Elimination of viruses, Elimination of viruses, viroids and phytoplasmas viroids and phytoplasmas

from grapevine germplasmfrom grapevine germplasm

Ivana GribaudoIvana Gribaudo, Danila Cuozzo, Giorgio Gambino, , Danila Cuozzo, Giorgio Gambino, Franco ManniniFranco Mannini

Plant Virology Institute, CNR (IVVPlant Virology Institute, CNR (IVV--CNR), CNR), Grugliasco UnitGrugliasco Unit

Via L. da Vinci 44, 10095 Grugliasco (TO), ItalyVia L. da Vinci 44, 10095 Grugliasco (TO), Italy

Grapevine viral diseases

Leafroll

Rugose wood

Fanleaf

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)

Grapevine viroids

Strong symptoms Strong symptoms with speckles tending with speckles tending

to gather along the to gather along the main veins main veins

Vein banding Vein banding symptoms in a vine symptoms in a vine doubly infected with doubly infected with yellow speckle viroid yellow speckle viroid and GRAPEVINE and GRAPEVINE FANLEAF VIRUSFANLEAF VIRUS

Scattered yellow Scattered yellow spots in a European spots in a European grape leaf, typical of grape leaf, typical of yellow speckle yellow speckle infectioninfection

GRAPEVINE PHYTOPLASMAS

Flavescence dorée (FD)Bois noir (BN)

NO THERAPY TO APPLY IN THE FIELD

PRODUCTION AND USE OF HEALTHYPROPAGATION 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:

1)do not lose accessions because of diseases

2)get correct data whena) describing ampelographic and

ampelometric featuresb) evaluating germplasm potentialities

GLRaV-3 +GVA

HEALTHY(Mannini e Argamante, 1996;

Mannini et al., 2000)

0 10 20 30 40 50 60 70 80 90 100

Calabria

Campania

Ischia

Basilicata

Puglia

Abruzzo

Marche

98,6

100

97,8

92,6

96,8

85,2

78,5

1,4

0

2,2

7,4

3,2

14,8

21,5

"Virus esenti" Infetti

SOUTH ITALY(Savino et al., 2002)

In areas of old, specialized grapevine culture, finding “healthy plants” can be difficult – expecially for minor cultivars

From: Avgelis e Grammatikaki, 2006Sanitary status of grapevine mother plants of local varieties

in Crete, Greece

Virus % infectionGFLV 95.8GLRaV-1 66.3GLRaV-3 94.7GFkV 65.3ArMV 0

From: Bertolini et al., 2010High 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 THROUGHMERISTEM 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 mmApical 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 GFLV98-100 % 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 100 %GVA 70.2 %GLRaV-1 25.1 %GLRaV-3 24.7 %GFkV 0 %

From: Panattoni et al., 2010

No. of lines obtained

RSPaV elimination

(%)

Meristem culture 38 28.9In vivothermotherapy

72 23.6

In vitrothermotherapy

44 9.1

(Gribaudo et al., 2006)

For some grapevine viruses the traditionaltechniques do not get good results (e.g. GRSPaV)

OTHER TECHNIQUES FOR VIRUS ELIMINATION:

MicrograftingChemiotherapy

ElectrotherapyCryotherapy

Fragmented apexes Somatic embryogenesis

MICROGRAFTING

Scion is a meristem, rootstock is a virus-free 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)

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

Somatic embryogenesis

Somatic embryos of Vitis vinifera

Virus eradication through somatic embryogenesis:

grapevine, citrus, sugarcane

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 (RT-PCR) assays: at least 3 assays in vitro and additional assays after transfer to greenhouse

Cultivar Viruses in mother plants

N° of regenerated

lines

Virus eradication

(%)Grignolino GVA + GLRaV-

146 100

Müller Thurgau, Brachetto d’Acqui

GLRaV-3 60 100

Brachetto GFkV 68 100Dolcetto GVA 6 100

Proviné, Cari, Roussan GFLV 72 94Albarola, Bosco,

Brachetto, Grignolino, Müller-Thurgau,

Rossese, Vermentino

GRSPaV 97 100

Virus eradication through somatic embryogenesis

Cultivar Sanitation method

GYSVd-1 HSVd

ProvinèSE In vitro plantlets 0/33 0/33SE 1 year-old plants 0/1 0/1SE 3 year-old plants 0/8 0/8

CariSE In vitro plantlets 0/16 0/16SE 1 year-old plants 0/7 0/7SE 3 year-old plants 0/10 0/10

Nebbiolo SE In vitro plantlets 0/21 0/21

Roussan

SE In vitro plantlets 0/13 0/13SE 1 year-old plants 0/2 0/2SE 3year-old plants 0/2 0/2T In vitro plantlets 34/34 34/34T 3 year-old plants 4/4 4/4

Incidence of VIROID infections in plants generated bysomatic embryogenesis (SE) or thermotherapy (T)

(n° of infected plants/n° of tested plants)

Presence of viroids in different culture phases

GYSVd-1 (cv Proviné)

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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

Grignolino VVS2 VVS5 VVMD5 VVMD6 VVMD7 VVMD21

VVMD24 VVMD25 VVMD27 VVMD28 VVMD31 VVMD32

VVMD36 VrZAG21 VrZAG25 VrZAG62 VrZAG64 VrZAG67

VrZAG79 VMC7h3

Dolcetto VVS2 VVS5 VVMD5 VVMD7 VVMD27 VVMD36

VrZAG62 VrZAG67 VrZAG79

3. AFLP/other analyses

1. Phenotypical analysis

2. SSR

303

EcoRI(AC)/MseI(ACC)

Use of micropropagation to eradicate phytoplasmas

Shoots from mother plantsinfected by da FD +/- LN

Micropropagation of axillary buds

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

Nested PCR assays + RFLP after 6/9 months-culture

Plantlets (with very short roots) showed

no significant damage when transferred to a medium + 50 mg.l-1

oxitetracyclin

Effect of oxitetracyclin in the culture mediaPreliminary assays on sensibility of grapevine explants

Proliferation mediumRooting medium

13 cvs26 clones

1 cv1 clone

10 cvs10 clones

4 cvs4 clones

1 cv1 clone

6 cvs19 clones

Virus eradication from grapevine germplasmthrough in vitro techniquesat IVV-CNR Grugliasco

CULTIVARN°

TREATED CLONES

VIRUSES IN THE MOTHER PLANTS TECHNIQUES USED

N°HEALTHY

LINESAlbarola 3 GVA, GLRaV-1, GLRaV-3 Meristem culture 24Albarola (biotipo Bianchetta genovese) 1 GVA, GLRaV-1, GLRaV-3 Meristem culture 3Albarossa 1 GVB In vitro thermotherapy, meristem culture 2Arvino 3 GVA, GLRaV-3 Meristem culture 8Asprinio 1 GVA, GLRaV-3 Meristem culture 4Barbera bianca 1 GVA, GLRaV-1, GLRaV-3, GFkV Meristem culture 1Bian ver 1 GVA, GLRaV-3 Meristem culture 1Bizzarria 1 GLRaV-1, GLRaV-3 Meristem culture 2Bosco 5 GVA, GLRaV-1, GLRaV-3 Meristem culture 23Bruciapagliaio 1 GVA, GLRaV-1 In vitro thermotherapy, meristem culture 11Cari 1 GFLV Somatic embryogenesis 6Carica l’asino 1 GFkV In vitro thermotherapy 6Castiglione 3 GVA, GLRaV-3 Meristem culture 3Frate Pelato 1 GVA, GLRaV-1 In vitro thermotherapy, meristem culture 9Gamba di pernice 1 GVA, GLRaV-1 Meristem culture 1Gaglioppo 9 GVA, GFkV Meristem culture 10Granaccia secolare 1 GVA, GLRaV-3 Meristem culture 1Guarnaccia bianca 1 GVA, GLRaV-1, GLRaV-3 Meristem culture 6Iuvarello 1 GVA, GLRaV-1, GLRaV-3 Meristem culture 3Lumassina 2 GFLV, GVA, GFkV In vitro thermotherapy + meristem culture 4Malvasia di Schierano 1 GVA, GLRaV-1, GFkV Meristem culture, somatic embryogenesis 21Massaretta 1 GLRaV-1, GLRaV-3 Meristem culture 2Mantonico 2 GVA, GLRaV-1, GLRaV-3 Meristem culture 6Moscatello di Taggia 1 GVA, GLRaV-1 Meristem culture 1Picabon 1 GVA, GLRaV-3 Meristem culture 2Prié blanc 1 GVA, GLRaV-1 Meristem culture 3Proviné 1 GFLV Somatic embryogenesis 33Rossese 5 GVA, GLRaV-1, GLRaV-3GFkV Meristem culture 53Rossese di Arcola 1 GLRaV-1 Meristem culture 2Rossese Campochiesa 1 GVA, GLRaV-1, GLRaV-3 Meristem culture 1Roussan 1 GFLV Somatic embryogenesis, in vitro thermotherapy 49L6 1 GVA, GLRaV-1, GLRaV-3 Meristem culture 1T1 1 GVA, GLRaV-1, GLRaV-3 Meristem culture 2P14 1 GVA, GLRaV-1, GLRaV-3 Meristem culture, in vitro thermotherapy 3N1 1 GVA, GLRaV-1, GLRaV-3 Meristem culture 3

Besides cultivation in

COLLECTION

VINEYARDS,

most sanitated lines

are also collected in

SCREEHOUSE and

IN VITRO

Thanks for your attention!