Elimination of viruses,
viroids and phytoplasmas
from grapevine germplasm
Ivana Gribaudo, Danila Cuozzo, Giorgio Gambino,
Franco Mannini
Plant Virology Institute, CNR (IVV-CNR),
Grugliasco Unit
Via L. da Vinci 44, 10095 Grugliasco (TO), Italy
Grapevine viral diseases
Fanleaf
Leafroll
Rugose
wood
GRAPEVINE VIRUSES
Someones very dangerous,
others do not kill the plant but affect yield and/or
quality
Diffusion on long/medium distances through infected
propagation materials (cuttings, graftings),
on the short distances through vectors
VIROIDS
Small, circular, non-protein-coding RNAs
Infected higher plants may develop
symptoms, resulting in economic losses
in several crops
Grapevine is one of the most
permissive, natural viroid host
•Grapevine yellow speckle viroid 1
(GYSVd-1)
•Grapevine yellow speckle viroid 2
(GYSVd-2)
•Hop stunt viroid (HSVd or HpSVd)
•Citrus exochortis viroid (CEVd
•Australian grapevine viroid (AGVd)
Strong symptoms
with speckles tending
to gather along the
main veins
Grapevine
viroids
Scattered yellow
spots in a European
grape leaf, typical of
yellow speckle
infection
Vein banding
symptoms in a vine
doubly infected with
yellow speckle viroid
and GRAPEVINE
FANLEAF VIRUS
GRAPEVINE PHYTOPLASMAS
Flavescence dorée (FD)
Bois noir (BN)
NO THERAPY
TO APPLY IN THE FIELD
PRODUCTION AND
USE OF HEALTHY
PROPAGATION
MATERIAL (=FREE FROM
THE MOST DAMAGING
VIRAL DISEASES)
Viruses, phytoplasma and some
other bacteria represent a real
risk for exploitation of grapevine
germplasm collections and - to a
certain extent - to their survival
Grapevine
germplasm
collection,
Grinzane Cavour,
Italy
How can this affect grapevine germplasm
collections?
We need clean materials to plant collection
vineyards in order to:
GLRaV-3 +GVA
1)do not lose accessions because of diseases
2)get correct data when
a) describing ampelographic and
ampelometric features
b) evaluating germplasm potentialities
HEALTHY
(Mannini e Argamante, 1996;
Mannini et al., 2000)
In areas of old, specialized grapevine
culture, finding “healthy plants” can be
difficult – expecially for minor cultivars
"Virus esenti"
Infetti
21,5
78,5
Marche
85,2
14,8
Abruzzo
96,8
3,2
Puglia
92,6
7,4
Basilicata
97,8
2,2
Ischia
100
0
Campania
98,6
1,4
Calabria
0
10
20
30
40
50
60
SOUTH ITALY
(Savino et al., 2002)
70
80
90
100
From: Avgelis e Grammatikaki, 2006
Sanitary status of grapevine mother plants of local varieties
in Crete, Greece
Virus
% infection
GFLV
95.8
GLRaV-1
66.3
GLRaV-3
94.7
GFkV
65.3
ArMV
0
From: Bertolini et al., 2010
High prevalence of viruses in table grape
from Spain detected by real-time RT-PCR
Common methods for virus
eradication
• meristem culture
• thermotherapy (in
vitro or in vivo)
NB: Efficacy is related to number and type of viral diseases
 Serological/molecular assays
(more than one!)
 Possibility of reinfection
VIRUS ERADICATION THROUGH
MERISTEM CULTURE
‘30-’40: it was observed that there is no
virus in meristems (true???)
‘50: in France the 1st plant (dahlia)
sanitated through meristem culture
Dimensions: 0.2-0.3 to 0.5-0.7 mm
Apical buds usually better than axillary
buds
Not for isodiametric viruses
MERISTEMS
MERISTEMS:
Virus eradication efficacy is
inversely proportional to the explant
size
The ability to resume growth
is related to the initial explant size
and to the genotype (problems in
meristem isolation, growth and
rooting)
GRAPEVINE
MERISTEM
ISOLATION
MERISTEM
GROWTH
FURTHER
MERISTEM
GROWTH …
ROOTING AND ACCLIMATIZATION
TRANSFER TO GREENHOUSE AND
VINEYARD
Results of virus eradication through meristem
culture (IVV-Bari):
40.8 % from GFLV
98-100 % from GLRaVs
8-9 to 12-15 months to obtain greenhouse plantlets
Results of eradication of phloem-limited virus
through meristem culture (IVV-Grugliasco):
Meristem development: from 5 to 76% (genotype
effect, and others)
Average success in eradicating viruses: 83.6%
(189 lines from 19 cultivars, 34 clones)
GVA is the most difficult virus to eradicate
THERMOTHERAPY
on potted plants (=in vivo)
37-39°C for 60-200 days
Then:
 Tip excision (herbaceous cuttings) to
root under mist
 Tip excision (1-20 mm) for in vitro
culture
 Meristem culture (0.3-0.7 mm)
THERMOTHERAPY on in vitro
plants
Lower temperatures (34°C)
Then:
 tip excision (1-3 mm) from apical buds
(plus eventually the youngest axillary buds)
for in vitro culture
 meristem culture (0.3-0.7 mm)
IN VITRO THERMOTHERAPY
(2-3 months)
CULTURE OF EXCISED
APICAL BUDS
Thermotherapy is more
effective on some viruses
GFLV
GVA
GLRaV-1
GLRaV-3
GFkV
100 %
70.2 %
25.1 %
24.7 %
0%
From: Panattoni et al., 2010
For some grapevine viruses the traditional
techniques do not get good results (e.g.
GRSPaV)
No. of lines
obtained
RSPaV
elimination
(%)
Meristem culture
38
28.9
In vivo
thermotherapy
72
23.6
In vitro
thermotherapy
44
9.1
(Gribaudo et al., 2006)
OTHER TECHNIQUES FOR VIRUS
ELIMINATION:
Micrografting
Chemiotherapy
Electrotherapy
Cryotherapy
Fragmented apexes
Somatic embryogenesis
MICROGRAFTING
 Scion is a meristem, rootstock is a virusfree seedling or cutting
 The rootstock acts as an intermediary
between the meristem and the medium
 This allows to overcome problems of slow
growth or rooting
 Used for citrus, apple, stone fruits
CHEMIOTHERAPY
•
Ribavirine
•
DHT (2,4-dioxoesaidro-1,3,5-triazina)
•
DHPA [(S)9 (2,3—diidrossipropil-adenina)]
•
Vidarabine
•
2-tiouracile
•
Oseltamir (neuramidase inhibitor)
Somatic embryogenesis
Usually or potentially adopted:
to regenerate plantlets in biotechnological breeding
programs
regeneration of transformants after gene
transfer
induction of somaclonal variation to increase
genetic variability
for separation of periclinal chimeras
in cryopreservation protocols
for virus eradication
Virus eradication through somatic
embryogenesis:
grapevine, citrus, sugarcane
Somatic embryos of Vitis
vinifera
10 cultivars (Vitis vinifera L.)
viruses: GLRaV-1, GLRaV-3, GVA, GRSPaV,
GFkV
single or mixed viral infections
266 lines regenerated from single somatic
embryos and micropropagated
serological (DAS-ELISA) and molecular (RTPCR) assays: at least 3 assays in vitro and additional
assays after transfer to greenhouse
Virus eradication through somatic embryogenesis
Cultivar
Viruses in
mother plants
N° of
regenerated
lines
Virus
eradication
(%)
Grignolino
GVA + GLRaV1
46
100
Müller Thurgau,
Brachetto d’Acqui
GLRaV-3
60
100
Brachetto
GFkV
68
100
Dolcetto
GVA
6
100
Proviné, Cari, Roussan
GFLV
72
94
Albarola, Bosco,
Brachetto, Grignolino,
Müller-Thurgau,
Rossese, Vermentino
GRSPaV
97
100
Incidence of VIROID infections in plants generated by
somatic embryogenesis (SE) or thermotherapy (T)
(n° of infected plants/n° of tested plants)
Cultivar
Provinè
Cari
Nebbiolo
Roussan
Sanitation
method
SE
SE
SE
SE
SE
SE
SE
SE
SE
SE
T
T
In vitro plantlets
1 year-old plants
3 year-old plants
In vitro plantlets
1 year-old plants
3 year-old plants
In vitro plantlets
In vitro plantlets
1 year-old plants
3year-old plants
In vitro plantlets
3 year-old plants
GYSVd-1
HSVd
0/33
0/1
0/8
0/16
0/7
0/10
0/21
0/13
0/2
0/2
34/34
4/4
0/33
0/1
0/8
0/16
0/7
0/10
0/21
0/13
0/2
0/2
34/34
4/4
greenhouse plants from somatic embryos
greenhouse plants from thermotherapy
GYSVd-1 (cv Proviné)
In vitro plants from somatic embryos
In vitro plants from thermotherapy
greenhouse plants from somatic embryos
in vitro plants from somatic embryos
somatic embryos
non-embryogenic calli from anthers
embryogenic calli from anthers
Presence of viroids in different culture phases
HSVd (cv Roussan)
At the moment, somatic embryogenesis seems to be the
most effective sanitation method to obtain healthy
grapevines, i.e. free from
viruses
viroids
(phytoplasmas?)
An experimental vineyard was planted
to check the performance of
embryo-derived grapevine plants
and to ascertain if any variation occurred
•
•
•
•
•
•
Grignolino
Dolcetto
Grüner Vertliner
Malvasia
Bosco
Albarola
1. Phenotypical analysis
2. SSR
Grignolino
Dolcetto
VVS2
VVS5
VVMD5
VVMD6
VVMD7
VVMD21
VVMD24
VVMD25
VVMD27
VVMD28
VVMD31
VVMD32
VVMD36
VrZAG21
VrZAG25
VrZAG62
VrZAG64
VrZAG67
VrZAG79
VMC7h3
VVS2
VVS5
VVMD5
VVMD7
VVMD27
VVMD36
VrZAG62
VrZAG67
VrZAG79
3. AFLP/other analyses
EcoRI(AC)/MseI(ACC)
303
Use of micropropagation to eradicate
phytoplasmas
Shoots from mother plants
infected by da FD +/- LN
Micropropagation of axillary buds
Nested PCR assays + RFLP
after 6/9 months-culture
No effect due to:
- time of culture beginning along the season
- position of the bud explant on the shoot, and of the
shoot on the mother plant
- presence or absence of plant growth regulators in the
culture media
Effect of oxitetracyclin in the culture media
Preliminary assays on sensibility of grapevine explants
Proliferation medium
Rooting medium
Plantlets (with very
short roots) showed
no significant damage
when transferred to a
medium + 50 mg.l-1
oxitetracyclin
Virus eradication
from grapevine germplasm
through in vitro techniques
at IVV-CNR Grugliasco
1 cv
1 clone
4 cvs
4 clones
10 cvs
10 clones
13 cvs
26 clones
1 cv
1 clone
6 cvs
19 clones
CULTIVAR
Albarola
Albarola (biotipo Bianchetta genovese)
Albarossa
Arvino
Asprinio
Barbera bianca
Bian ver
Bizzarria
Bosco
Bruciapagliaio
Cari
Carica l’asino
Castiglione
Frate Pelato
Gamba di pernice
Gaglioppo
Granaccia secolare
Guarnaccia bianca
Iuvarello
Lumassina
Malvasia di Schierano
Massaretta
Mantonico
Moscatello di Taggia
Picabon
Prié blanc
Proviné
Rossese
Rossese di Arcola
Rossese Campochiesa
Roussan
L6
T1
P14
N1
N°
TREATED
CLONES
3
1
1
3
1
1
1
1
5
1
1
1
3
1
1
9
1
1
1
2
1
1
2
1
1
1
1
5
1
1
1
1
1
1
1
VIRUSES IN THE MOTHER
PLANTS
GVA, GLRaV-1, GLRaV-3
GVA, GLRaV-1, GLRaV-3
GVB
GVA, GLRaV-3
GVA, GLRaV-3
GVA, GLRaV-1, GLRaV-3, GFkV
GVA, GLRaV-3
GLRaV-1, GLRaV-3
GVA, GLRaV-1, GLRaV-3
GVA, GLRaV-1
GFLV
GFkV
GVA, GLRaV-3
GVA, GLRaV-1
GVA, GLRaV-1
GVA, GFkV
GVA, GLRaV-3
GVA, GLRaV-1, GLRaV-3
GVA, GLRaV-1, GLRaV-3
GFLV, GVA, GFkV
GVA, GLRaV-1, GFkV
GLRaV-1, GLRaV-3
GVA, GLRaV-1, GLRaV-3
GVA, GLRaV-1
GVA, GLRaV-3
GVA, GLRaV-1
GFLV
GVA, GLRaV-1, GLRaV-3GFkV
GLRaV-1
GVA, GLRaV-1, GLRaV-3
GFLV
GVA, GLRaV-1, GLRaV-3
GVA, GLRaV-1, GLRaV-3
GVA, GLRaV-1, GLRaV-3
GVA, GLRaV-1, GLRaV-3
TECHNIQUES USED
Meristem culture
Meristem culture
In vitro thermotherapy, meristem culture
Meristem culture
Meristem culture
Meristem culture
Meristem culture
Meristem culture
Meristem culture
In vitro thermotherapy, meristem culture
Somatic embryogenesis
In vitro thermotherapy
Meristem culture
In vitro thermotherapy, meristem culture
Meristem culture
Meristem culture
Meristem culture
Meristem culture
Meristem culture
In vitro thermotherapy + meristem culture
Meristem culture, somatic embryogenesis
Meristem culture
Meristem culture
Meristem culture
Meristem culture
Meristem culture
Somatic embryogenesis
Meristem culture
Meristem culture
Meristem culture
Somatic embryogenesis, in vitro thermotherapy
Meristem culture
Meristem culture
Meristem culture, in vitro thermotherapy
Meristem culture
N°
HEALTHY
LINES
24
3
2
8
4
1
1
2
23
11
6
6
3
9
1
10
1
6
3
4
21
2
6
1
2
3
33
53
2
1
49
1
2
3
3
Besides cultivation in
COLLECTION
VINEYARDS,
most sanitated lines
are also collected in
SCREEHOUSE and
IN VITRO
Thanks for your attention!