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

Flanders’ agriculture, horticulture, food science and fisheries

Plant Sciences Plant Health

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

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

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

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)

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, …

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

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

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.

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.

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

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

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

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

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 65-75 cm from source. Drench: 15% of seedlings with Cmm on leaves at 65-75 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

Clavibacter michiganensis ssp. michiganensis Clavibacter michiganensis ssp. michiganensis. Secondary spread in nurseries. 10-100000 Cmm from contaminated seeds root inoculated Cmm in guttation droplets.: a = plants from contaminated seeds, b = plants root inoculated, c = plants shoot inoculated a: 29-37 days after sowing, 32-40 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 18-19 days. Symptoms 6 weeks after inoculation. shoot inoculated

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

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

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

old leaves ~ young leaves Clavibacter michiganensis ssp. michiganensis. Disease and age of the tomato plant. 15 days 75 days 100-130 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

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.

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)

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.

Clavibacter michiganensis ssp. michiganensis. Survival in crop residues and soil. Under greenhouse conditions in Argentina (Vega & Romero, 2015) In crop residues on the soil: 120-260 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)

Clavibacter michiganensis ssp. michiganensis. Critical management strategies. Prevent introduction: GSPP for all seed production seed treatment: 20-30 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

Clavibacter michiganensis ssp. michiganensis. 2010-2012 epidemic in Flanders. 2010 Cmm diagnosed on 12 tomato farms 7 tomato cultivars affected young plants from same nursery 2011-2012 Cmm resurgent on 3 tomato farms Judicial inquiry judicial inquiry on the introduction of Cmm no evidence of seed contamination in testing

Clavibacter michiganensis ssp. michiganensis. 2010-2012 epidemic in Flanders. 23 cultures of Cmm from 2010-2012 outbreaks in Flanders 8 cultures of Cmm from previous outbreaks in Flanders (1998-2008) 16 cultures of Cmm from outbreaks in the EU & Morocco (1970-2010) 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

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: 10-60 nucleotides During replication of DNA regions with repeats are not correctly duplicated : increase or decrease of number of repeats

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: 6-3-6-4-3-5-4-7 MLVA profile strain 2: 6-3-4-4-2-5-7-7

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

Clavibacter michiganensis ssp. michiganensis. Tracking and tracing by MLVA 23 strains from FL 2010-2012 clonal resurgent strains from farm one introduction new type/origin 8 strains from FL1998-2008 6 types/4 origins 1 with seeds from Taiwan strain from Morocco 2 strains from France 2010 in FL 2010-2012 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.

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.

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 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

Clavibacter michiganensis ssp. michiganensis.