CRITICAL COMPONENTS OF BACTERIAL CANKER SURVEILLANCE AND MANAGEMENT IN FIELD AND GREENHOUSE TOMATOES Sally A. Miller1, Melanie L. Lewis Ivey1, Fulya Baysal-Gurel1, Xiulan Xu1, Warren Arinaitwe2 and Michael E. Bledsoe3 1The Ohio State University, OARDC, Wooster, OH 2Makerere University, Kampala, Uganda 3Village Farms International, Inc., Heathrow, FL

Bacterial Canker • High consequence bacterial disease in temperate climates and greenhouses • Clavibacter

michiganensis subsp. michiganensis (Cmm) •  Seedborne •  Long-lived in environment •  Mechanically transmitted •  1°, 2° canker in open fields

Midwestern US outbreak 2011

GH tomato outbreak, 2010

Why Identify and Track Cmm Strains?

• Understand the movement of Cmm

•  Identify the source of outbreaks

•  Identify critical control points

•  Improve management practices

Production Flow Overview – GH Tomatoes

Germination*

Seedling*production

Grafting

Training*Dropping*

Trans6planting

Topping

De6lea7ing*

Seeding

Harvest*

Propa6gation

Crop*Work

Post6harvest*Handling***

Field Tomato Production Overview

Germination*

Seedling*production Pruning*

Staking*Spraying***

Trans6planting

Seeding

Harvest*

Propa6gation

Crop*Work

Post6harvest*Handling***

Development of a Multivariate Matrix for Cmm Surveillance • Rep-PCR fingerprinting

•  Historical Cmm types: ABCD •  Field tomatoes, Midwestern US, 1998 (Louws et al.)

•  Field tomato haplotypes: ABCD - Israel, 2008 (Kleitman et al.) •  Greenhouse tomato haplotypes: ABCD – Japan 2010 (Kawaguchi

et al.)

•  Gene sequence analysis – dnaA Replication Initiation Factor (RIF) (Schneider et al. 2011)

•  This study •  40 strains collected from North American GHs between 2006 and 2011

•  BOX-PCR and RIF analyses •  63 strains from processing field tomatoes in OH in 2011 – BOX PCR

Box PCR – Cmm Strains

RIF Sequence Analysis vs. Box-PCR: Clavibacter Strains

1 C 1 CG1 11 CG3 9 CG5

1 B 2 CG1 13 CG2 1 CG3 1 CG4 2 CG5 1 A 1 CG2

BOX PCR Clonal Groups

dnaA RIF

Cmm Strain Matrix GH 2006-2011 Loca%on' State'or'

Province' Country' Tomato'Type'' Scion'' Rootstock'' 'Phase' GH'ID' House' Row'

Number' Post' Week' Year' Strain' RepBPCR'Profile'

A' TX' USA' TOV' Grandella' Maxifort' 3' '' '' '' '' '' 2009' 09B158' CG1'A' TX' USA' TOV' Grandella' Maxifort' 3' '' '' '' '' '' 2009' 09B159'' CG1'A' TX' USA' TOV' '' '' 3' '' '' '' '' 37' 2008' 08B687' CG1''A' TX' USA' TOV' Trisha' Maxifort' 1' '' '' '' '' '' 2007' VF3B70B08' CG2'A' TX' USA' TOV' Trisha' Maxifort' 2' '' '' '' '' '' 2007' VF3B71B08' CG2'A' TX' USA' TOV' Grandella' Maxifort' '' S' 22' '' 8' '' 2009' VFB22SB09' CG2'

A' TX' USA' TOV' Grandella' Maxifort' '' S' 50' '' 19' '' 2009' VFB50SAB09' CG2'A' TX' USA' TOV' Grandella' Maxifort' '' S' 50' '' 19' '' 2009' VFB50SBB09' CG2'A' TX' USA' TOV' Grandella' Maxifort' '' S' 50' '' 19' '' 2009' VFB50SCB09' CG2'A' TX' USA' TOV' Grandella' Maxifort' 3' B' 72' 2' 10' 14' 2009' 09B165'' CG2'A' TX' USA' TOV' Grandella' Maxifort' 3' '' 72' 2' 12' 14' 2009' 09B166'' CG2'A' TX' USA' TOV' Grandella' Maxifort' '' N' 21' '' 4' '' 2009' VFB21NB09' CG2'B' TX' USA' TOV' Trisha' Maxifort' 1' '' '' '' '' '' 2007' VF6B7B08' CG2'B' TX' USA' TOV' Tricia' Maxifort' 2' 13' 3' 31' 2' 42' 2009' 42B3B1' CG2'B' TX' USA' Roma' Savantas' Maxifort' '' '' '' '' '' '' 2009' 09B198A' CG2'B' TX' USA' TOV' Komeet' Maxifort' 2' 16' 10' 100' 2' 12' 2010' SM157B10' CG2'A' TX' USA' TOV' Grandella' Maxifort' '' '' '' '' '' '' 2006' VF3B1B08' CG2''A' TX' USA' TOV' Grandella Maxifort 3 B 7 2008' VF6B8B08' CG3'A' TX USA TOV Grandella Maxifort S 8 8 17 2009 VFB8SB09' CG3 A' TX' USA' Beef' Starbuck' Maxifort' '' '' 2' '' '' 15' 2009' 09B177'' CG3'B' TX' USA TOV Tricia Maxifort 2 23 46 468 5 36 2009 SM101B09' CG3'

B' TX' USA' Roma' Savantas' Maxifort' '' '' '' '' '' '' 2009' 09B197A' CG3'B' TX' USA' Roma' Savantas' Maxifort' '' '' '' 242' '' '' 2009' 09B190A' CG3'C' BC' Canada' TOV' Idol' Maxifort' '' '' '' '' '' '' 2008' B6B78' CG3'C' BC' Canada' TOV' Idol' Maxifort' '' '' '' '' '' '' 2008' B6B79' CG3'C' BC' Canada' TOV' Trisha' Maxifort' '' '' '' '' '' '' 2009' 09B192B' CG3'

D' PA' USA' TOV' Castella'or'Grandella' Maxifort' '' '' '' '' '' '' 2008' 08B568' CG3'

D' PA' USA' TOV' Castella'or'Grandella' Maxifort' '' '' '' '' '' '' 2008' 08B567' CG3'

A' TX' USA' Beef' Starbuck' Maxifort' '' '' 2' '' '' 15' 2009' 09B176'' CG3''A' TX' USA' TOV' Grandella' Maxifort' 3' '' '' '' '' '' 2009' 09B157' CG4''E' ME' USA' TOV' Success' Maxifort' 1' '' '' 227' 1' 3' 2011' SM287B11' CG5'E' ME' USA' TOV' Success' Maxifort' 1' '' '' 227' 1' 3' 2011' SM288B11' CG5'E' ME' USA' TOV' Success' Maxifort' 1' R' '' 381' 12' 3' 2011' SM290B11' CG5'E' ME' USA' TOV' Success' Maxifort' 1' L' '' 286' 11' 3' 2011' SM291B11' CG5'E' ME' USA' TOV' Success' Maxifort' 1' L' '' 286' 11' 3' 2011' SM292B11' CG5'E' ME' USA' TOV' Success' Maxifort' 1' L' '' 286' 11' 3' 2011' SM293B11' CG5'E' ME' USA' TOV' Success' Maxifort' 1' R' '' 132' 11' 5' 2011' SM295B11' CG5'E' ME' USA' TOV' Success' Maxifort' 1' L' '' 103' 1' 4' 2011' SM297B11' CG5'E' ME' USA' TOV' Success' Maxifort' 1' R' '' 132' 16' 5' 2011' SM298B11' CG5'E' ME' USA' TOV' Success' Maxifort' 1' R' '' 381' 12' 3' 2011' SMB289B11' CG5'E' ME' USA' TOV' Success' Maxifort' 1' R' '' 132' 11' 5' 2011' SM296B11' CG5'

Location E - 2011

Loca%on' State'or'Province' Country' Tomato'

Type'' Scion*'' Rootstock*'' 'Phase' GH'ID' House' Row'Number' Post' Week' Year' Strain' RepBPCR'

Profile'

E' ME' USA' TOV' Success' Maxifort' 1' '' '' 227' 1' 3' 2011' SM287B11' CG5'

E' ME' USA' TOV' Success' Maxifort' 1' '' '' 227' 1' 3' 2011' SM288B11' CG5'

E' ME' USA' TOV' Success' Maxifort' 1' R' '' 381' 12' 3' 2011' SM290B11' CG5'

E' ME' USA' TOV' Success' Maxifort' 1' L' '' 286' 11' 3' 2011' SM291B11' CG5'

E' ME' USA' TOV' Success' Maxifort' 1' L' '' 286' 11' 3' 2011' SM292B11' CG5'

E' ME' USA' TOV' Success' Maxifort' 1' L' '' 286' 11' 3' 2011' SM293B11' CG5'

E' ME' USA' TOV' Success' Maxifort' 1' R' '' 132' 11' 5' 2011' SM295B11' CG5'

E' ME' USA' TOV' Success' Maxifort' 1' L' '' 103' 1' 4' 2011' SM297B11' CG5'

E' ME' USA' TOV' Success' Maxifort' 1' R' '' 132' 16' 5' 2011' SM298B11' CG5'

E' ME' USA' TOV' Success' Maxifort' 1' R' '' 381' 12' 3' 2011' SMB289B11' CG5'

E' ME' USA' TOV' Success' Maxifort' 1' R' '' 132' 11' 5' 2011' SM296B11' CG5'

Only Cmm CG5 was detected throughout large greenhouse 3-5 weeks after transplanting *Does not infer source of Cmm

Village Farms Ft Davis

CG2 and CG3 detected between 2007 and 2010 CG2 Detected in GH 10 in both 2009 and 2010 CG3 introduced in a specific variety in 2009 but affected many other varieties *Does not infer source of Cmm

Grower'or'Company'

Grower'Facility'' Region,'City'State,'Region,'or'Province' Country' Tomato'

Type'' 'Scion*' Rootstock*' Field'or'Phase' A/B'' House' Row'

Number' Post' Week' Year' CG''

Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' '' Trisha' Maxifort' 1''' '' '' '' '' 2007' CG2'Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' Roma' Savantas' Maxifort' '' '' '' '' '' '' 2009' CG3'Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' Roma' Savantas' Maxifort' '' '' '' 242''' '' 2009' CG3'Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' Roma' Savantas' Maxifort' '' '' '' '' '' '' 2009' CG2'Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' Roma' Savantas' Maxifort' '' '' '' '' '' '' 2009' CG2'Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' TOV' Tricia' Maxifort' 2' 15' 10' 101' 5' 38' 2009' CG2'Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' TOV' Tricia' Maxifort' 2' 13' 3' 31' 2' 42' 2009' CG2'Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' TOV' Tricia' Maxifort' 2' 23' 46' 468' 5' 36' 2009' CG3'Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' TOV' Tricia' Maxifort' 2' 14' 2' 26' 16' 42' 2009' CG3'Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' TOV' Komeet' Maxifort' 2' 16' 10' 100' 2' 12' 2010' CG2'Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' TOV' Komeet' Maxifort' 2' 16' 12' 128' 3' 12' 2010' CG2'Village'Farms'Ft'Davis' Fort'Davis' Texas' USA' TOV' Komeet' Maxifort' 2' 13' 9' 97' 9' 12' 2010' CG2'

2011 Processing Tomato Canker Outbreak, US Midwest

Two new, unique Cmm Clonal Groups

Primary Canker

Cmm Matrix (partial) GROWER

INFORMATION Crop Information PROPAGATOR SAMPLE INFORMATION

Farm State Year GPS Variety* Type Date

Received CG Source

Lab Source Lab Identifier

OSU Sample Name

A OH 2011 46 Processing 2 23#Aug' 8 OSU 59A#1' SM502#11'A OH 2011 46 Processing 2 23#Aug' 8 OSU 59A#2' SM516#11'A OH 2011 46 Processing 2 23#Aug' 8 OSU 59A#3' SM513#11'A OH 2011 46 Processing 2 23#Aug' 8 OSU 59B#1' SM559#11'A OH 2011 46 Processing 2 23#Aug' 8 OSU 59B#2' SM525#11'A OH 2011 46 Processing 2 23#Aug' 8 OSU 59C#1' SM555#11'A OH 2011 46 Processing 2 23#Aug' 8 OSU 59C#2' SM569#11'A OH 2011 46 Processing 2 23#Aug' 8 OSU 59C#3' ''A OH 2011 331 Processing 1 23#Aug' 8 OSU 60A#1' SM551#11'A OH 2011 331 Processing 1 23#Aug' 8 OSU 60A#2' SM570#11'A OH 2011 331 Processing 1 23#Aug' 8 OSU 60A#3' SM571#11'A OH 2011 331 Processing 1 23#Aug' 8 OSU 60B#3' SM542#11'A OH 2011 9704 Processing 1 23#Aug' 8 OSU 61A#2' SM539#11'A OH 2011 9704 Processing 1 23#Aug' 8 OSU 61A#3' SM541#11'A OH 2011 9704 Processing 1 23#Aug' 8 OSU 61B#2' SM552#11'A OH 2011 9704 Processing 1 23#Aug' 8 OSU 61C#2' ''B OH 2011 46 Processing 1 23#Aug' 8 OSU 65A#2' ''B OH 2011 46 Processing 1 23#Aug' 8 OSU 65A#3' SM553#11'B OH 2011 46 Processing 1 23#Aug' 8 OSU 65A#4' ''B OH 2011 9704 Processing 1 23#Aug' 8 OSU 64#2' SM524#11'B OH 2011 9704 Processing 1 23#Aug' 8 OSU 64#3' SM573#11'B OH 2011 9704 Processing 1 23#Aug' 9 OSU 64#1' SM524#11'C OH 2011 331 Processing 1 23#Aug' 8 OSU 58A#2' SM511#11'C OH 2011 331 Processing 1 23#Aug' 8 OSU 58A#3' SM509#11'C OH 2011 331 Processing 1 23#Aug' 8 OSU 58B#1' SM505#11'C OH 2011 331 Processing 1 23#Aug' 8 OSU 58B#4' SM510#11'C OH 2011 331 Processing 1 23#Aug' 8 OSU 58C#3' SM507#11'C OH 2011 331 Processing 1 23#Aug' 8 OSU 58D#1' SM499#11'C OH 2011 331 Processing 1 23#Aug' 9 OSU 58A#1' ''C OH 2011 331 Processing 1 23#Aug' 9 OSU 58B#2' SM498#11'C OH 2011 331 Processing 1 23#Aug' 9 OSU 58C#4' SM508#11'C OH 2011 9704 Processing 1 23#Aug' 9 OSU 61C#3' ''

*Does not infer source of Cmm

Distribution of Cmm Clonal Group 9 in Contiguous Tomato Fields in OH

WEST EAST Variety/Field 46 4007 331 401 9704

Greenhouse

2

2

1

2

1

Canker Incidence

5% 0 80% 0 85%

Canker Severity

1-2% 0 30% 0 50%

CG 9 - 9 - 9

August 23, 2011

Summary: Cmm Clonal Groups in Multivariate Matrices • Recently isolated strains different from historical groups

ABCD

• Strains/CGs from greenhouse and field outbreaks are different •  GH, field fresh market and processing varieties are produced by

different companies in many different locations

• Matrices detailing substantial information on GH and field outbreaks can provide clues to pathogen source and movement

Bacterial Canker Management • Early intervention is critical – GH and field systems

•  Seed •  Prevention •  Testing •  Treatment

•  Propagation •  Environment •  Sanitation •  Bactericidal treatments – Copper, streptomycin •  Testing?

•  Production •  Field – not much; long rotations necessary •  GH – sanitation, sanitation, sanitation

After 26 Years, What Have We Accomplished in Management of Bacterial Canker? •  No resistant varieties •  No highly effective bactericides •  A new GH tomato industry threatened •  Seeds are still a source of Cmm

•  We know a lot about the epidemiology of this disease •  But not the frequency and mechanism of seed infection

•  Cmm testing methods have improved significantly •  Standardization is still needed •  Effective sanitizing seed treatments are known but are generally not

adopted •  Field production practices have changed

•  The industry moved away from bare-root transplants but canker did not go away

•  Greenhouse environment for seedling production highly conducive to bacterial disease development

Thanks! • OSU

• Melanie Lewis Ivey • Xiulan Xu • Warren Arinaitwe •  Fulya Baysal Gurel • Xing Ma • David Francis

• Tomato Industry • Mike Bledsoe, VF • Erika Verrier, BYF • Steve Smith, Red Gold •  Jeff Unverferth, Hirzel

Farms

• USDA NIFA SCRI • OARDC SEEDS program