Elimination of viruses, viroids and phytoplasmas from grapevine germplasm

Transcription

1 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, Grugliasco (TO), Italy

2 Grapevine viral diseases Fanleaf Leafroll Rugose wood

3 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

4 VIROIDS Small, circular, non-protein-coding RNAs Infected higher plants may develop symptoms, resulting in economic losses in several crops

5 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)

6 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

7 GRAPEVINE PHYTOPLASMAS Flavescence dorée (FD) Bois noir (BN)

8 NO THERAPY TO APPLY IN THE FIELD PRODUCTION AND USE OF HEALTHY PROPAGATION MATERIAL (=FREE FROM THE MOST DAMAGING VIRAL DISEASES)

9 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

10 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)

11 In areas of old, specialized grapevine culture, finding healthy plants can be difficult expecially for minor cultivars "Virus esenti" Infetti Marche 21,5 78,5 Abruzzo 14,8 85,2 Puglia 3,2 96,8 Basilicata 7,4 92,6 Ischia 2,2 97,8 Campania Calabria 1,4 98, SOUTH ITALY (Savino et al., 2002)

12 From: Avgelis e Grammatikaki, 2006 Sanitary status of grapevine mother plants of local varieties in Crete, Greece

13 Virus % infection GFLV 95.8 GLRaV GLRaV 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

14 Common methods for virus eradication meristem culture thermotherapy (in vitro or in vivo) NB: Efficacy is related to number and type of viral diseases

15 Serological/molecular assays (more than one!) Possibility of reinfection

16 VIRUS ERADICATION THROUGH MERISTEM CULTURE 30-40: it was observed that there is no virus in meristems (true???) 50: in France the 1 st plant (dahlia) sanitated through meristem culture Dimensions: to mm Apical buds usually better than axillary buds Not for isodiametric viruses

17 MERISTEMS

18 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)

19 GRAPEVINE MERISTEM ISOLATION

20 MERISTEM GROWTH

21 FURTHER MERISTEM GROWTH

22 ROOTING AND ACCLIMATIZATION

23 TRANSFER TO GREENHOUSE AND VINEYARD

24 Results of virus eradication through meristem culture (IVV-Bari): 40.8 % from GFLV % from GLRaVs 8-9 to months to obtain greenhouse plantlets

25 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

26 THERMOTHERAPY on potted plants (=in vivo) C for days Then: Tip excision (herbaceous cuttings) to root under mist Tip excision (1-20 mm) for in vitro culture Meristem culture ( mm)

27 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 ( mm)

28 IN VITRO THERMOTHERAPY (2-3 months) CULTURE OF EXCISED APICAL BUDS

29 Thermotherapy is more effective on some viruses GFLV 100 % GVA 70.2 % GLRaV % GLRaV % GFkV 0 % From: Panattoni et al., 2010

30 For some grapevine viruses the traditional techniques do not get good results (e.g. GRSPaV) No. of lines obtained RSPaV elimination (%) Meristem culture In vivo thermotherapy In vitro thermotherapy (Gribaudo et al., 2006)

31 OTHER TECHNIQUES FOR VIRUS ELIMINATION: Micrografting Chemiotherapy Electrotherapy Cryotherapy Fragmented apexes Somatic embryogenesis

32 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

33 CHEMIOTHERAPY Ribavirine DHT (2,4-dioxoesaidro-1,3,5-triazina) DHPA [(S)9 (2,3 diidrossipropil-adenina)] Vidarabine 2-tiouracile Oseltamir (neuramidase inhibitor)

34 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

35 Virus eradication through somatic embryogenesis: grapevine, citrus, sugarcane Somatic embryos of Vitis vinifera

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39 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

40 Virus eradication through somatic embryogenesis Cultivar Grignolino Müller Thurgau, Brachetto d Acqui Viruses in mother plants GVA + GLRaV- 1 N of regenerated lines Virus eradication (%) GLRaV Brachetto GFkV Dolcetto GVA Proviné, Cari, Roussan GFLV Albarola, Bosco, Brachetto, Grignolino, Müller-Thurgau, Rossese, Vermentino GRSPaV

41 Incidence of VIROID infections in plants generated by somatic embryogenesis (SE) or thermotherapy (T) (n of infected plants/n of tested plants) Cultivar Sanitation method GYSVd-1 HSVd SE In vitro plantlets 0/33 0/33 Provinè SE 1 year-old plants 0/1 0/1 SE 3 year-old plants 0/8 0/8 SE In vitro plantlets 0/16 0/16 Cari SE 1 year-old plants 0/7 0/7 SE 3 year-old plants 0/10 0/10 Nebbiolo SE In vitro plantlets 0/21 0/21 SE In vitro plantlets 0/13 0/13 SE 1 year-old plants 0/2 0/2 Roussan SE 3year-old plants 0/2 0/2 T In vitro plantlets 34/34 34/34 T 3 year-old plants 4/4 4/4

42 Presence of viroids in different culture phases GYSVd-1 (cv Proviné) embryogenic calli from anthers non-embryogenic calli from anthers somatic embryos in vitro plants from somatic embryos greenhouse plants from somatic embryos In vitro plants from thermotherapy In vitro plants from somatic embryos greenhouse plants from thermotherapy greenhouse plants from somatic embryos HSVd (cv Roussan)

43 At the moment, somatic embryogenesis seems to be the most effective sanitation method to obtain healthy grapevines, i.e. free from viruses viroids (phytoplasmas?)

44 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

45 1. Phenotypical analysis 2. SSR 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 EcoRI(AC)/MseI(ACC) 303

46 Use of micropropagation to eradicate phytoplasmas Shoots from mother plants infected by da FD +/- LN Micropropagation of axillary buds

47 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

48 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

49 Virus eradication from grapevine germplasm through in vitro techniques at IVV-CNR Grugliasco 1 cv 1 clone 10 cvs 10 clones 4 cvs 4 clones 13 cvs 26 clones 1 cv 1 clone 6 cvs 19 clones

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

51 Besides cultivation in COLLECTION VINEYARDS, most sanitated lines are also collected in SCREEHOUSE and IN VITRO

52 Thanks for your attention!