Clavibacter michiganensis subsp. michiganensis (bacterial canker of tomato)
- Taxonomic Tree
- Distribution Table
- Risk of Introduction
- Hosts/Species Affected
- Host Plants and Other Plants Affected
- Growth Stages
- List of Symptoms/Signs
- Biology and Ecology
- Seedborne Aspects
- Pathway Vectors
- Plant Trade
- Detection and Inspection
- Prevention and Control
- Distribution Maps
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PicturesTop of page
IdentityTop of page
Preferred Scientific Name
- Clavibacter michiganensis subsp. michiganensis (Smith 1910) Davis et al. 1984
Preferred Common Name
- bacterial canker of tomato
Other Scientific Names
- Aplanobacter michiganensis (Smith) Smith 1914
- Bacterium michiganense Smith 1910
- Corynebacterium michiganense (Smith 1910) Jensen 1934
- Corynebacterium michiganense pv. michiganense (Smith) Dye & Kemp 1977
- Corynebacterium michiganense subsp. michiganense (Smith) Carlson & Vidaver 1982
- Erwinia michiganensis (=michiganense) (Smith) Jensen 1934
- Mycobacterium michiganense (Smith) Krasil'nikov 1941
- Phytomonas michiganensis (Smith) Bergey et al. 1923
- Pseudomonas michiganense (Smith) Stevens 1913
- Pseudomonas michiganensis (Smith) Stevens
International Common Names
- English: bird's eye of tomato fruit; vascular tomato wilt
- Spanish: cancer bacteriano del tomate; marchitamiento bacteriano del tomate; ojo de pajaro
- French: chancre bactérien de la tomate
Local Common Names
- Germany: Bakterien-: Tomate Krebs; Bakterien-: Tomate Welke
- Italy: cancro batterico
- CORBMI (Clavibacter michiganensis)
Taxonomic TreeTop of page
- Domain: Bacteria
- Phylum: Actinobacteria [phylum]
- Class: Actinobacteria
- Subclass: Actinobacteridae
- Order: Actinomycetales
- Suborder: Micrococcineae
- Family: Microbacteriaceae
- Genus: Clavibacter
- Species: Clavibacter michiganensis subsp. michiganensis
DescriptionTop of page
DistributionTop of page
Distribution TableTop of page
The distribution in this summary table is based on all the information available. When several references are cited, they may give conflicting information on the status. Further details may be available for individual references in the Distribution Table Details section which can be selected by going to Generate Report.Last updated: 30 Jun 2021
|Continent/Country/Region||Distribution||Last Reported||Origin||First Reported||Invasive||Reference||Notes|
|Algeria||Absent, Invalid presence record(s)|
|Congo, Democratic Republic of the||Absent, Unconfirmed presence record(s)|
|South Africa||Present, Widespread|
|South Korea||Present, Localized|
|Taiwan||Absent, Unconfirmed presence record(s)|
|Thailand||Absent, Unconfirmed presence record(s)|
|Vietnam||Absent, Unconfirmed presence record(s)|
|Belgium||Absent, Formerly present|
|Czechia||Present, Few occurrences|
|Finland||Absent, Invalid presence record(s)|
|Germany||Present, Localized||First reported: 193*|
|Ireland||Absent, Eradicated||First reported: 194*|
|Jersey||Absent, Formerly present|
|Netherlands||Present, Transient under eradication|
|Poland||Present, Few occurrences|
|Portugal||Present, Few occurrences|
|-Central Russia||Present, Widespread|
|-Southern Russia||Present, Widespread|
|Serbia and Montenegro||Present, Localized|
|Slovakia||Absent, Intercepted only|
|Slovenia||Present, Transient under eradication|
|Spain||Present, Few occurrences|
|-Canary Islands||Present, Transient under eradication|
|Union of Soviet Socialist Republics||Present|
|United Kingdom||Absent, Formerly present|
|-Channel Islands||Absent, Formerly present|
|-England||Absent, Formerly present|
|-Scotland||Absent, Formerly present|
|Martinique||Absent, Unconfirmed presence record(s)|
|United States||Present, Widespread|
|-North Dakota||Present, Few occurrences|
|-New South Wales||Present|
|New Zealand||Present, Localized|
Risk of IntroductionTop of page
Economic Importance High
Seedborne Incidence Moderate
Seed Transmitted Yes
Seed Treatment Yes
Overall Risk Moderate
Notes on Phytosanitary Risk
C. michiganensis subsp. michiganensis is an economically important pathogen that is seed transmitted. It should be considered of moderate phytosanitary risk due to its worldwide distribution and the availability of seed treatments to reduce seedborne inoculum.
Hosts/Species AffectedTop of page
Host Plants and Other Plants AffectedTop of page
Growth StagesTop of page
SymptomsTop of page
In advanced infection, vascular discoloration is seen as brown streaks on the stem and petiole. On cutting stems, petioles and peduncles, particularly at their junctions, a creamy-white, yellow or reddish-brown discoloration of vascular tissue and pith, and cavities within the pith will be evident. Sometimes, a very light pink discoloration of the vascular tissue may cause it to be confused with Verticillium or Fusarium wilt. Under certain circumstances, the brown streaks on the stem or petiole darken and split open as cankers, giving the disease its name. Secondary infections late in the growing season may cause marginal leaf necrosis. Fruits may remain small and fall prematurely, or ripen unevenly. They also often show external marbling and internal bleaching of vascular and surrounding tissue.
Under greenhouse conditions, the first symptom is a reversible wilting of leaves during hot weather, later becoming irreversible. The whole plant then desiccates. Leaves may show white interveinal areas, turning brown and necrotic generally before wilt symptoms appear.
The symptoms described are variable and are influenced by plant age, cultivar type and environmental factors. See Gleason et al. (1993) and Strider (1969) for descriptions and illustrations of canker symptoms.
List of Symptoms/SignsTop of page
|Fruit / lesions: scab or pitting|
|Leaves / necrotic areas|
|Leaves / wilting|
|Leaves / yellowed or dead|
|Stems / canker on woody stem|
|Stems / dieback|
|Stems / discoloration|
|Whole plant / dwarfing|
|Whole plant / plant dead; dieback|
Biology and EcologyTop of page
Although populations of the canker bacteria decline rapidly when the crop residue is decomposing in the soil, they seem to persist long enough in unbroken crop residue lying on the ground surface to initiate the disease in the following season on new plantings (Gleason et al., 1991; Chang et al., 1992a). Canker bacteria can also survive on volunteer tomato seedlings and alternative host species which can serve as sources of infection (Strider, 1969).
In the field, canker bacteria can spread from plants with primary infection to nearby plants by water splash, movement of machinery, or by people working when the field is wet resulting in marginal necrosis of leaves and fruit spotting (Ricker and Riedel, 1993). Canker bacteria also persist in epiphytic populations on the leaf surface of tomato plants. They cause wilting of leaves and yield reduction (Tsiantos, 1987; Gleason et al, 1991; Chang et al., 1992a).
Young plants have been shown to be more susceptible to canker (Van Vaerenbergh and Chauveau, 1985). Under natural conditions, tomato plants seem to be susceptible throughout their life (Rat et al., 1991). Variability in the length of incubation before symptom expression of bacterial canker depends on plant age, degree of resistance, temperature and inoculum concentration. In a quantitative study of these factors, it was shown that canker symptoms took longer to express and were less severe on older plants, on moderately resistant cultivars, when temperatures were cooler or warmer than 25°C, and under conditions of lower inoculum concentration. Conditions supporting rapid disease development also favour more severe symptoms (Chang et al., 1992b).
The bacterium is located in the xylem vessels (Leyns and De Cleene, 1983) where it can cause lysigenous cavities. Infected vessels contain viscous granular deposits, tylosis and bacterial masses (Marte, 1980). The pathogen produces a toxic glycopeptide which has biological activity (Miura et al., 1986). The pathogen is also reported to produce a heat-labile toxin and a heat-stable polysaccharide (Ueno et al., 1994). A high molecular weight extracellular polysaccharide produced by the bacterium in vitro was found to be similar to the one produced in planta and was suspected to be associated with the production of wilt symptoms in tomato plants (Bulk et al., 1991). Virulence of the canker pathogen has been shown to be associated with a plasmid DNA fragment accounting for the expression of the pathogenic phenotype (Meletzus et al., 1993).
Seedborne AspectsTop of page
The incidence of infection of tomato seeds by C. michiganensis subsp. michiganensis reported in the literature varies from <1 to 97% (Strider, 1969; Chang et al., 1991; Dhanvantari and Brown, 1993). This variability may be attributed to the age of seed stocks and seed storage conditions. In storage at room temperature, the viability of seed infection in naturally infected seeds harvested from the field declined from 82 to <1% in 18 months and to 0% in 2 years. In storage at 4°C and 60% RH, the general seed storage conditions and viability of seed infection declined from 100 to <5% in 3 years and 6 months (Dhanvantari, 1993). Indirect evidence from seed disinfection studies indicate that a small percentage of seed infection may be deep-seated within the seed tissue (Dhanvantari and Brown, 1993).
Effect on Seed Quality
Infected tomato seeds are usually indistinguishable from healthy seeds. Germination of infected seeds and the ensuing seedling stand also appear normal (Dhanvantari, 1989; Gitaitis et al., 1991; Chang et al., 1992a).
Studies on transmission of seedborne C. michiganensis subsp. michiganensis were carried out with seeds harvested from naturally-infected tomato plants (Dhanvantari, 1989); seed from artificially-inoculated plants (Dhanvantari, 1989; Chang et al., 1992a; Dhanvantari and Brown, 1993); and artificially-infested seeds (Tsiantos, 1987). Infection levels in plants grown from infected seeds varied from ca 1% (Tsiantos, 1987; Chang et al., 1992a), 24-53% (Dhanvantari and Brown, 1993) and 80-90% (Dhanvantari, 1989), depending upon the proportion of infected seeds in the seed batches and seed storage conditions (Dhanvantari, 1993).
More recently, Hadas et al. (2005) described from 0.05% to 4% incidence of bacterial canker in tomato seedlings grown from seed lots containing from 58 to 1,000 c.f.u./g seed, finding a high correlation between c.f.u./g seed and disease incidence. Concentrations of C. michiganensis subsp. michiganensis in individual seeds can be highly variable (Kaneshiro and Alvarez, 2003). Populations of 102-104 c.f.u./seed were estimated on naturally infected seeds (Fatmi and Schaad, 1988; Hadas et al., 2005) and 102 c.f.u./seed has been suggested as the probable threshold level for transmission of the pathogen (Kaneshiro et al., 2008). Real-time colonization studies of germinating seeds using a bioluminescent strain showed that C. michiganensis subsp. michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination (Xu et al., 2010).
Even low seed transmission rates may result in increased disease incidence as a result of grafting (Xu et al., 2010), trimming, packaging and transportation of transplants (Chang et al., 1991; Gitaitis et al., 1991); pruning and tying of trellis and staked tomato plants; or deleafing, suckering and tying operations in the greenhouse. Kritzman (1991) reported a seed health assay that enables the determination a minimal threshold of C. michiganensis subsp. michiganensis that is related to the percentage of diseased seedlings that develop from the same seed lot. Although the relative weight of contaminated seeds as a source of inoculum depends upon the secondary spread of the pathogen, it has been assumed that seed contamination rates as low as 0.01-0.05% (one to five seeds per 10,000) could be enough to initiate an epidemic of bacterial canker in production fields (Chang et al., 1991; Gitaitis et al., 1991).
The pathogen may survive in infected plant debris or on stakes and trellises for long enough to establish a resident population on newly planted tomato seedlings in the following season. This leads to infection via broken trichomes on the leaves or through wounds (Layne, 1967; Gleason et al., 1991; Shirakawa et al., 1991; Chang et al., 1992a). Entry of the bacteria into the leaf may also be gained by withdrawal of contaminated guttation drops through hydathodes (Carlton et al., 1992) or by injury caused by chemical sprays (Farley and Miller, 1973). The role of these local sources of inoculum in bacterial canker was supported by epidemiological studies using molecular typing of natural populations of C. michiganensis subsp. michiganensis. These indicate that once the pathogen has been introduced into a region via infected seeds or seedlings, plant debris could be the prime source of inoculum in subsequent years (Keitman et al., 2008; De León et al., 2009; Kawaguchi et al., 2010).
In nutrient film technique (NFT) culture of tomatoes growing in recirculating nutrient solution, it was shown that low populations of the bacterium are sufficient to infect the plants via roots at all stages of development (Griesbach and Lattauschke, 1991). Canker was shown to spread from infected transplants to other plants in recirculating NFT culture with a lag of ca 4 weeks before brown necrotic spots on the leaves and wilt symptoms began to appear on other plants (Dhanvantari, 1995).
Seed treatments for the eradication of C. michiganensis subsp. michiganensis from seeds include fermentation, hot water, hydrochloric acid, calcium hypochlorite and sodium hypochlorite, and o-hydroxydiphenyl (o-phenylphenol). Fermentation is routinely used to extract tomato seeds while reducing the population of the canker bacteria, but it can take as long as 96 h to eradicate them. Hydrochloric acid (HCl) has been reported to be consistently effective in eradicating the canker bacteria from tomato seeds. The higher the HCl concentration and the longer the period of treatment, the greater the effectiveness against C. michiganensis subsp. michiganensis, but seed germination can be negatively affected. HCl was used to treat the tomato pulp in seed extraction. This treatment, followed by drying the seeds for 3 h, achieved pathogen eradication (Thyr et al., 1973). In contrast, acid extraction by soaking pulp in an equal volume of 5% HCl for 10 min followed by washing did not entirely eliminate C. michiganensis subsp. michiganensis from naturally infected seeds (Pradhanang and Collier, 2009). HCl has also been used to soak dry seeds at 1.9% for 5-10 h (Shoemaker and Echandi, 1976; note that the concentration of HCl was erroneously mentioned as 5% in this report but corrected to 1.9% in an attachment to Plant Disease Reporter, 60:454); at 0.6 M (1.9%) for 1 h followed by rinsing in water (otherwise seed germination and seedling emergence would be reduced) (Dhanvantari, 1989) or at 0.1 M without the need for rinsing afterwards (Dhanvantari and Brown, 1993). Tomato pulp can be treated with pectinase-HCl to extract and disinfect the seeds in one step instead of fermentation which takes longer and produces inconsistent results (Dhanvantari, 1989).
In other seed treatments, 0.05% (w/v) o-phenyl phenol was as effective as HCl in disinfecting seed and reducing field incidence of canker (Dhanvantari, 1989; Dhanvantari and Brown, 1993). Calcium hypochlorite is favoured over sodium hypochlorite by the tomato seed industry as it is safe to handle and does not affect seed quality. However, neither treatment at 0.5% (w/v) completely eradicated the pathogen from the seeds. Hot-water treatment at 56°C for 30 min eradicated the pathogen from tomato seeds but resulted in 10-15% reduction in seed germination (Fatmi et al., 1991). Treating tomato seeds with hot water at 50°C for 25 min was effective in disinfecting without impairing seed germination and seedling emergence (Dhanvantari, 1994). Using an accurate thermometer and stirring the water and bags of seed continually to secure rapid penetration of heat to maintain uniform temperature in the seed batches are important procedures in hot-water treatment.
Seed Health Tests
Seed testing is an essential tool for the control of C. michiganensis subsp. michiganensis and is important for its regulation and control through phytosanitary certification and quarantine programs in the domestic and international seed trade (De León et al., 2011). Traditionally, tomato seed testing for C. michiganensis subsp. michiganensis detection was based on dilution plating of seed extracts on semi-selective media followed by pathogenicity tests as the most practical method to screen a large number of seed lots for canker contamination (Maddox, 1997). However, these protocols show weaknesses in several performance criteria, due to the time required, the interference of saprophytic bacteria and the low level of infection that may be present in naturally infected seeds (De León et al., 2011). Currently, these limitations have led to serological and PCR-based methods being introduced in seed health testing, as a complement to the culture-based standards.
Serological techniques used to detect and identify C. michiganensis subsp. michiganensis include agglutination tests, indirect immunofluorescence (IF) (van Vaerenberg and Chauveau, 1987; Franken et al., 1993), immunofluorescence colony staining (IFC) (Veena and van Vuuder, 2002), enzyme-linked immunosorbent assays (ELISA) (Kramer and Griesbach, 1995), lateral flow devices (e.g. immunostrips) and flow cytometry (Alvarez and Adams, 1999). In addition, immunocapture techniques such as immunomagnetic separation (IMS) can be used to capture target cells from seed extracts prior to plating (De León et al., 2006; De León et al., 2008). Polyclonal antibodies (PAbs) are commercially available for IF, ELISA, agglutination tests or immunostrips, for example, from Loewe, Neogen or Plant Research International.
Monoclonal antibodies (MAbs) have shown higher titer and specificity against C. michiganensis subsp. michiganensis than PAbs and the MAb Cmm1 (Alvarez et al., 1993; Kaneshiro et al., 2006) is available in commercial kits for ELISA and immunostrips from Agdia. The most frequently used serological test for the detection of C. michiganensis subsp. michiganensis in tomato seed is IF, with a detection limit of approximately 103 cells/ml. This threshold is improved up to 10-102 cells/ml by soaking the seed samples for three days at room temperature before IF (Olivier et al., 2010).
Different pairs of primers have been specifically designed for C. michiganensis subsp. michiganensis, such as primers CMM5/CMM6 (Dreier et al., 1995), CM3/CM4 (Sousa-Santos et al., 1997), or PSA-4/PSA-R (Pastrik and Rainey, 1999). It is however advisable to use more than one set of primers to obtain more reliable PCR results. Furthermore, DNA purification is necessary to avoid false negatives due to the presence of PCR inhibitors in seed extracts. In practice, the detection limit of C. michiganensis subsp. michiganensis by direct PCR is about 103 c.f.u./ml. Several strategies have been developed to increase this sensitivity, such as Bio-PCR, which involves previous multiplication of the putative pathogen on solid media and subsequent PCR amplification on washes from culture plates. According to Hadas et al. (2005), Bio-PCR was able to detect one contaminated seed in 10,000. In addition to conventional PCR, real-time PCR protocols have recently been developed (Bach et al., 2003; Zhao et al., 2007; Luo et al., 2008). Multiplex PCR has also been described for the simultaneous detection of C. michiganensis subsp. michiganensis together with other seedborne pathogens, using previously published primers and either conventional (Özdemir, 2009) or real-time PCR (Johnson and Walcott, 2012).
Standard protocols for tomato seed testing
For C. michiganensis subsp. michiganensis, standard seed test protocols are available from the European and Mediterranean Plant Protection Organization (EPPO, 2005) and the International Seed Federation (ISF) through the International Seed Health Initiative for Vegetable Crops (ISHI-Veg) (ISHI, 2011). The recommended sample size is 10,000 seeds, providing a 95% statistical probability of detecting a 0.03% level of contamination in the seed lot. Both protocols are based on the plating of seed extracts on semi-selective media to isolate the pathogen, followed by a pathogenicity assay. In addition, serological and PCR-based methods have been incorporated to current protocols for presumptive diagnosis and/or identification purposes. These protocols are revised periodically (a new version of the EPPO protocol will be published shortly) and they are available on the ISHI Webpage: http://www.worldseed.org/isf/ishi_vegetable.html and EPPO Webpage: http://archives.eppo.int/EPPOStandards/diagnostics.htm, with a full explanation of seed extract preparation, semi-selective media and pathogenicity tests, as well as the complete protocols for serological and PCR tests.
Published methods are listed as follows:
Semi-selective medium (Fatmi and Schaad, 1988)
Modified semi-selective medium (Waters and Bolkan, 1992)
Semi-selective medium/host plant inoculation (Valarini, 1995)
Immunofluorescence (Franken et al., 1993; van Vaerenbergh and Chauveau, 1987)
ELISA (Kramer and Griesbach, 1995)
PCR (Dreier et al., 1995; Sousa-Santos et al., 1997)
Liquid Plating Assay (Bolkan et al., 1997)
Extraction of the bacterium
1. Place 24 g of the seed sample (approximately 10,000 seeds) in a doubled plastic bag (20 cm x 25 cm and 0.15 mm thick) containing 150 ml sterile phosphate-Tween buffer (7.75 g/l of Na2HPO4 + 1.65 g/l KH2PO4 + 0.2 ml/l Tween 20), pH 7.4.
2. Incubate the plastic bag with its contents in a refrigerator at 4°C for 15 min.
3. After refrigeration, place the plastic bag with its contents in a stomacher (Lab Blender Model 400 Mark II) and blend for 15 min. Double bagging of the seed sample is recommended as insurance against breakage and loss of liquid.
Semi-selective medium for C. michiganensis subsp. michiganensis - SCM (Fatmi and Schaad, 1988)
1. Dissolve 2 g K2HPO4; 0.5 g KH2PO4; 0.25 g MgSO4.7H2O; 1.5 g boric acid; 10 g sucrose, 0.1 g yeast extract and 15 g of agar in 980 ml distilled water.
2. After autoclaving, cool to 45-50°C in a water bath and add 100 mg nicotinic acid (dissolved in 20 ml sterile distilled water); 30 mg nalidixic acid (sodium salt, dissolved in 1 ml of 0.1 M NaOH); 10 mg potassium tellurite (1 ml of 1% Chapman tellurite solution from Difco); and 200 mg cycloheximide (dissolved in 1 ml absolute methanol).
Modified semi-selective medium for C. michiganensis subsp. michiganensis - mSCM (Waters and Bolkan, 1992)
1. Dissolve 2.62 g K2HPO4.3H20; 0.5 g KH2PO4; 0.25 g MgSO4.7H2O; 1.5 g boric acid; 10 g mannose; and 0.1 g yeast extract in 980 ml distilled water.
2. Add 1 drop (1 ml pipette) of pourite (Baxter Healthcare Corporation, Scientific Division, McGaw Park, IL 60085, USA) and 12 g of agar.
3. After autoclaving, add 100 mg nicotinic acid 30 mg nalidixic acid and 200 mg cycloheximide as previously described for SCM media.
Note on methods: Distribute both media into Petri plates at the rate of 20 ml/plate and store at 4°C until needed.
Yeast extract-dextrose-CaCO2 agar - YDC (Schaad, 1988)
1. This contains 10 g yeast extract, 20 g light powder CaCO3 (Sigma, No. C-6763), 20 g glucose and 15 g agar per 1000 ml.
2. The glucose should be autoclaved separately and the medium cooled to 50°C before pouring plates (the final medium should be white throughout).
Culturing the bacteria
1. Pipette 0.1 ml of 0, 1:10, 1:100 dilutions (prepared using phosphate buffer without Tween) of each sample onto each of the three plates of SCM and mSCM media. Spread with an L-shaped glass rod and incubate at 26°C.
2. Examine the SCM plates after 10 days. C. michiganensis subsp. michiganensis colonies on SCM are convex, irregular, mucoid with internal black flecks.
3. Examine the mSCM plates after 7 and 10 days. At 7 days, C. michiganensis subsp. michiganensis colonies on mSCM are light grey, 2-3 mm in diam., translucent and easily distinguishable from other mucoid colonies by the presence of many internal flecks (specks). As incubation time increases the colonies become larger and the internal flecks become yellow whereas the non-C. michiganensis subsp. michiganensis colonies remain small and have no internal flecks.
4. Compare suspect colonies to a 7-10-day-old streak of a known culture of C. michiganensis subsp. michiganensis on mSCM and SCM media.
5. Remove suspected colonies with a sterile transfer loop and streak onto YDC agar and incubate at room temperature (24±1°C).
6. Examine for presence of yellow mucoid colonies (must compare to known culture of C. michiganensis subsp. michiganensis on YDC).
7. Confirm identity by immunofluorescence (IF), ELISA and/or pathogenicity test using single colonies.
Note on method: The source of antibodies is AGDIA, 30380 County Rd. 6, Elkhart, Indiana 46514, USA.
Identification of suspected colonies of C. michiganensis subsp. michiganensis by IF staining (Schaad, 1978)
1. Grow suspected colonies and a known strain of C. michiganensis subsp. michiganensis (control) on YDC for 24-48 h.
2. Make a suspension of cells from single YDC colonies using a loop of cells in a drop (0.1 ml) of saline-formalin solution (0.85% NaCl + 10% formalin).
3. After incubating for 5-10 min transfer 10 µl of the bacterial suspension to a fluorescence slide well (6 mm in diam., 8 wells/slides).
4. Flood with Kirkpatrick's solution (60% ethanol, 30% chloroform and 10% formalin) and incubate in moist chambers for 3 min.
5. Rinse with a fixative, drain and air-dry. Add one drop (1 ml pipette) of C. michiganensis subsp. michiganensis antisera and incubate in a moist chamber in the dark for 30 min.
6. Rinse once in saline then in phosphate buffered saline for 10 min. Rinse again in saline then in distilled water and air-dry.
7. Stain with a commercial anti-rabbit goat globulin FITC conjugate as above and mount in 0.5 M carbonate-buffered (pH 9) glycerin.
8. Examine under an epi-fluorescence microscope using 100x objective. Cells that are brightly fluorescent are considered to be positive C. michiganensis subsp. michiganensis cells.
Identification of suspected colonies of C. michiganensis subsp. michiganensis by ELISA (Kramer and Griesbach, 1995)
1. Grow suspected colonies on YDC for 24-48 h.
2. Prepare solutions from an Agdia ELISA reagent set containing peroxidase labelled conjugates (Agdia, Inc., 30380 County Road 6, Elkhart, IN 46514, USA) as follows:
PBS-Tween (pH 7.4: dilute 8 g NaCl, 0.2 g KH2PO4, 2.9 g Na2HPO4.12H20, 0.2 g KCl and 0.5 ml Tween 20 in 1 L of deionized water.
Coating buffer: dilute 1.59 g Na2CO3, and 2.93 g NaHCO3 in 1 L deionized water. Keep refrigerated.
Substrate buffer (pH 9.8): dilute 97 ml of diethanolamine in 800 ml deionized water and adjust pH with HCl. Keep refrigerated.
Extraction buffer: dilute 20 g PVP-40 (polyvinyl pyrrolidone) and 2 g BSA (bovine albumin) in 1 L PBS-Tween. Keep refrigerated.
3. Dilute the C. michiganensis subsp. michiganensis monoclonal antibodies in coating buffer (1:1000 dilution) and load plates by adding 0.2 ml/well.
4. Place plates in closed humid box and incubate at room temperature for 4 h or at 4°C overnight.
5. Remove the coating solution and wash plates by flooding wells with PBS-Tween. Repeat washing three times at 3 min intervals.
6. Dilute suspected colonies (one loop of cells) in 1 ml of extraction buffer and add to duplicate wells (0.2 ml/well). Use extraction buffer and a known culture as controls.
7. Incubate plates at room temperature (24±1°C) for at least 2 h or at 4°C overnight in a closed humid box.
8. Wash plates as previously described.
9. Dilute monoclonal antibodies in the extraction buffer (1:500 dilution) and load plates by adding 0.2 ml/well.
10. Incubate plates at room temperature for 2 h in a closed humid box.
11. Wash plates as previously described.
12. Dilute (1 mg/ml) o-phenylenediamine in substrate solution and load plates by adding 0.2 ml/well.
13. Incubate plates at room temperature in the dark for 15-30 min or until the positive controls develop a dark yellow-orange colour.
14. Stop the reaction by adding a drop of 3 M sulfuric acid to each well.
15. Measure optical density at 490 nm or evaluate visually. Colour intensity is proportional to bacterial concentration. The negative controls (extraction buffer) should be clear with no colour change.
Identification of C. michiganensis subsp. michiganensis by pathogenicity test
1. Grow suspected colonies on YDC for 24-48 h.
2. Sterile scissors dipped in inoculum of C. michiganensis subsp. michiganensis containing approximately 1 million cells/ml are used to cut the stem of each seedling just above the cotyledons.
3. The top part of the seedling is discarded and the inoculated plants are kept in a greenhouse at 25-27°C.
4. Symptoms such as a one-sided wilt of the leaflets on the inoculated side appear within 7-11 days.
Pathway VectorsTop of page
Plant TradeTop of page
|Plant parts liable to carry the pest in trade/transport||Pest stages||Borne internally||Borne externally||Visibility of pest or symptoms|
|Bulbs/Tubers/Corms/Rhizomes||Yes||Pest or symptoms usually visible to the naked eye|
|Flowers/Inflorescences/Cones/Calyx||Yes||Pest or symptoms usually invisible|
|Fruits (inc. pods)||Yes||Yes||Pest or symptoms usually visible to the naked eye|
|Growing medium accompanying plants||Yes||Pest or symptoms usually invisible|
|Leaves||Yes||Pest or symptoms usually visible to the naked eye|
|Roots||Yes||Pest or symptoms usually visible to the naked eye|
|Seedlings/Micropropagated plants||Yes||Pest or symptoms usually invisible|
|Stems (above ground)/Shoots/Trunks/Branches||Yes||Pest or symptoms usually visible to the naked eye|
|True seeds (inc. grain)||Yes||Yes||Pest or symptoms usually invisible|
|Plant parts not known to carry the pest in trade/transport|
ImpactTop of page
DiagnosisTop of page
Isolation of the causal organism can be attempted on nutrient dextrose agar or yeast peptone glucose agar (Lelliott and Stead, 1987). C. michiganensis subsp. michiganensis develops slow-growing, smooth, shining, round, yellow colonies with entire margins. However, white, pink, red and orange mutants do occur (Hayward and Waterson, 1964). Semi-selective media have been found useful for isolation. Popular media are modified versions of CNS agar (Vidaver and Davis, 1988) on which colonies appear in 6-7 days, SCM agar (Fatmi and Schaad, 1988) on which gray-to-black speckled colonies are formed, and m-SCM agar (Waters and Bolkan, 1992), a modification of SCM agar on which clear colonies with yellow flecks appear in 7-9 days. See Gleason et al. (1993) for details on these 'classical' semi-selective media for isolation of C. michiganensis subsp. michiganensis. New semi-selective media, as well as new improvement of SCM medium (Koenraadt et al., 2009), have further been reported, such as CMM1 agar (Kaneshiro et al., 2006) on which colonies of C. michiganensis subsp. michiganensis are yellow, mucoid and convex, and BCT agar (Ftayeh et al., 2011) on which typical colonies appear creamy to yellow in color, convex and shining.
Bacterial canker of tomato can be diagnosed by the symptoms described (see Symptoms) and by isolation of the causal organism on a non-selective medium or a semi-selective medium followed by a pathogenicity test on a 2- to 4-leaf-stage tomato seedling. The test is carried out by stab inoculation of the seedling at a node with a sterile toothpick charged with fresh inoculum from the selected colonies growing on the isolation plates. Variations of this technique include infiltrating a water-suspension of the inoculum (100,000,000 c.f.u./ml) with a syringe, or excising a leaflet on the first or second leaf with a pair of scissors contaminated with the inoculum (van Steekelenberg, 1985; Gitaitis et al., 1989). Leaf margin curling and one-sided wilting or withering of the leaves in the vicinity of inoculation will occur within 2-3 weeks if the inoculum used is virulent.
Following this approach, the European and Mediterranean Plant Protection Organization has published a protocol for diagnosis of C. michiganensis subsp. michiganensis in symptomatic plants (EPPO, 2005). According to the EPPO protocol, suspensions of affected vascular tissue, leaf or fruit spots should be plated on standard nutrient medium and on semi-selective medium. Presumptive colonies should be purified and identified by biochemical characteristics, serological tests or PCR. Pathogenicity of presumptive isolates is confirmed by inoculating tomato seedling. Suspensions of diseased tomato tissue can also be tested by rapid tests (IF and/or PCR) for presumptive diagnosis, but isolation is necessary for a positive detection. The detailed protocol, with full explanation about the preparation of plant tissue suspensions, dilution plating, media, rapid tests, identification of presumptive isolates and pathogenicity test, can be found on the EPPO Webpage: http://archives.eppo.int/EPPOStandards/diagnostics.htm.
Yasuhara-Bell et al. (2013) have developed a diagnostic method using loop-mediated amplification (LAMP) to detect Clavibacter michiganensis subsp. michiganensis.
Detection and InspectionTop of page
Media that were available earlier such as SCM agar (Fatmi and Schaad, 1988) and SMCMM agar (Shirakawa and Sasaki, 1988) may not be sensitive enough due to many antagonists present in the saprophytic flora, or may be too toxic, thus delaying the development of the colonies of the canker bacterium. Modified versions of semi-selective medium CNS agar from which lithium chloride and polymyxin b sulfate are deleted (Vidaver and Davis, 1988) and of SCM (m-SCM) with the deletion of tellurite and the replacement of sucrose with mannose (Waters and Bolkan, 1992), are being used by seed companies in the USA.
Serological methods are sensitive (Rat, 1984) but there are difficulties in obtaining sufficiently specific antisera. Specific and sensitive ELISA methods have been developed and it is claimed that they are useful in the routine analysis of latent infection (Gitaitis et al., 1991; Kramer and Griesbach, 1995). Immunofluorescence staining combined with bioassays have been described (van Vaerenbergh and Chauveau, 1987; Franken et al., 1993) which are specific and sensitive, but expensive and time-consuming. Other methods including fatty acid profiles (Gitaitis and Beaver, 1990), molecular hybridization (Thompson et al., 1989), protein profiles (Bruyne et al., 1987) and PCR methods (Ghedini and Fiore, 1995) have become available. Recently, PCR-based molecular probes specific for C. michiganensis subsp. michiganensis and its virulent strains have been reported (Dreier et al., 1995).
Prevention and ControlTop of page
Due to the variable regulations around (de)registration of pesticides, your national list of registered pesticides or relevant authority should be consulted to determine which products are legally allowed for use in your country when considering chemical control. Pesticides should always be used in a lawful manner, consistent with the product's label.Improved management of bacterial canker in tomato has been shown to be possible by the use of healthy seeds, seed treatment, appropriate cultural practices, chemical sprays where needed, hygiene and sanitation (Gleason et al., 1993).
Recent advances have introduced more efficient techniques to aid seed health testing, and many seed companies are using one or more of them. A substantial reduction of infection can be achieved by acid extraction of seeds or treatment of seeds with acid or other disinfectants or hot water (Shoemaker and Echandi, 1976; Dhanvantari, 1989; Fatmi et al., 1991; Dhanvantari and Brown, 1993; Dhanvantari, 1994).
Cultural practices such as deep ploughing to bury infected crop residue after harvest to accelerate decomposition, and crop rotation away from solanaceous crops for at least 2 years, are recommended to reduce the incidence of canker (Gleason et al., 1991). Production of tomato transplants in greenhouses planted in soilless medium in plastic trays, has been found to be feasible and more reliable than field-grown transplants for reducing the risk of bacterial canker (Gleason et al., 1993). They form part of the current production recommendations in Canada and the USA. Copper-based chemicals are usually sprayed on tomato for controlling bacterial diseases but their effect on canker is poorly documented. However, under conditions of frequent rainfall and prolonged wet periods, chemical sprays with copper-containing compounds have been found useful in reducing foliar blight and fruit spotting (Shoemaker, 1992).
Sources of resistance are available (Gardner et al., 1990; Poysa, 1993; van Steekelenberg, 1985) but have not yet been introduced into commercial cultivars.
In protected crops, strict hygiene measures such as early detection, isolation and eradication of infected plants, destruction of crop residues, rinsing hands/gloves and pruning tools with a disinfectant after working each row, and disinfection of structures and equipment are essential to manage canker. See Jarvis (1992) for general greenhouse hygiene.
ReferencesTop of page
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Sepúlveda Chavera G F, Salvatierra Martínez R, Sandoval Briones C, González Vásquez R, 2013. First report of tomato bacterial canker Clavibacter michiganensis subsp. michiganensis on tomato crops in Arica. IDESIA. 31 (2), 99-101. http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0718-34292013000200014&lng=es&nrm=iso&tlng=en
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