Post on 28-Feb-2021
National Institute for Agrarian and Veterinary Research (INIAV) (www.iniav.pt)
National Reference Laboratory for Plant Health
Phyl
bhr c
UW175UW070
UW167UW9P3 332R633LB1
NCPPB3987Ant307
BS025PD1939BR855UW224BR629
UW344CPBF833UW298CPBF915UW505PD1100JT516
CPBF568CPBF785
PD 1941CPBF850
CPBF894CPBF933
CPBF836BR113
CPBF914BS048UW365UW51WP306
BS024UW448MAFF301556UW152CPBF923PD441CPBF869UW296CPBF917CPBF793
CPBF759UW72
AWUW134ICMP7963K60CFBP2047
ICMP9600CPBF1192CPBF674
BS095UW469
DAR64836CFBP715CFBP712
BR574CFBP2958
0.99
1.00
1.00
Phyl
bhr c
UW175UW070
UW167UW9P3 332R633LB1
NCPPB3987Ant307
BS025PD1939BR855UW224BR629
UW344CPBF833UW298CPBF915UW505PD1100JT516
CPBF568CPBF785
PD 1941CPBF850
CPBF894CPBF933
CPBF836BR113
CPBF914BS048UW365UW51WP306
BS024UW448MAFF301556UW152CPBF923PD441CPBF869UW296CPBF917CPBF793
CPBF759UW72
AWUW134ICMP7963K60CFBP2047
ICMP9600CPBF1192CPBF674
BS095UW469
DAR64836CFBP715CFBP712
BR574CFBP2958
0.99
1.00
1.00
Phyl
bhr c
UW175UW070
UW167UW9P3 332R633LB1
NCPPB3987Ant307
BS025PD1939BR855UW224BR629
UW344CPBF833UW298CPBF915UW505PD1100JT516
CPBF568CPBF785
PD 1941CPBF850
CPBF894CPBF933
CPBF836BR113
CPBF914BS048UW365UW51WP306
BS024UW448MAFF301556UW152CPBF923PD441CPBF869UW296CPBF917CPBF793
CPBF759UW72
UW175UW070
UW167UW9P3 332R633LB1
NCPPB3987Ant307
BS025PD1939BR855UW224BR629
UW344CPBF833UW298CPBF915UW505PD1100JT516
CPBF568CPBF785
PD 1941CPBF850
CPBF894CPBF933
CPBF836BR113
CPBF914BS048UW365UW51WP306
BS024UW448MAFF301556UW152CPBF923PD441CPBF869UW296CPBF917CPBF793
CPBF759UW72
AWUW134ICMP7963K60CFBP2047
ICMP9600CPBF1192CPBF674
BS095UW469
DAR64836CFBP715CFBP712
BR574CFBP2958
0.99
1.00
AWUW134ICMP7963K60CFBP2047
ICMP9600CPBF1192CPBF674
BS095UW469
DAR64836CFBP715CFBP712
BR574CFBP2958
0.99
1.00
1.00
2
ILab Main activities Research on quarantine and quality phytopathogenic
bacteria of national and regional interest (phenetic diversity, diagnosis and epidemiology)
Support to national phtyossanitary authority (definition of national control plans for quarantine bacteria, sampling procedures and diagnostics)
Analysis of Q-bacteria (Ralstonia solanacearum, Clavibacter michiganensis subsp. sepedonicus, Erwinia amylovora, Pseudomonas syringae pv. actinidiae, Xylella fastidiosa; Huanlonbing)
Scientific and technical suport to official national/regional laboratories (diagnostic procedures, training)
Dissemination of knowledge to stakeholders and regional inpectors (lectures and technical courses)
Support to community (Plant Clinics)
Bacterial canker of kiwi caused by Pseudomonas syringae pv.
actinidiae in Portugal – Disease Importance and pathogen
characterization
22/04/2013
EAP Meeting - Copenhagen 23 October 2015
Leonor Cruz1,3, Joana Cruz1,3, Camila Fernandes1,4, Gisela Chicau2, Rogério Tenreiro3 1 Instituto Nacional de Investigação Agrária e Veterinária, UEIS-SAFSV – Laboratório de Fitobacteriologia, Quinta do Marquês, Oeiras; 2 DRAPN –Divisão de Apoio ao Sector Agroalimentar, Senhora da Hora, Porto; 3Universidade de Lisboa, Faculdade de Ciências, Centro de Biodiversidade Genómica Integrativa e Funcional (BioFiG); 4Universidade do Porto, Faculdades de Ciências, Campo Alegre, Porto.
leonor.cruz@iniav.pt
4
Symptoms
Leaves
Branches
5
Flowers
Symptoms
Presence of high levels of
inoculum
High number of orchards
Presence of very susceptible
cultivars (A. chinensis)
Temperature >15ºC during
blooming
Presence of polinators
High HR
Main risk factors in the area
Actinidia chinensis (summer kiwi, chinese kiwi)
Actinidia deliciosa (kiwi, Chinese gooseberry)
Actinidia arguta
Actinidia kolomikta
6
Bacterial canker of kiwi
Major and minor hosts
Bacteria Proteobacteria Gama-Proteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas sp. Classified in to 4 biovars based on phenotipic and genomic characteristics
7
Portuguese situation
Reynaud (2011)
Progression of P. syringae pv. actinidiae infections in Latina (Vanneste et al. 2011b). (circulo = 1km)
EPPO PRA Portugal Balestra et al. 2010d: disease incidence as high as 30% was noted in 2010 and incidence has increased up to 80% in 2011 (Renzi et al., 2011).
ano 2010 2011 2012 2013
nº total 10 175 71 293
pos 6 15 30 140
% 60% 9% 42% 48%
Portuguese Control Plan P. syringae pv. actinidiae
8
03/2010 – First identification (Santa Maria da Feira), in plant material imported sent by Direcção Regional de Agricultura do Norte (DRAPN)
Incidence
2010-2012 Orchards Nurseries neg pos freq. rel neg pos freq. Rel
TOTAL 44 42 48,84 156 6 3,70 Hayward 16 20 55,56 45 1 2,17 Bo Erica 4 0 0,00 15 0 0,00 Ciften 2 2 50,00 0 0 0,00 Summer kiwi 1 2 66,67 0 0 0,00 Tumori P1 3 2 40,00 38 2 5,00 p.e. D1 0 0 0,00 7 3 30,00 nd 18 16 47,06 7 0 0,00 Soreli 0 0 0,00 25 0 0,00 Belen 0 0 0,00 12 0 0,00 Tsechelidis 0 0 0,00 4 0 0,00
9
Portuguese situation
10
Kiwi area in north Portugal – 1500ha
Comission Decision n.º 2012/756/EU
Definition of portuguese contaminated and disease free areas
Definition of a sampling strategy
National Control Plan - surveillance of orchards, nurseries and garden centres
Training courses for farmers and inspectors
Research
Sampling
11
IDiagnosis Samples from orchards, nurseries and imported propagation materials
aprox. 1000 (2010-2013) 84 selected isolates from North and Center regions Diagnosis - internal method based on OEPP PM7/120(1)
Extraction from leaves, branches, fruits and roots Isolation on KMB Conventional PCR Scortichini et al., 2002 or Rees-Gerge et al., 2010 PAV 1 GGCGACGATCCGTAACTGGTCTGAGA 760 bp P 22 TTCCCGAAGGCACTCCTCTATCTCTAAAG Gallelli et al., 2011 KN-F (5’ – CACGATACATGGGCTTATGC – 3’) 492 bp KN-R (5’ – CTTTTCATCCACACACTCCG – 3’) AvrDdpx-F (5’ – TTTCGGTGGTAACGTTGGCA – 3’) 230 bp AvrDdpx-R (5’ – TTCCGCTAGGTGAAAAATGGG – 3’)
12
IIdentification PCR Scor 760 bp
PCR Gal 492 bp 230 bp
Biovar symptoms esculin
coronatin
phaseolotoxin PCR Scor PCR Gal
(bands)
1 ramos/folhas - - + + 2
2 ramos/folhas - + - + 2
3 ramos/folhas - - - + 2
4 folhas + - - + 1
13
IGenomic characterization
L1 –
1Kb
plu
s L2
– P
sa 1
365
L3 –
Psa
136
6 L4
– P
sa 1
367
L5 –
Psa
136
8 L6
– P
sa 1
369
L7 –
Psa
137
0 L8
– P
sa 1
371
L9 –
Psa
137
2 L1
0 –
1Kb
plus
L1
1 –
Psa
1373
L1
2 –
Psa
1374
L1
3 –
Psa
1375
L1
4 –
Psa
1416
L1
5 –
Psa
1377
L1
6 –
Psa
1393
L1
7 –
Psa
1395
L1
8 –
Psa
1396
L1
9– 1
Kb p
lus
Bv1 Bv2 Bv3 Bv4
(Cunty et al., 2015
14
IGenomic characterization
VGeographical Origin
Ponte de Lima
Ponte de Lima
Póvoa do Lanhoso
Fafe
Vagos
Penafiel
Penafiel
Vila Verde
Vila Verde
Cantanhede
Oliveira da Bairro
Anadia
Amarante
Celorico de Bastos
Arouca
Oliveira da Bairro
Cantanhede
Águeda
Póvoa do Lanhoso
Amares
Amares
Amares
Póvoa do Lanhoso
Amares
Felgueiras
Felgueiras
Felgueiras
Felgueiras
Felgueiras
Celorico de Bastos
Celorico de Bastos
Felgueiras
Felgueiras
Felgueiras
Felgueiras
Guimarães
Guimarães
Vila Verde
Vila Verde
Braga
Vila Verde
Maia
Gondomar
Maia
Gondomar
Braga
Felgueiras
Rio Tinto
Anadia
Maia
Oliveira do Bairro
Santa Maria da Feira
Castelo de Paiva
Castelo de Paiva
Oliveira do Bairro
Arouca
Felgueiras
Castelo de Paiva
Maia
Arouca
Arouca
Barcelos
Valongo
Castelo de Paiva
Santa Maria da Feira
Lousada
Vila Meã
Amarante
Amarante
Anadia
Braga
Amarante
Guimarães
box_todas (82 entries)
96
91
85
67
64
58
96
91
95
74
94
80
60
62
72
75
75
67
70
77
76
72
80
59
87
75
61
71
69
69
51
58
59
68
100
88
59
59
70
64
68
72
55
77
7192
68
83
85
70
74
86
BoxBox
CPB
1431
1432
1433
1430
1452
1427
1425
1428
1429
1445
1446
1444
1441
1424
1376
1450
1447
1449
1434
1438
1440
1439
1435
1437
1417
1421
1418
1419
1420
1422
1423
1412
1415
1411
1414
1397
1398
1407
1410
1406
1408
1399
1402
1400
1401
1405
1413
1362
1364
1368
1370
1367
1366
1365
1369
1374
1416
1372
1377
1373
1375
1395
1393
1371
1325
1327
1326
1328
1305
1322
1329
1330
1332
1356
1331
1359
1360
1442
1443
1436
1357
1396
Year of
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2012
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2013
2012
2012
2012
2012
2012
2012
2012
2012
2012
2013
2012
2012
2012
2012
2013
2013
2012
2011
2011
2011
2011
2010
2011
2011
2011
2011
2012
2011
2012
2012
2013
2013
2013
2012
2013
Box A1R Lows et al. (1994)
MW 1305 1322 1325 1326 1327 C- Pst
Coronatin production (Cfl) (Bereswill et al., 1994)
Lack of the specific band for the 84 strains of P. syringae pv. actinidiae tested C+ - Pst (CFBP 2212)
PCR Gal 492 bp 230 bp
Presence of strains from “biovar 4”
15
IDiagnosis
Decision scheme 1 . Sample preparation Orchard sub-samples of male and female plants 2 – Screening tests Isolation on KMB and conventional PCR (Scortichini et al 2002; Rees-George et al., 2010) 3 – Identification of colonies by Galleli et al. (2011) 4 – Confirmation by Real - time PCR (Gallelli et al., 2013) (EUPHRESCO II –
PSADID)
16
IResearch EUPHRESCO II - European Phytossanitary Research Coordination II
Development and harmonization of methods for diagnosis, detection and identification of Pseudomonas syringae pv. actinidiae (2013-2015) Resultados previstos: Implementation of new tools for the diagnosis of Psa in symptomatic a
asymptomatic plant material Validation of a sampling protocol Epidemiological knowledge of Pseudomonas syringae pv. actinidiae in
distinct areas of Europe
Partner Country
17
I Phylogenetic Characterization ICMP 19073 Biovar 2; Korea; 1998
CPBF 1428; Portugal; 2013 CFBP 7811 Biovar 3; New Zealand; 2010 CFBP 7287; Biovar 3; Italy; 2008 CFBP 4909; Biovar 1; Japan; 1984 NCPPB 3871; Biovar 1; Italy; 1992 CFBP 8052; Biovar 3; France; 2012 CPBF 1439; Portugal; 2013 CPBF 1367; Portugal; 2012 CPBF 1447; Portugal; 2013 CPBF 1364; Portugal; 2012 CPBF 1433; Portugal; 2013 CPBF 1411; Portugal; 2013 CPBF 1400; Portugal; 2013 CPBF 1329; Portugal; 2011 CPBF 1366; Portugal; 2012 ICMP 19439; Biovar 3; Chile; 2010 ICMP 19455; Biovar 3; Chile; 2010 CPBF 1371; Portugal; 2012 CPBF 1406; Portugal; 2013 CPBF 1444; Portugal; 2013 CPBF 1442; Portugal; 2013 ICMP 19457; Biovar 3; Chile; 2010 ICMP 19456; Biovar 3; Chile; 2010 CRA-FRU 10.22; Biovar 3; Italy; 2008 ICMP 19076; Biovar 3; New Zealand; 2011 ICMP 19072; Biovar 2; Korea; 1997 ICMP 18743; Biovar 3; Italy; 2010 ICMP 9855; Biovar 1; Japan; 1984 ICMP 18744; Biovar 3; Italy; 2010 ICMP 18745; Biovar 3; Italy; 2010 ICMP 19071; Biovar 2; Korea; 1997 ICMP 19068; Biovar 1; Japan; 1988 ICMP 19070; Biovar 1; Japan; 1987 ICMP 19069; Biovar 1; Japan; 1984 CPBF 1414; Portugal; 2013 CPBF 1372; Portugal; 2012 CPBF 1422; Portugal; 2013 CPBF 1421; Portugal; 2013 CPBF 1326; Portugal; 2011
ICMP 19440; Biovar 4; Australia; 2011 ICMP 19441; Biovar 4; Australia; 2011 ICMP 18882; Biovar 4; New Zealand; 2010 ICMP 19486; Biovar 4; Australia; 1990 CFBP 7905; Biovar 4; New Zealand; 2010 CFBP 8043; Biovar 4; France; 2011 CFBP 8045; Biovar 4; Australia; 1990 CFBP 8046; Biovar 4; Australia 2011
CFBP2212 Pseudomonas syringae pv. tomato
87
98
0.002
rpoD Neighbor-Joining phylogenetic tree using MEGA5 of 19 selected Portuguese isolates and 30 selected g worldwide strains according to Parkinson et al. (2011). Bootstrap test (1000 replicates) are shown next to the branches. Evolutionary distances were computed using the Jukes-Cantor method.
Biovares 1, 2 e 3
Biovar 4
Pst type strain
Italy 1st Focus – 1992 2nd Focus – 2008 Estirpes com características fisiológicas (produção de faseolotoxina e coronatina) e genómicas distintas (genes constitutivos e de virulência) Portugal 1st Identification – 2010 Identification of the bacterium in orchards with more than 10 years Sequenciação de 1 isolado Português obtido em 2010
18
IConclusions
Between 2010 and 2013 more than 100 isolates of Pseudomonas syringae pv.
actinidiae were collected from orchards nurseries and imported propagation materials.
The use of two conventional PCR protocols allowed identifying all known biovars of Pseudomonas syringae pv. actinidiae.
The use of primers directed to genes responsible for the production of the toxins coronatin (Cfl) and/or phaseolotoxin (argK) allow to exclude the presence of biovar 2 among Portuguese strains.
BOX PCR fingrprinting profiles were characteristic of biovar 3 for most of the strains tested
The phylogenetic tree generated by rpoD confirms the association of the selected strains as belonging to biovar 3.
The lack of avrD1 amplification indicates the presence of a small population of biovar 4 strains alocated recently to Pseudomonas syringae pv. actinidifoliorum.
Instituto Nacional de Investigação Agrária e Veterinária, I.P. Av. da República, Quinta do Marquês, 2780-157 Oeiras, Portugal Tel: (+ 351) 21 440 35 00 - Fax: (+ 351) 21 440 36 66 www.iniav.pt
Obrigada!