6 9 -1 6 ,1 7 4

PLUNGE, A1 George, 1928DEGENERATION OF GRAPEVINE, Vitis labrusca L.
’CONCORD’, IN MICHIGAN: DESCRIPTION OF THE
DISEASE AND THE ASSOCIATIONS OF VIRUSES AND
VECTOR SPECIES.
Michigan State University, Ph. D ., 1969
Agriculture, plant pathology

University Microfilms, Inc., Ann Arbor, Michigan

\

DEGENERATION OF GRAPEVINE, Vltls labrusca L. 'CONCORD', IN
MICHIGAN:

DESCRIPTION OF THE DISEASE AND THE

ASSOCIATIONS OF VIRUSES AND VECTOR SPECIES

By
_Gcc-t ciC -

A1 Plunge

A THESIS
Submitted to
M ichigan State U niversity
in p artial fulfillm ent of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Entomology
1969

ABSTRACT

DEGENERATION OF GRAPEVINE, Vitis labrusca L. 'CONCORD',
IN MICHIGAN: DESCRIPTION OF THE DISEASE AND THE
ASSOCIATION OF VIRUSES AND VECTOR SPECIES
By
A1 Plunge
Several pertinent observations of the grapevine Vitis labrusca L.
'Concord' th a t displayed symptoms of virus or v iru s-lik e d ise a s e s in
the Paw-Paw and Lawton, M ichigan areas are d escrib ed .

These w ere:

(1) delayed b u d-b reak , (2) malformed and discolored le a v e s , (3) mal­
formed canes and aty p ical vine growth, (4) berry sh ellin g , and (5)
vine d eclin e.

Since a ll of these were observed in a sso cia tio n with

one another, they were described a s (components of a syndrome)
herein named grapevine degeneration.

A 'Concord' grape vineyard

th at was approxim ately 75-y ears old and sev erely affected with grap e­
vine degeneration d ise a se w as used as the te s t vineyard in this rese arch .
M echanical sap -tran sm issio n s were attem pted from trap plants
(Petunia hvbrida Vilm .'Pink M agic') that were grown in relation to the
grapevine and from affected grapevine le a v e s .

TRSV virus was

iso lated w ith ease from trap p la n ts, w hereas, it was in itially d iffi­
cu lt to transm it the virus from the grapevine p la n ts.

Further tra n s ­

m ission te s ts with leaves of various ages and several buffers related
that sap ex tra cts from leav es soon after bud-break buffered with

A1 Plunge
se rie s

of young herbaceous p lan ts revealed th a t garden pea

Plsum sativum L. 1M idfreezer1 w as the d ifferen tial h o st.

No

reactions were obtained on 'M idfreezer' plants Inoculated with
PRMV, how ever, d istin c t system ic lines and rlngspottlng reactions
were noted on 'M ldfreezer' plants inoculated w ith TRSV.
Several v iru s-fre e , 1 yr-old V. labrusca 'Concord' plants
were m echanically-inoculated with a known strain of CMV (since
many of the petunia trap plants in the te s t vineyard became infected
w ith CMV from aerial vectors) to learn if grapevine w as a s u it­
able h o st.

It was found that CMV Infects the grapevine and b e ­

comes system ically estab lish ed ; how ever, CMV w as recovered from
the m echanically-inoculated grapevine by ad u lts of the green peach
aphid, Mvzus p erslcae (Sulzer), and not by m ech an ical-sap in o cu latio n s.

ACKNOWLEDGE MENTS

I w ish to thank the M ichigan Grape Grow er's A ssociation
, and their sta ff, who suggested th is project and aided in
estab lish in g a research p lo t.
This th e sis is ded icated to my co lleag u es:

to those p erso n s,

p a st and p resent, who have been a sso cia ted w ith me; for to each
and every one I owe much, and it is only the splendid cooperative
sp irit of this group that h as made this th e sis p o ssib le .
Finally, I would once more acknowledge my indebtedness
to my wife and p aren ts, p a st and p resen t, who have given much
help and encouragement in carrying the entire program through to
com pletion.

TABLE OF CONTENTS

Page
INTRODUCTION..........................................................................................

1-4

REVIEW OF THE LITERATURE ON VIRUS DISEASES
OF GRAPEVINE - THEIR SYMPTOMS, HOST RANGES,
DISTRIBUTIONS, AND MEANS OF TRANSMISSION.......................

5-14

OBSERVATIONS
(Description of the grapevine d eg en eratio n )......................

15-20

EXPERIMENTATION
GENERAL METHODS

AND MATERIALS.............................

21-26

Isolation of virus through the use of
a trap p la n t.............................................................................

27-29

Isolation of virus d irectly from
g ra p e v in e ...................................................................................

29-32

Identification of tobacco ringspot
v i r u s ..........................................................................................

33

Nematodes a sso cia ted with a degenerate
v in e y a rd ................................. ..................................................

33-35

Transm ission of TRSV by Xlphinema americanum
extracted from the root zone of degenerate
grap ev in es..................................................................................

36-38

EXPERIMENTS AND RESULTS

11

\

Page
Separation of TRSV and PRMV on the b a s is
of differen tial ho st r e a c tio n s ...........................................

38-43

Transm ission of peach ro sette m osaic virus
from m echanically-inoculated petunia p la n ts.............

44

Recovery of cucumber m osaic virus (CMV)
from m echanically-inoculated 'Concord'
g ra p e v in e ............................................................... ...................

45-49

Chem ical an a ly sis of grapevine leaves affected
with severe vein a n a s to m o s is ..........................................

49

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS . . . •

50-53

REFERENCES................................................................................................

54-59

ill

LIST OF TABLES

Table
1.

2.

3.

4.

5.

Page
Response of various plant sp e cie s when inoculated
with sap ex tracts of trap petunia p la n ts, that
were grown ad jacen t to grapevines affected with
grapevine d e g e n e ra tio n ..............................................................

28

Responses of various plants sp ecies when m echani­
ca lly Inoculated w ith extracts from leaves of g rap e­
v in e, Vitis labrusca L. 'C oncord', affected with
grapevine d eg en eratio n ...........................................................

31

Plant p arasitic nem atodes found in the root zone so il
area of grapevine, Vitis labrusca L. 'C oncord',
affected w ith grapevine d e g e n e ra tio n ...............................

35

Response of sev eral herbaceous plants to tobacco rin g spot virus (TRSV) and peach ro sette m osaic virus
(PRMV).............................................................................................

39

Transm ission of cucumber m osaic virus from m echani­
c a lly inoculated grapevine, Vitis labrusca L. 'C on­
c o rd ', by the green peach aphid, Mvzus perslcae
S ulzer...............................................................................................

48

iv

\

LIST OF FIGURES

Figure

Page

1.

Grape production area s of M ic h ig an ....................................

2.

'Fanleaf' condition on a 'Concord' grapevine leaf . . . .

16

3.

Virus-harboring 'Concord' grapevine leaves found
during the early growing seaso n ...................................

18

4.

'Shelling' condition on 'Concord' grapevines......................

20

5.

Normal 'Concord' grape b u n ch e s............................................

20

6.

A 'Concord' grape vineyard in South-W estern M ich­
igan showing d rastic vine d e c lin e .................................

23

Seedlings in ste riliz e d so il and granular calcined
pyrophyllite in 150-ml. b e a k e rs . The system
used for acq u isitio n of viruses by hand picked
nematodes w hich were introduced into b e a k e rs

37

Beginning stag es of ringspotting on the leaves of
Pisum sativum L. This plant w as m echanically
Inoculated w ith tobacco ringspot v ir u s .. .growth
of th is virus was sy ste m ic ................................................

40

Intermediate stag es of ringspotting on the terminal
leaves of Pisum sativum L. This plant w as
m echanically Inoculated w ith tobacco ringspot
v iru s . . .growth of this virus was sy stem ic................

41

The final stag es of ringspotting on the terminal
leaves of Pisum sativum L. This plant w as
m echanically inoculated w ith tobacco ringspot
v ir u s .. .growth of this virus was sy stem ic ................

42

7.

8.

9.

10.

v

2

INTRODUCTION
Grapes are an Important agricultural commodity In M ichigan.

The

S tate has ranked high in the Nation in grape production - 3rd in 1965
w ith a crop of 71,500 ton at a value of $6.5 million and 5th in 1967
w ith 39,000 ton a t $4.6 million (Michigan Department of Agriculture
1966, 1968).

All of the grapevines in Michigan are Vitis labrusca L.

(predominantly of the Concord variety) and are used primarily for juice
and w ine.

As of 1968, 16,000 acres were in production; 90% of the

acreage is in Berrien and Van Buren C ounties (in the southw estern
com er of the S tate), w ith the remainder in Kalamazoo, Ottawa and Kent
Counties (Fig. 1).
As one would ex p ect, M ichigan's grapevines have not been immune
to in se c t and d ise a se a tta c k .

The grape berry moth, Para lobes ia

viteana (Clem ens), and” the grape leafhopper, Ervthroneura comes (Say)
have been the m ost injurious, throughout the industry, of the in se ct
p e sts while the rose chafer, M acrodactvlus subsplnosus (F .), the grape
flea b e e tle . Altica chalbea (Illiger) and several sp ecies of climbing c u t­
worms in the family Noctuidae have been of a le ss e r problem.

Black

ro t, a destructive rot of the berry caused by the fungus Guianardia
bidw ellll (Ellis), and dead arm , a k iller of entire vines or single 'arm s'
caused by the fungus Phomopsis vitlcola (S acc.), have been the two
m ost damaging to grapevine in M ichigan.

2

d?

Grape production areas
of M ichigan

3
Powdery mildew, cau sed by UncInula necator (Schw.) and downy mildew,
caused by Plasm opara v ltlc o la . (Berlese and de Toni) are endemic to
M ichigan vineyards but th eir economic im portance, w hile recognized, is
undefined.

No virus d ise a se s have been identified in 'Concord' g rap e­

vine in M ichigan, but sev eral growers and research personnel have ob­
served a shelling problem (the berries drop off the c lu ste r prematurely)
and a delayed bud-break condition sin ce about 1950 and been suspicious
th at viruses have been the c a u s e .
Numerous u n su ccessfu l attem pts have been made a t various tim es by
pathologists in the M ichigan Agricultural Experiments Station to iso late
and identify agents from 'Concord* grapevines that break bud late an d /o r
s h e ll.

Failures a lso have been obtained in attem pts to iso late agents from

grapevine leaves displaying a fanleaf condition that first occurred about
1950 and w as feared as the grapevine fanleaf virus d ise a se of Pacific
C o ast v iney ard s.
As b e s t as could be determ ined, attem pts to transm it viruses from
M ichigan grapevine have involved (1) m echanical inoculations of sap e x ­
trac ts from d ise ase d grapevine to healthy grapevine (or o ccasio n ally to
p o te n tia lly -su sce p tlb le herbaceous weed h o sts), and (2) grafts of buds or
scio n s from d ise ase d grapevine to healthy grapevine.

Virus transm ission

experim ents to grapevine are hindered by a very long virus incubation period
(2-3 y r.) in the plant; th u s, symptoms and te s t resu lts are long delayed.
It appeared reasonable that ex ten siv e use of a lte rn a te , herbaceous host

plants a n d /o r trap (bait) plants would enable the tran sm issio n , is o ­
lation and identification of virus in cltan ts if they were present; further­
more, transm ission resu lts would be known in a few weeks or le s s .
Therefore, this research project was undertaken and the following
objectives were estab lish ed a t the onset:
1.

D escribe the array of disorders of unknown c a u s e .

2.

Transmit and iso late viruses from d ise a se d v in e s.

3.

Identify those iso lated v iru se s .

4.

Identify vector-virus relatio n sh ip s.

5.

Determine methods for a s s a y of grapevines and separation
of the 'resident* v iru se s .

Soon afte r the sta rt of this p ro ject, a few leaf sam ples from shelling
'C oncord' grapevines in Lawton, M ichigan were tested for virus by a
noted grapevine virus research er, H .F . D ias, a t the Vineland Research
S tation, S t. C ath erin es, O ntario.

He Isolated from those leaves peach

ro sette mosaic virus (Cation 1942) - a virus that he and D. Cation
(Michigan Agricultural Experiment Station) had su c cessfu lly grafted from
peach to 'Concord' grapevine and recovered through m echanical inoculation
methods (personal communication).

W d en Ewing Farm, Porter Township and Section 8 , Road M-119,
Lawton, M ichigan.

REVIEW OF THE LITERATURE ON VIRUS DISEASES
OF GRAPEVINE - THEIR SYMPTOMS, HOST RANGES,
DISTRIBUTIONS AND MEANS OF TRANSMISSION
GRAPEVINE FANLEAF
G rapevine fanleaf is the m ost d evastating virus d is e a se of g ra p e ­
vine in the world (Bovey 1958).

This d ise a se is cau sed by the g rap e­

vine fanleaf virus and has been a ls o called (1) the fanleaf d is e a s e of
grape, (2) urtlcado (Dias 1950 a ), (3) court noue (Vuittenez 1956), and
(4) component of degenerescence Infectiouse (Bovey 1958).
Symptoms. - Grapevine fan leaf virus ca u ses the primary veins of the
leaf to grow c lo se together resem bling the ribs of a p artially clo sed fan .
Furthermore, the sin u ses are lobed or d e e p ly -c u t, and the leaf blade is
asy m etttcal.

The leaves are m ottled in different shades of green (Hewitt

e t a l . 1956).
This virus affects the appearance of grapevine can es and c a u se s
malformations of varying types - short in tem o d es, double n o d es, f a s clatlo n , zig -zag growth betw een n o d es, and fla t can es (Hewitt and Gifford
1956).

The fruit se t is poor on fa n le a f-d lse a se d vines as the fruit 's h e ll'

profusely and s e t in very strag g ly b u n ch es.

In many v arie ties of g rap e­

v in e, fanleaf ca u ses decline and gradual d ia th of the vines (Dias 1963).

5

6
H ost ra n g e . - Amaranthus retroflexus L .: A. tricolor L. 'M olten
F ire'; Phaaeolus vulgaris L. 'Bountiful'; Cucumls satlv u s L .; Gomphrena
globosa L .; N lcotlana clev elan d ll Gray; N . atten tu ata Torr.; Chenopodlum am arantlcolor C oste & Reyn.; Chenopodlum qulnoa W llld .; Erodlum
macrophvllum Hook & A m .; Vitis vlnlfera L .; Vitis ru p estrls Scheele 'S t.
G eorge'; and Vitis labrusca L. 'C oncord' (Hewitt e t a l . 1962).
G eographic d istrib u tio n . - C alifo rn ia, New York, Portugal, Spain,
France, Sw itzerland, Italy (including Sicily) and Germany (Dias 1950 a ) .
Means of tran sm issio n . - H ew itt (1956) e stab lish ed the so il-b o rn e
nature of the d ise a se in C alifornia and a search for the mode of spread
led to the discovery of Xiphinema index Thome and Allen as the vector
(Hewitt e t a l . 1958).

Though 2 te s ts in th is report im plicated C rico-

nemoides xenoplax R aski. subsequent te s ts have been n eg ativ e.

G rape­

vines in C alifornia, a ls o are su b ject to a tta c k by a wide range of other
p la n t-p a ra sitic nem atodes (Raski and Lider 1959).
of th ese including another dagger nematode

R epresentatives of most

amerlcanum Cobb have been

te ste d as possib le vectors causing d is e a s e s of g rap ev in es, w ithout s u c c e s s .
Cadm an, D ias, and H arrison (1960) indicate the grapevine fanleaf is se ro ­
lo gically related to arab is m osaic virus (AMV).
Cadman, D ia s, and H arrison (1960) have shown in sero lo g ical te s ts
th a t raspberry yellow dwarf and straw berry yellow crinkle are strain s
of arabis m osaic v iru s .

Arabis mosaic virus occurs in the continent of

Europe and in w idely separated lo c alities in S cotland, England and
W ales; and the vector w as reported in 1959 as X. diversicaudatum
M icoletzky.
The soil-borne viruses th a t cau se fan leaf, yellow m o saic, and
vein banding are a ll m echanically sap -tran sm itted and produce very s i ­
milar symptoms in different herbaceous h o s ts .

Thus th ese v iruses can

only be separated on the b a sis of th eir affects on the grapevine p lan t.
The vectors for a ll 3 viruses are 3C. index (Raski e t a l. 1965).
GRAPEVINE YELLOW MOSAIC
G rapevine yellow mosaic is as vicious a grapevine k ille r a s g rap e­
vine fanleaf (Hewitt e t a l. 1962), in th at the vines gradually degen erate.
Yellow m osaic virus ca u ses th is d is e a s e , which has been a lso called
(1) paranchure (Vulttenez 1952); and (2) clorose lnfecclosa (Dias 1950 b).
G rapevine yellow mosaic virus is considered a s a strain of grapevine fan ­
leaf virus (Raski and H ew itt 1965), and only the symptoms of the g rap e­
vine w ill be d isc u sse d .
Symptoms. -

Yellow m osaic c a u se s eith er a few sm all irregular

yellow spots on some grape le a v e s, many yellow b lo tc h e s, or the leaves
are com pletely chlorotic (Hewitt and Delp 1953).
In grape v arieties of Vitis vlnlfera and V. ru p estrls Infected w ith
yellow m osaic v iru s, the grape clu sters are strag g ly , w ith sm all se e d le ss
b e rrie s, and are sm aller than those on non-lnfected v in e s .

GRAPEVINE VEIN BANDING
Vein banding d ise a se Is generally a sso c ia te d In the grapevine
w ith yellow m osaic and fanleaf and appears now th at a ll 3 d is e a se s
may be cau sed by strain s of the same virus (Hewitt e t a l 1962).

Vein

banding Is caused by the grapevine vein banding virus (Goheen and H e ­
w itt 1962).
Symptoms. - Vines affected w ith vein banding produce le s s fruit
than do normal v in e s, and the clu ste rs of the fruit are straggly with
sm all, se e d le ss b e rrie s.

The c h a ra c te ristic s of vein banding on the

grape leaf are chlorotic bands along the v e in s , e ith e r light green or
chrome yellow (Goheen and H ew itt 1962).
GRAPEVINE YELLOW VEIN
Grapevine yellow vein d is e a s e Is damaging to grapes but has been
confined primarily to C alifornia w ith a few sc a tte re d -in fe ctio n s In New
York of little economic significance (Gilmer e t a l. 1968).

The d ise a se

is caused by the yellow vein v iru s, a ls o known as tomato ringspot v iru s,
and w as firs t reported by H ew itt 0956).
Symptoms. - The leaves have chrom e-yellow veins and varying shades
of yellow .

The grapevines affected w ith yellow vein are larger than the

non-infected v in e s, and the c lu ste rs are straggly w ith larg e, seedberries

\

9
and sm all s e e d le s s berries (Hewitt 1956).
On o c c a sio n s, young leav es show a mild chlorotic mottle In an
oak leaf line pattern w hich fades a s the leaves a g e .

It is not un­

common for c lu ste rs of the grape to drop off after bloom (Gooding 1963).
H ost ra n g e s . - M echanical sap inoculations can be made to Chenopodlum am aranticolor C oste and Reyn, and Gomphrena alobosa L .;

Vitis

vlnlfera L .: Vitis labrusca L .; Vitis rupestris S cheele; and in many woody
plants such as elderberry, currant and raspberry (Gilmer and Kelts 1968).
G eographic d istrib u tio n . -

California and New York (Gilmer and

Kelts 1968).
M eans of tran sm issio n . - Breece and H art (1959) estab lish ed the
soil-borne nature of the d ise a se yellow bud m osaic strain and found the
vector to be X. am erlcanum .

H arrison (1960) has shown by serological

t e s ts , yellow bud mosaic is a strain of tomato ringspot virus and yellow
vein v iru s.

Fulton (1962) first dem onstrated the transm ission of tobacco

ringspot virus (TRSV) byJC. am erlcanum .

The ab ility of transm itting grape

yellow vein virus by X. amerlcanum w as estab lish ed by Teliz e t a l. (1966).
PIERCE'S DISEASE
P ierce 's d ise a se k ills m ost v arie ties of grape Vitis vlnlfera and
V. labrusca (Hewitt 1953).

It is the only virus d ise a se affecting g rap e­

vines th at is known to be spread by air-borne v ec to rs.

P ierce 's d ise a se

is caused by lucerne dwarf v iru s, a ls o referred to as alfalfa dwarf v iru s.

Symptoms. - The leaf symptomatology of P ierce 's d ise a se Is
characterized by scald s and b u rn s.

The leaf-burning moves In co n ­

centric z o n e s, and moves from the margins to the petiole of the le a f.
Fruits affected by this virus color prem aturely before drying or
w ithering.

C anes mature Irregularly and the leaves drop, w ith p etioles

s till attached to the can es (Hewitt e t a l . 1962).

Death of roots u s ­

ually follows clo sely the decline of the top (Hewitt 1953).
H ost ran g es. - A lfalfa. M edlcaqo satlv a L .; w hite sw eet clo v er,
Trlfollum lncam atum L .; crimson clover, T. pratense L .; Vitis vlnlfera
L .; and V. labrusca L. (Smith 1957).
Geographic d istrib u tio n . - Gulf c o a st s ta te s , C alifornia, Mexico
and Argentina (Hewitt e t a l . 1958).
Means of tran sm issio n . - The virus is not sa p -tran sm issib le but can
be transm itted by grafting.

The in sect vectors are a ll C icadellid le a f-

hoppers belonging to the subfam ily Amblycephalinae; the following s p e c ­
ies have been identified as vectors: (1) D raeculacephala mlnerva (Ball);
(2) C am eocephala fulalda (N ott.);

(3) C . triau ttata (N ott.); (4) H elo-

chara d elta (Oman); (5) Neokolla circellata (Baker); (6) N . qothica
(Sign.); (7) N . confluens (Uhler); (8) N. hieroglyphics (Say); and (9)
Cuema Occidents 11s (Oman and Beamer) (Smith 1957).
Freltag and Frazier (1954) have conducted te s ts to determine the
percentage of leafhopper vectors that were carrying the virus under
natural conditions in a number of different h a b ita ts.

In these te sts

p articular em phasis w as placed on 3 econom ically important vectors:

11
(1) the green sh arp -sh o o ter, D raeculacephala mlnerva (Ball); (2) the
red-headed sh arp -sh o o ter, C arneocephala fulalda (N ott.) and (3) the
blue green sh arp -sh o o ter, Neokolla clrce 11ata (Baker).

The resu lts

su g g est th at generally the virus occurs naturally wherever the 3
Important vectors are found (Smith 1957).
GRAPEVINE LEAFROLL
Grapevine leafroll affects vineyards prim arily in C alifornia and
Europe with the g re a te st sev erity being in Germany (Scheu 1936) .
Scheu estim ated th a t over 80 per cen t of a ll grapevines planted in
Germany were infected; some v arieties were so com pletely d ise ase d th at
the v arietal descriptions were based on symptoms of leafro ll-affected
p la n ts.

Grapevine leafroll virus is responsible for this d is e a s e , which

has been a lso called (1) Rollkrankheit (Scheu 1936); (2) w hite Emperor
(Harmon and Snyder 1946); and (3) grapevine leafroll (Harmon e t a l. 1956).
Symptoms. -

The first symptoms appear during the middle of the

growing season on the older leaves a t the b ase of the c a n e s .

V arieties

bearing w hite or green fruit affected with the leafroll virus have light
green leaves and the fruit does not color properly.

Burning of the

leaves is common on d ise ase d vines in the hot interior v alley of C a li­
fornia (Goheen, Harmon and W einberger 1958).

The ch a ra cte ristic in ­

ward rolling of the leaf margins toward the back of the lamina gives
the d ise a se its name.
Production is decreased w ith vines affected by leafroll and the
sugar content is lowered from 1-4 degrees Balling.
the fruit does not develop.

The full color of

12
H ost ran g es. - Vitis vlnlfera L. ; symptom le s s carrier Vltls rupe s trls Scheele 'S t. Georye' (Hewitt, G oheen, Raski and Gooding 1962).
Geographic d istrib u tio n . - C alifornia, New York, Portugal, Spain,
France, Sw itzerland, Ita ly , Bulgaria, C zechoslovakia and South Africa
(Gilmer e t a l . 1968).
Means of tran sm issio n . - H ew itt (1962), indicates
m ission of

the mode of tra n s ­

leafroll has been undeterm ined. Leafroll is not s a p -tra n s ­

mitted to herbaceous h o s ts .
GRAPEVINE ENATION
G rapevine enatlon d ise a se only occurs in southern Italy and very
little is known about the d ise a se other than the symptoms produced on
the grapevine Vltls vlnlfera L. (Granltl 1959).
Symptoms. - Leaves with enatlon are gen erally m isshapen, dwarfed,
and rough.

The veins appear to be larger than normal and are promin­

ent on the lower surface (Hewitt 1954).
H ost ran g e. - Vltls vlnlfera L.
G eographic distrib u tio n . - Italy (Granltl 1959).
Means of transm ission. - Grapevine enatlon d ise a se has no.known
vector, but can be graft-transm itted (Hewitt 1954).

GRAPEVINE CORKY BARK
G rapevine corky bark d ise a se only has been described a t D avis,
California as graft-transm itted V. rupestrls 'LN 33' from Vltls vlnlfera
'C arig n an e', 'French Colom bard', and 'G renache' vines th at did not

13
show symptoms.

The d is e a s e , cau sed by the corky bark v iru s,

Is not important (Hewitt e t a l. 1962).
Symptoms. - Since grapevine corky bark d ise a se was g ra ft-tra n s ­
mitted to 'LN 33' from vines th at revealed no symptoms, very little
is known about th is virus other than the symptoms th at developed on
'LN 3 3 '.

In can es of 'LN 3 3 ', the bark w as th ic k , soft and spongy

and often s p lit into longitudinal crack s w ith heal-form ing fissu res
(Hewitt e t a l. 1962).
H ost ra n g e s. - Vitis vlnlfera L .; and Vitis ru p estrls Scheele 'LN 33'
(Hewitt e t a l . 1962).
G eographic d istrib u tio n . - G raft-transm itted in California to 'LN 3 3 '.
Means of tran sm issio n . - Spread undetermined for the d ise a se
grapevine corky bark.
AN UNDESCRIBED DISEASE
This undescrlbed d ise a se is caused by tobacco m osaic virus which
produces no v isu a l symptoms in the grapevine and has no economic im­
portance in grape (Gilmer and Kelts 1968).

Tobacco mosaic virus only

has been detected in the grapevine by m echanical transm ission to host
plants and confirmed by serological testin g (Gilmer and Kelts 1965).
H ost ra n g e s. - Vitis vinifera L .; Vitis labrusca L. 'C oncord';
i

Chenopodium ouinoa, W llld .; N icotiana olutlnosa L .: N . tabacum L .;
P haseolus vulgaris L .; and many more too numerous to mention
(Smith 1957).

14

Geographic d istrib u tio n . - France, and New York (Gilmer and
Kelts 1968).
M eans of tran sm issio n . - The virus Is sap tran sm issib le and Is
one of the most Infectious of the plant v iru ses (Gilmer and Kelts
1968).
GRAPEVINE ASTEROID MOSAIC
Grapevine astero id m osaic is a d ise a se confined primarily to
C alifornia and Is not important (Hewitt and Goheen 1959).

The d ise a se

is caused by the grapevine astero id m osaic virus and has no other known
nam es.
Symptoms. - The leaf symptoms are represented by sm all, lu cid ,
third-order veins which often c o a lesce to centers to give the appearance
of sta r-lik e spots (Hewitt and Goheen 1959).

The affected vines produce

little or no fruit and are stu n ted .
H ost ra n g e s. - Vitis vlnlfera L .; and V. rupestrls Scheele 'S t.
G eorge' (Hewitt e t a l . 1962).
M eans of tran sm issio n . - The spread of grapevine astero id mosaic
virus is undeterm ined.

OBSERVATIONS
Every grape vineyard surveyed (50 or more) in the Paw Paw
and Lawton, M ichigan areas contained vines th at displayed symptoms
of virus or v iru s-lik e d is e a s e s .

In addition to the fan leaf, sh e llin g ,

and delayed bud-break symptoms of d ise a se th a t 'triggered' the sta rt
of this project, other virus or v iru s-lik e d ise a se symptoms were un­
covered.

These were the following:

(1) malformed and discolored

leaves w ithout the ty p ical vein anastom osis of the fanleaf condition,
(2) malformed canes and aty p ical vine grow th, and (3) vine d e c lin e .
M ost of the surveyed-vineyards appeared nearly h ealth y , ex cep t for
a w idespread occurrence of the fanleaf condition in early August.

This

ailm ent is m anifested in leaves w ith an open ventral s in u s , serrated
m argins, and vein anastom osis; the latter is m ost diagnostic and varies
from slig h t to severe to give the effect of an open to clo sed fan , r e ­
sp e ctiv e ly , as the leaf narrows w ith severity (Fig. 2).

Since th is malady

w as found in every vineyard surveyed w hether the other observed d ise ase
symptoms were evident or n o t, it w as judged a s a d ise a se unrelated to
the o th e rs.
Two vineyards* were c lo sely observed as they contained vines
*Arden Ewing Farm, Porter Township and Section 8 , Road M-119,
Lawton, M ichigan
lHarold W ilder Farm, Porter Township and Section 7, Valley Road,
Lawton, M ichigan
15

Figure 2 . - 'F anleaf' condition on a 'Concord' grapevine
le a f.

displaying a ll of the d ise a se symptoms th a t were recorded throughout
the survey and healthy vines as w e ll.

The rem ainder of the v in e­

yards were only sc arcely d ise a se d in relation to th ese two v in ey ard s.
Both of the vineyards w ere characterized by patches of dead or d e ­
clining vines in some area s of the vineyards and apparently healthy
vines in other a re a s; fanleaf occurred on nearly every vine by September
in 1966, 1967 and 1968.
Since a ll of the observed symptoms occurred on these two farm s,
it appeared likely th a t a ll of the symptoms (except the w idespread
fanleaf) were part of a syndrome th at could be b e st described as g rap e­
vine degeneration.
The grapevine degeneration syndrome appears to be as follow s:
D elaved b u d -b reak . - The dormant buds expand until a few m illi­
m eters in length, but then remain a t a sta n d still for 10-20 days in ­
stead of 'breaking*.

Vines affected w ith these m aladies develop slow er

than normal vines and fruit blooms a lso 'b reak ' la te .
Malformed and discolored le a v e s . - The leaves on young stem s
early in the seaso n are mottled with blotches of dark and light green
tis su e ; mottle fades out a s leaf a g e s .

Leaves acquire a shape much

like that described for the fanleaf condition, but serration of the leaf
margin and anastom osis of veins does not occur (Fig. 3).

The marginal

sin u ses on some leaves are d eep ly -cu t and the ap ical tip is o b literated .
Affected leaves u su ally fall e a rlie r than unaffected leav es; leaf drop
begins a t the b ase of the sh o o ts.

Malformed can es and a ty p ica l vine grow th. - Some can es are
a ty p ica l In structure and developm ent.

These can es are flat rather

than round (in c ro ss-se c tio n ) and often contain short ln tem o d es.
L aterals from th e se can es grow In a zig -zag manner a s they elo n g ate .
M aturity of th ese can es (when the outer tis su e hardens) occurs about
2-3 w eeks la ter than norm al.
Berry sh e llin g . - Soon afte r bloom sev eral of the berries In a
c lu ste r drop; some berries remain attach ed to the ped icel but do not
enlarge (sh o t-b errles).

Ripening of the remaining fruit Is delayed

sev eral days and the fruit do not a tta in full s iz e .

This alim ent leaves

vines w ith straggly berry clu ste rs of le s s than o n e-h alf the normal
number of fruit or w ith c lu ste rs of no fruit (Fig. 4 and 5).
Vine d e c lin e . - Shoots become dwarfed and contain malformed,
discolored and sm all leav es; growth is very s p a rse .

When growth is

reduced to only a few weak sh o o ts, the vine dies during or soon after
its next dormancy.

20

Figure 4. - 'Shelling' condition on 'Concord' grapevines

Figure 5

Normal 'Concord' grape b unches.

EXPERIMENTATION
GENERAL METHODS AND MATERIALS. - T est p la n ts , v iru s e s . and
v e c to rs. - To achieve a uniform crop of te s t p la n ts , the seed s were
sown In a pan of sterilized s o il.

Seedlings were transplanted 8-10

days la ter Into 4 Inch clay pots In ste riliz ed loam so il and used in
experim ents w ithin 2 w eeks afte r th ey were tran sp lan te d .

These plants

were grown in a sp e cia l room w ell iso lated from other rooms utilized
in this research greenhouse.
Black cow pea Vlana sin e n sis (Tomer) 'Black RS', re s is ta n t to to ­
bacco ringspot (TRSV) and su sc e p tib le to cucum ber m osaic virus (CMV);
black cowpea V. sin en sis (Tomer) 'Black SR', su sc ep tib le to TRSV and
re s is ta n t to CMV (De Zeeuw and Crum, 1963); Chenopodlum auinoa W illd .;
N lcotlana tabacum L. 'X aftthi-nc'; Petunia hvbrlda Vilm. 'Pink M agic';
Phaseolus vulgaris L .j Cucumis sa tiv u s L .j Pis urn sativum L. 'M id ­
fre eze r'; and Vitis labrusca L. 'C oncord'; were used ex clu siv ely a s te st
p la n ts .
The plants used in this study w ere eith er grown in or transferred to
the greenhouse and were held a t a temperature of about 22 C . , r e ­
lative humidity 60% and photoperiod 12 h r.

All rooms used for plant-

growing were fumigated each w eek w ith nicotine or parathlon smoke
bom bs.
21

22
The growth chambers u tilized in th is study were ad ju sted to a
tem perature of 22 C , 12 hr photoperiod and 50% relativ e hum idity.
G reen peach ap h id s, Mvzus p erslcae (Sulzer), used in th ese e x ­
periments to remove the CMV from the m echanically Inoculated 'C on­
cord1 grape le a v e s, were m aintained in the g reen h o u se.

The aphids were

allow ed to feed on te s t plants prior to a 2 -hr starvation feeding in
order to be sure no v iru ses were p resen t in the green peach aphids
used in this stu d y .
The v iru s-free nem atodes were obtained from an area where w hite
pine trees were growing and known as the Sandford W ood-Lot a t
M ichigan State U niversity.

This wood lot w as never u tilized for a g ri­

cultural crop production, thereby, elim inating the presence of grape
d is e a s e s .

Large numbers of v iru s-free nem atodes were obtained by pro­

ce ssin g many 1-p ln t sam ples of so il through the use of the technique
developed by C hristie and Perry (1951).
T est farm . - Petunia plants were planted ad jacen t to the grapevine,
affected with grapevine degeneration a t the farm of Mr. Arden Ewing,
Lawton, M ichigan.

Fig. 6 shows the resu lts of the grapevine degener­

ation d ise a se - a d rastic decline is quite evid en t.
M r. Ewing's vineyard w as approxim ately 75-years old and was re ­
planted w ith a new 'Concord' grapevine w herever old vines were rogued
o u t.

The grapevines were trained and pruned each year prior to bud

break, and fertilized w ith a com plete fe rtiliz e r.

A green-m anure or

cover crop w as planted in the fa ll to check grapevine grow th, thus
giving the vine time to mature its wood properly.

23

Figure 6 . - A 'Concord' grape vineyard in southw estern
M ichigan showing drastic vine d ec lin e .

24
No herbicides have ever been used in th is te s t vineyard,
how ever, a cultiv atio n program of keeping the grape vineyard free
from w eeds w as p ractice d .

In g e n e ra l, the grape spray program for

control of d ise a se s and in se c ts was conducted as outlined by the
extension se rv ic e.
Twelve rows of approxim ately 50 grapevines per row were present
in the te s t vineyard.

The vines were planted in a sandy-loam s o il,

located on a gradual slo p e .

The grape rows ran north and south and

on the south end of the field was a w ood-lot w hile on the north and
e a s t ends of the field were more g rap ev in es.
te s t vineyard was a productive asparagus fie ld .

On the w e st side of the
This te s t vineyard w as

an ex cellen t illu stratio n of grapevine degeneration.
M echanical in o c u latio n s. -

In the m echanical in o cu latio n s, other

than grape leaf tis su e e x tra c ts , disodlum phosphate .07 molar was used
a s a buffer.

Only enough buffer w as added to liquify the tis s u e ex tract

and plants were inoculated after 2 to 3 w eeks of growth using carb o r­
undum on the te s t plants as an abrasive and w ashing the leav es w ith
w ater immediately after the plant sap w as applied to the leaves by finger.
Those inoculations where young grape leaf tis s u e s were taken from
grape plants believed to be infected w ith grapevine degeneration were
thoroughly triturated in 2-3 ml of Kirkpatrick-Lindner buffer 3% nicotine
0.05 M K2HPO4 - 0.005 M cy stein e hydrochloride (1963).
Two to three leaves of the te s t plants were lightly dusted with
400-m esh Carborundum and inoculated w ith the grape leaf triturated
m aterial.

The plant sap was applied to the leaves by finger.

Surgical

25
gloves were used and w ashed a fte r each in o cu latio n .

The inocu­

lated leaves were rinsed 2 minutes afte r inoculation.

This prevented

the nicotine from entering the te s t plants and causing phytotoxicity.
The te s t plants were removed to an in sect-p ro o f greenhouse room to
aw ait symptom developm ent.

To guard a g a in st contam inating in se cts

from entering the room, a fumigation program was in itiated a t the g reen ­
house a s explained e a rlie r.

The te s t plants were undisturbed for 2 or

3 w eeks after inoculation before symptom readings were m ade.

After

the readings were tak en , the plants were m echanically back -in o cu lated
to th eir resp ectiv e p la n ts.
O uchterlonv D ouble-D iffusion Agar T e st. - Tobacco ringspot a n ti­
serum w as obtained from two so u rc e s.

One source w as from R. W .

Fulton, Department of Plant Pathology, U niversity of W isconsin and the
other source w as from H . A. S co tt, Department of Plant Pathology,
U niversity of A rkansas.

Both an tisera were p re -te ste d with a known to ­

bacco ringspot virus to make sure the antiserum s were properly recon­
stitu te d .
A 1% Bacto-Agar solution w as prepared and properly melted in a
w ater b a th .

After the solution was melted a 0.02% sodium azlde p re­

paration w as added to the Bacto-Agar in order to inhibit the growth of
m icro-organism s.
To each petri d ish th at w as used for the Ouchterlony te s ts , 20 ml.
of B acto-A gar.. .02% sodium azide solution was added and allow ed to
so lid ify .

A p la stic pattern of se v en , 7-mm rings in 3 rows w as used

26
to prepare the w ells for an tigen-antiserum re a c tio n s.

The edges of

each ring In the pattern were so spaced th a t the 2 outer rows of 2
w ere 1 cm from each ad jacen t ring.
The p etrl d ish of solidified ag ar was then placed over the pattern
and a flamed cork borer was used to punctr w ells In the ag ar representing
c irc le s of the p attern .
an a sp ira to r.

The agar In the w ells were removed by use of

Later a tran sfer cap illary pipette w ith melted agar was

used to place a droplet of agar In each w e ll.

This se ale d the bottom

of the solidified ag ar d is h .
Into the cen ter w ell, designated D, of the agar plate w as added
enough tobacco ringspot virus antiserum (1:8) to fill the w e ll.

Then to

the top 2 w ells d esig n ated A and B and the bottom 2 w ells designated
F and G were added petunia sap (diluted 1:8 w ith dlsodlum phosphate
ph 8.0) from the grapevine degeneration p la n ts.

To the remaining 2

ce n te r w ells labelled C and E were added known tobacco ringspot petunia
sap (diluted 1 to 8 with dlsodlum phosphate ph 8 . 0).
These agar g el plates were held at 22 C and 60% humidity properly,
m arked, and allow ed to incubate for 10 days w ith the tops on the petrl
d ish es (to allow for a m oist atm osphere).
were read and properly recorded.

After Incubation the samples

EXPERIMENTS AND RESULTS
Isolation of vim s through the use of a trap P lant.

- Two factors

encouraged the use of a trap plant in the iso latio n of virus (es) from
grapevine: (1) the pattern of degeneration in the te s t vineyard Indicated
the likelihood of a so il-b o rn e in c ita n t, and (2) grapevine w as known
to be a poor source plant from which to directly Iso late v iru ses through
m echanical means (Gilmer and K elts, 1967).
Petunia hvbrlda Vilm. w as se lec ted as the trap plant sp ecies as it is
su sc ep tib le to a wide range of v iru se s , including sev eral of grapevine.
Young petunia seed lin g s were planted ad jacen t to grapevine plants through­
out the te s t vineyard in May of 1967 and 1968; the two plantings were
id e n tic a l, except the plants in the la tte r planting were covered with
sa ran -screen ed cag es to exclude ae ria l in se c t contam ination.

Both p la n t­

ings encom passed grapevines varying in appearance from normal to se v e re ­
ly d eclin ed .

In early fall of each y ear, petunia plants th at showed any

Indication of virus symptoms were transplanted to large pots or tubs and
transferred to the g reen h o u se.

Then the plants were assay e d for v iruses

by m echanical Inoculations (in .07 M dlsodlum phosphate buffer) to
se v eral herbaceous te s t p la n ts.

27

28
TABLE 1.

Response of various plant sp e c ie s when inoculated

w ith sap ex tracts of trap petunia p la n ts, th at were grown ad jacen t
to grapevines affected w ith grapevine degenerationa

T est

T ransm issions^ and svmotoms0

Plants

1967

ChenoDodium auinoa W illd.

-

1968
8/13 RN

N icotlana tabacum L. 'X anthi-nc'

18/68 LP
14/68 CM

8/13 LP

Viana sin e n sis (Tomer) 'Black SR'

18/68 NLL

8/13 NLL

V. sin e n sis (Tomer) 'Black RS'

14/68 TMM

0/13 0

a Trap plants in 1967 were not protected from a e ria l in se c ts; plants
in 1968 were protected.
^Numerator=number of infections; denominator=number of tria ls .
Each trap petunia was assay e d once on the range of te s t p la n ts.
CM =chlorotic mottle; LP=line and oak leaf patterns; NLL=necrotic
local lesions w hich are reddish-brow n; 0=no symptoms; RN=rosetting
and necrotic lesio n s; TMM=trifoliate leaves mottled and mosaic; -= te st
plants not utilized for a s s a y .

29
Four different types of virus reactions were obtained (Table 1.)
The positive response of V. sin e n sis 'Black RS1 w hich is re sis ta n t
to tobacco ringspot virus and su scep tib le to cucumber m osaic virus
to the affected petunia sap suggested in the petunia plants were in ­
fected w ith CMV in the vineyard.
Another virus reaction w as the development of an oak c lu ste r on
the 'Xanthi-nc* p la n t.

This response is typical of a TRSV reac tio n .

A TRSV-susceptible reaction was a lso obtained on Vlona sin en sis
'Black SR'.

Primary lesio n s of necrotic spots appeared on the c o ty ­

ledons and w ithin sev eral days the virus became sy stem ic.
In e s s e n c e , two v iru ses were found in the petunia plants in te r­
planted during the 1967 se aso n in the affected v in e y a rd .. .CMV and
TRSV.
From stud ies made in 1967, Table 1 dem onstrates that the TRSV
w as soil-borne and p resen t in the affected vineyard during the 1968
growing se aso n and p o ssib ly in 1967 as w ell.
Isolation of virus directly from grapevine. - Several hundred
attem pts were made to iso late virus(es) from grape le a v e s, ste m s,
roots and fruit betw een June and Septem ber, 1967 w ith the u se of 2.5%
i

nicotine sulfate a s a buffer and C . aulnoa as an a s s a y p la n t.

Two

ex tracts made from the stem and term inal leaves of a degenerate g rap e­
vine on June 4 , 1967, during an in itial iso latio n attem pt, were infectious
and caused in itially a mosaic and finally a severe term inal stu n t and
chlorosis reaction on C . a u ln o a.
fectious in 1967.

None of the other ex tracts were in ­

30
The numerous failures obtained in 1967 led one to su sp e ct
th a t (1) another buffer system might more su itab ly sta b ilize the
inoculum than nicotine s u lfa te , and (2) the relativ e titre s of virus
and inhibitor might be optimum for m echanical virus transm ission a t,
or soon a fte r, b u d -b reak .

T hus, extractions in 1968 were begun a t

bud-break and made once a month th ereafter.

A late frost in May s e t

b ack most of the grapevines approxim ately 3 w eek s, therefore bu d break in 1968 did not occur u n til m id-June.

A lso, a t the suggestion

of R. M. Gilmer, Department of Plant Pathology, Cornell U niversity,
sap extracts were triturated in 2-3 ml of Kirkpatrick-Lindner buffer
(Kirkpatrick e t a l . , 1963).

TABLE 2.

Responses of various plants sp e cie s when m echani­

c a lly inoculated with ex tracts from leav es of grapevine, Vitis labrusca
L. 'C oncord', affected with grapevine degenerations

Plants

T ransm issions3 and symptoms*3

tested

22 June

6 Aug.

Chenopodium quinoa W illd.

21 RN

0

0

N icotiana tabacum L. 'X anthi-nc'

14 LP
7 EM

8 EM

0

Vicma sin e n sis (Torner) 'Black SR'

13 NLL

7 NLL

0

0 TMM

0

V. sin e n sis (Tomer) 'Black RS'

5 TMM

6 S ept.

a Number of transm issions per 22 grape plants tested ; the same
plants were assay ed on each d ate.
L

EM =epinasty and m osaic; LP=dine and oak leaf patterns; NLL=
necrotic local lesions which are reddish-brow n; 0=no symptoms; RN=
rosetting and necrotic lesio n s; TMM=trifoliate leaves have a mosaic
p a tte rn .

33

Identification of tobacco ringspot v iru s . - Twelve p lates of io n agar were prepared to a sc e rta in the presence of TRSV in th e-cag ed
petunia plants interplanted in the grapevine degeneration vineyard
during May, 1968, using the technique known as the Ouchterlony
agar double-diffusion te s t.

Known phosphate-buffered laboratory in ­

duced TRSV petunia ex tracts and phosphate-buffered ex tracts from the
caged trap petunias were placed in w ells in a g el diffusion p la te , and
the TRSV antiserum was placed in an adjoining w ell.

Both the 'caged

petunia' extracts and the known laboratory induced TRSV petunia ex tracts
reacted with the antiserum , and the preciptin zones co alesced in a
reaction of id en tity .

D efinite p recipitation zones were observed with

8 out of the 12 an tigen-antibody re a c tio n s.

This experim ent further

proves th at TRSV was present in the degenerate grapevine te s t vineyard.
Nematodes a sso c ia te d w ith a degenerate v in ey ard . - The likelihood
of a soil-borne in citan t in the te s t grape vineyard suggested the hypo­
th e sis that nem atodes were the v ec to rs.

A dditionally, some growers

were adam ant th a t the in citan t w as grapevine fan leaf, which is tra n s ­
mitted only by Xiphinema index Thorne and Allen in C alifornia (Hewitt
e t a l. 1958); this nematode is only known to occur a s far north as
southern Indiana (J. F erris, personal com m unication).

Thus, so il sam ples

were taken throughout the root zone of the grape plant to a depth of
12 inches and w ithin 2 ft. of the b ase of the plant w ith a so il probe to
asce rta in the nematode com position in the te s t vineyard.

These so il

sam ples were taken from the degenerate vineyard in May of 1966, 1967,
and 1968.

34

++

The Jenkin's (1964) sugar flotation technique was employed
to ex tract the plant p arasitic nematodes from the so il sam p les.

The

sugar flotation technique was used rather than the Chris tie-P erry
(1951) technique b ecau se some nem atodes, such a s , Criconem oides
s p . , are unable to wiggle through the cloth filter in the Baermann
funnels u tilized in the la tte r technique.
Soil sam ples were processed in 50 ml quantities and a ll nem a­
todes genera known or su sp ected to be p arasitic to grapevine were
sought in sample readings; only_X_. americanum Cobb and X. index
Thorne and A llen'w ere determined to the sp ecies lev el.

35

TABLE 3.

Plant p arasitic nem atodes found in the root zone

so il area of grapevine, Vitis labrusca L. 'C o n co rd ', affected with
grapevine degeneration

Nematodes

The number of nem atodes9 per 50 cc of
so il sampled in Mav of specified years

sought

1966

1967

1968

3

0

0

65

10

40

Hoplolaimus sp .

1

8

0

Lonaidorus sp .

0

2

0

Pratvlenchus sp .

3

3

5

Tricodorus sp .

2

0

0

Tvlenchus sp .

12

4

2

Xiphinema americanum

15

22

8

0

0

0

Aohelenchus s p .
Criconemoides sp .

Xiphinema index

a

Nematodes were extracted by the Jenkins (1964) sugar flotation
t

A

tech n iq u e.

36

R epresentatives of the 3 nematode genera (Lonqidorus. Tric hodorus and Xiphinema) known to transm it plant v iru ses were e x ­
tracted from the te st vineyard (Table 3).

However, only 2 individuals

of Lonqidorus and Trichodorus were obtained and on a single o ccasio n .
Xiphinema amerlcanum individuals were obtained in relative abundance
in each sam ple.

As might be expected no X. index individuals were

uncovered.
Transm ission of TRSV bv Xiphinema americanum extracted from the
root zone of degenerate g rap ev in es. -

Since TRSV w as iso lated from

b a it petunia plants that were grown in the te s t vineyard, it w as assum ed
that nematodes were the vector organism s.

This assum ption was te sted

through transm ission trials involving individuals of Xiphinema americanum
that were extracted from so il co llected from the root zone of degenerate
grapevines; sam ples were taken only from around vines that were free of
w e e d s.
Large numbers of nem atodes were obtained by processing many
1-p t. sam ples of so il through the use of the technique developed by
C hristie and Perry (1951).

Individual X_. americanum were hand-picked

into sm all vials of w ater in lots of 30-50 nem atodes.

These nematode

lots were introduced to the healthy te s t plants (petunia), w hich were
growing singly in 150 ml. beakers in a sterilized media of 1 part loam
so il to 1 part Turface, a granular calcined pyrophyllite (Fig. 7).

An

inoculation period of 14 days w as allo tted in a controlled environment
chamber; then, a ll petunia plants were indexed by m echanical inoculations
of expressed sap to V_. sin e n sis 'Black SR' and N. tabacum 'X anthi-nc '

37

Figure 7. - S e e d lin g s in s t e r il i z e d s o il and g ra n u la r

'

c a l c i n e d p y ro p h y llite in 150-ml b e a k e r s .
The s y s te m u se d for a c q u i s i ti o n of v iru s e s
b y hand p ick e d n em ato des w h ic h w e re in tr o ­
d u c e d in to b e a k e r s .

38

te st p lan ts.
S ixty p e rc e n t (12_ out of 2£ nem atode lo ts) tra n s m itte d TRSV to
p e tu n ia b a it p l a n t s , a s in d ic a te d by ty p ic a l TRSV r e a c tio n s on the 2
te s t p lan ts.

This confirm s th a t TRSV is p r e s e n t in the root zone of

the a ffe c te d t e s t v in ey a rd an d th a t X. am erican um a re c a r rie r s of the
virus .

S e p a ra tio n of TRSV an d PRMV on the b a s i s of d if f e re n tia l h o s t
rea ctio n s. -

W ith a t l e a s t 2 v ir u s e s p r e s e n t in 'C o n c o rd ' g ra p e v in e

in the t e s t v in e y a rd , su rv e y s w ill n eed to be made to d e te rm in e the
d is tr ib u tio n and e x te n t of b oth v i r u s e s , to g e th e r a n d / o r in d iv id u a lly .
An a s s a y te c h n iq u e w h ic h w ould perm it v iru s s e p a r a tio n on the b a s i s of
m e c h a n ic a l in o c u la tio n s of p la n t s a p to d if f e r e n tia l h e rb a c e o u s h o s t
p la n ts w ould be m ost b e n e f ic ia l; t h u s , t r ia ls w ere made to u n c o v er
su ch host p lan ts.
Sap e x tr a c ts of TRSV-infected to b a c c o and PRMV - in f e c te d p e a c h
w e re p rep ared from m ac era te d l e a v e s , tritu ra te d in 0 .0 7 M d isodium
p h o sp h a te b u ffe r, and m e c h a n ic a lly in o c u la te d to a s e r i e s of young
h e rb a c e o u s p la n ts (Table 4 ).
Chenopodium quinoa

did not r e v e a l a good d if f e re n tia l h o s t

r e a c tio n for the two v ir u s e s t e s t e d .

N ic o tia n a tabacum 'X a n th i-n c ',

P h a s e o lu s v u l g a r i s . Pis urn sa tiv u m 'M id fre e z e r' and Viqna s i n e n s i s
'B lack SR' and 'B lack RS' re v e a le d s p e c if ic d if f e re n tia l h o s t re a c tio n s
(Table 4).

H o w ev e r, Pisum sa tiv u m

'M id fre e z e r' illu s tr a te d a v e ry d i s t i n c t

s y s te m ic lin e and rin g sp o ttin g re a c tio n (Figures 8, 9 and 10) to

39

TABLE 4 .

R e sp o n se of s e v e r a l h e rb a c e o u s p la n ts to to b a c c o

rin g sp o t v iru s (TRSV) and p e a c h r o s e tte m o sa ic viru s (PRMV)

P lants

P la n t r e s p o n s e 9 to:

te s te d

TRSV

PRMV

Chenopodium q u in oa W illd .

DE

DE

N ic o tia n a tabacum L. 'X a n th i-n c '

LP

E

P h a se o lu s v u lq a ris L.

LP

CM

Pisum sa tiv u m L. 'M id fre e z e r'

LP

0

Viqna s i n e n s i s (Tomer) 'B lack SR1

NLL

TMM

V. s i n e n s i s (Tomer) 'B lack RS'

0

TMM

DE=dwarfing and e p in a s ty ; E = ep in a sty ; LP=line and oak le a f
p a tte rn ; C M ^ c h lo ro tic m ottle; NLL=necrotic lo c a l l e s io n s ; 0=no
sym ptom s; T M M = trifoliate le a v e s m ottled and m o s a ic .

40

Figure 8. - BEGINNING s ta g e s of rin g s potting on the le a v e s
of Pisum s a tiv u m L.

This p lan t w a s m e c h a n ic a lly

in o c u la te d w ith to b a c c o rin g s p o t v i r u s . . .g row th
of th is v iru s w a s s y s te m ic .

41

Figure 9 . - INTERMEDIATE s t a g e s or rin g sp o ttin g on the
term in a l le a v e s of Pisum sa tiv u m L.

This

p la n t w a s m e c h a n ic a lly in o c u la te d w ith t o ­
b a c c o r in g sp o t v i r u s . . .g row th of th is viru s
w a s s y s te m ic .

42

Figure 10. - The FINAL s t a g e s of rin g sp o ttin g on the term in al
le a v e s of Pisum sa tiv u m L.

This p la n t w a s

m e c h a n ic a lly in o c u la te d w ith to b a c c o rin g sp o t
v i r u s . . .g row th of th is v iru s w a s s y s te m ic .

44

T ra n s m is s io n of p e a c h r o s e tte m o sa ic v iru s from m e c h a n ic a lly in o c u la te d p e tu n ia p l a n t s . -

The o c c u rre n c e of p e a c h r o s e tte m o sa ic

viru s (PRMV) in the fie ld -g ro w n p e tu n ia trap p la n ts in th is s tu d y and
the rep o rte d r e la tio n s h ip s of PRMV to 'C o n c o rd ' g ra p e v in e (Klos e t a l . ,
1967 and H . F. D i a s , p e rs o n a l co m m u n ica tio n ), prom pted e x p e rim e n ta tio n
to d e te rm in e , und er c o n tro lle d c o n d it i o n s , w h e th e r X. am ericanu m is i n ­
deed a v e c to r of the v i r u s .

The t e s t s c o n d u c te d by Klos e t a l (1967)

w ere la rg e ly field tr ia ls and the t e s t p la n ts co u ld have b e e n c o n ta m in a te d
by o rg an ism s o th er th an n e m a to d e s .
A PRMV i s o l a t e from p e a c h w a s m e c h a n ic a lly in o c u la te d w ith e x tr a c t
buffered w ith diso d iu m p h o sp h a te to young p e tu n ia p la n ts th a t w ere grown
in the b e a k e r sy s te m of 1 p a rt loam s o i l to 1 p a rt T u rfa c e , d e s c r ib e d
e a rlie r.

About 14 d a y s la te r w h en p la n ts w ere show ing sym ptom s of

virus in fe c tio n , 5 0 -6 0 h a n d -p ic k e d , v i r u s - f r e e , X . am ericanum w ere
in tro d u c ed into the s o u rc e p la n t ro o t z o n e s .

A 10-day a c q u i s i ti o n period

w a s a ll o t te d in a c o n tro lle d env iron m ent c h a m b e r.
The a ff e c te d p e tu n ia p la n ts w ere rem oved from the b e a k e rs and
new v i r u s - f r e e p e tu n ia p la n ts w ere re p la n te d in th e sam e b e a k e r s .

The

p e tu n ia p la n ts rem ained in the b e a k e rs for 14 d a y s and w ere th e n i n ­
d e x ed to t e s t p la n ts u sing 0 .0 7 M disodium p h o s p h a te a s a b u ffer.
Eighty p e rc e n t (8 out of 10 nem atode lo ts) tra n s m itte d PRMV to
p e tu n ia b a it p l a n t s , a s in d ic a te d by ty p ic a l PRMV r e a c tio n s on th e t e s t
p lan ts.
This e x p erim e n t re v e a le d th a t X. am ericanum w a s an e x c e ll e n t v e c to r
for th is v i r u s .

Figure 11. - C ucum ber m o sa ic v iru s m e c h a n ic a lly in o c u la te d to
'C o n c o rd ' g rap e le a f ex em p lify in g p r o g r e s s io n of c u ­
cum ber m o saic v iru s from the le a f tip to the le a f c e n t e r .

Figure 12. - The l a tte r s ta g e of m e c h a n ic a lly in o c u la te d cu cu m b er
m osaic virus a ff e c te d 'C o n c o rd ' g rap e le a f c a u s in g le a f
d e a th .

47

a p h id s w ere rem o v ed , th e p la n ts w ere sp ra y e d w ith m a la th io n , and
3 w e e k s w ere a llo tte d b efore symptom re a d in g s w ere m ad e.
e x p erim e n t w a s c o n d u c te d on 2 o c c a s io n s w ith in one m onth.

The
After

the re a d in g s w ere m ad e, the p la n ts w ere m e c h a n ic a lly in o c u la te d to
v ir u s - f r e e t e s t p la n ts of th e sam e v a r ie ty an d the r e s u l ts of th e s e
in o c u la tio n s confirm ed th e f i n d i n g s .

\

49

G re en p e a c h a p h id s w ere v e ry e f f i c ie n t in the tr a n s m is s io n of
CMV from g ra p e l e a v e s to cu cum ber (Table 5).

In both t r i a l s , a l l

of the a p h id s t e s t e d a t a 23-m in a c q u i s i ti o n period b e ca m e in fe c tio u s
w h e re a s no tr a n s m is s io n s r e s u l te d from p e rio d s s h o rte r th an 10 m in.
T h e s e i n s e c t s a l s o pro ved t h a t the viru s b ecam e s y s t e m i c a l l y - e s t a b l i s h e d
in th e g ra p e v in e a s th e y re c o v e re d viru s from a n o n - in o c u la te d l e a f .
C h e m ic a l a n a l y s i s of g ra p e v in e le a v e s a ff e c te d w ith s e v e r e v e in
a n a s t o m o s i s . - The w id e s p r e a d o c c u rre n c e of th e le a f v e in a n a s to m o s is
or 'f a n le a f ' c o n d itio n in th e g rap e p ro d u ctio n a r e a s of M ic h ig a n s u g g e s te d
t h a t p e rh a p s the in c ita n t w a s the h e rb ic id e 2 ,4 -d ic h lo r o p h e n o x y a c e tic
a cid .

H o w ev e r, th is w a s c o n s id e r e d u n lik e ly a s 2 , 4 - D had no t b e e n

u s e d in the in v o lv e d c o u n tie s for over a d e c a d e .

A d d itio n a lly , no

v i r u s e s have b e e n i s o l a t e d from 'f a n - l e a v e s ' .
To t e s t the h y p o th e s is th a t 2 , 4 - D w a s the i n c ita n t of f a n le a f , an
a n a l y s i s w a s made of th e 2 , 4 - D c o n te n t of s e v e r e l y a ff e c te d g ra p e v in e
l e a v e s b y e le c tr o n c a p tu re g a s chrom ato grap hy (Erickson and H ie Id
1962).

G rape le a f t i s s u e from the t e s t v in ey a rd c o n ta in e d tra c e am ounts

(0.001 p . p . m . ) of 2 ,4 - E w h ic h p ro bably c o u ld n o t c a u s e the le a f v e in
a n a s to m o s is (Gilm er, p e rs o n a l c o m m u n ica tio n ).

T h u s, the in c ita n t for

the le a f v e in a n a s to m o s is or 'f a n le a f' c o n d itio n in th e g ra p e p ro d u ctio n
a r e a s of M ichig an w a s n ot lik e ly to b e the h e rb ic id e 2 ,4 - D .

DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS

From a v a i la b l e e v id e n c e p re s e n te d b o th h e re in and b y H . F.
D ia s (p erso n al c o m m u n ica tio n ), and Klos e t a l . (1967), i t is a p p a r e n t
th a t v in e s in th e t e s t v in e y a rd in v a rio u s s t a g e s of d e g e n e ra tio n a re
in fe c te d w ith p e a c h r o s e t te m o sa ic v iru s (PRMV), and to b a c c o r in g s p o t virus (TRSV).
In th is s t u d y , the s a p e x tr a c t s from g ra p e v in e th a t produced
p o s itiv e TRSV re a c tio n s on d if f e r e n tia l h e rb a c e o u s h o s ts w ere not t e s te d
s e r o l o g ic a l l y .

But s a p e x tr a c t s from p e tu n ia p la n ts growing in the sam e

ro o t e n viro nm ent g a v e i d e n t i c a l TRSV r e a c tio n s on the a s s a y p l a n t s ,
a nd t h e s e w ere s e r o l o g ic a l l y - p o s i ti v e for TRSV.

Some of t h e s e sam e

trap p e tu n ia p l a n t s , a l s o y ie ld e d s a p w h ic h t e s te d s e r o l o g ic a l l y - p o s i ti v e
for PRMV; th u s , the o rig in a l p rem ise th a t two or more v i r u s e s (PRMV and
TRSV) w ere p r e s e n t in the t e s t v in ey a rd w a s s u b s t a n t i a t e d .
The d if fe re n tia l h o s ts u n c o v ered for TRSV and PRMV s e p a r a tio n w ill
f a c i li t a t e su rv e y s of many v in e y a rd s in th e Paw Paw and L aw ton,
M ic h ig a n a r e a s to le a rn the d is tr ib u tio n in v in e y a rd s of s e v e r e , m oderate
an d mild d e g e n e ra tio n .

Surveys c o uld be made throughout the growing -

s e a s o n through the u se of sam p le c u ttin g s th a t a re s u b s e q u e n tly rooted
and p r e d is p o s e d w ith a 5 -m in per hr photoperiod regim e for 2 w e e k s
p rio r to s a p e x tr a c tio n .
50

Further te s tin g of the 'C o n co rd ' g ra p e p la n ts re v e a le d th a t
a f t e r m e c h a n ic a lly in o c u la tin g h e a lth y g ra p e le a v e s w ith CMV, the
v iru s in fe c te d the g ra p e v in e and b e c a m e s y s te m ic a l l y e s t a b l i s h e d .
S in ce CMV w a s re c o v e re d by the g re e n p e a c h a p h id , it is p o s s ib le
th a t th is c o s m o p o lita n a p h id , a l s o in o c u la te d fie ld -g ro w n 'C o n c o rd '
v in e s w ith CMV and t h a t CMV is a r e s i d e n t viru s in g ra p e v in e ; h o w ­
e v e r , CMV h as not b e e n e x tra c te d from fie ld -g ro w n g r a p e v in e .

E x p e ri­

m ents d e s ig n e d to rem ove CMV from th e fie ld -g ro w n g ra p e v in e by
m e c h a n ic a l m eans sh o u ld be i n v e s ti g a t e d .
Attempts sh o u ld now be made to d eterm in e 'c a u s e and e f f e c t'
r e la tio n s h ip s b e tw e e n th e v ir u s e s an d s p e c i f i c a s p e c t s of the d e g e n e ra tio n
sy n d ro m e .

Of param oun t im portance is a t e s t of the h y p o th e s is p r e ­

s e n te d h ere th a t PRMV a n d /o r TRSV c a n c a u s e b erry s h e llin g and p r e ­
mature b e rry d ro p .

A young 'C o n c o rd ' tr a n s p la n t p ro b ab ly w ill not fruit

u n til it is 4 yr old; t h u s , a long d e la y w ould o c cu r b efore in fe c tio n s of
TRSV or PRMV (cre ate d through m e c h a n ic a l or nem atode in o cu latio n ) c o u ld
be r e la te d to fruit s e t .
PRMV - or TRSV

Perhaps it w ould be more f e a s ib le to g ra ft

- in fe c te d buds in to v i r u s - f r e e 'C o n c o rd ' v in e s th a t

are w ith in 1-2 yr of m atu rity and a n ti c i p a te th a t in fe c tio n s w ould a f f e c t
fruit s e t w ith in 1-2 yr o f the g raftin g o p e ra tio n .
Klos e t a l .

(1967) in d ic a te d a c a u s a l r e la tio n s h ip of PRMV to d e ­

lay e d b u d -b re a k and s h e l li n g , b u t only 4 p la n ts w ere in v olv ed in th e
exp erim ent; only 1 's h e l l e d ' of the 13 in o c u la te d s e e d lin g s and it
occu rred 4 yr a f t e r in o c u la tio n .

No m ention w a s made of the c o n d itio n

of the p la n t or w h e th e r the g rafte d p la n ts becam e in f e c te d .

During th is

52

t h e s i s w o rk , s e v e r a l h e a lth y p la n ts have s h e l le d in and o u t of the
g re e n h o u s e an d s u s p ic i o n w a s c a s t on s o i l f e r tility during p o t - c o n fin em en t a s th e pro b ab le c a u s e .
It is lik e ly th a t a n y fa c to r th a t a f f e c t s g ra p e v in e n u tritio n w ill
a f f e c t b e rry s e t a n d m a tu rity .

N e arly a l l of the known g ra p e v in e v ir u s e s

have b e e n show n to a f f e c t fruit s e t , ra te of m aturity an d fru it s i z e .
H o w e v e r, f e r t i li z a t io n is known to b e h ig h ly c r i t i c a l in g rape p ro duction
and a n y s h o rta g e s of z in c a re known to a f f e c t the fruiting s ta g e (Shoe­
m ak er, 1955); t h u s , s t r e s s e s on the n u tritio n of th e p la n t c a u s e d b y
viru s in fe c tio n s co u ld p o s s ib l y e f f e c t fru it d e v e lo p m e n t.
v i r u s e s i s o la te d a re s u s p e c t c a u s e s .

Any of the

L ik e w is e , i t is r e a s o n a b le th a t

b u d - b r e a k is d e a ly e d in a p la n t of poor n u tritio n a s m ight be c a u s e d by
a resid en t v iru s.
M e c h a n ic a l m ethods of v iru s tr a n s m is s io n from g ra p e le a v e s w ere
d if f ic u lt.

Proper tim ing of e x tr a c tio n s an d the u s e of a p p ro p ria te b u ffer

s y s te m s w ere show n to be r e q u is ite for v iru s t r a n s m i s s io n .

O nly w hen

le a f sa m p le s from d e g e n e ra te 'C o n c o rd ' g ra p e v in e s w ere ta k e n 14 d a y s
a f te r b u d -b r e a k and th e K irkp atrick -L in dner buffer w a s u s e d w ere e x c e ll e n t
v iru s tr a n s m is s io n s o b ta in e d .
Further s tu d y sh o u ld be c o n d u c te d on the r e la tiv e titr e s of the
v i r u s e s in the g ra p e v in e an d the e lim in a tio n of the in h ib ito rs during
m e c h a n ic a l in o c u la tio n of the g rap e l e a f .

Varying the b u ffer c o n c e n ­

tra tio n an d the c h e m ic a l c o m p o s itio n of the buffer s y s te m s m ay e l i ­
m inate th e problem s e n c o u n te re d during m e c h a n ic a l in o c u l a t i o n s .

A lso ,

1

53

the ph oto p erio d for g rap e c u ttin g s sh o u ld be in v e s tig a te d a s to o p ­
timum an d minimum am ounts for proper m e c h a n ic a l in o c u la tio n s to be
made w ith th e g rap e v in e v i r u s e s (TRSV an d PRMV).

H . F . D ias

(p e rso n a l com m unication) h a s found th a t a 5 -m in p er hr pho toperiod
for 2 w e e k s p r e d is p o s e s th e g ra p e v in e fo r optimum s u c c e s s in v iru s
e x tr a c t i o n .
X. am erican um is c a p a b le of tra n s m ittin g PRMV and TRSV and
s in c e one or b oth of th e s e v i r u s e s is a lik e ly c o n trib u to r to the d e g e n ­
e ra tio n d i s e a s e it is r e a s o n a b l e , e v e n a t th is e a rly s ta g e of s t u d y , to
recom m end t h a t a l l new g ra p e v in e p la n tin g s be fum igated a n d th a t a l l
p ro p a g a tio n s b e made o nly from c e r tif ie d v ir u s - f r e e m other p l a n t s .
Embargoes sh o u ld be p la c e d on p la n ts p ro p ag a ted from n o n -in d e x e d
p la n ts.
For th o s e v in e y a rd s th a t a re p r e s e n tly in fe c te d w ith g ra p e v in e d e ­
g e n e r a tio n , a s a n ita r y program for d is in f e c tin g pruning s h e a r s a n d c u l t i ­
v a tio n sh o u ld be d e v e lo p e d b y the e x te n s io n s e r v i c e .

W here in d iv id u a l

g ra p e v in e re p la c e m e n t is n e c e s s a r y , a sim ple h a n d -g u n fum igator or
jiffy a p p lic a to r w ith p l a s t i c c o v erin g and fum igant c a n be e m p lo y e d .
Again v i r u s - f r e e re p la c e m e n t p la n ts a re a n e c e s s i t y .

Furtherm ore, a

v i r u s - f r e e g ra p e v in e re p la c e m e n t program sh ould be in s tig a te d b y the
s t a t e re g u la to ry d e p a rtm e n t.

REFERENCES

Bovey, R.

1958.

E tat a c t u e l d e s c o n n a l s s a n c e s su r le s m a la d ie s

a v iru s d e la v ig n e .

Vitis 1 : 2 3 7 -2 5 6 .

B re ec e, J. R . , and W . H . H a r t.

1959.

A p o s s ib l e a s s o c i a t i o n of

n e m a to d e s w ith the s p r e a d of p e a c h y e llo w bud m o sa ic v i r u s .
P la n t D i s e a s e R eptr.

43: 9 8 9 -9 9 0 .

C a d m a n , C . H . , H . F. D i a s , and B. D . H a r r is o n .

1960.

S ap -tran s­

m is s ib le v i r u s e s a s s o c i a t e d w ith d i s e a s e s of g r a p e v in e s in
Europe and North A m erica.
C a tio n , D .

1942.

N ature (London) 187: 5 7 7 - 5 7 9 .

The r o s e t te m osaic d i s e a s e of p e a c h .

E xp. S ta . T e c h . B ull.

180: 1 -2 4 .

C h r i s t i e , J. R. , and V. G . P erry.
so il.
C o b b , N . A.
c itie s.

1951. Removing n e m a to d e s from the

P ro c . H ilm inth S o c . W a s h .
1918.

M ic h . Agr.

18: 106-108.

N em ato des of th e s l o w - s a n d f ilte r b e d s of Am erican

C o n tr. S c i. N e m ato l.

C o o k , J. A . , a n d A. C . G o h e e n .

7: 189-212.
1961.

The e f f e c t s of a v iru s d i s e a s e ,

le a f r o ll, on th e m ineral c o m p o s itio n of g ra p e t i s s u e a n d a c o m ­
p a ris o n o f le a fro ll and p o ta s s iu m d e fic ie n c y sy m p to m s.
I n s t . B iol. S c i. P u b l.
D i a s , H . F.

1950 a .

N o . 8:

Amer.

3 3 8 -3 5 4 .

O 'N o -C u rto I n f e c c io s o ' da v id e ira (u rtic ad o ).

T h irtee n th C o n g r. L u so -E sp a n o l Prog. C ie n c ia s 5: 167-175.

55

D i a s , H . F. 1950 b .

C lo r o s e i n f e c c i o s s a d a v id e ira (panachure

on m o sa ic b ra n c o ) .
C i e n c ia s 5:
D i a s , H . F.

T h irte e n th C o n g r. L u s o - E s p a n o l.

Prog.

177-182.

1963.

H o s t rang e an d p ro p e rtie s of g ra p e v in e fa n le a f

and g ra p e v in e y e llo w m o sa ic v i r u s e s .

Ann. Appl. Biol.

51:

85 -95.
D i a s , H . F . , and B. D . H a r ris o n .

1963.

The r e la tio n s h ip b e tw e e n

g ra p e v in e f a n le a f , g ra p e v in e y ellow m o sa ic and a r a b i s an d
m o sa ic v i r u s e s .

Ann. Appl. B iol.

De Z ee u w , D . J . , and R. A. Crum*

51: 97-105.

1968.

I n h e rita n c e of r e s i s t a n c e

to t o b a c c o ring s p o t and cu cu m b er m o sa ic v i r u s e s in b l a c k c o w pea c r o s s e s .

P hy to p ath o lo g y 53: 3 3 7 -3 4 0 .

E ric k so n , L. C . , an d H . Z. H ie ld .

1962.

D e term in a tio n of 2 , 4 - d i -

c h lo ro p h e n o x y a c e tic a c i d b y e le c tr o n c a p tu re g a s c h ro m a to g ra p h y .
Agr. Food C h e m .

10: 204-207-

F r e ita g , J. H . , a n d N . W . F ra z ie r.

1954.

N a tu ra l i n f e c tiv ity of l e a f -

ho pper v e c to rs of P i e r c e 's d i s e a s e v iru s of g ra p e s in C a lif o r n ia .
Phyto p ath o lo g y 44:7-11 .
F ulto n, J. P.

1962.

T ra n s m is s io n of to b a c c o rin g s p o t v iru s by X ip-

hinem a a m e r ic a n u m .

P h y to p a th o lo g y 52: 3 75.

G iffo rd , E . M . , J r . , W . B. H e w itt, A. D . G rah am , and C . H .
L am oureaux.

1956.

in th e g r a p e v in e .

An in te rn a l sym ptom for id en tify in g fa n le a f
C a l i f . D e p . Agr. B ull.

G ilm er, R. M . , an d L. J. K e lts.

1965.

45: 3 6 8 -3 7 2 .

I s o la tio n of to b a c c o m osaic

56

v iru s
G ilm e r, R.

from g rap e fo lia g e and r o o t s .

P h y to path olo gy 55: 1283.

M . , a n d L. J. K e lts .

T ra n s m is s io n s of to b a c c o

1967.

m o saic v iru s in g rap e s e e d s .
G ilm e r, R. M . , and J. M . W il k s .

P h yto p ath o lo g y 57: 2 7 7 -2 7 8 .
1967.

to b a c c o v iru s in a p p le and p e a r .
G ilm e r, R.

M . , and L. J. K e lts .

v in ey a rd s.

Seed tr a n s m is s io n of

P h y to p a th o lo g y 57: 214-217.

1968.

G rap e v ir u s e s and New York

New York's Food and Life S c ie n c e s 1: 3 - 5 .

G o h e e n , A. C . , F. N . H arm on, and J. H . W e in b e rg e r.

1958.

(White Emperor d i s e a s e ) of g ra p e s in C a lifo r n ia .

L eafroll

P h yto p ath o lo g y

48: 51-54.
G o h e e n , A. C . , W . B. H e w i t t, an d C . J . A lley.
g rap e le a fro ll in C a lif o r n ia . Amer.
G r a n iti, A.

1959.

E nol. and V itic .

S tu d ie s of
10: 78 - 8 4 .

Prob abile p r e s e n z a d e ll a " M a la ttia d e lle e n a z io n e "

d e lla v ite in P u g lia .
G oo d in g , G . V.

1959.

1963.

N o tiz . m a la ttie p a in t e .

NS. 2 6 -2 7 : 140-143.

P u rific a tio n an d s e r o lo g y of a virus a s s o c i a t e d

w ith the g rape y ello w v e in d i s e a s e .
H arm on, F. N . , and E. S n y d e r.

1946.

Phytop atholog y 53: 4 7 5 -4 8 0 .
In v e s tig a tio n s of the o c c u r r e n c e ,

tr a n s m i s s io n , sp re a d and e ff e c t of w h ite fru it c o lo r in th e Em per­
or g r a p e .

P ro a . Amer. S o c . H o rt. S c i .

H arm on, F. N . , an d J. H . W e in b e rg e r.

1956.

47: 190-194.
F oliage burn of v in ife ra

g ra p e s a s a symptom of w h ite Emperor d i s e a s e .

P lan t D i s e a s e

Reptr. 40: 3 0 0 -3 0 3 .
H e w itt, W . B.
v in e.

1945.

A g r a f t - t r a n s m i s s ib l e m o sa ic d i s e a s e of g r a p e ­

P h y top atho log y 35: 9 4 0 -9 4 2 .

58

K irk patrick, H . C . , an d R. C . L indn er.

1963.

tr a n s m i s s ib l e v iru s l a te n t in a p p l e .

A m e c h a n ic a lly

P hy top atho lo gy

54: 229-231.
K lo s, E. J . # F. F ronek, J. A. Knierim, and D . C a tio n .

1967.

P e a c h r o s e t te m o sa ic tr a n s m is s io n s and c o n tro l s t u d i e s .
M ic h . Agr. Exp. S t a . Q u a r t. Bui.
O u c h te rlo n y , O .

1962.

cedures.
Perry, V. G .

In Progr.
1958.

49: 2 8 7 -2 9 3 .

S e ro lo g ic a l te c h n iq u e s , a g a r d iffu sio n p ro ­
A llergy.

6: 3 0.

P a ra s itis m of two s p e c i e s of d a g g er n em ato d es

(Xiphinema am erican um and X. c h am b ersi) to stra w b e rry .

P h y to ­

p a th o lo g y 48: 4 2 0 -4 2 3 .
R a sk i, D . J . , and L. L ider.
C a l i f . Agr.

1959.

N em atodes in g rap e p ro d u c tio n .

13(9): 13-15.

R a sk i, D . J . , an d W . B. H e w itt.

1960.

E xperim ents w ith Xiphinema

in d e x a s a v e c to r of fa n le a f of g r a p e v i n e s .

N em ato log ica

5: 166-170.
R a sk i, D . J . , W . B. H e w itt, A. C . G o h e e n , C . E. Taylor and R. H .
T ay lo r.

1965.

S u rvival of Xiphinem a ind ex and r e s e rv o rs of

f a n le a f viru s in fallo w ed v in ey a rd s o i l .
Scheu, G .

1936.

M ein W in z e rb u c h .

G . m. b . H . Berlin
S ho em ak er, J. S .

1955.

N em ato lo g ica 11: 3 4 9 -3 5 2 .

R e ic h s n a e h rs ta n d V erlags

1-2 74.
S m a ll-fru it c u ltu r e , p . 3-111.

M c G ra w -H ill

Book C o . , I n c . , New York, New York.
Sm ith, K. M .

1957.

A tex tb o o k of p la n t viru s d i s e a s e s , p . 5 9 7 -6 0 0 .

L ittle , Brown and C o . ,

B oston, M a s s .