Pseudomonas syringae

Transcrição

National Institute for Agrarian and Veterinary Research (INIAV)
(www.iniav.pt)
National Reference Laboratory for Plant Health
ILab Main activities
1.00
UW175
UW070
UW167
UW9
P3 332
R633
LB1
NCPPB3987
Ant307
BS025
PD1939
BR855
UW224
BR629
UW344
CPBF833
UW298
CPBF915
UW505
PD1100
JT516
CPBF568
CPBF785
PD 1941
CPBF850
CPBF894
CPBF933
CPBF836
BR113
CPBF914
BS048
UW365
UW51
WP306
BS024
UW448
MAFF301556
UW152
CPBF923
PD441
CPBF869
UW296
CPBF917
CPBF793
CPBF759
UW72
AW
UW134
ICMP7963
K60
CFBP2047
ICMP9600
CPBF1192
CPBF674
BS095
UW469
DAR64836
CFBP715
CFBP712
BR574
CFBP2958
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)
1.00
0.99
2
Phyl
bhr c
Bacterial canker of kiwi caused by
Pseudomonas syringae pv.
actinidiae in Portugal – Disease
Importance and pathogen
characterization
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
22/04/2013
Integrativa e Funcional (BioFiG);
4Universidade do Porto, Faculdades de Ciências, Campo Alegre, Porto.
[email protected]
EAP Meeting - Copenhagen
23 October 2015
 Symptoms
Leaves
Branches
4
 Symptoms
Flowers
Main risk factors in the area
 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
5
 Bacterial canker of kiwi
Major and minor hosts
Actinidia chinensis
(summer kiwi,
chinese kiwi)
Actinidia deliciosa
(kiwi, Chinese
gooseberry)
Actinidia arguta
Actinidia kolomikta
Bacteria Proteobacteria Gama-Proteobacteria
Pseudomonadales
Pseudomonadaceae
Pseudomonas sp.
Classified in to 4 biovars based on phenotipic and genomic characteristics
6
 Portuguese situation
Reynaud (2011)
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
Progression of P. syringae pv. actinidiae infections in Latina
(Vanneste et al. 2011b). (circulo = 1km)
7
 Incidence
03/2010 – First identification (Santa Maria da Feira), in plant material
imported sent by Direcção Regional de Agricultura do Norte (DRAPN)
2010-2012
Orchards
neg
pos
TOTAL
44
Hayward
16
Bo Erica
4
Ciften
2
Summer kiwi
1
Tumori P1
3
p.e. D1
0
nd
18
Soreli
0
Belen
0
Tsechelidis
0
8
Nurseries
freq. rel neg
pos
42
48,84
156
20
55,56
45
0
0,00
15
2
50,00
0
2
66,67
0
2
40,00
38
0
0,00
7
16
47,06
7
0
0,00
25
0
0,00
12
0
0,00
4
freq. Rel
6
3,70
1
2,17
0
0,00
0
0,00
0
0,00
2
5,00
3
30,00
0
0,00
0
0,00
0
0,00
0
0,00
 Portuguese situation
9
 Sampling
 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
10
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’)
11
IIdentification
PCR Scor
760 bp
PCR Gal
492 bp
230 bp
12
PCR Gal
Biovar
symptoms
esculin
coronatin
phaseolotoxin
PCR Scor
1
ramos/folhas
-
-
+
+
2
2
ramos/folhas
-
+
-
+
2
3
ramos/folhas
-
-
-
+
2
4
folhas
+
-
-
+
1
(bands)
L1 – 1Kb plus
L2 – Psa 1365
L3 – Psa 1366
L4 – Psa 1367
L5 – Psa 1368
L6 – Psa 1369
L7 – Psa 1370
L8 – Psa 1371
L9 – Psa 1372
L10 – 1Kb plus
L11 – Psa 1373
L12 – Psa 1374
L13 – Psa 1375
L14 – Psa 1416
L15 – Psa 1377
L16 – Psa 1393
L17 – Psa 1395
L18 – Psa 1396
L19– 1Kb plus
IGenomic characterization
13
Bv1 Bv2 Bv3
(Cunty et al., 2015
Bv4
IGenomic characterization
70
69
72
69
70
83
64
67
71
75
76
96
58
2012
Year of
2013
2013
2013
Geographical Origin
V
Arouca
2013
Box
Oliveira da Bairro
2013
2013
1376
Cantanhede
2013
Ponte de Lima
1450
Águeda
2013
CPB
1447
Póvoa do Lanhoso
2013
2013
1449
Amares
2013
Ponte de Lima
1434
Amares
2013
1431
1438
Amares
2013
1432
1440
Póvoa do Lanhoso
2013
2013
1439
Amares
2013
Póvoa do Lanhoso
1435
Felgueiras
2013
1433
1437
Felgueiras
2013
2013
1417
Felgueiras
2013
2013
1421
Felgueiras
2013
Fafe
1418
Felgueiras
2013
1430
1419
Celorico de Bastos
2013
2013
1420
Celorico de Bastos
2013
Vagos
1422
Felgueiras
2013
Penafiel
1423
Felgueiras
2013
1452
1412
Felgueiras
2013
2013
1415
Felgueiras
2013
Penafiel
1411
Guimarães
2013
1427
1414
Guimarães
2013
1425
1397
Vila Verde
2013
2013
1398
Vila Verde
2013
Vila Verde
1407
Braga
2013
1428
1410
Vila Verde
2013
2013
1406
Maia
2013
2013
1408
Gondomar
2013
Vila Verde
1399
Maia
2013
1429
1402
Gondomar
2012
2013
1400
Braga
2012
Cantanhede
1401
Felgueiras
2012
Oliveira da Bairro
1405
Rio Tinto
2012
1445
1413
Anadia
2012
1446
1362
Maia
2012
2013
1364
Oliveira do Bairro
2012
Anadia
1368
Santa Maria da Feira
2012
1444
1370
Castelo de Paiva
2012
2013
1367
Castelo de Paiva
Amarante
1366
Oliveira do Bairro
2013
1441
1365
2012
Celorico de Bastos
1369
Arouca
2012
1424
1374
Felgueiras
2012
95
1416
Castelo de Paiva
72
100
58
51
75
87
72
94
1372
77
75
80
74
96
91
67
60
59
88
2012
Maia
2013
Arouca
Arouca
2013
1377
1375
Barcelos
2012
1373
1395
Valongo
2011
2011
1393
Castelo de Paiva
1325
2011
1327
2011
2011
2011
2010
1326
1305
2011
1322
1329
2011
2012
1330
1442
1360
1359
Braga
Anadia
Amarante
Amarante
Vila Meã
2013
2012
2013
2013
2013
2012
2012
2011
1443
Amarante
1331
1356
1332
Lousada
1328
Santa Maria da Feira
1371
59
59
55
70
59
80
68
92
77
71
64
61
91
85
62
68
68
1436
Guimarães
Presence of strains from “biovar 4”
box_todas (82 entries)
74
85
1357
492 bp
230 bp
Lack of the specific band
for the 84 strains of
P. syringae pv. actinidiae
tested
C+ - Pst (CFBP 2212)
PCR Gal
Coronatin production
(Cfl) (Bereswill et al.,
1994)
MW 1305 1322 1325 1326 1327 C- Pst
Box
86
1396
14
Box A1R
Lows et al. (1994)
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)
15
IResearch
EUPHRESCO II - European Phytossanitary Research Coordination II
Partner
Country
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
16
I Phylogenetic Characterization
Italy
1st Focus – 1992
2nd Focus – 2008
Estirpes com características
fisiológicas (produção de
faseolotoxina e coronatina) e
genómicas distintas (genes
constitutivos e de virulência)
Biovares 1, 2 e 3
Portugal
1st Identification – 2010
Identification of the bacterium in
orchards with more than 10 years
Sequenciação de 1 isolado
Português obtido em 2010
87
Biovar 4
CFBP2212 Pseudomonas syringae pv. tomato
0.002
17
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
ICMP 19439; Biovar 3; Chile; 2010
98 CPBF 1406; Portugal; 2013
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 19071; Biovar 2; Korea; 1997
ICMP 19440; Biovar 4; Australia; 2011
ICMP 18882; Biovar 4; New Zealand; 2010
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
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.
Pst type strain
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.
18
Obrigada!
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

Pseudomonas syringae

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