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This is to certify that the

thesis entitled

INOCULATION AND INDEXING PRACTICES FOR USE IN SCREENING
SOUR CHERRIES FOR GENETIC RESISTANCE TO SOUR
CHERRY YELLOWS DISEASE

presented by

Majid Rahemi

has been accepted towards fulfillment
of the requirements for

M. S . degree in Horticulture

 

 

 

Date 10/28/77

0-7639

 

 

INOCULATION AND INDEXING PRACTICES FOR.USE IN SCREENING
SOUR.CHERRIES FOR.GENETIC RESISTANCE TO'SOUR

CHERRY YELLOWS DISEASE

Maj id Rahemi

‘A THESIS

Submitted to
Michigan State University
for the degree of
MASTER OF SCIENCE
Department of Horticulture

1977

INOGJIATION AND INDEXING PRACTICES FOR USE IN SCREENING
SOUR CHERRIES FOR GENETIC RESISTANCE '10 SOUR
CHERRY YELImS DISEASE

By

Maj id Rahemi

Virus indexing tests were conducted for necrotic ringspot
and prune dwarf viruses on 18 isolates from a wide geographic area
of Michigan's camercial sour cherry orchards. In 1976, 10 of 18

isolates showed positive reaction on Cucmnis sativus L. , a non-

 

differentiating indicator. In 1977, 47 isolates frcm 10 orchards

were indexed on Chempodium quinoa and Cucurbita maxima var . Butter-

 

 

cup, with 27 isolates having a positive reaction, 19 to necrotic
ringspot virus, 8 to prune dwarf virus and 4 to both. Serological
agar diffusion tests for the identification of necrotic ringspot
virus and prune dwarf virus were inconclusive in 1976 but showed
sane isolates to have both prune dwarf virus and necrotic ringspot

virus in 1977 tests. Methods of inoculation were evaluated to

NBjid Rahemi

establish their effect on virus infection and subsequent identifi—
cation. Budding diseased buds into one—year—old ' mntnorency ' on
Prunus mahaleb rootstock gave the greatest percent infection and
best identification results. It is suggested that this method
would be best for inoculation of one-year-old tests that propagates
in a breeding program for tolerance to prune dwarf and necrotic

ringspot viruses .

AQQMEMMENI‘S

I would like to express my sincere appreciation to the
following persons for assistance during the course of my studies:

'Ib Dr. Rabert Andersen as my major professor for his
suggestions and guidance in both my research and personal life.

To Drs. Allen Jones, Martin J. Bukovac, Frank G. Dennis,
Jr. , and Donald C. Ramsdell for their hopeful suggestions during
research.

it) Dr. George 'Ihottapilly for his helpful information
and suggestions during my laboratory work.

To many fruitgrowers win allowed me to use their orchards

for virus-indexing tests and graft inoculation experiments .

ii

TABLE OF CONTENTS

I. LIST OF TABLES ............. . ...... . .
II. INTRODUCTION ...... . . . . . . . . . ...... . .
III. LITERATURE REVIEW. . . . . . . . . . ...... . . . . .
History and Distribution. . . ...... . . . . . .
Effect of NRSV'and PDV on Yield and Growth . . . . . .
Detection of Viruses ........... . . . . .
Indexing ................ . . . . . . .
.Means of Transmission ............... . .
Genetic Tblerance to virus infection in woody Plants. .
IV' .MATERIALS AND METHODS ................. .
Identification ..... . . ..... . .
Serology Test . .................. . .
Inoculation of sour cherry by budding . ........
Indexing on Shirofugen. . . ........... . . .
V. RESUETS. . . . ........... . . .......
Herbaceous Indicator. ............ . . .
Serology Test ........ . . . ...... . . . .
Inoculation of sour cherry by budding . ...... I. .

Indexing on Shirofugen. . . . . . . . . . . . . . . . .

iii

Page

ll
l4
l6
19
21
22
23
25
26
27
28
31
36
39

44

Page
VI. DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 45
VII. SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . . . 57

VIII. LITERATURE CITED . . . . . . . . . . . . . . . . . . . . 60

iv

LIST OF TABLES

Table Page

1 . The identification and location of orchards
in Michigan which were sampled in the spring 29
of 1976 ...................

2. The type of symptoms lesions (L) or msaic
(M) on cucumber vars. Ohio MRl7 and Lemon
which were inoculated with isolates from old
orchards in the spring of 1976. . . . . . . . 30

3. The name and location of orchards were used
for sampling in the spring of 1977. . . . . . 32

4. The results of indexing of samples on squash
and Chenopodium in the spring of 1977 . . . . 33

5. Serological reaction of samples which showed
symptoms on cucumber in the spring of 1976. . 35

6. Serological reactions of samples from H.B.
orchard in 1977 which was used as a source of
diseased budwood for inoculation experiments. 35

7. The observed and expected values of trees per
each treatment which showed positive and
negative reaction when they were indexed for
PDV in the spring of 1977 .......... 37

8. The observed and expected values of trees per
each treatment which showed positive and
negative reaction when they were indexed for
NRSV in the spring of 1977 ........ . . 38

9. The observed and expected values of budding time
treatments which showed positive and negative
reaction when they were indexed for PDV and
NRSV in the spring of 1977. . . . ...... 39

Table

10.

ll.

12.

The observed and expected values of indexing
on Shirofugen flowering cherry in the early
summer of 1977 ........ y ........

The observed and expected values of trees in
each treatment which shoved reaction to at
least one of the 3 indexing tests in the
spring of 1977. . ..............

Cumulative results of trees per each treat-

nEKKLthat showed shock and yellow leaf symptom
under greenhouse condition in spring 1977 . .

vi

Page

40

41

42

INTKDDUCI'ION

Introduction

In Michigan and other Great lakes states where sour cherries are
grown, sour cherry yellows is a serious disease of 'antmorency' sour
cherry trees. The disease is important to the cherry growers because
it causes reduction of growth, premature defoliation, poor spur forma-
tion, poor fruit set, and ultimately decreases the profitable life of
the orchard. It has been reported that in 13 to 21 year-old trees
know to have been diseased for five years or more, the average
reduction in yield was approximately 50 to 62 per cent (52) .

Initially, sour cherry yellows was thought to be a physiological
condition within the tree. Keitt and Clayton (39) demonstrated that

SCY was graft transmissible. They reported finding yellows symptoms on

 

leaves in cherries (anus cerasus L.) and concluded that a virus caused
the disease. In 1948, more gt 31. showed mechanical transmission of a

virus from sour cherry to cucumber (Cucumis sativus L.) . Subsequent

 

work has shown herbaceous host range and symptom differences among
viruses isolated from sour cherry, indicating transmission of more than
one virus (22, 32) . It was soon recognized that prune dwarf virus PDV
was involved in the sour cherry yellows disease (37, 67) . The relation-
ship of necrotic ringspot virus 'NRSV to the sour cherry yellows disease

was to remain a problem for 20 years (26) .

Necrotic ringspot virus NRSV was known to occur widely among
P_r_ulu_s_ species (9) . It was reported that while NRSV could occur alone,
the yellows disease apparently was always associated with it (10),
and the yellows symptom usually followed NRSV infection by a year or
more (43) .

. Synergism, interference, and cross protection phenomena have all
been reported in research dealing with NRSV and PDV cherry viruses

(13, 46) . Thus, plant breeding for virus tolerance must take into
consideration both viruses .

PDV and NRSV are pollen born viruses and can be easily trans-
mitted by pollen from old orchards to the young trees in the new ones
(8, 14, 34, 61, 65) and through seed when infected seedling rootstock
are used (6, 31, 58). They are both graft transmissable (39) .

The rate of virus spread in young orchards is dependent upon the
age of the orchard, the proximity of older diseased trees and the amount
of disease within the orchard. Demski and Boyle (20) and Gilmer (32)
reported that more than 90% of old orchards were infected by NRSV and
PDV. The maximum rate of spread of NRSV can occur at any time after the
4th year while PDV does not spread rapidly until after the 10th year
(16) .

Since 1950, the use of virus-free budwood has been recommended
for the control of sour cherry yellows disease. This practice greatly
improved the quality of sour cherry nursery stock in New York State (5) .

In 1951 virus-free 'antmorency' budwood became available in mtario,

Canada and by 1953 most of the nursery stock being offered for sale
was propagated from virus- free budwood , but about 4% of the rootstocks
used carried virus.

Even though virus indexed, nursery trees are now camonly used
throughout the sour cherry industry, the disease has persisted as an
economically serious factor. This is due mostly to the infected pollen
being transferred from old orchards to the new ones, because of lack
of isolation (adequate distance).

No chemical substances (viricides) are available as yet for
controlling virus diseases of plants.

The need for the developrent of methods to control the spread
of virus in new orchards is obvious. (he method which has been
suggested for this purpose is the application of growth regulators to
delay flower bud formation in the young orchards . Another approach
to virus disease control is resistant (tolerant) varieties which could
tolerate infections by the viruses. In order to breed a tolerant variety,
a breeder needs to know virology techniques for identification of
virusorvirusesandhowtoseparatethem. Thenheneedstohave
knowledge of genetic variation between the isolates and knowledge of
genetic variation between cherry varieties in their symptam expression
from virus (es) infections. With the information, he ultimately chooses
tolerant parents for hybridization

This study was related to techniques to be used in a virus toler-

ance breeding program. Its specific purposes were (1) to become farmiliar

with virology methods needed for virus breeding; (2) to determine
whether or not NIBV and PDV were present in old orchards of Michigan
and; (3) to determine when and how to best inoculate virus free

'bbntrorency' sour cherry (P. cerasus) with buds from infected

trees which have both NRSV and PDV .

IITERATURE REVIEW

History and Distribution

 

The yellow leaf disease of sour cherry was observed by growers
in Michigan as early as 1920 (56) . It was also reported in New York
I in 1919 by Stewart (59) and again in 1928 by Gloyer and Glasgow (35) .
Since sour cherry yellows may be caused by a synergistic effect of
PDV and NRSV, it is appropriate to present a brief review of each.

In Chtario, Canada, NRSV was first observed in 1939 and its
virus nature was demonstrated in 1940, when typical symptoms were
expressed on 'lvbntmorency' sour cherry as a result of inoculations by
budding from infected trees. At about the same time NISV symptoms
on sour cherry were discovered independently in New York, Wisconsin,
Pennsylvania and Michigan. NRSV causes some degree of symptom develop-
ment in many species of Pig—us (28) . Distribution of this virus occurs
world wide in terperate regions (28) . Spread of NRSV is rapid in sour
cherry in Midwestern and Northeastern states, but relatively slow in
sweet cherry (63) . Dost evidence indicates that the place of origin
was (63) the Middle East or'Nestern Asia.

PDV was described as a virus disease by Thomas and Hildebrand
(60). It was soon recognized that PDV was involved in the sour cherry
yellows disease (37) . Chronic effects of PDV infection on the tree
habit and fruit production of sour cherry were perhaps observed first
in France in (1758) and in England in (1839) . The virus may have been

introduced into North America early in the 19th Century in 'Iarge

antmorency' sour cherry orchards (63) .

Effect of NRSV and PDV on Yield and Growth.

 

Sour cherry yellows appears to be economically the most important
known virus disease of sour cherry in the United States and Canada (62) .

It is known that the rate of spread of NRSV and PDV is related
to the age of orchard and the amount of disease within the orchard .

(16) . Gilmer (32) reported when an orchard was 12 years of age, 94% of
the trees had become infected with SCYV or PDV. Demski and Boyle (20)
concluded that the rapid increase of disease incidence was in the tenth
year, after 25% of the trees had becore infected. By the twelfth year,
over 91% of all trees in the orchard were infected. Both viruses can
spread over a considerable distance. NRSV at least 800 yds and SCYV
about 100 yds but most infections of both occur within 50 ft of a knom
source (16) . The effect of these two viruses on symptom expression,
yield, growth, and spur formation has been demonstrated.

Cropley 3t 31 (12) reported PDV and NRSV under East Malling
conditions were synergistic in 'IVbntmorency' and trees were infected with
PDV did not develop yellow symptoms in the absence of NIEV. Before they
reported their results, Berkely and Willison (1948) proposed that sour
cherry yellows was caused by a complex of PDV and NRSV. In further
studies (13) Cropley supported. the previous results . He inoculated
'Montmorency' trees on FlZ/l rootstocks by double-budding in August 1961

with either NRSV from P. malaleb seedlings. PDV from _P. mahaleb seedlings

or with buds from an established orchard tree containing these two

viruses .

He observed that NIEV alone did not cause sour cherry yellows

symptoms, PDV alone occasionally caused sour cherry yellows on only

a few trees while the yellows syndrore developed in all trees infected

with both viruses .

following table:

The result of this experiment are shown in the

Effects of NRSV and PDV on 'lbntmorency' cherry.

 

 

Inoculation Sour cherry Stemgirth

1961 1962 yellows % of control

- - 0/3 100
CH12 - 0/3 50
NRSV - 0/4 83
NRSV PDV 4/4 56
PDV - 3/10 72
PDV. NRSV 12/12 55

- Cle 3/3 55

- NRSV 0/3 85

- PDV 0/3 79

 

Interference between NIGV and PDV was also evident (12) . F12/l

cherry (_P. avium) trees were inoculated by buds from 'I‘Ibntmorency'

either with NRSV, PDV or both. The plants infected by NRSV showed very

severe leaf necrosis; plants infected with PDV showed very stall

necrotic spots on the leaves while plants infected with both viruses

developed intermediate symptoms. The presence of PDV suppressed
symptoms caused by NRSV.

Cross protection is a phenomenon in which plant tissues infected
with one strain of a virus are protected from infection by other strains
of the same virus (2) . The phenomenon has been reported by Marenaud
and Bernhard (46) with stone fruit viruses. They observed a cross-protection
effect between a mild, but not pure, source of NRSV and a severe and
probably pure isolate. They used horozygous and healthy peach seedlings

for this experiment. The results of their tests follow:

NBS severe strain (V. 566) alone 112 necrosis for 146 twigs observed
NRS + PD (mild strain) (8. 1174) + 3 necrosis for 56 twigs observed

N16 severe strain (V. 566)

Lewis (45) reported that the reduction of yield by NRSV was greatest
the first year symptoms appeared and related the severity of the symptoms.
Maximum yield reduction by yellows occurred several years after
infection (45) .

Moore ( 52) recorded corparative yield of yellows-infected and
yellows-free trees in 2 comercial orchards in Door County, Wisconsin for
several years. He observed that the rate of reduction in yield was little
or moderate in the first or second year following first observation of
symptoms and greater reduction occurred in the ensuing 2 or 3 year
period. He also reported that in the 13 year-old orchards, for the

trees knom to have been diseased for 5 years or more, the average

reduction in yield was approximately 50% and in 21 year-old orchards,
approximately 62% .

Cain and Parker (8) observed that yellows virus disease caused
reduction in yield, percentage of fruit set and spur formation of
FMontmorency' cherries. Spur fermation on trees with light infection
was nil. .Also, both percentage fruit set and number of spurs pro-
duced decreased progressively as the severity of yellows increased (8).

In.commercial sour cherry varieties, which are self-fertile, a
large proportion of the fruits are probably set by self-pollination,
therefore, fruit yields of yellows infected trees will be reduced by
self-pollination because most of the pollen available is from an
infected donor. Some of the infected pollenetubes:may burst during
fertilization and reduce fruit set (30).

way and Gilmer (66) pollinated two branches of a healthy English
.Morello cherry with healthy and yellows infected \Montmorency' donors.
Pollen from the healthy donor set fruits on 46 of 1057 flowers (4%)
but pollen from.the infected donor set fruits on only 2 of 685 flowers
(<l%). Therefore, they concluded the percentage of fruit set is reduced
when pollination achieved with pollen from yellows infected trees.

K105 and Parker (43) reported increased fruit size on yellows
trees over fruit on healthy trees in years fellowing initial infection
was probably due to less growth of fruit spurs and to less fruit set on
diseased trees. They also reported percentage of fruit set.was lower

on yellows affected trees and ringspot affected trees than on healthy trees.

_10_

The reported yellows-affected trees lose a large number of leaves early
in the season, which undoubtedly influences fruit bud formation for

the next season and probably accounts for poor fruit set (43) . They
said that the loss of leaves early in the season would reduce accumula-
tion of carbohydrates , which would be needed for good fruit set in the
following season. Defoliation of infected trees varies in different
years and different locations (56) . Cool weather induces yellow leaves
to appear in infected trees and subsequent defoliation (45) . The
maximum defoliation begins in late June and early July and continues for
two or three weeks (40) . The defoliation typically begins with older
leaves and extends towards the younger (40). The size of leaves is
dependent on the amount of defoliation. If light defoliation occurs, the
tree has normal leaf (normal size), normal spur and shoot growth; but
if the trees show heavy defoliation, they bear large leaves, few spurs
and long bare spaces occur on the twigs in subsequent years (56) .

The effect of NIEV and PDV on the growth of sour cherry trees and
the question of which virus has a greater retardation effect on the
growth has been studied by Davidson and George (17). NRSV and SCYV both
have significant effect on growth of young trees with SCYV having greater
retarding effect on growth than NRSV. The literature often refers to
SCYV in ways that may in sore cases refer to the complex of PDV and
NRSV and in others only refers to PDV. In this particular case, Davidson
and George must have been referring to PDV. They also reported the

growth rate of trees infected with both viruses was very similar to that

-11-

for trees with SCYV only and indicated that the predominance of this
virus in these combinations. They said when trees were infected with
only NRSV at l, 2, or 4 year-old, there was a 10 to 30% reduction in
growth and yield was reduced 36 to 56%. They recomended that growers
should try to keep new trees healthy during the first four or five

years by isolation and deflowering sprays. The main purpose of deflower-
ing would be to keep young orchardsfrom infection, thus creating a

heavy spur system and an increase in vigorous growth of crowns of trees.
Post-bloom spray of GA3 at high concentration in the range of 100 ppm will
greatly reduce bloom the fol lowing year in young cherry trees . Flower
bud initiation does not occur following the GA3 spray and increases

the number of vegetative buds. These vegetative buds develop into the
spurs which bear fruit in subsequent seasons. GA3 directly interferes
with bud formation rather than altering the vegetative-reproductive

cotpetition on terminal growth (11) .

A. Detection of viruses:

 

The first step in detection of sour cherry yellows is to be
familiar with the types of symptoms and factors which induce symptom
formation. NRSV and PDV, when inoculated in 'antmorency' separately,
have different symptoms and environmental requirements for expression.

Keitt and Clayton (39) reported yellow symptoms on the leaves

of sour cherries (Prunus cerasus L.) . They observed chlorotic areas

 

on only part of the leaf lamina. If the leaves persisted long enough

-12..

on the tree, sore of them became entirely yellow, the leaf symptoms
appeared 3 to 4 weeks after petal fall. They did not report the name
of the virus or viruses which caused the disease.

In 'lvbntmorency' sour cherry, the initial symptom of NRSV
is delayed foliation of individual limbs or entire trees. Leaves on
affected branches are reduced in size and before they unfold, they
may show light green spots and dark rings which very in size from y
1 mm or less up to 5 mm in diameter. Infected trees may have'sraller
leaves than healthy trees (63) . The first indication of infection by
NIGV is shock (16, 24) . "Shock" is manifested as a necrotic symptom
produced on the first new growth following infection with some viruses;
also called acute symptom (59) . Trees in shock due to NRSV soon
recover from this symptom and by the end of that season, terminal
elongation and lateral shoots are showing no shock (15) .

PDV causes a typical yellow leaf symptom during late June

followed by the casting of affected leaves (15) . Davidson and George
(15) reported tlat the secondary symptoms of sour cherry yellows;
"yellow leaf symptoms" appeared 2 years after shock symptoms (initial
symptoms of infected trees).

Davidson and George (15) reported the type and distribution of
initial symptoms of PDV and NRSV varied with time of inoculation. They
reported trees that were inoculated in April developed shock symptoms
at bud-break a few weeks later. The time of appearance and progress of

symptoms were identical for PDV and NRSV. Inoculation of cherry trees

-13-

with PDV during May, June, and July resulted in general shock symptoms I
at bud-break the following spring. Presumably, this virus moved down-
wards to the roots and did not become systemic until spring. Trees were
inoculated with NRSV during the same period developed secondary etch
symptoms the next season without any observable shock or retardation of
growth. Presumably, this virus in contact with PDV became systemic
before bud-break. August inoculation with PDV also resulted in shock
symptoms the following spring, but in sore cases adjacent branches were
symptomless. They concluded that the virus moved from inoculation point
into the surrounding tissue before growth ceased in the fall. Therefore,
they concluded that seasonal symptom variation revealed that the rate
of movement of the PDV differed from that of the NRSV.

It is known that temperature has a proformd effect on the
expression of symptoms and leaf casting of sour cherry yellows disease.
more and Keitt (52) reported that under greenhouse conditions, yellow
leaf symptom on 'antmorency' cherry could be expressed at 16°C and
NRSV symptoms over the range from 16° to 28°C. In another experiment
(53) they budded 'bbntmorency' trees with buds from yellows infected
trees. When a group of budded trees were placed at approximately 16°C,
they showed yellows while trees placed at 16°C after exposure to 24°C
(1-4 wks after budding) showed no symptoms.

The expression of symptoms and spread of NRSV in sour cherry
were delayed at temperature below 20°C and were very poor or lacking
at night terperature of 10°C ( 52) .

-14-

In Western North America, low night tetperature (10 to 16°C)
alternating with relatively high day tetperature (30 to 35°C) seems
to be favorable for leaf casting symptoms . Under favorable conditions
for development of symptom, 30 to 60 percent of leaves may drop but

leaf dropping gradually decreases as temperature increases (63) .

B. Indexing:

PDV and NRSV have been indexed on many herbaceous and woody
plant species and show different symptoms.

Cucumber has been used by many investigators to index sour
cherry yellows disease. [more _e_t a_1_ . (54) detonstrated that a virus
could be transmitted mechanically to cucumber from sour cherries which
were infected with necrotic ringspot. At first, identity of the
cucumber infecting virus was in doubt because of the inability to
transmitthevirus fromthecucumbertosourcherry. Itwasthen
confirmed by other workers that the virus transferred to cucumber
was NISV from sour cherry.

The first transmission by More _e_t a_l_. (54) of stone fruit virus
to cucumber (Cucumis sativus L.) showed that several viruses could be
transferred by sap inoculation from a m host to herbaceous plant
species.

Cropley _et a_l. (12) worked on the isolation of necrotic; ring-
spot and prune dwarf viruses in herbaceous indicators . They reported
NRSV and PDV can co-exist and multiply together in both Pr_1_1_n_u_s_ and

herbaceous hosts but prior infection of ' mntmorency ' trees with one

-15-

virus reduced the severity of shock symptom by another (interference

effect) .

Cucurbita maxima var. Buttercup has been used as a herbaceous

 

indicator. This plant shows a positive reaction to PDV (12, 14, 50, 63) .

Waterworth and Fulton (64) reported that Qlcurbita maxima L. var.

 

Buttercup was systemically infected by all the isolates which reacted
with PDV antiserum. Chlorosis as vein banding and mosaic are the most
frequently observed reaction (12 , 48 , 64) . Necrotic ringspot virus shows
large necrotic lesions on inoculated cotyledons of Buttercup squash but
no systemic infection (12) . Das and Milbrath (14) reported that squash
plants can be infected by pollen from plants infected with stone fruit
ringspot virus.

ChempOdium quinoa also has been used to index NRSV (12, 61) .
NRSV shows necrotic lesions on inoculated leaves and severe distortion
rings and lines on systemically infected leaves (12) .

Many woody plants have been used to index these two viruses.

Pine and Williams (55) used Prunus persica var. Rio Oso Gem peach

 

seedling as an indicator host for NRSV. Two bark chips from suspected
trees were placed in each test plant. Symptoms of NIBV appeared within
7-21 days. 'bbntmorency' sour cherry (P. cerasus) can be used as a host
indicator for SCYV or PDV in regions where the temperature is 650 or
below after bloom (32) .

Shirofugen flowering cherry (g. serrulata) has been used as an

indicator for sour cherry yellows disease by many investigators.

-16-

(36, 49, 55) but NRS and puv Cannot be distinguished with this indicator
(55) . Both of these viruses produce an intially localized necrotic
reaction with gumming in Shirofugen. The buds which show negative
results on Shirofugen are not a definite indication of virus free buds,
because they may contain laten virus quantities below the detection

threshold of the Shirofugen indicator (36) .

Means of transmission:

 

l. Pollen transmission: PDV is pollen-born in cherry and

 

infects previously healthy trees , in low percentage , when they are
pollinated with infected pollen (28) . NRSV is also pollen home in
cherry and infects trees when they are pollinated with virus-carrying
pollen (27) . '

Gilmer (34) pollinated healthy cherry (_P. _ayi_u_m_ L. cv Yellow
Glass) using SCYV infected sweet and sour cherry pollen (P_._ cerasus L.
cv 'Montmorency') and obtained tree to tree transmission which takes
place by pollen action. Sweet cherries which were infected via
pollination demnstrated that SCYV moved from the flowers or young fruit
into woody tissue. He also reported that interspecific transmission
of PDV is possible between sweet cherry and sour cherry which bloom at
the same time.

Pollen transmission also takes place between wild species and
cultivated species if bloom dates overlap. Wild m species can

transmit infected pollen to healthy cultivated plants. In Michigan,

-17..

there are several wild species of cherries from which infected pollen
transmission is possible in seasons when the bloom date of cultivated
cherries and wild cherries overlap. Bloom development on wild

(_P. _a_vi_u_m) is usually one or two weeks after sweet cherries have

bloomed. P. seratina and _P. virginiana and the other cornon wild

 

species usually bloom tm weeks later than sweet cherries and plum,

and one week later than sour cherries. However, the rate of dissem-
ination of NRSV and PDV in sweet cherry is very much slower than sour
cherry. In some years, when a warm period is followed by a cool condition
at the beginning of normal blossom season, a rush of late bloom occurs.
Under such conditions, pollen transmission between wild and cultivated .

species is possible or vice-verse (l9) .

2. Transmission through seed: NRSV and PDV are seed-borne

 

and it can be expected that sore wild B. 911.9“. should carry one or both
of then. Megahed and Moore (58) reported transmission of NRSV through
seed resulted in recovery ranging from 1% in Italian Prune seed to 91%
’from 'antmorency' sour cherry seed. With English bbrello recovery
was 50%, with Gold sweet cherry 37%, with _P_. mahaleb 70% and with

_P. pgnsylvanica 36° .

 

Inoculation from cotyledons of sour cherry and Italian prune
seedlings showed chlorotic symptoms and golden mottle on squash. With
Mahaleb cherry, cotyledon and seed coat infected both cucumber and
squash. Also, they reported that transmission occurred with both

mature and immature seeds of 'Nontmorency' .

-18-

Cation (6) took Mahaleb and 'Nontmorency' seeds from infected
trees and grew them to seedling stage and then indexed them on seedling
peach in the field. He reported that at least 10% of Mahaleb seeds
transmitted NRSV and at least 8.7% transmitted sour cherry yellows.
SCYV was not transmitted through the seeds of 'antmorency ' but 30%
of seeds carried NRSV. In 1952, he did another experiment wherein
he used seedlings from an infected Mahaleb tree with PDV and NRSV and
grew them in the greenhouse to seedling stage. These seedlings were
indexed on peach in the field and he found 2 to 1 ratio of PDV to
NRSV. 'tbntmorency ' seedlings showed 1 to 4 . He indicated that viruses
were not uniformly distributed through Mahaleb buds but some buds were
apparently virus free, however, it can be uniformly distributed through
Mazzard buds.

George and Davidson (31) reported that when SCYV symptoms
predominated as indicated by consistently strong yellows symptoms with
only mild etch, 80% of seed were infected. But when NRSV symptoms
were predominant, 99% of seeds carried virus. About one-third of the
seeds from trees with sour cherry yellows symptoms were aborted. Trees
with necrotic ringspot produce fruit with fewer aborted seeds as
corpared with sour cherry yellows SCY. The results of their experiment
stowed virus infected trees, especially with SCYV, telded to increase

the abortion rate of 'lVbntmorency' seed.

3. Transmission by vector: No insect vector is known for

 

-19-

PDV and NRSV (26, 27).

George and Davidson (31) introduced insects into growth chambers,
containing diseased and healthy trees. In just one of the chambers,
transmission took place by Thripidae or bees . When they introduced
bees alone in the chamber, the bees caused more transmission of the

virus than the Thripidae. This indicates rapid spread of NRSV is

 

probably related to an easy dissemination by an Arthropod vector.

Genetic tolerance to virus infection in woody plants:

 

Although health or vigor of host plants confers no resistance
on immunity to virus disease, breeding plants for hereditary resistance
to virus is of great importance and many plant varieties resistant to
certain virus diseases have already been produced (2) .

Study on the rootstock and scion varieties and rootstock-scion
combination for resistance to the virus disease of fruit has been done
by many investigators. For example, during the-past 25 years, virus
diseases have becore a critical factor in the citrus industry through-
out the world. The most damaging citrus viruses include tristeza,
psorosis and exocortis, and soretimes cachexia. Tristeza and exocortis
act primarily on specific rootstock-scion combinations, while psorosis
affects many scion varieties, (irrespective of rootstock. One of the
widely injurious virus reactions krown had been the destruction of
sweet orange on sour orange rootstock by tristeza. The trifoliate

orange was found as a tolerant rootstock to tristeza.

-20-

Studies on the reaction of cherry rootstocks and scion varieties
and rootstocks-scion combinations to either PDV, NRSV or both for
specific selection of parents with greater innate resistance is
necessary to improve the sour cherry and sweet cherry to the sour cherry
yellows disease.

_P_r__un_l_1_§ species have different genetic make-up because they
differ in number of chrorosotes (38) . They have stown different reactions
to either PDV, NRSV or both viruses (13) , and different reaction of
Prunus species to either PDV, NRSV or both. He reported that with
combination of both viruses, a synergistic effect on growth and symptom
expression occurred on 'tbntmorency' (P. cerasus L.) and an interference

effect on symptom expression of F12/l cherry (P. avium) .

MATERIALS AND METHODS

_‘)1_

MATERIALS AND MEIHOIB

Comercial 'tbntmorency' sour cherry orchards in the western
part of Michigan were used as a source of virus isolates. The ages
of the orchards were 15 to 53 years. They represented a broad geographic

sample of the entire 'lvbntmorency' sour cherry orchard industry.

I . Identification: Eighteen samples were taken from old

 

orchards before bloom time in March, 1976 and were placed in water and
forced to bloom under greenhouse conditions . The samples were corposed
of a few branches per tree per orchard. Several new leaves and blossoms
were taken from each sample, were put in a sterile mortar which contained
5 mls of phosphate buffer solution pH8, and ground with a sterile pestle
to a soupy consistency. The crude juice was applied to the cotyledons

of cucumbers (Cucumis sativus L. var. Lemon and Ohio MR1?) by rubbing

 

with cheese cloth. The cucumbers were dusted by carborundum before
inoculation to facilitate inoculation. Inoculated cucumber pots were
covered with wet paper towels in order to supply moisture for better
penetration of virus or viruses . After inoculation, they were trans-
ferred to an isolated greenhouse where they were kept under high light
intensity and high humidity conditions for a few days for symptom
evaluation. The humidity was provided by misting every ten minutes for
ten seconds in order to prevent collapsing of tissues of the inocu-v
lated cucumbers .

In April 1977, 5 orchards in Southwestern and West Central

-22-

-23-

and 5 orchards in Northwestern Michigan were sampled . Che sample was
taken from each of three adjacent trees in each orchard. The samples
were collected from swollen flower buds and new leaves. For a more
corprehensive study of a single orchard, the (F.P.) orchard in South-
western Michigan was used and 20 individual tree samples were taken from
two adjacent rows. The samples were kept in cool condition during
transport to East Iansing where indexing was accolplished.

Cucurbita maxima var. Buttercup and Cheopodium glinoa were

 

 

used as herbaceous plant indicators for this part of the experiment.
Theywere grown in four-inch pots in the greenhouse. A few flower
buds and new leaves from each sample were inoculated using the same

techniques as in 1976. Both squash and Chenopodium guinea plants

 

were kept under fluorescent lighting for 15 hours and below 80°F during

the experiment, but no intermittent misting was used in 1977.

II. Serology test: In 1976, tests were made to identify virus

 

entities present in field isolates. Flat bottom petri-dishes were
coated with 12 ml of agar solution. Agar solution was made by the
following method .85 9 NaCl added to 100 ml 0.01 M tris-solution

pH7 after sodium chloride was corpletely dissolved 0.75 grams Inoagar
was added and heated on the steam bath. in order to dissolve the
Inoagar in the solution. The agar solution was allowed to cool to
about 60°C, then 2 mls sodium azid was added to inhibit growth of fungi

and bacteria. Then 12 mls of warm agar solution was poured into each

-24-

petri dish and allowed to solidify. They were kept in moist, cool
condition for a few days (3) .

After the isolates stowed symptoms on cucumber cotyledons,
they were transferred for a few times on cucumbers to increase the
titer of virus. Then 10 - 12 infected cucumber cotyledons were
ground with 2 ml phosphate buffer pH8 . The sap material was extracted
and centrifuged at 104 rpm for 15 minutes, then centrifuged at 35 x
103 rpm for two hours. When the virus or viruses were purified and
concentrated, 10 drops of the buffer, p118, was added and put on the shaker
in about 40°F cool room overnight (29).

Patterns of three wells surrounding a central well, each well
being 3 mm in diameter and spaced 7 mm apart were prepared. They were
punched by sharpened brass tubing. The central wells were filled by
one drOp of PDV and/or NRSV antisera (1:50) and the outside wells were
filled up by concentrated virus or viruses for identification. The
petri dishes were placed in a moist chamber and covered with a plastic
bag to prevent the agar from drying and incubated for 24 hours when the
zones were visible.

In spring 1977, three samples from (H.B.) orchard, which was
the source of diseased budwood for inoculation experiment, were indexed
on cucumber (Cucumis sativus L. var. lemon) . After the symptoms appeared,
crude extracts of infected cotyledons were tested against antiserum of

PDV and NISV diluted at 1:50, 1:25 and undiluted antisera.

_25-

Ten to 12 infected cotyledons were ground with phosphate buffer
solution, 9H8 . The crude extract was prepared by squeezing through
cheese cloths. The rest of the test was followed as the method in

1976 except the crude extract was not concentrated by centrifugation.

III. Inoculation of sour cherry by budding: In this part of

 

the study, the experiment had three treatments. me-year-old m
mahaleb L. rootstock seedlings were obtained from virus certified
stocks of Hilltop Orchards and Nurseries, Inc. These were grown during
the summer of 1976 in soil placed in 30 pound frozen food containers
to be used as rootstocks for each treatment. Virus certified 'IVbnt-
morency' scion wood obtained from the "Dowd Orchards" was used for all
grafting to create trees for each treatment.

Diseased buds were taken from (H.B.) orchard that stowed
positive reaction to PDV antiserum, yellow symptom and leaf dropping

in June, 1976.

Treatment No. l: P. mahaleb rootstocks were 'I‘-budded with

 

diseased buiwood of 'antmorency' on August 3, 1976 and then they were
T-budded with virus certified budvood of 'antmorency' on September 3,

1976 .

Treatment No. 2: _P. mahaleb virus free rootstock were T-budded

 

by virus certified and diseased budvood of 'antmorency' sour cherry

at the same time on September 3 and 4, 1976.

-26-

Treatment No. 3: Prunus. mahaleb L. rootstock seedlings were

 

planted in cans in May 1976 in the greenhouse . They were chip-budded

in May with virus certified 'tbntmorency' buihwood. They were transferred
to the Horticulture Research Center (HRC) in East Iansing in early

sumer and were bud inoculated with diseased 'tbntmorency, HB strain'
budwood on September 3, 1976.

Budded trees of the three treatments were grown at the (HRC)
during fall, 1976 and winter, 1977. Then on March 3, 1977, 40 trees
from each treatment which appeared to have live buds were moved to the
greenhouse. These 120 trees were chosen on the basis of visual
inspection of the graft union knit. The trees were arranged in four
blocks . Each block contained three treatments with 10 plants per treat-
ment. After the buds which were initially clean started to grow,
(approximately 25 on) new leaves were taken from individual trees from

each treatment and indexed on squash (Cucurbitamaxima L. var Buttercup)

 

and Chenopodium quinoa for PDV and NRSV using the techniques previously

 

described. The symptore appeared after 6 to 8 days.

IV. Indexing on Shirofugen: The buds were taken from new growth

 

of buds which were initially clean, T-budded into Shirofugen flowering
cherry (P. serrulata) at the Michigan Department Of Agriculture "airport
repository". The results were recorded after 3 weeks on the basis of

gumming or non-gunning visual classifications.

RESULTS

-27-

RESULTS

I . Herbaceous indicator:

 

Cucumber: In 1976, the samples from the old orchards, Table l,

were indexed on cucumber (Cucumis sativus L. var. Leon and Ohio MR17) .

 

Both cucumber varieties stowed two types of symptoms 3 to 7 days after
inoculation. First, local lesions appeared on cotyledons followed by
systemic symptoms (mosaic) on new leaves after a few days, Table 2.
These symptoms were not used to differentiate between PDV and NRSV.

When the titer of virus or viruses was sufficiently high, the inoculated
cucumber seedlings collapsed immediately after local lesions became

visible on the cotyledons.

S_eparation of viruses: Table 3 shows the 10 orchards from the

 

southwest and northwest which were used for sampling in the spring of
1977 to determine the distribution of virus or viruses between and

within orchards .

_S_qu_a_sh: The isolates were indexed on squash (Cucurbita maxima

 

L. var. Buttercup). 'IWo types of symptoms developed after inoculation.
Sore isolates induced necrotic lesions on their cotyledons but no systemic
infection, others caused necrotic lesions followed by yellow areas and
vein clearing on systematically infected leaves. Necrotic lesions on
cotyledons were probably induced by strains of NIGV which the systemic

infection were probably due to PDV, and were similar to those reported

-28-

-29..

 

 

 

Table l . The identification and location of orchards in Michigan which
were sampled in the spring of 1976.
Date Name of orchard City and location Age
3/23/76 F. Pugsley (F.P.) Paw-Paw (S.W.) 15 yrs
3/23/76 D. Friday (D.F.) Hartford (S.W.) 31 yrs
3/23/76 R. Kinney (R.K.) Eau Claire (S.W.) 21 "
3/23/76 K. Weber (K.W.) Ooloma (S.W.) 30 "
3/23/76 H. Overhiser (H.O.) South Haven (S.W.) 26 "
3/23/76 R. Thomas (R.T.) Bangor (S.W.) 21 "
3/25/76 H. Brother (H.B.) Casnovia (W.C.) 25 "
3/25/76 Ch. Hill (C.H.) Bailey (W.C.) 22 "
3/25/76 R. Bull (R.B.) Frezont (W.C.) 25 "
3/25/76 G. Iewis (G.L.) New Era (W.C.) 25 "
3/25/76 F. Fox (F.F.) Shelby (W.C.) 36 "
3/25/76 V. Bull (V.B.) Shelby (W.C.) 25 "
3/25/76 A. Lister (A.L.) Ludington (W.C.) 25 "
3/29/76 W. Cox (W.C.) Williamsburg (N.W.) 53 "
3/29/76 B. McLachlan (B.M.C.) Kewadin (N.W.) 43 "
3/29/76 J. Galleager (J.G.) Traverse City (N.W.) 25 "
3/29/76 B. Deering (B.D.) Northport (N.W.) 30 "
3/29/76 B. Underwood (B.U.) Traverse City (N.W.) 35 "
3/29/76 A. Carroll (A.C.) Traverse City (N.W.) 30 "
W.C. = West Central
S.W. = Southwest
N.W. = Northwest

-30-

Table 2. The type of symptoms lesions (L) or mosaic (M) on cucurber'
vars. Ohio MR17 and lemon which were inoculated with

isolates from old orchards in the spring of 1976.

 

 

 

=

Name of Ohio MR17 Leron
isolate L M L M
H.B. + + +
J.G. + + +
D.F. + +
B.M.C.2 + +

8.0. + +

R.T. + +
V.B. + +

B.M.C. +

K.W. I +

F.F. +

 

31—

by the following investigators (12, 14, 48, 49, 62).

Chenopodium quinoa: The isolates were indexed on Chenopodium

 

M for NRSV. The new leaves of E. 912993 were inoculated by crude
sap of each isolate and necrotic lesions appeared on the inoculated
leaves. Same of the inocuhted Q. m seedlings srnwed systemic _
infection but they were not the result of the inoculum. The systemic
infection also developed on the healthy seedlings. Systemic symptams
did not appear at early stages of growth but developed when flowers
started to appear. The local lesion symptoms in this experiment
were similar to those reported in previous investigations (12 , 61) .

The results of indexing of isolates for NRSV and PDV on squash

and Chenopodiums have been tabulated in Table 4.

II . Serology test:

 

The results of reaction of purified virus or viruses to antisera
(PDV strain E) and (NRS Strain G) are shown in Table 5. None of the
samples in 1976 showed positive reaction to both antisera.

In the spring of 1977, the H.B. orchard isolate was indexed on
cucumber. Crude extracts of infected cotyledons were tested against
the antisera diluted at 1:50, 1:25 and non-diluted, Table S. The non-
diluted antisera showed, in same cases, reaction to both healthy and
infected cucumber cotyledon extracts. Diluted antisera 1:25 and 1:50
showed only reaction to the isolates. H81 showed positive reaction to
PDV antiserum in 1976 and positive reaction to NIEV antiserum in 1977

(Table 5) . H32 showed positive reaction only to non-diluted PDV and

-32-

Table 3 . The name and location of orchards were used for sampling

in the spring of 1977.

 

 

Date Name of orchard City and location ‘ Age
4/23/77 R.K. Eau Clare (S.W.) 21 yrs
4/23/77 K.W. Colcma (S.W.) 30 yrs
4/23/77 H.O. South Haven (S.W.) 26 yrs
4/23/77 H.B. Casnovia (W.C.) 25 yrs
4/23/77 F.P. Paw Paw (S.W.) 15 yrs
5/4/77 W.C. Williamsburg (N.W.) 53 yrs
5/4/77 B.U. Traverse City (N.W.) 35 yrs
5/4/77 B.M.C. Kewadin (N.W.) 9 yrs
5/4/77 A.C. Traverse City (N.W.) 30 yrs

 

-33..

Table 4. The results of indexing of samples on squash and Chenopodium

in the spring of 1977.

 

Region Isolate Squash var Buttercup

Chenopodium.quinoa

 

S.W.

W.C.
W.C.
W.C.

S.W.

S.W.

O
LUMP

as? aw”
-m:ha
(A)

222 mwm 000 223

WNH

MN?“

0
swmqmmwar-I

O O O O O
O O O O O
HHHHH
(11.5me

mmmwmwwmmwmwmmwwwmww

O
P
am

0 O C
O C
were
max:

WWWWWWWWWWWWFWWFWWWW WNW 2353.72

0
NH
0&0

local

local

local

local
local
local

local
local

local

local

local
local

local
local
local
local
local
local
local

lesions

lesions

lesion

lesion
lesion
lesion

lesion
lesion

lesion

lesion
lesion
lesion

lesion
lesion
lesion
lesion
lesion
lesion
lesion

systemic

systemic

systemic

local

local

local

local

local

local
local
local
local
local
local
local
local

lesions

lesion

lesion

lesion

lesion

lesion
lesion
lesion
lesion
lesion
lesion
lesion
lesion

systemic
systemic

systemic

systemic
systemic
systemic

systemic
systemic
systemic

systemic

systemic

-34-

Table 4 . The results of indexing of samples on squash and Chenopodium

in the spring of 1977. (continued)

 

Region Isolate Squash var Buttercup Chernpodium quinoa
‘ N.W. W.C. 1 Necrotic lesions systemic local lesion
" W.C. 2 Necrotic lesion - local lesion
" " W.C.3 - local lesion
N.W. J-Gl - -
" " J-G2 - - local lesion
" " J-G3 - - local lesion
N.W. A.C.l - - ' -
n " A.C.Z - - -
I! n A.Co3 _ _ _
N.W. B.M.C.l - - local lesion
" " B.M.C.2 - - -
" " B.M.C.3 local lesion -
N.W. B U'l - - -
B U 2
B U 3

 

-35..

Table 5 . Serological reaction of samples which showed symptans on

cucumber in the spring of 1976.

 

 

 

Isolate Antiserum (PDV) Antiserum (NRSV)
1:50 1:50
H.B. -
V.B.l - +
B.M.C.2 - +
B.U. + -
D.F. - +
J.G. + -
B.l\1.C.l - +
K.W. + -
F.F. + -
R.T. - -

 

Table 6. Serological reactions of samples from H.B. orchard in 1977
which was used as a source of diseased budwood for

inoculation experiments .

 

 

 

Antiserum HBl HB2 H33
PDV l : 25 - - -
PDV l : 50 - - -
PDV (nondiluted) - + +
NRSV l :25 + - +
NRSV l : 50 + - +
NRSV (non-diluted) + + +

 

-36-

NRSV antisera. HB slowed positive reaction to NRSV antiserum diluted .

3
at 1:25 and 1:50 and non-diluted NRSV and PW antisera.

III. Inoculation of sour cherry bLbudding:

 

Budded trees of the three treatments were forced in greenhouse
in spring 1977. After the new growth had developed fram thebuds which
were initially clean, new leaves of individual trees were used to

index on squash for PDV and Chenopodium quinoa for NRSV. The types

 

of symptoms on squash and Chenopodium were the same as samples which

 

were taken from the old orchards in 1977.

The amount of transmission of NRSV and PDV or both viruses,
from diseased buds to the buds which were initially clean were tested
using two way contingency tables.

Data tabled in (8) yielded X2 estimates which were calculated
to learn the difference between treatments for amount of transmission
of PDV from diseased buds to the new shoots which came fram clean buds.
Calculated X2 was significant at 5% and 1% levels indicating treatment

differences existed.

-37..

Table 7. The observed and expected values of trees per each
treatment.which showed positive and negative reaction

when they were indexed for PDV in the spring of 1977.

 

 

 

 

 

 

 

 

 

 

 

 

 

PDV NRSV
Treatment Total
4. -
T1 1 26 27 n3.
8.07 18.92
T2 9 31 40 n2.
11.96
T3 22 13 40 n1.
11.96 28.03
32 n.l 75 n.2 107 n..
2 " 2
Cal. x = 21.84“ Tab. :f = 5.99 Tab. x = 9.21
(2) .05 (2) .01 (2)
EXpected values and X2 were calculated by the following formula.
. 2
Eij = ni. x n.3. X2 = z (Qij Eij) = No. of rows

 

n. . (r-l) (c-l) ij No. of columns

 

-33....

FromlTable 8, Xz'was calculated to see if treatment.differences
were present for transmission of NRSV from diseased budsto the new
shoots that came from healthy buds. 'Calculated X2 was significant
at 5% and 1% level.

FrtntTable 9, X? was calculated to see the differences between
treatments for transmission of both PDV’and NRSV’from.infected to K
the new shoots which came from clean buds. Calculated )8 was signifi-

cant at 5% and 1% level.

Table 8 . The observed and expected values of trees per each treatment
which slowed positive and negative reaction when they were

indexed for NRSV in spring of 1977.

 

 

 

 

 

 

 

 

 

 

 

NRSV
Treatment Total
4. _
T1 3 24 27
9.58 27.41
T2 7 33 40
14.20 25.79
T3 28 12 40
14.20 25.79
- 38 69 107
4
Cal. X2 = 33.44** Tab. X2 = 5.99 Tab. X2 = 9.21

(2) .05 (2) .01 (2)

 

Table 9. The observed and expected values of budding timeetreatments

which showed positive and negative reaction when they were

indexed for PDN'and NRSV in the spring of 1977.

 

 

 

 

 

 

 

 

 

 

 

Treatment PDV AND NRSV No. of trees
p34
+ - treatment
T1 0 27 27
5.04 21.95
T2 4 36 40
7.47 32.52
T3 16 24 40
7.47 32.52
20 87 107
Cal. X2 = 20.14** Tab. X2 = 5.99 Tab. X2
(2) .05 (2) .01 (2)

 

-40..

IV. Indexing on Shirofugen:

 

In late spring 1977, buds from graft inoculated trees of each
treatment were T-budded on Shirofugen flowering cherry tree
(g. serrulata) . Infected buds showed localized necrotic reaction
with gunning in Shirofugen 21 - 23 days after budding. The results of
such indexing are shown in Table 10 .

From Table 10, X2 values were calculated to learn if treatment
differences were present for transmission of virus (es) frem diseased
buds to the new shoots which came from healthy buds. Calculated X"2
was not significant at 5% and 1% level. There were no significant
differences between treatments.

The results of indexing of individual trees in each treatment
on squash, Chenopodium, and Shirofugen were carpared to see which of
them had “shown positive reaction to at least one of 3 indexing tests.
The result of this carparison are shown in Table 11. From Table 11,

X2 values were calculated to see if treatment differences were present

for transmission of virus (es) frum diseased bud to the new sloots which
came from initially clean buds. Significant X2 at 5% level was obtained.
There were significant differences between treatments with regard

to the time of inoculation.

-41...

 

 

 

 

 

 

 

 

 

 

 

Table 10. The observed and expected values of indexing on Shirofugen
flowering cherry in the early summer of 1977.
Shirofugen (ginning) No. of trees
Treatment per
+ - treatment
T1 18 8 26
19.1 6.9
T2 27 13 40
29.33 10.67
T3 32 7 39
28.6 10.4
77 28 105
Cal. X2 = 2.42 n.s. Tab. X2 = 5.99 Tab. X2 = 9.21

(2)

.05 (2) .01 (2)

 

-42-

Table 11. The observed and expected values of trees in each treatment
which showed reaction to at least one of the 3 indexing

tests in spring of 1977.

 

 

 

 

 

 

 

 

 

 

 

 

Three indexing It» of trees
Treatment tests per
+ - treatment
T1 20 7 27
21.95 5.05
T2 29 ll 40
35.52 7.48
T3 38 2 40
32.52 7.48
87 20 107
2
Cal. X = 7.89* Tab. X2 = 5.99 Tab. X2 = 9.21

(2) .05 (2) .01 (2)

- 43 _

Table 12. Cumulative results of trees per each treatment that showed

shock and yellow leaf symptom.under greenhouse conditions in

 

 

spring 1977.
No. of Plt/ No. of Plt/ No. of Plt/
Ebmpmoms Date treatment1 treatment2 treatment3
Shock 4/15/77 0/27 0/40 11/40
Yellow leaf 4/15/77 0/27 0/40 1/40
Shock 5/26/77 1/27 2/40 8/40*
Yellow leaf 5/26/77 0/27 0/40 12/40
Shock 6/2/77 1/27 3/40 4/40*
Yellow leaf 6/2/77 8/27 20/40 33/40

 

*Leaf loss due to abscission accounts for gradual reduction of numbers

during spring observations.

V. Smtans:

The individual trees were checked for yellow leaf and "shock"
during the experiment. The leaf symptoms described below developed
after transmission by budding. The yellow leaf and "shock" symptate,
were observed on some of the trees of each treatment in the greenlouse
in the spring of 1977. The results are shown in Table 12. According
to Table 12, the maximmmn number of trees which showed shock and yellow
leaf symptoms were obtained from Treatment No. 3 and least from
Treatment No. l. Nbst shock symptans appeared by early spring. The
maximum amount of yellow leaf symptoms occurred in early June when the
terperature was around 70°F (21°C) . Chlorotic areas of light green
appeared on the leaf lamina, then chlorotic areas progressed in area of
leaves showing this symptom and developed to advance stage of yellow

color of whole lamina.

D
ISCUSSIG‘I

-45-

DISCUSS ICN

Herbaceous indicator:

 

The result of indexing of isolates from the old orchards in
1976 on cucumber are shown in Table 2. From the 18 orchards that were
used for sampling, 10 showed positive reaction on cucumber. Field
observation on amount of bloom growth condition, extent of yellow leaf
symptoms, and leaf dropping supported the hypothesis that a virus or
viruses were present in our samples, but for some reason some did not
transfer to cucumber.

One explanation for the failure of transmission of virus(es)
might be that too low of a concentration of virus existed in sate of the
samples. There is a possibility that there was non-equal distribution
of virus (es) within the trees.

The residue of plant material which was used for inoculation
was rot washed from the surface of inoculated cucumber cotyledons
after inoculation. Maybe the residue of plant material reduced trans-
mission of virus or viruses. Fulton and de Zoeten (29) reported the
residue of infected plant material on inoculated leaves may cause
damage to epidermal tissue and decrease infection.

At the time of inoculation, bud scales were not reroved from
the field isolate plant material . They could have reduced the rate of
transmission. Davidson and Rundans (18) found the presence of bud scales
slightly reduced the percentage of transmission. A virus inhibitor in
the bud scales can inactivate virus (es) and such inactivation can occur
in the interval between grinding the tissue and applying the extract
as inoculum (25) .

-45..

- 47-

Milbrath (47) says petals are favorable material for mechanical
transmission of virus in woody plants. Fulton (25) said viruses could
be transmitted mechanically from woody plants mostly by preparing
inoculum fram growing new leaves. Both believe that these plant materials
have fewer virus inactivators than other older tissues.

The lower than expected transmission of virus (es) to cucumber
could have been caused by using old buffer solution. The fresh buffer
solution contains 2-mercaptoethanol which gradually breaks down after
a few weeks . Mercaptoetharol can prevent oxidation of phenolic
carpounds when extracts are exposed to air (25) .

The results of indexing of isolates from the S.W. and W.C.
Michigan (spring 1977) on squash and Cheropodium were better than with
isolates from rorthwest Michigan (spring 1977) . Fresh phosphate buffer
was used for inoculation of all isolates and the inoculated squash
and Chenopodium were kept in the greenlouse in which the tetperature
was kept under 80°F . 'Therefore, the virus or viruses probably did rot
inactivate during inoculation because of using fresh buffer solution
and cool terperature in the greenhouse. The inoculum was cmposed of
the new leaves, petals, and pollen grains.

Failure to inoculate squash and Cheropodium by isolates fram
N. W. may have been due to various causes like low virus concentration
at the time of sampling, action of terperature during sampling, and
non-susceptibility of squash and Chenopodium.

Squash seedlings were good indicators for PDV and they were

not susceptible to other viruses. They grew very fast and a few

- 48-

days after planting, the seedlings were ready for inoculation . Chero-
podium seedlings were not a good indicator because they were susceptible
to a disorder which was suspected to be another virus. This disorder
resulted in symptoms appearing on non-inoculated leaves of inoculated
and uninoculated control plants. This suggests the systemic symptoms

may have been transmitted through seeds or by vectors such as aphids .

Serology test:

 

Serological techniques are very helpful in plant virus work
for several different purposes . Since serology reactions are highly
specific, they may be used to identify viruses and to determine relation-
ships between them. Serology is -a rapid method for detection of
specific virus or viruses in woody plants. Many factors interfere
with such tests such as low concentration of virus and denaturation of
virus by inhibitors during extracting of infected tissue (25) . Fresh
phosphate buffer solution can prevent inactivation of virus (es) during
extracting. The buffer solution which was used for mechanical trans-
mission and serology may not have been fresh enough in 1976 when poor
results were obtained. Buffer slould be changed every week to have
enough 2-mercaptoethanol to prevent inactivation of virus (es) (25) .

It was hypothesized that both viruses were present in the old
orchards and could be detected by serology tests. None of the 10
isolates in 1976 showed the presence of both viruses. Probably one of

two viruses which had less concentration was inactivated during extracting

because of using old phosphate buffer solution. In order to test for

the presence of both viruses, crude extracts of the infected cotyledons were
centrifuged to purify the virus (es) . The centrifugation was done by

two steps at different speeds. By each centrifugation, we may have

lost some percentage of virus(es) and ultimately the viruS'with the

low titer may not have shown a reaction in diffusion agar tests.

Another atterpt was made to increase the titer of virus (es)
by transfers in cucumbers. After initial symptoms appeared on cucumbers,
the isolates were transferred to other cucumbers fOr 5 to 6 times.

It is possible that during inoculation, one of the viruses could have
been lost by inactivation, nonrsusceptibility of cucumber, or the particles
of virus not.comdng in contact with a susceptible site during inoculation.

In 1977, by changing the techniques, both viruses were detected
in some of the samples from the H.B. orchard using serology tests.

After symptoms became visible on inoculated cucumber cotyledons, virus (es)
were transferred two times and fresh buffer was used for inoculation.

The crude extract.was used instead of centrifuged extract of infected
cotyledons.

No explanation is apparent for by HBl showed positive PDV
antiserum reaction in 1976 and negative in 1977. leaf drop symptoms
occurred in the field onHBl in 1976 and yellow leaves resulted from
grafting experiments so it does have sour cherry yellows.

Inoculation of sour cherry by budding;

Early reports by Keitt and Clayton (40) strongly suggested the

-50-

disease was bud transmissible and arother experiment which was done by
Davidson and Rmdans (18) , supported this idea that the PDV and NRSV
were present in dormant buds. The results of the present experiment
support the above results. Viruses can be transmitted from diseased
budtotlerewslootswhichonefromcleanbuds. Tables 7, 8, and9.

The trees of Treatment No. 3 (subsequent inoculation) grew very
uniform with high vigor in spring 1977 after breaking dormancy. The
results of indexing of the trees on squash and Cheropodimm for trans-
mission of PDV and NIEV from diseased buds to the clean buds, Tables
7 and 8, slowed that the trees had high concentration of viruses. In
contrast to heatment No. 3, Treatments No. l and 2 grew nonuniformily
with less vigor in the spring after breaking dormancy in the greenlouse.
Theresultsof indexingtreesofTreatmentsNo. landZonsquashand
Cheropodium showed lower transmission rate than Treatment No. 3. This
suggested there may have been a lower titer of viruses in Treatment
No. 1 and No. 2.

The different patterns of growth for the three treatments were
not related to the kind of rootstock (because they had the same rootstocks
(1:. mahaleb“, but were probably due to the time of holding or the
horticultural condition of the rootstocks at the time of budding. Since
they were budded at different times, they could rot be kept identical
at time of budding.

Calculated x2 from the results of indexing on Shirofugen, Table
10, did not show significant differences between treatments. This
suggests that the poor transmission of viruses from trees of fieatment

No. l and No. 2 may be caused by difficulties with techniques of testing

- 51-

of the presence of viruses. It was necessary to use older leaves
fromsoteofthetreesofTreatmentsNo. landNo. 2atthetimeof
sampling for indexing, probably the viruses were inactivated by
inhibitors. Fulton (24) said inhibitors such as phenolic corpounds
inactivate PDV and W. New leaves have less pherolic corpounds and
high concentrations of viruses (25) .

In arother statistical test, the results of indexing each tree
on squash, Cheropodium and Shirofugen were corpared to see how many
trees of each treatment slowed positive reaction to at least one of the
indicators. The reason for doing this corparison was to see low many
trees were completely virus free because see of the trees that slowed
no reaction on Shirofugen (gumming) had slown reaction on squash,
Cheropodium or both. From Table 11, calculated X2 was significant.
This result supported the calculated X2 values from Tables 7, 8, and 9
that there were significant differences between treatments. This
suggested that viruses were transmitted from diseased buds to the clean
buds but the titer of viruses was different in each treatment.

The trees of Treatment No. 3 probably had a higher titer of
viruses because one-year-old 'lbntmorency ' sour cherry trees were graft
iroculated in September and the viruses were able to move in the tree
and may have been localized in the dormant buds until spring. In
Treatment No. 2, g. mahaleb seedling rootstocks were budded by clean
buds and diseased buds at the same time. The transmission of viruses

from diseased buds to the clean buds slould have taken place through

g. mahaleb tissue. It is possible that diseased buds may have had less
titer of viruses by chance selection of the inoculating buds but poorer
transmission of viruses in rootstocks than in 'lbntmorency' also could
have occurred. This might also have been true in Treatment No. l. The
differences between the titer of viruses in each treatment may be due
to the different rate of inactivation of viruses by inhibitors which
may have differed in P_._ mahaleb and 'Montmorency' . Different levels
and types of pherolic compounds in g. mahaleb and 'antmorency' may
have inactivated viruses more in g. mahaleb than ’antmorency' . Dif-
ferent cherry varieties have different pherolic compounds. Yu and
Carlson (41) reported Mazzard (g. m L.) and Mahaleb (g. mahaleb L.)
were different in three phenolic groups: pherolic acids, coumarins,
and flavonoids. In anther experiment (42) , they found two rootstocks
were different in pherolic and coumarin corponents in the leaf, stems
and roots.

In neatment 1, g. mahaleb rootstock seedlings were budded with
diseased buds in August and clean buds in Septerber. In August, the
bark of g. mahaleb was not slipping erough for optimum building success
so it may be that the diseased buds did rot take well, and dried out
because of non-slipping bark and high terperature . Therefore, the
diseased buds may not have been present long enough to iroculate the

clean buds .

-53-

Alternatives to virus tolerance breeding:

 

According to the greenlouse and field evaluation of NRSV and
PDV in the old orchards in this study, it appears that sour cherry
yellows disease is a serious problem in Michigan. The field symptoms
of NIGV was observed in many young orchards and yellows disease
appeared in most old orchards. Therefore, it appears either a virus
tolerance breeding program or see other horticultural system may be
essential to the practice of keeping tart cherry orchards yielding
well . '

The literature indicates that sour cherry orchards of ages
10 to 12 are most vulnerable to PDV even though NRSV and PDV
can both occur naturally in the field at any time after flowering
commences (18) . Thus, the 10 to 12 year-old age may for sore reason
he the key time in the life of a 'bbntmorency' tart cherry tree when
it is most susceptible to PDV. Research is needed to understand why
PDV spreads rapidly so much later than NRSV. The literature also
indicates that when both viruses are present in the same tree, they often
have a synergistic effect on growth and yield.

Based on the rate of spread of both viruses in orchards, and
their synergistic effects on growth and yield of cherry orchards, we
slould attetpt' to optimize productivity of sour cherry orchards
between 4 to 12 years of age before yellows symptors becote wide spread.

If success can be gained in this practice, it might be possible to

54

escape most of the effect of the yellows and disease by discontinuing
sour cherry orchards after they reach about 10 to 12 years of age.

There are a few lorticultural practices which might help
bring the cherry orchard to the high fruit production between 4 to 12
years of age. One set of such practices is presently under research
in Michigan. It uses standard rootstocks, high fertility, optimum
soil moisture, gibberrellic acid sprays to prevent prerature flowering,
close planting, and summer and dormant season pruning to maintain
optimum fruiting wood surface in orchards.

Arother approach to this problem would be to use smaller trees
and induce precocity of such trees with dwarfing rootstocks or inter-
stems. This has been done in sweet cherry by using 'lVbntmorency' sour
cherry interstock between E. mahaleb rootstock and the scion cultivar
(38) . Very close planting would then be necessary to maximize early
production. Other lorticultural practices , such as branch bending ,
scoring, ringing and limiting fertilizer to induce flowering and early
fruit set, also deserve research in this type of system. .

Foliar application of gibberrelic acid has been effective in
stimulating growth of virus suppressed auxillary buds in sour cherry
disease which results in increased fruit production (2) . This practice
may be useful in prolonging the life of close planted orchards.

All of these practices may induce greater productivity of

orchards for a slort period of time. However, high cost of orchard

-55,-

establishment will probably cause growers to want to retain orchards
as long as possible. They will desire keeping them past age 10 to
12 years, so a need would still exist to breed trees for tolerance
to yellows disease.

Cross protection is an alternative approach to breeding for
scion variety tolerance to control the virus diseases. It may
warrant research consideration in sour cherry production. The
phenotenon las been tested on many crops. Salaman (60) observed this
phenomexon between mild strain and severe strain of potato virus X.
Kunkel (44) for the first time found a cross protection effect between
two strains of yellows virus, ordinary aster yellow strains and the
California aster yellow strain when transmitted by their leaflopper.
Aster yellows has recently discovered that it was caused by myccplasma
not virus.

Cross protection also has been found between NRSV in 13—91292
species. Cochran (10) found that mild strains of ringspot protected
peach trees against sore severe isolates from the same virus. Marenaud
and Bernhard (46) observed cross-protection effects between mild and
severe strains of NRSV. They also observed a transmissible effect
of cross-protection on growth of peach.

According to the above information, research still is necessary
to study the cross-protection effect between strains of NRSV and PDV

in sour cherries. "Virus breeding" may provide an opportunity to make

-56 .—

mild strains of both viruses available in all trees in order to protect
the new orchard from infection of the severe strains of isolates. It
has the desirable aspect that it would then rot be necessary to change
scions and rootstock cultivars as would be required in a virus tolerance
breeding program.

The literature indicates sour cherry yellows spreads slower
in sweet than in sour cherries. Research is necessary to study this
to be true. By crossing sour and sweet varieties, it may be possible
to establish a cultivar which reduces the rate of spread of sour cherry

yellows in young orchards .

SUVMARY AND CCNCIDSIONS

Eighteen samples were taken in March, 1976 from comercial
sour cherry orchards in Western Michigan to investigate the presence
of NIGV and PDV on a cucumber indicator. Ten of the eighteen
isolates slowed positive reaction. Field evaluation of growth
characteristics, yellow leaf symptoms , and leaf dropping supported
the idea that virus or viruses was present in samples; but for sole
reason, they were rot always transferred to cucumber. Failure of
transmission of virus (es) might be because a low concentration of
virus(es) existed in some of the samples, or inactivation of viruses
during transferring and iroculations.

Crude extracts of the isolates which developed symptots on
cucumber were centrifuged and they were tested against PDV and
NRSV antisera diluted at (1:50) . None of them showed positive re-
action to both antisera. Probably one of the viruses was inactivated
during extracting, centrifugation or during serial transfers to new
cucumbers.

In the spring of 1977, 47 isolates were taken from sour cherry
orchards in Western Michigan. They were indexed on squash and

Chenopodimm (Blinoa with 33 having a positive reaction. Twenty isolates

 

slowed local lesions, 10 showed systemic symptoms on squash, and 24
isolates slowed local lesions on Q. qui_roa_. The results of indexing

of isolates on herbaceous indicators shows that PDV and NFGV are

widespread within most bearing orchards in Western Michigan.
' Three treatments were applied to test the time and method
of inoculation and subsequent identification.

Teatment No. 1 (prior iroculation) showed very poor results
of indexing on squash and Cheropodium and fairly good indexing
results on Shirofugen.

Treatment No. 2 (simultaneous inoculation) slowed better
results of indexing on squash and Cheropodimm than Treatment No. l
but poorer than fieatment No. 3. The trees of Treatment No. 2
slowed good results of indexing on Shirofugen.

The poor indexing results of Treatments No. 1 and 2 for
(PDV and NIEV may be because of inactivation of viruses by inhibitors
during indexing or in the P. mahaleb rootstock seedlings.

Treatment No. 3 worked better than other treatments. The trees
in heatment No. 3 shoed better results of indexing on squash, Chero—
podiurm and Shirofugen. Probably the trees in fieatment No. 3 might
have had higher concentrations of viruses than other treatments because
disease buds were budded directly into one-year-old antmorency on
P. mahaleb rootstock. Probably different levels and types of pherolic
compounds (inhibitors) in _P. mahaleb and antmorency may have
inactivated viruses more in g. mahaleb than b‘bntmorency.

Another approach to higher rate of transmission of viruses

from trees of neatment No. 3 to herbaceous indicators may be related

_59-

to less' inactivation of viruses during indexing because new growing
tips were more available for indexing than other treatments.

Slock and yellow leaf symptoms were evaluated during the
spring of 1977 . Slock symptoms appeared at the early stages of
growth, but yellows leaf symptoms developed rapidly almost 2 months
after shock symptoms following a period of cool weather in early
June in the greenlouse.

In breeding programs, there is a need to index trees for
presence of virus. Many indicators can be used to evaluate the
presence of virus in the tree.

Herbaceous indicators can be used for indexing when new leaves
or petal and pollen grains are available. After bloom, when the leaves
becore old, rate of transmission will decrease due to the presence of
high levels of inhibitors in old leaves.

Shirofugen indicator trees can be used at any time of the
growing season because any age tissue can be grafted into it for
observation of gaming reaction. Shirofugen test does rot distinguish
clearly between PDV and NIEV.

Peach seedlings, Dbntmorency, and Italian Prune can be used as
specific plant indicators to detect the presence of PDV and NRSV

in the suspicious trees.

10.

ll.

12.

LI'I'ERA’IURECITED

Adrian, C., and B. Harrison. 1976. Plant virology, the principles.
John Wiley and Son, Inc., New York. 292 pp.

Agrious, G. A. 1969. Plant pathology. Academic Press, New York.
629 pp.

Ball, E. M. 1974. Serological tests for identification of plant
viruses. Aer. Phytopath. Soc. Plant Virology Committee.

31 pp.

Berkeley, G. H., and R. S. Willison. 1948. Yellows and necrotic
ring spot of sour cherry in Ontario iroculation experiments.
Phytopatlology 38: 509.

Brase, K. D., R. M. Gilmer and K. G. Parker. 1957. Quality of
cherry nursery trees in New York. Farm Res. 23 (4) 4-5.

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

Cation, D. 1949. Transmission of cherry yellows virus corplex
through seed. Phytopatlology 39: 37-40.

. 1952. Further studies on transmission of ring spot

 

and sour cherry yellow viruses through seeds. (Abstr.)
Phytopatlology 42: 4.

Cochran, L. C. 1946. Ringspot, a cormon contaminate of stone
fruit virus cultures. Phytopatlology 36: 396.

. 1952. Interference between forms of ringspot virus in

 

peach. Phytopatlology. 42: 512.

Coston, D. C. 1976. The use of 6A3 spray in altering flowering
and fruiting of the sour cherry (P. cerasus L. cv. bbntmorency) .
Ph. D. Thesis, Michigan State University, pp. 1—32.

Cropley, R., R. M. Gilmer, and A. F. Pasnette. 1964. Necrotic ring

spot and prune dwarf viruses in Prunus and herbaceous plants.
Ann. appl. Biol. 53: 325-332.

-60 -

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

- 61-

. 1968. Synergism and interference between necrotic

 

ringspot virus and prune dwarf virus in fruit trees. 'TagBer.
dt. Akad. IandWiss. Berl. 97: 139-142.

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

Davidson, T. R., and J. A. George. 1959. Symptoms of sour cherry
yellows and necrotic ringspot in relation to time of inocula-
tion. Can. J. Plant Sci. 36: 431-436.

, and . 1964. Spread of necrotic ringspot and

 

 

sour cherry yellows viruses in Niagara peninsula orchards .
Can. J. Plant. Sci. 44: 471-483.

, and . 1965. Effects of necrotic ringspot and

 

 

sour cherry yellows on the growth and yield of sour cherry
trees. Can. J. Plant Sci. 45: 525-535.

, and V. Rundans. 1972. Detection of (NRSV) and (PDV)

 

by indexing dormant cherry bud on herbaceous plant. Can . J.
Plant Sci. 52: 915-920. ’

, and . 1972. Incidence of (NRSV) and (PDV) in

 

 

wild Prunus species. Can. J. Plant Sci. 52: 907-913.

Demski, J. W., and J. S. Boyle. 1968. Spread of NRSV in a sour cherry
orchard. Plant Dis. Reptr. 52: 972-974.

Earl, T. H., and E. M. Hildebrand. 1936. A virus disease of prune.
Phytopatlology 26: 1145-1148. .

Fulton, R. W. 1957a. Cotparative lost range of certain mechanically
transmitted viruses of Prunus. Phytopatlology. 47: 215.

. 1957b. Properties of certain mechanically transmitted
virus of Prunus. Phytopatlology 47: 683.

 

. 1958. Identity of relationship among certain sour
cherry viruses mechanically transmitted to Prunus species.
Virology 6: 499-511.

 

. 1966. Mechanical transmission of virus of woody plant.
Ann. Rev. Phytopath. 4: 79-101.

 

26.

27.

28.

29.

30.

31.

32.

33.

. 34.

35.

36.

37.

38.

-62-

. 1968. Relationship among the ringspot of Prunus.

 

University of Wisconsin, College of Agric. ,Dept of Plant
Pathology. Madison, Wisc. pp. 124-138.

. 1970. Prunus necrotic ringspot virus. C.M.I. A.A.B.

 

Description of plant virus No . 5 .

. 1970. Prune dwarf virus. C.M.I. A.A.B. Description

 

of plant virus No. 19.

, and de Zoeten. laboratory manual for a course in plant
virology. Dept. of Patlology. University of Wisconsin,
Madison, Wisc. 99 pp.

 

George, J. A., and T. R. Davidson. 1963. Pollen transmission of
necrotic ringspot and sour cherry yellows from tree to tree .
Can. J. Plant Sci. 43: 276-288.

, and T. R. Davidson. 1966. Virus of seed from selected

 

Montmorency cherry trees. Can. J. Plant Sci. 46: 501-505.

Gilmer, R. M. 1961. The frequency of necrotic ringspot virus and
sour cherry yellows and green ring mottle viruses in
naturally infected sweet and sour cherry orchard trees.
Plant Dis. Reptr. 45: 612-615.

. 1961. The rapid determination of necrotic ringspot and

 

sour cherry yellows viruses of stone fruits. Plant Dis. Reptr.
45: 608.

. 1965. Additional evidence of tree to tree transmission

 

of sour cherry yellows virus by pollen. Phytopatlology
55: 482-483.

Gloyer, W. O., and H. Glasgow. 1928. Defoliation of cherry trees in
relation to winter injury. N. Y. State Agr. Expt. Sta. (Geneva)
Bul. 555. 27 pp.

Hampton, R. O. 1963. An attetpt to detect virus free buds in latent
virus infected sweet cherry. Plotopatlology 53: 718-719.

Hildebrand, E. M. 1944. Prune dwarf and cherry cotplex. Science
100: 147-148.

Janick, J., and N. more. 1975. Advance in fruit breeding. Purdue
University Press, West Lafayette, Indiana 623 pp.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

-63-

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

, and . 1942. A destructive disease of sour

 

 

cherry. Phytopatlology 33: 449—468.

Yu, 8., and R. F. Carlson. 1975. Paper chrotatographic
determination of pherolic corpounds occurring in the leaf bark
and root of Prunus avium and _13. mahaleb. J. Amer. Soc. Hort.
Sci. 100: 536-541.

 

, and . 1975 . Gas liquid chromatography deter-
mination of phenolic acid and coumanins in Mazzard and
Mahaleb cherry seedling. HortScience 10: 401-403.

 

 

Klos, E. J., and K. G. Parker. 1960. Yield of sour cherry affected
by (NRSV and (PDV). Phytopatlology 50: 412-415.

Kunkel, L. O. 1955. Cross-protection between strains of yellow
type viruses. Adv. in Virus Res. 3: 251-273.

Lewis, F. H. 1951. Tle effect of ringspot and yellows on the
yield of antmorency. Phytopatlology. 41: 24.

Marenaud, C., and R. Bernhard. 1968. Cross protection and inter-
ference with sore stone fruit viruses. TagBer. dt. Akad.
LandWiss. Berl. 97: 143-153.

Milbrath, J. A. 1953. Transmission of corponents of stone fruit
latent virus cotplex to cowpea and cucumber from cherry flower
petals. Phytopatlology 43: 479-480.

. 1956. Squash as a differential lost for strains of

 

stone fruit ringspot viruses. Phytopatlology 46: 638.

Mink, G. I., and J. L. Parsons. 1965. The prevalence of sore latent
viruses in sweet cherry of various age group in orchard in South
Central Washington. Plant Dis. Reptr. 49: 143-145.

. 1967. An indicator role of cytochrole oxidase in
loss of prune dwarf virus infection in squash tissue lmogerates.
Phytopatlology 57: 797-798.

 

Moore, J. D., and G. W. Keitt. 1946. Relation of temperature to

expression of symptors of sour cherry yellows and necrotic ring-
pot. Phytopatlology 36: 406-407.

52.

53.

54.

55.

56.

59.

60.

61.

62.

63.

64.

64-

. 1946. Elation of sour clerry to yield. Phytopatlology
36: 406-407.

 

, and G. W. Keitt. 1947. Terperature and seasonal develop-
ment of lost in relation to expression of leaf symptoms of sour

cherry yellows. Phytopatlology 37: 16.

, and , and J. S. Boyle. 1948. Wical trans-
mdssionofavirusdiseasetocucmberfromsourcleny.
Science 107: 623.

 

 

 

Pine, T. S., and H. E. Williams. 1960. Rio Oso Gem peach seedling
as indicator the Prunus ringspot. Plant Dis. Reptr. 44: 324-325.

Rasmussen, L. J., and D. Cation. 1942. A progress report on the
yellow leaf disease of sour cherry. Mich. State Hort. Soc.
Ann. Rept. 71: 100-105.

Salaman, R. W. 1933. Nature Land. 131: 468.

Seyed Megahad, E. L., and J. D. bbore. 1967. Differential mechanical
transmission of Prunus viruses from seed of viruses Prunus
and from different parts of the same seed. Phytopatlology
57: 821-822. (Abstr.)

Stewart, F. C. 1919. Notes on New York plant disease. N. Y. Agr.
Expt. Sta. Bul. 463: 157.

Thotas, H. E., and E. M. Hildebrand. 1939. A virus disease of
prune. Phytopatlology 26: 1145.

T'lottappilly, G. 1974. Summary of important cherry viruses.
Dept. of Ent., Mich. State Univ. 4 pp.

U.S.D.A. 1951. Virus disease and other disorders with virus-like
symptoms of stone fruit in North America. U. S. Gov. Printing
Office, Washington, D. C., Agr. Handbook 10: 152-158, and
164-170.

. 1976. Virus disease and non-infections disorders of
stone fruits in North America and U.S.D.A., Agric. Handbook
437: 103-132 and 179-190.

 

Waterworth, H. E., and R. W. Fulton. 1964. Variation among isolates
of necrotic ringspot and (PDV) isolates from sozr cherry.
Phytopatlology 54: 1155—1160.

-65-

65. Way, R. D., and R. M. Gilmer. 1958. Pollen transmission of necrotic
ringspot virus in cherry. Plant Dis. Reptr. 42: 1221-1224.

66. ,and . 1963. Reduction in fruit sets on cherry
trees and pollinated with pollen from trees with sour cherry
yellows. Phytopatlology 53: 399-401.

 

 

67. Willison, N. W. 1944. Strains of prune dwarf. Phytopatlology
34: 1037-1049.