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 phytoplasma (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
leafhopper Scaphoideus titanus S. titanus; Zeisner/AGES
Other leafhoppers harbouring FDp are
Dictyophara europaea
D. europaea; Strauss/AGES
Orientus ishidae
www.ages.at Reisenzein, AGES Orientus 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 insects 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 Austria in the years 2012 and 2013. for the presence of FDp in Austria, Total sample number/Number Latin name Locality of FD+ tested Switzerland, Slovenia, Italy, Spain Herbaceous plants Clematis vitalba 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 Carpinus betulus 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 Dictyophara 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 France); 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