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08 IOBC Grafdepi__Reisenzein 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. glutinosa as FD infection source and grapevine (cv. Scheurebe) Figure 1: Transmission trial set-up, Plexiglas cylinder with Alnus glutinosa twigs in water bottles and potted grapevines (cv. Scheurebe). www.ages.at 26 Results from transmission trial with S. titanus , A. glutinosa as 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.
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