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This is to certify that the
thesis entitled
ISOLATION AND CHARACTERIZATION OF TABACCO
RINGSPOT VIRUS FROM PEACH
presented by

CONSTANCE L. WI NANS

has been accepted towards fulfillment
of the requirements for

 

M.S. degree in Botany 5 Plant Pathology
MgoMessor
12-20-78

Date

0-7639

 

 

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ISOLATION AND CHARACTERIZATION
OF TOBACCO RINGSPOT VIRUS

FROM PEACH.

BY

CONSTANCE LARUE WINANS

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

Master of Science

Department of Botany and Plant Pathology

1978

ABSTRACT
ISOLATION AND CHARACTERIZATION OF TOBACCO RINGSPOT VIRUS FROM PEACH

BY

Constance L. Winans

A virus was isolated from dormant buds, blossoms, and leaves of
root suckers and top growth of declining Prunus persica. Trituration
of the tissue in 3% nicotine buffer and rub inoculation of cucumber
cotyledons induced local chlorotic lesions on cotyledons followed by
systemic chlorosis in primary leaves. Transfers were made from the

infected cucumbers to a host range of Chenopodium quinoa, Nicotiana

 

tabacum, Phaseolus vulgaris, and Vigna unguiculata. All inoculated

 

plants developed typical tobacco ringspot virus (TRSV) symptoms within

7 to 10 days. Sap from infected cucumbers reacted with TRSV but not to
tomato ringspot virus antiserum. The peach isolate of TRSV was purified
from cucumber and antiserum was produced which reacted specifically to
the peach TRSV isolate. Electron micrographs of purified virus showed
the particles to be isometeric, (ca. 28mm in diameter). Leaves of
etiolated healthy peach seedlings were mechanically inoculated with the
purified TRSV isolate. After 10 days triturated tissue from inoculated
peach seedlings induced TRSV symptoms in cucumber and expressed sap from
the cucumbers reacted with TRSV antiserum. This is the first complete

report of TRSV infection of Prunus persica.

 

TABLE OF CONTENTS

LIST OF TABLES... ...................... . ....... . ........ . ......... iii
LIST OF FIGURES...... ............. ............ ...... . ............ . iv
LIST OF ABBREVIATIONS.......... ........ . ..... .. ......... .......... v
MATERIALS AND METHODS......... ............ . ...... ... ....... . ...... 4

Field Study00000000000000000000000000000000000.000000000000000 4
Description and Identification of the Virus.......... ...... ... 6
Reintroduction of the Virus from Cucumber into Peach.......... 8

RESULTS .............. . .............. .............. ....... .. ....... 9
Field StUdy000000000000000000 ..... 00000000000000000000 ....... 0 9
Description and Identification of the Virus................... 11
Reintroduction of the Virus from Cucumber into Peach.......... 14

DISCUSSION ........... . ........ .. ........... . ..... . ..... .... ....... 18

REFERENCES 0000000000 0 ........ 000 ..... 0000000000000 ..... 00000000000 20

ii

Table

LIST OF TABLES

Page
Host range inoculated with tobacco ringspot virus
isolated from peach and symptom development....... .............. 12
Effect of inoculation method on infection in peach
seedlings using tobacco ringspot virus isolated
from peach................... ....... ......... .......... . ........ 17

iii

Figure

LIST OF FIGURES

Cucumbers showing local lesions on cotyledons and ,
systemic chlorosis on primary leaves 7 days following
inoculation with sap from peach infected with tobacco

ringspot Virus0000000000000000000000000.0000000000000000000

Reactions of Common cowpea plants to inoculation with

the isolate of tobacco ringspot virus from peach...........

The three componets of tobacco ringspot virus isolated

from peach, separated on a 10-40% sucrose density gradient..

Electron micrograph of a density-gradient preparation
of tobacco ringspot virus isolated from peach and

negatively stained with 0.5% ammonium molybdate ............

iv

Page

..... 10

..... 13

....15

..... l6

LIST OF ABBREVIATIONS

TRSV ........................................... Tobacco ringspot virus

TRSVpi .................................. .......Tobacco ringspot virus,
peach isolate

TomRSV........ ........ . ................. .......Tomato ringspot virus

INTRODUCTION

A declining peach (Prunus persica) orchard has been studied in
southwestern Michigan beginning in the spring of 1977. The decline
did not appear to be caused by either a fungus or a bacterium however
the nepovirus tobacco ringspot was consistently isolated from the
declining trees. This thesis is concerned with the isolation and ident-
ification of tobacco ringspot virus from declining peach trees.

Tobacco ringspot virus was suggested as the cause of a

disease of sweet cherry (Prunus avium L.) by Stace-Smith and Hansen (ll).

 

The symptoms of the disease were a'lighy-green zonal discoloration

and irregularity in leaf shape. TRSV was isolated from a sweet cherry
bud but the isolated virus was not returned to sweet cherry:

Three years later Uyemoto successfully isolated TRSV from sweet
(3. avium) and flowering (P. serrulata) cherries and returned it
to healthy Mazzard seedlings (14). The seedlings developed symptoms
corresponding to those described by Stace-Smith and Hansen (11) in
sweet cherry. After two dormancy periods TRSV was still transmissible
from the test plants. In subsequent isolation trials, TRSV was not
uniform in distribution in the tree nor could the virus be consistently
isolated from all infected trees.

Tobacco ringspot virus has also been reported as the causal agent
of a disease in grape (Vitis vinifera) (4). TRSV could easily be trans-
mitted from roots, leaf tissue and callus of infected grape vines, which

1

2
contrasts with the difficulty of isolation from Prunus. The symptoms
could in part be reproduced in healthy grape by mechanically inoculating

seedlings with the TRSV from grape. Xiphinema americanum, a TRSV vector,

 

was found in the soil around infected vines, and cucumber trap plants
became infected with TRSV.
Fulton, in 1962, reported positive evidence that tobacco ringspot

virus was transmitted to cucumber by Xiphinema americanum Cobb (3).

 

However attempts to transmitt TRSV or tomato ringspot virus (TomRSV)

into peach using 5. americanum have been inconclusive.

 

A great deal of work has been done with TomRSV in peach, the
causal agent of Prunus stem pitting. Stem pitting was described on

Prunus persica in detail by Barrat gt a; (2) in 1968. Mircetich gt al_(7)

 

reproduced stem pitting symptoms in healthy peach by grafting infected
material onto healthy peach seedlings. TomRSV was associated with

stem pitting symptoms in 1969 (10). Since that time TomRSV was isolated
from a Prunus stem pitted tree (6) and using cucumbers as bait plants,
TomRSV was found in soils around infected trees (9).

In a report by Smith and Stouffer (8) healthy peach seedlings were
mechanically inoculated with soil isolates and standard strains of TomRSV
and TRSV. The TomRSV inoculated seedlings developed pitting symptoms
while TRSV inoculated seedlings developed no symptoms. TRSV and TomRSV
were recovered from the mechanically inoculated peach seedlings up to
three weeks after inoculation. This was the only previously reported
case of TRSV in peach but as already pointed out, they did
not detect any symptoms of disease nor did they carry the work with
TRSV any further.

In this paper we report the isolation of virus from tissues of

3

peach buds, blossoms and leaves; identification of the virus according
to three critera; and reintroduction of the TRSV isolated from declining

peach trees into healthy peach seedlings.

MATERIAL AND METHODS

Field Study

 

In the hope of determining the cause of the decline of some 12
year old Glohaven peach trees in southwest Michigan, and attempt to
isolate a virus was undertaken. Young peach leaves were collected
early in the summers of 1977 and 1978 from the tips of suckers arising
from the base and from terminal and lateral shoots in the upper part of
the trees of apparently healthy and declining peach trees. Dormant
flower and leaf buds were also collected on April 12. 1978 from the
suckers and from the shoots of the declining trees. Flower petals were
collected at the full bloom stage of blossom bud development from the
declining trees.

The leaves were triturated in 3% nicotine buffer at pH 7.0 (5) and
the extract mechanically inoculated onto cotyledons of Cucumis sativus
(var. National Pickling) and onto young leaves of Chengpodium quinoa and

Nicotiana tabacumfvar. Wht. Burley). Dormant buds were tested in the same

 

manner after removal of the bud scales, as described by Davidson and
Rundans (2). Flower petals were ground in 0.5 M potassium phosphate
buffer, pH 7.0 and the sap rubbed onto cucumber cotyledons. The inocu-
lated plants were kept in the green house at 20 to 22 C. The virus was
maintained in cucumber and transferred every 10 to 14 days to maintain
a high titer.

The virus isolated from peach was tested serologically against
tobacco ringspot virus (TRSV) antiserum and tomato ringspot virus (TomRSV)

4

5
antiserum using double gel diffusion. The center wells were charged
wj4fl1the antisera and the peripheral wells contained either buffer or
extracts of cucumber which had been inoculated with TRSV peach isolate
(TRSVpi), TRSV (Gooding), TomRSV (Uyemoto), healthy peach sap or 3%
nicotine buffer alone.

Partially purified extracts from leaves of trees suspected of being
infected with TRSV were also tested serologically. Leaves were ground
in 3% nicotine buffer (pH 7.3) in a waring blender, :hi homogenate
expressed through cheese cloth and the sap placed the a freezer overnight.
Thawed extract was centrifuged on Sorvall RCZ-B with a no. 30 rotor at
10,000 rpm for 30 minutes. The supernatant fluid was dialized against
an ammonium sulfate solution(200g/1) for 24 hours, then centrifuged for
10 minutes. The resulting concentrated peach sap was tested in double
gel diffusion plates against TRSV and TomRSV antisera. Similarily pre-
pared extract from healthy peach leaves was used as a control.

Soil fibrous root samples were taken from the upper root zones of
apparently healthy and declining peach trees and assayed for nematodes.
The samples (about 2 Kg in wt) were divided and a portion of each sample

was examined for the presence of the nematode Xiphinema americanum. The

 

remaining portion of each sample was placed in sterilized eight inch clay
pots with 5 to 6 cucumber seeds planted per pot as trap plants. A11 clay
pots were kept on a greenhouse bench that was designed to prevent nema-
tode movement from pot to pot. Sap was expressed from cucumber plants
that developed virus-like symptoms and was applied to the cotyledons of
healthy cucumbers. Five days after the application of the sap from the
suspect plants, these second cucumber plants were tested serologically

for the presence of TRSV and TomRSV.

6
To complete the field study all trees in the area of the TRSV
infection were examined for Prunus stem pitting symptoms. This involved
removal of soil around the scion and rootstock union to about 1 meter
below the soil line and checking the bark for the thickening character-
istic of stem pitting. Following removal of outer bark the exposed

cambial layer was then examined for pitting and grooving symptoms.

Description and Identification of the YEEEE.

Sap from cucumber seedlings infected with the virus isolated from
declining peach trees was passed through a 0.45 u Millipore filter and
rub-inoculated onto the following herbaceous test plants: Cucumis
sativus, Chenopodium quinoa, Nicotiana tabacum, Phaseolus vulgaris,

Vigna unguiculata (S), and Z, unguiculata (SY). Plants rubbed with

 

buffer served as controls. The test plants were examined daily and the
sequence of symptom development was recorded. Return inoculations to
healthy cucumber were made from all test plants.

The peach isolate of the suspected TRSV was partially purified by
a modification of the procedure developed by Stace-Smith ES a1 (13).
Infected cucumber plants were harvested two weeks after inoculation and
the tissue (lKg/liter) was homogenized in a waring blender with 0.5 M
borate buffer at pH 6.8. The homogenate was expressed through cheese-
cloth and frozen overnight. The thawed sap was centrifuged in a Sorvall
RCZ-B at 9,000 g for 30 minutes. The supernatant fluid was dialyzed
against 5 volumes of ammonium sulfate solution (200g/1) for 24 hours and
centrifuged at 9,000 g for 10 minutes to remove precipitated plant
protein. The dialyzed solution was centrifuged in a Beckman Model L

ultracentrifuge with a no. 30 rotor at 28,000 rpm for 120 minutes.

7
The pellet was resuspended by shaking for 12 hours in 0.01% EDTA,
pH 7.0, and was then centrifuged in a no. 40 rotor 15,000 rpm for 10
minutes and the pellet discarded. The remaining solution was further
centrifuged in the no. 40 rotor at 38,000 rpm for 60 minutes. The final
resulting pellet was resuspended and clarified as before by another low
speed centrifugation.

The suspended partially purified virus was layered on 10-40%
sucrose density gradient, centrifuged in a SW 27 rotor at 23,000 rpm for
150 minutes. The separate bands were collected in a ISCO density-
gradient fractionator and infectivity for the bands was determined using
a local lesion assay on cowpea half-leaves. The bottom band was dialyzed
against 0.01 M tris acetate buffer, pH 7.0.

Antiserum to the bottom component was produced in a New Zealand
white rabbit by dntra-muscular injections of 0.5 ml of 0.8 mg/ml virus
emulsified in 0.5 m1 Freund's incomplete adjuvant at seven-day intervals
for 6 weeks. Antiserum was obtained starting two weeks after the initial
injection. Its titer was measured by determining the dilution end point
when it was tested against the antigenic peach virus at 0.252 mg/ml in
double diffusion plates.

Particle size and shape of the peach isolate virus was determined
by layering purified virus on formvar-coated 300-mesh grids, staining
with 0.5% ammonium molybdate and examining the grids in a Philips 300
electron microscope.

Biophysical characteristics of TRSV were determined by usual methods
with crude sap from cucumber. The dilution end point, thermal inactiva-
tion and longivity in_vi££g_were determined using infected cucumber sap

and cowpea half-leaves to assay for infectivity.

8

Reintroduction of the Virus from Cucumber into Peach

 

 

Thirty virus-free peach seedlings (var. Siberian C) with an
average height of 250 cm were placed in a light-proof chamber and
exposed to 10 minutes of light each hour to stimulate growth. The
inoculation of seedlings invloved three treatments. Inoculum for two
treatments was purified TRSVpi, undiluted and diluted 1:2 in phosphate
buffer and inoculum for the third treatment was expressed sap from
cucumbers infected with TRSVpi. For each of the first two treatments,
seven carborundum-dusted Siberian C seedlings were rubbed with virus
inoculum on etiolated leaves only. In thethird treatment seven
seedlings were removed from the soil, the whole seedling rub-inoculated
with TRSVpi infected sap and then returned to sterilized soil. Nine
seedlings were rubbed with buffer solution to serve as controls.

Ten days after inoculation two samples from each seedling were
collected, inoculated and non-inoculated new leaves. The peach tissue
was triturated in 3% nicotine buffer, rubbed onto cucumber and in four
days the cucumber sap was tested against TRSVpi antiserum. This assay

for infection was continued for 90 days.

RESULTS

I -

Field Study

 

Leaf extract from three trees induced local lesions on cucumber
cotyledons (see figure 1) followed by a systemic mottling of the primary

leaves. No symptoms developed on Chenopodium quinoa or on Nicotiana

 

tabacum when they were directly inoculated with peach extract. In one
tree extract from 23 out of 25 leaf samples induced TRSV symptoms when
rubbed on cucumber with isolation of the virus from suckers being equal
to isolation from top growth. For the remaining two trees extract from
10 out of 20 leaf samples were shown to be infected with TRSV. The
high infectivity of the peach isolate was easily maintained in cucumber.

In isolation trials during spring of 1978, the virus was also
obtained from 14 out of 30 bud samples and 7 out of 10 flower samples
from the main source tree. Virus was not recovered from the buds and
flowers of the other trees sampled.

Partially purified leaf extract from the virus-infected trees and
sap from cucumbers inoculated with the peach virus gave a homologous
reaction with antiserum to TRSV (Ramsdell) but no reaction with antiserum
to TomRSV (Uyemoto).

Soil from around the roots of declining peach trees was infested

with Xiphinema americanum. Cucumber trap plants grown in subsamples of

 

soil developed chlorotic local lesions within 14 to 24 days, followed by
systemic symptoms. Serological tests on expressed cucumber sap indicated

9

10

 

Figure 1. Cucumbers showing local lesions on cotyledons and
systemic chlorosis on primary leaves 7 days following inoculation
with sap from peach infected with tobacco ringspot virus.

11
that both TomRSV and TRSV were present. Soil from an adjacent peach

orchard without declining peach trees was not infested with §. americanum

 

nor did cucumbers planted in the soil develop virus symptoms.

Five peach trees exhibited stem pitting symptoms that is, develop-
ment of spongy bark at the ground line and pitting of the cambial layer.
This group of trees included the two which were minor TRSV sources
while the major source tree for TRSV did not, as of summer of 1978, have

stem pitting symptoms.

Description and Identification g: the Virus,

 

When a limited host range was inoculated with sap from cucumbers
infected with the virus from the ailing peach trees all genera developed
symptoms which are characteristic of TRSV (see Table 1). In cucumber,
chlorotic lesions were followed by necrosis of the cotyledons and systemic
mottling, stunt, and systemic necrosis develoyed on the primary leaves.
Chenopodium quinoa developed necrotic lesions but none of the expected
apical necrosis. Nicotiana tabacum developed yellow, local lesions with
necrotic rings. Leaves of N, tabacum which developed after inoculation
were symptomless but back inoculation to cucumber showed that virus was
present. Necrotic local lesions developed on several cultivars of

Phaseolus vulgaris. Common cOWpea developed necrotic local lesions with

 

yellow halos (see figure 2) in 4 days followed by apical necrosis. Cow-
pea strain (SY) did not exhibit symptoms and back inoculations to cucumber
were negative. Check plants rubbed with the buffer remained symptomless.
The purified preparation of the TRSVpi was infectious. It was
obtained with a yield of 60 to 70 mg virus per Kg tissue. Separation of

partially purified virus on a sucrose density gradient produced two

12

Table 1. Host range inoculated with tobacco ringspot virus
isolated from peach and symptom development

 

 

Host

Symptoms

 

Chenopodium quinoa

Cucumis sativus
(var. National pickling)

Nicotiana tabacum
(var. Burley)

Phaseolus vulgaris
(vars. Monroe, Coche, Scotia)

Vigna unguiculata
(S)

Vigna unguiculata
(SY)

Sparse necrotic lesions

Chlorotic local lesions, systemic
mottling, dwarfing, necrosis

Yellow, local lesions with some
necrotic ring formation

Local necrotic spots

Apical necrosis, necrotic local
lesions with yellow halo

No symptoms

 

13

 

Figure 2. Reactions of Common cowpea plants to inoculation
with the isolate of tobacco ringspot virus from peach

14
visible bands. Examination of the ultraviolet absorbance pattern from
the 1800 fractionator indicated three bands (see Figure 3). The bottom
band was the major component containing 90% of the infectivity, the
middle band with 10% and the top band was non-infective. The dialyzed
bottom component's 260nm/280nm ratio was 1.78.

Antiserum produced from intramuscular injections of the purfied
virus into a rabbit yielded a single band of precipitate to the antigenic
preparation from cucumber sap. There was no precipitate formed between
the antiserum and healthy cucumber extract. In double diffusion plates
the TRSVpi antiserum titers ranged from 1:64 to 1:256.

In electron micrographs of the purified virus, isolated from peach-
infected cucumber, the particles were polyhedral and 28 to 30nm in dia-
meter (see Figure 4).

For the properties of TRSV in cucumber extract the dilution end
point of the virus in crude sap was 10-4, the thermal inactivation
point was 64 to 66 C and the virus remained infective is crude sap for

10 days at 22 C.

Reintroduction of the Virus from Cucumber £3 Peach

 

 

Within ten days, leaf extract from each TRSVpi inoculation treat-
ment, but not buffer control, induced local lesions on cucumber cotyle-
dons. Expressed sap from infected cucumbers reacted with TRSVpi anti-
serum. In the first two treatments, if the etiolated growth was
infected the adjacent new growth was also infected and in the third
treatment virus was transmissible from the whole seedling. The virus

continued to be transmissible for 90 days following inoculation.

15

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Figure 4. Electron micrograph of a density-gradient prep—
paration of tobacco ringspot virus isolated from peach and
negatively stained with 0.5% ammonium molybdate.

17

Table 2. Effect of inoculation method on infection in peach
using tobacco ringspot virus isolated from peach

 

 

No. TRSVpi infected/
'Treatment No. inoculated

Mechanical inoculation of etiolated leaves
Purified TRSV(.82mg/ml), undiluted 6/7

Purified TRSV(.82mg/ml), diluted 1:2
in phosphate buffer 4/7

Mechanical inoculation of entire seedling

Expressed sap from cucumber infected
with TRSV 7/7

 

DISCUSSION

Tobacco ringspot virus was isolated from buds of Prunus persica

 

by Smith gt El (8), but the isolate was not characterized nor was the
isolation repeated. We have confirmed the presence of TRSV in peach

by isolating TRSV from a mature peach tree, by identifying the virus with
host range, serology, and electron microscopy, by purifying the virus
and producing antiserum, by mechanically inoculating peach seedlings
with TRSVpi and by isolating TRSV from these inoculated seedlings.

In cherry, sweet cherry and grape, TRSV was not consistantly
distributed in the plant nor could it be isolated consistantly from
season to season (4, 11, 14). Our findings demonstrated that the TRSV
distribution in peach was relatively equivalent between buds, blossoms,
and leaves, with leaves being the best source of virus for transmission
to herbacious indicators. The TRSV isolation from these peach trees was
repeated throughout two growing seasons with all trees infected the
first season still being TRSV transmissible the second season. This
is a sharp contrast to tomato ringspot virus which has only been sap
transmitted from peach to herbacious host on one occassion (7).

Smith 35 a1_mechanically inoculated healthy peach seedlings with
TRSV and could isolate TRSV from these seedlings up to three weeks after
inoculation. In our study TRSV was isolated from inoculated seedlings
for 90 days, up to the time when they were put in cold storage. The
inoculated seedlings did not develop consistant leaf symptoms upon

18

19
inoculation, as did TRSV when inoculated to cherry (14).

Our evidence demonstrates fairly conclusively the presence of TRSV
in declining peach trees but it is still unproven that TRSV is the cause
of the decline. The answer to the latter question must await demonstra-
tion of the development of the decline symptoms in TRSV-inoculated peach
seedlings. In other words, this virus must persist in the seedlings
over several dormant periods and cause a similar decline to that origi—
nally observed before the virus can be determined as the cause of decline.
However TRSV has already been shown to be pathogenic in other fruit
crops such as cherry, sweet cherry and grape. Therefore those working

with nepoviruses in peach probably should anticipate that TRSV is

indeed a cause of disease and a potential hazard.

10.

11.

12.

13.

14.

REFERENCES

BARRAT, J.G., S.M. MIRCETICH, and H.W. FOGLE. 1968. Stem pitting
of peach. Plant Dis. Reptr. 52:91-94.

DAVIDSON, T.R., and V. RUNDANS. 1972. Detection of necrotic ring-
spot and prune dwarf viruses by indexing dormant cherry buds on
herbaceous plants. Can. J. Plant Sci. 52:915-920.

FULTON, J.P. 1962. Transmission of tobacco ringspot virus by
Xiphinema americanum. PhytOpathology 57:535-537.

GILMER, R.M., J.K. UYEMOTO, and L.J. KELTS. 1970. A new grapevine
disease induced by tobacco ringspot virus. Phytopathology 62:1294-1302.

KIRKPATRICK, R.C., P.W. CHENEY, and R.C. LINDER. 1964. Mechanical
transmission of plum line pattern virus. Plant Dis. Reptr.48:616-6l8.

MIRCETICH, S.M. and E.L. CIVEROLO. 1972. Relationship between
stem pitting in peach and in other prunus species. Phytopathology
62:1294-1302.

MIRECTICH, S.M., H.W. FOGLE, and E.L. CIVEROLO. 1970. Peach stem
pitting: transmission and natural spread. Phytopathology 60:1329-1333.

SMITH, S.H., R.F. STOUFFER, and D.M. SOULEN. 1973. Induction of
stem pitting in peaches by mechanicalinoculation 'with tomato ringspot
virus. Phytopathology 63:1404-1406.

SMITH, S.H., and R.F. STOUFFER. 1970. Isolation of tomato and
tobacco ringspot viruses from soil around stem pitted Prunus trees.
Phytopathology 60:1017-1018.

SMITH, S.H., and J.A. TRAYLOR. 1969. Stem pitting of yellow bud
mosaic virus-infected peaches. Plant Dis. Reptr. 53:666-667.

STAGE-SMITH, R., and A.J. HANSEN. 1974. Occurance of TRSV in
sweet cherry. Can. J. Bot. 52:1647-1651.

STAGE-SMITH, R. 1970. Tobacco ringspot virus. no. 17 in Descript-
ions of plant virusesl Commonw. Mycol. Inst., Assoc. Appl. Biol.,
Kew, Surrey, England. (Unpaged).

STAGE-SMITH, R., M.E. REICHAMAN, and N.S. WRIGHT. 1965. Purification
and properties of TRSV and two RNA-deficient components. Virology
25:487-494.

UYEMOTO, ..K., M.F. WELSH, and E. WILLIAMS. 1977. Pathogenicity
of TRSV in cherry. Phytopathology 67:439-441.

20