1. The case of Clavibacter michiganensis ssp. michiganensis
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. The case of Clavibacter michiganensis ssp. michiganensis.
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
Cmm infection in nursery and on tomato farm
Critical Cmm management issues
Tracking and tracing of Cmm

5. Clavibacter michiganensis ssp. michiganensis. Key features.
tomato, pepper/paprika (eggplant)
not motile
through wounds & natural openings
in vascular tissues
wilt , cankers & fruit symptoms
seedborne & seed transmitted
Natural openings: hydathodes = end of leaf veins

6. Clavibacter michiganensis ssp. michiganensis. Key features.
regulated EU/EPPO

7. Clavibacter michiganensis ssp. michiganensis.
Why disease in tomato plants?
PAI
pCM1
pCM2
PAI
pCM1
pCM2
PAI
chp/ppa
tom
pCM1
cel
pCM2
pat-1
Natural situation = no disease.
Two processes in tomato plant: colonization + infection
Genetic nature is not well known.
Chromosome and two small DNA molecules (plasmids)
Tomatinase = neutralise tomatine = natural defence in tomato plant
Cmm100 = no plasmids = colonization but no disease
Cmm27 = no PAI = poor colonization = poor disease (e.g. only canker at site of inoculation)

8. Clavibacter michiganensis ssp. michiganensis. Worldwide.
No formal records of many countries in Asia: e.g. provinces in China, Vietnam, Thailand, … and in South America: e.g. Bolivia, Paraguay, Venezuela, …
16 EU countries
South America
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. Clavibacter michiganensis Family tree.
C.m. ssp. californiensis
C.m. ssp. chilensis
Cm-like bacteria from seeds: no disease on tomato. True not pathogenic? Many different plant species would need to be tested.
New taxa to make difference with Cmm.
Cm chilensis = from Chile and India (seed
Other new Clavibacter michiganensis = on bean (subsp. phaseoli).
C.m. from Korea
Yasuhara-Bell & Alvarez, 2015

10. Clavibacter michiganensis ssp. michiganensis Seed contamination.
Cmm that produces a protein to make it fluorescent under UV.
A-D: brushing of Cmm on immature fruits. Lesions develop on the fruit skin and Cmm migrates to vascular tissues in the fruit (red arrows).
E-H: inoculation of inflorescences (drawing). Systemic infection and Cmm migrates to vascular tissues in the fruit (red arrows).
I-L: water inoculations. White arrows = vascular tissues.
Matthew A. Tancos et al. 2013

11. Clavibacter michiganensis ssp. michiganensis Seed contamination.
External inoculation of tomato fruits with subsequent seed colonization.
A and B. Vascular infection of Cmm leading up to several discoloured tomato seeds (red arrows).
C. Thin section of the discoloured seed from B with xylem leading into the seed.
D. Magnified view of seed in C with eGFP-labeled Cmm colonizing the cells surrounding the xylem tissue.
Section of seed corresponding to the same anatomical structure is labeled with a red “X”.
White arrows = several Cmm.

12. Clavibacter michiganensis ssp. michiganensis Seed contamination.
Systemic inoculation of peduncle resulting in colonization of xylem vessels within the funiculus of a developing tomato seed.
Xylem vessels leading into the developing seed.
Magnification of A showing xylem with Cmm colonization (red arrows).
Cmm on developing seed.
Cmm seed contamination at different layers (external --- internal). Effect on seed decontamination.

13. Clavibacter michiganensis ssp. michiganensis Seed transmission.
ILVO study indicated a distribution of Cmm from the contaminated seed to the cotyledons/first few leaves and to the roots.
Cmm on cotyledons produce blister spots under saturated air humidity.
Cmm on roots resulted in systemic infection and wilting.
At the time of study (late 1980’s), grafting was not common and seedlings were not kept under high humidity.
Cmm on the leaves did not contribute to disease

14. Clavibacter michiganensis ssp. michiganensis Seed transmission.
Bioluminescence of Cmm onto tomato seeds. Bacteria were observed on seed coats throughout the germination period and colonized other seedling components as they emerged.
The number at the bottom of each image indicates the day of germination. The rainbow scale represents photon counts (photons/s).
(B) Distribution of the bioluminescent Cmm strain on tomato seedlings on the fifth day of germination: Cmm on all parts of the seedling.
Each bar represents the mean bacterial count for 12 of each seedling component with standard deviations.
Xiulan Xu et al. 2010

15. Transmission from seed → seedling → seedling → transplant
Clavibacter michiganensis ssp. michiganensis
Transmission and spread in the nursery.
Transmission from seed → seedling → seedling → transplant
3 phases in the nursery
1. transmission from seed to seedling
2. Primary spread from seedling to seedling
3. Secondary spread from seedling to transplant: grafting, contact, water, aerosols

16. Clavibacter michiganensis ssp. michiganensis
Clavibacter michiganensis ssp. michiganensis. Secondary spread in nursery.
after 17 days
Black square = 9 seedlings with Cmm on roots.
a = overhead water supply (nozzles) , b = tray drench water supply.
Symptoms of systemic infection during second week after inoculation.
Disease severity (blister spots on leaves) after 17 days – 4 levels. Cmm found at 30 cm from the source in both systems of water supply
c = overhead vs. Drench. Disease decreased with distance from source. Overhead water supply significantly more disease than drench but the rate of decrease is not significantly different.
How did Cmm arrive on the leaf surface? Through guttation.
Frenkel et al., 2016

17. Clavibacter michiganensis ssp. michiganensis
Clavibacter michiganensis ssp. michiganensis. Secondary spread in nurseries.
Colonization of seedlings after 21 days
Comparison overhead vs. Drench after spray inoculation
Colonization of seedlings 21 days post inoculation = presence of Cmm on leaves.
Overhead: still 30% of seedlings with on leaves at cm from source.
Drench: 15% of seedlings with Cmm on leaves at cm from source.
Conditions: foliar wetness, t°, seedling age.
Spray inoculation.
T: optimum at ~28°C. Not below 14°C and not above ~36°C.
Seedling age: highly significant
Leaf wetness: not significant
Frenkel et al.,2016

18. Clavibacter michiganensis ssp. michiganensis
Clavibacter michiganensis ssp. michiganensis. Secondary spread in nurseries.
Cmm
from contaminated seeds
root inoculated
Cmm in guttation droplets.: a = plants from contaminated seeds, b = plants root inoculated, c = plants shoot inoculated
a: days after sowing, days in all plants. No symptoms. All plants colonized 104 – 105 viable Cmm/gram tissue.
b: 5-8 days after inoculation: 57-70% of the plants after 30 days. Symptoms 5-6 weeks after inoculation. ~20% of the leaves had cmm on surface. Mean = 1.6 cmm/gram
c: 3-6 days after inoculation: 55-77% of the plants after days. Symptoms 6 weeks after inoculation.
shoot inoculated

19. Clavibacter michiganensis ssp. michiganensis
Clavibacter michiganensis ssp. michiganensis. Secondary spread in tomato cultivation.

20. plants with Dry/Wet leaves
Clavibacter michiganensis ssp. michiganensis. Secondary spread in tomato cultivation.
V1/V2 = with knife, not disinfected and disinfected. Assessment after 6 weeks = symptoms of systemic infection. NS = 3.6 plants, S = 0.5 plants.
V3/B1/B2/B3 = contact by crop interventions, when plants were wet or dry.
Assessment of disease = blister spots and hydathode necrosis. All plants contact when wet developed symptoms.
knife
plants with Dry/Wet leaves

21. Clavibacter michiganensis ssp. michiganensis
Clavibacter michiganensis ssp. michiganensis. Movement in the tomato plant.
Cmm in stem of 3 week old tomato seedlings (inoc. at cotyledons).
above inoculation site
4 cm below inoculation site
In the xylem: rapid upward and lateral spread. Cmm moves freely with water stream.
In phloem: no freely movement. Through phloem parenchym
Cmm in stem at cotyledons in 3 week old tomato seedlings.
After 3 days: moved 4 cm in the stem.
After 7 days: moved 6 cm in the stem.
After 15 days: at the top
Below inoculation site: moved 4 cm after 3 days

22. old leaves ~ young leaves
Clavibacter michiganensis ssp. michiganensis.
Disease and age of the tomato plant.
15 days
75 days
days
late
early
Cmm inoculated at different age of the tomato plant.
Late infection (29/30 leaves): 60% of the plants without symptoms.
Most vulnerable for disease = 7/8 leaves.
Leaf age tested of tomato plants with 19/20 leaves.
old leaves ~ young leaves

23. Clavibacter michiganensis ssp. michiganensis.
Colonization and age of the tomato plant.
Tomato plants with 7/8 leaves and with 19/20 leaves.
Inoculated at the two youngest leaves.
Density of Cmm in the tomato plant at LS = stem at soil level, IS = at inoculation site, apex.
After one week: Cmm is already at the stem basis and at the top of the plant! Compare with delay for symptoms.

24. Clavibacter michiganensis ssp. michiganensis.
Age related resistance of the tomato plant.
Tomato plants with 7/8 leaves and with 19/20 leaves.
Inoculated at the two youngest leaves.
Expression of virulence proteins in older plants; poor in cel (disease) and chp (colonization)

25. Clavibacter michiganensis ssp. michiganensis.
Survival in crop residues and soil.
Genes of Cmm very similar to soil bacteria
long survival in tomato crop residues
Cmm is very sensitive to antibiosis of soil bacteria
How deep goes Cmm in soil?
roots of tomato plant
Determined by field conditions (Fatmi & Schaad, 2002)
tomato stems on the soil: 132 days (Aït Melloul)
194 days (Melh Zhar)
2 years in Iowa, USA
Cmm survives well in tomato tissue but not unprotected in the soil.

26. Clavibacter michiganensis ssp. michiganensis.
Survival in crop residues and soil.
Under greenhouse conditions in Argentina (Vega & Romero, 2015)
In crop residues on the soil:
days after winter crop (ending in July)
45-75 days after summer crop (ending in December)
In crop residues at 10 cm in the soil:
45-75 days after winter crop
All sudies rely on viable cells on semi-selective culture media
sensitivity?
Cmm in other condition?
Argentina: two tomato cultivations per year. One ending in July (winter crop) and one ending in December (summer crop)

27. Clavibacter michiganensis ssp. michiganensis.
Critical management strategies.
Prevent introduction:
GSPP for all seed production
seed treatment: min. at 52°C - 56°C in water
effect on seed germination
Prevent dissemination:
GSPP for nurseries
During cultivation:
avoid conditions for guttation
avoid contact with plants when wet
delay colonization ↔ symptoms
Crop residues
temperature in soil → 45°C/4 weeks
soil amendments
composting: ~70 days > 45°C

28. Clavibacter michiganensis ssp. michiganensis.
epidemic in Flanders.
2010
Cmm diagnosed on 12 tomato farms
7 tomato cultivars affected
young plants from same nursery
Cmm resurgent on 3 tomato farms
Judicial inquiry
judicial inquiry on the introduction of Cmm
no evidence of seed contamination in testing

29. Clavibacter michiganensis ssp. michiganensis.
epidemic in Flanders.
23 cultures of Cmm from outbreaks in Flanders
8 cultures of Cmm from previous outbreaks in Flanders ( )
16 cultures of Cmm from outbreaks in the EU & Morocco ( )
reference cultures of Cmm from worldwide (culture collections)
Cm-like bacteria from tomato seeds
Method for tracking and tracing:
Multilocus Variable Number Tandem Repeats Analysis: = MLVA (VNTR)
Discriminate individual strains in epidemics of bacteria present in food (E. coli, Salmonella) and bacteria pathogenic to humans (Streptococcus pneumoniae, Mycobacterium tuberculosis).
Tracking transmission in hospitals, in livestock, … Used in forensic science to analyse DNA from crime scenes
Restriction: MLVA is different for each bacterial species

30. Clavibacter michiganensis ssp. michiganensis.
Tracking and tracing by 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: nucleotides
During replication of DNA regions with repeats are not correctly duplicated : increase or decrease of number of repeats

31. Clavibacter michiganensis ssp. michiganensis.
Tracking and tracing by 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
Strain 2
Strain 3
Strain 4 10 3 5 7
MLVA profile strain 1:
MLVA profile strain 2:

32. Clavibacter michiganensis ssp. michiganensis.
Tracking and tracing by MLVA.
MLVA process is standardized and can be done in any lab with

33. Clavibacter michiganensis ssp. michiganensis.
Tracking and tracing by MLVA
23 strains from FL
clonal
resurgent strains from farm
one introduction
new type/origin
8 strains from FL
6 types/4 origins
1 with seeds from Taiwan
strain from Morocco
2 strains from France 2010
in FL group
same tomato variety
in cluster P with strain from PT (1998)
Minimum spanning tree based on 8 VNTR loci.
Each circle is a MLVA type (haplotype). The surface of the circle corresponds to the number of strains in the MLVA type.
Circles connected by a thick solid line differ from each other by one VNTR locus.
Circles connected by a thin solid line differ from each other by two VNTR loci.
Circles connected by dashed/dotted lines differ from each other by 3 or more VNTR loci.
Grouping in 1 large clonal group and 5 clusters which differ by 2 loci at most = from the same ancester / geographic origin.

34. Clavibacter michiganensis ssp. michiganensis.
Virulence of newly introduced exotic strains.
1998, MLVA_E
2007, MLVA_U
2010, MLVA_W
New exotic strains are highly virulent/contagious.
Twitching motility as indicator for xylem colonisation.
In tomato plantlets (cv. Moneymaker) at cotyledons (5 µl of ~106 c/ml).
Colonization after 72 hours. Stem segments of 0.5 cm dilution plating.

35. Clavibacter michiganensis ssp. michiganensis.
Tracking & tracing of exotic strains.
MLVA
highly discriminative for individual strains
relatively simple (PCR, sequencing)
transferable to other labs
reference strains in database
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. Clavibacter michiganensis ssp. michiganensis.