Bacterial Canker of Tomato. Gitta Coaker Associate Professor Department of Plant Pathology University of California, Davis

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1 Bacterial Canker of Tomato Gitta Coaker Associate Professor Department of Plant Pathology University of California, Davis

2 Bacterial canker disease symptoms Leaf yellowing/necrosis Necrotic lesions Bird s-eye spots Pith discoloration Stem cankers Causal agent: Clavibacter michiganensis subsp. michiganensis (Cmm) Proliferates in the xylem

3 Other Clavibacter subsp. can infect diverse plants subsp. sepedonicus Potato ring rot subsp. capsici Bacterial canker on pepper subsp. insidiosus Bacterial wilt on alfalfa subsp. nebraskensis Goss s wilt of corn subsp. tessellarius Bacterial mosaic on wheat

4 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

5 Nonpathogenic Clavibacter are frequently associated with tomato CASJ002 (Pathogen) CASJ009 (Non-Pathogenic)

6 Specific Cmm detection is challenging ImmunoStrips (Agdia) High rate of false positives Cross-reacts with other Clavibacter PCR Cmm 5/6 does not detect all pathogenic Cmm strains Cmm 3/4 potential for cross-amplification with Clavibacter subsp. infecting alfalfa

7 Workflow for generating a specific Cmm detection platform Sequence many Clavibacter genomes isolated from tomato (Pathogenic and Nonpathogenic) Genome comparisons Identify genes found only in pathogenic Cmm Develop specific Cmm detection platform (multiplex PCR) Sequence data for other Clavibacter subsp. infecting different plants

8 Geographical distribution of Cmm strain collection Strains Continents Decades Years > MLST phylogeny used to identify strains for sequencing

9 Genome sequencing of Clavibacter strains Sequenced 12 Cmm strains and one nonpathogenic strain collected in CA +4 additional nonpathogenic strains, +34 pathogenic Clavibacter strains

10 Variation in Cmm growth, virulence Cmm titers 14dpi (tomato stems) Canker formation 14dpi Log CFU/100 mg NCPPB382 CASJ009 CASJ001 CASJ002 CASJ003 CASJ004 CASJ005 CASJ006 CASJ007 CASJ008 CAYO001 CA00001 CA00002

11 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

12 The pcm2 plasmid is not found in all pathogenic Cmm strains Disease symptoms Bacterial growth 14 dpi 10 b b 8 Nonpathogenic CASJ009 pcm1 - pcm2 - pcmsap1 + Pathogen CASJ001 pcm1 + pcm2 - pcmsap1 - Pathogen NCPPB382 pcm1 + pcm2 + pcmsap1 - LOG CFU/100mg a 0 CASJ009 CASJ001 NCPPB382

13 Identification of conserved targets for PCR: The pcm1 plasmid is found in all pathogenic Cmm Disease symptoms Pathogenicity: Strain: Plasmids: Pathogen CASJ001 pcm1 Nonpathogenic CASJ001 pcm1 None pcm1 is required for disease development

14 Identification of conserved targets for PCR: cela on the pcm1 plasmid is found in all pathogenic Cmm Pathogenicity: Strain: Plasmids: Pathogen CASJ001 pcm1 Non-pathogenic CASJ001 pcm1 phn16(ev) Weakly pathogenic CASJ001 pcm1 phn16(cela)

15 Cmm detection via multiplex PCR Bacteria # strains Sab cela 16S Cmm Nonpathogenic Clavibacter Cms Other strains Cmm NP Cms Pst cela Sab 16S

16 Multiplex PCR assay detects Cmm from summer canker outbreak

17 Phylogeny P P P 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)

18 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?

19 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)

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