VALI DATI ON OF DI AGNOSTI C VALI DATI ON OF DI AGNOSTI C PROTOCOLS - PowerPoint PPT Presentation

Phytoplasm as and viruses m anagem ent in Grapevine Collections ACTI ON FA0 8 0 7 ACTI ON FA1 0 0 3 for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2 VALI DATI ON OF DI AGNOSTI C VALI DATI ON OF DI AGNOSTI C PROTOCOLS FOR THE DETECTI ON OF PROTOCOLS FOR THE DETECTI ON OF GRAPEVI NE VI RUSES COVERED BY GRAPEVI NE VI RUSES COVERED BY PHYTOSANI TARY RULES PHYTOSANI TARY RULES F. Faggioli, F. Anaclerio, E. Angelini, G. Bianchi, P. A. Bianco, M. Cardoni, P. Casati, R. Credi, E. De Luca, G. Durante, C. Gianinazzi, G. Gam bino, A. Luvisi, U. Malossini, F. Mannini, P. Saldarelli, F. Terlizzi, E. Triolo, N. Trisciuzzi 1

Phytoplasm as and viruses m anagem ent in Grapevine Collections ACTI ON FA0 8 0 7 ACTI ON FA1 0 0 3 for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2 W orking group- ARNADI A CAV - Faenza/ Am pelos I ASMA – San Michele all’Adige CNR-I VV Bari I PAD - Lodi CNR-I VV Grugliasco UNI BO – University of Bologna CRA-PAV Rom a UNI MI - University of Milano CRA-VI T Conegliano UNI PI - University of Pisa CRSA - Locorotondo VCR – Vivai Cooperativi Rauscedo ERSA - FVG REGI ONAL PHYTOSANI TARY SERVI CES I NVOLVED I N THE I NTER-LABORATORY RI NG TEST SFR Trentino ( Laim burg) SFR Em ilia Rom agna SFR Lom bardia SFR Valle d’Aosta SFR Friuli Venezia Giulia ( ERSA) 2

Phytoplasm as and viruses m anagem ent in Grapevine Collections ACTI ON FA0 8 0 7 ACTI ON FA1 0 0 3 for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2 W HAT’S THE MEANI NG OF THE W ORK DONE? A protocol validation is the evaluation of a process to determ ine its fitness for a particular use. A validated assay yields test results that identify the presence of a specific target The aim of W G has been to produce validated reference protocols allow ing for the harm onization of the diagnosis of the selected grapevine viruses. The choice of the protocols to validate has been m ade taking into consideration their reliability, efficiency, tim e consum ing, cost effective and large scale use 3

Phytoplasm as and viruses m anagem ent in Grapevine Collections ACTI ON FA0 8 0 7 ACTI ON FA1 0 0 3 for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2 SELECTED TARGETS ( VI RUSES)  Pathological im portance  Diffusion  I nclusion in phytosanitary rules ( EU and I taly)  Availability of large scale diagnostic tools I n this view has been selected eight grapevine viruses: GLRaV 1 GLRaV 1 GLRaV 2 GLRaV 2 GFkV GFkV GLRaV 3 EU GVA GLRaV 3 EU GVA ArMV ArMV GVB GVB GFLV GFLV 4

Phytoplasm as and viruses m anagem ent in Grapevine Collections ACTI ON FA0 8 0 7 ACTI ON FA1 0 0 3 for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2 METHODS UNDER VALI DATI ON COAT PROTEI N VI RUS STRUCTURE VI RUS STRUCTURE NUCLEI C ACI D ( RNA) ELI SA: to detect the viral coat protein RT-PCR: to detect the viral nucleic acid 5

Phytoplasm as and viruses m anagem ent in Grapevine Collections ACTI ON FA0 8 0 7 ACTI ON FA1 0 0 3 for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2 SAMPLI NG The vegetal m atrix to use in all the protocols is the scraped phloem tissue obtained from w oody m aterial collected during the w inter Period: all the dorm ant season Source of m aterial: lignified shoots one year old Type of sam ple: 2 w oody portions collected from the basal part of the shoots. The w oody sam ples m ust be intact and m ust not show changes due to abiotic or biotic factors Maintenance of the sam ple: The plant m aterial m ust be dry, m ust be placed in plastic bags to be stored at low tem peratures or in a cool place such as to avoid possible dehydration Traceability of the sam ple: Each sam ple m ust be properly signed on the envelope and on the plant Shipm ent of sam ples: The sam ples m ust reach the laboratory w ithin 7 2 hours, preferably in cool bags 6

Phytoplasm as and viruses m anagem ent in Grapevine Collections ACTI ON FA0 8 0 7 ACTI ON FA1 0 0 3 for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2 ELI SA Antisera from : Agritest ( 8 ) , Bioreba ( 1 0 ) , Sediag ( 9 ) Viruses: GLRaV 1 , 2 , 3 , GVA, GVB,GFLV, ArMV, GFkV, GLRaV 1 + 3 , ArMV+ GFLV The tests w ere conducted follow ing all instructions provided by the Com panies for each antiserum Moreover, com parative tests of extraction w ere perform ed for all antisera, using the follow ing m ethods: 1 . Use of plastic bags and hom ogenizer 2 . Use of m ortar and pestle w ith or w ithout liquid nitrogen 3 . Use of m illing m achines 7

Phytoplasm as and viruses m anagem ent in Grapevine Collections ACTI ON FA0 8 0 7 ACTI ON FA1 0 0 3 for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2 ELI SA PLATE SCHEME 1 2 3 4 5 6 7 8 9 10 11 12 A Buff Buff er er B C D E F G buff buffe er r H 8

Phytoplasm as and viruses m anagem ent in Grapevine Collections ACTI ON FA0 8 0 7 ACTI ON FA1 0 0 3 for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2 MULTI PLEX RT-PCR Ref. Gambino G. and Gribaudo I. 2006. Phytopathology 96 (11): 1223-1229) Prim ers: GLRaV 1 , 2 , 3 , GVA, GVB,GFLV, ArMV, GFkV, 1 8 S ic The tests w ere conducted follow ing the above reported protocol w ith som e m odifications ( ie ArMV prim ers) Moreover, also in this case, com parative tests of extraction w ere perform ed, using the follow ing m ethods: 1 . Silica extraction 2 . CTAB extraction 3 . McKenzie ( 1 9 9 7 ) + Com m ercial KI T ( RNeasy m ini plant Kit – Qiagen) 9

Phytoplasm as and viruses m anagem ent in Grapevine Collections ACTI ON FA0 8 0 7 ACTI ON FA1 0 0 3 for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2 SAMPLES UTI LI ZED I N THE VALI DATI ON Phloem tissue obtained from the bark 1 0 2 reference sam ples 6 7 infected 3 5 healthy ( target) ( not target) 5 5 varieties 1 2 rootstocks 1 7 varieties 1 8 rootstocks Plus 2 0 pool sam ples com posed of 5 plants( 1 infected + 4 healthy) 10

Sam ples list ( 1 2 2 ) and their sanitary status 1 GLRaV 1 + GLRaV 2 + GLRaV 3 + 4 6 SANO 9 1 GFLV, GLRaV 3 GVA+ GFkV 4 7 SANO 9 2 GLRaV 1 -3 e GVA 4 8 POOL DA 1 8 ( ArMV) 2 GVA 9 3 GFLV 4 9 SANO 3 GVB 9 4 SANO 5 0 GLRaV 3 , GFkV, GFLV 4 SANO 9 5 ArMV 5 1 SANO 5 SANO 9 6 GVA 5 2 sano 6 SANO 9 7 SANO 5 3 POOL DA 4 4 ( GLRaV1 ) 7 GLRaV3 + GVA 9 8 sano 5 4 SANO 8 GFLV 9 9 sano 5 5 GVA + GFkV + GLRaV-3 + GLRaV2 9 GFkV 1 0 0 SANO 5 6 GFLV 1 0 POOL DA 4 2 ( GLRaV1 , GVA,GFLV) 1 0 1 SANO 5 7 GLRaV3 1 1 GLRaV 3 , GFLV 1 0 2 GFLV – GFkV – GLRaV-3 5 8 SANO 1 2 GVA, GLRAV1 , GLRaV3 1 0 3 GLRaV 2 ( isolato BD) 5 9 GVA+ GLRaV1 + GFLV 1 3 ArMV, GFLV, GLRaV1 1 0 4 GFkV, GLRaV3 6 0 sano 1 4 GLRaV-1 1 0 5 GFKV 6 1 sano 1 5 POOL DA 1 0 7 ( GLRaV1 ) 1 0 6 GFkV 6 2 Sano 1 6 sano 1 0 7 GLRaV1 6 3 GLRaV 2 + GLRaV 3 + GVA 1 7 POOL DA 1 0 6 ( GFKV) 1 0 8 GVA 1 8 ArMV 6 4 POOL DA 1 2 ( GVA,GLRaV1 -3 ) 1 0 9 sano 6 5 SANO 1 9 ArMV ( GRSPaV) 1 1 0 SANO 6 6 GLRaV 2 , GLRaV 3 GVA 2 0 GVA, GRLaV2 , GLRaV3 1 1 1 GLRaV 3 + GVA 6 7 SANO 2 1 GLRaV-3 + GVA 1 1 2 GLRaV 3 , GVA, GFLV 2 2 GLRaV-1 , GFkV, GFLV 6 8 GLRaV 2 ( isolato BD) 1 1 3 GFKV 6 9 GFLV – GFkV – GLRaV-3 2 3 SANO 1 1 4 GLRaV 2 ( isolato RG) 7 0 GFLV 2 4 SANO 1 1 5 POOL DA 7 0 ( GFLV) 7 1 GVA – GFkV – GLRaV-3 2 5 GVA + GFLV 1 1 6 POOL DA 1 1 2 ( GLRaV3 , GVA, GFLV) 2 6 GLRaV-1 GLRaV-3 GVA 7 2 GFkV+ GLRaV 2 + GLRaV 3 + GVA 1 1 7 POOL GFLV 7 3 GFkV 2 7 POOL DA 9 3 ( GFLV) 1 1 8 POOL DA 7 2 ( GFKV, GLRaV2 - 7 4 POOL DA 1 ( GLRaV1 -2 - 2 8 GFkV 3 ,GVA) 3 ,GVA,GFkV) 2 9 sano 1 1 9 POOL DA 9 5 ( ArMV) 7 5 ArMV, GFKV 3 0 GLRaV 3 1 2 0 POOL DA 2 5 ( GVA,GFLV) 7 6 GLRaV2 GLRaV3 3 1 GVA 1 2 1 POOL DA 7 1 ( GVA, GFKV,GLRaV3 7 7 GFkV + GLRaV2 3 2 SANO 1 2 2 POOL DA 1 1 3 ( GFKV) 7 8 GFLV, GFKV 3 3 sano 3 4 SANO 7 9 GLRaV3 3 5 ArMV, GFLV, GFKV, GLRaV1 , GVA 8 0 GLRaV1 3 6 POOL DA 6 3 ( GLRaV2 -3 , GVA) 8 1 SANO 3 7 GLRaV 2 + GFkV 8 2 Sano 3 8 POOL DA 6 6 ( GLRaV2 , GLRaV3 ,GVA) All sam ples w ere 8 3 SANO POOL 3 9 GLRaV- 1 8 4 SANO 4 0 ArMV 8 5 sano analyzed in blind 4 1 GVA – GFkV – GLRaV- 3 8 6 Sano 4 2 GFLV + GLRaV1 + GVA 8 7 GVA, GRLaV2 , GLRaV1 4 3 POOL SANO 11 4 4 GLRaV1 8 8 GLRaV2 ( isolato BD) 4 5 GLRaV3 8 9 SANO 9 0 GLRaV-1 e GVA