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)