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

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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

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Phytoplasm as and viruses m anagem ent in Grapevine Collections 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

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

W orking group- ARNADI A

CAV - Faenza/ Am pelos CNR-I VV Bari CNR-I VV Grugliasco CRA-PAV Rom a CRA-VI T Conegliano CRSA - Locorotondo ERSA - FVG I ASMA – San Michele all’Adige I PAD - Lodi UNI BO – University of Bologna UNI MI - University of Milano UNI PI - University of Pisa VCR – Vivai Cooperativi Rauscedo

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)

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Phytoplasm as and viruses m anagem ent in Grapevine Collections 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?

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 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

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Phytoplasm as and viruses m anagem ent in Grapevine Collections 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 3 GLRaV 3 GLRaV 2 GLRaV 2 ArMV ArMV GFLV GFLV GFkV GFkV GVA GVA GVB GVB EU EU

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

METHODS UNDER VALI DATI ON

ELI SA: to detect the viral coat protein

COAT PROTEI N NUCLEI C ACI D ( RNA)

VI RUS STRUCTURE VI RUS STRUCTURE

RT-PCR: to detect the viral nucleic acid

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

SAMPLI NG

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 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

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

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

ELI SA

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

buffe r buff er

G F E D C B

Buff er Buff er

A 12 11 10 9 8 7 6 5 4 3 2 1

ELI SA PLATE SCHEME

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

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)

MULTI PLEX RT-PCR

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Phytoplasm as and viruses m anagem ent in Grapevine Collections 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

3 5 healthy ( not target) 1 0 2 reference sam ples 6 7 infected ( target) 1 8 rootstocks 1 7 varieties 1 2 rootstocks 5 5 varieties

Plus 2 0 pool sam ples com posed of 5 plants( 1 infected + 4 healthy)

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Sam ples list ( 1 2 2 ) and their sanitary status

1 GLRaV 1 + GLRaV 2 + GLRaV 3 + GVA+ GFkV 2 GVA 3 GVB 4 SANO 5 SANO 6 SANO 7 GLRaV3 + GVA 8 GFLV 9 GFkV 1 0 POOL DA 4 2 ( GLRaV1 , GVA,GFLV) 1 1 GLRaV 3 , GFLV 1 2 GVA, GLRAV1 , GLRaV3 1 3 ArMV, GFLV, GLRaV1 1 4 GLRaV-1 1 5 POOL DA 1 0 7 ( GLRaV1 ) 1 6 sano 1 7 POOL DA 1 0 6 ( GFKV) 1 8 ArMV 1 9 ArMV ( GRSPaV) 2 0 GVA, GRLaV2 , GLRaV3 2 1 GLRaV-3 + GVA 2 2 GLRaV-1 , GFkV, GFLV 2 3 SANO 2 4 SANO 2 5 GVA + GFLV 2 6 GLRaV-1 GLRaV-3 GVA 2 7 POOL DA 9 3 ( GFLV) 2 8 GFkV 2 9 sano 3 0 GLRaV 3 3 1 GVA 3 2 SANO 3 3 sano 3 4 SANO 3 5 ArMV, GFLV, GFKV, GLRaV1 , GVA 3 6 POOL DA 6 3 ( GLRaV2 -3 , GVA) 3 7 GLRaV 2 + GFkV 3 8 POOL DA 6 6 ( GLRaV2 , GLRaV3 ,GVA) 3 9 GLRaV- 1 4 0 ArMV 4 1 GVA – GFkV – GLRaV- 3 4 2 GFLV + GLRaV1 + GVA 4 3 POOL SANO 4 4 GLRaV1 4 5 GLRaV3 4 6 SANO 4 7 SANO 4 8 POOL DA 1 8 ( ArMV) 4 9 SANO 5 0 GLRaV 3 , GFkV, GFLV 5 1 SANO 5 2 sano 5 3 POOL DA 4 4 ( GLRaV1 ) 5 4 SANO 5 5 GVA + GFkV + GLRaV-3 + GLRaV2 5 6 GFLV 5 7 GLRaV3 5 8 SANO 5 9 GVA+ GLRaV1 + GFLV 6 0 sano 6 1 sano 6 2 Sano 6 3 GLRaV 2 + GLRaV 3 + GVA 6 4 POOL DA 1 2 ( GVA,GLRaV1 -3 ) 6 5 SANO 6 6 GLRaV 2 , GLRaV 3 GVA 6 7 SANO 6 8 GLRaV 2 ( isolato BD) 6 9 GFLV – GFkV – GLRaV-3 7 0 GFLV 7 1 GVA – GFkV – GLRaV-3 7 2 GFkV+ GLRaV 2 + GLRaV 3 + GVA 7 3 GFkV 7 4 POOL DA 1 ( GLRaV1 -2 - 3 ,GVA,GFkV) 7 5 ArMV, GFKV 7 6 GLRaV2 GLRaV3 7 7 GFkV + GLRaV2 7 8 GFLV, GFKV 7 9 GLRaV3 8 0 GLRaV1 8 1 SANO 8 2 Sano 8 3 SANO POOL 8 4 SANO 8 5 sano 8 6 Sano 8 7 GVA, GRLaV2 , GLRaV1 8 8 GLRaV2 ( isolato BD) 8 9 SANO 9 0 GLRaV-1 e GVA 9 1 GFLV, GLRaV 3 9 2 GLRaV 1 -3 e GVA 9 3 GFLV 9 4 SANO 9 5 ArMV 9 6 GVA 9 7 SANO 9 8 sano 9 9 sano 1 0 0 SANO 1 0 1 SANO 1 0 2 GFLV – GFkV – GLRaV-3 1 0 3 GLRaV 2 ( isolato BD) 1 0 4 GFkV, GLRaV3 1 0 5 GFKV 1 0 6 GFkV 1 0 7 GLRaV1 1 0 8 GVA 1 0 9 sano 1 1 0 SANO 1 1 1 GLRaV 3 + GVA 1 1 2 GLRaV 3 , GVA, GFLV 1 1 3 GFKV 1 1 4 GLRaV 2 ( isolato RG) 1 1 5 POOL DA 7 0 ( GFLV) 1 1 6 POOL DA 1 1 2 ( GLRaV3 , GVA, GFLV) 1 1 7 POOL GFLV 1 1 8 POOL DA 7 2 ( GFKV, GLRaV2 - 3 ,GVA) 1 1 9 POOL DA 9 5 ( ArMV) 1 2 0 POOL DA 2 5 ( GVA,GFLV) 1 2 1 POOL DA 7 1 ( GVA, GFKV,GLRaV3 1 2 2 POOL DA 1 1 3 ( GFKV)

All sam ples w ere analyzed in blind

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

For the protocol validation has been calculated the follow ing pa For the protocol validation has been calculated the follow ing pa ram eters, according to ram eters, according to UNI / CEI / EN I SO/ EC 1 7 0 2 5 and 1 6 1 4 0 UNI / CEI / EN I SO/ EC 1 7 0 2 5 and 1 6 1 4 0 – – EPPO EPPO – – Diagnostics PM7 / 7 6 and PM7 / 9 8 : Diagnostics PM7 / 7 6 and PM7 / 9 8 :

Diagnostic sensitivity: ability of the method used to detect the presence of the pathogen

in the samples surely infected by the pathogen in question - true positive

Diagnostic specificity: ability of the method used to NOT detect the presence of the

pathogen in samples not infected by the pathogen in question - true negative

Accuracy: the average of diagnostic sensitivity and specificity
Analytical sensitivity: the smallest amount of infectious entities that can be identified by

the diagnostic method

Repeatability or accordance: degree of conformity of the results obtained in replications
f the method, made at short intervals of time, using the same reference sample and in the

same working conditions i.e. equipment, operator, laboratory

Reproducibility or concordance: degree of conformity of the results obtained using the

same method with the same reference samples in different laboratories

VALI DATI ON PARAMETERS

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

PERFORMANCE CRI TERI A

Obtained negative / expected negative ( negative agreem ent)

D B

Obtained negative / expected positive ( negative deviation) Obtained positive / expected negative ( positive deviation)

C A

Obtained positive / expected positive ( positive agreem ent)

% SENSI TI VI TY: A/ ( A+ B) % SPECI FI CI TY: D/ ( C+ D) % ACCURACY: A + D/ ( A+ B+ C+ D) Analytical sensitivity: the sm allest am ount of infectious entities that can be identified by diagnostic m ethod ( in the case of plant viruses, w hich cannot be quantified in vitro, corresponds to dilution lim it of initial extract in w hich, the used m ethod, is able to identify the pathogen

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

For the REPEATABI LI TY or ACCORDANCE has been chosen 4 targets ( 3 infected and one healthy) and m ade tw o dilutions. The sam ples w ere analyzed by the sam e person, w ith the sam e reagents, three tim es on the sam e day. The values w ere calculated by checking how m any tim es the sam e result w as repeated regardless of w hether they w ere infected or not % REPEATABI LI TY or ACCORDANCE C/ N C = concordant results N = total sam ples For REPRODUCI BI LI TY or CONCORDANCE has been applied the sam e m ethod

repeatability,

that analyses w ere perform ed in different laboratories, using the sam e reagents, the sam e protocol and the sam e standards. % REPRODUCI BI LI TY or CONCORDANCE ΣC/ ΣN SC = sum m ation of concordant results for each sam ples SN = sum m ation of num ber of laboratories that analyzed the sam e sam ple

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

I NTERPRETATI ON OF READI NGS W I TH PHOTOMETER

Background ( A) = average of the values of the absorbance of the negative controls ( healthy and blank - m ax 0 .2 OD) Threshold ( B) = A x 2 ,5 ( I f this value w as greater than or equal to 0 .1 OD, otherw ise the threshold value w ill be equal to 0 .1 OD) . Reading of sam ple: ≥ B = positive Reading of sam ple: < B = negative I n the event that the tw o replicas w ere not both above or below the threshold B, the sam ple w as considered doubtful and analyzed again, using the sam e hom ogenate, w hen stored in the refrigerator w ithin 4 8 hours of its preparation,

therw ise a new extract.

ELI SA RESULTS EVALUATI ON

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

ELI SA - OBTAI NED RESULTS

GLRaV1 GLRaV2 GLRaV3 GFKV ArMV GFLV GVA GVB 89 86 81 85 64 75 74 86 97 100 100 100 85 96 100 100 93 90 84 88 74 80 80 92 100 94 100 100 100 100 95 100 92 88 98 92 94 87 92 100 85 32 66 72 64 65 73 nt 100 95 94 100 94 100 100 nt 89 59 75 84 81 79 81 nt 100 94 100 100 100 100 95 nt AGRITEST Reproducibility Sensitivity pool Specificity pool Accurancy pool Repeatability pool Accurancy Repeatability Parameter Sensitivity Specificity

GLRaV1 GLRaV2 GLRaV3 GFKV ArMV GFLV GVA

GLRaV 1+3 ArMV+GFLV

94 67 90 90 48 82 45 84 88 100 100 100 100 95 92 100 100 62 96 77 92 92 71 84 58 90 82 100 92 100 100 100 100 100 100 100 91 82 91 86 93 91 73 92 93 61 38 97 47 42 90 30 81 79 100 100 100 100 100 93 75 94 62 73 64 98 70 76 91 43 85 73 100 86 94 100 100 86 86 100 86 Repeatability pool Parameter BIOREBA Repeatability Reproducibility Sensitivity Specificity Accurancy Sensitivity pool Specificity pool Accurancy pool

GLRaV1 GLRaV2 GLRaV3 GFKV ArMV GFLV GVA

ArMV+GFLV

96 87 97 30 50 77 87 67 100 100 100 100 96 92 96 67 98 91 97 46 72 81 89 67 100 92 100 95 100 100 100 96 92 82 92 88 96 91 88 74 77 21 100 42 43 79 82 47 100 95 93 100 97 94 100 67 84 53 98 67 74 85 87 53 100 92 100 95 100 100 100 83 Specificity Accurancy SEDIAG Parameter Sensitivity Accurancy pool Repeatability pool Repeatability Reproducibility* Sensitivity pool Specificity pool

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

ELI SA – Som e considerations arising from the

analysis of the results of different param eters

Method 1 : Use of plastic bags and hom ogenizer Method 2 : Use of m ortar and pestle w ith or w ithout liquid nitrogen Method 3 : Use of m illing m achines

Extraction m ethods Tim e reading of the results

I t w as not possible to establish an optim um tim e for reading regardless viruses and antisera. I t seem s to be absolutely dependent only from the laboratory. Method 1 resulted less sensitive ( 5 -8 % ) of m ethod 2 and 3 in detection of GFLV, ArMV and ( 2 -4 % ) GVA. Method 2 and 3 resulted equivalent betw een them

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

Rootstocks

No difference highlighted for GLRaV 1 , 2 , 3 and GFkV betw een the sam ples of European varieties and rootstocks. Sm all and not alw ays statistically significant differences ( in negative for rootstocks) for ArMV, GVA and GFLV. No difference betw een the different Com panies for the sam e antiserum ,

Pool sam ples

Generally good results by the pool sam ples. Surely the accuracy w as found to be low er ( 1 0 -1 5 percentage points) for GLRaV 1 , GLRaV 2 and GFkV com pared to individual sam ples. No statistically significant difference for others.

Antisera com parison

All Antisera behaved absolutely equivalent in the diagnosis of GLRaV 1 , 2 , 3 , GFLV, ArMV. Only tw o antisera ( GFkV of Sediag and GVA of Bioreba) have given results less valid than the respective antisera of other Com panies. Good results w ere obtained by m ixed antisera ( GLRaV 1 + 3 and GFLV + ArMV) by Bioreba, w hile m ixed antiserum GFLV + ArMV by Sediag proved less perform ant.

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

9 0 % 1 0 0 % 9 5 % 9 8 % 8 9 % TOTAL 9 95 5% % 100% 100% 10 10 -

9 95 5% % 9 95 5% % 95 95% % GFkV 76 76% % 100% 100% 10 10 -

90 90% % 100% 100% 68 68% % GFLV 10 100% 0% 100% 100% 10 10 -

98 98% % 99 99% % 92 92% % ArMV 10 100% 0% 100% 100% 10 10 -

100% 100% 100% 100% 100% 100% GVB 94 94% % 100% 100% 10 10 -

98 98% % 99 99% % 96 96% % GVA 100% 100% 100% 100% 10 10 -

95 95% % 93 93% % 100% 100% GLRaV-3 8 83 3% % 95 95% % 10 10 -

85 85% % 98 98% % 84 84% % GLRaV-2 70 70% % 100% 100% 10 10 -

9 94 4% % 100% 100% 74 74% % GLRaV-1

Reproducibility Repeatability Analytical sensitivity Accuracy Specificity Sensitivity VI RUS/ PARAMETER

MULTI PLEX RT-PCR - OBTAI NED RESULTS

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

MULTI PLEX RT-PCR

results evaluation w ith regards to: Extraction m ethods

Method 1 : Silica extraction Method 2 : CTAB extraction Method 3 : McKenzie ( 1 9 9 7 ) + Com m ercial KI T ( RNeasy m ini plant Kit – Qiagen) THE THREE METHODS resulted equivalent am ong them , w e suggest the use of the METHOD 3 since is foresees the use of a com m ercial KI T, giving m ore assurances about the standardization of the m ethodology

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Phytoplasm as and viruses m anagem ent in Grapevine Collections for Germ plasm Conservation, Mobilization and Evaluation Sofia 8 -9 May 2 0 1 2

89% 99% 10-1 87/86/86 98/99/97% 81/75/77% ELISA

Virus Diagnostic protocol Sensitivity Specificity Accuracy Analytical sensitivity Repeatability Reproducibility ArMV Multiplex RT-PCR

92 % 99 % 98 % 10-2 100% 100 %

ELISA – A/B/S

64/48/50% 85/95/96% 74/72/72% 10-2 100% 95%

GFLV Multiplex RT-PCR/

68 % 100% 90 % 10-3 100% 76%

ELISA – A/B/S

75/82/77% 96/92/92% 80/84/81% 10-2 100% 90%

GFkV Multiplex RT-PCR

95% 95% 95% 10-2 100% 95%

ELISA – A/B/S

90/90/30% 100% 92/92/46% 10-1 98% 88%

GVA Multiplex RT-PCR

96 % 99 % 98 % 10-2 100% 94 %

ELISA – A/B/S

77/45/87% 100/100/96% 83/58/89% 10-1 98% 82%

GVB Multiplex RT-PCR

100% 100% 100% 10-2 100% 100%

ELISA – A/B/S

86% 100% 92% 100 (2-2) 100% 85%

GLRaV 1 Multiplex RT-PCR

74 % 100 % 94 % 10-2 100% 70 %

ELISA – A/B/S

89/94/96% 100% 93/96/980% 10-2 100% 92%

GLRaV 2 Multiplex RT-PCR

84% 98% 85% 10-2 95% 83%

ELISA – A/B/S

86/67/87% 100% 93/96/98% 100 (2-2) 93% 84%

GLRaV 3 Multiplex RT-PCR

100 % 93 % 95 % 10-3 100% 100 %

ELISA – A/B/S

81/90/97% 100% 84/92/97% 10-3 100% 94%

MULTIPLEX RT-PCR

89% 98% 95% 10-2 100% 90%

MOLECULAR vs ELI SA

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REFERENCE SAMPLES COLLECTI ON

GLRaV 3,GVA, GVB ELISA 28/ 2007 23 GLRaV 3,GVA, GVB ELISA 17/ 2007 22 GLRaV 2 66 MLI 63 P8 21 GLRaV 2 85 ALB 027 20 GVB

camp. 31635

17 GVB

camp. 31637

16 GFKV 157/ 11 15 GFKV 161/ 049 14 GVA, GFkV,GLRaV 3 Pizzutella 2 13 sano 1103 Paulsen P.38 12 GFLV Traminer 920 vm 11 ArMV Muller Th. 8013 10 ArMV Pinot nero 189 9 GFLV Traminer 921 vm 8 GFLV Gold Traminer 7 GLRaV 3 Gold Traminer 6 GLRaV 1 Pinot nero 5 sano P6/ K-S 4 sano P4/ K-S 3 GLRaV 3 Sagrantino 2 GLRaV 1 Sagrantino 1 Sanitary status Variety/ origin Ref. GLRaV 3 147/ 19/ 1 Madelaine Vialette 44 GLRaV 2, GFkV 4/ 9/ 2 Vitis Coignetiae 43 GVA 152/ 11/ 2 Corazon de Angel 42 GFLV 1/ 7/ 2 Riparia Baron 41 GLRaV 3, GVA 151/ 14/ 5 Cereza 40 GLRaV 2 145/ 20/ 3 Red Globe 39 GLRav 1, GFLV, GVA 142/ 19/ 4 Terra Promessa 38 GLRaV 2, GLRaV3, GVA 2/ 9/ 4 riparia Scribner 37 sano Neg 13 36 sano Neg 8 35 GFkV Sangiovese Ceppo G2 34 ArMV, GFKV Riesling 33 GLRav 1, GFLV, ArMV varieta europea 32 GVB Albarossa 31 GVA Moscato 30 30 GFkV Nebbiolo 185 29 ArMV Berla Grossa 28 GLRaV 2 Pecorello 27 GLRaV 1 Scimiscià 26 sano Piedirosso 4-19-034 25 sano Piedirosso 4-19-019 24 Sanitary status Variety/ origin Ref.

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CONCLUSI ON

For the first tim e are available harm onized and

validated reference diagnostic protocols for the m ain grapevine viruses

The efficiency and robustness of the protocols has

been proved using a large num ber of sam ples in a high num ber of labs

For the first tim e a reference sam ples collection

( targets and not targets) has been established

The use of these diagnostic tolls w ill im prove the

quality of grapevine germ plasm for collections, for m obilization or for sanitary selection purposes

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CRA - Centro di Ricerca per la Patologia Vegetale CRA - Plant Pathology Research Center

Francesco Faggioli Francesco Faggioli

e e-

mail:

mail: francesco.faggioli@entecra.it francesco.faggioli@entecra.it

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