Grapevine leafroll-associated viruses and grapevine virus A inselectedVitis vinifera cultivars in northern Italy

R. CREDI and L. GIUNCHEDIIstituto di Patologia Vegetale, Universita` degli Studi, 40126 Bologna, Italy

The occurrence of grapevine leafroll-associated virus 1 (GLRaV-1), grapevine leafroll-associated virus 3(GLRaV-3) and grapevine virus A (GVA) was demonstrated in a viticultural region of northern Italy (Emilia-Romagna) using immunoelectron microscopy. Virus incidence was subsequently assessed using ELISA. Atotal of 60.6% of the 150 clone selections tested, from 18 localVitis vinifera cultivars, were found to beinfected. ELISA did not reveal the presence of grapevine leafroll-associated virus 2 (GLRaV-2) or grapevineleafroll-associated virus 5 (GLRaV-5). GLRaV-1, GLRaV-3 and GVA were found individually and invarious combinations. The most common findings were GLRaV-1 alone (25.3%) and associated with GVA(33%). Serological data confirmed that the majority (91%) of the clones known to be affected by grapevineleafroll (GLR), on its own or in association with rugose wood (RW), contained viruses. On the other hand,where the RW phenomenon was present on its own, only 40% of these clones were ELISA-positive. Theimplications for the biology of GLR and RW are discussed and the complex aetiology of these grapevinediseases is confirmed.

INTRODUCTION

Grapevine is susceptible to several graft-transmis-sible diseases caused by viruses and virus-likepathogenic agents. Amongst these diseases, grape-vine leafroll (GLR) and rugose wood (RW), whichrepresent a great economic threat to growers, havehigh incidences in all the viticultural regions of theworld. The causal agents of GLR and RW have notbeen clearly established. In recent years, however,different clostero-like particles (flexuous, filamen-tous), ranging in modal length from about 800 to2200 nm, have been found associated with sympto-matic vines (Martelli, 1993). GLR is apparentlyinduced by a complex of viral agents, known asgrapevine leafroll-associated viruses (GLRaVs).On the other hand, a key role in the developmentof RW abnormalities, particularly the Kober stemgrooving (KSG) syndrome, may be played by thepresence of grapevine virus A (GVA) (Hu et al.,1990; Zimmermannet al., 1990; Digiaroet al.,1994; Garauet al., 1994; Chevalieret al., 1995;Bosciaet al., 1995).

These disorders often show diagnostic symptomsin the field. However, the only reliable identifica-tion method is the use of woody indexing tests.Nevertheless, with the production of antibodiesagainst a pool of GLRaVs (Huet al., 1990;

Zimmermannet al., 1990) and GVA (Contiet al.,1980; Milneet al., 1984; Bosciaet al., 1992), it isnow possible to perform rapid and sensitiveserological tests such as immunosorbent electronmicroscopy (ISEM) and enzyme-linked immuno-sorbent assay (ELISA).

This paper reports the results of the use of someof these antisera with the ISEM and decorationtechniques. It also reports the results of ELISA usedon a larger scale to determine the incidence of theviruses, their relative frequency and to analyse thecorrelation between their presence and the GLR andRW conditions.

MATERIALS AND METHODS

Plant material

The grapevine samples used in this study were froma foundation vineyard set up by the Dipartimento diColture Arboree, University of Bologna, Italy. Thiscollection was established in 1980 with severalclones of most of the localVitis vinifera cultivarsgrown in Emilia-Romagna (north Italy), developedfrom a single mother vine selection (Table 1). Allthe original individual mother vines and thedaughter clonal lines were inspected for symptomexpression of virus and virus-like diseases for manyyears. Moreover, most of them were, or are

Plant Pathology(1996)45, 1110–1116

Accepted 1 July 1996.

Grapevine leafroll-associated viruses 1111

Tab

le1

Inci

denc

ean

dfr

eque

ncy

ofG

LRaV

-1,

GLR

aV-3

and

GV

Ain

fect

ions

incl

ones

ofV

.vi

nife

racu

ltiva

rsse

lect

ed

inth

eE

mili

a-R

omag

nare

gion

asse

ssed

byE

LIS

A

Num

ber

ofcl

ones

cont

aini

ngvi

ruse

sa

Num

ber

GLR

aV-1

+of

clon

esG

LRaV

-1+

GLR

aV-1

+G

LRaV

-3+

GLR

aV-3

+C

ultiv

arte

sted

GLR

aV-1

GLR

aV-3

GV

AG

LRaV

-3G

VA

GV

AG

VA

Tot

alcl

ones

infe

cted

Alb

ana

203

04

26

11

17A

lionz

a4

01

00

10

02

Bia

ncal

e10

20

02

10

05

Bru

gnol

a2

00

01

00

12

Can

ino

30

10

10

00

2C

arga

rello

40

10

00

00

1F

orta

na7

00

10

00

01

Gra

ppel

lo3

10

00

20

03

Lanc

ello

tta4

10

00

20

03

Lam

brus

co35

72

12

90

021

Mon

tuni

111

03

02

11

8M

osca

to4

00

00

30

03

Pig

nole

tto4

00

00

00

00

Rib

olla

40

00

21

00

3R

ossi

ola

21

00

00

00

1S

angi

oves

e18

32

00

33

213

Tre

bbia

noR

.12

32

00

00

05

Tre

bbia

noC

.3

10

00

00

01

Tot

als

150

23(2

5. 3%

)9

(9. 9

%)

9(9

. 9%

)10

(11%

)30

(33%

)5

(5. 5%

)5

(5. 5

%)

91(6

0. 6%

)

a Sam

ples

wer

era

ted

posi

tive

byE

LIS

Aif

abso

rban

ceva

lues

wer

eat

leas

tth

ree

times

the

aver

age

read

ing

ofhe

alth

ygr

apev

ine

extr

acts

.

currently, subject to extensive indexing usingherbaceous and woody indicator plants (Credi &Babini, 1987; unpublished data). Consequently,based on the field symptoms and on theexamination of indexing data from these grape-vine sources, they are known to be either GLRand/or RW-affected or free from these diseases.

Extraction and electron microscopyidentification of viruses from grapevine tissues

Grapevine leafroll-associated viruses and GVAwere extracted from infected grapevine tissueusing the method of Gugerli (1987). Proceduresfor negative staining of crude extracts, ISEMand decoration tests, were similar to thosedescribed elsewhere (Milne, 1984). Virus particleswere trapped for 10–30 min at room temperatureusing grids coated with an antiserum dilution of1:1000 and decorated with an antiserum dilutionof 1:50. All the preparations were finallynegatively stained in 2% aqueous uranyl acetatebefore observation with a Philips CM10 electronmicroscope.

Antisera and serological assays

A conventional double-antibody (DAS) ELISA(Clark & Adams, 1977) was used for GLRaV-1(Gugerli et al., 1984) and GLRaV-3 (Zeeet al.,1987) with coating and conjugate antibody pre-parations purchased from Sanofi Phyto-Diagnostics(Marnes-La-Coquette, France) and Bioreba AG(Basle, Switzerland). Biotin-DAS-ELISA wasused for GLRaV-2 (Huet al., 1990; Zimmermannet al., 1990; Bosciaet al., 1995) and GLRaV-5(Zimmermannet al., 1990) using kits also preparedby Sanofi Phyto-Diagnostics. The presence of GVA(Conti et al., 1980) in grapevine samples wasassayed with polyclonal and enzyme-conjugatedmonoclonal antibodies supplied by Dr D. Boscia(Dipartimento di Protezione delle Piante, Bari,Italy). The antibody-trapped antigen form ofELISA was used following a protocol developedby Bosciaet al. (1992).

For electron microscopy investigations, poly-clonal antisera to other GLRaV-1, GLRaV-3 andGVA isolates were included. The former wasobtained from Dr P. Gugerli (Station Fe´derale deRecherches Agronomiques de Changins, Nyon,Switzerland). For the second virus, one antiserumwas supplied by Dr D. Gonsalves (Department ofPlant Pathology, New York State AgriculturalResearch Station, Geneva NY, USA) and theother was produced locally. The third antiserum

was supplied by Dr M. Conti (Istituto di Fito-virologia Applicata del CNR, Turin, Italy).

ELISA tests were carried out with a fewmodifications, as described in literature (Clark &Adams, 1977; Gugerliet al., 1984; Rosciglione &Gugerli, 1986; Telizet al., 1987; Zimmermannet al., 1988) and in accordance with the manu-facturer’s instructions. Duplicate samples werecollected from each vine for two consecutiveyears. Leaf samples (13-mm-diameter discs takenfrom the main veins of the oldest nonsenescentbasal leaves) and/or bark scrapings of dormantcanes were collected, frozen in liquid nitrogen,ground with a pestle and mortar, mixed inextraction buffer (0.2-M Tris/HCl pH 8.2 contain-ing 0.8% NaCl, 2% PVP and 0.05% Tween 20)with a ratio of 1:10 (weight/vol.) and groundagain. The extracts were filtered through twolayers of muslin prior to adding 200-�L samplesto duplicate wells on micro-ELISA plates (Dyna-tech). Samples of virus-free grapevine tissues,from vines previously subjected to heat therapy,were also added to each plate as negative controls.Optical density (A 405 nm) was measured with anELISA reader. Absorbance values three timesgreater than those of the mean healthy controls(0.03) and equal to or greater than 0.10 wereconsidered as positive. Samples with an absorb-ance close to the positive–negative thresholdwere assayed again and/or checked by electronmicroscopy.

RESULTS

Virus detection by electron microscopy

Physical identification of the virus particles byelectron microscopy was performed on crudegrapevine extracts. ISEM with preparations madefrom tissues of symptomatic vines, subsequentlyfound to be ELISA-positive, always showedflexuous rod viral particles, exhibiting a distinctcross-banding structure (Fig. 1 a,c,e). On the otherhand, samples collected from asymptomatic vines,ELISA-negative for the viruses, revealed no similarparticles. Decoration tests with the antisera andhomologous antigens provided the definitive con-firmation of virion serological identity (Fig. 1 b,d,f).When viewing preparations from plants withmultiple infection (assessed by ELISA), stronglydecorated and undecorated particles were oftendetected, as expected. Moreover, virus particles, inpart decorated, were also observed in some cases(Fig. 1 g).

R. Credi & L. Giunchedi1112

Grapevine leafroll-associated viruses 1113

Fig. 1 Electron microscopy of virus particles. Micrographs of GLRaV-1 and GLRaV-3 (a, c), decorated by the homologousantisera, respectively (b, d). Bars = 100 nm. Undecorated (e) and decorated (f) GVA particle and a particle from a grapevineextract with multiple infection, partially decorated after treatment with GLRaV-1 antiserum (g). Bars = 100 nm. Thepreparations were stained with 2% uranyl acetate.

R. Credi & L. Giunchedi1114

Tab

le2

Cor

rela

tion

ofG

LRaV

-1,G

LRaV

-3an

dG

VA

infe

ctio

nsw

ithth

ele

afro

llan

dru

gose

woo

dco

nditi

ons,

prev

ious

lyas

cert

aine

din

the

sele

cted

V.v

inife

racl

ones

ofE

mili

a-R

omag

nacu

ltiva

rs

GLR

aV-1

+T

otal

Num

ber

GLR

aV–1

+G

LRaV

-1+

GLR

aV–3

+G

LRaV

-3+

clon

esD

isea

sest

atusa

ofcl

ones

GLR

aV-1

GLR

aV-3

GV

AG

LRaV

-3G

VA

GV

AG

VA

infe

cte

d

Leaf

roll

(GLR

)33

7(b)

41

68

13

30(9

1%)

Rug

ose

woo

d(R

W)

257

11

00

10

10(4

0%)

GLR

+R

W45

52

34

223

241

(91. 1%

)G

LRan

dR

Wne

gativ

e47

42

40

00

010

(21

. 3%

)T

otal

s15

023

99

1030

55

91(6

0. 6

%)

a San

itary

stat

eac

cord

ing

tofie

ldsy

mpt

oms

and/

orin

dexi

ngon

spec

ific

indi

cato

rpl

ants

.b N

umbe

rof

clon

espo

sitiv

efo

rvi

ruse

sou

tof

the

num

ber

ofcl

ones

with

orw

ithou

tdi

seas

esy

mpt

oms.

Incidence of viruses amongst grapevines

In this study, where grapevine samples from a totalof 150 selected clones were processed, GLRaV-2and GLRaV-5 were not found with ELISA. On thecontrary, an overall 60.6% of clones were infectedby GLRaV-1, GLRaV-3 and GVA, the onlyexception being the few selections of cv. Pignoletto.The incidence of viruses in the other cultivarselections varied; most had more than 50%infection. Single GLRaV-1 infections were detectedin 25.3%, GLRaV-3 in 9.9% and GVA in 9.9% ofthe clones. Multiple infections of these viruses werealso found. GLRaV-1 with GLRaV-3 was present in11%, GLRaV-1 plus GVA in 33%, GLRaV-3 withGVA in 5.5% and, finally, the combined presence ofthe three viruses was detected in 5.5% of thegrapevine sources tested (Table 1).

Correlation of virus infections with GLR andRW diseases

Table 2 shows the incidence of viruses in the cloneswhich were, or were not, affected by GLR and RW.The ELISA data revealed that 30 (91%) of the 33GLR-diseased clones were infected by viruses. Themost prevalent infection was that of GLRaV-1alone and in combination with GVA or GLRaV-3.Only 10 (40%) of the 25 RW-affected clonesrevealed viruses. Again the most prevalent one wasGLRaV-1. Forty-five clones were known to containmultiple GLR and RW diseases, and 41 of these(91.1%) were ELISA-positive, the majority con-taining the GLRaV-1 and GVA association. Finally,10 (21.3%) of the 47 clones rated free from thesediseases also appeared to contain viruses.

DISCUSSION

The occurrence of filamentous virus-like particles inclones of severalV. viniferacultivars, selected in aregion in north Italy, was first demonstrated byelectron microscopy observations. In ISEM decora-tion studies, they were coated by the GLRaV-1,GLRaV-3 or GVA antisera. Hybrid-like particleswere sometimes observed in grapevine tissueextracts, subsequently found to contain multipleviruses with ELISA. A similar phenomenon hasalready been reported by Milneet al. (1984) as apossible end-to-end aggregation of two different virusparticle serotypes.

The widespread presence of these viruses in thefoundation planting, and their incidence, wassubsequently demonstrated with ELISA. GLRaV-1 was the most frequent of the single-virus

infections. This virus combined with GVA wasalso the most prevalent of the mixed infections.These findings differed from those reported for thesouth of Italy by Digiaroet al. (1994) who, on thecontrary, found higher GLRaV-3 levels. GLRaV-2and GLRaV-5 were not detected, indicating thatthey do not commonly infect grapevine in thisviticultural region.

ELISA data also made it possible to analyse therelationship between the presence of GLRaV-1,GLRaV-3 and GVA with the GLR and RWconditions, previously determined on the basis offield symptoms and/or traditional woody indexing.In particular, the study confirmed the strongassociation (91%) of these viruses with GLR, aswell as multiple GLR and RW diseases. On theother hand, only 40% of the clones affected by RWon its own were ELISA-positive. Recently, theclose association between GVA and KSG wasreported (Garauet al., 1994; Chevalieret al., 1995).To explore the significance of these findings, morerefined woody indexing tests with our RW-affectedsources are needed to differentiate between thesyndromes now considered to be different compo-nents, or diseases, of the RW complex (Martelli,1993). Moreover, the possible occurrence of grape-vine virus B (GVB), reported by Bosciaet al.(1993), and a similar but serologically distinct onecalled grapevine virus C (GVC), reported byMonette & James (1991), must also be investigated.

In our survey, screening of grapevine sources,found to be negative for GLR and RW withbiological indexing, also revealed some virusinfections. One of the explanations of these positiveELISA reactions may be that woody indexing testsare not completely reliable as results can be affectedby various factors. On the other hand, the absenceof positive reactions in a few extracts of GLR-affected grapevines is not surprising, because otherclostero-like virus particles may be involved. Forexample, the possible occurrence of GLRaV-4 (Huet al., 1990) and GLRaV-6 (Bosciaet al., 1995)remains unknown as they were not investigated.

In conclusion, the study demonstrates the aetio-logic complexity of GLR and RW. From a practicalpoint of view, however, the use of the three antiserain ELISA seems to be reliable enough to detect thefirst disease, singly or in association with the secondone. For almost all the samples, the results ofELISA testing and indexing on woody indicatorswere almost identical. The opposite was true whenRW phenomena were found in the absence ofGLR. Research is needed to provide more informa-tion on the biology of these grapevine infectiousdiseases.

Grapevine leafroll-associated viruses 1115

ACKNOWLEDGEMENTS

The authors wish to thank Drs D. Boscia, M. Conti,D. Gonsalves and P. Gugerli for their kindassistance with antisera.

The research supported by the National ResearchCouncil of Italy, Special Project RAISA, Subpro-ject no. 2, Paper No. 2789.

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