National Institute for Agrarian and Veterinary Research (INIAV) · 2018. 5. 11. · UW51 WP306...

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


CPBF568CPBF785

PD 1941CPBF850

CPBF894CPBF933

CPBF836BR113



CPBF759UW72



BS095UW469


BR574CFBP2958

0.99

1.00

1.00

Phyl

bhr c

UW175UW070

UW167UW9P3 332R633LB1

NCPPB3987Ant307

BS025PD1939BR855UW224BR629


CPBF568CPBF785

PD 1941CPBF850

CPBF894CPBF933

CPBF836BR113



CPBF759UW72

UW175UW070

UW167UW9P3 332R633LB1

NCPPB3987Ant307

BS025PD1939BR855UW224BR629


CPBF568CPBF785

PD 1941CPBF850

CPBF894CPBF933

CPBF836BR113



CPBF759UW72



BS095UW469


BR574CFBP2958

0.99

1.00



BS095UW469


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.

[email protected]

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!

National Institute for Agrarian and Veterinary Research (INIAV) · 2018. 5. 11. · UW51 WP306...

Documents

Transcript of National Institute for Agrarian and Veterinary Research (INIAV) · 2018. 5. 11. · UW51 WP306...