Global Surveillance of Bacterial Phytopathogens from Seeds: Impact on Seed Companies and Nurseries

1. Global surveillance of bacterial phytopathogens from seeds affects both seed companies and nurseries. The case of Clavibacter michiganensis ssp. michiganensis

2. Flanders’ agriculture, horticulture, food science and fisheries

3. Plant Sciences Plant Health

4. Global surveillance of bacterial phytopathogens from seeds affects both seed companies and nurseries. The case of Clavibacter michiganensis ssp. michiganensis. • Outline • Key features of Cmm • Cmminfection in nursery and ontomato farm • CriticalCmm management issues • Tracking and tracing of Cmm

5. Clavibactermichiganensisssp. michiganensis. Key features. • tomato, pepper/paprika (eggplant) • notmotile • throughwounds & naturalopenings • in vascular tissues • wilt , cankers & fruit symptoms • seedborne & seedtransmitted

6. Clavibactermichiganensisssp. michiganensis. Key features. • regulated EU/EPPO

7. Clavibactermichiganensisssp. michiganensis. Whydisease in tomatoplants? pCM1 pCM2 PAI PAI pCM1 pCM2 PAI chp/ppa tom pCM1 cel pCM2 pat-1

8. Clavibactermichiganensisssp. michiganensis. Worldwide. 16 EU countries SouthAmerica Asia present in countries where tomato seeds / young plants are produced However, … no formal records of many countries in Asia: e.g. many provinces in China, Vietnam, Thailand, … and in South America: e.g. Bolivia, Paraguay, Venezuela, …

9. Clavibactermichiganensis Family tree. C.m. ssp. californiensis C.m. ssp. chilensis C.m. from Korea Yasuhara-Bell & Alvarez, 2015

10. Clavibactermichiganensisssp. michiganensis Seedcontamination. Matthew A. Tancos et al. 2013

11. Clavibactermichiganensisssp. michiganensis Seedcontamination.

12. Clavibactermichiganensisssp. michiganensis Seedcontamination. C C

13. Clavibactermichiganensisssp. michiganensis Seedtransmission.

14. Clavibactermichiganensisssp. michiganensis Seedtransmission. B Xiulan Xu et al. 2010

15. Clavibactermichiganensisssp. michiganensis Transmission and spread in the nursery. Transmission from seed → seedling → seedling → transplant

16. Clavibactermichiganensisssp. michiganensis. Secondary spread in nursery. after 17 days Frenkelet al., 2016

17. Clavibactermichiganensisssp. michiganensis. Secondary spread in nurseries. Colonization of seedlings after 21 days Frenkelet al.,2016

18. Clavibactermichiganensisssp. michiganensis. Secondary spread in nurseries. 10-100000 Cmm root inoculated from contaminated seeds shoot inoculated

19. Clavibactermichiganensisssp. michiganensis. Secondary spread in tomatocultivation.

20. Clavibactermichiganensisssp. michiganensis. Secondary spread in tomatocultivation. knife plantswith Dry/Wet leaves

21. Clavibactermichiganensisssp. michiganensis. Movement in the tomato plant. Cmm in stem of 3 week old tomato seedlings (inoc. at cotyledons). aboveinoculation site 4 cm belowinoculation site

22. Clavibactermichiganensisssp. michiganensis. Disease and age of the tomato plant. 15 days late 75 days early 100-130 days oldleaves ~ youngleaves

23. Clavibactermichiganensisssp. michiganensis. Colonization and age of the tomato plant.

24. Clavibactermichiganensisssp. michiganensis. Agerelatedresistance of the tomato plant.

25. Clavibactermichiganensisssp. michiganensis. Survival in cropresidues and soil. • Genes of Cmmverysimilar to soilbacteria • long survival in tomatocropresidues • Cmm is very sensitive to antibiosis of soilbacteria • HowdeepgoesCmm in soil? • roots of tomato plant • Determinedby field conditions (Fatmi & Schaad, 2002) • tomatostemson the soil: 132 days (AïtMelloul) • 194 days (MelhZhar) • 2 years in Iowa, USA

26. Clavibactermichiganensisssp. michiganensis. Survival in cropresidues and soil. • Under greenhouse conditions in Argentina (Vega & Romero, 2015) • In cropresidueson the soil: • 120-260 daysafter winter crop (ending in July) • 45-75 daysaftersummercrop (ending in December) • In cropresidues at 10 cm in the soil: • 45-75 daysafter winter crop • All sudiesrelyonviablecellsonsemi-selective culture media • sensitivity? • Cmm in othercondition?

27. Clavibactermichiganensisssp. michiganensis. Critical management strategies. • Prevent introduction: • GSPP forall seedproduction • seedtreatment: 20-30 min. at 52°C - 56°C in water • effect onseedgermination • Prevent dissemination: • GSPP fornurseries • Duringcultivation: • avoidconditionsforguttation • avoid contact withplantswhen wet • delaycolonization↔ symptoms • Cropresidues • temperature in soil→ 45°C/4 weeks • soilamendments • composting: ~70 days > 45°C

28. Clavibactermichiganensisssp. michiganensis. 2010-2012 epidemic in Flanders. • 2010 • Cmmdiagnosedon 12 tomato farms • 7 tomato cultivars affected • youngplantsfromsamenursery • 2011-2012 • Cmmresurgenton 3 tomato farms • judicialinquiryon the introduction of Cmm • noevidence of seedcontamination in testing

29. Clavibactermichiganensisssp. michiganensis. 2010-2012 epidemic in Flanders. • 23 cultures of Cmmfrom 2010-2012 outbreaks in Flanders • 8 cultures of Cmmfrompreviousoutbreaks in Flanders (1998-2008) • 16 cultures of Cmmfromoutbreaks in the EU & Morocco (1970-2010) • reference cultures of Cmmfromworldwide (culture collections) • Cm-likebacteriafromtomatoseeds • Methodfortracking and tracing: • MultilocusVariableNumber Tandem RepeatsAnalysis: = MLVA (VNTR)

30. Clavibactermichiganensisssp. michiganensis. Tracking and tracingby MLVA MLVA is based on the natural variation in the number of tandem repeated DNA sequences found in the microbial genome of most bacterial species ...ATAGTATTC GAATCGG GAATCGG GAATCGG GAATCGG CCGATTTGC... Microsatellites: less than 10 nucleotides Minisatellites: 10-60 nucleotides

31. Clavibactermichiganensisssp. michiganensis. Tracking and tracingby MLVA. • Because of this variation such loci are designated as • variable number of tandem repeat(VNTR) loci The number of repeats in a particular locus may differ between strains Strain 1 10 Strain 2 3 Strain 3 5 Strain 4 7 MLVA profilestrain 1: 6-3-6-4-3-5-4-7 MLVA profilestrain 2: 6-3-4-4-2-5-7-7

32. Clavibactermichiganensisssp. michiganensis. Tracking and tracingby MLVA.

33. Clavibactermichiganensisssp. michiganensis. Tracking and tracingby MLVA • 23 strainsfrom FL 2010-2012 • clonal • resurgentstrainsfrom farm • oneintroduction • new type/origin • 8 strainsfrom FL1998-2008 • 6 types/4 origins • 1 withseedsfrom Taiwan • strainfromMorocco • 2 strainsfrom France 2010 • in FL 2010-2012 group • sametomatovariety • 2 strainsfrom France 2010 • in cluster P withstrainfrom PT (1998)

34. Clavibactermichiganensisssp. michiganensis. Virulence of newlyintroducedexoticstrains. 1998, MLVA_E 2007, MLVA_U 2010, MLVA_W

35. Clavibactermichiganensisssp. michiganensis. Tracking & tracing of exoticstrains. • MLVA • highly discriminative for individual strains • relatively simple (PCR, sequencing) • transferable to other labs • reference strains in database • 2010-2012 FL → China • Other typing methods • AFLP (naktuinbouw, NL) • MLST (France) • whole genomes (cost ↓) • Introduction of exotic strains with tomato seed • hypervirulent • strains associated with place of production • known not to occur ↔ not known to occur

36. Clavibactermichiganensisssp. michiganensis.