IOBC meeting, 2015 Vienna

Epidemiological studies on flavescence dorée (FD)

Helga Reisenzein, AGES

Euphresco Partners: G.Pasquini, CRA-PAV, G. Strauß, AGES, S. Schaerer, CH-ACW, A. Bertaccini, DISTA Patologia Vegetale, B. Bagnoli, E. Gargani, CRA-ABP, M. Dermastia, N. Mehle, NIB, A.Batlle, IRTA, E. Angelini, CRA-VIT Centre for Research in Viticulture

www.ages.at Austrian Agency for Health and Food Safety Current state of knowledge

HOSTS

Main hosts for FD (FDp) are

Vitis vinifera and Vitis riparia

Known wild species infected by FDp are

Clematis vitalba Alnus sp. Ailanthus altissima

www.ages.at 2 Reisenzein, AGES Current state of knowledge

VECTORS

The only known natural vector of FDp is

Scaphoideus titanus S. titanus; Zeisner/AGES

Other harbouring FDp are

Dictyophara europaea

D. europaea; Strauss/AGES

Orientus ishidae

www.ages.at Reisenzein, AGES ishidae; Strauss/AGES 3 Current state of knowledge

FDp STRAINS

16Sr V

Map gene : FD 1, FD 2 and FD 3

16S gene : FD-D, FD-C, FD-70…. FD-D FD-C STOL

www.ages.at Reisenzein, AGES 4 ERA-NET EUPHRESCO-Project:

Epidemiological studies on reservoir hosts and potential vectors of Grapevine flavescence doree (FD) and validation of different diagnostic procedures for FD (GRAFDEPI)

Work package 2: Epidemiological studies

Graziella Pasquini, CRA-PAV Santiago Schaerer, CH-ACW Bruno Bagnoli, Elisabetta Gargani, CRA-ABP Elisa Angelini, CRA-VIT

Marina Dermastia, NIB

Assunta Bertaccini, University of Bologna Helga Reisenzei, Gudrun Strauß, AGES

www.ages.at Assumpició Batlle, IRTA 5 Insights in

New possible New potential Prevalence of reservoir host vectors of FD FD strains plants of FDp

www.ages.at Reisenzein, AGES 6 Methodological approach

Outbreak investigations

Surveillance, risk communica- tion Hypothesis on trans- mission Environ- ment analysis Testing in the lab

Field- monitoring

Guidelines based on the CDC (American Centers for Human Disease Control) – adapted for FD

www.ages.at Methodological approach

Case investigations

• FDp strain characterization of selected samples (different grape samples, wild plants and commonly present inside or outside the vineyards)

• Molecular characterization of FDp isolates using different genes (MLST)

16S gene sec Y gene tuf gene

www.ages.at Reisenzein, AGES 8 Results from transnational work – step 3: Environment analysis

Reservoir host plants of FDp

Different wild plants were tested Table: Wild plants and grapevines examined for the presence of FDp at different sites in in the years 2012 and 2013. for the presence of FDp in Austria, Total sample number/Number Latin name Locality of FD+ tested , Slovenia, , Herbaceous plants 3/3 Glanz Clematis vitalba 0/2 Bairisch Kölldorf Convolvulus sepium 0/1 Bairisch Kölldorf Convolvulus arvensis 0/1 Glanz Urica dioica 0/1 Glanz Stachys sylvatica 0/1 Glanz Salvia glutinosa 0/1 Glanz Persicaria maculosa 0/1 Bairisch Kölldorf Shrubs New reservoirRosa canina host 0/3 Bairisch Kölldorf Euonymus sp. 0/2 Bairisch Kölldorf Salix sp. 0/2 Bairisch Kölldorf plant speciesCorylus could avellana 0/1 Bairisch Kölldorf Ligustrum vugare 0/1 Bairisch Kölldorf Rubus sectio Rubus 0/1 Bairisch Kölldorf not be detectedRubus sectio Rubus 0/2 Glanz Sorbus aucuparia 0/1 Glanz Cornus sanguinea 0/5 Glanz Sambucus nigra 0/1 Glanz Trees Alnus glutinosa 1/1 Glanz Alnus glutinosa 3/3 Bairisch Kölldorf Acer campestre 0/1 Glanz Prunus avium 0/1 Glanz Fraxinus excelsior 0/1 Glanz Fraxinus excelsior 0/1 Bairisch Kölldorf 0/1 Bairisch Kölldorf Ailanthus altissima 0/6 Bairisch Kölldorf Quercus petraea 0/2 Bairisch Kölldorf www.ages.at Juglans regia 0/1 Bairisch Kölldorf 9 Results from transnational work – step 3: Environment analysis

Reservoir host plants of FDp

Results from Slovenia, Italy, Austria,

Clematis vitalba Alnus glutinosa Ailanthus altissima

picture: Follak/AGES

Country Infection FDp- Country Infection FDp-Strains Country Infection FDp- rate Strain rate rate Strains SLO 60% FD-C SLO 90% FD-C, FD-D, SLO 5% FD-C, FD-70, ALY FD-D AT 60% FD-C AT 100% FD-C AT 0% IT 22% FD-C

www.ages.at 10 Results from transnational work – step 3: Environment analysis

Vectors

Results from Austria, Switzerland, Slovenia, Italy, Spain

1. Scaphoideus titanus (AT, IT, SLO, CH – 0 to 8 % infection rates)

2. Orientus ishidae

Country Infection rate FDp-Strain

SLO 62% FD-C, FD-D, FD-70, ALY AT 47% FD-C

CH 20% -

IT 0 -

3. Oncopsis alni

Country Infection rate FDp-Strains SLO 50% FD-C, FD-D, FD-70, ALY

www.ages.at 11 Results from transnational work – step 3: Environment analysis

Vectors

Results from Austria

4. Phlogotettix cyclops

5. Psylla alni

www.ages.at 12 Results from transnational work – step 3: Environment analysis

Vectors

What about europaea ?

Country Numberof Number of caught FDp positive individuals Slovenia 2 0

Austria 2 0

Switzerland 0 0

Italy 5 0

Spain 16 0

• D. europaea could be rarley found in SLO, AT, CH and IT, frequently in Spain • D. europaea did not harbor FDp

www.ages.at 13 Case investigations

Results of molecular characterization of the FD strains (IT)

Varieties 16S SecY Gene 2012 Gene Tru1I TaqI AluI TaqI Albarola FD-C c+d c c Albarola FD-C c - - Chardonnay FD-C - - - Chardonnay FD-C d d c Ciliegiolo FD-C d d c Pollera P. FD-C - - - Pollera P. FD-C d d c Pollera P. FD-C d d c Vermentino FD-C d d c Vermentino FD-C d d c

-, amplification negative d, RFLP profile referable to strains belonging to FD-D c, RFLP profile referable to strains belonging to FD-C

www.ages.at 14 Case investigations

Distribution map of FDp strains hosted in grapevines

www.ages.at 15 Austrian situation and results Team: Strauß, G., Altenburger, J., Berger N., Suarez-Mahecha, B. Reisenzein, H.

4 established focus and saftey zones

First finding FD: 2009 Established populations of S. titanus

www.ages.atFirst finding S. titanus: 2004 16 Potential new vectors of FDp

Phlogotettix cyclops

picture: Altenburger/AGES

Psylla alni

picture: Strauss/AGES www.ages.at 17 Preliminary results of strain typing

Psylla allni

FDp detection: real time PCR; (Christensen et al. 2004, Angelini et al. 2007) Strain typing: Direct generic PCR followed by nested generic PCR (16r758f/M23Sr primer); RFLP analysis with Taq I restriction enzyme

Psylla alni Vitis v. Alnus incana.

16SrV-C 33% infection rate 16SrV-C 16SrV-D

. Lanes: 100bp ladder, 1 to 9 Psylla alni ; 10 V. vinifera cv. Sämling 88 (isolate from Austria); 11 grapevine (FD 2 reference isolate from ); 12 Alnus incana (isolate form Austria) www.ages.at Reisenzein, H., 18.3.2014 18 Preliminary results of strain typing

Phlogottetix cyclops

FDp detection: real time PCR; (Christensen et al. 2004, Angelini et al. 2007) Strain typing: Direct generic PCR followed by nested generic PCR (16r758f/M23Sr primer); RFLP analysis with Taq I restriction enzyme

Phlogottetix cyclops Vitis v.

16SrV-C 32% infection rate 16SrV-D

. Lanes: 100bp ladder, 1 to 8 Phlogottetix clyclops ; 9 – V. vinifera cv. Sämling 88 (isolate from Austria); 10 grapevine (FD 2 reference isolate from France); www.ages.at Reisenzein, H., 18.3.2014 19 Hypothesis on transmission mechanisms and dynamics of FD

2011

2010 2012

2011 2011 2011

www.ages.at ARZ-GFD-Zagreb/March, 2013, Reisenzein 20 Hypothesis on transmission mechanisms and dynamics of FD

Monophagous species are responsible for the spread of FDp strains within alder trees Oncopsis alni

FD- C, D, 70

Psylla alni Natural epidemiological cylce

FD- C, D, 70 FD-C

Alder trees

www.ages.at Hypothesis on transmission mechanisms and dynamics of FD

O. ishidae Clematis plants

FD- FD-C C, D, 70 Polyphagous species are Alder trees responsible for the spread P. cyclops FD- C, D, of FDp strains within 70 FD-C

reservoir host plants Tree of heaven

D. europaea

FD- C, D, 70 FD-C www.ages.at Natural epidemiological clycles brancing to a cultivated plant

Single affected Vitis plants O. ishidae plants

P. cyclops Coincidental presence of S.titanus – a start off for an epidemic disease outbreak Natural host reservoir for FDp S. titanus

Grapevines Grapevines Grapevines D. europaea Epidemic

www.ages.at outbreak 23 Hypothesis on transmission mechanisms and dynamics of FD

Infected P. cyclops on clematis plants 2011

All isolates (grape, 2010 2012 insects, alder, 2011 clematis) harbored 2011 FD-C 2011 strains Infected O. ishidae on alder trees

www.ages.at ARZ-GFD-Zagreb/March, 2013, Reisenzein 24 www.ages.at www.ages.at Institute for Plant Health 25 Results from transmission trial with S. titanus, A. glutinosaas FD infection source and grapevine (cv. Scheurebe)

Figure 1: Transmission trial set-up, Plexiglas cylinder with Alnus glutinosatwigs in water bottles and potted grapevines (cv. Scheurebe).

www.ages.at 26 Results from transmission trial with S. titanus, A. glutinosaas FD infection source and grapevine (cv. Scheurebe)

FDp status of potted tested number of FDp positive replicate grapevines (cv. S. titanus S. titanus Scheurebe) cylinder 1 3 1 adult negativ cylinder 2 16 0 negative cylinder 3 5 0 negativ cylinder 4 5 1 larva negativ cylinder 5 13 2 adults Positive zylinder 6 12 0 Negativ In total: 54 3 adults 2 positive grapevines 1 larva

www.ages.at Institute for Plant Health 27