Upload
others
View
8
Download
1
Embed Size (px)
Citation preview
Bacterial Canker of Tomato
Gitta CoakerAssociate Professor
Department of Plant PathologyUniversity of California, Davis
Bacterial canker disease symptoms
Stem cankers
Necrotic lesionsBird’s-eye spots
Pith discoloration
Leaf yellowing/necrosis
• Causal agent: Clavibacter michiganensis subsp. michiganensis (Cmm)• Proliferates in the xylem
Other Clavibacter subsp. can infect diverse plants
subsp. sepedonicusPotato ring rot
subsp. nebraskensisGoss’s wilt of corn
subsp. insidiosusBacterial wilt on alfalfa
subsp. capsiciBacterial canker on pepper
subsp. tessellariusBacterial mosaic on wheat
Bacterial Canker Disease control
• Systemic disease, seed-borne, water-borne, long lived in GH environment
• Seed disinfestation with HCl
• Sanitation procedures during production
• Properly dispose of vines and residuals
• No genetic resistance, no robust chemical control
Nonpathogenic Clavibacter are frequently associated with tomato
CASJ009(Non-Pathogenic)
CASJ002 (Pathogen)
Specific Cmm detection is challenging
ImmunoStrips (Agdia)High rate of false positivesCross-reacts with other Clavibacter
PCRCmm 5/6 – does not detect all pathogenic Cmm strains
Cmm 3/4 – potential for cross-amplification with Clavibacter subsp. infecting alfalfa
Workflow for generating a specific Cmm detection
platform
Sequence many Clavibacter genomes isolated from tomato
(Pathogenic and Non-pathogenic)
Sequence data for other Clavibacter subsp. infecting different plants
Genome comparisons
Identify genes found only in pathogenic Cmm
Develop specific Cmm detection platform
(multiplex PCR)
Geographical distribution of Cmm strain collection
Strains Continents Decades Years
>100 6 6 1946 - 2016
MLST phylogeny used to identify strains for sequencing
Genome sequencing of Clavibacter strains
Sequenced 12 Cmm strains and one nonpathogenic strain collected in CA
+4 additional nonpathogenic strains, +34 pathogenic Clavibacter strains
Variation in Cmm growth, virulence
456789
1011
NCP
PB38
2CA
SJ00
9CA
SJ00
1CA
SJ00
2CA
SJ00
3CA
SJ00
4CA
SJ00
5CA
SJ00
6CA
SJ00
7CA
SJ00
8CA
YO00
1CA
0000
1CA
0000
2
Log
CFU
/100
mg
Cmm titers 14dpi (tomato stems) Canker formation 14dpi
Criteria for diagnostic PCR targets
Present in all pathogenic Cmm strains
Absent in related Clavibacter unable to cause disease on tomato
Required for disease development
One target should be plasmid-borne Increased copy number per cell than chromosomal genes Enhanced sensitivity for detection
The pCM2 plasmid is not found in all pathogenic Cmm strains
Bacterial growth 14 dpiDisease symptoms
0
2
4
6
8
10
CASJ009 CASJ001 NCPPB382
LOG
CFU
/100
mg
a
bb
pCM1 -pCM2 -pCMSap1 +
NonpathogenicCASJ009
Pathogen CASJ001
Pathogen NCPPB382
pCM1 +pCM2 -pCMSap1 -
pCM1 +pCM2 +pCMSap1 -
Identification of conserved targets for PCR: The pCM1 plasmid is found in all pathogenic Cmm
Disease symptoms
Pathogen CASJ001pCM1
Pathogenicity:Strain:
Plasmids:
NonpathogenicCASJ001∆pCM1None
pCM1 is required for disease development
Identification of conserved targets for PCR: celA on the pCM1 plasmid is found in all pathogenic Cmm
Pathogenicity: Pathogen Strain: CASJ001
Plasmids: pCM1
Non-pathogenicCASJ001∆pCM1pHN16(EV)
Weakly pathogenicCASJ001∆pCM1pHN16(celA)
Cmm detection via multiplex PCR
Bacteria # strains Sab celA 16S
Cmm 82 + + +Nonpathogenic Clavibacter 6 - - +Cms 10 - - +Other strains 20 - - -
Cmm NP Cms Pst
celASab16S
Multiplex PCR assay detects Cmm from summer canker outbreak
Phylogeny
• Non-pathogenic strains isolated from tomato cluster with subsp. causing other bacterial diseases
• Implications for seed production/greenhouse production/crop rotation• Highlights need for specific Cmm detection
MLSA of six housekeeping genes (maximum likelihood)
PP
P
Summary
Utilized genome sequences to develop specific multiplex PCR detection platform.
More specific Cmm detection platform should be implemented for seed detection.
Nonpathogenic Clavibacter strains on tomato are very diverse and some cluster with subsp. infecting other crops. Could Cmm cycle in other plants in a related way, influencing
disease persistence?
Acknowledgements
• Shree Thapa (UC Davis)• Bob Gilbertson (UC Davis)• Mike O’Leary (UC Davis, Gilbertson Lab)• Marie-Agnes Jacques (INRA)• Eugene Miyao (CE Advisor, ANR)