GWT 09 03 Final Report

7/30/2019 GWT 09 03 Final Report

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Attendance at the 16t

Meeting of theInternational Council forVirus and virus-likeDiseases of Grapevine,Dijon, France.

TRAVEL GRANT REPORT toGRAPE AND WINE RESEARCH & DEVELOPMENT CORPORATION

Research Organisation: Department of Primary IndustriesVictoria

Date: 22nd September 2009

Principal Investigator: Fiona Constable

Project Number: GWT 09/03


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Executive Summary

Dr. Fiona Constable attended the 16th Meeting of the International Council for the Study of Virus and Virus-likeDiseases of the Grapevine (ICVG), Dijon, France August-September 2009 as part of a travel grant awarded by theGrape and Wine Research and Development Corporation (GWRDC). During the conference Dr. Constablepresented results from the GWRDC funded project entitled Development and validation of diagnostic protocols for

the detection of endemic and exotic pathogens of grapevines (Project number: DPI05/04). Two papers werepresented: A survey for endemic and exotic viruses of Australian grapevines by Fiona Constable, Phil Nicholas,

Narelle Nancarrowand Brendan Rodoni Development and validation of sampling strategies for the detection of endemic viruses of Australian

grapevines by the same authors.

Both presentations generated considerable interest in the area grapevine virus diagnostic techniques. In particular,there was interest from various researchers about the sampling strategies, extraction technology and PCRtechniques for grapevine virus detection that were developed within the GWRDC funded project. Contact wasmade with many other scientists engaged in grapevine virus and virus-like disease research and collaborativeopportunities explored.

The knowledge gained as a result of this travel will be considered for inclusion within the current GWRDC fundedproject, where relevant. Future projects will be developed where applicable. Publication of a review article aboutthe highlights of the 16th ICVG is being written in collaboration with Dr. Nuredin Habili (Waite Diagnostics) and Dr.Judit Monis (STA Laboratories, Gilroy, CA, USA) and will be published, locally and internationally, in non-peerreviewed grower journals.

Highlights from the meeting include: There are now 60 taxonomically recognized viruses, 10 phytoplasmas and six viroids reported to infect

grapevines world-wide. There may be a rationalisation of the grapevine leafroll associated virus taxonomy, resulting in less formally

recognised species Several of the new virus species but their association with disease is not known and this has important

implications for quarantine and certification programs.

Full length infectious clones of GRSPaV and GVA are being constructed and will be used as a tool in thefunctional genomics of grapevines and to elucidate the roles of specific virus genes in grapevine-virusinteractions.

Links were forged and potential collaborations were discussed with Dr. Nicola Fiore and Dr. BaozhongMeng who work in the areas of grapevine virus diagnostics and grapevine-virus interactions respectively.

Dr. Constable was asked to represent Australasia to assist with the ICVG publications list.


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PROJECT OVERVIEWTravel detailsAttendance at the16th Meeting of the International Council for the Study of Virus and Virus-like Diseases of theGrapevine (ICVG), Dijon, France August-September 2009

Name and Contact Details of Travel Grant Recipient

Name Dr F E ConstablePostal Address Department of Primary IndustriesPrivate Bag 15Ferntree Gully Delivery CentreVictoira, 3156Telephone 03 9210 9222Fax 03 9800 [email protected]

Itinerary

Date Travel and Meeting Activities Day #

27th-28th August 2009 Air travel Melbourne to Paris 1-2

29th August 2009 Train travel to Dijon visit with Colleagues at INRA 3

30th August - 4thSeptember 2009

16th Meeting of the International Council for the Study ofVirus and Virus-like Diseases of the Grapevine (ICVG), Dijon

4-9

5th September 2009 Weekend 10

6th-7th September 2009 Air travel Paris to Melbourne 11-12

Funds provided by GWRDC2000/2001GWRDC $4290

This amount covered registration for the 16th Meeting of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine (ICVG), Dijon and international fares. Accommodation and personal expenses werecovered from other sources.
mailto:[email protected]:[email protected]:[email protected]:[email protected]


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OBJECTIVES:

1. Attend the 16th meeting of the ICVG in Dijon with the purpose of gaining up-to-date information andobtaining the latest research findings of international researchers.

2. Present the latest findings of the GRWDC funded project entitled Development and validation of diagnosticprotocols for the detection of endemic and exotic pathogens of grapevines.

3. Visit with colleagues at INRA to discuss phytoplasma and virus diseases of grapevines with the purpose of(i) Capability/competence development.(ii) Development of collaborations for future projects.

Outcomes

Dr Constable attended and presented the results from the GWRDC funded project entitled Development andvalidation of diagnostic protocols for the detection of endemic and exotic pathogens of grapevines (Projectnumber: DPI05/04) at the 16th ICVG. Two papers were presented:

A survey for endemic and exotic viruses of Australian grapevines by Fiona Constable, Phil Nicholas,Narelle Nancarrowand Brendan Rodoni

Development and validation of sampling strategies for the detection of endemic viruses of Australiangrapevines by the same authors.

Dr Constable forged and consolidated ties with colleagues at INRA, Dijon and also with colleagues at otherinstitutions world wide, who are currently at the forefront of research into phytoplasmas, viroids and plant viruses ofgrapevines. In particular linkages were established with Baozhong Meng, Assistant Professor, Department ofMolecular and Cellular Biology, Guelph Grape and Wine Research Group,University of Guelph, Canada. Dr. Menghas considerable expertise in the development of full length infectious clones to study plant-virus interactions with afocus on grapevine Rupestris stem pitting-associated virusand is prepared to collaborate with us in this area forfuture projects. He is attending the Australasian plant virology workshop, Melbourne, November 2009 as an invitedguest speaker.

Up-to-date information about grapevine viruses and virus-like organisms was also gained, particularly with relation

to virus discovery, virus taxonomy, diagnostic technologies, and plant-virus interactions.


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Background

Many viruses, phytoplasmas and some viroids have been associated with disease of grapevines. Some of thesediseases are associated with significant yield loss, reduced fruit quality, graft incompatibility, vine decline and deathin grapevines. As a consequence The International Council for the Study of Virus and Virus-like diseases of thegrapevine (ICVG) was founded in 1964. It was initiated by plant pathologists from North America and Europe who

recognized the importance of these diseases and the benefits of sharing knowledge amongst the internationalscientific community and passing on information about the diseases, diagnostic tests and control strategies to theglobal viticultural industry. The ICVG organises scientific meetings every three years to promote global researchand collaboration related to virus and virus-like diseases of grapevine.

GWRDC provided funding for airfares and conference registration so that Dr Fiona Constable could attend the 16thMeeting of the ICVG which was held in Dijon, France from 31st August 4th September 2009. The program for themeeting is attached in appendix 1. Dr Constable gave two presentations at the meeting about the results from theGWRDC funded project entitled Development and validation of diagnostic protocols for the detection of endemicand exotic pathogens of grapevines (Project number: DPI05/04):

Oral presentation: Development and validation of sampling strategies for the detection of endemic viruses

of Australian grapevines by Fiona Constable, Phil Nicholas, Narelle Nancarrow

and Brendan Rodoni Poster presentation: A survey for endemic and exotic viruses of Australian grapevines by Fiona

Constable, Phil Nicholas, Narelle Nancarrowand Brendan Rodoni

The abstracts, which have been published in le Progrs Agricole et Viticole: "Proceedings of the 16th Meeting ofthe International Council for the Study of Virus and Virus-like Diseases of the Grapevine (ICVG) and will be madeavailable on the ICVG website in the near future, are attached in Appendix 2.

Attendance at the conference allowed me to consolidate ties with colleagues currently at the forefront of researchinto phytoplasmas, viroids and viruses of grapevines and gain up to date information about grapevine virology.

Summary of meeting themes

More than160 scientists from most of the viticulture regions around the world participated in the ICVG meeting andgave presentations around the following themes:

1. Detection, plant material and virus sources2. Fanleaf, Fleck and other spherical viruses3. Epidemiology Survey of vineyards4. Phytoplasmas5. Molecular biology New technologies6. Virus effects Control Crop performances7. Viruses of the Leafroll disease complex8. Rugose wood complex Viruses9. Emerging diseases and diseases of unclear aetiology

There are now 60 taxonomically recognized viruses, 10 phytoplasmas and six viroids reported to infect grapevinesworld-wide. Several of the new virus species have been identified through novel sequencing technology.Consequently the biological properties of these viruses, particularly if they are cryptic or associated with disease,are not understood. The identification of such viruses has important implications for both quarantine andcertification programs - should these be controlled if they are not associated with diseases of grapevines? Shouldall grapevine material entering Australia undergo virus eradication to reduce the risk of these viruses to grapevinesand other potential hosts? This highlights the need for studies to determine Kochs postulates for cause of diseasesfor many grapevine viruses.

Following is a brief summary of the research presented during each session, with an emphasis on information thatmay be relevant to Australian viticulture:

1. Detection, plant material and virus sources

Molecular techniques, particularly RT-PCR techniques remain the preferred method for virus detection. Advanceshave been made in the area with the development of quantitative PCR methods for virus detection. Many of theseRT-qPCR techniques are based on Taqman probes, which were shown to be highly sensitive and in someinstances detected viruses when other molecular and serological tests could not. Taqman assays fort he detectionof phytoplasma were also reported during the phytoplasma session. However, I remain apprehensive about the use


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of this technology for routine diagnostic testing as the increased specificity of the tests may result in false negativeresults.

Antibodies are still being raised against grapevine viruses by various researchers worldwide for serology-basedstudies and detection. Not all of these antibodies are available commercially for use in ELISAs for virus detectionand this makes comparison of test results at a global level difficult. Despite the restraints of reliable source of

quality antisera, serological-based detection systems remain an important part of grapevine virus taxonomy. Thereactivity of viruses to the various available antibodies is still considered an important property when viruses arebeing assigned a taxonomic position.

2. Fanleaf, Fleck and other spherical viruses

Much of the work reported during this session related to the molecular biology of nepoviruses, primarily Grapevinefan leaf virus (GFLV) and Arabis mosaic virus(ArMV). Work included identification of genes involved in to vectorspecificity, and symptom determinants, recombination with GFLV isolates and between GFLV and ArMV andgenetic diversity. The genetic variability was not always associated with differences in symptom expression. Coatprotein-mediated transgenic resistance is considered a promising strategy to control fanleaf disease. It was alsoshown that mycorrhizal infection significantly reduced numbers of Xiphenema index, the vector of GFLV, in the soilaround grapevines and on grapevine roots, indicating that this may assist in reducing the spread of GFLV. Plant

biomass was also significantly greater in mycorrhizal colonized plants compared to un-colonized plants.3. Epidemiology Survey of vineyards

Surveys of native Vitis species in the USA indicated that these may be hosts of viruses that infect cultivatedgrapevines. In particular Grapevine rupestris stem pitting associated virus(GRSPaV), Grapevine leafroll associatedvirus 2(GLRaV-2) and Grapevine virus B (GVB) were detected in V. californica. Several previously undescribedviruses were identified by cloning and sequencing of dsRNAs isolated from native Vitisspecies found in a naturalenvironment; however their biological significance to viticulture is not understood.

In many countries, including Australia (see the attached poster), GLRAV-3 and GLRaV-1 were the most frequentlydetected leafroll viruses. Based on our results GLRaV-9 is the third most frequently detected leafroll virus inAustralia (Poster attached in Appendix 1). Grapevine fleck virus (GFkV) was often found in mixed infections with

various leafroll viruses. Interestingly Grapevine virus A (GVA) was frequently found to co-infect grapevines withGLRaV-1 and it is thought that GLRaV-1 acts as a helper virus and increases the efficiency of GVA transmission bymealybug and scale vectors.

4. Phytoplasmas

Most presentations in the phytoplasma session focussed on Flavesence dore (FD) and Stolbur (Bois Noir, Stol)phytoplasmas.

Several phylogenetic analyses for strain variation and differentiation were presented for FD and Stol phytoplasmasand the implications for their epidemiology were discussed. Phylogenetic analysis of five genes showed that FDphytoplasmas and Alder yellows (AldY) phytoplasma belong to the 16SrV group and have a high sequencesimilarity and that they have a common origin in alders. It was been proposed that FD and AldY phytoplasmas be

classified into a new species known as Candidatus Phytoplasma cauldwelli. Strain variation of the Stolphytoplasmas is closely linked to its epidemiology: there are two Stol phytoplasma strains, each with differentalternative hosts and are transmitted by specific genotypes of the vector Hyalesthes obsoletus, which havedifferent biology and timing of lifecycles. As a consequence of these differences slightly different control strategiesare required to prevent the spread of Stol phytoplasma into vineyards.

Studies showed that wild rootstocks may be a reservoir for FD phytoplasma vector, Scaphoideus titanus, and maycounteract control strategies to reduce the spread of FD. Studies showed that recovered FD affected grapevineswere less likely to be a source of phytoplasma for acquisition by S titanusor for transmission through propagation.However transmission in propagation material can occur and hot water treatment remains a recommended controlstrategy to prevent further spread of FD and Stol phytoplasmas in Europe. Remission of Bois Noir disease wasinduced by the application of various commercially available chemicals such as Aliette, which induces natural hostdefences, through abiotic tress associated with partial uprooting, pollarding and by summer pruning.

Proteomic analyses and gene expression studies are being used to elucidate the biochemical pathways involved inplant-phytoplasma interactions for both FD and Stol phytoplasmas. Alteration in carbohydrate metabolism,photosynthesis and the phenylpropanoid pathway were reported with the suggestion that some alterations may beinvolved in host defence responses.


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The detection of an Aster yellows phytoplasma in the Southern Cape of South Africa was also reported.

5. Molecular biology New technologies

New high throughput sequencing technologies are being used to identify previously undetected anduncharacterized viruses and to determine the prevalence of viruses within vineyards. The aim of the research was

to understand the hostpathogen interactions and the association between virus and disease. The use ofrecombinant GFLV tagged with autoflourescent markers is being used to determine how viral proteins mightinteract with the plant and take over cellular processes for replication. The application of GVA-based virus inducedgene silencing in several cultivars is being developed for analysis of gene function.

6. Virus effects Control Crop performances

Control:The use of transgenic rootstocks, which contain the GFLV coat protein gene, to reduce the spread of GFLV isbeing assessed. So far research indicated that the transgenic plants were not resistant but the rate of infection intransgenic plants was much slower compared to the controls. Results also indicated that, transgenic plants did notlead to GFLV variants that overcome resistance, and there was no transfer of CP mRNA or coat protein betweenrootstock and scion. Consequently the use of transgenic grapevines to reduce the rate of spread of GFLV seems

promising.

More traditional control strategies to reduce their spread of GLRaV-3 in South Africa were reported and includedthe integrated use of certified planting material, rouging and judicious use of insecticides.

Crop Performance:The effect of virus on field performance and oenological characteristics was variable and depended on the virusspecies and also on the grape variety. Variable effects were also observed between virus infected scion woodgrafted onto different rootstocks, indicating that rootstock tolerance to virus is an important factor to consider inrootstock-scion combinations.

7. Viruses of the Leafroll disease complex

Many of the leafroll virus species have highly variable genomes and several new but unclassified molecularvariants have been described. However, based on molecular phylogenetic analyses, these variants and speciescluster together to form a subgroup within the Ampelovirus genus. Therefore it was suggested, as per the 15 thICVG meeting, that the some of these new variants, such as GLRaV-4,-5,-6 and -9, should be assigned to a singletaxon (species; probably GLRaV-4), especially as they have similar biological traits such as vectors, symptomexpression and some cross reactivity with antisera that has been raised to the individual species or strains withinthis group.

GLRaV-7 has been completely sequenced. It is not closely related to the other characterised leafroll viruses and ismore closely related to the viruses in the genus Crinivirus. The vector for this virus is unknown; howevercriniviruses are often transmitted by the glasshouse whitefly. The possibility that GLRaV-7 is transmitted by theglasshouse whitefly is being investigated.

The existence of Grapevine leafroll associated virus 8 has been questioned and it is likely to be removed from thelist of viruses infecting grapevines. This virus has only been reported once and its existence was based only on apartial sequence (273bp) of the putative coat protein gene. It has since been discovered that fragment was in factamplified by PCR techniques from the genome of Vitis vinifera. As a consequence it is advisable that this virusshould be considered for removal from the AQIS list of quarantineable grapevines viruses.

Leafroll viruses in the genus Ampelovirus (GLRaV-1,-3,-4,-5,-6 and -9) can be transmitted by mealybugs and scaleinsects. Two previously unreported soft scale vectors, Coccus hesperidumand C. longulus, which both occur inAustralia, were shown to transmit GLRaV-3. However they may not play a significant role in the spread of the virusas they are poorly adapted to grapevine. The citrus mealybug, Planococcus citri, which also occurs in Australia,was reported as an additional vector for GLRaV-1.

8. Rugose wood complex Viruses

The rugose wood viruses comprise of GVA, GVB, Grapevine virus D(GVD), GRSPaV and Grapevine inner berrynecrosis virus. In 2008, Grapevine virus E (GVE) was described and shown to be transmitted by Psuedococcuscomstockibut was not associated with a specific disease in grapevine. GVE has not been reported in Australia.


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Vein necrosis is a disease that affects the rootstock 110 Richter. Research indicated that this disease may beassociated with two specific subgroups of GRSPaV.

Full length infectious clones of GRSPaV are being produced with the aim of being used as a tool in the functionalgenomics of grapevines. Full length infectious clones have also been constructed for GVA and these are beingused to elucidate the roles of specific genes within the GVA genome.

9. Emerging diseases and diseases of unclear aetiology

The association between viruses and Shiraz disease in South Africa and Syrah decline in Europe remains unclear,although GRSPaV and GVA have been implicated in previous studies. There was no evidence for an associationbetween these diseases nor were known viroids or phytoplasmas detected in symptomatic material.

There were two field trips to vineyards of Ctes de Beaune and Mconnais regions of Bourgogne and the Chteau-Chalon region of the Jura to observe viticulture, certification programmes and virus and phytoplasma associateddisease.

Report on Outcomes

(i) Attendance at the meeting and information gathering

The sessions were informative and relevant to the research strategies of our GWRDC funded grapevinediagnostics project. Where appropriate information and technologies that were presented at the meeting will beincorporated into the current project and will be used to develop further projects for grapevine virus and virus-likedisease of grapevines.

(ii) Presentations

The latest research from the GWRDC funded project entitled Development and validation of diagnostic protocolsfor the detection of endemic and exotic pathogens of grapevines (Project number: DPI05/04) were presented atthe 16th ICVG. Two papers were presented and the abstracts and the poster presentation are attached in Appendix2. These results were received positively by many researchers at the meeting. There was interest from various

researchers about the sampling strategies, extraction technology and PCR techniques for virus detection ingrapevines that have been developed within our project and the protocols for sampling will be passed on.

(iii)Collaborative research links

Existing collaborative links were strengthened and new linkages were made. Discussions were held with manyresearchers regarding the grape diagnostics research project during the conference. Specifically the goals of thegrapevine diagnostics project were discussed with Dr. Nuredin Habili (Waite Diagnostics, Australia), Dr. AdibRowhani (USDA, California) and Dr. Nicola Fiore (University of Chile). In particular we discussed which techniqueswere most appropriate to detect strains of grapevine viruses and we discussed other viruses that might be animportant focus for future projects and diagnostic development. Dr. Nicola Fiore expressed an interest indeveloping collaborative research about virus and virus like diseases of horticultural crops as many of DPIs andhis research objectives are similar.

I also met with Dr. MaryJo Farmer and Dr. Elisabeth Boudin-Padieu to discuss phytoplasmas. They also discussedsome collaborative work about the chaperonin gene as an additional taxonomic marker for phytoplasmas.

I was asked, as a representative for Australasia to take part in gathering information for the ICVG publications list.This role will assist in forging ties with the ICVG.


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APPENDIX 1Conference program

ICVG XVI DIJON, France, 31 Aug 4 Sept 2009

16th Meeting of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine

PROGRAMME

Sunday 30 August Reception hall of the SNCF Railway station of Dijon Ville welcome and registration

Monday 31 August Grand Dijon

8.00 registration continued mounting of posters

8.45 Opening session

9.30 Coffee break

10.00 - Oral Session I Introductory Key notes

Chair : Edna Tanne - Elisabeth Boudon-Padieu

10.00 Martelli G. P.

Grapevine Virology Highlights 2006-09

10.30 Gugerli P. 25 years of serological identification of grapevine leafroll-associated viruses: antiserum and monoclonalantibodies to GLRaV-1 to GLRaV- 9

11.00 Dolja V. V. New developments in understanding gene functions and evolution of the grapevine Closteroviruses

11.30 Oral Session II Detection, plant material and virus sources

Chair : Raymond Johnson Sandrine Rousseaux

11.30

Invited lecture : Sabanadzovic S. Viruses of native Vitis germplasm in the Southeastern united states

12.00 Comparison of High Throughput Low Density Arrays, Rt-PCR and Real-Time Taqman RT-PCR in the detection ofgrapevine viruses Osman F., C. Leutenegger, D.A. Golino, A. Rowhani

12.15 Grapevine viruses in Chile: Multi-parallel detection based on metagenomic strategies Engel E. A., P. F. Escobar, P.Rivera, P. D.T. Valenzuela.

12.30 Grapevine virus collection at Nyon: A contribution to a putative network of a worldwide grapevine virus referencecollection Gugerli P., J-J. Brugger, M-E. Ramel, S. Besse.

12.45 - Lunch

13.45 - Oral Session III Fanleaf and other spherical viruses

Chair : Olivier Lemaire Thierry Wetzel

13.45 Invited lecture Demangeat G. Nepovirus transmission by Longidoridae nematodes with special emphasis on the pairGrapevine fanleaf virus/Xiphinema index

14.15 Identification of plasmodesmata located receptors involved in the movement of Grapevine fanleaf virus (GFLV) and otherviruses employing tubule-guided movement Amari K., E. Boutant, C. Hofmann, L. Fernandes-Calvino, C. Schmitt-Keichinger, P.Didier, C. Thomas, A. Lerich, M. Heinlein, Y. Mely, A. Maule, C. Ritzenthaler

14.30 Symptom determinants on the Arabis mosaic nepovirus genome Dupuis L., C. Himber, P. Dunoyer, A. Bassler, M. Keller,T. Wetzel

14.45 Recombination events in Rna-2 of Grapevine fanleaf virus and Arabis mosaic virus in grapevines affected by Yellowmosaic Jawhar J., A. Minafra, P. La Notte, C. Pirolo, P. Saldarelli, D. Boscia, V. Savino, G. P. Martelli

15.00 The specificity of GFLV and ARMV transmission by their respective nematodes: from structure to function SchellenbergerP., G. Demangeat, P. Andret-Link, C. Keichinger, M. Bergdoll, P. Bron, S. Trapani, B. Lorber, C. Sauter, A. Marmonier, D.Esmenjaud, O. Lemaire, M. Fuchs, C. Ritzenthaler


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15.15 Partial RNA-2 Sequence of Grapevine Bulgarian latent virus Elbeaino T., F. Fallanaj, M. Digiaro, S. Kuzmanovi

15.30 Characterization of Raspberry bushy dwarf virus isolates from grapevine Mavri Pleko I., M. Virek-Marn, S. irca, G.Urek

15.45 Isolation of movement protein gene by the use of degenerate primers from Iran isolates of Grapevine fanleaf virus andassessment of the genetic diversity Sokhandan Bashir N., A. Delpasand Khabbazi

16.00 Coffee break

Official photo

16.45 - Oral Session IV Epidemiology Survey of vineyards

Chair : Giuseppe Belli Adib Rowhani

16.45 Development and validation of sampling strategies for the detection of endemic viruses of australian grapevinesConstable F., P. Nicholas, N. Nancarrow, B. Rodoni

17.00 Preliminary results on the quantification of different grapevine viruses in a typical northwestern italian cultivar Pacifico D.,P. Caciagli, F. Mannini, F. Veratti, C. Marzachi

17.15 Current status of grapevine viruses in the pacific northwest vineyards of the United States Rayapati N.A., M.A. Tefera,A..J. Olufemi, J. Sridhar, K. Gandhi, L.R. Gutha, R.R. Martin

17.30 Survey of wild grapes, weed and cover crop species for grapevine viruses Golino D.A., S.T. Sim, F. Osman, R. Aldamrat,V. Klaassen, A. Rowhani

17.45 Contrasting epidemiologies of grapevine viruses depending on appellation and variety. Legorburu F.J., E. Recio, E.Lopez, M. Larreina, F. Aguirrezabal, J.F. Cibriain, R. Gonzalez- Rodriguez, C. Cabaleiro

18.00 - Poster session

ICVG Steering Committee Meeting

19.00 Departure to the official reception Dijon Town Hall

Evening free

Tuesday 1 September - Grand Dijon

8.00 Oral session V Phytoplasma diseases

Chair : Antoine Caudwell A. Bertaccini

08.00 Invited lecture : Foissac X.

Ecology and taxonomy of Flavescence dore phytoplasmas: the contribution of genetic diversity studies Malembic-Maher S., P.Salar, P. Carle, X. Foissac

08.25 Molecular identification and geographic distribution of Flavescence dore phytoplasma strains Bertaccini A., S. Paltrinieri,

F. Dal Molin, J. Mitrovi, B. Duduk

08.40 Comparison of prevalence, geographical distribution and biological properties of two Flavescence dore phytoplasmastrains Salar P., D. Clair, X. Foissac, E. Boudon-Padieu, S. Malembic-Maher

08.55 Genetic polymorphism of stolbur phytoplasma in grapevine, wild plants and insects Filippin L., E. Tonon, V. Forte, M.Zottini, G. Santovito, M. Borgo, E. Angelini

09.10 Aspects of the interaction of Stolbur phytoplasma, vectors and host plants in the two epidemic systems of Bois noirMaixner M., J. Johannesen, A. Seitz

09.25 On host races and co-evolution of the grapevine yellows vector Hyalesthes obsoletus and stolbur phytoplasmaJohannesen J., A. Seitz, N. El Sayar, M. Maixner

9.40 - Coffee break10.15 - Oral Session V Phytoplasma diseases (continued)

Chair : Michael Maixner Djana Skoric


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10.15 Molecular markers among stolbur phytoplasma (16SrXII-A) strains and their association with natural ecologies ofgrapevine Bois noir in Italy Quaglino F., Y. Zhao, P.A. Bianco, W. Wei, G. Romanazzi, S. Murolo, M.R. Silletti, V. Savino, P.Casati, G. Durante, R.E. Davis

10.30 Long term effects of Stolbur phytoplasma on grapevines in Israel Zahavi T., R. Sharon, M. Mawassi, V. Naor

10.45 Bois noir phytoplasma induces significant reprogramming of genes involved in carbohydrate metabolism and

photosynthesis in the field grown grapevine Dermastia M., M. Hren, P. Nikoli, A. Rotter, N. Terrier, M. Ravnikar, K. Gruden

11.00 Detection and identification of a new phytoplasma disease in grapevines in the Western Cape Engelbrecht M., J. Joubert,J.T. Burger

11.15 Biochemical pathways in phytoplasmaplant interactions in symptomatic and recovered leaves of Bois noir - affectedgrapevines Landi L., G. Romanazzi

11.30 - Poster Session

12.45 Lunch

14.00 - Oral Session VI Molecular biology New technologies

Chair : Johan Burger Pascale Seddas

14.00 Invited lecture : Sela I. A universal vector for expression and silencing in plants (including grapevine) Peretz Y., R.Mozes-Koch, E. Tanne, I. Sela

14.30 Expression/silencing of GVA components in grapevine and N. benthamiana using the IL-60 system Peretz Y., I. Sela, E.Tanne

14.45 Virome of a vineyard: Ultra deep sequence analysis of diseased grapevines Coetzee B., M-J. Freeborough, H.J Maree, J-M. Celton, D.J.G. Rees, J.T. Burger

15.00 Deep sequencing analysis of viral short RNAs from Pinot noir clone ENTAV 115 Pantaleo V., P. Saldarelli, L. Miozzi, A.Giampetruzzi, A. Gisel, S. Moxon, T. Dalmay, J. Burgyan

15.15 High-Throughput sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple

virus infection that includes a novel virus Al Rwahnih M., S. Daubert, D.A. Golino, A. Rowhani15.30 Characterisation of the genomic and subgenomic RNA of Grapevine leafroll-associated virus 3 (GLRaV-3) Maree H.J., E.Jooste, D. Stephan, M-J. Freeborough, J.T. Burger

15.45 Grapevine fanleaf virus replication: illuminating the way Schmitt-Keichinger C., M-C. Herranz-Gordo, F. Courte, R.Elamawi, S. Chapuis, P. Didier, Y. Mely, C. Ritzenthaler

16.00 Coffee break

16.30 - Oral Session VII Virus effects Control Crop performance

Chair : Deborah Golino Marc Fuchs

16.30 Transgenic rootstocks expressing GFLV coat protein gene in a three years field trial; resistance assessment, impact onGFLV diversity and exchanges between rootstock and scion Hemmer C., E. Vigne, V. Goldschmidt, V. Komar, A. Marmonier, L.Valat, G. Demangeat, S. Vigneron, J.E. Masson, O. Lemaire

16.45 A case study of control of grapevine leafroll disease spread on Vergelegen wine estate, South Africa, 2002-2008Pietersen G., N. Spreeth, T. Oosthuizen, A. Van Rensburg, D. Lottering, D. Tooth, N. Rossouw

17.00 Tentative analysis of the economic impact of grapevine leafroll disease in the vineyard of Valais (Switzerland) Besse S.,C. Rutsche, P. Gugerli

17.15 Field performances and wine quality modification in a clone of Dolcetto (Vitis vinifera L.) after GLRaV-3 eliminationMannini F., A. Mollo, D. Cuozzo, R. Credi

17.30 A mild strain of Grapevine leafroll-associated virus 3 is present in desirable clones of Crimson seedless table grapes inwestern Australia Habili N., I. Cameron, J. Randles


18.30 General Assembly of the ICVG

Evening free


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Wednesday 2 September - Grand Dijon

9.00 Oral session VIII Viruses of the leafroll complex

Chair : Etienne Herrbach Paul Gugerli

09.00 Invited lecture : Cabaleiro C. Current advances in the epidemiology of Grapevine leafroll disease

09.30 First epidemiological study of ampeloviruses in Turkish vineyards Buzkan N., S. Karadag, V. Oztekin, A. Kaya, A. Minafra,Y. Ben-Dov

09.45 Distribution and diversity of Leafroll-associated ampeloviruses in viruliferous vectors and grapevine hosts Fuchs M., P.Marsella-Herrick, G.M. Loeb, T.E. Martinson, H.C. Hoch

10.00 Identification and distribution of three divergent molecular variants of Grapevine leafrollassociated virus - 3 (GLRaV-3) inSouth african vineyards Jooste E., H.J. Maree , G. Pietersen , D.E. Goszczynski, J.T. Burger

10.15 Taxonomy, complete nucleotide sequence and genome organization of grapevine leafrollassociated virus - 7

Mikona C., C. Turturo, B. Navarro, W. Menzel, A. Minafra, M.E. Rott, G.P. Martelli, W. Jelkmann

10.30 Coffee break


12.45 Lunch

14.00 Oral session VIII Viruses of the leafroll complex (continued)

Chair : Filomena Fonseca Ivana Gribaudo

14.00 Comparison of phylogenetic inference based on HSP70, HSP90 and CP genes of GLRaV- 3: implications for detectionand taxonomy Teixeira Santos M., F. Esteves, M. De Lurdes Rocha, J.E. Eiras-Dias, F. Fonseca

14.15 The role of seasonality on mealybug transmission of Grapevine leafroll-associated viruses: an ecological hypothesisAlmeida R.P.P., C-W. Tsai, K.M. Daane

14.30 Spread of GVA, GLRaV-1 and -3 and role of the mealybug vector Heliococcus bohemicus in a vineyard of Langhe(Northwestern Italy) Bertin S., F. Mannini, D. Bosco, G. Gambino, D. Cuozzo, I. Gribaudo

14.45 Transmission of Grapevine leafroll-associated virus 3 (GLRaV-3) by three soft scale insect species (Hemiptera: Coccidae)and notes on their developmental biology on grapevine Kruger K., N. Douglas

15.00 Monitoring of Grapevine leafroll-associated virus 1 (GLRaV-1) dispersion by the mealybug Phenacoccus aceris LeMaguet J., E. Herrbach, G. Hommay, M. Beuve, E. Boudon-Padieu, O. Lemaire

15.15 Transmission trials of grapevine viruses by the mealybug Planococcus ficus (Hemiptera:

Pseudococcidae) Credi R., F. Terlizzi, L. Martini, S. Borsari

15.30 Coffee break

16.00 - Oral session VIII Viruses of the leafroll complex (end)

Chair : Kerstin Krger Nuredin Habili

16.00 Transmission of Grapevine leaf roll-associated virus-1 and -3 (Ampelovirus) and Grapevine virus A (Vitivirus) by naturalpopulations of soft scales and mealybugs in the North-Eastern French vineyard Hommay G., J. Le Maguet, V. Komar, O.Lemaire, E. Herrbach

16.15 Rapid spread of leafroll disease in Cabernet Sauvignon grapevines in Napa Valley, California Golino D.A., E. Weber, S.T.Sim, A. Rowhani

16.30 Sequence variation in Grapevine leafroll-associated virus-3 (GLRaV-3) New Zealand isolates Chooi K.M., M.N. Pearson,D. Cohen, J.C.H. Pong

16.45 Last Poster Session

19.00. Removal of posters

19.30 departure to the Official dinner Chteau de Saulon la Rue


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Thursday 3 September

Full day trip to vineyards of Ctes de Beaune and Mconnais

8.00 - Departure

12.30 - Lunch at the "Maison des Vins" in Mcon

19.00 - Return to Dijon

Evening free

Friday 4 September Conseil regional de Bourgogne

8.30 - Oral session IX- Rugose wood complex

Chair : Dirk Stephan Alberto Materazzi

8.30 Invited lecture: Saldarelli P. Flexiviruses: a grapevine point of view

9.00 Different variants of GRSPaV are associated to diverse diseases in grapevine Borgo M., N. Bertazzon, F. Anaclerio, E.Angelini

9.15 Molecular characterization of biologically divergent strains of GRSPaV Bouyahia H., M. Della Bartola, A. Materazzi, E.Triolo

9.30 Toward development of Grapevine rupestris stem pitting-associated virus into a VIGS vector Meng B., C. Li, W. Wang

9.45 Characterisation of ORF5 of three South African Grapevine virus A variants as pathogenicity determinant in Nicotianabenthamiana Blignaut M., D. Stephan, J. Du Preez, S. Haviv, M. Mawassi, J.T. Burger

10.00 Coffee break

10.30 Chair : Piero Bianco Keramatollah Izadpanah

10.30 - Oral session IX- Rugose wood complex (continued)

10.30 Towards the elucidation of Grapevine virus A ORF 2 gene function Du Preez J., D. Stephan, D.E. Goszczynski, M.Muruganantham, M. Mawassi, J.T. Burger

10.45 Single domain antibodies for detection of GVB Masri S., H. Rast, N. Habili

11.00 Oral session X Diseases of unclear etiology

11.00 Viral sanitary status and genetic diversity of Rupestris stem pitting associated virus in French Syrah clones exhibitingvarious susceptibility levels to decline Beuve M., B. Moury, A-S. Spilmont, L. Sempe-Ignatovic, C. Hemmer, O. Lemaire

11.15 Discussion on Posters 91-95

11.30 General conclusions

Chair : Paul Gugerli Elisabeth Boudon-Padieu

12.15 Farewell drink offered by the Conseil rgional de Bourgogne

12.45 Lunch

Half day trip to Jura vineyards

14.00 Bus Departure

19.00 Return to Dijon

POSTERS

Topic II Detection Plant material and Virus source

P 01 Grapevine virus Q: The first plant virus with a permuted active site of RNA-dependent RNA polymerase Sabanadzovic S.,N. Abou Ghanem-Sabanadzovic, A. E. Gorbalenya

P 02 Production of monoclonal antibodies to Grapevine leafroll- associated virus 9 (GLRaV-9) Gugerli P., S. Rigotti, M-E.Ramel, N. Habili, A. Rowhani, W. Bitterlin, S. Besse.


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P 03 Detection of viruses in seeds of infected grapevines with an emphasis on Grapevine rupestris stem pitting-associated virusHabili N., R. Davies, J. Randles.

P 04 Real time PCR for sensitive reliable grapevine virus detection Malan S., M-J. Freeborough, J. Burger

P 05 Prevalence of seven grapevine viruses in table grapes from Vinalop (Alicante) determined by ELISA and Real-Time RT-PCR Bertolini E., C. Martinez, J. Garcia, J. Juarez, A. Martinez, E. Vidal, A. Yuste, M. Albiach-Marti, M. Cambra, A. Olmos.

P 06 Virus-derived sequences in the nuclear genome of grapevine Bertsch C., M. Beuve, V. V. Dolja, M. Wirth, F. Pelsy, E.Herrbach, O. Lemaire

P 07 How widespread is the presence of Grapevine leafroll-associated virus 8 (GLRaV-8) putative capsid protein genesequences in DNA and RNA obtained from grapevine varieties? Esteves F., M. Teixeira Santos, M. De Lurdes Rocha, J. E.Eiras-Dias, F. Fonseca

P 08 The occurrence of viruses in the clonal selection vineyards in Czech republic Holleinova V., L. Blahova, K. Barankova

P 09 Contribution of new certified clones to the improvement of the italian table grape industry La Notte P., C. Pirolo, P.Giannini, G. Bottalico, A. Campanale, V. Savino, G. P. Martelli

P 10 Clonal selection and sanitary status of local grapevine germplasm in Serbia Mandi B., . Ivanovi, L. Susca, G. Bottalico,M. Starovi, D. Jaki, S. Kuzmanovi, D. Ivanievi, V. Gavrilovi, Kora N., Digiaro M., P. La Notte

P 11 Old grapevine varieties vineyards: a window over a pre sanitary selection era and a source of viruses Teixeira Santos M,M. De Lurdes Rocha, A. Guedes Da Silva, F. Esteves, F. Fonseca, J. E. Eiras-Dias

Topic III - Fanleaf and other spherical viruses

P 12 Real-time PCR of Grapevine fanleaf virus Blahova L., M. Pidra

P 13 First records of Arabis mosaic virus (ARMV) on grapevine in Spain Abelleira A., J. P. Mansilla, V. Padilla, I. Hita, C.Cabaleiro, A. Olmos, F. J. Legorburu

P 14 Absolute quantification of Grapevine fanleaf virus in Chenopodium quinoa using real-time RT-PCR assays Ulloa S., C.Ritzenthaler, P.D.T. Valenzuela , C. Bruno

P 15 Detection of Virus like particles (Vlps) by ISEM in transgenic grapevines expressing different GFLV CP-constructsGottschamel J., M. Castellano, F. Maghuly, M. Laimer

P 16 Distribution of Grapevine fanleaf virus (GFLV) in grapevines during the season epin U., S. Krsmanovi, M. Pompe-Novak, M. Ravnikar

P 17 Comparisons of genomic and pathological features among barley and grapevine infecting-isolates of Arabis mosaic virusMarmonier A., E. Vigne, V. Komar, G. Demangeat, S. Besse, P. Gugerli, 0. Lemaire

P 18 Nicotiana benthamiana plants expressing viral small interfering RNA show differential resistance level against GFLV BrunoC., P.D.T. Valenzuela

P 19 Genetic diversity of the coat protein gene of Chilean isolates of Grapevine fanleaf virus Zamorano A., A.M. Pino, N Fiore

P 20 Genetic variability within the coat protein gene of Grapevine fanleaf virus (GFLV) isolates from South Africa Liebenberg A.,M-J. Freeborough, C. J Visser, D.U. Bellstedt, J.T. Burger

P 21 Mycorrhiza reduce development of the nematode vector of Grapevine fanleaf virus in soil and root systems Hao Z., L.Fayolle, D. Van Tuinen, X. Li, V. Gianinazzi-Pearson, S. Gianinazzi

P 22 Evidence of recombination in 2A gene from a Grapevine fanleaf virus isolate Nourinejhad Zarghani S., M. Shams-Bakhsh,M. Pazhohande, N. Sokhandan Bashir

Topic IV Epidemiology Survey of vineyards

P 23 A preliminary survey of grapevine viruses in kurdistan province (West of Iran) Afsharifar A.R., M. Kamali, B. Harighi, S.Bahrami Kamangar, V. Roumi, K. Izadpanah

P 24 Occurrence of grapevine leafroll-associated virus-1 and 3 in Croatian autochthonous grapevine varieties from Dalmatia

Voncina D., E. Dermic, B. Cvjetkovic, E. Maletic, I. Pejic, J. Karoglan KonticP 25 Survey of major grapevine virus diseases in the vineyard of Valais (Switzerland) Besse S., P. Gugerli

P 26A survey of grapevine viruses in native cultivars in old plantations of Stefnesti Arges vineyard, Romnia Buciumeanu E-C., I.C. Gu, F. Semenescu


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P 27 A survey for endemic and exotic viruses of Australian grapevines Constable F., P. Nicholas, N. Nancarrow, B. Rodoni

P 28 Frequence of occurrence of grapevine virus disease-complexes based on over 30 years indexing results in Hungary LazarJ., M. Terjek

P 29 Emerging diseases of the grapevine at the Ukraine Milkus B.N., I.D. Zhun'ko, N.V. Limanska, L.A. Konup

P 30 Sanitary status of authochthonous minor wine grape varieties: an unusual diffusion of Arabis mosaic virus Materazzi A., C.D'Onofrio, H. Bouyahia, E. Triolo

P 31 The virus status of grapevine mother plantations in Cyprus Neophytou G., K. Sykas, N. Ioannou

P 32 Presence of GLRaV -1, -2, -3, -4 and -6 in Spanish vine material according to different ecosystems. Padilla V., I. Hita, B.Garcia de Rosa, C.V. Padilla, E. Salmeron, N. Lopez, S. Lucas

Topic V - Phytoplasma diseases

P 33 Proteomic analysis of grapevine (Vitis vinifera L. cv. Nebbiolo) midrib tissues infected by flavescence dore phytoplasmaMargaria P., S. Palmano

P 34 Study of rplP gene for characterization and phylogenetic analysis of phytoplasma strains within the 16Sr-V group DuranteG., E. Boudon-Padieu, D. Clair, F. Quaglino, P. Casati, P.A. Bianco

P 35 Real-time PCR assay for diagnosis of Flavescence dore (16Sr-V) Durante G., M. Calvi, A. Colombo, P.A. Bianco, P.Casati

P 36 Detection of grapevine Flavescence dore and Bois noir phytoplasmas by multiplex Real-time PCR (Taqman) Terlizzi F.,C. Ratti, C. Poggi Pollini, A.. Pisi, R. Credi

P 37 Detection of phytoplasmas associated with grapevine yellows in rootstocks Borgo M., L. Filippin, N. Bertazzon, E. Angelini

P 38 High occurrence of Scaphoideus titanus on wild rootstocks in North Eastern Italy Forte V., M. Borgo, L. Dalla Cia, E.Angelini

P 39 Spatial correlation of Scaphoideus titanus Ball adults on European grapevine at a plot scale: a case study Lessio F., E.Borgogno Mondino, A. Alma

P 40 Do Scaphoideus titanus larvae aggregate for feeding ? Chuche J., A. Boursault, D. Thiery

P 41 Acquisition of Flavescence dore phytoplasma by Scaphoideus titanus Ball from recovered and infected grapevines ofBarbera and Nebbiolo cultivars Galetto L., C. Roggia, C. Marzachi, A. Alma, D. Bosco

P 42 Graft transmission trials of Flavescence dore phytoplasma from recovered grapevines Carraro L., N. Loi, P. Ermacora, M.Martini, M. Borgo, P. Casati, R. Osler

P 43 Microbial diversity in healthy, yellows infected and recovered grapevines Bulgari D., P. Casati, L. Brusetti, F. Quaglino, D.Daffonchio, P.A. Bianco

P 44 Flavescence dore in the Empord (Catalonia): results of Scaphoideus titanus containment through aerial treatmentsTorres E., J. Rahola, J. Bech, S. Paltrinieri, A. Bertaccini

P 45 Epidemics of Flavescence dore disease in Portugal Sousa E., C. Sa, F. Cardoso, M. Mesquita, A. Oliveira, P. Casati,P.A. Bianco

P 46 Grapevine cultivar Plovdina is it really an indicator plant for flavescence dore disease ? Kuzmanovi S., D. Joi, .Ivanovi, M. Starovi, S. Stojanovi, G. Aleksi, M. Toi

P 47 Phytoplasma diffusion through grapevine propagation material and hot water treatment Mannini F., N. Argamante, G.Gambino, A. Mollo

P 48 Towards strain differentiation among grapevine bois noir phytoplasmas Contaldo N., B. Duduk, S. Paltrinieri, M. Kolber, I.Ember, A. Bertaccini

P 49 Genetic diversity among Bois noir phytoplasma populations in Italy: new 16Sr subgroups and distinct SNP genetic lineagesQuaglino F., Y. Zhao, P.A. Bianco, W. Wei, G. Romanazzi, S. Murolo, P. Casati, G. Durante, R.E. Davis

P 50 Preliminary results on the variability of Bois noir isolates in a vineyard system in Sicily Tessitori M., C. Oliveri, F. Veratti, V.Cavalieri, R. La Rosa, C. Marzachi

P 51 Incidence of Bois noir phytoplasma in different viticulture regions of Spain and stolbur isolates distribution in plants andvectors Batlle A., J. Sabate, A. Lavina


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P 52 Photosynthesis and transpiration in grapevines affected by Bois noir and recovered Murolo S., G. Romanazzi, D. Neri

P 53 Abiotic stress and treatment with elicitors for the control of Bois noir in Chardonnay vineyards Romanazzi G., S. Murolo, A.Masciulli, D. DAscenzo, V.A. Prota, R. Garau

P 54 Reaction of different grapevine varieties to summer pruning measures for combating Bois noir Ipach U., E. Muller, L. Kling,B. Helmstatter

P 55 Can pruning measures combat bois noir disease of grapevine ? Riedle-Bauer M., K. Hanak, F. Regner, W. Tiefenbrunner

P 56 Anaceratagallia ribauti (Oss., 1938) (Hemiptera, Auchenorrhyncha, Agalliinae) transmits a stolbur type phytoplasmaRiedle-Bauer M., A. Sara

P 57 Vectorphytoplasma relationships during natural infection of Hyalesthes obsoletus, Euscelis lineolatus, Neoaliturusfenestratus and Psammotettix alienus captured in vineyard agro-ecosystems in the Marche region (Central-Eastern Italy) LandiL., N. Isidoro, P. Riolo

P 58 Further data on occurrence of grapevine yellows-associated phytoplasmas in vineyards of Veneto region (north-easternitaly) Quaglino F., N. Mori, P. Casati, A. Zorloni, G. Zanini, P.A. Bianco

P 59 Survey of phytoplasma diversity in heavily grapevine yellows affected areas of Croatia eruga Musi M., D. kori, .Budinak, I. Krianac, I. Mikec

P 60 Identification of stolbur-related phytoplasmas in grapevine showing decline symptoms in Iran Karimi M., N. Contaldo, B.Mahmoudi, B. Duduk, A. Bertaccini

P 61 New high incidence of Australian grapevine yellows and evidence on pathogenesis following natural heat therapy inseverely infected South Australian vineyards Magarey P.A., N. Habili, J. Altmann, J.D. Prosser

Topic VI Molecular biology New technologies

P 62 Vacuum-agroinfiltration of different V. vinifera cultivars and application of VIGs in the cv. Sultana Stephan D., J. Du Preez,C. Stander, M. Vivier, M. Muruganantham, M. Mawassi, J.T. Burger

Topic VII - Virus effects Control Crop performances

P 63 Detection and elimination of grapevine fanleaf virus in callus, somatic embryos and regenerated plantlets of grapevineGambino G., R. Vallania, I. Gribaudo

P 64 Virus elimination in infected clones of four Greek grapevine wine cultivars (Roditis, Xinomavro, Baftra and savatIano) usingin vitro thermotherapy and tip meristem culture Grammatikaki G., A. Avgelis

P 65 Virus effects on vine growth and fruit components of three California heritage clones of Cabernet Sauvignon Golino D.A.,J. Wolpert, S.T. Sim, J. Benz, M. Anderson, A. Rowhani

P 66 Virus effects on vine growth and fruit components of Cabernet Sauvignon on six rootstocks Golino D.A., J. Wolpert, S.T.Sim, J. Benz, M. Anderson, A. Rowhani

P 67 Comparative study of in vitro behaviour of grapevine (V. vinifera l, Feteasc neagr cv.) under the influence of variousvirus infections Gu I.C., E-C. Buciumeanu, E. Visoiu

P 68 A study on LN33 grapevine infected with five viruses Kominek P., M. Kominkova

P 69 Effect of Grapevine fanleaf virus (GFLV) and Grapevine leafroll-associated virus 3 (GLRaV-3) on red wine qualityLegorburu F.J., E. Recio, E. Lopez, J. Baigorri, M. Larreina, A. Remesal, J.F. Cibriain, L. Caminero, J. Suberviola, F.Aguirrezabal

P 70 Leafroll symptoms caused by GLRaV-3 are associated with an increase of resveratrol in grapevine leaves Bertazzon N.,O. Repetto, M. De Rosso, R. Flamini, E. Angelini, M. Borgo

P 71 Effects of gva elimination on physiological, agronomic and oenological characteristics of a V. vinifera Marzemino cloneMalossini U., L. Zulini, A. Vecchione, E. Decarli, P. Bianchedi, R. Moscon, G. Nicolini

P 72 Effects of GLRaV-1 elimination on physiological, agronomic and oenological characteristics of two cv. Marzemino clonesMalossini U., L. Zulini, G. Nicolini, A. Vecchione, E. Decarli, P. Bianchedi, R. Moscon

P 73 Control of grape berry mechanical properties modifications due to GFLV by mean of texture analysis Santini D., L. Rolle, F.Mannini

P 74 2-amino-6-mercaptopurine: a preliminary study of a novel chemical group for grapevine antiviral therapy Luvisi A., A.Panattoni, E. Triolo


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P 75 Advances on the eradication of Grapevine rupestris stem pitting associated virus (GRSPaV) from Vitis vinifera explantsSkiada F., V.I. Maliogka, E.P. Eleftheriou, N.I. Katis

Topic VIII Viruses of the leafroll complex

P 76 A real-time RT-PCR assay for the detection and quantification of Grapevine leafroll-associated virus 3 (GLRaV-3) in Vitisvinifera L. (Vitaceae) and Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae) Douglas N., G. Pietersen, K. Kruger

P 77 Advances in molecular detection of GLRaV-1 Bertazzon N., I. Bazzo, O. Repetto, M. Borgo, E. Angelini

P 78 Development of an ELISA for the simultaneous detection of Grapevine leafroll-associated virus 4, 5, 6, 7 and 9 Besse S.,W. Bitterlin, P. Gugerli

P 79 Serological and molecular characterisation of Grapevine leafroll-associated virus 2 variants occurring in Switzerland BesseS., C. Balmelli, V. Hofstetter, P. Gugerli

P 80 Serological detection of Grapevine leafroll associated viruses 1, 2 and 3 Cabaleiro C., A.M. Pesqueira, J.R. Pan, I. Cortez

P 81 Serological relationships between grapevine infecting ampelovirus from Argentina Gomez Talquenca S., M. Lanza Volpe,C. Munoz

P 82 Detection of leafroll GLRaV-1, 2 and 3 on grapevine rootstocks varieties Beccavin I., M. Beuve, O. Lemaire, S. Grenan

P 83 Partial molecular characterization of Grapevine leafroll associated virus-1 isolate from Iran Roumi V., A.R. Afsharifar, K.Izadpanah

P 84 Genetic diversity of Portuguese isolates of Grapevine leafroll-associated virus 1 (GLraV-1) based on the capsid proteinEsteves F., M. Teixeira Santos, M. De Lurdes Rocha, J.E. Eiras-Dias, F. Fonseca

P 85 Genetic variability of Grapevine leafroll-associated virus 2 (GLRaV-2) in a Portuguese vineyard, based on the HSP70h andcapsid protein genes Esteves F., M. Teixeira Santos, M. De Lurdes Rocha, J.E. Eiras-Dias, F. Fonseca

P 86 RFLP analysis of RT-PCR products to evaluate the genetic variability of CP ORF in GLRaV-2 isolates Lanza Volpe M., O.Gracia, S. Gomez Talquenca

P 87 Assessment of GLRaV-3 variants occurring in Portuguese grapevine varieties according to the coat protein gene Gouveia

P., F. Esteves, M. Teixeira Santos, F. Fonseca, J.E. Eiras-Dias, G. NolascoP 88 Grapevine leafroll disease in Vitis vinifera cv Menca Pesqueira A.M., R. Gonzalez-Rodriguez, C. Cabaleiro

P 89 Further data on the sensitivity of different rootstocks to the graft incompatibility associated with GLRaV-2 infection PiroloC., D. Boscia, P. La Notte, A. Cardone, G.P. Martelli

P 90 Acquisition and transmission of GLRaV-1 (Grapevine leafroll associated ampelovirus 1) by Planococcus citri (Risso)(Hemiptera: Pseudococcidae) Scotto C., B. Conti, A. Panattoni, E. Triolo

Topic IX Rugose wood complex

P 91 Biological, serological and molecular characterization of Grapevine virus A isolates infecting vines in the Marche region,Central-Eastern Italy Murolo S., D. Boscia, G. Romanazzi, V. Savino

P 92 A novel vitivirus, Grapevine virus E Nakaune R., S. Toda, M. Mochizuki, M. Nakano

Topic X Diseases of unclear etiology

P 93 Preliminary investigations on a Syrah decline in Central Italy Bianco P.A., A. Zorloni, C. De Biasi, G. Belli

P 94 Shiraz disease and grapevine yellows in South Africa Pietersen G., T. Oosthuizen, E. Jooste, L. Filippin, N. Bertazzon, E.Angelini

P 95 Syrah decline: no evidence for viroid etiology Renault-Spilmont A-S., L. Bernard, P. Serra, N. Duran-Vila

P 96 Identification and partial characterization of grapevine viroids in Southern Iran Zaki-Aghl M., K. Izadpanah

.


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APPENDIX 2Abstracts of the oral and poster presentation and the poster presentation


19/23

Fiona CONSTABLE1*, Phil NICHOLAS2, Narelle NANCARROW1 and Brendan RODONI1

1Department of Primary Industries, Knoxfield, Private Bag 15, Ferntree Gully Delivery Centre, Victoria 3156, Australia.

2South Australian Research and Development Institute, Loxton Research Centre Loxton South Australia 5333, Australia.

*Corresp. author :Fiona Constable, Telephone: +61 3 920 9222, Fax: +61 3 9800 3521, Email:[email protected]

Summary

To determine the best time of year for the detection ofviruses replicate field trials were established in a hot climateregion and a cool climate region. Chardonnay and Shiraz

grapevines were inoculated with either Grapevine leafrollassociated virus 2 (GLRaV-2), Grapevine leafroll associated virus3 (GLRaV-3), Grapevine virus A (GVA) or Grapevine fleck virus(GFkV). Preliminary results indicate that testing may be reliablyconducted from late spring to early autumn for GLRaV-2,GLRaV-3 and GFkV. So far GVA has not been detected in any of

the inoculated grapevines. Preliminary results also indicate thatthe PCR tests that we have developed are more sensitive than

ELISA and should reduce the risk of obtaining false negativeresults.

INTRODUCTION

Although ELISA and RT-PCR are commonly andglobally used for the detection of grapevine viruses therehave been few comprehensive, systematic studies todetermine the reliability of these tests in comparison witheach other. Where comparative studies have been done,

most results have indicated that RT-PCR is more sensitivethan ELISA for detection of grapevine viruses, especiallyon symptomatic plants. Studies have indicated thatdetection of GLRaV-3 by ELISA and RT-PCR in

symptomless plants was erratic (Chen et al. 2003). ELISAand RT-PCR were shown to be reliable for the detection ofGLRaV-3 from bark scrapings of field symptomatic ornon-symptomatic infected grapevines throughout one

season but not for flowers and fruits (Ling et al. 2001). Incontrast, Rowhani et al. (1997) showed that GLRaV-3

maybe unevenly distributed within the same grapevine andcould lead to inconsistent test results. One study has shownthat ELISA may be more sensitive than RT-PCR for

detection of GLRaV-1 and -3 (Cohen et al. 2003).

No studies have been conducted to show the reliability

of RT-PCR and ELISA for the detection of grapevineviruses over time on replicates of grapevines of differentvarieties that are inoculated by the same source of virus andmaintained in different climatic conditions. In Australia,

grapes are grown in diverse environmental and climaticconditions. One of the main aims of our project is toidentify diagnostic protocols for the detection of a range of

endemic pathogens under Australian conditions. Wecompared ELISA and RT-PCR for the detection of GLRaV-2, GLRaV-3, GFkV and GVA in grapevines grown in two

climates to determine at what time of year these tests aremost reliable for virus detection in Australia.

MATERIALS AND METHODS

Field sites: Replicate trials were established in a hot

climate region (Sunraysia, Victoria) and a cool climate

region (Yarra Valley, Victoria) in 2006. Each trial containstwo varieties (Shiraz and Chardonnay) and for each varietythere are five treatments each consisting of five replicate

grapevines. The five treatments include un-inoculatedgrapevines as a control and grapevines inoculated withGLRaV-2, GLRaV-3, GVA or GFkV. These grapevines

were chip bud inoculated, using two virus infected buds per

grapevine, in October 2006 (Sunraysia) and November2006 (Yarra Valley).

Sampling: Each grapevine has been sampled and tested

monthly by ELISA and PCR since December 2006(Sunraysia) and January 2007 (Yarra Valley). Wherepossible green tissue, particularly petioles, were used.During dormancy phloem scapes of lignified canes were

used. Six hundred milligrams of tissue was taken from eachsample, finely chopped and divided equally into twoseparate grinding bags to be used for ELISA or RT-PCR. In

2007 Virus testing was only done by PCR in July atSunraysia and August at the Yarra Valley. Testing was notdone in September 2007 and 2008 as the grapevines had

been pruned.

ELISA: The ELISA kits used in this experiment were

from Bioreba (GLRaV-2 and GLRaV-3) or AGRITEST(GVA and GFkV) and the tests were done according to themanufacturers instructions. Extracts from infected

grapevines were used as a positive control for each virusand buffer controls were also included.

Nucleic Acid extraction: Total RNA was extractedfrom green grapevine tissue using a modified lysis buffer(MacKenzie et al. 1997) and a protocol developed by us foruse on the QIAxtractor (Qiagen). Extracts from phloemscrapes of woody tissue could not be done using theQIAxtractor due to the precipitation of substances that

blocked the capture plate and a CH3:IAA extractionprocedure was used.

RT-PCR: Primers for the detection of malate dehydrogenase

(MDH) mRNA were used to determine the quality of theextracted RNA (Table 1). The PCR primers used to detectGLRaV-2, GLRaV-3, GVA and GFkV are given in Table1. The SuperScript III One-Step RT-PCR System

(Invitrogen) was used for detection viruses and MDHmRNA. The total reaction volume was 12.5 l for MDHmRNA and 20 l for each virus. After amplification, 8 L

of each PCR reaction was run on a 2% agarose gel in 05

Tris-borate-EDTA, stained with ethidium bromide andvisualised on a UV transilluminator.

RESULTS AND DISCUSSION

GLRaV-3 and GFkV were detected 6-7 weeks post-

inoculation in both Chardonnay and Shiraz at Sunraysia in

Development and validation of sampling strategies for the detection of endemic viruses of

Australian grapevines


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December 2006. GLRaV-3 was detected by ELISA and

PCR and GFkV was detected by PCR only. By January2007 positive results were obtained for GLRaV-2,-3 andGFKV at both sites.

The June and July/August 2007 season samples wereonly tested using PCR as there was not enough material to

perform ELISA testing as well. In the 2006/07 season

(November 2006-August 2007) no uninoculated grapevinehas tested positive for GLRaV-2, GLRaV-3, GVA or GFkVat each site. GVA was not detected in any of the 10inoculated grapevines at each site. In most months at bothsites, more positive results were obtained by RT-PCR than

by ELISA, particularly for GLRaV-2 and GFkV. At eachsite 20 of the 30 Shiraz and Chardonnay grapevines thatwere inoculated with either GLRaV-2, GLRaV-3 or GFkV

tested positive for virus by RT-PCR and/or ELISA.

Table 1. Primers used in PCR and RT-PCR for detection of housekeeping

genes of grapevines and GLRaV-2, GLRaV-3, GVA and GFkV.

Pathogen Primer name Reference

RNA house-keeping gene

- malate dehydrogenase

MDH-H968Nassuth et al. 2000.

MDHC1163

Grapevine leafroll

associated virus 2

(GLRaV-2)

V2dCPf2 senseBertazzon and Angelini

2004V2CPr1 antisense

Grapevine leafroll

associated virus 3

(GLRaV-3)

P3U/

Turturo et al 2005P3D

Grapevine virus A (GVA)H587 Minafra and Hadidi

1994C995

Grapevine fleck virus

(GFkV)

GFkV-U279 Sabanadzovic et al

2001GFkV-L630

In the 2007/08 season (October 2007- August 2008) no

uninoculated grapevine tested positive for GLRaV-2,GLRaV-3, GVA or GFkV at each site. GVA was still notdetected in any of the 10 inoculated grapevines at each site.

In most months, at both sites, more positive results forGLRaV-2, GLRaV-3 and GFkV were obtained by RT-PCRthan by ELISA. October was the least reliable month for

virus testing by ELISA and RT-PCR.

At Sunraysia, in 2007/08, 27 of the 30 Shiraz and

Chardonnay grapevines that were inoculated with eitherGLRaV-2, GLRaV-3 or GFkV tested positive for virus byRT-PCR and ELISA. More grapevines tested positive forvirus by RT-PCR in February, March and May than in any

other month by RT-PCR or ELISA. All the chardonnay andShiraz grapevines inoculated with GLRaV2 and GLRaV-3

tested positive in most months during the testing period. AllShiraz grapevines and 2/5 Chardonnay grapevinesinoculated with GFkV also tested positive in most months.

At the Yarra Valley, in 2007/08, 29 of the 30 Shirazand Chardonnay grapevines that were inoculated with either

GLRaV-2, GLRaV-3 or GFkV tested positive for virus byRT-PCR and ELISA. More grapevines tested positive forvirus by RT-PCR in December and January than in anyother month by RT-PCR or ELISA All GLRaV-2 and

GLRaV-3 inoculated Chardonnay and Shiraz grapevinestested positive in most months during the testing period. All

Shiraz grapevines and 4/5 Chardonnay grapevinesinoculated with GFkV also tested positive in most months.GVA has not been detected.

It was also observed that the GLRaV-2 and GFkVELISAs were slow to develop a positive reaction comparedwith the GLRaV-3 ELISA. For the GLRaV-3 ELISA, clear

positive results were observed after three hours of platedevelopment. For the GLRaV-2 ELISA clear positiveresults were often only obtained after overnightdevelopment of the plates. A similar result was also

observed for the GFkV ELISA, especially later in theseason, particularly from January to June.

In conclusion, the results of this study show that RT-PCR is more sensitive and reliable for virus detection

compared to ELISA for Australian grapevines. In Australia,grapevine viruses can be detected reliably as early asDecember in each growing season and green tissue can beused.

LITERATURE

BERTAZZON N., ANGELINI E. 2004. Advances in the detection of

grapevine leafroll associated virus 2 variants. Journal of Plant

Pathology, 86, 283-290

CHEN J.J., LIU C.H., GU,Q.S., PAN X., CAO, Z.Y. 2003. Comparativestudies on DAS-ELISA, RT-PCR and IC-RT-PCR for detecting

grapevine leafroll-associated virus-3. Journal of Fruit Science, 20,

173-177.

COHEN D., VAN DEN BRINK R., HABILI, N. 2003. Leafroll virus

movement in newly infected grapevine. Extended abstract, 14 th

Meeting of the ICVG, Locorotondo, 2003. p39

LING K.S. ZHU H.Y. PETROVIC, N., GONSALVES, D. 2001.

Comparative effectiveness of ELISA and RT-PCR for detecting

grapevine leafroll-associated closterovirus-3 in field samples.

American Journal of Enology and Viticulture, 52, 21-27.

MACKENZIE D.J., MCLEAN M.A., MUKERJI S., GREEN M. 1997.

Improved RNA extraction from woody plants for the detection of

viral pathogens by reverse transcription-polymerase chain reaction.Plant Disease, 81, 222-226.

MINAFRA A., HADIDI, A. 1994. Sensitive detection of grapevine virus

A, B, or leafroll-associated III from viruliferous mealybugs and

infected tissue by cDNA amplification. Journal of Virological

Methods, 47, 175187.

NASSUTH A., POLLARI E., HELMECZY K., STEWART S.,

KOFALVI S.A. 2000. Improved RNA extraction and one-tube RT-

PCR assay for simultaneous detection of control plant RNA plus

several viruses in plant extracts.Journal of Virological Methods, 90,

3749

ROWHANI A., UYEMOTO J.K., GOLINO, D.A. 1997. A comparison

between serological and biological assays in detecting grapevine

leafroll associated viruses. Plant Disease, 81, 799-80.

SABANADZOVIC S., ABOU GHANEM-SABANADZOVIC N.,

SALDARELLI P., MARTELLI G. P. 2001. Complete nucleotidesequence and genome organization of Grapevine fleck virus. Journal

of General Virology, 82, 2009-2015.

STEWART S., NASSUTH, A. 2001. RT-PCR based detection of

Rupestris stem pitting associated virus within field-grown

grapevines throughout the year. Plant Disease, 85, 617-620.

TURTURO C., SALDARELLI P., YAFENG D., DIGIARO M.,

MINAFRA A., SAVINO V., MARTELLI, G.P. 2005. Genetic

variability and population structure of Grapevine leafroll-associated

virus 3 isolates.Journal of General Virology, 86, 217224.

ACKNOWLEDGEMENTS

This research is funded by the Grape and Wine Research and

Development Corporation and DPI Victoria. The authors wish to thank the

Australian Vine Improvement Association, the Victorian and Murray

Valley Vine Improvement Association and Orths Nursery for their

assistance in establishing the field trials. The authors would also like to

thank Julian Connellan (DPI NSW) Cathy Taylor (DPI, Vic) and Tony

Bass (SARDI) and Nuredin Habili (Waite Diagnostics) for their assistance

and advice.


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Fiona CONSTABLE1*, Phil NICHOLAS2, Narelle NANCARROW1 and Brendan RODONI1

1Department of Primary Industries, Knoxfield, Private Bag 15, Ferntree Gully Delivery Centre, Victoria 3156, Australia.2South Australian Research and Development Institute, Loxton Research Centre Loxton South Australia 5333, Australia.

*Corresp. author :Fiona Constable, Telephone : +61 3 920 9222, Fax : +61 3 9800 3521, Email: [email protected]

Summary

We are currently conducting a project to improve diagnostictesting procedures for endemic and exotic grapevine viruses of

Australian grapevines. The aim of the project is to increase thesensitivity for detection of the viral and some bacterial pathogensof grapevines that are tested for during post entry quarantine andwithin certification schemes in Australia. Prior to their use as the

standard protocols for Australian diagnostic laboratories and

which are accepted worldwide, the methods we are developingwill be validated by surveying key grape growing regions inAustralia.

INTRODUCTION

More than 50 viruses have been reported to infectgrapevines worldwide (Martelli 2003) and 11 of those havebeen reported in Australian grapevines. Consequently,Australia suffers few of the serious virus associateddiseases that affect grapevines in other countries. The 11grapevine viruses reported to infect Australian grapevines

include Grapevine leafroll associated virus (GLRaV-) 1,GLRaV-2, GLRaV-3, GLRaV-4, GLRaV-5 and GLRaV-9,

Grapevine virus A (GVA) , Grapevine virus B (GVB)Grapevine rupestris stem pitting associated virus(GRSPaV), Grapevine fleck virus (GFkV) and Grapevine

fanleaf virus (GFLV) (Krake et al. 1999; Habili andRowhani 2002). Although GFLV has been reported in

Australia, it was contained in the Rutherglen region and isstill considered a quarantineable pathogen (Krake et al.1999). GVB is not associated with corky bark disease inAustralia and the disease is considered quarantineable.

Grapevine material is imported into Australia mainlyas canes or, less frequently, as tissue cultured plantlets. Thismaterial is propagated and maintained in quarantinefacilities operated by the Australian Quarantine and

Inspection Service (AQIS), whilst in quarantine the materialundergoes mandatory testing for a range of diseases thathave not been reported or have been contained and

eradicated in Australia. Testing is not required for thegrapevine viruses known to occur in Australia, althoughimporters can choose to test for these for a fee.

During post entry quarantine (PEQ), importedgrapevines are inspected weekly for symptoms of bacterial,

fungal, phytoplasma and viral diseases and additionaltesting is done if symptoms are observed. The primarydiseases that are actively tested for include degeneration

and decline associated with nepoviruses and corky bark

disease. Currently testing for nepoviruses is done viabiological indexing using the herbaceous indicators

Chenopodium quinoa and Cucumus sativus (cucumber) andcorky bark disease testing is done by biological indexingusing the sensitive indicator LN33. Imported material isalso grown and observed for unusual symptoms and if

necessary electron microscopy, ELISA or PCR techniquesare used to confirm the presence of virus associated with

any symptoms observed on the indicators or importedgrapevines.

The AQIS PEQ process can take 2-3 years and if noexotic diseases or pathogen are detected the material can

legally enter Australia. Over time the number of grapevineviruses has increased and not all of the reported viruses areactively tested for in PEQ. As a consequence it is possiblethat some undetected grapevine viruses have entered

Australia.

After entry into Australia grapevine material may enter

certification schemes, which provide high health plantingmaterial to the viticultural industry. These schemes requirereliable, efficient diagnostic protocols, which are cost

effective, for routine pathogen testing for a range ofimportant bacteria, fungi, phytoplasmas and viruses that areknown to occur in Australia.

We are currently developing improved diagnostic

testing procedures for endemic and exotic grapevine virusesof Australian grapevines. Before incorporation intoAustralian quarantine protocols and certification schemes itis vitally important that the diagnostic tests be validated

under Australian conditions. To complete the validation ofthe diagnostic test, we are conducting an Australia widesurvey for exotic and endemic viruses of Australian

grapevines. The purpose of the survey is two-fold: (i) toupdate the disease status for each pathogen and (ii) to testprotocols under local conditions and identify any

potential false positives or organisms that can makeinterpretation of results difficult.

MATERIAL AND METHODS

Sampling: Sampling began in December 2008 and will

be completed by May 2009. Four shoots, ca. 50 cm longand with leaves attached, were collected from the majoreach grapevine. More than 200 grapevines will be sampled

from grape growing regions across Australia. Oldergrapevines will be selected in preference to more recentimportations as these are less likely to have come through

certification schemes which actively test for endemicgrapevine viruses. Diseased grapevines were selected inpreference to healthy grapevines.

Nucleic Acid extraction : Total RNA is extracted fromgreen grapevine tissue using a modified lysis buffer

(MacKenzie et al. 1997) and a protocol developed by us foruse on the QIAxtractor (Qiagen).

RT-PCR & PCR: Primers for the detection of malatedehydrogenase (MDH) mRNA are used to determine the

A survey for endemic and exotic viruses of Australian grapevines


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quality of the extracted RNA prior to virus testing (Nassuth

et al. 2000).

A literature search was conducted to identify the most

appropriate PCR primers for virus detection of endemicviruses and viruses of quarantineable significanceincluding: GLRaV-1-7 and -9, GVA, GVB, GVD, RSPaV,

GFKV, GRGV nepoviruses and tombusviruses known to

infect grapevines. Where possible bioinformatic analysis ofpathogen sequences were used to support the selection ofspecific PCR primer pairs. The primers were compared tothe aligned sequences of strains of each pathogen todetermine their specificity and chance of success in

detecting all strains. Specific tests for most viruses andsome generic tests for virus genera or groups have beenselected for assessment and validation in the survey.

The SuperScript III One-Step RT-PCR System(Invitrogen) are used for detection viruses and MDH

mRNA. The total reaction volume is 12.5 l for MDHmRNA and 20 l for each virus. PCR is done in 20 l

reactions using Platinum Taq (Invitrogen). Afteramplification, 8 L of each PCR reaction was run on a

2% agarose gel in 05 Tris-borate-EDTA, stained withethidium bromide andvisualised on a UV transilluminator.

RESULTS AND DISCUSSION

During development of RT-PCR tests for endemicgrapevine viruses we assessed a number of published RT-

PCR tests for the detection of endemic grapevine virusesagainst a range of grapevines found to be infected withthese viruses using one or more of WI, ELISA or RT-PCR

or which were suspected of having a virus infection. Thereis variation in detection by the different RT-PCR assays for

GLRaV-1, -2, -3 -5, GVA, and GVB and some RT-PCRassays for each virus do not detect each virus strain. Ourresults have also allowed us to confirm infection ofgrapevines by strains of GVA, GLRaV-2 and GLRaV-3

that were not previously detected by biological indexing,ELISA or other PCR tests. For GLRaV-1 two RT-PCR testsare required to detect all known Australian isolates.

The results of the survey for endemic and exoticgrapevine viruses will be used to update the disease status

for each pathogen and determine area freedom withinAustralia. It will also indicate the extent of grapevineviruses in Australia. The most accurate of the validated tests

for each virus will be recommended for incorporation intoPEQ protocols and grapevine certification schemes toensure that Australia is using the most up to date and

reliable methods. These methods will assist in limiting theentry of exotic viruses into Australia by providing AQISwith accurate tools for virus detection. Development ofrapid diagnostics for exotic pathogens will shorten post

entry quarantine periods and offer quicker access of newgermplasm/varieties to industry. Rapid PEQ diagnosticswill improve preparedness in case of an incursion of an

exotic grapevine pathogen. The Australian viticultureindustry will benefit through the production of high healthmaterial that has been reliably tested for the endemicpathogens that have a significant negative impact on thequality of fruit for wine production. A direct outcome ofthis project is a worlds best practice diagnostic capability

that will support PEQ and grapevine certification schemes.

LITERATURE

HABILI N., ROWHANI A. 2002. First detection of a new virus,

Grapevine leafroll-associated virus type 9, in a popular clone of

Cabernet Sauvignon in Australia. The Australian and New Zealand

Grape grower and Winemaker 30th Annual Technical Issue 461a,

102-103.

KRAKE L.R., STEEL-SCOTT N., REZAIAN M.A., TAYLOR R.H.1999. Graft Transmitted Diseases of Grapevines. CSIRO Publishing,

Collingwood, Victoria.

MACKENZIE D.J., MCLEAN M.A., MUKERJI S., GREEN M. 1997.Improved RNA extraction from woody plants for the detection of

viral pathogens by reverse transcription-polymerase chain reaction.Plant Disease, 81, 222-226.

NASSUTH A., POLLARI E., HELMECZY K., STEWART S.,KOFALVI S.A. 2000. Improved RNA extraction and one-tube RT-

PCR assay for simultaneous detection of control plant RNA plus

several viruses in plant extracts.Journal of Virological Methods, 90,

ACKNOWLEDGEMENTS

This research is funded by the Grape and Wine Research and

Development Corporation and DPI Victoria. The authors wish to thank the

Australian Vine Improvement Association and the Australian viticultural

industry in general for assisting us with this survey. The authors would

also like to thank Julian Connellan (DPI NSW) Tony Gerlach (SARDI)

and Tony Bass (SARDI) and Nuredin Habili (Waite Diagnostics) for theirassistance and advice.


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