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

DATE DUE DATE DUE DATE DUE ‘

W0\ A

     

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU ls An Affirmative Action/Equal Opponunlty Institution

;..-...-_-. ”EM—— _:—1"""'--- -

 

A STUDY OF THE RELATIONSHIP OF PRUNUS NECROTIC RINGSPOT
VIRUS, PRUNE DWARF VIRUS AND TOMATO RINGSPOT VIRUS TO SOUR

CHERRY DECLINE IN SOUTHWEST MICHIGAN

BY

Susan Mae Morrissey

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

1988

502220013

ABSTRACT
A STUDY OF THE RELATIONSHIP OF PRUNUS NECROTIC RINGSPOT

VIRUS, PRUNE DWARF VIRUS AND TOMATO RINGSPOT VIRUS TO SOUR
CHERRY DECLINE IN SOUTHWEST MICHIGAN

BY

Susan Mae Morrissey

The symptoms of stone fruit decline (SFD) in sour cherry
include reduced life expectancy and reduced fruit production.
The relationship of Prunus necrotic ringspot virus (PNRSV),
prune dwarf virus (PDV), and tomato ringspot virus (TmRSV) to
SFD was investigated in twelve commercial cherry orchards in
southwestern Michigan. At each orchard, leaf and cambial
tissue was tested for PNRSV, PDV and TmRSV by enzyme-linked
immunosorbent assay. Levels of infection by all three
viruses were significantly higher in the decline orchards
than in the non—decline orchards. Both TmRSV and PNRSV were
associated with poor tree vigor, trunk damage and high
orchard levels of tree death and replanting. Distribution of
TmRSV within sour cherry tissues was irregular; the cambial
tissues at or below the graft union and the roots were the
most reliable source of virus. Infection by TmRSV was not
well associated with pitting symptoms, vector nematodes or

weed hosts.

To the cherry trees and cherry tree growers of Michigan,
whose cooperation made this research possible

'iii

ACKNOWLEDGMENT S

I express my gratitude for the guidance and support of
Dr.Donald Ramsdell, my major professor.

I thank the other members of my guidance committee,
Drs.Clyde Burton and Alfred Saettler, for their support and
assistance in the preparation of this manuscript.

I am grateful for the technical assistance and
friendship of Jerri Gillett. I wish to thank the other
members of the laboratory for their friendship, and the many
students who assisted me in laboratory and field work.

Special thanks go to my husband, Jay Sandweiss, for his
encouragement and companionship, and to the members of my
family for their love and understanding.

Finally, my gratitude goes to my colleague and friend,
Nancy Schulte-Pason. Her support, suggestions and unrelenting
encouragement made the completion of this work possible.

iv

TABLE OF CONTENTS

LIST OF TABLES ......................................... vii

LIST OF FIGURES ...................................... ... ix

INTRODUCTION ........................................... 1

CHAPTER I LITERATURE REVIEW ......................... 6

Prunus Necrotic Ringspot Virus and Prune Dwarf Virus. 6

Transmission of PNRSV and PDV ..................... 8

Host Symptoms of PNRSV and PDV .................... 10

Effects of PNRSV and PDV on Growth and Yield ...... 13

Detection of PNRSV and PDV ........................ 16

Tomato Ringspot Virus ................................ 18

TmRSV and Prunus Stem Pitting ..................... 20

TmRSV and Apple Union Necrosis & Decline .......... 26

TmRSV and Prunus Brownline Disease ................ 30

Association of TmRSV with Nematodes and Weed Hosts 31

Detection of TmRSV ................................ 33
CHAPTER II PRUNUS NECROTIC RINGSPOT VIRUS AND PRUNE

DWARF VIRUS ............................... 41

MATERIALS AND METHODS ................................ 41

Field and Nursery Survey for PNRSV and PDV ........ 41

Plot Design .................................... 41

Sampling ....................................... 42

ELISA Procedures ............................... 42

Tree Evaluation ................................... 45

RESULTS .............................................. 46

Field Survey for PNRSV and PDV .................... 46

Prunus Necrotic Ringspot Virus ................. 46

Prune Dwarf Virus .............................. 46

Tree Evaluation ................................... 51

Decline vs. Non—Decline ........................ 51

Prunus Necrotic Ringspot Virus ................. 58

Nursery Survery for PNRSV and PDV ................. 64

DISCUSSION ........................................... 67

CHAPTER III TOMATO RINGSPOT VIRUS ..................... 77

MATERIALS AND METHODS... ............................. 77

Field and Nursery Survey for TmRSV ................ 77

Tree Evaluation .................................... 78

V

vi

Distribution of TmRSV within Cherry Trees ......... 78
TmRSV, Dandelion and Nematode Study ............... 79
Symptom Expression Study .......................... 80
ELISA Procedure for TmRSV ......................... 81
Assay Comparisons for TmRSV Detection ............. 83
Virus Preparation ............................... 83
Antiserum Production ............................ 85
Immunoglobulin Preparation ...................... 86
Conjugation of Immunoglobulin ................... 87
DAS-ELISA ....................................... 88
HADAS ELISA ..................................... 88
F(ab')2 ELISA ................................... 89
Dot-ELISA ....................................... 90
RESULTS ............................................... 91
Field and Nursery Survey for TmRSV ................. 91
Tree Evaluation ................................... 91
Distribution of TmRSV within Cherry Trees .......... 101
TmRSV, Dandelion and Nematode Study ................ 104
Symptom Expression Study ........................... 108
Assay Comparisons for TmRSV Detection .............. 108
DISCUSSION ........................................... 113
SUMMARY .............................................. 127
LITERATURE CITED ..................................... 130

LIST OF TABLES

Table 2.1 Incidence of trees ELISA-positive for Prunus
necrotic ringspot virus in six declining and six
non-declining sour cherry orchards in southwest Michigan..47

Table 2.2 Chi-square analysis of PNRSV incidence between
decline and non-decline orchards .......................... 48

Table 2.3 Incidence of trees ELISA-positive for prune
dwarf virus in six declining and six non-declining
sour cherry orchards in southwest Michigan ................ 50

Table 2.4 Chi-square analysis of PDV incidence between
decline and non-decline orchards .......................... 52

Table 2.5 Percentage of stunting and blindwood for each

of six decline and six non-decline orchards and mean
incidence of stunting and blindwood for the decline and
non-decline orchards overall .............................. 56

Table 2.6 Tree vigor ratings for six decline and six
non-decline orchards ...................................... 57

Table 2.7 Percent tree replant, stunting, blindwood,

and tree death for trees ELISA-positive for PNRSV and trees
ELISA-negative for PNRSV between and within decline and
non-decline orchards ...................................... 60

Table 2.8 Percent trunk damage on trees ELISA-positive
and ELISA negative for PNRSV between and within
decline and non-decline orchards .......................... 62

Table 2.9 Tree vigor ratings for trees ELISA-positive and
ELISA-negative for PNRSV between and within
decline and non-decline orchards .......................... 63

Table 2.10 One-year-old trees and rootstocks ELISA-positive
for PNRSV and PDV at four nurseries ....................... 65

Table 3.1 Incidence of trees ELISA-positive for tomato
ringspot virus in six declining and six non-declining sour
cherry orchards in southwest Michigan ..................... 92

Table 3.2 Chi-square analysis of TmRSV incidence between
decline and non-decline orchards ......................... 93

vii

viii

Table 3.3 Percent tree replant, stunting, blindwood, and
tree death for trees ELISA-positive for TmRSV and trees
ELISA negative for TmRSV between and within decline

and non-decline orchards .................................. 95

Table 3.4 Tree vigor ratings for trees ELISA-positive for
TmRSV and trees ELISA-negative for TmRSV between and within
decline and non-decline orchards ......................... 97

Table 3.5 Percent trunk damage on trees ELISA-positive
for TmRSV and trees ELISA-negative for TmRSV between
and within decline and non-decline orchards .............. 99

Table 3.6 Percent PNRSV and PDV ELISA—positives among

trees ELISA-positive for TmRSV and trees ELISA-negative

for TmRSV between and within decline and non-decline
orchards ................................................. 100

Table 3.7 TmRSV distribution in cambial tissues of
11-year-old naturally infected cherry trees .............. 102

Table 3.8 TmRSV distribution and detection'in 'Montmorency'
trees on 'Mahaleb' rootstock nine months post-inoculation
with TmRSV-positive wood chip ............................ 103

TABLE 3.9 TmRSV detection in cambial tissues from five
naturally infected cherry trees at intervals over a
68—day period ............................................ 105

TABLE 3.10 Mean vigor of cherry trees and mean percent
TmRSV-positive dandelions and nematode density surrounding
30 cherry trees in three decline and three non-decline
orchards ................................................. 107

Table 3.11 Expression of cambial pitting and grooving
symptoms vs. TmRSV infection in 'Montmorency' cherry ...... 109

Table 3.12 The relative sensitivity of different

immunoassay methods and enzyme labelled gamma-globulins

in detection of tomato ringspot virus in infected

cherry tissue ............................................. 110

Table 3.13 Relative sensitivity of F(ab')2 ELISA and
dot-Blot immunoassay in detecting TmRSV ................... 111

LIST OF FIGURES

Figure 1.1 Location of major sour cherry growing regions
in Michigan. (Michigan Agricultural Statistics, 1987) ...... 2

Figure 2.1 Percentage of trees ELISA—positive for PNRSV
by orchard (200 trees per orchard) showing mean values of
infection for decline and non-decline categories .......... 49

Figure 2.2 Percentage of trees ELISA-positive for PDV
by orchard (200 trees per orchard) showing mean values of
infection for decline and non-decline categories ......... 53

Figure 2.3 Percentage of trunk damage for 967 decline
and 1193 non-decline orchard trees ....................... 55

Figure 2.4 Percentage of PNRSV and PDV infection in 1200
trees by tree age ........................................ 59

Figure 2.5 Percentage of nursery tree samples
ELISA-positive for ilarvirus (either PNRSV or PDV) ....... 66

Figure 3.1 Percentage of trees ELISA-positive for TmRSV
by orchard (50 trees per orchard) showing mean values of
infection for decline and non-decline categories ......... 94

Figure 3.2 ELISA values for five 11 year-old previously

ELISA-positive (+) cherry trees tested five times over
a 68 day interval ........................................ 106

ix

INTRODUCTION

Sour or tart cherry (Prunus cerasus L.) is an important
commerical fruit crop in Michigan. Michigan has long been
the national leader in sour cherry production, with
approximately 75 percent of the nations' sour cherry crop
produced in the state. Nationally, the 300 million sour
cherry trees in production brought 60 million dollars to
growers in 1986. The 33,000 acres of sour cherry in Michigan
produced a 170 million pound crop in 1986, valued at 38
million dollars (Mich.Ag.Statistics, 1987). Other important
sour cherry producing states are New York, Pennsylvania,
Wisconsin and Utah.

In southwest Michigan the sour cherry growing region is
centered in Van Buren and Berrien counties. In west-central
Michigan and northwest Michigan, the production centers are
in Oceana and Grand Traverse/Leelanau Counties respectively
(Figure 1.1).

Ninety percent of all the sour cherry planted consists
of a 'Montmorency' cultivar budded onto a 'Mazzard' (P.avium
L.) or 'Mahaleb'(P. mahaleb L.) rootstock. Typically, trees
will begin to bear a crop six years after planting and be
productive for a minimum of 20 years, with some plantings
producing well into their 3rd decade.

In the past 20 to 30 years growers and extension agents
1

 

 

 

      
      

 

 

 

 

 

 

 

 

THOUSANDS or TART CHERRY TREES ,Z/fl Q

3 00 i
m :5: ..300 J
.m so - I50 ‘
:1 o - 49

 

 

 

 

 

 

 

 

 

Van Buren

 

 

Berrien

 

 

 

Figure 1.1 Location of major tart cherry growing

regions in Michigan (Michigan Agricultural Statistics,
1987)

in all cherry growing regions have reported a field and
production decline of mature cherry orchards that has
steadily worsened. Trees which produced for up to 35 years
in the past die or decline in yield and vigor in 20-25 years
or even less. In addition to decreased life expectancy,
growers report declining or inconsistent yields and decreased
tree vigor beginning at only 8 years of age. Trees
apparently healthy one year may suddenly decline in yield or
vigor and may die in the early spring of the following year.
Trees may break easily at the graft union, or develop bare,
thin vegetative growth. Trees usually begin to show signs of
decline as they come into their bearing years, and the
resulting decline in or loss of productivity can seriously
affect the growers ability to turn a profit on an often
substantial intial investment.

The diverse symptoms reported for decline suggest a
complex, multi-faceted cause. Researchers in Michigan have
approached the investigation of the problem in a
multi-disciplinary fashion. Present research in Michigan
includes investigation of a number of factors which may be
limiting tree growth and production. As possible causal
agents to the decline syndrome many biological factors are
being investigated. These include nematodes, bacterial
canker, mycoplasma caused X-disease, tree borers and the soil
pathogens Phytophthora, Pythium and Armillaria. Cultural and
environmental factors such as soil structure, harvesting

equipment damage, and frost and winter injury are also being

4
studied. Long term research includes the investigation of
genetic resistance to decline and development of accelerated
production strategies for sour cherry. Research is also in
progress on tree decline in peach and plum, which includes
varietal studies, biocontrol of Cytospora canker and
investigations of various cultural practices.

This research was undertaken as part of the
multi-disciplinary program. Its purpose was to investigate
the role and the relationship of several plant pathogenic
viruses to the declining sour cherry orchards in southwest
Michigan. '

Several viruses are known to infect and adversely
affect sour cherry. Two are classified in the Ilarvirus
group. These are Prunus necrotic ringspot (PNRSV) and prune
dwarf virus (PDV). They are pollen borne and have been
reported in Wisconsin, New York, Oregon, Washington and
Ontario (Keitt and Clayton, 1943; Mink, 1980). All have been
shown to cause biological stresses on sour cherry trees
resulting in reduced tree vigor and lowered fruit production.

Tomato ringspot virus (TmRSV), a nepovirus, has been
associated with poor production in a number of stone and pome
fruits. Prunus stem pitting (PSP) in peach and nectarine,
apple union necrosis and decline (AUND) in apple, and the
Prunus brownline (PBL) disorders of plum and prune have all

been found to be caused by TmRSV infection.

The purpose of this work was to determine the extent
to which PDV, PNRSV, and TmRSV exist in southwest Michigan

plantings of sour cherry; to investigate nurseries as the

5
source of these viruses; and evaluate the contribution of

these three viruses to sour cherry decline in Michigan.

I. LITERATURE REVIEW

WWW

Prunus necrotic ringspot virus (PNRSV), and prune dwarf
virus (PDV), are both included in the category known as
'ilar' (icose-hedral labile ring spotting) viruses. In the
present study, these two viruses were considered as possible
influencing agents in stone fruit decline. The effects that
PDV and PNRSV infection produce in sour cherry include yield
reduction, yield fluctuation and reduced tree vigor.

Serologically unrelated, PNRSV and PDV may occur
together in sour cherry and other Prunus species. Prunus
necrotic ringspot virus causes a necrotic leaf ringspot
disease in many species of Prunus. In cherry, infection by
PDV causes sour cherry yellows (SCY), the most economically
damaging disease of sour cherry in the Great Lakes fruit belt
(Jones, 1982).

Often PDV infection is accompanied by PNRSV infection.
This relationship, a crossover in some host symptomatology
between the two viruses, and the fact that both viruses may
be latent in cherry has caused some confusion in the
literature (Willison et al., 1957; Fulton, 1958 & 1960;
Berkeley, 1948; Pine, 1964). Prior to antibody-specific
testing the two viruses were sometimes inaccurately and
inefficiently identified.

Prunus necrotic ringspot virus virions are
quasi-spherical and range in diameter from 22 to 27 nm. The
protein coat consists of subunits with a molecular weight of

approximately 2.5 x 104 daltons (Barnett and Fulton, 1969).
6

7
Prunus necrotic ringspot virus is an RNA virus with the
nucleic acid accounting for about 16 percent of the total
particle weight. Loesch and Fulton (1975) found three
species of particles that have sedimentation coefficients of
728, 908 and 955. Alone, the bottom particles are slightly
infectious while the middle and top particles are
noninfective. Mixing the bottom and middle particles
markedly increases infectivity. Particle molecular weights
range from 5.2 to 7.3 x 106 daltons. The A260/280nm ratio
for PNRSV is 1.56. The virus is unstable in crude, undiluted
sap; infectivity is lost within a few minutes at room
temperature. In diluted sap maximum longevity is 9 to 18
hours. When the virus is in a stabilizing buffer, the
thermal inactivation point (10 minute exposure) ranges from
55 to 62° C, depending on the isolate of the virus
(Waterworth and Fulton, 1964; Fulton, 1970b).

Prune dwarf virions are also quasi-spherical, with a
diameter of approximately 19 to 35 nm. Halk and Fulton
(1978) found that PDV sedimented in sucrose density gradients
as five closely spaced zones of about 75, 81, 85, 98, 1135.
The composition of the virus protein coat has not been
determined. The virus contains 14 percent RNA. Only the
faster sedimenting particles are infective. The A260/A280nm
ratio is 1.56. The virus is unstable and loses one half its
infectivity within 30 seconds in undiluted cucumber sap. The
virus is stable in EDTA and salt solutions of 0.03 to 0.3 M.
Infectivity is lost quickly in solutions of divalent cations.

The thermal inactivation point of PDV (after 10 minutes

8

exposure with infectivity stabilized) is 45 to 540 C
depending on the isolate (Fulton, 1970a).

Prunus species other than P. cerasus are susceptible to
PDV and PNRSV. Some of the more important species are; P.
fasiculata (Torr.) Gray, P. dulcis (Mill) D.A.Webb, P.
persica L. (Batsch), P. mahaleb L., P. pumila L., P.
virginiana L., P. americana Marsh, P. bessyi Bailey, P.
cerasifera Ehrh., P. fruticosa Pall., P. serotina Ehrh., P.
pennsylvania L. and P. salicina L. Genera other than Prunus
that are susceptible to these two viruses are as follows:
Malus, Humulus, Rosa, and Rubus (Fridlund, 1970; Cochran and
Hutchins, 1941; Keitt et al., 1951; Kirkpatrick et al., 1962;

Bock, 1966).

I . . E EEK I EHESH

Both PDV and PNRSV are pollen-borne in cherry. Gilmer
and Way (1960) presented conclusive evidence for pollen
transmission when a healthy parent cherry inoculated with
diseased pollen produced diseased seeds. Extensive
pollination studies involving caged trees, bud removal, and
bee and hand pollination established pollen transmission of
PNRSV and PDV from tree to tree (George and Davidson, 1964;
Davidson, 1976). Prune dwarf virus was transmissible at
lower rates than PNRSV.

Cole et al., (1982) have found that PNRSV is carried on
the exterior of the pollen grain and they proposed that
pollen germination may not be required for virus

transmission. Other researchers demonstrated that mechanical

9
transmission of PDV and PNRSV does occur, both through
foliage contact and from pollen to the host plant during
fertilization (Hamilton et al., 1984; Swanson and Carpenter,
1961; Das and Milbrath, 1961).

Megahed and Moore (1967) have reported that up to 70
percent of the seed from Prunus species infected with PNRSV
will carry the virus, and 70 to 80 percent of the seed from
PDV infected trees will carry PDV. 'Mazzard' and 'Maheleb'
seed and seedlings were found to have both viruses at
appreciable levels and may have been the initial source of
infection in some orchards (Davidson and George, 1965; Gilmer
and Kamalsky, 1962). Mink and Aichele, (1984) tested the
levels of both viruses in the seed of eight Prunus species.
High levels of PDV and PNRSV were found in 'Mazzard' and
'Maheleb' seed, yet much lower levels were found in seedings
from the same seed lot. This suggested that seed infected
with these viruses often did not germinate or did not produce
acceptable seedlings.

Prune dwarf virus and PNRSV have no known insect
vector. This fact was established after extensive insect
tests involving 130,293 individuals including 30 aphid, 18
leafhopper and 12 other insects species (Swenson and
Milbrath, 1964). Nematode transmission has been reported

(Fulton, 1970a and 1970b).

10

WW

Researchers in North America and Ontario began to
report on a ringspot-causing disease in cherry and other
hosts around 1940. (Cochran and Hutchins, 1941; Keitt and
Clayton, 1939; Hildebrand, 1943; Willison et al.,1948).
Typical symptoms of ringspot on sour cherry include delayed
foliation and blooming and small (1mm) dark necrotic rings on
emerging leaves. The necrotic rings may fall out leaving a
shot hole or shredded leaf appearance. The affected leaves
are small but remain normal in color and are not dropped from
the tree. Symptoms are most evident 1 - 3 weeks post bloom
and can affect a few leaves, a branch or the entire tree.
Affected branches may show stunted shoot growth and terminal
necrosis. There is a wide range of symptom severity with
milder symptoms even disappearing in midsummer. Symptoms on
an individual tree may vary greatly year to year. The first
year is typically the most severe, and is considered the
"shock" or acute stage (Lewis, 1947; Berkeley and Willison,
1948). Lack of symptoms after the initial stage often masks
the incidence of the virus and the seriousness of the losses
it can produce (Berkeley & Willison, 1948; Cochran et al.,
1950). More ringspot disease is found with increased tree
age.

Early studies noted that similar ringspot symptoms
were occurring in widely separated locations and in related
species. A peach ringspot strain was transferred to almond,
myrobalan plum, and 'Mahaleb' and 'Mazzard' cherry, producing

typical ringspot leaf symptoms (Cochran and Hutchins, 1941).

11
Parker and Cochran (1951) reported on transmission of
ringspot inducing virus strains from New York, California and
Oregon as well as a strain from peach to 'Montmorency' and
'Mazzard' cherry. It was determined that all the strains
were actually one virus, eventually termed Prunus necrotic
ringspot virus (Berkeley et al., 1951).

In 1919, Stewart described the symptoms of a chlorosis
problem in sour cherry. Twenty years later Keitt and Clayton
(1939) reported a yellows condition occurring throughout
cherry orchards in Wisconsin. Other researchers had noted a
similar problem in New York and Oregon (Rasmussen, 1938;
Gloyer and Glasgow, 1928). Keitt and Clayton (1943),
established the viral nature of this yellows condition and
ruled out fungal or bacterial causes. Citing the conspicuous
yellow leaves produced, they suggested the name sour cherry
yellows.

Yellows symptoms develop in sour cherry 3 to 4 weeks
after petal fall, and progress from older to younger leaves.
Typical leaf symptoms include conspicuous chlorotic areas of
lighter than normal green, variable in size and shape.

Leaves are relatively large in size and either drop off or
change from paler green to bright yellow. Little or no
necrosis occurs. Major defoliation (as high as 50 percent)
occurs in late June or early July (Keitt and Clayton, 1939,
1943). The spur system of the tree may be reduced due to an
initial overproduction of flower buds, leaving a willowy type
of growth associated with long bare spaces on twigs which is

sometimes termed "blindwood" (Lewis, 1947). Sour cherry

12
yellows may also produce "shock" symptoms similar to, yet
milder than, those produced by PNRSV (Fulton, 1970).
Overall, however, the disease tends to occur gradually and
does not "kill the trees outright but...impairs their health
and fruitfulness and probably tends to shorten their lives"
(Keitt and Clayton, 1943).

Hildebrand (1943), after a nine year study, noted that
at least one strain of the SCY virus was in fact PDV, first
described as a disease of prune in 1936 (Thomas and
Hildebrand, 1936). Later researchers confirmed that PDV was
the SCY causal agent (Fulton, 1960; Gilmer and Brase, 1963;
George and Davidson, 1964).

Berkley (1948) and Cameron and Moore (1956) both noted
the high incidence of PNRSV infection on PDV infected trees,
although work by Fulton and Hamilton (1960) and Tremaine et
a1. (1964) clearly establishes the two viruses as
serologically unrelated. Fulton (1970) and Willison et al.
(1957) using serological and electron microscopy techniques
found no evidence that SCY requires more than one virus for
expression.

Symptom expression of sour cherry yellows and ringspot
was found to be particularly temperature dependent. Mills
(1946) concluded that the chief factor in determining SCY
symptom severity is temperature during the 30 day period
following petal fall. Keitt and Moore (1943) found from
greenhouse experiments that yellows symptoms developed freely
on potted 'Montmorency' that were in greenhouses which

fluctuated from 12 to 16° C night temperatures and 24 to 28 °

13
C day temperatures, with the best symptoms developing at 16°
C. At constant temperatures of 20° C or greater no symptoms
developed on yellows infected 'Montmorency'. Moore (1946)
observed that ringspot symptoms were expressed over the
entire 16 to 28° C temperature range, with more rapid symptom
development and necrosis occurring at the higher
temperatures. Best leaf symptom expression occurred at 20 to

24° c.

KW

The effects of both PDV and PNRSV on cherry growth and
yield are well documented. Keitt and Clayton considered
yellows an "unfruitful condition" as early as 1939 with their
experiments showing a range of 16.7 to 41.6 percent yield
reduction due to virus. Rassmussen and Cation (1942) showed
SCY diseased trees had only 47 percent of the production of
healthy trees while a 1946 report (Moore) noted yield
reduction due to sour cherry yellows could be as high as 62
percent. The largest yield reductions occurred several years
after infection (Lewis, 1951; Keitt and Clayton, 1943).
Fruit produced on yellows infected trees had the same sugar
content as fruit from healthy trees yet tended to be larger
in size (K103 and Parker, 1960; Keitt and Clayton, 1943).

Lewis (1951) indicated that ringspot infection can

cause up to 35 percent yield reduction, although it was noted
that in subsequent years after initial infection, the bearing
capacity of ringspot infected trees can approach that of

healthy trees. K103 and Parker (1960) obtained results in

14

agreement with Lewis. The yield reduction caused by ringspot
was considered important even at low levels, however, due to
the sheer prevalence of the disease (Cochran et al., 1950).

Cain and Parker (1951) and Way and Gilmer (1963)
noted that it was the decreased fruit set, possibly due to
decreased pollen viability, that accounted for decrease in
production for both yellows and ringspot infected trees.
Nyeki and Vertesy (1974) showed morphological and
physiological effects on pollen to be respOnsible for the
lower rates of fertilization and fruit set. Infected pollen
grains were smaller in size, variable in shape and had
decreased viability and incomplete pollen tube formation.

In a study of ringspot effects on growth, Milbrath
(1950) noted that virus-free trees were taller and sturdier
than those with ringspot infection. Parker et al.(1959) also
reported growth reduction for ringspot infected trees. Spur
production was reduced for trees infected with ringspot
(Keitt and Clayton, 1943) and yellows (Cain and Parker, 1951)
where infection was associated with terminal extension (Klos
and Parker, 1960). Cain contrasted this with a healthy
condition:

" The type of growth resulting when terminal
extension continues with a reduced formation of
spurs and lateral branches is commonly associated
with trees infected with cherry yellows. The
lateral buds on these shoots are largely flower buds
which set a few fruits the second year and are
completely barren thereafter. In contrast, on
vigorous healthy shoots one or more of the basal
buds are vegetative, forming spurs the second year

and a fruit cluster the third year. These spurs
then remain productive for several years."

15

Young tree growth was also affected by ringspot. A
comparison of ringspot-inoculated young trees and virus-free
trees showed that ringspot induced serious growth reduction.
The largest difference in growth was in numbers of branches
formed; the tree heights were approximately the same
(Millikan, 1955). Davidson and George (1965) inoculated
young trees with PDV, PNRSV, or both. Inoculations of trees
at 1,2,4, and 6, years of age retarded growth. The younger
the tree was at the time of inoculation the more severe was
the growth reduction. Prune dwarf virus inoculation caused
up to 60 percent growth reduction, with effects especially
severe 2 to 5 years after inoculation. Prunus necrotic
ringspot virus infected trees had less reduction in growth
but yield reduction was substantial, as high as 56 percent
less than healthy. Trees infected with both viruses had
reductions in yield and growth similar to those only
inoculated with PDV.

Prunus necrotic ringspot virus and PDV also affect the
growth and yield of peach trees. A study in California
showed up to 51 percent reductions in growth and up to 32
percent reductions in yield on peach trees infected by PNRSV
and PDV'(Pine, 1964). Work done in Bulgaria from 1975 to
1981 showed a decrease in leaf size, limb circumference,
fruit set and fruit size in peach trees infected with PNRSV
and PDV (Topchiiska, 1982). In addition, yields were reduced
from 37-99 percent and high rates of tree death were seen in
the infected trees. Estimates of the economic damage PNRSV

causes to peach crops in California suggest that an intial

16
infection level of four percent could, after fourteen years,
cost up $2200.00 per hectare (Heaton et al.,1981). Current
research in Pennsylvania, Georgia and West Virginia show
PNRSV to be prevalent in peach orchards and nurseries, and
possibly related to slow decline in peach (Jaffee et al.,

1986; Wells et al., 1986; Barrat and Otto, 1985).

W

Originally detection methods for PDV and PNRSV
exploited the characteristic reactions of different Prunus
species upon graft inoculation with infected bud tissue
(Berkeley, 1948; Moore and Keitt, 1949; Berkeley et al.,
1951; Hampton et al., 1966). For example, the necrotic
reaction of 'Shirofugen' flowering cherry (P.5errulata
Lindl.) when inoculated with tissue infected with PDV and/or
PNRSV can be used to determine the presence of and sometimes
distinguish between these viruses. Such bud inoculation is
an inefficent methodology, however, especially in detecting
early PNRSV or PDV infections. Hampton (1966) for example,
showed that there was up to a 63 percent chance of failing to
detect PDV using this method.

Later, more rapid virus detection was achieved by
mechanical inoculation of virus infected tissue to different
herbaceous hosts (Moore and Keitt, 1941; Fulton, 1957;
Gilmer, 1961). Host reactions could distinguish virus
presence in 10 to 14 days. In 1964, Waterworth and Fulton
developed a PDV and PNRSV detection protocol using a

combination of serology (micropreciptin or gel diffusion

17
tests), host range, thermal inactivation point and symptoms
on selected hosts.

With the advent of enzyme linked immunosorbent assay
(ELISA) PNRSV and PDV were detected and discriminated more
rapidly and conveniently than possible with host range,
grafting, or gel diffusion (Voller et al., 1976; Clark and
Adams, 1977). ELISA was more discriminating than precipitin
tests in distinguishing between serologically related strains
of these viruses (Barbara et al., 1978).

Experiments by Torrence and Dolby (1984) report that
detection of PNRSV and PDV by ELISA is possible throughout
the year, yet the ELISA test is most sensitive early in the
season. No evidence was found for uneven virus distribution
in trees, yet it was recommended that multiple samples should
be taken from any given tree to increase the chances of

detecting recent infections.

T | E' | fl'

Tomato ringspot virus (TmRSV) has often been associated
with systemic disease in perennial plants and has been
present as a limiting factor in stone fruit longevity and
production in particular. Tomato ringspot virus has been
shown to cause Prunus stem pitting (PSP), apple union
necrosis and decline (AUND), and Prunus brownline. All of
these disorders display some symptoms that are similar to
those noted in declining cherry orchards and this suggested
TmRSV as a part of the cause of stone fruit decline.

Tomato ringspot virus was described by Price in 1936.
Tomato ringspot virus is an RNA-containing virus with
isometric particles about 28 nm in diameter. A purified
preparation of TmRSV has three major classes of particles,
designated top (T), middle (M), and bottom (B). The
particles have sedimentation rates of 538, 1198 and 1268,
respectively. The T particles consist of protein subunits
and lack RNA. M and B particles contain the infective
nucleoprotein. Forty-one percent of the particle weight of M
and 44 percent of the particle weight of B is RNA
(Stace-Smith, 1984).

In tobacco sap, the virus loses infectivity after 10
minutes at 58°C, 2 days at 20°C, 3 weeks at 4°C, or several
months at -20°C. Dilution end point of sap from inoculated
tobacco leaves or cucumber cotyledons is 10'3, that of sap
from systemically infected tobacco is less than 10‘1

(Stace-Smith, 1970; 1984).
18

19

Tomato ringspot virus is both seed transmitted (Kahn,
1956; Mellor and Stace-Smith, 1963) and transmitted by adults
and three larval stages of the dagger nematode Xiphinema
americanum Cobb,1913. The nematodes can acquire the virus
within 1 hour and transmit it into healthy plants within 1
hour. Single nematodes can transmit the virus (Teliz et al.,
1967). Tomato ringspot virus is also transmitted by X.
rivesi and X. californicum (Forer and Stouffer, 1982; Forer
et al., 1984; Bonsi et al., 1984; Hoy et al., 1984). Insect
transmission has not been reported.

Cadman and Lister (1961) found through plant protection
and serological tests that peach yellow bud mosaic (PYBMV),
reported to cause serious diseases in almond, nectrine, plum
and sweet cherry, (Schlocker and Traylor, 1976) was
antigenically no different than TmRSV. Civerolo and
Miricetich (1972) found the stem pitting isolate of TmRSV was
related to, but distinct from PYBMV.

Tomato ringspot virus is similar in size, shape, physical
properties, host reaction, and geographic distribution to
other nematode-borne viruses. Tomato ringspot virus is
especially similar to tobacco ringspot (TRSV), yet the two
viruses are serologically unrelated (Tall et al., 1949).

Tomato ringspot virus has been reported in a wide variety
of plants, although mostly in ornamental and woody or
semiwoody species. Affected species include Vitus vinifera
and french hybrid grape (in combination with TRSV), geranium,
cymbidum orchid, strawberries, raspberries, blueberries,

passion fruit, gladious, and tobacco. (Uyemoto, 1975;

20
Christensen and Paludan, 1978; Goff and Corbett, 1977;
Convese, 1981; Stace-Smith, 1962; Johnson, 1972; Koening and

Fribourg, 1986; and Stace-Smith, 1970).

W

Wood or stem pitting was first observed in 1956
(Guengerich and Millikan) as being associated with a disorder
of Virginia crab apple. The disorder was found to be graft
transmittable to healthy tissues which suggested a viral
cause. The first description of pitting in peaches was by
Barrat in 1964. He noted ”the pitting was of the rough,
ridged type with fissures up to 1/8" deep, with occasional
wood enations." The first report of stem pitting in cherry
was by Lott in 1967, which he termed a "xylem abberation.”
Stouffer et al. (1969) gave a fuller description of pitting
in several species of Prunus including pitting on the
'Montmorency' cultivar on 'Mahaleb' rootstock. He described
a "prominent union, invaginated, with pockets of brown
necrotic tissues embedded. This suggests union
incompatibility and weak root systems, possibly inducing
winter injury."

Other researchers described pitting on other species of
cherry, peach, wild peach, apricot, nectarine, several plum
varieties, Italian prune and pear (Mircetich and Fogle, 1969;
Stouffer et al., 1969; Aldwinkle and Arneson, 1972; Dowler et
al., 1968; Mircetich, et al., 1968; Cameron, 1968; Millecan
et al., 1964). Pitting was noted in eastern Europe, Greece,

and in many areas of the United States (Zagaja and Millikan,

21
1972; Gavalas, 1970; Agrios, 1971). Early reports listed
TmRSV in the following states: New York, Delaware, Maryland,
Pennsylvania, West Virginia, New Jersey, North Carolina,
South Carolina and later, in states further west - Illinois,
Ohio, Michigan, and California (Aldwinkle and Arneson, 1972;
Miricetich and Fogle, 1976; Mircetich et al., 1968; Dowler et
al., 1968; Thornberry, 1970; Jones and Janeson, 1969; Smith
and Traylor, 1969). Thornberry (1970) suggested movement was
from the east by vector movement in wet soil.

Barrat et al. (1968) first named this stem pitting
disorder in peach as stem pitting of peach. Later, the
disorder was renamed to Prunus stem pitting (PSP). Barrat
described the complex of symptoms on peach. The foliar
symptoms were listed as girdling, brachytic terminal growth,
leaf curl, droop, chlorosis, and early leaf abscission. Also
mentioned were early fruit ripening and drop. The lower
trunk showed enlargement at or below ground level, with
extremely thickened bark (2 to 4 times thicker than normal)
and wood pitting. Some trees broke off at or below ground
level. Disorganized tissues were found in the woody
cylinder, and xylem elements were abnormal. The trees did
not appear to recover and it was noted there were no signs of
incompatibility since the union was normal.

In 1968, Lewis et al., documented the decline problem on
peach in Pennsylvania and Maryland. The foliar and pitting
symptoms were similar to those described by Barrat et
al.(1968). Pitting was found to extend to the roots and up

to 12 inches above the union on the scion, although sometimes

22

the pitting was limited to the rootstock. Lewis et al. found
pitting on over 90 percent of the declining peach trees but
not on vigorously growing trees. The symptoms were found in
trees as early as 3 months post-budding, but 3 to 5 years
post-budding was most common. Symptomatology led the
researchers to suggest that a combination of virus, winter
injury and drought was causing the problem. Also in 1968,
Mircetich et al., described a pitting disorder on peach,
apricot and nectarine in east coast regions. A detailed
description of the pitted wood showed that the secondary
tissues produced by the affected cambium were highly
disorganized. Phloem tissue was thickened and spongy, with
enlarged rays. The woody cylinder was disorganized, and
inclusions of large wide xylem rays and phloem islands were
noted. Lignification was incomplete, resulting in a weak
woody cylinder. The pitting was noted as affecting trees
from one to 20 years in age, and the point was made that some
of the trees were affected well after maturity had been
reached (10 to 15 yrs). This indicated that the causative
factor must be there or needed to have certain environmental
factors present for expression. Nematode involvement was
suggested. Mircetich and Fogle (1969), after noting symptoms
on several fruit species supported the theory that PSP was
more than just a graft union incompatibility problem.

In transmission studies in the early 1970's, Mircetich et
al.(1970) found the disorder to be graft-transmissible and
noted that the causal agent may be present before

"macroscopic" stem pitting develops. This was supported by

23
the fact that some healthy trees inoculated with symptomless
tree roots developed pitting. No correlation was found
between leaf symptoms and stem pitting. In further work on
the peach stem pitting problem in Pennsylvania, Stouffer and
Lewis, (1969) reported that there seemed to be no association
between the pitting and PNRSV infection. This was later
verified by Mircetich et al.(1971).

Research in 1971 (Mircetich et al.) and 1972 (Mircetich
and Civerolo) showed that the PSP agent was graft transmiss-
ible across Prunus species, indicating the same agent was
involved in all pitted species. Tomato ringspot virus was
recovered from plum, cherry and apricot seedlings inoculated
with PSP root chips from naturally infected peach and apricot
trees. Root chips of affected trees grafted into healthy
trees were found to be the most effective tranmission method.
Symptoms were reproduced in five months, including pitting
and grooving in the woody cylinder, cambial and bark
necrosis, trunk enlargement and disorganization of xylem.
Researchers suggested at that time that the stem pitting
agent was not uniformly distributed, citing graft
transmittable viruses of fruit trees (green ring mottle and
apple mosaic virus) that were known to have localized
distribution within the plant. Soulen et al., (1972) also
found the PSP agent to be graft transmissible, and found high
rates of graft transmission were possible.

Mircetich showed in 1972 that the spread of the disorder
was from tree to tree in an apricot to sour cherry

transmission study. A soil agent was suspected to be

24

involved and a virus was recovered that was related to TmRSV
and peach yellow bud mosaic virus (PYBMV). Stouffer et al.
(1972) soon after proved PSP was transmitted through the
soil.

Other workers in the same year (Civerolo and Miricetich,
1972) serologically compared the stem pitting (SP) isolate
and PYBMV isolate and found them to be related, yet distinct.
No correlation was found between the virus associated with
the pitting symptom or the experimentally infested trees, yet
seedlings planted in infected peach sites were showing
symptoms of stem pitting within 5 months. They attributed
the lack of association between TmRSV and stem pitting, to
the virus not being present in all trees, or to inefficient
detection methods.

In 1970, Smith and Stouffer had used cucumber baiting to
show that tomato and tobacco ringspot viruses were present in
the soil around pitted trees, including sour and sweet
(Prunus avium L.) cherry. In 1973, Smith et al.,
mechanically inoculated TmRSV into peach and reproduced PSP
symptoms - thick bark, elongated pits and grooves, reduced
terminal growth and chlorotic, or red/purple leaves. No
pitting was found to be associated with TRSV or PNRSV. This
was the first concrete experimental evidence positively
linking TmRSV and the PSP problem.

Other workers associated stem pitting and grooving with
the presence of the dagger nematode, Xiphinema spp., known to
be a virus vector (Bloom et al., 1972). Stouffer, working

with chemical (fumigant) control, experimentally confirmed

25
the soil/nematode nature of PSP spread and showed that
control of Xiphinema reduced stem pitting and increased
yields (Stouffer et al., 1975; Stouffer and Mowery, 1980).

By 1977, Mircetich et al., working in California, found a
consistent association of stem pitting and decline in several
Prunus species. Variations were found in the nature and
severity of rootstock pitting, with 'Mazzard' and 'Napoleon'
showing little or no pitting. 'Mahaleb' rootstock exhibited
short and relatively shallow pits without cambial necrosis
and 'Bing' in contrast, showed extensive, severe pitting. No
stem pitting was found on trees with severe yellow bud
mosaic, suggesting that only specific viral strains cause the
stem pitting phenomenon.

Extensive work involving over 22,000 sweet cherry trees
in Washington state revealed that reduced vigor was
associated with severe pitting only for trees with
'Montmorency' interstocks (Mink and Howell, 1980). Although
it was noted that sweet cherry orchards planted with
certified virus-tested trees showed less stem pitting, there
was little consistent overall association of thick bark and
unthrifty tree growth with pitting. Stem pitting was noticed
on trees that were otherwise normal. In some but not all
cases, the symptomology recorded for sweet cherry was similar
to stem pitting recorded in peach. Graft transmission of the
sweet cherry pitting agent was not successful.

Other work with sweet cherry in Washington involving an
attempt to transmit a stem pitting agent, was unsuccessful as

well (AlMusa et al., 1980). There were Xiphinema spp.

26
present, yet TmRSV could not be isolated from trees, bait
plants, or weeds. No pitting developed on peach or cherry
seedlings that were grown in infested soil or experimentally
inoculated. Again, although it was noted that pitting was
present, it was not associated with an overall decline;
normal growth was observed.

Lister et al., (1980), in work with detection of TmRSV in
apple and peach found that the virus was somewhat localized
at the base of the trees, yet led to impairment of
translocation resulting in dwarfing and death of the canopy.

In 1984 Barrat et al., noted that PSP remains a serious
problem in peach orchards in West Virginia and Pennsylvania.
Surveys of peach trees and dandelion showed that TmRSV is
widespread throughout these areas.

Prunus stem pitting was found to be caused by TmRSV
infection via transmission by Xiphinema spp., yet the strengh
of the connection between symptomatology, virus, and nematode
varies with Prunus species. Even though programs have been
implemented to control virus dissemination through infected
stock, reservoir plants and nematodes have established the
disease in some areas. Under these conditions complete
control is not feasible, and hope for the future in
eradicating PSP lies in TmRSV resistant germ plasm or cross

protection with mild strains of TmRSV.

T ESM I E 1 fl . H . E 1'
In 1976, Stouffer and Uyemoto reported that girdling-type

symptoms had been occurring in northeastern United States

27
apple orchards. Symptoms included leaves that were sparse,
small and chlorotic. Terminal growth was reduced and twigs
exhibited a bunchy type growth. Fruits were small and highly
colored. Many trees had cracked or broken off at the graft
union. Tissue at the graft union was abnormally thick and
spongy and a distinct scion/stock necrotic line was often
present. The 'Delicious' trees on Malling-Merton 106
rootstocks were found to be the most severely affected.
Initially, the disease was reported from Pennsylvania and
Virginia, but later, reports came from all apple growing
regions of the eastern United States.

Researchers isolated TmRSV from apple rootstocks from
Pennsylvania, Virginia and New York (Stouffer and Uyemoto,
1976; Stouffer et al., 1977). A common etiology for PSP and
the apple disorder was shown when 100 percent of 37 peach
trees inoculated with wood chips from the affected apple
trees developed PSP (Stouffer and Powell, 1979). Later
reports showed TmRSV had been isolated from trees with the
apple disorder in the western United States as well (Parish
and Converse, 1981).

Further work in the eastern United States (Rosenberger et
al., 1983) found the disease (now termed AUND) was prevalent
in orchards in the Hudson river valley of New York. Although
the virus was found to be unevenly distributed in the trees,
in one experiment, TmRSV was found in 89 percent of trees
with graft union symptoms. It was theorized that the
rootstocks were susceptible or tolerant to TmRSV and the

scions were sensitive or hypersensitive, resulting in the

28
graft union symptoms. Rosenberger et al., (1983) observed
that the stressed trees (heavily fruited, waterlogged, bark
injured) were more susceptible to TmRSV infection and /or
developed AUND symptoms quicker than non-stressed trees. It
was noted that the combination of high levels of Xiphinema
spp. and high numbers of TmRSV infected dandelions was
favorable for the spread of the virus from dandelions to
trees.

The researchers theorized that initially the virus
entered the orchard via dandelion seed. If the nursery had
been the primary source of TmRSV, a higher incidence of the
problem should have been seen then, not in the eastern Hudson
Valley, but in western New York where more of the susceptible
MM106 rootstocks had been planted. Reasons suggested for the
differences noted in eastern and western New York included
the fact that there were different strains of virus/vector,
different percentages of infected weeds, and differences in
environmental conditions.

As observed with other TmRSV fruit tree infections, it
was noted that apple tree infection seemed to depend on more
than just susceptible plants, virus presence and nematode
vectors. For example, very little PSP was found in New York
and often AUND symptoms were found next to an unaffected
peach. Yet there was Stanley constriction (also caused by
TmRSV) of plum in New York, in areas where there was little
or no AUND and PSP.

Histological investigation showed that the disturbed

union was not a hypersensitive reaction in apple, but was

29
associated with increased production of ray and axial
parenchyma and decreased vessel and fiber cells. Apparently
the union disorder occurs after the plant switches from
vegetative to reproductive phase with a loss in ability of
cambium to differentiate normally (Tuttle & Gottleib, 1982).

In 1985 Rosenberger et al., continued research on AUND in
the Hudson river valley of New York. The AUND syndrome was
noted on seven scion cultivars propagated on MM106 rootstocks
although different symptoms were noted for the different
cultivars. In four orchards, dandelions and trees were
tested for TmRSV and the soil was tested for Xiphinema spp.
In the trees there was a high correlation of AUND symptoms
with virus infection. The combined tree, dandelion and
nematode results supported the theory that the TmRSV came in
on dandelion seed and was spread by nematodes from dandelion
to tree.

Studies reported in 1985 (Rosenberger et al.)
experimentally provided evidence that AUND can be reproduced
by inoculating trees with TmRSVeinfected bark, although it
was indicated that particular strains of TmRSV may not be
capable of causing AUND.

Control of AUND has been partially achieved through
removal of MM106 rootstocks and removal of weed hosts. It
has been postulated, however, that the totality of the AUND
problem has yet to be realized. In the years 1965-1975, 40
percent of the apples planted in New York were on susceptible
MM106 rootstocks. It has been speculated that it is only a

matter of time before many of these trees become infected

30
with TmRSV and develop AUND, but the numbers of trees
involved preclude wholesale tree removal as a preventative

measure (Bitterlin et al., 1984).

TBS}! 12 E J' E'

In the late 1950's, researchers noted a constriction
problem in 'Stanley' plum (Brase and Parker, 1955;
Kirkpatrick et al., 1958). Symptomatology led them to
suspect virus and/or winter injury as the cause. Later work
by Parker and Gilmer (1976) supported the winter injury
theory, suggesting that a mild union incompatibility
predisposed the trees to injury in the first place.

A similar hypersensitive necrotic reaction at the union
of prune and peach was noted by Mircetich and Hoy (1981) who
named the problem prune brownline disease. They detected
TmRSV in rootstocks of both species and noted that detection
was high from all tissues early in the onset of the disease
and lower when the trees were dormant or dying. Serological
tests indicated that the TmRSV strain was related, but not
identical to, the strain causing PSP, yet was the same as the
TmRSV-PYBMV strain.

Comparative studies by Hoy et al.(1984), showed that
inoculation of prune with similar isolates - PYBMV, PSP,
cherry leaf mottle and California PSP - did lead to union
infection. Hoy et al. also noted that Xiphinema califbrnicum
could transmit the virus and that one plum rootstock,
'Marianna' 2624, did not become infected with TmRSV.

Noting the similarity of 'Stanley' constriction and

31
decline and prune brownline, Cummins and Gonsalves (1986)
suggested the use of the name Prunus Brownline (PBL). As
with the AUND syndrome, the researchers thought that PBL was
caused by interaction of TmRSV infection, presence of an
efficient vector, and use of a susceptible but tolerant
rootstock with a resistant scion cultivar. They presented a
scenario of original nursery infection leading to infected

trees becomming widely dispersed in the orchard industry.

E . l' E T ESM 'II N | I i H i H |

The PYBMV strain of TmRSV, was first associated with
Xiphinema spp. in 1959 (Breece and Hart). Nearly ten years
later PYBMV was transmitted experimentally to apple and peach
by Xiphinema americanum (Teliz et al., 1967). This was an
important finding, because Xiphinema has worldwide
distribution and virus vector capability (Lownsberry and
Maggenti, 1963).

Braun et al.(1966), considered nematode populations in
relation to apple vigor. They found a positive relationship
of Xiphinema and another nematode, Pratylenchus, to tree
vigor, although there was no investigation of virus on the
site. Pratylenchus had been previously associated with
serious damage to cherry and other tree fruits in New York
and Michigan (Parker and Mai, 1956; Knierim, 1964; Davidson,
1957; Mai and Parker, 1967). Control of Pratylenchus
resulted in a decrease in a sour cherry decline associated
with 'Mazzard' and 'Mahaleb' rootstock (Edgerton and Parker,
1958).

32

Two Xiphinema species, Xiphinema rivesi in Pennsylvania
and Xiphinema californicum in California, were also shown to
transmit TmRSV to stone and pome fruit trees with the same
efficiency as x; americanum (Forer and Stouffer, 1981; Forer
et al., 1984; Bonsi, 1984; Boy et.al., 1984).

In 1981, Powell et al., noted that dandelion was
associated with persistence of TmRSV-caused diseases. Later,
seed transmission of TmRSV was established in dandelion,
proving that dandelions can be a source ongmRSV inoculum and
a major factor in orchard and inter—orchard movement of the
virus (Murant and Lister, 1967; Lister and Murant, 1967;
Mountain et al., 1983). This is especially true since the
nematode movement is slow and nematodes can only retain TmRSV
for a short time. Weed seeds are one initial source of virus
and infected weeds are the source for nematodes reacquiring
the virus (Murant and Lister, 1967). This can explain why
some viruses like TmRSV are widely distributed in a scattered
and localized way even though nematodes can not move over
much distance.

Research has shown that dandelion, chickweed, henbit,
creeping sorrel, common plaintain, strawberry, sorrel and red
clover, can all be infected with TmRSV (Miller,1980; Powell
et al., 1982). The most frequently infected, ubiquitous weed
is dandelion. Work by Powell et al. (1984a) established
criteria for a weed to be an important virus reservoir and
showed that only dandelion fulfills these criteria.

Powell et al. (1984b) compared the population of

Xiphinema and the presence of dandelions, with the incidence

33
of TmRSV and PSP. More dandelions infected with TmRSV were
found in orchards with PSP than orchards without PSP or
non-orchard sites. This work supported the hypothesis that
TmRSV originates in the nursery or in dandelion seed and is
often well established in weed hosts.

Current data indicate, however, that dandelion density
and dagger nematode density are not the limiting factors to
TmRSV spread or tree infection. Tomato ringspot virus
initially comes in on nursery stock, then low tillage
facilitates virus movement. As weed infection increases, the
probability of tree infection also increases, on both an

orchard and on a regional level.

Detention_of_TmBSY

Initally, researchers detected TmRSV in plant tissues by
combining a number of detection methods, including host range
and serology. In 1953, Yarwood first mechanically
transmitted the PYBMV strain of TmRSV from peach tissue to an
herbaceous host plant. In the years following, various
herbacous hosts were tested and the symptoms that TmRSV
induced were recorded. Typically, tissue from infected trees
was sap inoculated to selected herbaceous hosts, and then
this infected tissue was tested for presence of virus using
serological methods such as micropreciptin and gel diffusion
(vanSlogteren, 1955; Crowle, 1961). Slower methods of
detection were also used, such as inoculations of infected
fruit tree tissue into a healthy fruit tree, which was then

"read" for symptoms after 3 to 4 months (Soulen et al.,

34
1972). As previously noted, Mircetich et al.(1971) found
root chips were the most efficient tissue to use for this
type of test.

In the 1970's the use of enzyme linked immunosorbent
assay (ELISA), originally developed for use in the medical
field, was applied to plant virus detection (Engvall and
Perlman, 1971; Voller et al., 1976; Clark and Adams, 1977).
ELISA has since been valued as the method of choice for
efficiently detecting plant viruses, often at below the
concentrations detected by many other standard procedures.
For detection of TmRSV in woody hosts in particular, ELISA
simplified the investigation of etiology and epidemiology of
TmRSV induced diseases like PSP and AUND (Lister et al.,
1980).

Converse (1978), working with naturally infected
raspberry tissue, first used ELISA to detect TmRSV. He found
that ELISA detected partially purified TmRSV at
concentrations as low as 41 ng/ml. In 1981,-Hoy et al.,
compared the abilities of ELISA, radio-immunosorbent assay
(RISA) and mechanical transmission (MT) to detect TmRSV in
Prunus species. When Prunus species were inoculated with a
stem pitting strain of TmRSV, the virus was detected in 95
percent of the trees by ELISA and only in 13 percent by MT.
Tests of naturally infected prune found MT detected TmRSV in
12 percent of the trees tested, while ELISA detected the
virus in 51 percent of the trees, and RISA detected 97
percent TmRSV infection.

ELISA.can be used to detect and assay both the virus

35
(direct method) and the antibody prepared against the virus
(indirect method) (Voller, 1977). Both direct and indirect
ELISA techniques can detect TmRSV, with indirect techniques
slightly more sensitive (Powell, 1984). Indirect ELISA has
less viral strain specificity (Van Regemortel and Burckard,
1980; Koenig, 1981) and the benefit of using one enzyme
conjugate for detection of more than one virus (Lommel et
al., 1982).

A commonly used direct ELISA is the double antibody
sandwich (DAS) form of ELISA which uses immunoglobulin
purified from an antiserum prepared in an animal to trap
viral antigen. The antigen is then detected though use of an
enzyme-labeled immunoglobulin which attaches to the the bound
antigen and is then reacted with a substrate. The substrate
initiates a color reaction upon degradation of the enzyme and

this reaction is measued spectrophotometrically.

F'(ab)2-based indirect ELISA uses pepsin derived

F'(ab)2 fragments of immunoglobulin from specific antiserum

to trap virus. Trapped virus is then reacted with intact

immunoglobulin. Enzyme-labeled Protein A that reacts
specifically with the FC portion of the intact immunoglobulin

is added and then reacted with a substrate to give a
characteristic measurable color reaction (Clark, 1981;
Barbara and Clark, 1982).

Another indirect ELISA, heterologous antibody double
antibody sandwich (HADAS) uses immunoglobulin prepared in two

animals for virus detection. The second immunoglobulin in

36
the 'sandwich' is from a different animal species than the
species used to prepare the first immunoglobulin. This
second immunoglobulin is then reacted with an enzyme labeled
anti-immunoglobulin and the color reaction is measured
spectrophotometrically upon the addition of substrate
(Bar-Joesph, 1982).

Dot-Blot immunoassay or dot-ELISA is similar to the DAS
form of ELISA except this system uses a nitrocellulose
membrane (NCM) to bind either the antigens or the virus
specific antibodies. The NCM has a better binding capacity
than the polystyrene binding surface used in the other forms
of ELISA (Banttari and Smith, 1984; Banttari and Goodwin,
1985).

It is a concern in the use of ELISA that not all antisera
can detect all isolates of a virus. For TmRSV in
particular, serological differences among isolates could
complicate diagnosis (Bitterlin and Gonsalves, 1988; Lister
et al., 1980). Bitterlin and Gonsalves tested 30 isolates of
TmRSV and found they fell into four serogroups. Antiserum to
one isolate, PYBMV, detected 30/33 isolates of TmRSV when
using direct ELISA, and 32/33 when an indirect ELISA
technique using partially purified antigen was employed.
Antisera to three other TmRSV strains were also tested.
Results indicate that proper antisera must be used to assure
efficient detection of isolates which belong to various
serogroups. Bitterlin and Gonsalves suggest that antibodies
to both the PYBMV and the 'Chickadee' isolate be used to

assure TmRSV detection if the direct DAS method of ELISA is

37
used.

Though simplifying work with detecting TmRSV, ELISA has
not eliminated the fact that TmRSV is often irregularly
distributed in woody tissues. Various researchers have
tested different tissues in attempts to locate the most
reliable and efficient detection methods.

Lister et al.(1980) found that TmRSV is not systemic in
apple, and that root pieces (2 to 4mm long) were the best
tissue sources for detection. In another study, however,
apple roots were an erratic source of TmRSV, while leaves
(when a representative sample was taken), and bark tissues
were more consistent (Bitterlin et al., 1984).

Barrat et al.(1984), found that sampling the the
root/trunk transition zone in peach was an inefficient
detection methodology, since only 48 percent of the peach
trees with PSP symptoms were TmRSV positive when tested in
this manner.

Recent studies, however, contradict Barrat's finding.
Bitterlin et al. (1988) found that TmRSV and pitting symptoms
were irregularly distributed within peach trees but mostly
concentrated at and below soil line. Their experiments
investigated distribution of TmRSV and pitting symptoms in
7-year-old peach trees and in 'Halford' (a peach rootstock)
seedlings. Both orchard trees and seedlings were extensively
sampled at roots, stem, leaves and branches. The ELISA test
results indicated that the cambium on the trunk below the
soil line was the most reliable sampling location for

detection of TmRSV. Root tissues and the cambial tissues

38
above the soil line were less efficient for detection and no
virus was detected in leaves or cambial tissues from the tree
branches. To evaluate the use of pitting symptoms as a
detection method, the results of TmRSV detection by ELISA
were also compared to the pitting symptoms present in the
orchard trees and seedlings. Pitting symptoms were similarly
distributed in most trees, with most pitting occurring at the
stem below the soil line. All symptomatic (pitted) tissues
were ELISA positive for TmRSV, yet some tissues were positive
that were not symptomatic.

Cummins and Gonsalves (1986) performed similar work in
plum to determine the appropriate sampling methods for field
surveys of TmRSV infection. Trees were sampled around the
trunk circumference at the graft union and brownline symptoms
were noted. Symptoms and virus distribution were found to be
non-uniform. It was determined that detection of TmRSV
through symptoms was possible visually if readings were taken
from at least four locations around the graft union.

Reliable detection of TmRSV by ELISA was possible if at least
three locations at the graft union were sampled. Sampling
was most efficient in July and August, when the bark was
'slipping' and was easily separated from the TmRSV-infected
vascular tissues.

The contradictions in sampling techniques between species
most likely are due to irregular distribution of TmRSV in
individual trees. Bitterlin et al.(1988), noted that the age
of the trees, the mode of inoculation, particular sampling

techniques and environmental conditions could also have

39
affected results. In addition, they noted that particular
isolates of TmRSV differ in their abilities to infect
tissues, with PYBMV the most likely strain to systemically
infect fruit trees. The possible effects of the mode of
inoculation were noted by Bitterlin et al. (1984) in one
study where TmRSV was detected in the roots in 62 percent of
nematode inoculated trees, yet in no bud inoculated trees.
Bitterlin et al. (1988) when testing cambial tissues noted
that positive and negative samples were sometimes only 1 cm
apart. In their studies they used larger and less diluted
samples than Lister et al.(1980) and Barrat et al.(1984), who
by using smaller and fewer samples may have missed the
”pockets" of virus infection.

Cummins and Gonsalves (1982), noted that transmission,
maintenance and reproduction of TmRSV within plant tissues,
as well as the recovery of TmRSV from plant tissues, appears
to be influenced by a number of factors. Upon initial
grafting of tissues between stock and scion there may be
variations in the establishment of new vascular tissue.
Particular cultivars could differ in the capacity to support
reproduction of TmRSV, or to destroy TmRSV upon infection.
The physical status of host plants within a given clone may
also produce different responses. Plant defense mechanisms,
in response to the relatively slow influx of TmRSV may
temporarily or permanently inhibit the movement of the virus.
Whatever the cause or influencing factors, the irregular
distribution of TmRSV, especially in woody tissues, must be

considered in all field sampling for TmRSV and in

40

implementing any control measures, such as cross protection.

II. PRUNUS NECROTIC RINGSPOT VIRUS AND PRUNE DWARF VIRUS

MATERIALS AND METHODS

WW

Eth_Design. During the 1983 and 1984 growing seasons,
twelve sour cherry orchards in Van Buren and Berrien counties
of southwest Michigan were indexed for prunus necrotic
ringspot virus (PNRSV) and prune dwarf virus (PDV). Six
decline orchards and six non-decline orchards were selected.
The decline-affected or decline-free status was agreed upon
after conferring with growers and extension agents. A11
trees consisted of a 'Montmorency' cultivar budded onto
'Mazzard' or 'Mahaleb' rootstocks. Trees ranged from 6 to 12
years of age and the average tree age was 8.5 years. Within
each of the twelve orchards a rectangular plot of 200 trees
was randomly selected for the study. Plots were mapped and
individual tree ages and orchard cultural characteristics
were noted.

For each of the orchards studied, the nursery source of
the trees was determined and samples were then taken at these
nurseries for virus testing. Three of the four nurseries
studied (nurseries A,B, and C) were located in southwest
Michigan and sampling was done at the nursery site. One
nursery (D) was located outside of Michigan. This nursery
was tested for virus by testing recently purchased,
field-planted trees. At nursery A, 120 'Montmorency' /

'Mazzard' and 120 'Montmorency' / 'Mahaleb' 1-year-old

41

42
"liners" were tested. Sixty 'Mazzard' and 60 'Mahaleb'
rootstocks were also tested. At nursery B, 160-IR2 project
and BOO-Michigan Department of Agriculture (MDA) certified
virus-free bud wood trees were tested. In addition, 100
'Mazzard', 100 'Mahaleb' and 30 'Turkish Mahaleb' rootstocks
and 50 'Montmorency'/'Mahaleb' l-year-old "liners" were
tested. Two hundred 1-year-old 'Montmorency'/'Mahaleb' trees
were tested at nursery C. The fourth nursery (D), was tested
by sampling 46 2-year-old 'Montmorency'/'Mazzard' trees
received from the Sierra Gold nursery of Yuba City,
California within the 10 days prior to testing. In all
cases, representative samples of the field selection were
taken.

Sampling. At all twelve orchard sites and the four
nurseries, the trees were tested for PNRSV and PDV by enzyme
linked immunosorbent assay (ELISA). Spring buds, flowers, or
young leaf tissue samples were taken from each tree. For the
orchard sites and the nursery budwood trees, samples
Consisted of tissue from eight locations on each tree,
equally distributed around the canopy. The eight subsamples
were combined to make one sample per tree. In the nurseries,
two to four subsamples were taken from each 1-2 year old tree
tested. Samples were placed in plastic bags and stored at 5°
C until assayed for virus. Samples were always processed
within 10 days.

ELISA_£rgcedures. Virus assay was performed following
the double antibody sandwich ELISA procedures of Clark and

Adams (1977). Samples of 0.5 g to 1.0 g per tree were

43
diluted 1:10 (v/w) in cold extraction or PBS-Tween /PVP
buffer (phosphate buffered saline, pH 7.4, containing .05 %
Tween 20, 2 % polyvinylpyrrolidone MW 40,000, 0.2 % ovalbumin

and .02 % sodium azide). The composition of PBS per liter

was 8 g NaCl, 0.2 g KH2P04, 1.15 g NazHPO4, 0.2 g KCL, 0.2 g

NaN3. Samples were ground with a Tekmar Tissumizer

(SDT-182EN shaft, Tekmar Co., Cincinnati, OH 45237) for 30 to
60 seconds at approximately 60 percent of maximum speed
(20,000 rpm) and then strained through 1 to 2 layers
cheesecloth. The filtrates were stored at 5° C for no more
than 4 hours.

Rabbit antiserum prepared against the G isolate of
PNRSV and antisera to PDV was obtained from American Type
Culture Collection, (ATCC, 2301 Parklawn Dr., Rockville,
Maryland 20852). Anti-virus IgG (coating antibody) was
purified from the antiserum by ammonium sulfate precipitation
and by passing through a DEAE—22 cellulose (Sigma, St Louis,
Mo 63178) column (Clark and Adams, 1977).

Flat-bottomed polystyrene Microelisa® substrate plates
(ImmulonG’I, Dynatech Lab Inc., Alexandria, VA 22305) with 96
wells per plate were used for the ELISA evaluations. For
each virus the procedure consisted of the following steps: 1)
200 pl of 1.0 ug/ml purified IgG (PNRSV or PDV) in sodium
carbonate buffer, pH 9.6 was placed in each of the 96 wells.
The plates were then incubated in plastic bags for 4 hours at
37° C. 2) Plates were emptied and rinsed with phosphate

buffered saline solution, pH 7.4 with 0.5 % (v/v)

44
polyoxyethylene sorbitan monolaurate (Tween 20). This rinse
procedure was repeated at least three times with at least 3
minutes between rinses. 3) A 200 ul sample of tissue extract
was placed in each plate well. Each plate included five to
10 healthy controls wells, two to four diseased control wells
and one to two wells containing extraction buffer. The
remaining wells were filled with test samples, which were
each replicated twice on the same Microelisa‘® plate. Filled
plates containing sample extract were stored in plastic bags
at 5° C overnight. 4) After rinsing as per step 2, 200 ul
of a solution containing alkaline phosphatase-conjugated-IgG
(anti- PNRSV or PDV) diluted 1:800 in extraction buffer was
added to each well. Plastic covered plates were then
incubated for 3 to 6 hours at 37° C. 5) After rinsing as per
step 2, 200 pl of a solution containing p-nitrophenyl
phosphate(Sigma no. 104, Sigma Chemical Co, St Louis, MO
63178), freshly prepared at a concentration of 1.0 mg/ml in
substrate buffer [10 % diethanolamine (v/v), pH 9.8, in
distilled water containing .02 % sodium azide (w/v)] and was
added to each well. Plates were incubated at room
temperature for 15 to 30 minutes for PNRSV, and 30 to 60
minutes for PDV. The wells were rated visually for color
change and absorbance was measured at 410 nm with a
Microelisa® minireader (Dynatech Lab Inc., Alexandria, VA
22305). Plates were zeroed on an empty well and at least two
readings were made of each plate. For visual readings, a
distinct yellow color was considered ELISA-positive, and a

colorless well, ELISA negative. Photometrically, samples

45
were considered ELISA-positive if the mean absorbance at 410

nm of each of the duplicate sample wells was greater than the
mean A410 nm of the healthy control samples plus three

standard deviations.

WM

All the trees in the six decline and the six
non-decline plots were evaluated for various characteristics
in 1985. The presence of natural wounds, gummosis, borer
injury, blindwood and stunting was noted for each tree.
Mechanical harvesting damage to the trunk portion of the
tree, if present, was noted as mild or severe. Trees were
also rated for overall vigor. A rating of 1 was given to a
non-vigorous, stunted or sparsely foliated or fruited tree,
often with excessive bare spaces on the branches. A rating
of 5 was given to an extremely vigorous tree with a even
ratio of shoot growth to fruit spur formation. In all twelve
orchards the incidence of replanted and dead trees within the

plots was also noted.

RESULTS

W

Prunus Necrotic Ringspot Virus. The 1983 survey
revealed widespread infection of commercial sour cherry
plantings in Van Buren and Berrien counties by PNRSV. Prunus
necrotic ringspot virus was detected by ELISA in all six
decline and all six non-decline orchards in the study.
Infection levels per orchard ranged from 1.02 percent to 99.4
percent of the trees tested, with eight of the twelve
orchards having over 25 percent of the trees infected (Table
2.1). Within the selected ZOO-tree plots, the six decline
orchards had an average of 62.48 percent of the trees testing
ELISA-positive for PNRSV while the non-decline orchards had
an average of 23.05 percent of the trees testing
ELISA-positive for PNRSV. Chi-square analysis found the
incidence of trees testing ELISArpositive for PNRSV in the
decline and non-decline orchards to differ significantly
(2;.001) from the expected 1:1 ratio (Table 2.2). Figure 2.1
shows the percentage of the individual trees infected with
PNRSV in each orchard and the overall mean percentage of
trees that were detected as ELISA-positive for PNRSV in the
six decline and six non-decline orchards.

Prune Dwarf Virus. Table 2.3 shows the levels of trees
ELISArpositive for PDV for each of the twelve orchards

tested. Prune dwarf virus was present in seven of the twelve

46

47

Table 2.1 Incidence of trees ELISArpositive for Prunus
necrotic ringspot virus in six declining and six
non-declining sour cherry orchards in southwest Michigan.

 

# of Trees PNRSV positive

 

 

Orchard Orchard Incidence of
Number Status # of Trees Sampled Virus (%)
1 Decline 167/187 89.30
2 Decline 48/186 25.81
3 Decline 135/182 74.18
4 Decline 79/200 39.50
5 Decline 172/194 88.66
6 Decline 115/197 58.38
7 Non Decline 3/200 1.50
8 Non Decline 197/198 99.49
9 Non Decline 2/196 1.02
10 Non Decline 12/200 6.00
11 Non Decline 5/200 2.50
12 Non Decline 56/199 28.14

48

Table 2.2 Chi-square analysis of PNRSV incidence
between decline and non-decline orchards.

 

 

 

Treatment
Comparison Observeda Expectedb X2 P
D J- . II D J. C
PNRSV positive 716:275
PNRSV negative 430:913 1:1 370.489 <.001

 

aObserved values are for ELISA positives (+) and ELISA
negatives (-) for the compared treatments summed over six
decline plots and the six non decline plots.

bNull hypothesis: The number of diseased and healthy
trees are independent of the decline and non-decline status..

CDecline treatments consisted of 1146 individual
'Montmorency' trees on 'Mazzard' or 'Mahaleb' rootstock from
six declining orchards. Decline treatments consisted of 1193
individual 'Montmorency' trees on 'Mazzard' or 'Mahaleb'
rootstock from six non-declining orchards.

dA significant X2 indicated that PNRSV incidence is not
independent of decline and non-decline status.

49

mofiuomoumo majooolco: pom wagooo mom coauooug mo mogul, some mcfizonm Aoumnouo
nod moouu com. oumnouo >2 >mmzm now ozuflmomIdeqm moon» mo ommucoouwm TN ousmfim

"5ng 59:52
2 3 a m l. 2&5 m n v m m HAIIO Emceo

 

‘ ‘ ‘
woo...

 

OH
om

 

om
ov

om

mZMm>

 

om

69

Oh

QO<m0mo
MZHAOWQIZOZ

om

MZHAUMD

 

om

 

 

L
I

OOH

50

Table 2.3 Incidence of trees ELISA-positive for prune dwarf
virus in six declining and six non-declining sour cherry
orchards in southwest Michigan.

 

# of Trees PDV positive

 

 

Orchard Orchard Incidence of

Number Status # of Trees Sampled Virus (%)
1 Decline ' 8/187 4.28
2 Decline 2/186 1.08
3 Decline 0/182 0.00
4 Decline 3/200 1.50
5 Decline 3/194 1.55
6 Decline 28/197 14.21
7 Non Decline 0/200 0.00
8 Non Decline 13/198 6.57
9 Non Decline 0/196 ' 0.00
10 Non Decline 0/200 0.00
11 Non Decline 0/200 0.00

12 Non Decline 8/199 4.02

51
orchards tested. Prune dwarf virus was found at much lower
levels than PNRSV, with 14.21 percent PDV infection being the
highest incidence of the virus in any one orchard. Within the
selected ZOO-tree plots, the six decline orchards had an
average of 3.84 percent of the trees testing ELISA-positive
for PDV while the non-decline orchards had an average of 1.76
percent of the trees testing ELISA-positive for PDV.
Chi-square analysis found the incidence of trees testing
ELISA-positive for PDV in the decline and non-decline
orchards to differ significantly (P=.01) from the expected
1:1 ratio (Table 2.4). Figure 2.2 shows the percentage of
the individual trees infected with PDV in each orchard and
the overall mean percentages of the trees that tested
ELISA-positive for PDV in the six decline and six non-decline

orchards.

Wraluation

Decline vs. NOn-decline. It is useful to first compare
some characteristics of the the tree evaluation data between
decline orchards and the non-decline orchards without regard
to virus infection. The total number of replanted trees in
the decline orchards was 23.29 percent of the total number
sampled, significantly higher than the non-decline figure of
2.96 percent (P =.001, chi-square procedure). Similarly,
within the six 200 tree plots of the decline orchards a
significantly higher percentage (4.8 percent) of the trees
were dead, while in the six non-decline orchards only 0.75

percent of the trees were dead (2:.001, chi-square

52

Table 2.4 Chi—square analysis of PDV incidence
between decline and non-decline orchards.

 

 

 

Treatment
Comparison Observeda Expectedb X2 P
D J I . II D J o C
PNRSV positive 44:21
PNRSV negative 1102:1172 1:1 8.85d <.o1

 

aObserved values are for ELISA positives (+) and ELISA
negatives (-) for the compared treatments summed over six
decline plots and the six non decline plots.

bNull hypothesis: The number of diseased and healthy
trees are independent of the compared decline and non-decline
status.

cDecline treatments consisted of 1146 individual
'Montmorency' trees on 'Mazzard' or 'Mahaleb' rootstock from
six declining orchards. Decline treatments consisted of 1193
individual 'Montmorency' trees on 'Mazzard"or 'Mahaleb'
rootstock from six non-declining orchards.

dA significant X2 indicated that PDV incidence is not
independent of decline and non-decline orchard status.

53

mwfiuomoumo mafiaomolooc pom moaaooo How c0fiuommcfl mo mosam> some mcfizocm Apumnouo
Hod momma oomv oumnouo an >om you m>aufim0d1<qum mommy mo mmmucoouwm N.m onomam

 

 

 

 

 
 

mag 32> .3 =52
zen: 2 S 2 e e 2. 24m: e n e m N HAITPEPO
" l l l r o
1N
.. e
l m >
o
.m m
- S e
mgmomo ..w mommmomo . . NH
mquomouzoz mm. mquoma
been?” ..uu .. l l vH

 

54
procedure). Figure 2.3 compares the decline and non-decline
orchards in terms of the amount of trunk damage. At all three
levels - damage 1 (mild), damage 2 (severe), and total damage
(mild and severe combined), the decline orchards had
significantly more damage than the non-decline orchards
(2:.001, chi-square procedure). In addition, the two tree
evaluation characteristics of borer incidence and trunk or
branch gummosis were significantly more prevalent (P =.001,
chi-square procedure) in decline orchards than in the
non-decline orchards.

The levels of blindwood and stunting in the decline and
non-decline orchards are shown in Table 2.5. The decline
orchards had significantly more blindwood than the
non-decline orchards. The reverse was found to be true for
stunting; the non-decline orchards were shown to have
significantly more stunted trees than the decline orchards.

Comparisons of the mean vigor rating and the five
individual vigor ratings, per orchard and between the decline
and non-decline orchards, are shown in Table 2.6. No
differences were seen between decline and non-decline in
terms of the mean vigor rating. The percentage of trees
given the lowest and the medium vigor rating (1 and 3) was
significantly higher in the decline orchards. Significantly
more trees given the high vigor ratings (4 & 5) were found in
the non-decline orchards than in the non-decline orchards.
When all the trees given the two‘low vigor ratings (1 & 2)
are combined significance is shown at the P = .001 level

(higher incidence for the decline orchards) and when all the

55

moonu oumnouo ocHHooplco: mmHH pom ooHHoop Smm How omnEmo Manna m.N ouslo

 

 

madame gonna ouo>om w pHHz mousse xcsua ouo>om ommemo gonna UHHZ
N a H oumemo w N omMEmn w H ommaoo w

V.4’l.4.1 4.4.4.0.; o

cocoooooox

enononououenonono».
.-m
.-0H
I .l mH
.uom

NM.HN %

-mN

 

WZHAUMHDIZOZ E mZHHUmo I

 

 

 

D‘CS‘COWQ

0P

56

Table 2.5 Percentage of stunting and blindwood for each of
six decline and six non-decline orchards and mean incidence
of stunting and blindwood for the decline and non-decline
orchards overall.

 

 

Orcharda % Of Trees % Of Trees
Number with Stunting with Blindwood
1 2.67 -

2 1.61 0.00

3 6.04 76.4

4 4.50 3.50

5 0.00 89.2

6 1.52 89.3

7 5.53 0.00

8 1.51 59.1

9 0.00 18.0

10 13.0 2.00

11 11.5 0.00

12 5.50 2.00

Orchard Statusb W W
Decline 3.23 51.62<-001>
Non-Decline 6.18C(-01) 13.45

 

aEach orchard plot = 200 trees rated in 1983. Orchard 1 was
not rated for blindwood.

bDecline orchards include orchards 1 to 6; non-decline
orchards include orchards 7 to 12.

cValue significantly greater than the corresponding value
(Penumber in parenthesis) when incidence of tree evaluation
characteristic is compared over orchard type by chi-square
analysis.

57

Table 2.6 Tree vigor ratingsa for six decline and six
non-decline orchards.

 

% Trees % Trees % Trees % Trees % Trees
Mean Vigor Vigor Vigor Vigor ‘Vigor
Orcha rd Vigor Rat ing Rat ing Rat ing Rat ing Rat ing
Numberb Rating "1" "2" "3" "4" "5"

 

1 2.88 25.65 8.38 41.88 0.00 24.08
2 3.38 0.00 0.00 81.11 0.00 18.89
3 3.40 13.92 8.76 38.14 2.06 37.11
4 3.71 6.63 3.57 44.39 2.55 42.86
5 _ _ .. - - -

6 2.91 14.21 0.00 75.96 0.00 9.84
7 4.02 1.00 1.00 45.50 0.00 52.50
8 2.84 18.69 1.52 68.18 0.00 11.62
9 3.93 0.00 0.51 52.53 0.00 46.97
10 4.00 5.50 6.00 24.50 11.50 52.50
11 2.94 13.64 4.04 69.19 3.03 10.10
12 3.93 3.02 6.03 45.23 0.00 50.25
Orchard statusc

Decline 3.2 12.2d<-°°1) 4.2 55.7mm 0.1 27.0
Non _

Decline 3.6 7.0 3.2 50.8 2.4<-°5>37.4<-01)

 

aVigor rating system: 1 = tree excessively stunted; branches
sparsely foliated,few fruit spurs 2 = tree slightly stunted;
low fruit spur/shoot growth ratio 3 = tree growth
average;slightly more shoot growth to fruit spur growth 4 =
tree growth average to above average; nearly equal ratio of
fruit spur/shoot growth 5 = tree growth above average;
branches full; fruit spur/ shoot growth equal or better.

bEach field plot consisted of 200 trees; all live trees were
rated for vigor in 1986. Orchard # 5 was not rated for vigor.

CDecline orchards (orchards 1 to 6) included 944
'Montmorency' trees on 'Mazzard' or 'Mahaleb' rootstock.
Non-Decline orchard (orchards 7 to 12) included 1193
'Montmorency' trees on 'Mazzard' or 'Mahaleb' rootstock.

dValue is significantly greater (2; number in parenthesis)
than corresponding value when the total incidence of each
vigor level is compared over orchard status by chi-square
analysis.

58
trees given the two higher vigor ratings (4 & 5) are
combined, significance is also shown at the 2,8 .001 level
(higher incidence for non-decline orchards).

Prunus Necrotic Ringspot Virus. The same tree
evaluation characteristics were considered with reference to
the PNRSV survey data. The incidence of trees singly
infected with PDV was too low overall to statistically
associate the presence of PDV with any of the tree evaluation
data. In addition, the age of the trees was considered in
regard to virus infection. Figure 2.4 shows the variation of
infection by both PNRSV and PDV viruses with age. These data
were taken from six orchards in the study which had a
variation in tree ages present.

Table 2.7 compares the stunting, blindwood,tree death
and replant percentages for four different categories. One
category, (A) compares the trees that tested ELISA-positive
for PNRSV to the trees that tested ELISA-negative for PNRSV
in the decline orchards. Another catergory, (B), compares
the trees that tested ELISA- positive for PNRSV to the trees
that tested ELISA-negative for PNRSV in the non-decline
orchards. A third category, (C), compares all the decline
orchards to all the non-decline orchards without regard to
virus infection. The fourth category,(D), compares all the
trees (decline or non-decline) that tested ELISA-positive for
PNRSV to all the trees that tested ELISA-negative for PNRSV.

As Table 2.7 indicates, blindwood incidence was
consistently and significantly higher for the decline

orchards and for the PNRSV-ELISA positive trees. Similarily,

59

wow wow» >3 moouu oomH cH coHuoomcH >om pom

AHHIGH m on m

C)
O .-

.mmsac mom
e 9pm

I

I.
u Iril
‘

 

>8 .0. >mmzm -o.

 

>mmzm mo mmmucmouom

m on v

C {J-

v.m musmHm

v v
8.0
:82:
..ooem
moeem
1.8.3
.098
.098
.82:
.098
18.8
..oo.ooH

l

 

>H0fiDm

60

Table 2.7 Percent tree replant,stunting,blindwood,and tree
death for trees ELISA-positive for PNRSV and trees
ELISA-negative for PNRSV between and within decline and
non-decline orchards.

 

 

i_Qf_TreeS_with: 3_Qf_1reeSi
Category Stunting Blindwood Dead Replanted
(A)
+a/declineb 2.65 96.17C<JW1) 2.76 18.20
-/decline 2.33 25.51 2.20 25.25‘J5)
(B)
+/non-decline 2.91 44.36(-°°1) 2.32(-°5) 13.31
-/non-decline 7.19<-05> 4.25 0.11 2.94
(C)
Decline
PNRSV + & - 2.53 56.69(°°°1) 2.46<-°°1) 20.74<-001>
Non-Decline
PNRSV + a - 6.20<JW1) 13.50 0.42 3.10
(D)
All PNRSV 2.73 78.83<-001) 2.32<-01> 13.31<-05)
positives
All PNRSV 5.63<JW1> 10.24 0.75 9.80
negatives

 

3+ - PNRSV-ELISA positive, - - PNRSV-ELISA negative.

bDecline orchards included 944 'Montmorency' trees on
'Mazzard' or 'Mahaleb' rootstock;Non-decline orchards
included 1193 'Montmorency' trees on 'Mazzard' or 'Mahaleb'
rootstock.

CValue is significantly greater than the corresponding values
(2; number in parenthesis) when total incidence of a
characteristic is compared within categories by chi—square
analysis.

61
the amount of tree death per orchard was significantly higher
for the decline orchards and for the trees with PNRSV when
compared to the non-decline orchards and the PNRSV negative
trees. Since the virus data and the tree evaluation data
were taken in subsequent years, it was possible to record
deaths of trees known to have tested ELISA-positive for
PNRSV. The numbers of replanted trees was also found to be
significant, with the numbers of replants in the declining
orchards higher than in the non-declining orchards, but the
relationship of replants to PNRSV ELISA-positive or
ELISA-negative trees was inconsistent. Table 2.7 also
indicates that incidence of stunting was significantly higher
in the non-decline orchards and that the incidence of
stunting was significantly higher for all the trees without
PNRSV infection.

The comparison of trunk damage within the four
categories is shown in Table 2.8. In terms of trunk damage,
the non-decline orchards were significantly healthier (more
trees rated damage = 0) than the decline orchards and
significantly less damage was present in PNRSV-negative
trees. The total damage incidence (mild and severe damage
combined) showed damage to be positively associated with both
decline orchards and with the presence of PNRSV infection.

Vigor ratings given to the orchard trees are compared
within the four categories in Table 2.9. The incidence of
tree vigor rating 3 was only significant in comparison (C),
with the decline orchards having significantly more trees

rated at this level than the non-decline orchards. The

62

Table 2.8 Percent trunk damagea on trees ELISA-positive and
ELISA-negative for PNRSV between and within decline and
non-decline orchards.

 

 

Category % Trees % Trees % Trees Total
Damage Damage Damage Damage
Category Category Category Category
non "1" "2" "1 & 2"

(A)

+b/declinec 70.22 8.45 18.7sd<-001> 27.20<-001>

-/decline 85.53(-001> 5.63 6.86 12.49

(B)

+/non-decline 95.27 2.18 2.18 4.36

-/non-decline 95.32 2.29 1.85 4.14

(C)

Decline

PNRSV + & - 75.05 7.08(-001) 13.34(-001)20.42<-001>

Non-Decline

PNRSV + & - 95.31<-001) 2.26 1.93 4.19

(D)

All PNRSV

positives 78.63 6.34(~01) 13.18<-001)19.52<-001>

All PNRSV

negatives 92.31(-°°1) 3.32 3.39 6.71

 

aT‘Trunk damage rating system : 0 = no visible trunk damage,
1 a visible mild trunk damage, 2 = visible severe trunk
damage.
b'+ I PNRSV-ELISA positive; - = PNRSV-ELISA negative.
C Decline orchards included 944'Montmorency' trees on
'Mazzard' or 'Mahaleb' rootstock. Non-Decline orchards
included 1193 'Montmorency' trees on 'Mazzard' or 'Mahaleb'
rootstock.

Value is significantly greater (P = number in parenthesis)
than the corresponding value when damage classes were
compared within category by chi-square analysis.

63

Table 2.9 Tree vigor ratingsa for trees ELISA-positive and
ELISA-negative for PNRSV between and within decline and
non-decline orchards.

 

 

% Trees % Trees

Mean ‘Vigor % Trees Vigor

Vigor Ratings Vigor Ratings
Category Rating 1 & 2 Rating 3 4 & 5
(A)
+b/declinec 2.23 22.61d(-°°1) 51.84 22.06
-/decline 3.38 7.1 57.35 31.62h0U
(B)
+/non-decline 3.11 17.46<-001) 51.27 23.27
-/non-decline 3.73 3.15 48.04
44.55LOMJ
(C)
Decline
PNRSV + & - 3.13 15.60<-°01) 52.98 (~05) 25.56
Non-Decline
PNRSV + & - 3.59 6.87 48.78
39.55(.001)
(D)
All PNRSV
positives 3.00 20.88(°°m“ 51.65 22.47
All PNRSV
negatives 3.62 4.76 50.94
40.57(.001)

 

a Vigor rating system:l - tree excessively stunted; branches sparsely
foliated,few fruit spurs
shoot growth ratio 3 - tree growth average;slightly more shoot growth to
fruit spur growth 4 - tree growth average to above average; nearly
equal ratio of fruit spur/ shoot growth 5 - tree growth above average;
branches full; fruit spur/ shoot growth equal or better.
b + - PNRSV-ELISA positive, - - PNRSV-ELISA negative.

C Decline orchards included 944'Montmorency' trees on 'Mazzard' or
'Mahaleb' rootstock. Non-Decline orchards included 1193

'Montmorency' trees on 'Mazzard' or

'Mahaleb' rootstock.

2 - tree slightly stunted; low fruit spur/

d Value is significantly greater (P,- number in parenthesis) than the
corresponding value when vigor classes were compared within category by
chi-square analysis.

64
incidence of poor tree vigor (ratings 1 and 2) was
significant for the categories (C) and (D). Significantly
more decline than non-decline trees and more trees
ELISA-positive for PNRSV than trees ELISA-negative for PNRSV
were rated at these lower levels. The incidence of the high
vigor ratings (4 and 5) was shown to be significant in all
four categories. Categories (A),(B) and (D) all indicated
that significantly more PNRSV-free trees were given these
high vigor ratings. Category (C) indicated that
significantly more non-decline trees were given a 4 or 5

rating.

W
The results of the ELISA surveys for PNRSV and PDV at

the four nurseries are shown in Table 2.10. Prunus necrotic
ringspot virus or PDV was detected at three out of the four
nurseries tested, though at generally lower levels than what
was detected in the commercial orchards. Overall, 4.4
percent of the 1086 one—year old trees and rootstocks tested
at the four nurseries were ELISA positive for either PNRSV or
PDV. As in the commercial orchards, the majority of these
(95.8 percent) were PNRSV-ELISA positive while considerably
less (4.2 percent) tested PDV-ELISA positive. Figure 2.5
indicates the percentages of nursery samples ELISA-positive
for either PNRSV or PDV. In addition, one out of the 460
total budwood source trees sampled at nursery B tested

ELISA-positive for PDV.

65

Table 2.10 One-year old trees and rootstocks ELISA-positive
for PNRSV or PDV at four nurseries.

 

 

 

 

Number PNRSV Number PDV
positive positive
Number Percent PNRSV Percent PDV
Nursery Sampled Variety positive positive
"A" 120 Mont.a/Mazzard 0/0.0 0/0.0
120 Mont./Mahaleb 0/0.0 0/0.0
6O Mazzard O/0.0 0/0.0
60 Mahaleb 0/0.0 0/0.0
"B" 100 Mazzard 0/0.0 1/1.0
100 Mahaleb 0/0.0 0/0.0
30 Turkish Mahaleb 0/0.0 0/0.0
259 Mont./Mahaleb 0/0.0 0/0.0
"C" 200 Mont./Mahaleb 35/17.50 0/0.0
"D" 46 Mont./Mahaleb 11/23.91 1/2.17

 

a:Mont. = 'Montmorency'

66

‘P

can no >mmzm nocueov no.3.»
HmHH How m>HuHmom|<mHHm moHdEmm menu >uomus: mo ommucoouom m.m ousmHm

Nmmmmbz

4

 

mv\NH

 

 

 

mo.mm

 

oom\mm

‘-

 

 

om.hH

HN.o

omv\H

d-

00.0

owm\o

 

oo.o
oo.N
oo.v
oo.m
oo.m
oo.OH
oo.NH
oc.vH
oo.mH
oo.mH
oo.om
oo.NN
oo.vm
oo.mm
oo.mm

>ihiazt>01

DISCUSSION

Chi-square analyses indicated that there is a
relationship between the decline and non-decline orchards and
level of infection by two ilarviruses. The exact nature of
this relationship however, was not entirely defined by the
results of this study. For example, consider the level of
PNRSV in field #8 in Figure 2.1. Although this was a fairly
vigorous, productive non-declining orchard, the level of
PNRSV infection was the highest of all twelve fields tested.
While noting that this orchard could be an exception, it
indicates, along with the presence of PNRSV in the other
non-declining orchards, that PNRSV infection is probably not
the sole factor in tree decline. Overall levels of PDV
infection, while significantly higher in the decline orchards
than in the non-decline orchards, were simply too low to
account for the scope of the decline problem.

In addition, the age data presented in Figure 2.4 show
how both the PNRSV and the PDV incidence of infection
increase with tree age. This would be expected with any
pollen-borne virus prevalent in the growing region. Aside
from relatively high infection in the youngest tree age group
(< than 5 years old and due to nursery infection) the older a
tree is the more likely it is to be infected with either
virus. In these six orchards at least, and for PNRSV in
particular, any tree over 8 years of age was as likely to be

infected as it was not.

67

68

The tree evaluation data give further insight to the
possible nature of the relationship of PNRSV to decline. By
comparing all decline and all non-decline orchards (Figure
2.3, Tables 2.5,2.6) it is seen that the overall health of
the decline orchards is lower than that of the non-decline
orchards, as would be expected. More trees in the decline
orchard had died and had been replanted. Decline orchards
also contained more trees of low vigor and less trees of high
vigor than the non-decline orchards and more trees exhibiting
blindwood. Levels of trunk damage were also significantly
higher in the decline orchards than in the non-decline
orchards. Incidence of borer injury and trunk gumosis, both
factors which could be associated with trunk damage, were
also significantly higher in the decline orchards than in
the non—decline orchards. All of these findings validate the
orchard selection and they characterize the overall decline
and non-decline conditions.

When the tree evaluation data is considered in terms of
the PNRSV survey, the relationship of virus to decline can be
seen. To fully understand the results, however, some aspects
of the four way comparison tables should be noted.

The comparison category (C) gives information on a
given characteristic's relationship to the orchard decline or
non-decline status. If, for any characteristic, the entire
decline group is significantly different from the entire
non-decline group, we can regard the decline status as being
associated with the characteristic in some way. The

comparisons of all the PNRSV positives to all the PNRSV

69
negatives without regard to decline or non-decline status
[category (D)] gives similar information in terms of the
influence of viral infection. It must be realized, however,
that this last category can be biased. The group of trees
ELISA-positive for PNRSV will in general contain more trees
from the decline orchards since they have been shown to have
a higher rate of infection with this virus. Any significance
seen in the comparison of decline to non-decline [category
(C) ] could then be a contributing factor or 'tag along' in a
comparison of the PNRSV positive group to PNRSV negative
group [category (D)]. The other two comparison categories of
PNRSV infection within decline [category(A)], and PNRSV
infection within non-decline orchards [category(B)], can
avoid this situation. In the decline comparison (A), for
example, all trees are from the same orchard type; the only
known difference is virus infection. A significance in this
category then could indicate that PNRSV infection or lack of
PNRSV infection could be associated with a particular
characteristic. We can not, however, rule out other factors
that may be occurring and contributing such as macro- and
micro-site factors, environmental influences and various
pathogenic interactions, viral or otherwise.

Tables 2.7 and 2.9 indicate that the characteristics of
blindwood and tree vigor were both positively associated with
PNRSV presence. Keitt and Clayton (1943) noted that PNRSV
infection reduced spur formation in sour cherry. Blindwood -
an excess of sparsely foliated or fruited growth - could be a

similar effect of PNRSV infection. Other factors such as

70
pruning methods, nutrient status, and environmental
conditions must be considered, however in interpreting the
association of blindwood and PNRSV infection.

Overall reduction in vigor, in terms of reduction of
growth and yield has been noted in sour cherry by Lewis
(1951), Way and Gilmer,(1963) and Davidson and George (1965).
Our results indicate that PNRSV infection was associated with
those trees in the study that were of low vigor and lack of
PNRSV infection was one characteristic of those trees that
were very healthy and vigorous. It is likely the decline in
vigor shown by the tree evaluation data for the
PNRSV-positive trees and in the decline orchards is a part of
the total decline syndrome. Perhaps the virus first initates
lowered spur/fruit production and reduces overall vigor. The
reduced vigor could then predispose the trees to invasion by
other pathogens and weaken the ability of the tree to
withstand unfavorable abiotic conditions.

To look directly at possible yield reduction, another
characteristic often associated with PNRSV (and PDV)
infection in sour cherry, was out of the scope of this
project. The best indirect way to approach yield through the
context of this study was through the combination of
blindwood incidence and overall tree vigor. High
blindwood/low vigor could indicate that the fruiting system
of the tree was reduced. The incidence of high blindwood and
lower vigor occurred significantly in both the decline versus
the non-decline orchards overall and in the PNRSV positive

trees from decline orchards versus the PNRSV negative trees

71
from decline orchards. The decline orchards and the trees
that tested positive for PNRSV would then be expected to have
lower fruit production than the non-decline orchards or the
trees that tested negative for PNRSV. This lowered
production would have resulted from the decreased fruit set
on infected trees or the high incidence of trees of low
vigor, which had a higher rate of vegetative growth than
fruit spur formation.

The tree evaluation data also reveal interesting
information about trunk damage. Overall, the decline
orchards had significantly more trees with trunk damage than
the non-decline orchards. In addition, the overall damage
that occurred in the decline orchards was more severe that
the overall damage to trees in the non-decline orchards.
These findings indicate that trunk damage could be a part of
the decline syndrome. Since within both the decline and
non—decline categories there was significantly higher
incidence of trunk damage for the trees with virus; trunk
damage was also associated with PNRSV infection.

The trunk damage rated for this study was damage caused
by mechanical harvesting machines. Brown et al. (1982) have
shown that damage to sour cherry by harvesting equipment is
due to improper clamp pressures, decreased bark strength and
exacerbated by high water levels in the bark. Although it is
not known to do so, physiologically, PNRSV may have affected
the trees in such a way as to promote trunk damage.
Similarly, the virus may have induced lowered vigor which

then decreased the trees ability to withstand any damage.

72
Yet, it is also possible that the lower vigor seen for the
decline orchards and for the PNRSV infected trees is partly
due to the overall higher levels of trunk damage from these
same groups. Without a full investigation of many
interacting factors on a tree by tree basis it is not
possible to separate all the implications of trunk damage and
its' relationship to virus and vigor. Perhaps, other
research in progress on this issue will further elucidate the
relationship of these factors and decline.

Stunting was positively associated with the trees
without PNRSV infection as well as with the non-decline
orchards. It should be noted however, that the total number
of trees affected was relatively low. The declining orchards
would have been expected to have more stunting than the
non-decline orchards. It is possible that more of the
stunted trees in the decline orchards had been removed, since
the levels of replants for the decline orchards were much
higher. The association of stunting with PNRSV-negative
trees is not in agreement with Milbrath (1950) who showed an
association of virus free trees with taller and sturdier
growth. Millikan (1955) however, noted that although a
growth reduction occurred with PNRSV infection, it was due to
a reduction of the number of branches and not due to
stunting. Further investigation of micro-site factors - soil
type and structure, soil and root levels of nematodes - as
well as concise differences in branch growth and tree height
would be needed to reveal other causal factors relevant to

this finding.

73

In considering PNRSV and PDV in relation to the
decline problem it is important to note that both viruses
have been known to be present in Michigan cherry plantings
since the trees were first planted in the state. The decline
phenomenon is only recent, however, and this indicates that
other factor(s) may be invloved. Although early decline
symptoms and high levels of infection by PNRSV may occur at
relatively the same age in cherry, currently there is no
evidence to directly and singularily correlate this virus
with the decline syndrome, mostly due to other possible
influencing/ interfering characteristics.

It has been proven that both PDV and PNRSV have a
deleterious effect on tart cherry growth and yield
(Rassmussen and Cation, 1942; Parker et al.; 1959; Cochran et.
al., 1950). It was shown by this study that the declining
orchards are more severely infected with both viruses than
were the non-declining orchards, which suggests that the
presence of virus and declining orchard status is related. It
is likely that virus - PNRSV particularily - plays a part,
perhaps as a predisposition agent, in the decline problem.
Prunus necrotic ringspot virus, acting to induce overall low
vigor, could also be an important contributing factor in a
cumulative disease process. Perhaps what alone is a slight
problem or strain to a tree becomes, in conjunction with
enviromental, cultural or other pathogenic factors, a more
lethal component.

There was a large variation (from zero to 26.09

percent) of virus infection levels between the nurseries

74
tested. At nursery A, no virus was detected in the 240
1-year-old trees tested or in 120 rootstocks, while nursery D
had a relatively high incidence of virus infection of 12 out
of a total of 46 l-year-old trees tested. When questioned,
the nursery operators reported that a variety of virus
control measures (including none) were practiced at each
nursery when obtaining budwood for propagation purposes,
which could account for the wide variation in virus
infection. Operators at nursery C, for example, reported a
complete lack of testing of budwood for either PNRSV or PDV
prior to selection and the same was suspected but unconfirmed
for nursery D. One of the primary sites of budwood for
Nursery C was an orchard included in the field survey of this
study which had a 74 percent PNRSV infection rate. The
nursery operators claimed to use only healthy trees from this
orchard for budwood but the variability of, or the complete
lack of symptomatic tissue on PNRSV-infected trees would make
it difficult to avoid infected trees. In general, all of the
trees in commercial orchards that were planted from nursery
stock from either nursery C or D would be expected to have
higher PNRSV or PDV infection at an earlier age, since the
infected stock would serve as a local inoculum source.

The nursery controls at nursery A and B, in contrast,
included testing the budwood trees at regular intervals and
obtaining budwood trees from virus-free certified nurseries.
Equally important was that these nurseries controlled virus
in their rootstocks and/or purchased similarily certified

roostock materials. As shown in a study by Fleisher et al.

75
(1964) of 'Mazzard' and 'Mahaleb' rootstocks, certification
of rootstock is important for control of these viruses. In
that study, levels of infection of rootstock by PNRSV, for
example, varied from 0.2 to 35 percent in unregistered
seedlings while the highest level of PNRSV detected in
registered seedlings was 2.7 percent.

The orchard industry's general premise that all sour
cherry plantings in southwest Michigan will eventually be
infected with PNRSV may be somewhat true, yet as shown in
this study the amount of infection at earlier ages is
markedly different for some orchards. The orchards planted
with infected stock are disadvantaged since both viruses have
been shown to cause reduced vigor and stunting in young
trees. Both Millikan (1955) and George and Davidson (1965)
have shown that the effect on growth and vigor is more severe
the younger the trees are at the time of infection. Cleaning
up all nurseries would improve the chances of giving these
trees a better start, even though the current levels of virus
in established plantings cannot ensure a virus-free life.
Controls such as purchasing virus free nursery stock and
treating young trees with gibberellin in the first years of
flowering to prevent early infections (Hull and K103, 1962)
should be implemented. The most effective control of
planting disease resistant stock is not yet available but
preliminary work in Washington state by Howell and Mink
(1988) may eventually lead to biological control of PNRSV.
These researchers have begun studies in sweet cherry with an

apparently non-pathogenic strain of PNRSV which may offer a

76
form of natural protection from PNRSV infection. Such a
possibility increases the importance of attempts to prevent

virus infection in the nursery and in the orchard.

III. TOMATO RINGSPOT VIRUS

MATERIALS AND METHODS

21W

Experiments were conducted to investigate the level of
TmRSV in declining and non-declining cherry trees in
southwest Michigan. Within each of the twelve 200 tree plots
selected for PNRSV and PDV testing, fifty trees were selected
in a grid pattern for TmRSV testing. Cambial samples were
taken from each tree at or below the graft union. A 1.0 cm
diameter boring tool was used to remove circular bark pieces
from each tree. Four samples per tree were taken at
equidistant locations around the tree. The borer cylinder
was hammered through the bark and the cylinder of tissue was
removed. The cylinder of tissue consisted of an outer bark
piece which was discarded and the cambial tissues which were
collected. In addition any cambial tissue remaining on the
exposed wood of the tree trunk was collected. Samples were
‘ rinsed with water and the four samples per tree were combined
and placed in cold PBS-Tween/PVP buffer [approximately a 1:5
dilution (v/w)] and stored on ice.

At three area nurseries, cherry trees were tested for
the presence of TmRSV. At one nursery, 60 trees consisting
of 'Montmorency' on 'Mazzard' rootstock and 60 trees
consisting of 'Montmorency' on 'Mahaleb' rootstock were
tested. At the second nursery two hundred trees consisting
of 'Montmorency' on 'Mahaleb' rootstock were tested and at a

third, 200 trees consisting of 'Montmorency' on 'Mazzard'
77

78
rootstock and 200 trees consisting of 'Montmorency' on
'Mahaleb' rootstock were tested. The trees were all 1 or 2
years old, and sampling was done with a 0.5 cm diameter

boring tool due to the small diameter of the trees.

W

As noted in Chapter 2, each tree was evaluated for
various growth and health characteristics. Trees were rated
for presence of blindwood and stunting; mechanical harvesting
damage to the trunk portion of the tree, if present was noted
as mild or severe. Trees were also rated for overall vigor,
with a rating of 1 given to a non-vigorous, stunted or
sparsely foliated or fruited tree, often with excessive bare
spaces on the branches. A rating of 5 was given to an
extremely vigorous tree with an even ratio of shoot growth to
fruit spur formation. In each orchard the number of dead and
replanted trees was noted. These various features of each
tree were then compared in terms of orchard status (decline

and non-decline) and TmRSV presence.

8' l .1 l' E I ESM 'I1' :1 I

Studies were initiated to investigate the distribution
of TmRSV within individual cherry trees. Six trees that had
tested ELISA-positive for TmRSV were selected for testing for
TmRSV distribution. On each tree, cambial samples were
removed from the north, south, east and west sides of the
trunks, at or below the graft union. Samples were also taken

in 5 cm increments along the trunk with five samples taken

79
above and five samples taken below the graft union. Each
sample was assayed separately. In addition, six to eight
young leaf samples and four root samples were taken from each
tree. If available, sucker leaf tissue was also sampled.

In another distribution test, 150 greenhouse-grown
2-year-old trees that had been inoculated with a TmRSV
infected bark chip were tested 9 months post-inoculation.

The infected bark chips were obtained from mature cherry
trees naturally infected with TmRSV. On each inoculated
2-year-old tree, four 0.5 cm diameter cambial tissue samples
were taken near the graft location; four to six leaf and
flower bud samples and one to three root samples were also
taken.

In another study, five trees that had previously tested
TmRSV-positive were re-tested at regular intervals over a
period of 68 days. Four cambial tissue samples were combined

for each tree on each test date.

WWW

At each of three decline and three non-decline orchard
plots, five TmRSV-positive and five TmRSV-free trees were
selected to study the relationship between the infection of
trees and surrounding dandelion by TmRSV and the population
density of Xiphinema species. At each orchard, the dandelion
populations around the 10 trees were sampled for ELISA
testing for TmRSV. Six dandelions at the dripline of the
tree canopy and four near the trunk of the tree were selected

at each site. One to five leaf and flower samples and one to

80

three root samples were combined for each dandelion tested.

Soil and tree root samples were also taken at each
tree. Young feeder roots were taken from three separate
locations approximately 6 to 12 inches below ground and
tested by ELISA for TmRSV. Soil samples for each tree were
made up of eight subsamples equally distributed around the
tree. In the laboratory, 100 cc-portions of each soil sample
were processed for nematode counts by the density gradient
centrifugation-flotation technique (Jenkins, 1959).
Extracted nematodes of the species Xiphinema, Criconemella,
and Pratylenchus were identified and counted with the aid of
a dissecting microscope. Endoparasitic nematodes were
extracted by the Shaker technique (Southey, 1970), detailed
as follows: One gram of washed roots was suspended in a 50 ml
mix of mecuric chloride (10ppm) and dihydrostreptomycin
sulfate (50ppm) and then shaken at 100 rpm for 48 hours. The

extracted Pratylenchus species were identified and counted.

WW

Three decline fields were studied to compare symptom
expression with detection of TmRSV. In each of the three
fields, the fifty trees that were tested for TmRSV were
examined for typical stem pitting and grooving symptoms
associated with PSP in peach. An oval patch of bark
approximately 3 cm by 5 cm was cut out from each tree and the
bark peeled back to reveal the inner bark. Invaginated

pitting and longitudinal grooves were recorded if present.

81

W

In all cases the samples to be tested for TmRSV were
kept cold throughout the collection, storage and virus assay
procedure. All bark, root and leaf samples were assayed
within 6 hours of collection. Bark and root samples were cut
into small pieces, diluted 1:5 (w/v) in PBS—Tween/PVP buffer
(extraction buffer), and ground up with a Tekmar Tissumizer
or with a mortar and pestle, and then strained through
cheesecloth. Cherry orchard dandelion leaf samples were
diluted 1:10 (w/v) in extraction buffer and ground up with a

Tissumizer.
Samples were assayed for TmRSV by F(ab')2 ELISA

(Barbara and Clark, 1982), in flat-bottomed polystyrene
Microelisag’substrate plates with 96 wells per plate.
Antiserum to TmRSV was prepared from virus purified from

infected raspberry tissue (See page 85 for details). The

F(ab')2 ELISA procedure consisted of the following steps. 1)

200 pl of 1.0 mg/ml TmRSV-IgG-F(ab')2 fragments in sodium

carbonate buffer, pH 9.6, were placed in all 96 wells. The
plates were then incubated in plastic bags for 4 hours at 370
C. 2) Plates were emptied and rinsed with a phosphate
buffered saline solution, pH 7.4 with 0.5 % (v/v)
polyoxyethylene sorbitan monolaurate (Tween 20). This
procedure was repeated at least three times with at least 3
minutes between rinses. 3) A 200 pl sample of tissue extract
was placed in each plate well. Each plate included four to

eight healthy control wells, two to three diseased control

82
wells and one to two wells containing extraction buffer only.
The remaining wells were filled with test samples, which were
each replicated twice on the same Microelisa® plate. Filled
plates containing sample extracts were stored in plastic bags
at 5° C overnight. 4) After rinsing as per step 2, 200 ul of
a solution containing 1.5 ug/ml of TmRSV IgG in extraction
buffer was placed in each well. The plates were then
incubated in plastic bags for 3 hours at 37° C. 5) After
rinsing as per step 2, 200 pl of horseradish
‘ peroxidase-labeled protein-A conjugate (Zymed Laboratories,
So.San Francisco, CA 94080) at a concentration of 2.0 ug/ml
in extraction buffer was added to each well. The plates were
then incubated in plastic bags for 3 hours at 370 C. 6)
After rinsing as per step 2, 200 ul of a solution containing
freshly prepared O-phenylene diamine (dihydrochloride) (Sigma
P-3808 Sigma Chemical Co, St Louis, MO 63178) at a

concentration of 0.7 mg/ml in substrate buffer (7.3 g
Na2HP04, 5.11 g citric acid, 0.4 mls H202 ) was added to each

well. Plates were incubated at room temperature and scored
visually and spectrophotometrically for an amber color
reaction. Absorbance was measured at 490 nm with a
Microelisa® minireader. Plates were zeroed on an empty well
and readings were made of each plate at 30 minutes and 60
minutes after substrate addition. Photometrically, samples
were considered ELISA-positive if the mean absorbence at 490

nm of each of the duplicate sample wells was greater than the

mean A490 nm value of the healthy control samples plus three

83

standard deviations.

WWW

Variations of enzyme linked immunosorbent assay (ELISA)
were evaluated for use in detecting TmRSV in plant tissues.
In 1985, the double antibody sandwich (DAS) form of ELISA
(Clark and Adams, 1977) was compared to the heterologous
antiglobulin double antibody sandwich (HADAS) form of ELISA

(Voller et al, 1978; Bar-Joesph and Malkinson, 1980) and to
an indirect method of ELISA using F(ab')2 fragments of
immunoglobulin (Barbara and Clark, 1982). In 1987, the
F(ab')2 method was compared to the dot—ELISA (Banttari and

Goodwin, 1985) method using antibody coated nitrocellulose
membranes.

(Virns_Ereparatignl Tomato ringspot virus was obtained
from field-infected raspberry (Bnhufi idaens L.) plants.
Cultures of TmRSV were maintained under greenhouse conditions
in 'National Pickling' cucumber (Cucumis sativus L.) and
Chenopodium quinoa Willd.

Tomato ringspot virus was purified for ELISA and serum
production using 'National Pickling' cucumber as the
propagation host. The purification procedure used was
developed from a variation of a protocol by Dias (1975). All
procedures were carried out at 40 C. One hundred grams of
infected tissue was homogenized in a cold waring blender in
250 ml of 0.05 M phosphate buffer, pH 8.0, containing .02 M

2-ME and .02 M ascorbic acid. The homogenate was then

84
squeezed through four layers of cheesecloth and the mixture
was then centrifuged in an IEC no 872 rotor at 10K rpm for 20
minutes (low speed centrifugation). Ammonium sulfate (15
percent w/v) was added to the supernatant. The mixture was
stirred overnight and was then given a second and third low
speed centrifugation. The virus particles in the supernatant
were then pelleted by ultracentrifugation in a Beckman no. 30
rotor at 28K rpm for 2.5 hours. Pellets were resuspended in
1.0 ml of 0.01 M phosphate buffer, pH 7.5, and stored at 4° C
overnight. The virus was further purified by another low
speed centrifugation, and clarified by a high speed
centrifugation (Beckman 40 rotor ,38K rpm 90 min) of the
resulting supernatant. The resulting pellets were then
resuspended in 200 #1 of 0.05 M phosphate buffer, pH 7.5 and
stored at 4° C overnight. Linear log sucrose density
gradients (0 -30 %) were prepared with buffer and left to
equilbrate overnight. Sucrose gradients were loaded with 0.6
ml of virus preparation per tube and centrifuged in Beckman
SW-41 rotor at 38 K rpm for 90 minutes at 4° C. Gradients
were fractionated and the virus band collected using an ISCO
density gradient fractionator and UV analyzer
(Instrumentation Specialities Co., Lincoln NE 68504). The
sucrose fractions containing virus were diluted 3:1 (v/v)
with .01M sodium phosphate buffer and centrifuged in Beckman
no.40 rotor at 38K rpm for 4 hours. Pellets were resuspended
in 0.01 M phosphate buffer pH 7.5, using a glass rod and

allowed to stand overnight. Concentration of the virus

85
preparation was determined at A250nm using a Gilford

spectrophotometer.

Root and cambial tissues of sour cherry used in these
experiments were sampled as previously mentioned (pages 77
and 78). Leaf and flower tissue from Chenopodium quinoa,
'National pickling' cucumber and field grown dandelions and
roots from dandelions were also sampled. Samples consisted
of 0.5 to 1.0 gm of tissue in various dilutions of cold
extraction (for ELISA) or grinding buffer (for dot-ELISA).
The tissue was ground by hand with a mortar and pestle or
with a Tissuemizer. After grinding, samples were strained
and stored at 4° C until further use.

Antisernm_2rnduction. Tomato ringspot virus antiserum
was produced in New Zealand white rabbits or in White Leghorn
hybrid laying hens. Rabbits were immunized by intramuscular
injections of 1.0 ml solutions (0.5 mg/ml TmRSV) emulsified
(1:1 v/v) in Freund's complete adjuvant (Difco Products Co.,
Detroit, Mi 48232) for the first injection and Freund's
incomplete for subsequent injections. A total of three
injections were made on a 10-12 day schedule. Rabbits were
bled from the marginal ear vein one week after the last
injection and at 7 to 10 day intervals for a total of four
bleedings. Rabbit serum used was from the third or fourth
bleeding. The blood was collected and left for 3 hours at
room temperature. The clear serum was pipetted off and
stored at 4° C until further use. Serum titers were

determined by gel double diffusion tests [8% agarose (Sigma

86

type 1)] using TmRSV infected cucumber as the test antigen.

Chickens were immunized by two separate wing vein
injections (70 ug/ml and 100 ug/ml TmRSV respectively ) on
day 1 and day 6 and one intramuscular breast injection on day
13 (700 ug/ml TmRSV, using Freunds' Incomplete adjuvant). A
total of 10 eggs were collected on days 17, 20, 23, 28, 31,
32, 35, 37 and 39. Antibody used was from the yolk of eggs
collected on days 32, 35 and 37 and combined.

1mmnngglghnlin_2;eparatign+ Anti-TmRSV-globulins (IgG)
from immunized rabbits were prepared by precipitation from
antisera with ammonium sulfate (Clark and Adams, 1977). Ten
ml of saturated ammonium sulfate was added dropwise to 1:10
dilution (v/v) of serum in distilled water and mechanically
stirred for 30 to 60 minutes. After centrifugation at 6K rpm
(Beckman no.40 rotor) for 5 minutes, the pellet was collected
and resuspended in 2.0 ml (v/v) half-strength PBS. This
globulin fraction was dialyzed three times against 500 ml
half-strength PBS and passed through a 5 cm column of
DEAE-cellulose (Sigma) in a 10 ml glass pipette.
Half-strength PBS was used to elute the gamma-globulin
fraction from the column. Two ml protein fractions were

collected by monitoring the effluent spectrophotometrically

at A280nm- The first fractions with a peak absorbing at

A280nm were collected and spectrophotometrically adjusted to

a 1.0 mg/ml concentration.
Anti-TmRSV-chicken globulins (IgG) were obtained from

the eggs of immunized hens as described by Bar-Joesph and

87
Malkinson (1980). Egg yolks were mixed with PBS, (20ml/yolk)
and centrifuged (10K, 20min). The supernatant was mixed 1:1

(v/v) with glycerol for storage at -20°C.
The F(ab')2 fragments of immunoglobulin were prepared

by pepsin digestion. Rabbit-produced anti-TmRSV-IgG (lug/ml
in 0.07 M sodium acetate pH 4.0 containing 0.05 M NaCl) was
digested by the addition of pepsin (Sigma) diluted

1:1000(v/v) in distilled water (45 mg/ml) and incubated
overnight at 37° C. The F(ab')2 fragments were separated

from the products of digestion by dialysis against three
changes of PBS.

ggnjngatign_gf_1mmungglghnlin+ Alkaline phosphatase
(AP) (Sigma Type VII-S) and hydrogen peroxidase (HRP)(Sigma)
was coupled to gamma-globulin by the gluteraldehyde method of
Avrameas (1969) as described by Clark and Adams, (1977). A
1.0 ml aliquot of AP or HRP was centrifuged at 6K rpm in a
Beckman no. 40 rotor for 5 minutes. To the pellet, 1.0 ml of
purified anti—TmRSV gamma-globulin was added followed by
dialysis three times against PBS, pH 7.4. Conjugation of
enzyme to the IgG-fraction was facilitated by the addition of
glutaraldehyde (Sigma,electron microscope grade) to a final
concentration of 0.05 % (v/v) for 4 hours at room
temperature. Glutaraldehyde was removed by dialysis against
PBS three times and conjugated-anti-TmRSV-IgG was stored with
1.0 % bovine serum albumin (BSA) (w/v) at 40° C.

Hydrogen peroxidase-labeled rabbit-anti-chicken-IgG was

obtained from U.S. Biochemical Co. (Cleveland, OH 44122).

88
Protein A, (from Staphylococcus aureus) conjugated to HRP and
AP was purchased from Cooper Biomedical (Malvern, PA 19355)
and Zymed Laboratories (So.San Francisco, CA 94080),
respectively.

DAS:ELISA. Assays were performed as described on page
42, however, both AP- and HRP-conjugated-IgG systems were
tested. For the AP system, the coating antibody was used at
0.5 pg/ml, and for the HRP system the coating antibody was
used at 1.0 ug/ml. Both conjugates were used at a dilution
of 1/800 (v/v) in extraction buffer. The substrate for the
HRP-labeled antibody was O-phenylene diamine in OPD substrate
buffer. Plates were read after 15 and 30 minutes using a
Dynatech Microelisa® minireader with a 410 nm (AP) or 490 nm
(HRP) filter.

HADASrELISA. The assay procedure was carried out in
Microelisa® polystyrene plates and consisted of the following
steps: 1) A 200 ul solution containng 1.0 ug/ml
rabbit-produced anti-TmRSVeIgG in sodium carbonate buffer, pH
9.6 was placed in each well. The plate, wrapped in plastic
was incubated at 37° C for 1.5 hours. 2) Plates were emptied
and rinsed with a phosphate buffered saline solution, pH 7.4
with 0.5 % (v/v) polyoxyethylene sorbitan monolaurate (Tween
20). This procedure was repeated at least three times with
at least 3 minutes between rinses. 3) A 200 ul sample of
tissue extract was placed in each plate well. Each plate
included four to eight healthy controls wells, two to three
diseased control wells and one to two wells containing

extraction buffer only. The remaining wells were filled with

89
test samples, which were each replicated twice on the same
Microelisa‘® plate. Filled plates containing sample extracts
were stored in plastic bags at 5° C overnight. 4) After
rinsing as per step 2, a 200 ul sample containing a 1:500
(w/v) dilution of chicken-produced anti-TmRSV-IgG in PBS
containing 2.0 % BSA was placed in each plate well. The
plate, wrapped in plastic was incubated at 37° C for 1.5
hours. 5) After rinsing as per step 2, a 200 pl sample
containing HRP-conjugated rabbit anti-chicken-IgG diluted
1:1000 (v/v) in PBS was placed in each plate well. The
plate, wrapped in plastic was incubated at 37° C for 1.5
hours. 6) After rinsing as per step 2, a 200 pl sample
containing a solution of O-phenylene diamine (0.7 mg/ml) in
OPD substrate buffer was place in each plate well. Plates
were allowed to incubate and after 15 and 30 minutes were
read using a Dynatech Minireader equipped with a 490nm
filter. Photometrically, samples were considered

ELISArpositive if the mean absorbance at 490nm of the
duplicate sample wells was greater than the mean A490 nm

value of the healthy control samples plus three standard

deviations.
Elab112_ELISA. The protocol for the F(ab')2 assay was

reported on page 81. In addition, a similar system using
AP-conjugated protein-A was tested. The enzyme substrate for
the AP-protein—A moeity was a solution of p-nitrophenyl
phosphate (1.0 mg/ml) in substrate buffer [10% diethanolamine
(v/v) ,pH 9.8].

90

DQL;ELISA. The method of Bantarri and Goodwin (1985)
was used. The assay was carried out on nitrocellulose
membranes (NCM BioRad 4.5 Hm, BioRad Laboratories Richmond,
CA 94804). All wash steps consisted of five six-minute
rinses on a gyratory shaker in a solution of Tris-buffered
saline containing 0.05 % Tween 80 (TBS-T80 buffer). All
incubations were in a covered pyrex dish. The NCM was
handled with forceps or with gloves. Assay steps were as
follows: 1) The NCM was incubated four hours at room
temperature with a solution of 60 ul anti-TmRSV-IgG in 60 ml
sodium carbonate buffer, pH 9.6.(a 1:1000 v/v dilution) 2)
The NCM was washed and then transferred to 60 ml TBS°T80
containing 3.0 % BSA (w/v) (blocking step) and incubated 1.5
hours at 37° C. 3) Sample extracts [tissue ground in TBS-T80
containing 10 % PVP (grinding buffer)] were added to the NCM
which was placed in a BioRad Bio Dot® apparatus. Samples
were incubated overnight at 4° C. 4) After removal from the
apparatus, the NCM was washed and then transfered to 60 ml of
a 1:800 (v/v) solution of AP-conjugated anti-TmRSv—IgG in
TBS-T80 and incubated 3 hours at room temperature. 5) The
NCM was again washed, then incubated for 30 minutes to 1 hour
in 60 ml of a substrate solution [6.0 mg/ml fast red salt
(Sigma)] in 0.2M Tris HCL pH 8.2, filtered,combined in a 1:1
ratio with 0.1 % AS-MX phosphate (Sigma) in 0.2 M Tris HCL.
6) The NCM was then rinsed with distilled water, air dried or
blotted dry and read visually for color reaction. Samples
were considered positive if a red to pink dot appeared and

negative if no dot appeared.

RESULTS

WSW

Tomato ringspot virus was detected in trees from eight
out of the twelve southwest Michigan orchards sampled. The
highest level of infection was in orchard number three, a
declining orchard, where 50 percent of the trees tested
ELISA-positive for TmRSV. Incidence of TmRSV for the
individual orchard blocks is shown in Table 3.1. The six
decline orchards had an average of 23.67 percent of the trees
that tested ELISA-positive for TmRSV while the non-decline
orchards had an average of 9.67 percent of the trees that
tested ELISA-positive for TmRSV. Chi-square analysis found
the incidence of trees testing ELISA-positive for TmRSV in
the decline and non-decline orchards to differ significantly
(2?.001) from the expected 1:1 ratio (Table 3.2). Figure 3.1
shows the percentage of trees infected in individual orchards
and the overall mean percentage of trees that were detected
as ELISA-positive for TmRSV in the six decline and six
non-decline orchards.

No TmRSV was detected in any of the 720 nursery trees

tested at the three area nurseries.

muraluation
The individual tree evaluation data can be considered
in relation to TmRSV infection. Table 3.3 compares the

stunting, blindwood, tree death and tree replant percentages

91

92

Table 3.1 Incidence of trees ELISA-positive for tomato
ringspot virus in six declining and six non-declining sour
cherry orchards in southwest Michigan.

 

Number of Trees
TmRSV positive

 

 

Orchard
Block Orchard Number of Incidence
Number Condition Trees Sampled of Virus(%)
1 Decline 23/50 46.00
2 Decline 9/50 18.00
3 Decline 25/50 g 50.00
4 Decline 2/50 4.00
5 Decline 8/50 16.00
6 Decline 4/50 8.00
7 Non Decline 4/50 8.00
8 Non Decline 8/50 16.00
9 Non Decline 0/50 0.00
10 Non Decline 0/50 0.00
11 Non Decline 10/50 20.00

12 Non Decline 7/50 14.00

93

Table 3.2 Chi-square analysis of TmRSV incidence
between decline and non-decline orchards.

 

 

 

Treatment

Comparison Observeda Expectedb X2 P
Decline/ +71 29 1:1 20.17d <.001
Non-DeclineC -229:271

 

aObserved values are for ELISA positives (+) and ELISA
negatives (-) for the compared treatments summed over the
sampling dates.

bNull hypothesis: The number of diseased and healthy trees
are equal between the compared treatments.

CDecline treatments consisted of 300 individual 'Montmorency'
trees on 'Mazzard' or 'Mahaleb' rootstock from six declining
orchards. Decline treatments consisted of 300 individual
'Montmorency' trees on 'Mazzard' or 'Mahaleb' rootstock from
six non-declining orchards.

dA significant X2 indicated that TMRSV incidence is
not independent of decline and non-decline treatments.

94

moHHoooumo DSHHooplco: pom ocHHoop How coHuooucH mo mo=Hm> some 9539:.“
3.3598 won moouu om. pumnouo >n >939 How 0>HuHmOQ|¢mHHw moouu mo ommucoouom H.m 393nm

osHm> oon> 50:52
can: NH 2 2 a. e a can: a m e m N. H Al nonzero
- 88

 

 

 

oo.m

oo.OH

oo.mH

oo.o~

oo.mm

5421a3033>

oo.om

0P

oo.mm

oo.ov

oo.mv

 

oo.om

mQMdmomo mZHHUMQIZOZ mamdmomo MZHHUHQ

95_

Table 3.3 Percent tree replant,stunting,blindwood,and tree
death for trees ELISA-positive for TmRSV and trees
ELISA-negative for TmRSV between and within decline and
non-decline orchards.

 

 

Category % Trees % Trees % Trees % Trees
with with that were that were
Stunt ing Blindwood Dead Replanted

(A)

+a/declineb 0.00 57.14 3.17 12.69

-/decline 3.74 58.28 2.67 10.16

(B)

+/non-decline 3.45 6.90 0.00 0.0

-/non-dec1ine 5.54 12.92 0.37 1.85

(C)

Decline

TmRSV + & - 2.80 58.00CH-001) 2.80(-05> 10.8<-001)

Non-Decline

TmRSV + & - 2.67 6.17 0.17 0.83

(D)

All TmRSV 1.08 41.30 2.17 8.69

positives

All TmRSV 4.80 31.44 1.31 5.24

negatives

 

517- TmRSV-ELISA positive, - - TmRSV-ELISA negative

bDecline orchards included 250'Montmorency' trees on
'Mazzard' or 'Mahaleb' rootstock. Non-Decline orchards
included 300 'Montmorency' trees on 'Mazzard' or 'Mahaleb'
rootstock.

cValue is significantly greater than the corresponding values
(E,= number in parenthesis) when total incidence of a
characteristic is compared within categories by chi-square
analysis.

96
in four different categories. One category, (A) compares the
trees that tested ELISA-positive for TmRSV to the trees that
tested ELISA-negative for TmRSV in the decline orchards.
Another category, (B) compares the trees that tested
ELISAepositive for TmRSV to the trees that tested
ELISA-negative for TmRSV in the non-decline orchards. A
third category, (C) compares all the decline orchards to all
the non-decline orchards without regard to TmRSV infection.
The fourth category, (D), compares all the trees (decline or
non-decline) that tested ELISA-positive for TmRSV to all the
trees that tested ELISA-negative for TmRSV. According to
chi-square analyses, the decline versus non-decline category
was the only category that was shown to have significant
differences in terms of the levels of these four
characteristics. The levels of blindwood, tree death and
replants were all significantly higher in the decline
orchards than in the non—decline orchards. Levels of tree
stunting between decline and non-decline orchards did not
differ significantly.

Vigor ratings given to the orchard trees are compared
within the four categories in Table 3.4. In comparison
category (A), significantly more decline trees that tested
ELISA-positive for TmRSV than decline trees that tested
ELISA-negative for TmRSV were given the low vigor ratings 1
and 2. Similarly, in the overall decline versus non-decline
category (C), the decline trees had significantly more trees
given the low vigor ratings 1 and 2 than did the non-decline

trees. Also when the trees that tested ELISA-negative for

97

Table 3.4 Tree vigor ratingsa for trees ELISA-positive for
TmRSV and trees ELISA-negative for TmRSV between and within
decline and non-decline orchards.

 

 

Mean % Trees % Trees % Trees
Category Vigor Vigor Vigor Vigor
Rating Rating 1 & 2 IRating 3 Rating 4 & 5
(A)
+b/declineC 2.41 38.09d<JW1> 49.20 9.5
-/decline 2.05 18.18 63.10(-°5) 16.04
(B)
+/non-decline 3.03 10.35 79.31‘45) 10.34
-/non-decline 3.55 9.6 50.92 38.70(-WU
(C)
Decline
TmRSV + & - 3.78 23.20<-0¢U 59.60 14.40
Non-Decline
TmRSV + & - 3.51 9.67 53.67 36.00<-0¢H
(D)
All TmRSV
positives 2.61 29.34(-0°1) 58.69 9.78
All TmRSV
negatives 3.29 13.10 55.89 29.47(-°W“

 

aVigor rating system: 1 = tree excessively stunted; branches
sparsely foliated,few fruit spurs; 2 - tree slightly
stunted; low fruit spur to shoot growth ratio; 3 = tree
growth average;slightly more shoot growth to fruit spur
growth; 4 = tree growth average to above average; nearly
equal ratio of fruit spur to shoot growth; 5 = tree growth
above average; branches full; fruit spur to shoot growth
equal or better.

b+ . TmRSV-ELISA positive, - a TmRSV-ELISA negative.

CDecline orchards included 250'Montmorency' trees on
'Mazzard' or 'Mahaleb' rootstock. Non-Decline orchards
included 300 'Montmorency' trees on 'Mazzard' or 'Mahaleb'
rootstock.

dValue is significantly greater (P = number in parenthesis)
than the corresponding value when vigor classes were compared
within category by chi-square analysis.

98
TmRSV were compared to the trees that tested ELISA-positive
for TmRSV [comparison category (D)], the latter had
significantly more trees rated at these two lower vigor
levels. The incidence of high vigor ratings 4 and 5 was
shown to be significant in three of the four categories.
Categories (B) and (D) indicated that significantly more
TmRSV-free trees were given these high vigor ratings.
Category (C) indicated that significantly more non-decline
trees were given a 4 or 5 rating.

Levels of trunk damage within the same four comparison
categories are indicated in Table 3.5. As was true in
previous trunk damage comparisons, the decline orchards
overall had significantly more trunk damage than the
non-decline orchards. Trees that tested ELISAepositive for
TmRSV also had significantly more trees rated with damage
than did the trees that tested ELISA-negative for TmRSV.
Tomato ringspot virus was also significantly associated with
total damage within the non-decline orchards.

Another comparison that can be made for the
TMRSV-positive trees is that of incidence of PNRSV and PDV.
These comparisons are shown in Table 3.6. Overall, PNRSV
infection was significantly higher in the trees from the
decline orchards than in the trees from the non-decline
orchards,[category (C)] as was previously shown. In
addition, the TmRSV-positive trees had significantly more
trees also infected with PNRSV than did the trees without
TmRSV [category(D)]. Within the decline orchards as well

[category (A)], significantly more trees that were

99

Table 3.5 Percent trunk damagea on trees ELISA-positive for
TmRSV and trees ELISA—negative for TmRSV between and within
decline and non-decline orchards.

 

 

Category 7% Trees % Trees % Trees *Total
Damage 0 Damage 1 Damage 2 Damage 1 & 2

(A)

+b/declineC 69.87 6.34 23.80 30.14

-/decline 74.86 9.62 14.43 24.05

(B)

+/non-decline 89.66 3.45 6.90 10.3sd<-05>

-/non-decline 94.46 2.58 1.85 4.43

(C)

Decline

TmRSV + & - 73.60 8.80<-0°1) 16.80<-°°1) 25.6<-001>

Non-Decline

TmRSV + & - 94.00(-°¢U 2.67 2.33 5.00

(D)

All TmRSV

positives 76.08 5.43 18.47< 05) 23.9o<-01)

All TmRSV

negatives 86.46h05) 5.45 6.98 12.43

 

aTrunk damage rating system : 0 = no visible trunk damage; 1
- visible mild trunk damage; 2 = visible severe trunk damage.

b+ - TmRSV-ELISA positive; - = TmRSV-ELISA negative.

CDecline orchards included 250'Montmorency' trees on
'Mazzard' or 'Mahaleb' rootstock. Non-Decline torchards
included 300 'Montmorency' trees on 'Mazzard' or 'Mahaleb'
rootstock.

dValue is significantly greater (P_= number in parenthesis)
than the corresponding value when damage classes were
compared within category by chi-square analysis.

100

Table 3.6 Percent PNRSV and PDV ELISA-positivesa among trees
ELISA-positive for TmRSV and trees ELISA-negative for TmRSV
between and within decline and non-decline orchards.

 

 

Category % PNRSV % PDV
(A)

+b/declinec 80.2ed(-°5) 0.00
-/decline 63.76 3.93
(B)

+/non-decline 37.93 0.0
-/non-decline 23.62 1.11
(C)

Decline

TmRSV + & — 67.67(-°01> 3.00
Non-Decline

TmRSV + & - 12.50 0.50
(D)

All TmRSV

positives 68:001-0m” 0.00
All TmRSV

negatives 42.00 2.40

 

aAll trees tested by DAS-ELISA in 1983, 1984.
b+ - TmRSV-ELISA positive; - = TmRSV-ELISA negative.

CDecline orchards included 250‘Montmorency' trees on
'Mazzard' or 'Mahaleb' rootstock. Non-Decline orchards
included 300 'Montmorency' trees on 'Mazzard' or 'Mahaleb'
rootstock.

dValue is significantly greater (P = number in parenthesis)
than the corresponding value when PNRSV and PDV
ELISA-positives were compared within category by chi-square
analysis.

101
ELISA-positive for TmRSV were ELISA-positive for PNRSV than

were the trees that tested ELISA-negative for TmRSV.

Dil'll' ETBSIE ill) [I] I

The distribution of TmRSV in cambial tissues around
the union of six naturally infected TmRSV positive trees is
reported in Table 3.7. When the cambial samples from the
four locations on the tree were combined, in all six cases
TmRSV was detected. Individually, the four cambial samples
from a given tree were not always positive. No cambial
samples were positive for tree C when individually tested,
although the combined sample was positive for TmRSV. Trees
B,D,E had one of the four samples positive individually while
trees A and F had three of the four positive individually.
No virus was detected in the leaf and root or sucker samples
taken from these six trees. Of the samples taken above and
below the graft union only those five centimeters below the
union were positive and not in all six trees. No samples
taken more than five centimeters below the graft union or
those taken above the graft union were positive for TmRSV.

Table 3.8 shows the results of the testing of 150
greenhouse grown TMRSV-bark chip inoculated 2-year old trees.
Overall, the incidence of virus detected was relatively low,
with 4.16 percent the highest level of virus in any of the
tissues tested. The sucker and cambial tissues had the most
TmRSV positive samples.

When five trees that had tested ELISA-positive for

TmRSV were tested repeatedly over a three month period, TmRSV

102

Table 3.7 TmRSV distribution in cambial tissues of
11-year-old naturally infected cherry trees.

 

 

 

SAMELEa

N-S-E-W Healthy
TREE North South East West Combined Mean
A .15b(+)c 15 (+) .15 (+) .05 H .13 (+) 0.04
B .10(+) .06(-) .05(-).04(-) .14(+) 0.04
C .06(-) .06(-) .06( ).07(-) .13(+) 0.04
D .06(-) .05(-) .06( ).11(+) .13(+) 0.04
E .02(-) 03(-) .03( ).10(+) .18(+) 0.02
F .02(-) 18(+) .26( ).11(+) .14(+) 0.02
5North, South, East, West samples consisted of 2 cm plug of

cambial tissue from respective location on trunk.

bNumber is A405nm value of the sample.

c(+)

- positive for TmRSV,
ELISA.

(-)

- negative for TmRSV, by
Samples were considered positive if the mean A405nm

value of the of two replicate wells exceeded two times the

mean value of five healthy sample wells.

103

Table 3.8 TmRSV distribution and detection in 'Montmorency'
trees on 'Mahaleb' rootstock nine months post-inoculationa
with TmRSV-positive wood chip.

 

IISSHE_IXEE

Cambial Leaf/Flower/Bud. Root. Sucker

 

Number Positive b

 

Number Sampled 3/150 1/150 0/150 3/72

Virus Incidence (%) 2.00 0.66 0.00 4.16

 

aTrees were inoculated at one year of age by inserting at the
graft union two 1.5 centimeter wood chips taken from the
graft union of a tree that had tested ELISA-positive for
TmRSV.

bSamples considered positive if the mean A405nm value of two

replicate wells was over two times the mean of healthy
controls.

104
detection was variable (Table 3.9). One tree (B), although
positive before the study, failed to yield a TmRSV-positive
response at any of the five sampling dates. The other four
trees were positive on at least two but not more than four of
the five sampling dates. On any given sampling date the
highest rate of TmRSV detection was four out of the five
trees tested. Figure 3.2 graphically illustrates the
varibility of virus detection over the three month period and

within the five trees.

Wild!

Table 3.10 compares the characteristics of this study
for the TmRSV-positive and the TmRSV-negative trees within
the decline and non-decline groups selected for this
experiment. In addition, the overall decline and non-decline
groups without regard to virus infection are compared and the
TmRSV-positive group is compared to the TmRSV-negative group.
The characteristics shown in this table are mean vigor, the
mean percent positive dandelion for each group, and soil and
root nematode levels measured. It is only in the category of
overall decline versus non-decline status [category(C)], that
statistical significance is seen. The trees in the decline
group had significantly more of the surrounding dandelions
TmRSV-positive than did the non-decline group. The
non-decline group however, had significantly more
criconemella nematodes and significantly more Pratylenchus

per 100cc of soil.

105

Table 3.9 TmRSV detection in cambial tissues from five
naturally infected cherry trees at intervals over a 68-day
period.

 

 

SAMELEa
TREE Day 1 Day 11. Day 20 Day 34 Day 68 Totals
Healthy
Mean 0.0851) 0.09 0.06 0.09 0.11
A O.56(+)CO.24(+) 0.18(+) 0.30(+) 0.17(-) 4/5
B 0.10(-) 0.12(-) 0.09(-) 0.14(-) 0.15(-) 0/5
C 0.21(+) 0.68(+) O.12(-) 0.20(+) 0.09(-) 3/5
D 0.19(+) 0.43(+) 0.16(+) 0.27(+) 0.09(-) 4/5
E 0.20(+) 0.69(+) 0.07(-) 0.11(-) 0.09(-) 2/5
Day Total 4/5 4/5 2/5 3/5 ' 0/5

 

3Each sample consisted four cambial samples taken at four
equidistant locations at or below graft union, combined. All
trees were tested positive for TmRSV at least once before day
1.

bNumber = A405nm value, each value is average of two
replicate wells

C(+) - positive for TmRSV,'(-) _ negative for TmRSV : by
ELISA. Samples were considered positive if the mean A405nm

value of two replicate wells exceeded two times the mean of
value of five healthy sample wells.

 

 

s DAY 34 D DAY 68

S DAY11

I DAY1

l DAY 20

 

 

106

 

 

 

 

 

 

I
P
O

I l l l’ I I
L0 I!) Q' m N H
O C) O O O O

<Qmounmcoo mu ¢om CE

Figure 3.2 ELISA values for five 11 year-old previously ELISA-positive (+) cherry trees
tested five times over a 68 day interval

107

Table 3.10 Mean vigor of cherry trees and mean percent
TmRSV-positive dandelions and nematode density surrounding 30
cherry trees in three decline and three non-decline orchards.

 

 

WC
Mean %
Mean Positive Xiphinema Criconemella
Pratylenchus
Category Vigora Dandelionb soil soil soil root
+°/decline° 2.87 2.00 % 20.27 0.80 0.13 18.73
-/decline 2.50 1.73 % 25.93 0.80 1.80 3.80
+/non-decline 3.20 0.33 % 51.00 10.33 2.87 2.47
-/non-decline 3.00 1.27 % 29.93 5.87 3.07 1.53
Decline
TmRSV + & - 2.68 1.87 %f<-°1) 23.10 0.80 0.97 11.27
Non-Decline
TmRSV + & - 3.10 0.80 % 40.47 8.10‘J1’ 3.0“°“2.00
All TmRSV
positives 3.03 1.17 % 35.63 5.57 1.50 10.60
All TmRSV
negatives 2.75 1.50 % 27.93 3.33 2.43 2.67

 

a Vigor rating system:1 - tree excessively stunted; branches sparsely
foliated,few fruit spurs; 2 - tree slightly stunted; low fruit spur to
shoot growth ratio; 3 - tree growth average;slightly more shoot growth
to fruit spur growth; 4 - tree growth average to above average; nearly
equal ratio of fruit spur to shoot growth; 5- tree growth above
average; branches full; fruit spur to shoot growth equal or better.

b 10 dandelions / per tree were tested for TmRSV by F(ab)'2 ELISA.

cPer 100cc soil or 1 gram root.

d+ - TMRSV-ELISA positive, - . TMRSVéELISA negative.

eDecline treatment included 30 (15 TmRSV positive/15 TmRSV
negative)'Montmorency' trees on 'Mazzard' or 'Mahaleb’ rootstock.
Non-Decline treatment included 30 'Montmorency' trees (15 TmRSV
positive/15 TmRSV negative) on 'Mazzard' or 'Mahaleb' rootstock.
fValue is significantly greater than the corresponding values (2,-
number in parenthesis) when total incidence of a characteristic is
compared within categories by chi-square analysis.

108

W

The results of the comparison of symptom expression to
TmRSV detection are shown in Table 3.11. Approximately
one-half of the trees that were ELISA-positive for TmRSV
infection also showed cambial pitting and grooving symptoms.
Approximately one-third of the trees ELISA-negative for TmRSV
infection, in contrast, did have cambial pitting and grooving

symptoms.

WM

Tables 3.12 and 3.13 reveal the results of the two
assay comparisons. Table 3.12 indicates the relative
sensitivity of different immunoassay methods and conjugated
enzymes in detecting TmRSV. While the DAS-ELISA alkaline
phosphatase system could detect TmRSV in cucumber at the
highest dilution (1:10000) it failed to detect TmRSV in

infected tree cambial tissues. All three indirect ELISA
systems - F'(ab)2 alkaline phosphatase and horseradish

peroxidase, and HADAS-ELISA, could detect TmRSV in cucumber
and cambial tissues with a similar sensitivity.

Table 3.13 shows the relative sensitivities of
F'(ab)2-ELISA and dot-Blot immunoassay in detecting TmRSV,
both purified and in various tissues. Both systems could
detect 0.01 pg of TmRSV but the F'(ab)2-based system was more

sensitive at detecting TmRSV diluted in healthy sap.

Dot-ELISA did not detect TmRSV in cherry roots and was not as

109

Table 3.11 Expression of cambial pitting and grooving
symptoms vs. TmRSV infection in 'Montmorency' cherry.a

 

SXMEIQMS

NumheLSamnledGronmng Wing

(percent)
POSITIVE 57/150 23/57 34/57
for TMRSVb (38.0%) (40.3%) (59.6%)
NEGATIVE 93/150 35/93 58/93
for TMRSV (62.0%) (37.6%) (62.4%)

 

a150 'Montmorency' on 'Mazzard' or 'Mahaleb' rootstock sour
cherry trees.

bTrees tested for TmRSV by F'(ab)2 ELISA in 1985 . One sample
equaled four 5 cm cambial discs taken at or below the graft
union. Samples were considered positive if the mean A405

value of two replicates exceeded the mean of four replicates
of healthy controls.

110

Table 3.12 The relative sensitivity of different immunoassay
methods and enzyme labelled gamma-globulins in detection of
tomato ringspot virus in infected cherry tissue.

 

The highest sample

dilution at which

TmRSV was detecteda
Immunosorbent assay =
methodb and enzyme

Inner bark and
cambium

 

Number
A405nm mail-.118

 

used for y-globulin CucumberInner bark healthy'Number
conjugation leavesC and cambiumd-mean sampled
DAS-ELISA,peroxidase 600 5 .125 2/11
DAS-ELISA,alkaline

phosphatase 10000 — .16 0/11

F' (ab) 2-ELISA,

peroxidase 3000 10 .075 6/11
F’(ab)2-ELISA alkaline

phosphatase 3000 10 .115 2/11
HADAS-ELISA,peroxidase 4000 10 .18 2/11

 

aELISA absorbance values at least twofold higher than the
absorbance values of comparable controls (TmRSV-free tissue)

on the same microtiter plates.

bDAS-ELISA = double antibody sandwich method of ELISA;
F'(ab)2 = F'(ab)2-based indirect ELISA; HADAS = heterologous

double antibody sandwich method of ELISA.
CArtificially inoculated with TmRSV.

dNaturally infected with TmRSV.

111

Table 3.13 Relative sensitivity of F(ab')2 ELISA and
dot-Blot immunoassay in detecting TmRSV.

 

DETECTION LIMIT5:;

 

VIRUS SOURCE dot-Blot F'(ab)2

 

Purified TmRSV .01 pg/ml .01 pg/ml

Purified TmRSV in sap

of healthy cherry root 100 pg/ml .01 pg/ml
Cherry roots no detection 1:10b
Dandelion roots 1:100 1:1000
Cucumber leaves 1:10,000 1:100,000

 

aLevel at which the ELISA absorbence value (A405nm) was at

least two times the comparable healthy tissue value or where
red pigment was seen on the dot-Blot nitrocellulose membrane.

b Ratios indicate the dilution level of sample:extracting
buffer (gm:ml).

112
sensitive as F'(ab)2-ELISA in detecting TmRSV in dandelion

roots and cucumber leaves.

DISCUSSION

In the twelve orchards sampled for this study, the
levels of TmRSV infection found were variable. Infection
levels overall were lower than the orchard infection levels
of PNRSV but higher than orchard infection levels of PDV.

The orchards tested ranged in levels of TmRSV infection from
zero to 50 percent of the fifty trees per orchard tested.
All six of the decline orchards tested had some TmRSV
infection present, and the decline orchards had over twice
the TmRSV infection levels than did the non-decline orchards.
While overall the level of TmRSV found in this study
was_not extremely high, the levels found could be important
due to the potentially serious effects of TmRSV on tree
health. Even though TmRSV is somewhat localized at the base
of infected fruit trees, studies in apple and peach show the
virus can cause serious translocation problems as a result of
tissue disorganization at the graft union (Tuttle and
Gotleib, 1982). This can result in the death of the canopy,
weakened root systems, and related winter injury (Lister et
al., 1980; Stouffer, 1969). Associations have also been made
with willowy, sparse growth habits (Stouffer et al., 1969),
although other researchers have not found this to be a
consistent feature of TmRSV infection. In our study there
were both decline and non—decline orchard trees found to be
infected with TmRSV. A preponderance of the TmRSV infected

trees were of decline status, however, pointing to an

113

114
association of viral infection and poor tree health.

As noted by Cummins and Gonsalves (1982), there are
many factors that can influence the transmission,
maintenence, reproduction and recovery of TmRSV and some of
these factors could have affected the results of the TmRSV
field survey. In particular, tree to tree differences in
establishment of vascular tissues after grafting, and
variations in defense mechanisms could have affected the
susceptibility of trees to TmRSV, as well as the recovery of
TmRSV from plant tissues. Trees that were all equally
exposed to the virus in the field may, due to individual
variability, show differences in infection levels. Mircetich
and Boy (1981) noted that TmRSV was easily detected in the
early stages of infection with Prunus brownline disease in
plum and prune, and less efficiently detected as the trees
became symptomatic and were dying. This phenomenon may also
be true with TmRSV infection in cherry.

In addition, methods of TmRSV detection in the orchard
trees may have affected the results. In this study, the fifty
selected trees in each orchard were tested once. This was
done to obtain a 'moment in time' test for virus in the
trees, and to ascertain virus levels in a relative sense.
Since TmRSV is known to be irregularly distributed, it can be
speculated that repeated testing of the trees for TmRSV could
have resulted in higher infection levels in the orchards
sampled.

The distribution of the TmRSV-infected trees within

the individual orchard blocks appears to be random in nature.

115
Many more samples, perhaps the entire 200 tree block instead
of the chosen 50, would need to be taken in order to clearly
establish any viral distribution patterns. When the
pre-plant fumigation status for each orchard was noted, it
was found that both of the orchards that had zero percent
infection with TmRSV had been pre-plant fumigated. The
orchard with 46 percent TmRSV infection, however, also had
been pre-plant fumigated. Perhaps a inefficient fumigation
job was done in this case. If one assumes an equal
efficiency of the fumigation in all cases, this could
indicate that TmRSV levels in the orchards may be associated
with factors other than original Xiphinema levels in the soil
at planting.

The lack of TmRSV infection at the three nurseries may
indicate that at least some of the TmRSV infection in
southwest Michigan is due to field infection and not from
propagative sources. Due to the irregular distribution of
TmRSV in cherry, and the primary localization of virus at or
below the graft union, the spread of this virus through
budding of scion wood to rootstocks is probably relatively
infrequent. It should also be noted in this study that the
total sample size was rather small (250 trees). The sampling
method used, in effect, sacrificed the trees tested, so a
larger sample size was not feasible.

In considering the individual tree evaluation data, it
is important to note that this TmRSV study represents a
subsample of the PNRSV/PDV test group, with fifty and not two

hundred trees virus tested per orchard. The tree replant,

116
tree death, and blindwood data that were shown to be
significant in Table 3.3 were also significant for the
decline and non-decline fields overall, when two hundred
trees were rated at each orchard. These factors seem to be
positively associated mainly with the decline status of the
orchard and not in particular with TmRSV (or PNRSV/PDV)
infection of the trees. At the least, these results verify
that declining orchards have more dead, replanted and poor
appearing trees than non-decline orchards.

The vigor of the trees, in contrast, appears to be
somewhat virus related. More of the trees rated at the lower
vigor levels of 1 and 2 were decline trees than were
non-decline trees. Also more TmRSV-positive trees (in decline
and non-decline orchards combined) were rated at 1 and 2 than
were TmRSV-free trees.

At the higher vigor levels as well, there appears to
be an influence of TmRSV infection on vigor. When compared
to the TmRSV-positive trees, the TmRSV-free trees show
significantly more trees at the higher vigor levels 4 and 5.
Tomato ringspot virus infection in other species (peach,
apple, plum) is known to affect vigor by reducing terminal
growth, blocking effective vascular conduction, and affecting
root development (Kirkpatrick et al., 1958; Lott, 1967; Lewis
et al., 1968; Barratt et al., 1968; Stouffer and Uyemoto,
1976). Our studies on cherry indicate that TmRSV infection
probably does not cause noticable changes in average orchard
vigor. However, TmRSV presence may be a factor in the

orchard trees that are extremely unhealthy, and the lack of

117
TmRSV seems to be a characteristic of the orchard trees that
are considered extremely healthy.

Trunk damage, similar to what was seen with the PNRSV
infection data, was positively associated with TmRSV-infected
trees. It is interesting to note that even though the
TmRSV-negative group is over four times as large, (450 trees)
the smaller TmRSV-positive group (100 trees) still has twice
as much trunk damage. As with PNRSV infection/trunk damage
associations, no significant conclusion can be drawn from
these results without further investigation on a tree by tree
basis. Speculation may be made on the influence of any
compromised health status (such as virus infection or trunk
damage from harvesting) and the effect of any further stress,
(such as harvesting damage or viral infection, respectively)
on trees. Tomato ringspot virus, which can affect the
strength of the union in some fruit trees, may, through
effects on the conductive tissues or on cambial/bark strength
promote or predispose the the trees to trunk damage. Damaged
cherry trees may be predisposed to or less likely to recover
from viruses due to their weakened condition, as Rosenberger
et al., (1983) found to be the case with apple. Perhaps
TmRSV and harvesting machine mechanical damage combine with
still other factors to create an interaction that puts the
trees in a overall stressed/declining state.

Prunus necrotic ringspot virus infection was also
significantly associated with TmRSV infection, and again,
only speculation can be made on this association without

further investigation. There is nothing in particular about

118
these two viruses that would predict their association in
nature. Orchards with high numbers of TmRSV-susceptible
dandelions located nearby older PNRSV infected fields would
certainly encourage the development of trees with multiple
infections. One virus may induce a weaker condition overall
and then allow increased infection and disease development by
the other virus. Prunus necrotic ringspot virus has been
shown to significantly affect the vigor and growth of trees
especially when infected while young. Tomato ringspot virus,
through field infection, may possibly occur on these already
weakened trees with greater frequency. Specific tests
designed to deduce the true relationship would need to be
done to determine whether this virus to virus relationship is
random or truly significantly related.

The experiment which investigated the distribution of
TmRSV at the union of TmRSV infected trees revealed what
appears to be an uneven distribution pattern of the virus
within the trees. In these naturally infected trees, it
appears that samples a few centimeters apart can be different
in virus presence. In addition, no TmRSV was found in the
roots or the leaves of these same trees. Bitterlin et
al.(1988), Cummins and Gonsalves (1986), and Lister et
al.(1980), found similar distribution phenomena of TmRSV in
other species. As Mircetich et al.(1971) noted, several
plant pathogenic graft-transmissible viruses are known to
have local distribution in infected fruit trees. Apple,
grape, plum, as well as small fruits, e.g. blueberry and

raspberry, are known to have irregular distribution of this

119
virus in particular. Our study agrees with Cummins and
Gonsalves (1986) in that it indicates that cambial sampling
for TmRSV in cherry should consist of samples from a number
of locations at or below the graft union, with the actual
number depending on the diameter of the tree trunk.

In the trees that were infected artificially with a
wood chip from a TmRSV-positive tree, little viral transfer
was found after nine months. The sucker and cambial tissues
were the most reliable of the four tissues tested, although
overall, little virus (4 percent of all the trees tested) was
detected. One reason for the low detection could have been
the length of time after inoculation. Another, more likely
reason may have been that the wood chips used were subject to
the same irregular distribution pattern shown in the graft
union Study. Further work that has been done with this virus
in our laboratory used a direct bark-slash inoculation method
(Bitterlin et al., 1987) with purified virus for artificial
infection. It appears from both the wood chip and the
purified virus studies, however, that successful rates of
TmRSV inoculation of cherry may be much lower than that
reported for peach, apple and plum.

The studies on detection of TmRSV over time may also
have been affected by an uneven distribution of the virus in
the tissues. The study itself was limited by the sampling
technique, and over-sampling of a finite virus reservoir may
also have affected the results. This particular study
indicated that detection of TmRSV in cambial tissues is

inconsistent. Other work with artifically inoculated trees

120
has shown that although the variablity of detection of TmRSV
in the cambium is also somewhat true in regard to root
tissue, the roots are the least invasive and most reliable
method of repeated testing, at least in young sour cherry.
Studies on virus spread or studies in which the presence of
TmRSV in tissues is important would have to account for this
variability in detection, possibly through repeated sampling
a minimum of four times.

It is possible that TmRSV infection and distribution
is unique in each individual tree. The implications of this
would have to be considered in any investigations involving
this virus. Only extensive testing such as that done by
Bitterlin and Gonsalves (1988) in peach or Cummins and
Gonsalves (1986) in plum can determine the precise locations
of virus within plant tissues and the probabilities of
detection in specific tissues. In comparison to our studies
it appears that the distribution of TmRSV in cherry is
similar to the distribution of TmRSV in plum and peach. In
all cases TmRSV is best detected in the tissues below the
soilline. In peach and plum, as in naturally infected
cherry, the cambial tissues at or below the graft union are
the most consistent source of TmRSV. Root tissues seem to be
most consistent in detection of the virus in young,
artificially inoculated cherry and a satisfactory source of
virus in naturally and artificially inoculated peach trees.
In contrast to TmRSV detection studies in apple, (Bitterlin
et al., 1984) TmRSV is rarely detected in cherry leaf tissue

or rootstock sprouts.

121

In the TmRSV infected orchard tree, dandelion and
nematode study, the limiting factor seems to be the virus
itself. Tree infection is, however, dependent upon actual
nematode feeding. Other research would suggest, especially
in the absence of known nursery infection, that
TmRSV-positive trees would be associated with increased
vector nematode populations and increased density of
TmRSV—positive dandelions. However, in this study,
population levels of nematodes were not positively correlated
with the viral status of either the trees or the dandelions.
Perhaps if more trees had been tested it would have
elucidated a clearer relationship. In this study the number
of TmRSV-positive dandelions were higher in decline orchards
than in the non-decline orchards. Yet overall, the number of
TmRSV-positive dandelions was lower near individual trees
that were TmRSV-positive than near the trees that were
TmRSV—negative. Powell (1984) had found more dandelions
infected with TmRSV in orchards with trees having
TmRSV-induced PSP symptoms than in orchards without PSP or in
non-orchard sites. Barrat et al., (1984) suggested that a
low rate of correlation between trees and adjacent weeds was
likely due to a low rate of virus detection in the trees and
this may have been a factor in our work as well. Overall,
Barrat did find that orchard health was not correlated with
infected dandelion levels. Rosenberger et a1. (1983)
suspected that the uneven distribution of virus in the
dandelions below trees is due to variation in the weeds that

survived herbicide application.

122

The nematode population was not significantly higher
in the decline sites, or near TmRSV-positive trees. In fact
the Xiphinema population was lower than in non-decline sites
or in the TmRSV-negative trees. One possible reason that the
nematodes were not present is that the declining or
TmRSV-infected trees and/or roots may be less vigorous and
therefore less appealing to the nematodes than are the
non-declining/non-TmRSV-infected roots. Powell (1984) also
found no difference in Xiphinema levels between the PSP
infected and PSP non-infected sites. Though Braun et al.,
(1966) found a positive relationship between levels of
Xiphinema and Pratylenchus and tree vigor, when this was
investigated no such association in cherry was found. In
addition, the density of Pratylenchus or Criconemella spp.
alone was not associated with the health of the tree,
although other studies (Parker and Mai, 1956; Okie and
Reilly, 1984)have shown that Pratylenchus is pathogenic to
cherry.

It is not clear from our study that TmRSV infection in
trees is always associated with TmRSV infection in dandelion
and with nematode presence. Undoubtably, the dandelions do
play a role in viral spread since the nematodes do lose virus
upon moulting and the dandelions are a source of
re-infection. A current study (Ramsdell et al, unpublished
data) shows rapid infection of healthy dandelion (through
nematode feeding) when in proximity of diseased dandelion,
which in an orchard setting could be a major factor in virus

spread. Certainly some tree infection by TmRSV does occur

123
from dandelion through nematodes. Considering the combined
seed and nematode transmission of TmRSV in dandelions,
orchard control of this weed should be considered in
controlling TmRSV, especially when other options such as
nematode control are difficult and sometimes prohibitively
expensive.

Rosenberger et al., (1983) and others have suggested
that there does seem to be more involved in TmRSV tree
infection than just virus presence. In our work as well,
there were several trees surrounded by dandelions infected
with TmRSV (up to 70 percent of the nearby population) and
high populations of Xiphinema, yet the trees were free of
TmRSV. Just what factors are at work in situations as these
are yet to be determined. Individual genetic and
physiological tree varibility, plant defense mechanisms,
nematode transmission specificity, TmRSV detection limits and
environmental differences may all be playing a role.

The study of symptom expression shows that cambial
pitting and grooving is simply not well correlated with TmRSV
infection in sour cherry. The large variation in symptom
expression in cambial tisssues combined with the
aformentioned virus distribution and detection problems make
pitting and grooving symptoms useless as an indicator of
TmRSV infection in cherry trees. Virus was either not in
the tissues that had symptoms or the detection methods were
inefficient. In addition, it is possible that early
infections are present in some trees and yet these trees are

not showing symptoms, or a particular stress is required for

124
symptom expression. In other cases, early symptoms could be
occuring, yet there is extremely low virus titer in these
trees.

Mink and Howell (1980) and AlMusa et al.(1980), also
showed pitting on otherwise normal (virus free) cherry trees.
Our study is also in partial agreement with Bittlerlin and
Gonsalves (1988) who showed that, in peach, TmRSV was in all
the symptomatic stem pitted tissues tested, but also in some
non-symptomatic trees.

Lewis et al. (1968), in work with peach showed that
pitting was present from the root up to 12 inches above the
union. In contrast to our work, they found that ninety
percent of the declining/PSP-affected trees had the pitting
symptom. Rosenberger et al. (1983) noted that the best
symptoms in apple were indeed pitting along with necrosis at
the graft union. Although there was a failure rate of 11
percent (trees with symptoms, yet not virus) they attributed
this to uneven virus distribution or low titer.

Mircetich et al.(1977) speculated that pitting was only
produced by some strains of TmRSV. This is probably not a
major consideration in our study, for there is no reason to
believe that more than one strain of TmRSV is involved in
tree infection in southwest Michigan. What may be occuring
is a species difference, with cherry reacting differently,
for whatever reason, to TmRSV infection than does peach, plum
or apple. The latter two are found with pitting but also
with a consistent brown line of necrosis that is rarely found

in cherry trees. It is possible that the pitting produced in

125
cherry is related to TmRSV but not a consistent symptom of
the virus in this species.
Assay comparisons showed that all of the systems tested

were capable of detecting TmRSV in plant tissues. The
F(ab')2- ELISA peroxidase system, however, gave the lowest 1
background (non-infected tissues) levels and detected TmRSV
in more cases than the other systems. F(ab')2- ELISA/

peroxidase was the assay of choice based on speed, ease of
use for large scale testing and ease of quantification. Hoy
et al.(1984) found indirect ELISA to be most reliable in the

detection of TmRSV. Barbara and Clark (1982), also reported
that F(ab')2- ELISA improved detection of virus due to

lowered background. As noted by Powell (1984), indirect
ELISA is more sensitive when viral concentration is a factor
- which was important in our studies due to the irregular
distribution of TmRSV in cherry tissues.

Dot-ELISA is often more sensitive or as sensitive as
direct or indirect ELISA for detecting plant viruses
(Banttari and Smith, 1984). In this study, there seemed to
be an interference by the cherry root extract and this may
have affected the results. The root extract tended to stick
to the NCM which could have interfered with subsequent
binding steps. Intial attempts to reduce this interference
by increased washings with TBS-T80 and adding Trition X to
the wash solution were not successful. Further work on this
problem as well as use of anti-TmRSV antibodies cross-reacted

with healthy cherry antigens may increase the detection

126

efficiency of dot-ELISA in detecting TmRSV.

SUMMARY

The decline syndrome in sour cherry is a dynamic and
complex disorder. Trees in an orchard are subject to a
variety of biological, environmental and cultural stresses at
all stages of growth. Young trees are susceptible to soil
pathogens and affected by poor soil structure, while older,
maturing trees suffer cankers, mycoplasma-caused X-disease,
borer insects, cold injury and damage from mechanical
harvesting machines. Any number of interactions of these and.
other factors may be occuring in decline, with a synergistic
total effect on tree health.

Viruses can infect trees at all ages and can reduce their
capacity to carry out physiological functions. While rarely
the cause of outright death, viral induced reductions in tree
health and vigor may lower tree survival when the trees are
subjected to other biotic and abiotic stress. Prunus
necrotic ringspot and PDV have been shown to reduce the
growth and yield of sour cherry trees. Tomato ringspot virus
has been implicated in many serious diseases of tree fruits,
primarily through effects on the conductive systems of the
affected trees. Our surveys have shown that the incidence of
all three of these viruses is significantly higher in
declining southwest Michigan orchards than in non-declining
southwest Michigan orchards. Further, our studies indicate
that an association may be occuring between viral infection

(by PNRSV and TmRSV) and poor tree vigor, blindwood, tree

127

128
death and tree replant incidence. Interestingly, both
viruses are also associated with trunk damage. While the
correlative nature of the data precludes determination of the
precise nature of the associations that are occuring, these
results indicate some ways that these two viruses may be
acting as contributors to the decline phenomenon.
Significant associations of any characteristic with viral
presence also indicates areas where further studies, with
individual trees in controlled environmental conditions (as
opposed to orchards in varying natural conditions) could be
focused.

Peach tree short life (PTSL), a serious problem in peach
cultivation is similar to decline in its complexity. Detailed
studies have led to the conclusion that PTSL results from a
complex interaction of abiotic and biotic factors. Use of
susceptible cultivars, production factors e.g. pruning and
soil fumigation, presence of nematodes, and individual
orchard site characteristics all increase the susceptibility
of peach tress to injury and pathogens. Important to the
control of PTSL has been a continuing cooperative effort in
the southeastern United States that has resulted in a
10-point program for controlling the problem and for
increasing overall longevity of economically productive peach
orchard (Ritchie and Clayton,1981; Reilly et al,1986).

A similar combined effort on the part of many researchers
may eventually result in a similar plan for decline-free
cherry cultivation in Michigan. Attempts are in progress to

associate the known biological, environmental and cultural

129
contributers to stone fruit decline so that a Decision
Support System may be developed for growers to use as part of
their management strategy to help reduce decline incidence in

orchards.

L ITERATURE CITED

L ITERATURE C ITED

Agrios, G.N. 1971. Premature foliation, cambial zone
discoloration, and stem pitting of peach and apricot in
Greece. Plant Dis. Reptr. 55:1049-1053.

Aldwinckle, H.S., and P.A. Arneson. 1972. Stem pitting of
peach in New York. Plant Dis. Reptr. 56:616-617.

Almusa, A.M., G.I. Mink, and J.L. Parsons. 1980. Attempts to
transmit the causal agent of a cherry stem pitting disorder
in Washington sweet cherry trees. Plant Dis. 64:1081-1083.

Banttari, E.E.and P.H. Goodwin.1985.Detection of potato
viruses S, X, and Y by Enyzme-linked immunosorbent assay on
nitrocllulose membranes (dot-ELISA)Plant Dis. 69: 202-205.

Barbara, D.J., M.F. Clark. 1982. A simple indirect ELISA
using F(ab')2 fragments of immunoglobulin. J. Gen Virol.
58:315-322.

Barbara, D.J., M.F. Clark, and J.M. Thresh. 1978. Rapid
detection and serotyping of Prunus necrotic ringspot virus in

perennial crops by enzyme-linked immunosorbent assay. Ann.
Appl. Bio. 90:395-399.

Bar-Joseph, M. and M. Malkinson. 1980. Hen egg yolk as a
source of antiviral antibodies in the enzyme-linked
immunosorbent assay (ELISA): A comparison of two plant
viruses. J.Virol. Meth. 1:179-183.

Barnett, O.W. and R.W. Fulton. 1969. Some chemical properties
of Prunus necrotic ringspot and tulare apple mosiac viruses.
Virology 39:556-561.

Barrat, J.G. 1964. Problems of young peach trees. Hort. News,
New Jersy Hort. Soc. 45:14-18.

Barrat, J.G., S.M. Mircetich, and H.W. Fogle. 1968. Stem
pitting of peach. Plant Dis. Reptr. 52:91-94.

Barrat, J.G., R. Scorza, and B.E. Otto. 1984. Detection of
tomato ringspot virus in peach orchards. Plant Dis.
68:198—200.

Barrat J.G. and Otto, B.E. 1985. Prunus necrotic ringspot
virus infection and canker.pp.114-122 In: 1984 Stone fruit

130

131

Berkeley, G.H. 1948. Cherry yellows and necrotic ring spot of
sour cherry in Ontario. No. 1. The value of Prunus persica
and P.domestica var. Italian prune as index hosts.
Phytopathology. 37:2-3.

Berkeley,G.H., D.Cation, E.M. Hildebrand, G.W. Keitt and J.D.
Moore. 1951. pp. 164-170 In: Virus diseases and other
disorders with viruslike symptoms of stone fruits in North
America. USDA Handbook No. 10. 276 pages.

Berkeley, G.H., and R.S. Willison. 1948. Yellows and necrotic
ringspot of sour cherries in Ontario: inoculation
experiments. Phytopathology 38:509-518.

Bitterlin, M.W., D. Gonsalves. 1988. Serological grouping of
tomato ringspot virus isolates: Implications for diagnosis
and cross-protection. Phytopathology 78:278-285.

Bitterlin, M.W., D. Gonsalves, and J.G. Barrat. 1988.
Distribution of tomato ringspot virus in peach trees:
Implications for viral detection. Plant Dis. 72:59-63.

Bitterlin, M.W., D. Gonsalves, and J.N. Cummins. 1984.
Irregular distribution of tomato ringspot virus in apple
trees. Plant Dis. 68:567-571.

Bitterlin, M.W., D. Gonsalves, and R. Scorza. 1987. Improved
mechanical transmission of tomato ringspot virus to Prunus
seedlings. Phytopathology 77:560-563.

Bloom, J.R., S.H. Smith, and R.F. Stouffer. 1972. Evidence
that Xiphinema americanum transmits the causal agent of
Prunus stem pitting. Phytopathology 62:667-668.

Bock, K.R. 1966. Arabis mosaic and Prunus necrotic ringspot
viruses in hop (Humulus lupulus L.)Ann. Appl. Bio.
57:131-140.

Bonsi, C., R. Stouffer, and W. Mountain. 1984. Efficiency of
transmission of tomato ringspot virus by Xiphinema americanum
and Xiphinema rivesi. Phytopathology 74:626.

Brase, K.D., and K.G. Parker. 1955. Decline of Stanley prune
trees. Plant Dis. Reptr. 39:358-362.

Braun, A. J., D.H. Palmiter, and J.A. Keplinger. 1966.
Nematodes found in apple orchards in the Hudson Valley
1956-1965. Plant Dis. Reptr. 50:783-786.

Breece, J.R., and W.H. Hart. 1959. A possible association of
nematodes with the spread of peach yellow bud mosaic virus.
Plant Dis. Reptr. 43:989-990.

Brown, G.K. J.R.Frahm, R.L. Ledebuhr, B.E.Cargill. 1982. Bark
damage when trunk shaking cherry trees. ASAE paper No.

132

82.1557. Annual meeting of American Society Agric. Engineers.
Chicago, Il.

Cadman, C.H., and R.M. Lister. 1961. Relationship between
tomato ringspot and peach yellow bud mosaic viruses.
Phytopathology 51:29-31.

Cain, J.C., and K. G. Parker. 1951. A preliminary report on
the response of virus-infected 'Montmorency' cherry trees to
nitrogen fertilizer. Phytopathology 41:661-664.

Cameron, H.R. 1968. Stem pitting of Italian Prune. Plant Dis.
Reptr. 53:4-6.

Cameron, H.R., and J.D. Moore. 1956. Prune dwarf virus and
the sour cherry viruses. Phytopathology 46:635.

Christensen, O.V., and N. Paludan. 1978. Growth and flowering
of pelargonium infected with tomato ringspot virus. J. Hort.
Sci. 53:209-213.

Civerolo, E.L., and S.M. Mircetich. 1972. A peach isolate of
Prunus necrotic ringspot virus. Phytopathology 62:529-532.

Clark, M.F.1981. Immunosorbent Assays in plant pathology. In:
Ann.Rev.Phytopathology 19:83-106.

Clark, M.F., and A.N. Adams. 1977. Characteristics of the
microplate method of enzyme-linked immunosorbent assay for
the detection of plant viruses. J. Gen. Virol. 34:475-483.

Cochran, L.C., and L.M. Hutchins. 1941. A severe ringspot
virosis on peach. Phytopathology 31:860.

Cochran, G.W., B.N. Wadley, G. H. Kaloostian, and B. L.
Richards. 1950. Ring spot cause of tree degeneration in Utah
orchards. Farm and Home Sci. 11:20-21.

Cole, A., G.I. Mink, and S. Regev. 1982. Location of Prunus
necrotic ringspot virus on pollen grains from infected almond
and cherry trees. Phytopathology 72:1542-1545.

Converse, R.H. 1978. Detection of tomato ringspot virus in
red raspberry by enzyme-linked immunosorbent assay (ELISA).
Plant Dis. Reptr. 62:189-192.

Converse, R.H. 1981. Infection of cultivated strawberries by
Tomato ringspot virus. Phytopathology 71:1149-1152.

Crowle, A.J. 1961. Immunodiffusion. Academic Press., New
York, 333p.

Cummins, J.N., and D. Gonsalves. 1982. Recovery of tomato
ringspot virus from inoculated apple trees. J. Amer. Soc.
Hort. Sci. 107:798-800.

133

Cummins, J.N., and D. Gonsalves. 1986. Irregular distribution
of tomato ringspot virus at the graft unions of naturally
infected Stanley plum trees. Plant Dis. 70:257—258.

Das, C.R., and J.A. Milbrath. 1961. Plant-to-plant transfer
of stone fruit ringspot virus in squash by pollination.
Phytopathology 51:489-490.

Davidson, J.H. 1957. Preplant soil fumigation treatments for
improving growth of fruit trees. Ann.Rept.State
Hort.Soc.Mich. No. 87:93-97.

Davidson, T.R. 1976. Field spread of Prunus necrotic ringspot
in sour cherries in Ontario. Plant Dis. Reptr. 60:1080-1082.

Davidson, T.R., and J.A. George. 1965. Effects of necrotic
ringspot and sour cherry yellows on the growth and yield of
young sour cherry trees. Can. J. Plant Sci. 45:525-535.

Dias, H.F. 1975. Peach Rosette Mosaic Virus. Desriptions of
Plant Viruses. No. 150. Commonweath Mycol Inst./Assoc. Appl.
Biol. Kew, Surrey, England. Unpaged.

Dowler, W.M., R.K. Jones, and R.J. Ferree. 1968. Stem pitting
of peach in South Carolina. Plant Dis. Reptr. 52:907-910.

Edgerton, L.J. and K.G. Parker. 1958. Effects of nematode
infestation and rootstock on cold hardiness of Montmorency
cherry trees. Proc.Amer.Soc. Hort. Sci. 72:134-138.

Engval,E. and P. Perlmann. 1971. Enzyme linked immunosorbent
assay (ELISA): Quantitative assay of immunoglobulin
Jrnl.Immunochemistry 8:871-74.

Fleisher, Z. and E.C. Blodgett and M.D. Aichele. 1964.
Presence of virus (necrotic ring spot group) in Mazzard and
Mahaleb cherry seedlings grown in the Pacific Northwest from
various seed sources. Plant Dis. Reptr. 48:280-281.

Forer, L.B. and R.F. Stouffer. 1982. Xiphinema spp.
associated with tomato ringspot virus infection of
Pennsylvania fruit crops. Plant Dis. 66:735-736.

Forer, L.B., C.A. Powell, and R.F. Stouffer. 1984.
Transmission of tomato ringspot virus to apple rootstock
cuttings and to cherry and peach seedlings by Xiphinema
rivesi. Plant Dis. 68:1052-1054.

Fridlund, P.R. 1970. Systemic invasion of Prunus budsticks by
the green ring mottle virus. Plant Dis. Reptr. 54:91.

Fulton, R.W. 1957. Comparative host ranges of certain
mechanically transmitted viruses of Prunus. Phytopathology
47:215-217.

Fulton, R.W. 1958. Identity of and relationships among

134

certain sour cherry viruses mechanicallly transmitted to
Prunus species. Virology 6:499—511.

Fulton, R.W. 1970a. Prune dwarf virus. Descriptions of Plant
Viruses. No.19. Common Wealth Mycol Inst./Assoc. Appl. Biol.
Kew, Surrey, England. Unpaged.

Fulton, R.W. 1970b. Prunus necrotic ringspot virus.
Descriptions of Plant Viruses. No.5. Common Wealth Mycol
Inst./Assoc. Appl. Biol. Kew, Surrey, England. Unpaged.

Fulton, R.W., and R.I., Hamilton. 1960. Serological
differentiation of prune dwarf and sour cherry necrotic
ringspot viruses. Phytopathology 50:635-636.

Gavalas, N.A. 1970. Symptoms of stem pitting on peach trees
in Greece. Plant Dis. Reptr. 54:73-75.

George, J.A., and T.R. Davidson. 1964. Further evidence of
pollen transmission of necrotic ring spot and sour cherry
yellows viruses in sour cherry. Can. J. Plant Sci.
44:383-384.

Gilmer, R.M. 1961. The rapid determination of necrotic ring
spot and sour cherry yellows viruses of stone fruits. Plant
Dis. Reptr. 45:608-611.

Gilmer, R.M., and K.D. Brase. 1963. Non-uniform distribution
of prune dwarf virus in sweet and sour cherry trees.
Phytopathology 53:369-370.

Gilmer, R.M., and L.R. Kamalsky. 1962. The incidence of
necrotic ring spot and sour cherry yellows viruses in
commercial mazzard and mahaleb cherry rootstocks. Plant Dis.
Reptr. 46:583-585.

Gilmer, R.M., and R.D. Way. 1960. Pollen transmission of
necrotic ringspot and prune dwarf viruses in sour cherry.
Phytopathology 50:624-625.

Gloyer, W.O. and H. Glasgow. 1928. Defoliation of cherry
trees in relating to winter injury. New York (Geneva) Agr.
Exp. Stat. Bull. 555:27pp.

Goff, L.M., and M.K. Corbett. 1977. Association of tomato
ringspot virus with a chlorotic leaf streak of cymbidium
orchids. Phytopathology 67:1096—1100.

Guengerich, H.W., and D.F. Millikan. 1956. Transmission of
the stem pitting factor in apple. Plant Dis. Reptr.
40:934-938.

Halk, E.L. and R.W. Fulton. 1978. Stabilizatioon and particle
morphology of prune dwarf virus. Virology 91:434-443.

Hamilton, R.I., C. Nichols, and B. Valentine. 1984. Survey

135

for Prunus necrotic ringspot virus and other viruses
contaminating the exine of pollen collected by bees. Can. J.
Plant Pathol. 6:196-199.

Hampton, R.O. 1966. Probabilities of failing to detect prune
dwarf virus in cherry trees by bud indexing. Phytopathology
56:650-652.

Hampton, R.O. and M.D. Aichele and E.C. Blodgett. 1966.
Prunus effusa 'Krassa Serera‘ as a prune dwarf virus index
host. Phytopathology 56:65-69.

Heaton, C.R., J.M. Ogawa, and G. Nyland. 1981. Evaluating
economic losses causes by pathogens of fruit and nut crops.
Plant Dis. 65:886-888.

Hildebrand, E.M. 1943. Prune dwarf and the cherry virus
complex. Phytopathology 33:2-10.

Hoy, J.W., and S.M. Mircetich. 1984. Prune brownline disease:
susceptibility of prune rootstocks and tomato ringspot virus
detection. Phytopathology 74:272-276.

Howell, W.E.and G.I. Mink, 1988. Natural spread of cherry
rugose mosaic disease and two Prunus necrotic ringspot virus
biotypes in central Washington sweet cherry orchard. Plant
Dis. 72:636-40.

Hoy, J.W., S.M. Mircetich, and B.F. Lownsberry. 1984.
Differential transmission of Prunus tomato ringspot virus
strains by Xiphinema californicum. Phytopathology 74:332-335.

Hoy, J.W., S.M. Mircetich and R.J. Shepherd. 1981.
Immunosorbent assays for tomato ringspot virus in stone fruit
trees. Phytopathology 71:227.

Hull, E. and E.J. Klos, 1962. Effect of gibbberellin on
virus-infected Montmorency cherry trees. Proceedings of the
5th European Symp.on Fruit Tree Virus Diseases. Bolonga.

Jaffee, B.A., C.A. Powell and M-A. Derr. 1986. Incidence of
Prunus necrotic ringspot virus in some Pennsylvania peach
orchards and nurseries. Plant Dis. 70:688-689.

Jenkins,W.R. 1964. A rapid centrifugal-flotation technique
for extracting nematodes form soil. Plant. Dis. Reptr.48:692.

Johnson, F. 1972. Tomato ringspot virus of blueberries in
Washington. Phytopathology 62:1104.

Jones, A.L. 1982 Diseases of Tree Fruit. North Central Ext.
Pub. No.45. 34pp.

Jones, E.M. and B.F. Janson. 1969. New peach disease in Ohio
orchards. Ohio Report 54(1):11-13.

136

Kahn, R.P. 1956. Seed transmission of the tomato ringspot
virus in the Lincoln variety of soybeans. Phytopathology
46:295.

Keitt, G.W., and C.N. Clayton. 1939. A destructive
bud-transmissible disease of sour cherry in Wisconsin.
Phytopathology 29:821-822.

Keitt, G.W., and C.N. Clayton. 1943. A destructive virus
disease of sour cherry. Phytopathology 33:449-468.

Keitt, G.W. and J.D.Mooore. 1943. Masking of symptoms of
sour cherry yellows by temperature effects. Phytopathology
33:1213-1215.

Kirkpatrick, J. D., K.G. Parker, and E.G. Fisher. 1958.
Decline of 'Stanley' plum accentuated by winter injury. Plant
Dis. Reptr. 42:65-70.

Kirkpatrick, J.D., K.G. Parker, and E.G. Fisher. 1962. Effect
of ringspot, yellows, and rootstock on the macronutrient
element composition of leaves and on growth of sour cherry.
Phytopathology 52:419-423.

Klos, E.J., and K.G. Parker. 1960. Yields of sour cherry
affected by ring spot and yellows viruses. Phytopathology
50:412-415.

Knierim J.A. 1964. A soil fumigation test in relation to the
cherry replant problem.Q. Bull.Mich St. Univ. Agric. Exp.
Stn. 46:527-532.

Koenig, R. 1981. Indirect ELISA Methods for the broad
specificity detection of plant viruses. J. Gen. Virol.
55:57-62.

Koenig, R. and C.E. Fribourg. 1986. Natural occurence of
tomato ringspot virus in Passifora edutis from Peru. Plant
Dis. 70:244-245.

Lewis, F.H. 1947. Notes on two virus diseases of sour
cherries. Penn. State Hort. Assoc. Proc. 24:65-71.

Lewis, F.H. 1951. The effect of ring spot and yellows on the
yield of MDntmorency cherry. Phytopathology 41:24.

Lewis, F.H., R.F. Stouffer, and F.N. Hewetson. 1968. A

serious new disorder of peach trees. Plant Dis. Reptr.
52:292-294.

Lister, R.M., and AIF. Murant. 1967. Seed-transmission of
nematode-borne viruses. Ann. Appl. Biol. 59:49-62.

Lister, R.M., W.R. Allen, D. Gonsalves, A.R. Gotlieb, C.A.
Powell, and R.F. Stouffer. 1980. Detection of tomato ringspot
virus in apple and peach by ELISA. Acta Phytopathology

137
Academiae Scientiarum Hungaricae 15:47-55.

Loesch, L.S. and R.W. Fulton. 1975. Prunus necrotic ringspot
virus as a multicomponent system. Virology 68:71-78.

Lommel, S.A., A.H. McCain, and T.J. Morris. 1982. Evaluation
of indirect enzyme-linked immunosorbent assay for the
detection of plant viruses. Phytopathology 72:1018-1022.

Lott, T.B. 1967. Xylem aberration, a transmissible disease of
stone fruits. Can. Plant Dis. Surv. 47:74-75.

Lounsbery, B.F., and.A.R. Maggenti. 1963. Some effects of
soil temperature and soil moisture on population levels of
Xiphinema americanum. Phytopathology 53:667-668.

Mai, W.F. and K.G. Parker. 1967. Root diseases of fruit trees
in New York state. I. Poulations of Pratylenchus penetrans
and growth of cherry in response to soil treatments with
nematocides. Plant Dis. Reptr. 51:398-401.

McLean, D.M. 1962. Differentiation of tobacco ringspot and
tomato ringspot viruses by experimental host reactions. Plant
' Dis.Reptr. 46:877-881.

Megahed E. and J.D. Moore. 1967. Differential mechanical
transmissionof Prunus viruses from seed of various Prunus
spp. and from different parts of the same seed.
Phytopathology 57:821.

Mellor,F.C. and R. Stace-Smith. 1963. Reaction of strawberry
to a ringspot virus from raspberry. Can J.Bot. 41:865-870.

Michigan Agricultural Statistics. 1987. USDA-NASS. Michigan
Dept. Agric. 80pp.

Milbrath, J.A. 1950. The effect of latent viruses on the
growth of cherry trees. Phytopathology 40:19.

Miller, P.M. 1980. Reproduction and survival of Xiphinema
americanum on selected woody plants, crops, and weeds. Plant
Dis. 64:174-175.

Millecan, A.A., G. Nyland, and C.W. Nichols. 1964. A
wood-pitting symptom in pear; its occurrence, distribution
and association with certain pear virus diseases.
Phytopathology 54:1435 (Abstr.)

Millikan, D.F. 1955. The influence of infection by ring spot
virus upon the growth of one-year-old Montmorency nursery
trees. Phytopathology 45:565-566.

Mills, W.D. 1946. Temperature effects on the expression of
the yellows virus in sour cherries. Phytopathology
36:353-358.

138

Mink, G.I. and M.D. Aichele. 1984. Detection of Prunus
necrotic ringspot and prune dwarf in Prunus seedlings by
enzyme-linked immunosorbent assay. Plant Dis. 68:378-381.

Mink, G.I., and W.E. Howell. 1980. Occurrence and
distribution of stem pitting of sweet cherry trees in
Washington. Plant Dis. 64:551-552.

Mircetich, S.M., and E.L. Civerolo. 1972. Relationship
between stem pitting in peach and in other Prunus species.
Phytopathology 62:1294-1302.

Mircetich, S.M., and H.W. Fogle. 1969. Stem pitting in Prunus
spp. other than peach. Plant Dis. Reptr. 53:7-11.

Mircetich, S.M., and H.W. Fogle. 1976. Peach Stem Pitting.
pp.77-85 In: Virus Diseases and Non-infectious Disorders of
Stone Fruit in North America. USDA Handbook no 437. 433
pages.

Mircetich, S.M., and J.W. Hoy. 1981. Brownline of prune
trees, a disease associated with tomato ringspot virus
infection of Myrobalan and peach rootstocks. Phytopatology
71:30-35.

Mircetich, S.M., E.L. Civerolo, and H.W. Fogle. 1971.
Relative differential efficiency of buds and root chips in
transmitting the causal agent of peach stem pitting and
incidence of necrotic ringspot virus in pitted trees.
Phytopathology 61:1270-1276.

Mircetich, S.M., H.W. Fogle, and J.G. Barrat. 1968. Further
observations on stem pitting in Prunus. Plant Dis. Reptr.
52:287-291

Mircetich, S.M., H.W. Fogle, and E.L. Civerolo. 1970. Peach

stem pitting: transmission and natural spread. Phytopathology
60:1329—1334.

Mircetich, S.M., W.J. Moller, and G. Nyland. 1977. Prunus
stem pitting in sweet cherry and other stone fruit trees in
California. Plant Dis. Reptr. 61:931-935.

Moore, J.D. 1946. Relation of sour cherry yellows to yield.
Phytopathology 36:406.

Moore, J.D. and G.W. Keitt. 1944. Host range studies of
necrotic ringspot and yellows of sour cherry. Phytopathology
34:1009.(Abstr.)

Moore, J.D. and G.W. Keitt. 1949. An indexing method for
necrotic ring spot and yellows of sour cherry. Phytopathology
39:15-16

Mountain, WL., C.A. Powell, L.B. Forer, and R.F. Stouffer.
1983. Transmission of tomato ringspot virus from dandelion

139
via seed and dagger nematodes. Plant Dis. 67:867-868.

Murant, A.F., and R.M. Lister. 1967. Seed-transmission in the
ecology of nematode-borne viruses. Ann. Appl. Biol. 59:63-76.

Nyczepir, A.F., E.I. Zehr, SA. Lewis, and D.C. Harshman.
1983. Short life of peach trees induced by criconemella
xenoplax. Plant Dis. 67:507-508.

Nyeki, J., and J. Vertesy. 1974. Effect of different ringspot
viruses on the physiological and morphological properties of
Montmorency sour cherry pollen II. Acta Phytopathologica
Academiae Scientiarum Hungaricae 9:23-29.

Okie, W.R., and C.C. Reilly. 1984. Effect of the ring
nematode on growth and physiology of peach rootstocks under
greenhouse conditions. Phytopathology 74:1304-1307.

Parish, C.I., and R.H. Converse. 1981. Tomato ringspot virus
associated with apple union necrosis and decline in western
United States. Plant Dis. 65:261-263.

Parker, K.G., K.D. Brase, G. Schmid, T.H. Barksdale, and W.R.
Allen. 1959. Influence of ring spot virus on growth and yield
of sour cherry. Plant Dis. Reptr. 43:380-384.

Parker, K.G., and L.C.Cochran. 1951. Similarities of symptoms
produced by the viruses causing ring spot of peach and
necrotic ringspot of sour and sweet cherry. Phytopathology
41:42.

Parker, K.G., and R.M. Gilmer. 1976. Constriction disease of
Stanley prune. pp.268-273 In: Virus Diseases and
Non-infectious Disorders of Stone Fruit in North America.
USDA Handbook no.437.433 pages.

Parker, K.G. and W.F. Mai. 1956. Damage to tree fruits in New
York by root lesion nematodes. Plant Dis. Rptr. 40:694-99.

Pine, T.S. 1964. Influence of necrotic ringspot virus on
growth and yield of peach trees. Phytopathology 54:604-605.

Price, W.C. 1936. Specificity of acquired immunity from
tobacco ringspot diseases. Phytopathology 26:665-674.

Powell, C.A. 1984. Comparison of enzyme-linked immunosorbent
assay procedures for detection of tomato ringspot virus in
woody and herbaceous hosts. Plant Dis. 68:908-909.

Powell, C. A., and R.F. Stouffer. 1979. Induction of Prunus
stem pitting in peach with union necrosis-affected apple
rootstock buds. Phytopathology 69:1046.

Powell, C.A., L.B. Forer, and R.F. Stouffer. 1982. Reservoirs
of tomato ringspot virus in fruit orchards. Plant Dis.
66:583-584.

140

Powell, C.A., W. Mountain, and L.B. Forer. 1981. The role of
dandelion in the epidemiology of tomato ringspot
virus-induced diseases of tree fruit. Phytopathology 71:900.

Powell, C.A., L.B. Forer, R.F. Stouffer, J.N. Cummins, D.
Gonsalves, D.A. Rosenberger, J. Hoffman, and R.M. Lister.
1984a. Orchard weeds as hosts of tomato ringspot and tobacco
ringspot viruses. Plant Dis. 68:242-244.

Powell, C.A., W.L.Mountain, T.Dick, Forer, L.B. Derr, M.A.
Lathrop, L.D. and R.F.Stouffer. 1984b. Distribution of
tomato ringspot virus in dandelion in Pennsylvania. Plant
Dis. 68:796-798.

Rasmussen, E.J. 1938. Causes of premature defolation in sour
cherry. Org.State Hort.Soc.Ann.Rep. 30:131-137

Rasmussen, E.J., and D. Cation. 1942. Experiments on the
yellows disease of sour cherry (Prunus cerasus).
Phytopathology 32:15.

Reilly, C.C., A.P. Nyczepir, R.R. Sharpe, W.R. Okie, and P.L.
Pusey. 1986. Short life of peach trees as related to tree
physiology, environment, pathogens, and cultural practices.
Plant Dis. 70:538-541.

Ritchie, D.F. and C.N, Clayton. 1981. Peach tree short life.
Plant Dis. 65:462-469.

Rosenberger, D.A., J.N. Cummins, and D. Gonsalves. 1985.
Development of apple union necrosis and decline in apple
trees inoculated with tomato ringspot virus. Phytopathology
75:1312.

Rosenberger, D.A., M.B. Harrison, and D. Gonsalves. 1983.
Incidence of apple union necrosis tomato ringspot virus , and
Xiphinema vector species in Hudson Valley orchards. Plant
Dis. 67:356-360.

Schlocker, A. and J.A. Traylor. 1976. Yellow Bud Mosaic. pp
156-165 In: Virus Diseases and Non-Infectious Disorders of
Stone Fruits in North America. USDA Handbook no 437.433
pages.

Slogteren, D.M. Van. 1955 pg.51-54 1n Serological
microrelation with plant viruses under paraffin oil. Proc 2nd
Confer.on Potato Virus Diseases. Lissee-Wageingen.

Smith, F.D. and Banttari, B.E. 1984. Dot-ELISA on
nitrocellulose membranes for detection of potato leaf roll
virus. Phytopathology 74:847. (Abstr.)

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

141

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.(Abstr.)

Smith, S.H., R.F. Stouffer, and D.M. Soulen. 1973. Induction
of stem pitting in peaches by mechanical inoculation with
tomato ringspot virus. Phytopathology 63:1404-1406.

Soulen, D.M., R.F. Stouffer, and S.H. Smith. 1972. Root chip
inoculation, an indexing method for Prunus stem pitting.
Plant Dis. Reptr. 56:785-788.

Southey, J.F. 1970. Laboratory methods for work with plant
and soil nematodes. Tech. Bull. I. Ministry of Agric.
Fisheries and Foods, Her Majesty's Stationary Office. London
148 pages.

Stace-Smith, R. 1962. Studies on Rubus virus diseases in
British Columbia IX. Ringspot diseases of red raspberry. Can
J. Bot. 40:905-912.

Stace-Smith, R. 1966. Purification and properties of tomato
ringspot virus and an RNA deficient component. Virology
29:240-247.

Stace-Smith, R. 1970. Tomato ringspot virus. Descriptions of
Plant Viruses. No.18. Common Weath. Mycol Inst./Assoc. Appl.
Biol. Kew, Surrey, England. Unpaged.

Stace-Smith, R. 1984. Tomato ringspot virus. Descriptions of
Plant Viruses. No.290 (18 revised). Common Weath. Mycol
Inst./Assoc. Appl. Biol. Kew, Surrey, England. Unpaged.

Stewart, F.C. 1919. Notes on New York plant diseases, II. New
York (Geneva) Agr. Exp Stat. Bull. 463:157-188.

Stouffer R.F. and F.H. Lewis. 1969. The present status of
peach stem pitting in Pennsylvania. Plant Dis Reptr.
53:429-434.

Stouffer, R.F. and P.D. Mowery 1980. Effect of preplant soil
fumigation on the control of Prunus stem pitting and the
growth of late sunhaven peach trees. Acta Phytopathologica
Academiae Scientiarum Hungaricae 15:247-250.

Stouffer, R. F.and J.R. Uyemoto. 1976. Association of tomato
ringspot virus with apple union necrosis and decline. Acta
Hort. 67:203-208.

Stouffer, R.F., K.D. Hickey, and M.F.Welsh. 1977. Apple union
necrosis and decline. Plant Dis. Reptr. 61:20-24.

Stouffer, R.F., F.H. Lewis, and D.M.Soulen. 1969. Stem
pitting in commerical cherry and plum orchards in
Pennsylvania. Plant Dis. Reptr.53:434-438.

142

Stouffer, R.F.,D.M. Soulen and S.H. Smith. 1975. Spread and
control of Prunus stem pitting. Acta Hort. 44:101-102.

Stouffer, R.F., S.H. Smith, F.H. Lewis, and D.M. Soulen.
1972. Soil transmission of Prunus stem pitting.
Phytopathology 62:672-673.

Swenson, K.G. and G.P. Carpenter. 1961. Transmission of the
ringspot virus of stone fruits form squash to squash by
foliage contact. Phytopathology 51-68-69.

Swenson, K.G., and J.A. Milbrath. 1964. Insect and mite
transmission tests with the Prunus ringspot virus.
Phytopathology 54:399-404.

Tall, M.G., W.C. Price, and K. Wertman 1949. Differentiation
of tobacco and tomato ringspot viruses by cross immunization
and complement fixation. Phytopathology 39:288-299.

Teliz, D., B.F. Lownsbery, R.G. Grogan, and K.A. Kimble.
1967. Transmission of peach yellow bud mosaic virus to peach,
apricot, and plum by Xiphinema americanum. Plant Dis. Reptr.
51:841-843.

Thomas, H.F. and E.M.Hildebrand. 1936. A virus disease of
prune. Phytopathology 26:1145-1148.

Thornberry, H.H. 1970. Peach stem pitting in Illinois. Plant
Dis. Reptr. 54:67-69.

Torrance, L., and C.A. Dolby. 1984. Sampling conditions for
reliable routine detection by enzyme-linked immunosorbent
assay of three Ilarviruses in fruit trees. Ann. Appl. Biol.
104:267-276.

Topchiiska, M.L. 1982. Effect of prunus necrotic ringspot
virus (PNRV) and prune dwarf virus (PDV) on some biological
properties of peach. Acta Hort. 130:307-312.

Tremaine, J.H., W.R. Allen, and R.S. Willison. 1964.
Serological detection and differentiation of prune dwarf and
necrotic ringspot viruses in Prunus tissue extracts.
Phytopathology 54:995-998.

Tuttle, M.A., and A.R. Gotleib. 1982. Histological study of
graft union necrosis in apple assoiciated with TMRSV
infection. Phytopathology 72:267.(Abstr.)

Uyemoto, J.K. 1975. A severe outbreak of virus-induced
grapevine decline in cascade grapevines in New York. Plant
Dis. Reptr. 59:98-101.

Van Regenmortel, M.H.V., and J. Burckard. 1980. Detection of
a wide spectrum of tobacco mosaic virus strains by indirect
enzyme-linked immunosorbent assays (ELISA). Virology

143
106:327-334.

Voller, A., A. Bartlett, D.E. Bidwell 1978. Enzyme
immunoasssys with special reference to ELISA techniques.
Clin. Path. 31: 507-520.

Voller, A., A. Bartlett, D.E. Bidwell, M.F.Clark and A.N.
Adams. 1976. The detection of viruses by enzyme-linked
immunosorbent assay.(ELISA) J.Gen. Virol. 33:165-167.

Voller, A. and D.E. Bidwell. 1977. Enzyme Immunoassays and
thier potential in diagnostic virology. pp. 449-457 In
Comparative diagnosis of viral diseases. Vol II. Kurstack and
Kurstak eds. Academic Press: New York.

Waterworth, H.E. and R.W. Fulton. 1964. Variation among
isolates of necrotic ringspot and prune dwarf viruses
isolated from sour cherry. Phytopathology 54:1155-1160.

Way, R.D. and Gilmer R.M. 1963. Reductions in fruit set on
cherry trees pollinated with pollen from trees with sour
cherry yellows. Phytopathology 53:399-401.

Wells, J.M., H.C. Kirkpatrick and C.L. Parish. 1986.
Symptomatology and incidence of Prunus necrotic ringspot
virus in peach orchards in Georgia. Plant Dis. 70:444-447

Willison, R.S., G.H. Berkeley and G.C. Chamberlin. 1948.
Yellows and necrotic ring spot of sour cherrries in Ontario -
distrbution and spread. Phytopathology 38:776-792.

Willison, R.S., M.Weintraub and J.D. Ferguson. 1957.
Purification and electron microscopy of viruses causing
cherry yellows and related diseases. Can J.Bot 34:86-103.

Yarwood, C.E. 1953. Quick virus inoculation by rubbing with
fresh leaf discs. Plant Disease Reptr. 37:501-502.

Zagaja, S.W., and D.F., Millikan. 1972. Stem pitting on sour
cherry in East Europe. Plant Dis. 56:462.