Embed Size (px)
Citation preview
Marie-Agnès Jacques
Diversity of seed-borne bacteria and consequences for detection
strategies
Exclusion of the pathogens:
-Chemical treatments: hot water, chlorine treatment
Managment strategies for seed-borne diseases
Exclusion of the pathogens:
-Chemical treatments: hot water, chlorine treatment
-Seed health testing
Managment strategies for seed-borne diseases
Reliable seed health tests
Diversity of seed-borne bacteria and consequences for detection strategies
Specificity of Clavibacter michiganensis subsp. michiganensis detection methods and existence of look-alikes
Transmission of Cmm look-alikes to tomato seeds
Bacterial wilt and canker of tomatoClavibacter michiganensis subsp. michiganensis
– Actinobacteria• Microbacteriaceae
-Enters the plant by wounds and stomata-Gets into the xylem: systemic infection-Plugs the xylem vessels causing the wilting of the leaves-May affect growth of leaves and petioles-Induces canker on the stems
Seedborne pathogenInfected seeds = major inoculum source and the cause for Cmm outbreaks Quarantine pathogen in EU (A2)
CFBP 7559
CFBP 7572
CFBP 7508
CFBP 7478
CFBP 7311
NCPPB 1064
CFBP 1940
CFBP 5
CFBP 5842
CFBP 7318
CFBP 7471
CFBP 7567
CFBP 7568
CFBP 1465
CFBP 1714
CFBP 7487
CFBP 7488
CFBP 1462
CFBP 2108
CFBP 2500
CFBP 5843
CFBP 1460
CFBP 1461
CFBP 1463
CFBP 1464
NCPPB 382
CFBP 7569
CFBP 7574
CFBP 2495
CFBP 2496
CFBP 2497
CFBP 2499
CFBP 2501
CFBP 7506
CFBP 7158
CFBP 7594
CFBP 7597
CFBP 7313
CFBP 7562
CFBP 7578
CFBP 7312
CFBP 7507
CFBP 7551
CFBP 7310
CFBP 7316
CFBP 7309
CFBP 7314
CFBP 4999
CFBP 7317
NCPPB 2034
CFBP 7464
CFBP 7586
CFBP 7590
CFBP 7591
CFBP 7439
CFBP 7555
CFBP 7599
CFBP 7504
CFBP 2493
CFBP 2492
CFBP 2494
CFBP 2498
CFBP 6885
CFBP 7315
CFBP 7444
CFBP 7449
CFBP 7584
CFBP 7589
CFBP 7606
CFBP 3559
CFBP 2409
CFBP 3560
CFBP 7500
CFBP 7575
CFBP 7495
CFBP 7505
CFBP 7492
CFBP 6488
CFBP 6492
CFBP 2404
CFBP 7577
CFBP 3521
CFBP 2405
CFBP 7553
CFBP 7576
CFBP 3496
CFBP 3494
CFBP 3499
CFBP 807
100
85100
51100
100
100
96
83
53
100
99
100
53
44
50
93
70
99
100
100
84
58
34
48
69
33
71
62
61
35
24
76
43
16
100
41
95
26
25
94
62
31
4924
11
5226
90
76
36
3250
48
533
33
35
0.02
C. m. sepedonicus
C. m. tessellarius
Cmm Look-alikes
C. m. insidiosus
C. m. nebraskensis
C. m. michiganensis
Cmm Type strain, CFBP 499 T
Rathayibacter iranicus
Look-alikes of Cmm isolated from tomato
Jacques et al., 2012
Look-alikes- are clearly distinct from Cmm- form several phyla
atpD, dnaK, gyrB, recA, rpoB, ppkML, 1000 bootstraps
Characterization of Clavibacter-like strains isolated from tomato
Pathogenicity on tomato5 PCR-based identification tests IF: 2 antisera compared
Jacques et al., 2012
Characterization of Clavibacter-like strainsisolated from tomato
Nb of
strains
Pathogenicity
IF PCR-based identification
IF (PRI) IF (Loewe) ZTO Cmm F/R Psa PsaR/8 Ptssk
129 + + + + + + + +
11 +/- + + + + + + +
1 (+/-) + + + + + + +
1 - + + + + + - -
1 - + + + + - + -
2 - + + + + - - -
6 - + + + + trace - -
2 - + + - - trace + -
4 - + + - - - - -
4 - + + - - trace - -
2 - - + - - - - -
2 - - - + + - - -
32 - - - - - - - -
141 Cmm
56 avirulentStrains: look-alikes
(small) cankerNo wilting
CankerWilting
197 strains
+
+/-(+/-)
Jacques et al., 2012
Characterization of Clavibacter-like strainsisolated from tomato
Nb of
strains
Pathogenicity
IF PCR-based identification
IF (PRI) IF (Loewe) ZTO Cmm F/R Psa PsaR/8 Ptssk
129 + + + + + + + +
11 +/- + + + + + + +
1 (+/-) + + + + + + +
1 - + + + + + - -
1 - + + + + - + -
2 - + + + + - - -
6 - + + + + trace - -
2 - + + - - trace + -
4 - + + - - - - -
4 - + + - - trace - -
2 - - + - - - - -
2 - - - + + - - -
32 - - - - - - - -
Manycross-
reactionswith look-
alikes
36%
39%
(Nunhems, 2008; Rijlaarsdam et al, 2004; Pastrik & Rainey, 1999; Berendsen et al., 2011)
Cmm
Look-alikes
197 strainsJacques et al., 2012
Characterization of Clavibacter-like strainsisolated from tomato
Nb of
strains
Pathogenicity
IF PCR-based identification
IF (PRI) IF (Loewe) ZTO Cmm F/R Psa PsaR/8 Ptssk
129 + + + + + + + +
11 +/- + + + + + + +
1 (+/-) + + + + + + +
1 - + + + + + - -
1 - + + + + - + -
2 - + + + + - - -
6 - + + + + trace - -
2 - + + - - trace + -
4 - + + - - - - -
4 - + + - - trace - -
2 - - + - - - - -
2 - - - + + - - -
32 - - - - - - - -
Ptssk : specific and
sensitive
21%
<1%
2%
(Nunhems, 2008; Rijlaarsdam et al, 2004; Pastrik & Rainey, 1999; Berendsen et al., 2011)
Cmm
197 strains
Look-alikes
Manycross-
reactionswith look-
alikes
Jacques et al., 2012
1. In vitro tests to select look-alikes that interfere with Cmm2. Transmission to seed for candidate look-alikes
individually, and in mix with the pathogen, C144
Role of look-alikes in seed transmission of pathogens
Look-alikes were isolated from seeds.No report of crossed contaminations.
Do look-alikes have a role in seed transmission of pathogenic strains?antagonist or facilitator ?
22 “Cmm look-alikes”
In vitro antibiosis tests
Cmm: C144 CFBP 7494CFBP 7496CFBP 7497
Role of look-alikes in seed transmission of pathogens
Look-alike aloneLook-alike in mix
Cmm aloneCmm in mix
Log
cfu/
ml
In vitro competition test
Antibiosis of C144 toward 21 look-alikesCFBP 7496 is not sensitive to C144
Spray inoculation (1 × 106 ufc/mL)
Flower
Flower bud
Fruit
Analysis 2 mo afterinoculation
Role of look-alikes in seed transmission of Cmm pathogenic strain
Pathogen alone
Look-alike alone
Mixtures P-NP
5 plants / treatment
3 flower bud/ plant
Cmm: C144 749474967497
0
1
2
3
C144/7494 C144/7496 C144/7497
Popu
latio
n si
zes
(Log
10 C
FU/g
of
see
d)
Pathogenic strain alone
Non-pathogenic strain alone
Pathogenic strain in mix
Non-pathogenic strain in mix
Detection threshold
M1 M2 M3
aa
a
a
a a
b b b b b b
Contamination rate of seedlots by Pathogenic strain(C144)
C1447494
C1447496
C1447497
Detection threshold
Contamination rate0,5% 0,5% 0,5% 0,5% 0,5% 0,5%
Pathogen alone
Look-alike alone
Pathogen in mix
Look-alike in mix
No transmission of any look-alikesNo effect of look-alike on pathogen transmission
Role of look-alikes in seed transmission of Cmm pathogenic strain
Look-alikes- are responsible for false positive results in identification tests- are phylogenetically diverse, but could be closely related to pathogens- are not transmitted to seeds of their host of isolation- do not interact with pathogens for transmission to seeds
Some look-alikes are commensals
Summary
Look-alikes- are responsible for false positive results in identification tests- are phylogenetically diverse, but could be closely related to pathogens- are not transmitted to seeds of their host of isolation- do not interact with pathogens for transmission to seeds- are efficient xylem colonizers
Some look-alikes are commensals
Could other look-alikes be pathogens carried by non-host material?
Summary
Identification of C. michiganensis pathogenic on bean(Gonzalez and Trapiello, 2014)
Identification of C. michiganensis pathogenic on bean
Some look-alikes are very close to this new bean pathogen
But, pathogenicity on bean is difficult to evidence
Cmp LPPA 982Cm CFBP 8016
P. vulgaris, cv. Flavert, 5 plants/strainStem injection + sponge inoculation on leaves
Identification of C. michiganensis pathogenic on bean
Some look-alikes are very close to this new bean pathogen
But, pathogenicity on bean is difficult to evidence
Cmp LPPA 982Cm CFBP 8016
Test other bean cv.
Carriage of pathogen by seeds of non-host plants(Darrasse et al., 2010)
Acknowledgments
Armelle DarrasseJean-François Guimbaud
Karine DurandAnne Préveaux
INEM
Perrine PortierGéraldine Taghouti
Thank you for your attention
