systemically-invaded leaves of virus-in-
fected plants and comparable leaves of healthy plants
were sectioned and viewed under light and phase micro-
scopy.

The resistant barriers in tomato were breached as
shown by inclusion formation by (a) certain natural, wild
strains, (b) host-selected substrains and (c) repeated
passage of a virus strain through the host. The types
and numbers of cytoplasmic inclusions produced in both
varieties of hosts were classified and should serve to

expand Group BV of McWhorter's virus inclusion classifica-

Arthur S. Allen

tion. All six TMV strains produced the typical hexagonal
crystal, four of the strains produced dense X-bodies and
each strain produced specific inclusions or combination of
inclusions in the susceptible host. Only rarely, if at
all, were inclusions formed in the resistant host and then
only under the aforementioned treatments (a,b,c,) were in-
clusions produced to an appreciable degree. Specific in-
clusions found in Bonny Best were short needles, long
coiled fibers, cuboidal crystals, rectangular crystals,
irregular crystals, thin grey plates, banded rectangular
crystals and dendritic clusters of short needles.
Cytological examination by phase-contrast procedures
revealed for the first time the presence of TMV in in-
clusions in lesion marginal zones of the local lesion host
-- Nicotiana tabadum var. Xanthi-nc but not in Nicotiana
glutinosa, each infected with the JSB strain of TMV.
Through the use of a specific staining procedure, the
presence of plasmodesmata between adjacent trichomal cells
of Bonny Best tomato was clearly demonstrated for the first
time. Subseduent width measurements of the stained plasmo-
desmata indicated sufficient space to enable TMV particles

but not inclusions to migrate from one cell to the next.

CYTOPATHOLOGY OF INCLUSIONS CAUSED
BY SIX STRAINS OF TOBACCO MOSAIC
VIRUS IN LEAF CELLS OF RESISTANT

AND SUSCEPTIBLE LYCOPERSICON
ESCULENTUM MILL.

y ("u
{'er I.

Arthur Sf Allen

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology
1968

ACKNOWLEDGMENTS

I wish to express my profound gratitude to Dr. H. H.
Murakishi for his patience, advice and encouragement dur-
ing this period of professional development. I extend my
appreciation to the following members of the guidance com-
mittee: Dr. W. J. Hooker for critical evaluation of the
work and use of his phase-contrast photographic equipment,
Dr. A. Kivilaan for the translation of pertinent chapters
from Goldin's book on inclusions, Dr. R. C. Olien for infor-
mative discussions on crystal formation and Dr. W. B. Drew
for taxonomic information on the varieties of the genus
Lycopersicon. My special thanks goes to Dr. J. E. Cantlon
who willingly reviewed the final manuscript in the absence
of Dr. W. B. Drew.

I thank Mrs. Kayla Ager for the hours spent typing
the manuscript.

I wish to recognize the members of the Michigan
State University Photographic Laboratory for their profess-
ionalmassistancein.the preparation of the photographs from
the original slides.

I bestow my deepest praise upon my wife, Marlene,
for her steadfastness and ability to create a pleasing home
atmosphere and upon my sons, Steven and Christopher, who

have made my life more meaningful as a father.

ii

DEDICATION

This dissertation is sincerely dedicated to the
memory of my mother, an understanding teacher, who sacri-
ficed much to impart an incentive for education to her

sons and to my father who introduced early in my life the

world of nature.

iii

ACKNOWLEDGEMENTS
DEDICATION . .
LIST OF TABLES
LIST OF FIGURES
INTRODUCTION .

LITERATURE REVIEW
MATERIAL AND METHODS
EXPERIMENTAL RESULTS

TABLE

0

OF CONTENTS

Symptomatology . . . . . .

Development of Inclusions
Special Substrains
Old Virus-infected
Local Lesion Isolates from
Chemical Tests on Inclusion
Local Lesion Plants
Local Lesion Isolate of SA .
of Virus Throu h H

Cytology:
Cytology:
Cytology:
Cytology:
Cytology:
Cytology:
Cytology:
Plasmodesmata
Photographs

DISCUSSION . .
SWRY o o o 0
LITERATURE CITED

0

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ii

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vii

26
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41
43
45
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51
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72
82

84

LIST OF TABLES
Table Page

1. Symptom development on virus-infected
tomatoes under greenhouse conditions, Feb.-
Aprb , 1967 O O O O O O O O O O O O O O O O 0 30

2. Symptom development on virus-infected
tomatoes under greenhouse conditions, Sep.-
OCt.’ 1967 O O O O O O O O O O O O O O O O O 32

3. Bioassay of tomatoes infected with six
Strains Of TMV O O O O O O O O O O O O O O 0 34

4. Survey of inclusions in tomato trichomes
systemically infected by strains of TMV . . 35

5. Survey of inclusions in tomato trichomes
systemically infected by strains of TMV . . 37

6. Survey of inclusions in tomato seedlings
inoculated with special substrains of
three TMV strains . . . . . . . . . . . . . 40

7. Survey of inclusions in tomato plants
systemically infected for 170 days . . . . . 42

8. Single lesion isolates made from in-
fected Nicotiana tabacum var. Xanthi-
nc transferred to cotyledons of 2-week
old Bonny Best seedlings . . . . . . . . . . 44

9. Cytochemical tests on inclusions within
systemically infected Bonny Best tri-
Chomes O O O O O C O O O O O O O C O O O O C [+6

10. Survey of inclusions in the local lesion
host Nicotiana tabacum var. Xanthi-nc
inoculated with JSB from Bonny Best
tomato . . . . . . . . . . . . . . . . . . . A7

11. Survey of inclusions in the local lesion
host Nicotiana glutinosa inoculated with
JSB from Bonny Best tomato . . . . . . . . . 49

12- Comparative study of inclusions produced
by SA and a local lesion isolate in
Nicotiana tabacum var. White Burley
May,l9€7..o.........:..oo50

Table Page

13. Survey of inclusions in virus-infected,
symptom-bearing tomatoes, Bonny Best and
N32 after repeated passage of the virus
through the host . . . . . . . . . . . . . . . . 52

vi

LIST OF FIGURES

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vii

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ix

Errata

Throughout the sections entitled EXPERIMENTAL
RESULTS and DISCUSSION, the work cuboidal should read

cubical.

INTRODUCTION

One of the most conspicuous cytological phenomena
associated with plant virus diseases is the formation of
virus inclusions in virus-infected plant cells. The
importance of this statement is neither reduced nor
altered by the fact that these inclusions may sometimes
be all virus, partly virus, or proteinaceous bodies
produced by cells in response to viral infection. As a
result of classifying the various forms of viral in-
clusions more plant virologists should be able to
identify a particular virus by one or more of the assoc-
iated inclusions.

It is interesting to note that inclusions are con-
fined to plants and animals infected with viruses; no
other known entities of disease parallel this. Approxi-
mately 30 animal-infecting viruses, inclusions (Negri
bodies) are diagnostically important. The simple ob-
servation of inclusions in living tissues indicates that
‘virus is present; however, the converse is meaningless.

This investigation was undertaken to clarify the
Cytological descriptions of TMV inclusions in resistant
and susceptible tomato cells. The following objectives
served as a means for the development of this thesis:
(1) to expand the details of McWhorter's virus inclusion

Classification, group BV, type TMV section; (2) to relate

virus inclusions to strain of TMV in the host plant and
relate virus inclusions to syndrome; (3) to add information
on site of virus assembly as related to development of

virus inclusions.

LITERATURE REVIEW

nggoduction. Two nearly simultaneous announcements
in 1903, set the stage for investigations of virus inclu-
sions. Negri (1903) described the numerous inclusions in
neurons of brains in animals afflicted with rabies.
Iwanowski (1903) described the inclusions in tobacco
plants affected with tobacco mosaic virus. During the
next twenty years, early researchers of plant virus diseases
confused inclusions with the cause, rather than the result
of infection and, further, even attempted to animate the
inclusions. Finally, among the pertinent papers published
in the United States and England in the twenties, the
publication by Goldstein (1924) produced a basic under-
standing of TMV inclusions in living cells. Her study
centered about the elusive amorphous inclusions of TMV,
coining the term "X-bodies", that is still employed in
today's literature. The investigations of Goldstein
placed anatomic studies of plants infected with viruses
on a modern level of science and became significant in
understanding the relationship between virus and host.

Establishment 9§_virus inclusions. According to
McWhorter's (1965) recent review, three basic types of
Virus inclusions have been proposed based primarily on
molecular arrangement and chemical constituents. These
are (a) crystalline-bodies with internal, regular, re-

Peating atomic arrangements and external plane faces;

3

(b) paracrystalline-bodies with molecular units arranged
to give a two-dimensional figure, but lacking the signifi-
cant third dimension of depth; and (c) amorphous-bodies
without complex organization but with variable shapes.
Although Iwanowski (1903) suggested that the amorphous
inclusions were breakdown products from the nucleus of
the infected cell, Goldstein (1924), through her staining
procedure involving thionin, gave evidence to the contrary.
She further reported that the X-bodies did not cause any
change in appearance of the nuclei. Again, Goldstein
(1926) published more detailed information on the mode
of X-body development and multiplication. In this same
paper she reported the occurrence of other forms of
inclusions: crystalline striate bodies, hexagonal plates
and irregular plates; tannin inclusions with vacuolar net-
‘work; and cuboidal bodies found in the nuclei. X-bodies
‘were observed in cells of tobacco (N, tabacum var. Con-
neticut) stems, leaves, roots, leaf and branch primordia,
and flowers. Goldstein apparently initiated the correla-
tion of inclusions with symptoms of virus mosaic disease.
During the late twenties and through the thirties,
TMV was crystallized, more studies of TMV inclusions were
conducted and the first important paper of TMV in tomato
leaf cells was reported. In 1928, Smith produced the

first major report of inclusions produced by a TMV strain

in tomatozaucuba mosaic virus of tomato. It is extremely
difficult to positively trace the exact virus strain used
by Smith and later by Sheffield since there is another TMV
strain known as tomato aucuba mosaic virus. In my opinion
I believe that the TMV strain currently called yellow
aucuba mosaic virus had its origin from Bewley's (1924)
aucuba mosaic virus of tomato and was named Yellow Tobacco
Mosaic Virus by Smith (1928). Sheffield (1931) expanded
the knowledge of intracellular inclusions utilizing Bewley's
aucuba mosaic virus in several hosts including tomato.

She found that in infected Solanum nudiflogum, the X-bodies
were insoluble in water, ethyl alcohol, xylol, acetone

and chloroform. They were soluble in 10% KOH and in
strong concentrated mineral acids. The crystalline in-
clusions (hexagons) in tomato trichomes, found only very
rarely, became striate when weak acids (no designation of
acids given) were introduced under the cover slip. Later,
in 1934, Sheffield published further information on in-
clusions in tomato produced by aucuba mosaic virus and
Johnson's tobacco virus 1. She was able to describe
subtle differences between the X-bodies produced in
tomato trichomes by these two strains of TMV. Since
Iwanowski's discovery of viral inclusions in plant cells,
Sheffield was the first person to clearly establish the
diagnostic value of intracellular inclusions. The next

year, 1935, Stanley made the discovery that TMV could

be purified as long, needle-like proteinaceous crystals.
Beale (1937) was able to follow up this work and show
the relation of Stanley's crystalline tobacco virus
protein to intracellular crystalline particles. Dilute
HCl, HNO

and CH COOH (@ pH = 1.3) caused needles to

3 3
form from hexagonal crystals; however, the crystals
denatured without forming needles @ pH 1.0 and pH 11.0-
11.8. Beale concluded that Stanley's preparation gave
needle-like paracrystals because the acidity and alka-
linity used to denature protein would also dissolve any
true crystals. Livingston and Duggar (1934) went a step
further to show that (a) the inclusions of TMV were lo-
cated primarily, if not solely, in the protoplasm of the
virus-infected cell and (b) attempted, with some success,
to indicate that the occurrence of intracellular bodies
was related to relatively high virus concentration. Bernal
and Fankuchen (1937) proved that ip_yit§g paracrystals
were made up of virus rods composed in hexagonal two-
dimensional array perpendicular to the long axis of the
paracrystals, but could not show a predictable array
along their lengths. Bawden and Sheffield (1939) pointed
out that some viruses do not cause formation of intra-
cellular inclusions;cmhers cause production of amorphous
bodies only, and still others produce both amorphous and
crystalline inclusions. In a second publication in 1939

Sheffield provided evidence that the X-bodies found in

aucuba mosaic virus-infected hair cells of Solanum
nodiflorum could be isolated and assayed for infectivity
on leaves of Nicotiana glutinosa L. The X-body sus-
pension produced necrotic local lesions indicating that
these inclusions definitely contained virus. Unfortu-
nately, she was not able to isolate or assay the crystal—
line inclusions of either aucuba or tobacco mosaic virus
due to the extreme delicate nature of the inclusions.
Sheffield (1936) overcame this problem by electron micro-
scopy and reported that TMV and hexagonal plates were
composed of rod-shaped particles of 15,» diameter.
Similar results were obtained with the aucuba strain of
TMV.

Though the period from 1940-1949 was not one of
outstanding contributive papers on plant virus inclusions,
several investigators accumulated more information about
TMV inclusions in both tobacco and tomato cells. Esau
(1941a) produced clear evidence for the presence of TMV
(Johnson TMV, strain 1) X-bodies and striate material in
the guard cells of stomata of Nicotiana tabacum L., thus
invalidating Sheffield's (1936) conclusions about their
eabsence. Esau (1941b) also investigated the effects of
‘tobacco mosaic virus on the phloem of tobacco and ob-
Ensrved that inclusions could be found in all types of
CEEIIS of the vascular elements. Because of the close

anatomical similarity between tobacco and tomato, she

found the tomato showed the same degenerative changes as
tobacco when infected by Johnson's tobacco mosaic virus
strain 1.

The research of Kassanis and Sheffield (1941) pointed
out, apparently for the first time, the occurrence of a
third type of TMV inclusion: paracrystalline fibers.
Ordinary TMV and aucuba mosaic virus produced not only
crystalline and amorphous inclusions, but also fibrous
bodies ranging from spike-like to needle-shaped to long,
curved "figure eight" fibers. In the establishment of
the three basic types of TMV inclusions, all reports
prior to 1948 indicated that inclusions were confined
strictly to the cell cytoplasm. However, Woods and Eck
(1948) provided evidence that TMV "nuclear strain" 10
in addition to crystalline and amorphous inclusions, in-
duced the formation of intranuclear fibrous and crystalline
inclusions in three varieties of tobacco (but failed with
N. glutinosa), and one variety each oprepper and tomato.

The literature pertaining to TMV inclusions between
1924-1949, although largely descriptive in nature, laid
the basic foundation for renewed and increased interest
:in plant virus inclusions. In 1950 two independent in-
\restigations elucidated the particulate nature of the ip
Q19 hexagonal crystals of TMV. Wilkins, _e_I_:.. 2;... (1950),

Enablished results stating that when the crystals were

viewed near extinction with crossed nicols with a bright
light source, a distinctly laminated composition was
observed. Since each lamination measured 3a 3000 A0 in
width, it was proposed that the crystal consisted of iso-
tropic rod-shaped particles lying parallel to the hexagonal
axis. Thus, the hexagonal crystal was constructed of
palisade-like layers of TMV rods. Another publication

by Rubio-Huertos (1950), remained unnoticed by most re-
searchers for the next fifteen years. His electron
photomicrograph proved beyond any doubt the parallel rod
arrangement within the hexagonal crystalline inclusion.
Max:0f the credit for this discovery has been unduly
attributed to Steere and Williams (1953) who utilized the
freeze-drying technique, followed by electron microscopy
of the isolated crystals from virus-infected hair cells,
and showed that these crystals consisted of very numerous,
infective TMV particles (rods).

During the 1950-1959 period, a number of the present-
day investigators began publishing more noteworthy re-
search on the chemical nature as well as the morphology
Of viral inclusions. Zech (1952) corroborated the ob-
servations of Esau (1941a) and found that TMV crystals
in the stomatal cells of Nicotiana tabacum, were excellent
indicators of virus infection. Zech (1954) measured the

light absorbance of TMV X-bodies, crystals and fibrils in

10

order to determine index values in comparison to normal
cell constituents, i.e. nucleus and cytoplasmic organelles.
One significant advancement in crystalline inclusion
studies was made independently in Madrid, Spain and
Berkeley, California. Rubio-Huertos (1954) removed a
complete aucuba mosaic virus hexagonal crystal from a
tobacco cell and showed by electron microscopy that the
hexagonal crystal was composed of TMV. Steere and Williams
(1953) extracted hexagonal crystals of TMV by a freeze-
drying procedure, followed by electron microscopy, and
showed that these crystals consisted primarily of particles
of TMV and a volatile solvent. The electron micrographs
presented by Wehrmeyer (1957, 1959) distinctly illustrated
that the TMV hexagonal crystal was composed of bands of
parallel-oriented TMV particles measuring 300 mp in length.
Unfortunately, it is difficult to equate his observations
with others since his selected strains of TMV have not
apparently received intensive investigation, e.g. one

green strain-Vulgare and two yellow strains-G2 and G7.

The timing of events leading to the formation of
inclusions in host cells, of interest since Goldstein's
study (1924), was not successfully measured until Zech
(1955, 1956) developed an arbitrary time scale based on
infection by TMV in single hair cells of N. tabacum var.

White Burley and N. glutinosa. He found that of the nine

ll

infection, periods (IP), X-body formation occurred during
the fifth IP, whereas cubicle and hexagonal microcrystals
occurred during the seventh IP. Hence, these studies in-
dicated that TMV X-body formation preceded the formation
of TMV crystals. Two years later, Hirai (1959) fully
substantiated Zech's conclusions about the sequential
development of inclusions. Working with detached Turkish
tobacco leaves inoculated with CTMV and floated on a
nutrient solution, he reported the following sequence of
inclusion formation: granular X-body = 2 days, vacuolate
X-body = 4 days, incomplete crystal 2 4 days, and complete
crystal = 6 days. In a series of delicate experiments,
Rubio-Huertos (1956) detected compositional differences
between X-bodies and crystals of TMV—infected epidermal
cells of Turkish tobacco. The common strain of TMV
produced X-bodies with very few, if any, detectable virus
particles and appeared to be composed mainly of amorphous
material. In contrast, however, the aucuba mosaic strain
of TMV produced X-bodies which contained much virus and
clearly showed masses of long parallel fibrils approxi-
mately the width of TMV particles. The crystals of both
sflxains were almost entirely composed of rod-shaped virus
particles. Rubio-Huertos advanced the theory, based on
EM work and phloxine staining, that the inclusions were

intimately associated with plastids and was most likely

12

the site of origin for inclusions. Rawlins, Weierich

and Schlegel (1956) partly confirmed the results of Rubio-
Huertos by cytochemical means. They found that the in-
clusions of the common strain of TMV stained differen-
tially with the Sakaguchi color reaction test for arginine:
the crystals gave a very strong reaction but the X-bodies
gave no detectable reaction. Furthermore, the X-bodies

of the aucuba strain of TMV differed from those associated
with CTMV because crystals and long fibrils formed within
the boundaries of the X-bodies. The study of Matsui (1958),
conducted with CTMV on N. tabacum L. var. Xanthi-nc under
conditions whereby the plant showed slight symptoms of
systemic infection, produced no evidence for chloroplasts
being the site of virus formation. Thus, the observations
of Matsui were in direct disagreement with those of Rubio-
Iiuertos. Shalla (1959) employed ultra thin tissue-sec-
'tioning for the electron microscope and was able to show
tfllat CTMV-inoculated tomato (Improved Pearson) failed to
bfiield.rod-shaped particles of TMV except in cells containing
CIfiystalline inclusions.

The increased research conducted during the period
159Ek3-1968 probably produced more controversy about the
Ilat-ure, origin and significance of inclusions than anytime
Siruze Iwanowski's discovery in 1903. Three major reviews

anfil a.book.were published in this 8-year period that served

13

to consolidate the available information on inclusions.

The Russian investigator, Goldin (1963), published the

first known book on plant Viral inclusions, approaching

the subject by considering inclusions to be functions of
'various viruses as they develop in plant families. The
(Eerman investigator, Mundry (1963), although reviewing

Inuch of the biochemical-physiologica1 work on plant virus-
luast cell relations, pointed out the nature and cell loca-
tion of crystalline inclusions. Two American investigators,
IAcWhorter (1965) and Esau (1967), published reviews on
'uypes and a classification system of plant virus inclusions,
respectively.

More variable forms of inclusions were noted and
:studied during recent years. Wehrmeyer (1960), working
Imith the G2 strain of TMV, observed the formation of intra—
Iflxasmic strands (trabeculae) across the lumina of tobacco
Celjjsin.inoculated secondary leaves of Samsun and White
Ihirley tobacco. Trabeculae formed in leaf mesophyll cells
Of"both upper and lower epidermis. More recently, Hoefert
Enid Gifford, Jr. (1967), reported trabeculae in parenchyma
CEElls within and adjacent to vascular bundles of young
Cahes and leaves of grapevine infected with leafroll virus.
G<Dildin (1960) unsuccessfully attempted to introduce the
teI‘m."Iwanowski" crystal in lieu of the term hexagonal

Crystal and later (1963, 1966) he introduced the "Kazakhi"

14

strain of TMV which apparently is capable of producing an
array of various forms of inclusions. I should however
interject at this point that through personal communica-
tion with F. P. McWhorter (1968), I found that he, too,

had been unsuccessful in obtaining the "Kazakhi" strain
from Goldin; hence, to my knowledge no one else has been
able to experiment with this particular strain. The forms
of inclusions and conditions leading up to their develop-
Inent in virus-infected host cells has recently received
Imore attention from qualified investigators. Solberg and
IBald (1962) accomplished an excellent comparison between
tflie cytoplasm of healthy and TMV-infected living hair cells
arui reported that crystals precipitated from dense areas
<Df cytoplasm, while X-bodies formed from regions of co-
Eugulated cytoplasm. Working with petunia ringspot virus
le cells of Petunia hybrida Vilm, N. glutinosa L., N.
;tabacum L., and Vigig'fapg L., Rubio-Huertos (1962) ob-
fisrved crystalline inclusions of different shapes, ranging
fI‘Omhexagons to octahedrons, apparently developed from
'qu X-bodies. He continued his observations (Rubio-Huertos,
15964) on severe etch virus in cells of White Burley tobacco
arui reported both intranuclear crystalline inclusions as
'Well.as intracytOplasmic X-bodies. Solberg and Bald
(196%) reported that an iodine-iodide fixative preserved,

‘Without distortions, both crystalline and amorphous in-

l5

clusions of TMV-infected Turkish tobacco and N. sylvestris.
Thus the study of inclusions was enhanced, especially in
preparing material for fine structure analysis by electron
microscopy. Chandra and Hildebrandt (1965) were the first
to report inclusions in fresh "Havana 38" tobacco callus
cells derived from a virus-infected plant as well as in
callus cells cultivated through two transfers in culture.
'They further noted the presence of abundant inclusions in
some cells while others had none. An interesting observa-
tion by Edwardson (1966), on TEV in tobacco cells, showed
that one type of cytoplasmic inclusion, when sectioned in
(tifferent planes, can produce configurations easily mis-
ixflken for another type of inclusion. Rubio-Huertos, gt.
5&1. (1967), based on electron micrographs of turnip yellow
nuisaic virus-infected Chinese cabbage leaves, proposed
'Unat X-bodies corresponded to clumped and broken chloro-
EHJHStS mixed with cytoplasmic material, osmiophilic
{granules, and arrays of virus particles.

Origin 2; inclusions. One of the most controversial
E31lbjects in today's literature is the origin of inclusions
j~11 virus-infected plant cells. Two schools of thought
hei‘Ve developed, each with attending experts, thus only
C0ntinued meticulous experimentation will finally resolve
the question of whether the original material for in-

clusion formation derives from the nuclear or cytoplasmic

16

constituents.

Bald and Solberg (1961a) reported two important
findings: (a) TMV-U1 virus caused production of "grey
plates" consisting of several monolayers that eventually
assumed the familiar hexagonal crystals, and (b) there
was a release of a viral substance from the host cell
nucleus that initiated the formation of inclusions in
the cytoplasm. They further reported (Bald and Solberg,
1961b) that the first appearance of aggregating TMV-U1
in an infected cell was preceded by and apparently assoc-
iated with intense activity around the nucleus. In 1963,
Hooker and Bald, working with TNV, detected the emission
of a substance, presumably RNA, from nucleolus into
nucleus. However, Hooker (1964) was unable to detect
any release of either virus or virus RNA from the nucleolus
into the nucleus of PVX-X5 infected cotyledons of Datura
'tatula L. Pyronin Y-methyl-green-staining RNA masses were
Lasually closely associated with the nucleus and these
i.nclusions progressively stained more intensly throughout
'the period of observation. Bald (1964) was finally able
to demonstrate in virus-infected tobacco leaf cells, that
TRW%U1-RNA appeared in the nucleolus and passed into the
cytoplasm where it was detected in material from which
the X-bodies originated. Using TMV-U1 infected tomato

Stem epidermal strips, Hirai and Wildman (1963) recorded

17

events of virus multiplication within the trichome cells.
They concluded that as viral RNA was released from the
nucleus, it was surrounded by sphaerosomes, transformed
into granular bodies, then transported to other regions
of the cell via cyclosis; hence, the X-bodies could have
resulted from aggregation of these sphaerosome-TMV com-
plexes. The following year, Hirai and Hirai (1964) pre-
sented evidence for TMV synthesis in the nucleus of in-
jected, living tomato trichomes. Use of a fluorescent
TMV antibody showed the presence of the virus protein in
the nucleus 6 to 18 hours after injection and as time pro-
gressed, staining of the cytoplasm became more prominent.
Mundry (1963) denoted in his review that material, possibly
RNA, from the nucleus was transported into the cytoplasm
where assembly of the virus occurred. He remarked that
crystals were always found in distended regions of the
endoplasmic reticulum and always surrounded by a membrane
and ribosomes. Reddi (1964a, 1964b, 1964c) published a
series of rather convincing papers on the biosynthesis
of TMV based on chemical, cytological and physical tests.
He concluded that TMV is synthesized in the nucleus.

0n the other hand, prominent investigators have
arrived at the opposite conclusion that the material for
TMV assembly and formation of inclusions originates in

the cytoplasm. Wettstein and Zech (1962) made observations

18

on the structure of nucleus and cytoplasm in trichomes
during TMV multiplication and reported that the appear-
ance of the mature TMV particles within inclusions im-
plicated virus formation in restricted cytoplasmic areas.
Under cyto-chemical tests conducted by Takahashi (1962),
the nuclei of TMV-infected tobacco cells remained un-
changed while crystalline cytoplasmic and guard cell
inclusions stained red with pyronine - a stain for RNA.
Shalla (1964) studied tomato mesophyll cells infected by

a strain of TMV similar to U1 and his evidence indicated
that TMV was assembled in the ground cytoplasm, the
particles sometimes becoming enveloped by distorted chloro-
plasts. Furthermore, he found X—bodies in the cytoplasm
contained mitochondria, TMV particles and numerous mem-
branes. Based upon an apparent absence of particles in
any other part of the cell in early infection, Shalla
concluded that TMV assembly occurred in the ground cyto-
plasm. Zech, Kolemainen and Wettstein (1965) found hexa-
gonal crystals in ctyoplasm of N. tabacum cells systemically
infected by a flavum strain of TMV, as well as Bald's
"grey plates" and some tubular inclusions. They con-
curred that assembly of inclusions most likely took place
in the cytoplasm in the regions of intensely-folded endo-
plasmic reticulum and the ribosomes. Milne (1966) reached

similar conclusions as Shalla and Zech founded on his ob-

l9

servations with CTMV in Turkish tobacco leaf palisade
cells. Warmke and Edwardson (1966) have produced the
most definitive electron micrographs Whidielucidate the
crystalline inclusions in hair cells of Turkish tobacco
inoculated with TMV (ATCC, #AC-Z). They found no evidence
of crystals in nuclei or chloroplasts but, due to intimate
association of aggregates with the ground cytoplasm, sug-
gested that formation of inclusions was cytoplasmic.
Inclusions gp local lesion hosts. The second area
of recent controversy has developed over the presence
or absence of inclusions in leaf cells of local lesion
hosts. Yogoslavic researcher Miligié (1962), reported
inclusions within the chlorotic halos surrounding necrotic
local lesions in TMV-infected Datura stramgnigm L. and
Chenqpodium amaranticolor. If the local lesion was
chlorotic, Milifié found inclusions throughout the lesion
site. Confirmational observations came from Hayashi
and Matsui (1963) when they presented an electron micro-
graph showing TMV particles within necrotic lesion areas
on N. glutinosa. The early observations of Shalla (1959)
on.N5 glutinosa infected by CTMV, produced no evidence
ofIEMV particles in cells adjacent to necrotic lesions.

Hrgel and Brggk (1964), conducting observations on the

TMV-infected, local lesion host, N. tabacum L. var. Xanthi-

20

no, recorded the absence of any rod-like particles or
crystalline inclusions from the local lesions or surround-
ing cells. The Canadian investigators, Weintraub and
Ragetli (1964), corroborated the findings of Shalla and
on the basis of their work with TMV-infected N, glutinosa,
they emphatically stated that no structures were observed
at any stage that could be identified as TMV particles.
Intracellular movements 9: inclusions and organelles.
Visual cytoplasmic alterations in virus-infected plant
cells have attracted the attention of cytologists since
the publication of Goldstein (1924). Benda (1959) made
an interesting observation upon a leaf trichome cell of
N. langsdorffii Weinm. He found, after the_cell was
punctured near the apical cross-wall by a glass needle,
that oftentimes the nucleus migrated toward the wound
site. Upon repeated experiments, he calculated that this
movement occurred between 52 and 230 minutes after wounding
and at approximately 342 minutes the nucleus began to move
away from the wound. Resconich (1961) noted that TMV in-
clusions in epidermal cells of Prince bean, systemically
infected with TMV-5P (Lister and Thresh strain), in spite
of their large size, were freely carried about in the cyto-
plasmic stream. Nuclear migration was noted by Hirai and
Wildman (1963) in TMV-infected tomato stem trichomes, in-

oculated in such a manner that infection of the trichome

21

cells progressed from the base cell to apical cell. 0n
the third day of infection, the nuclei had migrated to-
wards the transverse wall of the basal cells, apparently
the site of infection. The nuclear migration was again
observed by Hirai and Hirai (1964) in TMV-infected
tomato trichome cells injected by a fine glass needle.
They stated that the first evidence of TMV infection
was migration of the nucleus towards the point of entry
of the needle and virus. Singh and Hildebrandt (1966)
actually mapped, through still cinephotomicrography, the
movement of several types of TMV inclusions within
tobacco callus cells (N. tabacum L.) varieties Havana 38
and Bright Yellow.

intracellular movements 2; virus and inclusions. For
approximately 40 years, plant virologists and cytologists
have pondered the question of how plant viruses entered
and spread through cells of their hosts. Although there
is general agreement that the ectodesmata and plasmodesmata
are the avenues of virus distribution, the published evi-
dence is still strongly circumstantial. A brief account
of the important papers will serve as background for this

phase of the ensuing report.

The first description of plasmodesmata was made by
Tangl (1879) and prompted botanists to study these struc—

tures. However, due to improper observations and staining

22

techniques, reports have been considered misleading until
the work of Crafts (1931) became recognized. In the same
year that Stanley made his discovery, Livingston (1935)
published his observations on the nature and distribution

of plasmodesmata in the tobacco plant. He reported numerous
plasmodesmata (ave. no. : 40 per 100 sq.,p) on basal septa
of N. tabacum var. Wisconsin Havana #142 trichomes. Samuel
in 1934 and Sheffield in 1936 postulated the role of plasmo-
desmata in the translocation of virus even though the morph-
ology of the TMV particle was unknown to them. Johansen's
text (1940), Plant Microtechnique, reported the procedure
for demonstrating plasmodesmata although the technique was
developed by Crafts (1931).

Three German investigators laid the foundation for the
study of ectodesmata in the epidermal cells of many plants.
Schumacher (1942), Lambertz (1954) and Schnepf (1959) pro-
duced photographs and diagrams with supporting evidence
for the presence, types and locations of these canals. The
German school of investigators expanded the knowledge of
ectodesmata and plasmodesmata. Franke (1962, 1964a, 1964b,
1964c) provided excellent information on the nature and
structure of ectodesmata, giving evidence for their proto-
plasmic origin. He also showed (1961) that leaf structures
such as guard cells, trichomes and epidermal cells adjacent

to the leaf veins consistently contained large numbers of

23

ectodesmata. Schumacher and Kollmann (1962), using the
phloem of Metasequoia glyptostroboides, announced that the
plasmodesmata were internally lined with elements of the
endoplasmic reticulum (ER) and passed through the plasmo-
desmata from one parenchyma cell to the next. Schnepf
(1964), Dolzmann (1964, 1965) confirmed the ER concept of
the plasmodesmata in other plants.

The relation of virus to the protoplasmic pathways,
gnstmlatedtw’Sheffield (1936), was revived through elec-
tron micrographs of Shalla (1959). His photos supported
the view that plasmodesmata were continuous protoplasmic
connections between adjacent cells of tobacco leaves and
were of sufficient magnitude to allow passage of TMV in
the form of intact nucleoprotein rods. The plasmodesmata
in young tobacco leaf cells measured at least 20 mp in
width while others ranged up to 200 mp. Kontaxis (1961),
working with detached leaves of N. glutinosa infected with
TMV, indicated (and indirectly implicated the ectodesmata)
that TMV penetrated the epidermal strips into the palisade
layer in less than 3 hours. Later, Kontaxis and Schlegel
(1962) and Herridge and Schlegel (1962), based on Clglabeled
TMV-infected leaf cells of N. glutinosa and N. tabacum,
advanced the hypothesis that when the trichome was broken
during mechanical inoculation, virus was deposited on the

basal septum which was coated with remnants of the trichome

24

cytoplasm. Furthermore, they stated that this cytoplasm
was in direct contact with the cytoplasm of the living
foot cell through the many plasmodesmata in the basal
septum. Hence the entrance of virus into uninjured host
cells was created and infection occurred.

The publications of Brants (1964, 1965, 1966) demon-
strated that in TMV-infected plants the resulting increase
:in the number of visible ectodesmata was correlated with
ean increase in the number of local lesions. Esau (1966)
Ireported the presence of beet yellows virus particles in
‘the minor veins of bean leaves. These particles were
].ocated in the wall channels that connected the sieve
eelements with one another, sieve elements with parenchyma
czells and parenchyma cells with one another. She proposed
tihat phloem movement of virus particles took place in the
txranslocation stream as complete particles. The review by
bfundry (1963) presented the conclusion that ectodesmata
bee appreciated as the principle infectible sites on tobacco
lxaaves and probably also on other leaves. Bawden (1964)
Eitrongly suggested the plasmodesmata assisted in the dis-
‘tribution of virus-infected cells, although the mechanism
<Df the movement has not yet been established.

The foregoing rules out any possibility of most TMV
inclusions being transported via the plasmodesmata due pri-

nuxrily to size of inclusions. There are two reports of

25

inclusions moving from cell to cell but in a rather differ-
ent manner. Goldin (1963) and Singh and Hildebrandt (1966)
showed clear evidence that the stiff, sharp-pointed, TMV
paracrystalline fibers oftentimes punctured the walls of

adjoining cells when pushed by cytOplasmic streaming.

MATERIALS AND METHODS

TMV inocula were cultured in tomato, Lycqpersicon
esculentum Mill. var. Bonny Best (Burgess Seed Company)
in the greenhouse. All TMV strains were systemic in Bonny
Best. A second variety of tomato, N32 (Spartan Red x
Holmes P. I. #235673), developed at Michigan State
University, was resistant to TMV. The following six
strains of TMV were obtained for this study: (a) Holmes
Rib-Grass (HRG), from Dr. F. 0. Holmes in dried tobacco
leaves, 1958; (b) Holmes Distorting Strain (HDS) from
Dr. F. O. Holmes in dried Samsun tobacco, 1958; (c) John
S. Boyle (JSB) tomato internal browning strain from Dr.

J. S. Boyle in Rutgers tomato, 1958; (d) 5P from Dr. C. E.
Yarwood in Prince bean, 1961; (e) Yellow Aucuba (YA) from
The Virus Laboratory, Berkelqg in dried tobacco leaves,
1960; (f) Strain 4 (S4) from Dr. L. J. Alexander, Wooster,
in tomato leaves, 1965.

Plants were grown in a sterilized soil mix, a mixture
of 50 percent loam, 30 percent sand, 15 percent peat moss
and 5 percent Vermiculite, supplemented twice a week with
a commercial nutrient solution (Plant Marvel) composed of
12, 31 and 14 percent of N, P and K respectively at the
rate of one tablespoon per gallon of water. Plants were
grown with and without supplemental light in the greenhouse

. . o .
and maintained near 20 C except during summer months when

26

27

daytime temperatures sometimes approached 2800. During
the winter artificial lighting (50 percent mixture of
40-W cool white and Sylvania Gro-Lux fluorescent lamps)
was supplied on a 16-hour cycle followed by 8 hours of
darkness.

Stock inocula were prepared from young Bonny Best
leaflets by grinding them with a mortar and pestle,
utilizing a dilution of 1:10 (v/v) with distilled water.
Inoculations of selected uniform tomatoes, dusted with
400 mesh silicon carbide, were made by rubbing the virus
suspension over the upper leaf surface with a cube of
polyurethane and a polyurethane pad for leaf support
or with a gound glass spatula and a folded paper towel
for leaf support. Control plants were rubbed with dis-
tilled water. Symptom development of the infected
plants was recorded at time intervals after inoculation.

Virus-infected leaves were removed from greenhouse
plants at intervals after inoculation for cytological
observations. Para-epidermal strips and free-hand
transverse sections of the leaflets were made in such
fashion as to retain the trichomes. An experimental
microtome (Hooker, 1967) was also used to obtain trans-
verse sections of tomato leaflet tissue.

Observations of prepared tissue were performed with

an American Optical Microstar Research Microscope, achro-

28

matic objectives 10x, 43x and 97x, equipped with a Zeiss
Ikon 35mm camera, 12.5x ocular tube. Phase contrast
observations were conducted with a Wild-Heerbrugg M20
Microscope, Fluotar objectives 40x and 100x (NA 0.75 and
1.30 respectively) equipped with a Kodak Pony II 35mm
camera. Photographs were made using 35mm Kodak Ektachrome
X, Kodak High Speed Ektachrome Type B and Kodak Panatomic
X film. Negatives for black and white prints in this
study were prepared from the Ektachrome X using Kodak
Panatomic X film.

Examination of all inclusions were carried out in
the absence of any stains since these compounds did not
enhance the clarity of the observations nor the nature of
the inclusions. The staining procedure used to demonstrate
the ectodesmata and plasmodesmata followed the work of

Lambertz (1954) and Schnepf (1959).

EXPERIMENTAL RESULTS

Symptomatology: Experiment 1. The development of
symptoms on virus-inoculated susceptible (BB) and resis-
tant (N32) tomato is presented in Table 1. Seven days
after inoculation, S4 in N32 was the first to show symp-
toms of infection in the form of excessive decurvature of
the young petioles. By day 11, interveinal clearing was
noted and by day 12, symptoms were still not detected on
any other plants. On day 13 initial symptoms were re-
corded on all BB and in addition, epinasty was noted on
N32 infected with HDS. A general intensification of symp-
toms were noted through day 65 at which time it was prac-
tically impossible to discern symptom differences between
HRG, JSB, HDS, S4 in BB; but YA and 5P in BB showed symp-
toms that allowed respective identification. The unusual
eventduring this experiment was the apparant breakdown of
the resistant host, N32, against HDS, expressed by epinasty
of the youngest leaflets and deformity of the older leaf-
lets. YA and 5P were considered to be the least virulent
of the six strains on BB.

Experiment 2, conducted in exactly the same manner
but at a different time of the year, produced somewhat
similar results as presented in Table 2. In comparison
the symptoms of both BB and N32 occurred one week earlier

than in experiment 1. HDS at day 5 was the first to pro-

29

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33

duce symptoms in N32, followed at day 7 by S4. YA and 5P
at day 5 were the first to produce symptoms in BB. The
symptoms recorded on day 7 for all strains in BB progress-
ively intensified through day 18. The symptoms produced
in BB by HRG, JSB, HDS and S4 were very closely similar,
but the symptoms produced in BB by YA and 5P could be
separated from the previous four and from each other.

The results of experiment 2 generally paralleled those

of experiment 1, except for the chronological development
of symptoms.

Experiment 3 was conducted to check on any possible
contamination of stock control plants and to observe the
effects of stock inocula on a local lesion host. The re-
sults appear in Table 3, indicating transmission of all
strains in BB and no transmission from any strains in
N32 nor healthy control BB or N32.

Development 9; inclusions. Results of cytological
examination of the leaflet trichomes from virus-inoculated
and non-inoculated plants (experiment 1) are summarized
in Table 4. Inclusions were observed in all trichomes of
virus-treated BB plants and no inclusions were observed in
any trichomes of virus-treated N32 plants save for HDS.

A normal trichome from healthy BB leaflet is presented in
Fig. 1A. No inclusions were ever observed in any of the

healthy control plants. Irregular crystals of BB-S4 are

34

Table 3. Bioassay of all plants
used in Experiment number 2. Extracts from donor plants
98 days after inoculation. Greenhouse conditions with
l6-hr illumination per day, Jan., 1968. Young succulent
N. tabacum var. Xanthi-nc used as local lesion test

plant.(a)

 

All TMV strains from infected Bonny Best gave local

lesions.

 

All TMV strains from inoculated N32 gave no local lesions.

 

All healthy Bonny Best and N32 extracts gave no local

lesions.

 

(a)

Youngest three leaves per plant were treated.

35

Table 4.

Survey of inclusions in

tomato trichomes systemically infected by strains of TMV.
Observations based on 3 cross sections of symptom-bearing

young leaflets supporting approximately 100 trichomes.

Healthy controls:

no inclusions.

 

Type of Inclusion

 

 

 

 

Host-Virus Day 20(a) Day so
scattered hexagons, few needles, cubical
BB-HRG numerous irregular numerous crystals,
rectangular crystals, scattered X-bodies
few X-bodies
BB-JSB numerous irregular numerous rectangular
rectangular crystals crystals
BB-HDS numerous irregular numerous rectangular
rectangular crystals and irregular crys—
tals, numerous needles
BB-S4 scattered large "grey numerous large hexa-
plates", scattered gons
hexagons, numerous
irregular rectangular
crystals, numerous
microcuboidal crys-
tals
BB-YA few irregular rec- scattered hexagons,
tangular crystals few irregular rec-
tangular crystals
BB-5P numerous micro- numerous microcuboidal
cuboidal crystals crystals, scattered
irregular crystals
N32-HRG none observed none observed
N32-JSB none observed none observed
N32-HDS none observed numerous microcuboidal
crystals
N32-S4 none observed none observed
N32-YA none observed none observed
N32-5P none observed none observed
(a) . .
Days after inoculation.

36

shown in Fig. 1B; hexagon in Fig. 10; rectangular (hexa-
gons on edge) crystals in Fig. 1D; large, few-layered
"grey plates" in Fig. 1E; perfect hexagon in glandular
hair cell in Fig. 1F. Measurement of a similar hexagon
in a basal cell of a guard hair gave the following:
length = 18.7p, width = 10.7p, depth = 2.0p. Rectangular
crystals of BB-HDS are pictured in Fig. 2A and 2B. Rec-
tangular crystals of BB-JSB near nucleus shown in Fig. 2C.
The cubical crystals and a large X-body of BB-HRG are
presented in Fig. 2D and 2E respectively. An hexagonal
crystal in a subsidiary cell of a trichome, BB-S4, is
noted in Fig. 2F, while a similar crystal appears in an
epidermal cell of BB-S4 in Fig. 3A. Rectangular crystals
of BB-5P are found in Fig. 3C. Irregular, rectangular
and paracrystalline fiber of BB-HDS are photographed in
Fig. 3D and 3E. Needle (paracrystalline fiber) of BB-
HRG shown in Fig. 3F; oval unit is nucleus. Cubical
crystals and a "split" hexagonal crystal are shown in
Fig. 4A, 4B, and 4C. Cubical crystal passing by the
nucleus in BB—HRG is observed in Fig. 5A, while more of
the irregular (collection of hexagons?) crystals of BB-
HRG appear in Fig. 5B. The nucleus with attendant trans-
lucent cytoplasm of N32-JSB is shown in Fig. 6.

The results of cytological examination during

experiment 2 are presented in Table 5. These results

37

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38

 

 

 

 

 

 

 

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39

were rather similar to those of experiment 1 except in
the following respect: (a) X-bodies were located in
BB-HDS, BB-S4, BB-YA, BB-5P and (b) no inclusions were
observed in N32-HDS. An inclusion body, time-lapse se-
quence was made on a trichome basal cell of BB-5P in
order to show the movement of inclusions. Figs. 7A,
7B, 7C, 7D depicts this movement within the flowing
cytoplasm. Hexagons of BB-5P, face and edge views,
were photographed within the same cell as the X-bodies
and appear in Figs. 7E and 7F.

Cytology: special substrains. Two substrains,
A and B, of strains JSB, S4 and YA were selected for
cytological examination. These substrains were chosen
from their symptomatic reaction on either BB or N32 or
both. The results, found in Table 6, show the inclusions
observed with the host-virus system. The "mushy" bodies
of N32-JSB-B are shown in Fig. 4D. A tipped hexagon of
N32-YA-B appears in Fig. 4E, while a dense X-body and
numerous short paracrystalline fibers show in Fig. 4F.
An imperfect hexagon of BB-S4-A is shown in Fig. 8A.
A hexagonal "doublet" of N32-S4-B is presented in Fig.
8B. Another hexagon of N32-S4-B found in a subsidiary
cell of a guard hair is depicted in Fig. 8C. Two imper-
fect hexagons of BB-JSB-A are photographed in Fig. 8D

and a large X-body of BB-YA-A is observed in Fig. 8E.

40

Table 6. Survey of inclusions in

tomato seedlings inoculated with special substrains (A
and B) of three TMV strains obtained from T. A. Zitter,
Michigan State University. Greenhouse conditions, 28°C,
and supplemental lighting.

All substrain A produces symptoms on BB, but not on
N32 (no virus present).

All substain B produces symptoms on both BB and N32.

A and B substrains from single-lesion isolates on

 

 

Xanthi-nc.

Host Substrain Inclusions

BB-JSB-A scattered, small irregular crystals,
few hexagons.

N32-JSB-B few poorly-defined "mushy" bodies,
scattered irregular crystals.

BB-S4-A scattered irregular crystals, few
hexagons.

N32-S4-B numerous hexagons.

BB-YA-A scattered X-bodies.

(a)N32-YA-B numerous X—bodies, few rectangular

crystals, numerous needles.

 

(a)Unusually severe symptoms.

41

When the YA substrain B virus became systemically estab-
lished in N32 tissue, it produced unusually severe symp-
toms and accordingly, more variable and more numerous in—
clusions.

Cytology: inclusions in o;g_y;;us-infected.plants.
Experiment 5 was conducted to ascertain the fate of in-
clusions in old, systemically-infected plants. Contrary to
Bawden's textbook information, inclusions could still be
observed in tissues of tomatoes and were just as prevalent
as in experiment 1 and 2. The results of long-term in-
fection are shown in Table 7. As before, no inclusions
were noted in any of the virus-inoculated N32 nor in
healthy BB or N32 plants. Two trends seem noteworthy
from these data: (a) there was a marked decrease in the
presence of X-bodies in the first five strains (Table 7),
whereas (b) X-bodies were rather common in BB-5P. Further-
more, irregular and hexagonal crystals were found in all
virus-infected BB plants.

The inclusions recorded in experiment 5 follow. The
long, doubled-over, paracrystalline fiber of BB-S4 is
shown in Figs. 8F, 9A and 9B, while also showing banded
crystalline bodies. Numerous irregular, angular crystals
of BB-YA can be seen in Fig. 9C. A long paracrystalline
fiber (needle) of BB-JSB appears in Fig. 9D and at the
Other end of this same needle appears 2 X-bodies as shown

:in Fig. 9E. Two X-bodies and 2 banded rectangular crystals

42

Table 7. Survey of inclusions in
tomato plants systemically infected for 170 days. Observa-
tion based on three cross sections of symptom-bearing young
leaflets supporting approximately 100 trichomes. Green-
house conditions with supplemental lighting, March, 1968.
Healthy controls and all N32 inoculated plants yielded no

 

 

 

 

 

 

 

inclusions.

Host-Virus Inclusions

BB-HRG scattered hexagons, few
needles.

BB-JSB scattered irregular crystals
and cuboidal crystals, few
needles and X-bodies.

BB-HDS numerous hexagons, scattered
cuboidal crystals.

BB-S4 numerous imperfect near-
hexagons, long needles and
rectangular crystals,
scattered banded bodies.

BB-YA several clusters of irregular,
angular crystals.

BB-5P scattered hexagons (rectangu-

lar crystals?), X-bodies
common.

 

43

of BB-5P are clearly defined in Fig. 9F, while imperfect
hexagons in another trichome cell is recorded in Fig. 10A.
Perfect cube crystals of BB-HDS and an imperfect hexagon
are presented in Figs. 10B and 10C, respectively. Imper-
fect hexagons and rectangular crystals of BB-HRG are photo-
graphed in Fig. 10D and a stout paracrystalline fiber is
shown in Fig. lOE.

Cytology: tomato inoculated with local lesion isolates
from tobacco. Experiment 6 was performed for the purpose
of detecting any possible cross-contamination and/or mix-
tures of virus strains in tomato by transferring the virus
to a local lesion host (tobacco) then transferring back to
tomato, the systemic host. Cytological examination for
inclusions were made on tomato that received the local
lesion isolates from tobacco. The results are tabulated
in Table 8 and bear out the "purity" of the virus strains.
One new inclusion occurred in this experiment which in-
volved the production of dendritic clusters of needles in
BB-JSB.

The inclusions recorded in experiment 6 follow. The
cell nucleus, three X-bodies and a dendritic cluster of
BB-JSB needles appear in Figs. 10F and 11A. A fusiform
packet of paracrystalline fibers of BB-YA is presented in
Fig. 11B. Several coiled, long paracrystalline fibers of
BB-JSB appear in Fig. 11C. An irregular hexagon of BB-S4

in face View and edge View is displayed in Figs. 11D and

44

Table 8. Single lesion isolates
made from infected Nicotiana tabacum var. Xanthi-nc
transferred to cotyledons of 2-week old Bonny Best
seedlings. Bonny Best virus-infected stock served as
inocula for Xanthi-nc. Inclusion survey made 4 weeks
after inoculations to Bonny Best. Greenhouse conditions
with 16-hr. illumination per day, April, 1968.

 

Host-Virus Inclusions

 

BB-HRG numerous hexagons, small
cuboidal crystals and X-
bodies, scattered long needles.

 

BB-JSB numerous hexagons, irregular
crystals and X-bodies, den-
dritic cluster of needles,
long, curved fibers.

 

 

 

BB-HDS ‘ scattered hexagons, very
numerous clustered cuboidal
crystals.

BB-S4 numerous perfect hexagons,
irregular crystals, scattered
X-bodies.

BB-YA scattered hexagons, numerous

rectangular crystals, clusters
of needles.

 

BB-5P scattered very irregular
crystals and clusters of hexa-
gons, scattered dense X-bodies.

 

45

11E, photographed only 30 seconds apart. A perfect
hexagon in face view and another in edge view of BB-S4
appear in Fig. 11F. A paracrystalline fiber and a hexa-
gon of BB-YA are shown in Fig. 12A. Several cubical
crystals of BB-HDS are recorded in Fig. 12B. A very
irregular cluster of imperfect hexagons of BB-5P can be
seen in Fig. 120. Clusters of cubical crystals of BB-
HRG can be observed in Fig. 12D and enlarged in Fig. 12E.
Cytology: chemical tests on inclusions. Experiment
7 was designed to confirm the nature and the reaction of
inclusions to an acid and a base. The 0.1N HCl, pH = 1.1,
did not cause dissolution of any inclusions; however, the
0.1N NaOH, pH = 13.0, caused dissolution without formation
of paracrystals. The HCl solution failed to produce para-
crystalline fibers from hexagonal crystals as noted by
Beale (1937) and this was attributed by her to the differ-
ence in pH, thus affecting the process of denaturation.
The results of this experiment are recorded in Table 9.
Cytology: local lesion plants. Experiment 8 was
established to ascertain the absence or presence of in-
clusions within or around the site of local lesions on
Nicotiana tabacug var. Xanthi-nc leaves. The virus se-
lected for use, the treatments and the results are item—
ized in Table 10. Only treatment number 3 produced in-
clusions and then only in the chlorotic halo zone sur-

rounding the lesion.

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47

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pee: QOHeeH Heooa ens CH macamsaosfl mo hebhsm .OH eases

 

 

48

A description of the inclusions found in experiment 8
follows. The distorted nucleus in a trichome cell of a
local lesion Xanthi-nc infected with Xan-JSB on day 3 of
infection appear in Fig. 12F. A cubical crystal of Xan-
JSB on day 4 is shown in Fig. 13A. A virus-infected JSB ,
trichome cell from Xanthi-nc, showing nucleus, nucleolus
and chloroplasts is presented in Fig. 13B. Three hexagonal
crystals of Xan-JSB in sub-apical cell of glandular tri—
chome on day 5 are recorded in Fig. 13C and enlarged in
Fig. 13D. A string of chloroplasts near an X-body (out of
focus) and the same X-body (in focus) of Xan—JSB are given
in Figs. 13E and 13F, respectively.

Experiment 9 followed essentially the same design as
experiment 8, but was conducted on a related local lesion
host plant, Nicotiana glutinosa. An exhaustive search,
performed on plants grown under two treatments, produced
absolutely nothing that could positively be recognized as
an inclusion. The results of experiment 9 are offered in
Table 11. During several periods of observation, very
small thin, rectangular plates demonstrating Brownian
motion were observed in trichome cell from halo zones;
however, upon careful inspection of healthy, virus-free
trichomes these same plates, acting with the same movement,
were clearly smgned. Thus NG-JSB plates were descredited
as any possible virus-associated inclusion but are, how-

ever, recorded in Figs. 14A and 14B for the interest of

49

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neon seamed Heeoa esp ca macameaosfl mo he>Hem .HH NHQNE

50

Table 12. Comparitive study of
inclusions produced by S4 and a local lesion isolate in
Nicotiana tabacum var. White Burley, May, 1967. S4
(Alexander's tomato strain) produces local lesions on
White Burley, but LL-S4-4 (local lesion isolate) pro-

duces no local lesions on White Burley.

Host-Virus Inclusions

 

WB-S4 numerous large thick hexagons
and rectangular crystals,

often several per cell.

 

WB-LL-S4-4 numerous aggregates of thin
imperfect hexagons, scattered

small irregular crystals.

 

51

future research.

Cytology: a loca; lesion isolate ofégg on White
Burley tobacco. Experiment 10 was designed to compare
possible differences in inclusions between the S4 virus
in White Burley (WB) tobacco and a local lesion isolate,
LL-S4-4. The results of experiment 10 are noted in
Table 12.

A comparison of the inclusions from experiment 10
follows. A group of roughly hexagonal crystals of WB-S4
appears in Fig. 14C and a more general view of the same
group is given in Fig. 14D. A cluster of hexagons of WB-
S4 is recorded in Fig. 14E. A collection of thin, elongate
hexagons of WB-LL-S4-4 is presented in Fig. 14F and a more
general view is found in Fig. 15A.

Cytology: repeated passage of virus through host.
Experiment 11 was conducted on two virus strains, each
infecting both N32 and BB, to identify any differences in
inclusions. The results of experiment 11 are given in
Table 13. Three of the host-virus associations produced
a definite increase in the quantity of inclusions, namely,
BB#5-YA, BB#l-S4, and N32#1-YA. The remaining host-
virus association, N32#2-S4, produced only a few crystals.

The inclusions reported in experiment 11 follow.
Dense X-bodies of BB#5-YA appear in Figs. 15B, 15C and 15D,
the latter showing a cell nucleus near wall.. Fig. 150

also shows a cuboidal crystal in close proximity to a

52

Table 13. Survey of inclusions in
virus-infected, symptom-bearing tomatoes, Bonny Best (BB)
and N32 after repeated passage of the virus through the
host. Greenhouse conditions, 28°C, April-May, 1967.

Material obtained from T. A. Zitter, Michigan State

 

 

 

 

 

University.

Host-Virus Inclusions

BB#5-YA very numerous cuboidal crys-
tals, numerous hexagons,
numerous needles, scattered
X-bodies; in one case all
three types found within one
cell.

BB#l-S4 very numerous large perfect
hexagons and small irregular
crystals.

N32#l-YA very numerous small irregular
crystals, numerous X-bodies
and needles.

N32#2-S4 few angular crystals.

 

BB = 4th passage of virus through Bonny Best, 7 weeks
after inoculation.

N32 = 3rd passage of virus through N32, 7 weeks after
inoculation.

53

large X-body. A paracrystalline fiber (needle) and a
dense X-body of N32#l-YA are recorded in Figs. 15E and
15F, respectively. These two photographs are of the same
cell, the first to clearly show the needle, the second to
show the X-body. A small angular crystal of N32#2-S4 is
recorded in Fig. 16A which also shows the nucleus near
wall and three large foreign particles of unknown origin.
A large imperfect hexagon of BB#l-S4 appears in Fig. 16B.
Three types of inclusions formed in one trichome cell of
BB#5-YA are recorded in Figs. 160, 16D and 16E, hexagons
on edge, X-body and needle respectively.

Plasggdesmata. Experiment 12 was conducted to demon-
strate, if present, the plasmodesmata which connect the
cytoplasm of adjacent cells in many plant tissues. Follow-
ing frustrating work to stain and photograph these fine
strands, one tissue section of Bonny Best gave positive
results. These plasmodesmata are presented in Figs. 17A
and 17B. A diagramatic representation is given in Fig.
17C to indicate which of these strands were selected to
obtain measurements of width. The widths of the plasmo-

desmata ranged from 0.3p to 0.5p.

 

A x 125 B x 125

 

Fi9.1

Fig. l. (A) BB healthy trichome; (B) BB-S4 irregular crystals;
(C) BB-S4 hexagon; (D) BB—S4 hexagons on edge; (E) BB-S4 few-
layered "grey plates"; (F) BB-S4 perfect hexagon in glandular
trichome.

54

 

Fig.2

Fig. 2. (A) BB-HDS rectangular crystals; (B) BB-HDS rectangu-
lar crystals enlarged; (C) BB-JSB rectangular crystals below
nucleus; (D) BB-HRG cubical crystal in front of nucleus; (E)
BB-HRG same cell showing a large X-body; (F) BB-S4 hexagonal
crystal in subsidiary cell at base of trichome.

55

 

Fig. 3. (A) BB-S4 hexagons in epidermal cells of leaflet;
(B) BB-5P rectangular crystals; (C) BB—5P irregular crystals;
(D) BB—HDS irregular crystal; (E) BB—HDS rectangular crystals
and paracrystalline fiber; (F) BB-HRG paracrystalline fiber
near nucleus.

 

Fig. 4. (A) BB-HRG cubical crystals; (B) BB-HRG split hexa-
gon; (C) BB—HRG split hexagon enlarged; (D) N32-JSB-B "mushy"
bodies; (E) N32-YA-B tipped hexagon; (F) N32-YA-B dense X-
body and numerous short paracrystalline fibers.

57

B

 

Fig. 5. (A) BB-HRG cubical crystal in front of nucleus, X-
body above and irregular crystal upper left; (B) BB—HRG ir-
regular (collection of hexagons?) crystals.

c; O
,U

 

K 1500

Fig.6

Fig. 6. N32-JSB nucleus with normal-appearing translucent
cytoplasm.

\)
\O

 

Fig. 7
Fig. 7. All BB-5P within same cell. (A) through (D) showing

movement of three X-bodies at 15 sec. intervals; (E hexagons
on edge; (F) hexagons in face View.

60

 

Fig. 8.

A) BB-S4-A imperfect hexagon; (B) N32-S4-B hexagon
doublet; (C) N32—S4—B hexagon is central area of subsidiary
cell; (D) BB-JSB—A doublet of imperfect hexagons; (E) BB-YA-A

large dense Xebody; (F) BB—S4 long, looped-over paracrystalline
fiber.

 

Fig. 9

Fig. 9. (A) BB-S4 basal portion of looped-over fiber; (B)
BB-S4 medial segment of looped-over fiber with banded bodies;
(C) BB-YA many irregular, angular crystals; (D) BB-JSB para-
crystalline fiber at cross wall; (E) BB-JSB other end of same
fiber two X-bodies of different size; (F) BB-5P two X-bodies
with two banded rectangular crystals.

 

Fig. 10

Fig. 10. (A) BB-5P poorly-formed hexagon; (B) BB—HDS perfect
cube crystals; (C) BB-HDS imperfect hexagon in glandular tri-
chome; (D) BB-HRG imperfect hexagons and true rectangular
crystal; (E) BB-HRG cluster of hexagons and stout paracrystal-
line fiber; (F) BB-JSB dendritic cluster of fibers below
nucleus and two X-bodics.

63

 

.1. . i A
‘tl LQKUK’

Fig.11

(A) BB—JSB same as 10F to show K-bodies; (B) BB-YA

fusiform packet of paracrystalline fibers; (C) BB—JSE several
coiled, long para rystalline fibers; (D) BB—S4 imperfect hexa—
gons in face and edge View;

Fig. 11.

(E) BB-S4 same lower hexagon on
edge; (F) BB-S4 perfect hexagons in face and else View.

(3L1.

 

[-73.12

Fig. 12. (A) BB-YA paracrystalline fiber end he"

u ciagon; (B)
BB-HDS cluster of cubical crystals; (C) BB-EP highly irregular
composite of hexagons; (D) BE—HRG clusters of cubical crystuls;
(E) BE-KRG same cluster of crystals enlarged (F) Xanthi-JSB
distorted nucleus on day three after infection.

 

E X FOO phase F

X 400 phase
Fig.1?)

Fig. 13. (A) Kanthi-JSB cubical crystal; (B) Xanthi JSB
nucleus, nucleolus, chloroplasts; (C) Xanthi-JSB three hexa-
gons in subapical cell; (D) Xanthi—JSB same enlarged; (E)
Xanthi-JSB string of chloroplasts; (F) Xanthi—JS; same to
show X—body on left margin.

 

Ficj.14

(A) HG-JSB false plate inclusions; (B) NG-JSB false
plate inclusions; (C) WB-Sq large, rough hexagons; (D) flB-Sq

general View of same; (E) dB-Su cluster of smaller hevacons;

(F) WB-LL-Sq-u thin hexagons.

4.V.Q

Fig. 14.

67

 

C X 537 D x 125

 

E x 125 I F x 125
Fig. 15

Fig. 15. (A) ‘1'4'B—LL-511—b, general View of 14F; (B) BB-S-YA
dense X-bodies; (C) BS-B-YA close-up of single X-body; (D)
BB-E-YA to show X—body with nucleus in profile in third cell
from tip; (E) LIBE—l-YA paracrystalline fiber in focus; (F)
N32-l-YA same to show X-body in View.

cm
Co

 

Fig. l6

Fig. 16. (A) 1132-2-54 small angular crystal to extreme right
with nucleus in upper left and three particles of unknown
origin; (13) 1313-1-34 imperfect hexagon; (C) BB—E-YA hexagons On
edge; (D) 313-75—Y1X same cell showing; X-body; (13) BB-S—YA same
cell showing paracrystalline fiber. '

69

 
  

cg)! um”

 

C X 220:) F'cj'17
Fig. 17. (A) Bonny Best plasmodesmata connecting two adjacent
cells of a trichome; (3) enlargnent of same; (C) diagramatic
representation of (A) and (E) to show approximate location of
width measurements.

70

71

LIST OF HOST PLANTS MENTIONED
OR EMPLOYED IN THIS STUDY

Chenopodium amaranticolor (hybridum) L.

Datura stramonium L.
Datura tatula L.

Lycepersicon esculentum Mill. var. Bonny Best

Lyc0persipon esculentum Mill. var. N32
F(Spartan Red x Holmes P. I. 235673)

Metasequoia glyptostroboides Hu and Cheng

Nicotiana glutinosa L.

Nicotiana
Nicotiana
Nicotiana
Nicotiana
Nicotiana
Nicotiana

Nicotiana

Nicotiana
Nicotiana
Nicotiana

Petunia hybrida Vilm.

langsdorffii Weinm.

sylvestris Spegaz.

tabacum
tabacum
tabacum
tabacum
tabacum
tabacum

tabacum

tabacum

L

t"t"t"t"‘t"t"t"

var 0
var 0
var o
var 0
var 0
var o
var o

Solanum nudiflorum Dun.

Vicia faba L.

 

Bright Yellow
Conneticut

Havana 38

Samsun

White Burley
Wisconsin Havana 38
Xanthi-nc

DISCUSSION

Symptomatologl. It is well-known that growing con-
ditions prevailing before and during the expression of
symptoms in virus-inoculated plants greatly affect the
severity and rate of development of the symptoms (Bawden,
1964). The results of experiment 1 compared to experiment
2, clearly indicate the modifying effect of winter-growing
conditions versus those of late summer. Plants grown
during the late summer period, inoculated with virus,
showed symptoms one week earlier than similar plants during
the winter period and became more severe in a shorter period
of time. Bawden (1964) has noted this general reaction in
TMV-infected plants and others. The symptoms as expressed
on the susceptible host, Bonny Best tomato (BB) agree with
the above observations; however, the resistant tomato (N32)
did not follow the general pattern of symptom expression.
Strain 4 (from Alexander's tomato leaves) in N32 caused
slight symptoms as early as the seventh day after inocula-
tion under conditions of both experiment 1 and 2, but
reached the symptom peak on day 23 in experiment 1 and
day 14 in experiment 2. This was a rather slow develop-
ment of symptoms. HDS in N32 produced mild symptoms on
day l} and the symptoms were mild throughout the duration
of the experiment. YA in N32 did not produce symptoms
during either experiment, indicating the resistant nature

of the host. YA in BB danflxmmd rapidly under summer con-

72

73

(fitidm3(experiment 2), expressing symptoms by day 5
and reaching maximum expression by day 14. YA-BB may
reflect an environmental association that favors the de-
velopment of this strain during the summer period. The
least virulent host-strain combination, SP-BB, may re-
flect the same environmental association as YA—BB.
Approaching the final analysis, three factors
appeared. outstanding: (a) BB was susceptible in varying
degrees toward the six strains of TMV, (b) the resistant
nature of N32 can be penetrated by $4 and HDS, and (c)
YA and SP are the least virulent on BB. Finally, the
diagnostic value of the expressed symptoms should be
noted. Subtle differences may be detected between the
four severe strains in BB: HRG, JSB, HDS and SA; how-
ever, for practical reasons, these are not considered
diagnostically significant. On the other hand YA and
5P in BB are easily separable from the previous four
and from each other. A mild mosaic of the leaves was
characteristic of YA and 5P which served to separate
these two from the previous group of four. YA-BB dis-
plays yellowing and puckering of the leaflets with pro-
duction of fern-like leaves, whereas SP-BB showed mild
mosaic and cupping of the leaflets. Only two strains
produced symptoms in N32. HDS-N32 caused epinasty,
puckering and leaflet deformity, while Sh-NBZ yielded

decurved petioles, mild mosaic, puckering and fern-leaf.

74

Thus, these described symptoms are of some practical
value but should not be used entirely alone for diagnos-
tic purposes.

Inclusions associated,flith symptoms. Several
factors are generally important in the relationship of
inclusions in virus-infected plants: (a) not all plants
produce inclusions (Bawden, 1964), (b) not all tissues of
a plant produce inclusions (Kassanis, 1939), (c) not all
cells of a particular tissue produce inclusions (Chandra
and Hildebrandt, 1965), (d) inclusions may be present one
year and not in the following year or years (Kassanis and
Sheffield, 1941), (e) inclusions may be observed at about
the same time as the appearance of the first symptoms,
(f) inclusions are usually prominent when symptoms are
maximum (Bawden, 1964), (g) inclusions are usually assoc-
iated with symptom-bearing portions of affected plants
(Livingston and Duggar, 1934) and (h) age of the plant at
time of inoculation plays a role in the development of
inclusions as well as symptoms (Bawden, 1964). In spite
of the foregoing, the data in Tables 4 and 5 indicate
that certain tomato-TMV strain combinations produce pre-
dictable types and quantities of inclusions. Bonny Best,
the susceptible host, produced inclusions rather character-
istic of the infecting TMV strain. This agrees closely
with McWhorter (1965). In BB, the strains of TMV could be

identified if the types and quantities of inclusions were

75

noted together with the symptoms (Tables 4 and 5). It is
obvious from the reaction of N32 toward the six strains
of TMV, that the host must be also identified, even to
variety. In a personal communication McWhorter states,
"Besides the specificity of inclusions to virus strains,
the host effect is also important". The inclusions pro-
duced by HDS-N32 could be easily confused with those of
5P-BB or JSB-BB; therefore, plant variety and the ex-
pressed symptoms are important. YA and SF in BB produced
inclusions at a slower rate than the other four strains
and apparently closely followed the development of symp-
toms. The complete lack of any observed inclusions in
S4-N32 is indeed puzzling and no ready answer is avail-
able. Perhaps the failure lies in the observation tech-
nique, although it performed well with all other host-virus
combinations. Symptoms were recorded, no inclusions were
sited and no transmission was detected via local lesion
bioassay; hence, the virus may not have multiplied to an
appreciable extent, consequently produced no inclusions.
Cytology. Based upon an English translation of
Goldin's (1963) book, the only major publication on in-
clusions, a theoretical scheme has been presented in an
attempt to place order in the chaotic array of inclusions.
Goldin has recently surveyed inclusions produced in virus-
infected members of seven dicotyledonous families and two

monocotyledonous families. The following scheme has

76
developed from his observations on TMV found in the

Solanaceae which includes Nicotiana and Lycopersicon.

Stepwise develOpment of X-body Iwanowski
inclusion body; specific (hexagonal)
and characteristic for semispecific crystals;
different strains for strains nonspecific
of TMV and common to

all TMV strains
This scheme developed specifically from his observations
on the following strains of TMV: Yellow Strain G2,
Cyphomandra Strain, Petunia Strain, Green Strain, White
Strain, Enation Strain and Kazakhi Strain. None of these
strains have apparently been made available to researchers
in the U. S. A. I believe that based upon my observations
and data presented herein, Goldin has proposed a workable
theory that now should receive more attention since it fits
the information on TMV inclusions and the inclusion classi-
fication, Group BV, produced by McWhorter (1965).

Upon examination of six to eight-celled trichomes of
tomato leaflets, one fact became increasingly evident as
observations increased: there was a continuous stepwise
evolution of a particular inclusion. Since in a system-
ically-infected host the process of cellular infection
must be unidirectional from base cell to apical cell, then
the mature forms of inclusions are found in the basal cell,
the more immature forms found in the intermediate cells
and finally the more immature (early) are located in the

apical cell. Previous to the translation of Goldin (1963)

77

I thought this was an original observation; however, the
same observation is depicted in Fig. 13, p. 22 of Goldin's
book. He states that X-bodies gave rise to typical crys-
tals in the tomato trichome and unfortunately the design
of the present study did not yield information that would
support or refute this supposition.

In relation to the special substrains, A and B, of
strains JSB, S4 and YA (Table 6), substrain A infects BB
but not N32 whereas substrain B infects both BB and N32.
Since substrain Awmusnot able to infect the resistant host,
it theoretically may be considered incapable of producing
inclusions or produced fewer types of inclusions or fewer
numbers of inclusions. It was obvious from cytological
work that indeed far less numbers of inclusions are pro-
duced in BB. This reduction in quantity was apparently
true with substrain A of all three strains tested in BB.
As regards the situation with substrain B of JSB, S4 and
YA on N32, the resistant host, a different explanation is
in order. Substrain B of JSB and S4 were able to breach
the host barriers of resistance but in so doing produced
fewer types of inclusions and fewer numbers of inclusions.
On the other hand substrain B of YA in N32 produced un-
usually severe symptoms coincidental with the production
of varied types of inclusions and more numerous inclusions,
especially X-bodies and needles. Therefore, when host

resistance was successfully overcome, inclusions were pro-

78

duced in types and numbers comparable to the susceptible
host.

Upon examination of another host-virus system (Table
12), S4 in White Burley tobacco, a similar reaction was
obtained. The isolate WB-LL-S4-4 produced fewer inclusions
and fewer types of inclusions. Furthermore, inclusions of
a particular type were imperfectly formed in relation to
shape and thickness.

Repeated passage of a virus strain through a host plant
(Table 13) produced a marked increase in quantity of inclu-
sions but not types of inclusions. This measurement was
supported in three of the four host-virus combinations,
namely, BB#5-YA, BB#1-S4 and N32#l-YA. N32#2-S4, again,
did not produce an appreciable quantitative increase of
inclusions, thus supporting the observation that S4 was not
able to completly breakdown the resistance offered by N32.

The search for inclusions in or near the lesion site
of infected local lesion hosts has resulted in conflicting
statements (Weintraub 1964, Shalla 1964, M111€16 1962 and
Hayashi and Matsui 1963). The observations of the present
study presented the first report of inclusions in N. tabacum
var. Xanthi—nc; however, no inclusions were found in N.
glutinosa. Hence, in my opinion, in order to clarify this
controversy, TMV strains must be employed in both of the
selected standard local lesion hosts and maintained under
similar growing conditions.

All the cytological observations in this study reflect

79

upon the possible site of inclusion assembly. Considering
the experimental procedure herein, no evidence of inclusion
formation within the nucleus could be detected. All inclu-
sions were formed in the cytoplasm, oftentimes, near the
nucleus, thus agreeing with McWhorter (1965), Bald (1961)
and Hooker (1964). One reason for this rests upon the fact
that with the exception of the "nuclear strain 1C" (Woods
and Eck, 1948) and Goldin's Kazakhi strain (1963) all
strains of TMV produce cytoplasmic inclusions.

The evidence given in this study suggests several
possible hypotheses about the nature and role of inclusions.

First, formation of inclusions represent part of the
cell's defensive reaction against viral invasion. This
hypothesis is based upon (a) general absence of inclusions
in resistant hosts created by activation of the hypersensi-
tive reaction; (b) the clumping of cellular material
(sphaerosomes) around TMV constituents in the susceptible
host shows subtle differences in appearance; and (c) hexa-
gons are aggregations of TMV rods and such clustering prob-
ably lowers the dispersal of rods within the cell as well
as lowering probability of TMV rods (perhaps even viral
RNA) moving through the plasmodesmata to adjacent cells.
Second, the stepwise development from the sphaerosome --
TMV complex to form amorphous, paracrystalline and crystal-
line inclusions represents a mal-functioning of a normally
successful virus defense mechanism that was corrupted and

has been nurtured by man's selection for various yield char-

80

acteristics in his domesticated plants. This hypothesis is
one I consider more plausible than the others. Third, in—
clusions are simply condensation products of high viral con-
centration that increasingly preceipitate in hexagonal form
as viral titer increases. Fourth, inclusions are merely by-
products of viral infection and as sphaerosomes (perhaps
lysosomes?) coalesce with these by-products, inclusions
form.

The nature of ectodesmata is currently a battleground
among leading investigators in this country and Europe.
Personal communication with the three following researchers
provides an insight into the problem of ectodesmata (also
plasmodesmata) and virus entry into plants. Esau believes
that we must establish whether ectodesmata actually exist
as cytoplasmic structures and if they do, then the mode of
virus entry becomes an important problem for research.
Brants states that ectodesmata should more than likely occur
in tomato leaves and that ectodesmata have a protoplasmic
nature. Franke has demonstrated ectodesmata in epidermal
cells of tomato (trichomes were not mentioned), however,
strongly doubts that ectodesmata are plasmic strings con-
necting the protoplasts of epidermal cells with the cuticle.
If these strings are non-protoplasmic, filled with water
and water-soluble substances, then the virus particles must
traverse a path of non—living substance. Experiments by
Brants indicate that TMV possesses such resistance and

passes the non-living substance successfully. Personally,

81

since it is common practice to create a suspension of virus
particles by addition of distilled water (oftentimes sim-
ple phosphate buffers) and since in nature TMV is so easily
transmitted via non-protoplasmic substances, I believe that
the complete TMV rod can most certainly pass the supposed
non-protoplasmic nature of the plasmodesmata.

The tomato plant presents a cytological system that
lends itself easily to the study of both ectodesmata and
plasmodesmata since these channels are found in many types
of tomato cells, including the roots as illustrated by
Scott (1965). She further implied that it appears possible
that viruses may be swept along the plasmodesmata from cell
to cell like brush in a fast-moving current. The present
study confirmed the presence of plasmodesmata in the tri-
chomes of the susceptible host, Bonny Best tomato, and
width measurements show room for passage of virus particles,
but not inclusions. In summation, Behnke (1966) states
that there is sufficient space in the lumen of plasmodesmata
to enable viruses to migrate from one parenchyma cell to
the next; however, there is still no evidence that viruses
do migrate through plasmodesmata, nor do we know how
viruses move. This phase of the current study has at least
implicated the importance of the plasmodesmata in the cell-

to-cell distribution of tobacco mosaic virus.

SUMMARY

Cytopathology of six strains of TMV (HRG, JSB, HDS,
S4, YA and 5P) in a resistant (N32) and a susceptible
(Bonny Best) variety of Lycopersicon esculentum Mill. was
studied by the use of light and phase-contrast microscopy.
The gross foliar symptoms expressed by the virus-infected
hosts were periodically recorded and tabulated along with
simultaneous cytological observations for inclusions
formed within leaflet trichomal cells. Neither symptoms
nor inclusions considered separately gave any practical
diagnostic value, however, when symptoms and inclusions
were correlated, identification of infecting strains of
TMV was possible and consequently supplied diagnostic
significance.

Types and quantities of inclusions were found to be
characteristic of the TMV strain-host combinations and
were further modified by environmental growing conditions
and host-selected substrains. The typical hexagonal
(Iwanowski) crystal was produced by all six strains in
the susceptible host, thus this type of crystal was con-
sidered nonspecific. Four of the strains in the sus-
ceptible host produced X-bodies, thus, this type of in-
clusion was labelled semispecific. Specific inclusions
were produced by all six strains in the susceptible host
which included paracrystalline fibers, short needles,

cubical crystals, rectangular crystals, irregularly-

82

83

shaped crystals, Bald's "grey plates", banded crystals

and dendritic fibers. Inclusions produced in the re-
sistant host, occurring occasionally with only four strains,
gave somewhat similar results although inclusions pro-
duced were fewer in type and quantity. The resistant
barriers in tomato were breached as shown by inclusion
formation by certain natural, wild strains, host—selected
substrains and repeated passage of a virus strain through
the host.

The presence of inclusions in a local lesion host --
Nicotiana tabacum var. Xanthi-nc -- was clearly demon-
strated in the chlorotic halo zones around the lesion
sites. A closely related local lesion host -- Nicotiana
glutinosa -- gave absolutely no evidence of structures
that could be called inclusions.

Plasmodesmata were shown to be present between ad-
jacent cells in the trichome and subsequent measurements
indicated that virus particles could pass transverse septa
via the plasmodesmata, thus migrating from basal cell to
apical cell. In contrast, however, even the smallest in-
clusions could not pass through plasmodesmata due to the

size of the inclusions.

3‘

10.

ll.

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