Invasive Species Compendium

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Datasheet

Clavibacter michiganensis
(bacterial canker of tomato)

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Datasheet

Clavibacter michiganensis (bacterial canker of tomato)

Summary

  • Last modified
  • 16 November 2021
  • Datasheet Type(s)
  • Invasive Species
  • Pest
  • Natural Enemy
  • Preferred Scientific Name
  • Clavibacter michiganensis
  • Preferred Common Name
  • bacterial canker of tomato
  • Taxonomic Tree
  • Domain: Bacteria
  •   Phylum: Actinobacteria [phylum]
  •     Class: Actinobacteria
  •       Subclass: Actinobacteridae
  •         Order: Actinomycetales
  • Summary of Invasiveness
  • Since the first report of the disease in the USA in 1910, bacterial canker has spread throughout the world and causes serious losses to both greenhouse and field tomato (Solanum lycopersicum) crops either by killing the young plants or re...

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Pictures

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PictureTitleCaptionCopyright
Clavibacter michiganensis (bacterial canker of tomato); diseased greenhouse tomato plants.
TitleSymptoms
CaptionClavibacter michiganensis (bacterial canker of tomato); diseased greenhouse tomato plants.
Copyright©B.N. Dhanvantari
Clavibacter michiganensis (bacterial canker of tomato); diseased greenhouse tomato plants.
SymptomsClavibacter michiganensis (bacterial canker of tomato); diseased greenhouse tomato plants.©B.N. Dhanvantari
Clavibacter michiganensis (bacterial canker of tomato); Severe infection of tomato plants with the bacterial canker pathogen Clavibacter michiganensis leading to overall wilting and plant death. August 2020.
TitleField symptoms
CaptionClavibacter michiganensis (bacterial canker of tomato); Severe infection of tomato plants with the bacterial canker pathogen Clavibacter michiganensis leading to overall wilting and plant death. August 2020.
Copyright©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Severe infection of tomato plants with the bacterial canker pathogen Clavibacter michiganensis leading to overall wilting and plant death. August 2020.
Field symptomsClavibacter michiganensis (bacterial canker of tomato); Severe infection of tomato plants with the bacterial canker pathogen Clavibacter michiganensis leading to overall wilting and plant death. August 2020.©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Distant view of tomato plants showing bacterial canker symptoms, which were grown from Clavibacter michiganensis-infected seed lots. August 2020.
TitleField symptoms
CaptionClavibacter michiganensis (bacterial canker of tomato); Distant view of tomato plants showing bacterial canker symptoms, which were grown from Clavibacter michiganensis-infected seed lots. August 2020.
Copyright©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Distant view of tomato plants showing bacterial canker symptoms, which were grown from Clavibacter michiganensis-infected seed lots. August 2020.
Field symptomsClavibacter michiganensis (bacterial canker of tomato); Distant view of tomato plants showing bacterial canker symptoms, which were grown from Clavibacter michiganensis-infected seed lots. August 2020.©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Interveinal necrosis and wilting of tomato plant infected with C. michiganensis.  June 2020.
TitleField symptoms
CaptionClavibacter michiganensis (bacterial canker of tomato); Interveinal necrosis and wilting of tomato plant infected with C. michiganensis. June 2020.
Copyright©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Interveinal necrosis and wilting of tomato plant infected with C. michiganensis.  June 2020.
Field symptomsClavibacter michiganensis (bacterial canker of tomato); Interveinal necrosis and wilting of tomato plant infected with C. michiganensis. June 2020.©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Typical symptoms (extended canker on stem) of the bacterial canker of tomato caused by Clavibacter michiganensis in the final stages of infection leading to plant death. August 2020.
TitleSymptoms
CaptionClavibacter michiganensis (bacterial canker of tomato); Typical symptoms (extended canker on stem) of the bacterial canker of tomato caused by Clavibacter michiganensis in the final stages of infection leading to plant death. August 2020.
Copyright©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Typical symptoms (extended canker on stem) of the bacterial canker of tomato caused by Clavibacter michiganensis in the final stages of infection leading to plant death. August 2020.
SymptomsClavibacter michiganensis (bacterial canker of tomato); Typical symptoms (extended canker on stem) of the bacterial canker of tomato caused by Clavibacter michiganensis in the final stages of infection leading to plant death. August 2020.©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Typical symptoms (extended canker on stem) of the bacterial canker of tomato caused by Clavibacter michiganensis in the earlier stages of infection. August 2020.
TitleSymptoms
CaptionClavibacter michiganensis (bacterial canker of tomato); Typical symptoms (extended canker on stem) of the bacterial canker of tomato caused by Clavibacter michiganensis in the earlier stages of infection. August 2020.
Copyright©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Typical symptoms (extended canker on stem) of the bacterial canker of tomato caused by Clavibacter michiganensis in the earlier stages of infection. August 2020.
SymptomsClavibacter michiganensis (bacterial canker of tomato); Typical symptoms (extended canker on stem) of the bacterial canker of tomato caused by Clavibacter michiganensis in the earlier stages of infection. August 2020.©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Symptoms of the bacterial canker of tomato caused by Clavibacter michiganensis in the interior part of ripening fruit. Discoloration of placenta from white to deep yellow indicates the infection. August 2020.
TitleFruit symptoms
CaptionClavibacter michiganensis (bacterial canker of tomato); Symptoms of the bacterial canker of tomato caused by Clavibacter michiganensis in the interior part of ripening fruit. Discoloration of placenta from white to deep yellow indicates the infection. August 2020.
Copyright©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Symptoms of the bacterial canker of tomato caused by Clavibacter michiganensis in the interior part of ripening fruit. Discoloration of placenta from white to deep yellow indicates the infection. August 2020.
Fruit symptomsClavibacter michiganensis (bacterial canker of tomato); Symptoms of the bacterial canker of tomato caused by Clavibacter michiganensis in the interior part of ripening fruit. Discoloration of placenta from white to deep yellow indicates the infection. August 2020.©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); bird's eye-spot of tomato fruit.
TitleSymptoms
CaptionClavibacter michiganensis (bacterial canker of tomato); bird's eye-spot of tomato fruit.
Copyright©B.N. Dhanvantari
Clavibacter michiganensis (bacterial canker of tomato); bird's eye-spot of tomato fruit.
SymptomsClavibacter michiganensis (bacterial canker of tomato); bird's eye-spot of tomato fruit.©B.N. Dhanvantari
Clavibacter michiganensis (bacterial canker of tomato); unilateral leaf wilt of an inoculated tomato plant.
TitleSymptoms
CaptionClavibacter michiganensis (bacterial canker of tomato); unilateral leaf wilt of an inoculated tomato plant.
Copyright©B.N. Dhanvantari
Clavibacter michiganensis (bacterial canker of tomato); unilateral leaf wilt of an inoculated tomato plant.
SymptomsClavibacter michiganensis (bacterial canker of tomato); unilateral leaf wilt of an inoculated tomato plant.©B.N. Dhanvantari
Clavibacter michiganensis (bacterial canker of tomato); stem canker and leaf wilt of an inoculated tomato plant.
TitleSymptoms
CaptionClavibacter michiganensis (bacterial canker of tomato); stem canker and leaf wilt of an inoculated tomato plant.
Copyright©B.N. Dhanvantari
Clavibacter michiganensis (bacterial canker of tomato); stem canker and leaf wilt of an inoculated tomato plant.
SymptomsClavibacter michiganensis (bacterial canker of tomato); stem canker and leaf wilt of an inoculated tomato plant.©B.N. Dhanvantari
Clavibacter michiganensis (bacterial canker of tomato); interveinal chlorosis of tomato leaves.
TitleSymptoms
CaptionClavibacter michiganensis (bacterial canker of tomato); interveinal chlorosis of tomato leaves.
Copyright©B.N. Dhanvantari
Clavibacter michiganensis (bacterial canker of tomato); interveinal chlorosis of tomato leaves.
SymptomsClavibacter michiganensis (bacterial canker of tomato); interveinal chlorosis of tomato leaves.©B.N. Dhanvantari
Clavibacter michiganensis (bacterial canker of tomato); Pure culture and single colonies of Clavibacter michiganensis on YPGA medium. September 2020.
TitleAgar Culture
CaptionClavibacter michiganensis (bacterial canker of tomato); Pure culture and single colonies of Clavibacter michiganensis on YPGA medium. September 2020.
Copyright©Dr Ebrahim Osdaghi, University of Tehran, Iran
Clavibacter michiganensis (bacterial canker of tomato); Pure culture and single colonies of Clavibacter michiganensis on YPGA medium. September 2020.
Agar CultureClavibacter michiganensis (bacterial canker of tomato); Pure culture and single colonies of Clavibacter michiganensis on YPGA medium. September 2020.©Dr Ebrahim Osdaghi, University of Tehran, Iran

Identity

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Preferred Scientific Name

  • Clavibacter michiganensis (Smith 1910; Davis et al. 1984) Li et al. 2018

Preferred Common Name

  • bacterial canker of tomato

Other Scientific Names

  • Aplanobacter michiganensis (Smith) Smith 1914
  • Bacterium michiganense Smith 1910
  • Clavibacter michiganensis subsp. michiganensis (Smith 1910) Davis et al. 1984
  • 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

EPPO code

  • CORBMI

Summary of Invasiveness

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Since the first report of the disease in the USA in 1910, bacterial canker has spread throughout the world and causes serious losses to both greenhouse and field tomato (Solanum lycopersicum) crops either by killing the young plants or reducing marketable yields. C. michiganensis is an economically important pathogen that is seed transmitted and one of the most destructive seedborne agents of tomato worldwide. The disease occurs in many countries on tomato and pepper (Capsicum) with a particular importance in regions characterized by rainy and humid summers, or areas where sprinkler irrigation is used. As a seedborne pathogen, C. michiganensis is included in the A2 (high risk) list of quarantine pathogens by European and Mediterranean Plant Protection Organization; hence, it is under strict quarantine control and zero tolerance. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning and harvesting.

Taxonomic Tree

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  • Domain: Bacteria
  •     Phylum: Actinobacteria [phylum]
  •         Class: Actinobacteria
  •             Subclass: Actinobacteridae
  •                 Order: Actinomycetales
  •                     Suborder: Micrococcineae
  •                         Family: Microbacteriaceae
  •                             Genus: Clavibacter
  •                                 Species: Clavibacter michiganensis

Notes on Taxonomy and Nomenclature

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Clavibacter michiganensis, the causal agent of tomato bacterial canker, was first reported by Erwin F. Smith in Grand Rapids, Michigan (USA) in 1909. Accordingly, the disease was named at that time as ‘the Grand Rapids tomato disease’ and the causal agent was named as Bacterium michiganense (Smith, 1910). The name was later changed to Aplanobacter michiganense, which described the non-motile, rod-like bacterium isolated from tomato (Solanum lycopersicum) plants in Michigan (Smith, 1910; Smith, 1914; Bryan, 1930). Subsequently, Bergey et al. (1939) transferred Gram-positive plant pathogenic bacteria into the genus Phytomonas and renamed the bacterial canker of tomato pathogen Phytomonas michiganensis. However, as the genus Phytomonas encompassed both Gram-negative, motile, green-fluorescent bacteria (now known as Pseudomonas spp.) and Gram-positive, non-motile, yellow/orange-pigmented bacteria (now known as Clavibacter spp.), the proposed reclassification was not accepted by most bacteriologists at that time. Thus, Dowson (1942) transferred the Gram-positive coryneform plant pathogenic bacteria into the genus Corynebacterium (‘club’ bacterium) (Lehmann and Neumann, 1896) and the bacterial canker of tomato pathogen was named Corynebacterium michiganense. Finally, based on its unique 2,4-diaminobutyric acid content in the peptidoglycan layer, the pathogen was transferred to the genus Clavibacter and named C. michiganensis subsp. michiganensis as one of the five subspecies within the species (Davis et al., 1984).

Following the emergence of high throughput molecular-phylogenetic techniques, many Clavibacter spp. strains which have often previously been misidentified based on phenotypic features, were assigned into novel taxa. Using phylogenetic analysis and polyphasic characterization of C. michiganensis strains isolated from tomato seeds, Jacques and her co-workers (2012) showed that non-pathogenic strains were distinct from tomato pathogenic strains of C. michiganensis. Subsequently, tomato-associated non-pathogenic members of C. michiganensis sensu lato were assigned into two new subspecies, C. michiganensis subsp. californiensis and C. michiganensis subsp. chilensis (Yasuhara-Bell and Alvarez, 2015). Furthermore, non-pathogenic, peach-coloured strains isolated from the tomato phyllosphere were reported to be distinct from the tomato pathogenic members of Clavibacter spp. (Osdaghi et al., 2018a).

Recently, a reclassification of Clavibacter spp. into several new species was proposed based on genomic information, e.g. average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) indices (Li et al., 2018). The original subspecies of C. michiganensis sensu lato were elevated to the species level and the bacterial canker of tomato pathogen was designated C. michiganensis (C. michiganensis sensu stricto). Further complete genome sequence-based investigations, i.e. comparative genomics and phylogenetic analyses using all the publicly available genome sequences of the genus, have confirmed the new taxonomic changes and showed that the new taxon C. michiganensis should encompass only the tomato pathogenic strains of the genus (Ansari et al., 2019; Osdaghi et al., 2018b, 2020).

Description

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C. michiganensis is an aerobic, non-motile, Gram-positive, non-spore producing, curved rod. For a full description, see Davis et al. (1984), Bradbury (1986), Sneath et al. (1986) and Evtushenko and Takeuchi (2006).
 

Distribution

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Bacterial canker was first identified on tomato in Michigan (USA) in 1909 and is now widespread throughout Africa, Asia, Europe, North America, Oceania and South America. The disease is important in countries with a rapidly-growing tomato industry e.g. Iran and Turkey (Ansari et al., 2019). Among the top-ten tomato producing countries, severe outbreaks of bacterial canker disease have been recorded in the USA, Turkey and Iran. Widespread occurrence of the disease has also been reported in Europe, e.g. in Italy and Belgium, as well as in South America, e.g. Chile and Uruguay (de León et al., 2011). Since the mid-twentieth century, the pathogen has spread widely through the international tomato seed trade. Ansari et al. (2019) have investigated the worldwide population of the bacterial canker pathogen using MLSA/MLST and suggested multiple introductions of C. michiganensis from the New World into the Old World which was inferred from the haplotype arrangements among the global population of the pathogen.

See also CABI/EPPO (1998, No. 253).

Distribution Table

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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: 12 May 2022
Continent/Country/Region Distribution Last Reported Origin First Reported Invasive Reference Notes

Africa

AlgeriaAbsent, Invalid presence record(s)
Congo, Democratic Republic of theAbsent, Unconfirmed presence record(s)
EgyptPresent
KenyaPresent
MadagascarPresent
MoroccoPresent, Localized
South AfricaPresent, Widespread
TanzaniaPresent, Localized
TogoPresent
TunisiaPresent, Localized
UgandaPresent
ZambiaPresent
ZimbabwePresent, Localized

Asia

ArmeniaPresent
AzerbaijanPresent
ChinaPresent
-LiaoningPresent
-XinjiangPresent
IndiaPresent, Localized
-Andhra PradeshPresent
-KarnatakaPresent
-Madhya PradeshPresent
-MaharashtraPresent
-Tamil NaduPresent
IndonesiaPresent, Localized
-JavaPresent
IranPresent1988
IsraelPresent, Widespread
JapanPresent
-HonshuPresent
JordanPresent
LebanonPresent
PakistanPresent
South KoreaPresent, Localized
SyriaPresent, Localized
TaiwanAbsent, Unconfirmed presence record(s)
ThailandAbsent, Unconfirmed presence record(s)
TurkeyPresent, Widespread
UzbekistanAbsent, Eradicated
VietnamAbsent, Unconfirmed presence record(s)

Europe

AustriaAbsent, Eradicated
BelarusPresent
BelgiumAbsent, Formerly present
BulgariaPresent, Localized
CyprusPresent, Localized
CzechiaPresent, Few occurrences
FinlandAbsent, Invalid presence record(s)
FrancePresent, Widespread
GermanyPresent, LocalizedFirst reported: 193*
GreecePresent, Widespread
-CretePresent
HungaryPresent, Localized1959
IrelandAbsent, EradicatedFirst reported: 194*
ItalyPresent
-SardiniaPresent
-SicilyPresent
JerseyAbsent, Formerly present
LatviaPresent, Localized
LithuaniaAbsent, Eradicated1930
NetherlandsPresent, Transient under eradication
NorwayAbsent, Eradicated
PolandPresent, Few occurrences
PortugalPresent, Few occurrences
RomaniaPresent, Localized
RussiaPresent, Widespread
-Central RussiaPresent, Widespread
-Southern RussiaPresent, Widespread
-Western SiberiaPresent
SerbiaPresent, Localized
Serbia and MontenegroPresent, Localized
SlovakiaAbsent, Intercepted only
SloveniaPresent, Transient under eradication
SpainPresent, Few occurrences
-Canary IslandsPresent, Transient under eradication
SwedenAbsent, Eradicated
SwitzerlandPresent, Widespread
UkrainePresent, Localized
Union of Soviet Socialist RepublicsPresent
United KingdomAbsent, Formerly present
-Channel IslandsAbsent, Formerly present
-EnglandAbsent, Formerly present
-ScotlandAbsent, Formerly present

North America

BelizePresent
CanadaPresent, Widespread
-AlbertaPresent
-British ColumbiaPresent
-ManitobaPresent
-Nova ScotiaPresent
-OntarioPresent
-QuebecPresent
-SaskatchewanPresent
Costa RicaPresent
CubaPresent
DominicaPresent
Dominican RepublicPresent
GrenadaPresent
GuadeloupePresent
MartiniqueAbsent, Unconfirmed presence record(s)
MexicoPresent, Localized
PanamaPresent
United StatesPresent, Widespread
-AlabamaPresent
-ArkansasPresent
-CaliforniaPresent
-ColoradoPresent
-ConnecticutPresent
-FloridaPresent
-GeorgiaPresent
-HawaiiPresent
-IllinoisPresent
-IndianaPresent
-IowaPresent
-KentuckyPresent
-MainePresent
-MarylandPresent
-MassachusettsPresent
-MichiganPresent
-MinnesotaPresent
-MontanaPresent
-NebraskaPresent
-New HampshirePresent
-New JerseyPresent
-New YorkPresent
-North CarolinaPresent
-North DakotaPresent, Few occurrences
-OhioPresent
-OklahomaPresent
-OregonPresent
-PennsylvaniaPresent
-South DakotaPresent
-UtahPresent
-VermontPresent
-WyomingPresent

Oceania

AustraliaPresent, Widespread
-New South WalesPresent
-QueenslandPresent
-South AustraliaPresent
-TasmaniaPresent
-VictoriaPresent
-Western AustraliaPresent
FijiPresent
GuamPresent
New CaledoniaPresent
New ZealandPresent, Localized
TongaPresent

South America

ArgentinaPresent
BrazilPresent
-PernambucoPresent
-Sao PauloPresent
ChilePresent, Localized
ColombiaPresent
EcuadorPresent, Localized
PeruPresent
UruguayPresent, Widespread

History of Introduction and Spread

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Bacterial canker was first identified on tomato in Michigan (USA) in 1909, and is now widespread in Africa, Asia, Europe, North America, Oceania and South America. C. michiganensis is a seedborne pathogen and since the mid-twentieth century, international tomato seed trade has spread the pathogen within and between continents. Ansari et al. (2019) have investigated the worldwide population of the bacterial canker pathogen using MLSA/MLST and suggested multiple introductions of C. michiganensis from the New World into the Old World which was inferred from the haplotype arrangements among the global population of the pathogen.

Risk of Introduction

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Risk Criteria Category

Economic: Importance High
Distribution: Worldwide
Seedborne Incidence: Moderate
Seed Transmitted: Yes
Seed Treatment: Yes

Overall Risk: Moderate to high depending on the environmental conditions, cultivar susceptibility status and availability of seed testing facilities.


Notes on Phytosanitary Risk

C. 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.

Habitat List

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CategorySub-CategoryHabitatPresenceStatus
Terrestrial ManagedCultivated / agricultural land Present, no further details
Terrestrial ManagedProtected agriculture (e.g. glasshouse production) Present, no further details
OtherHost Present, no further details

Hosts/Species Affected

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The main host of economic importance is tomato (Solanum lycopersicum) but natural infection has also been reported on pepper (Capsicum annuum) (Lai, 1976; Moffett and Wood, 1984; Latin et al., 1995); and on the wild plants Solanum douglasii, S. nigrum [S. americanum] and S. triflorum (Bradbury, 1986). A number of solanaceous plants are susceptible on artificial inoculation (Thyr et al., 1975). Stamova and Sotirova (1987) have claimed to have produced leaf wilt by artificial inoculation on maize (Zea mays), wheat (Triticum aestivum), barley (Hordeum vulgare), rye (Secale cereale) and other hosts, but it has not been confirmed by others.

Recently, Ignatov et al. (2019) reported pathogenicity of C. michiganensis on potato (Solanum tuberosum) under natural conditions in Russia. The strains were distinct from the potato pathogen, C. sepedonicus, and were pathogenic in both tomato and potato under greenhouse conditions. Since the pepper-pathogenic strains of the former C. michiganensis species complex have recently been reclassified as a novel species (C. capsici), caution should be taken in the identification of the pathogen where it is isolated from non-tomato hosts (Li et al., 2018).

Growth Stages

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Flowering stage, Fruiting stage, Seedling stage, Vegetative growing stage

Symptoms

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Infected tomato (Solanum lycopersicum) seeds and transplants raised in the greenhouse do not show visible symptoms of the disease; seed germination and seedling stand also appear to be normal (Dhanvantari, 1989; Gitaitis et al., 1991; Chang et al., 1992a). Unilateral wilting or withering of leaflets on one side of the leaf and bird's eye-spot lesions (raised white spot developing a necrotic centre) on the fruit surface are the outstanding features diagnostic of tomato bacterial canker. The first symptoms include desiccation of the edge of the leaflets which may overlap with plant wilt in severe infections. Plants infected as young seedlings become stunted and wither rapidly. Numerous small, whitish or tan pustules may appear on leaf veins, petioles and peduncles.

In advanced infection, vascular discolouration 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 discolouration of vascular tissue and pith and cavities within the pith will be evident. Sometimes, a very light pink discolouration 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/Signs

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SignLife StagesType
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 Ecology

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Clavibacter michiganensis is a seedborne pathogen that colonizes tomato (Solanum lycopersicum) vascular tissues and fruits. Contaminated seeds are the main source of inoculum in both greenhouse- and field-grown tomatoes (Fatmi and Schaad, 1988). The disease spreads to other seedlings and plants by practices such as clipping and packaging for transplant production; tying and staking of trellis tomatoes; deleafing, suckering and tying in greenhouses and generally by water splashing caused by rain, overhead irrigation, or chemical sprays (EPPO, 2016). Vascular infection caused by the handling operations leads to stunting of growth, withering, wilting and early plant loss (Peritore-Galve et al., 2020).

Although populations of the pathogen 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). C. michiganensis can survive on volunteer tomato seedlings and alternative host species which can serve as sources of infection (Strider, 1969).

Conversely, a number of Gram-positive, yellow-pigmented actinobacterial species are reported to epiphytically colonize the tomato phyllosphere and could be misidentified as the canker pathogen on general culture media (Osdaghi et al., 2018c, d). Fatmi and Schaad (2002) have investigated the survival of C. michiganensis under natural field conditions in the USA (California, Ohio) and Morocco using semi-selective agar medium. The pathogen survived significantly longer in tomato stems left on the soil surface than in stems buried in the soil at all locations studied. The pathogen was recovered from tomato stems left on the soil surface for 314 days in Ohio and California, and for 194 days in Morocco. It was also recovered from stems buried in the soil for up to 314 days in Ohio, up to 240 days in California, and up to 60 days in Morocco. In Argentina, C. michiganensis survived in tomato debris left on the soil surface for 120-260 days for crop production cycles that ended in winter and 45-75 days for those that ended in summer, while this period was 45-75 days for stems or roots buried in winter (Vega and Romero, 2016).

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 (Tsiantos, 1987; Gleason et al., 1991; Chang et al., 1992a).

Frenkel et al. (2016) have found that the pathogen dispersed spatially from root-inoculated source seedlings and colonized the leaf surfaces of surrounding seedlings to distances of 65-75 cm. Under natural conditions, tomato plants seem to be susceptible throughout their life (Rat et al., 1991). The period of vulnerability of tomato seedlings to C. michiganensis ranges from transplanting to the 17- to 18-leaf stage, while no significant changes in disease incidence were observed when leaves of different ages were inoculated within this period. Plants inoculated after this period expressed disease symptoms but did not wilt or die. Yield accumulation was significantly reduced in plants inoculated within this window of vulnerability compared with those inoculated after this period (Sharabani et al., 2013). Later researchers have also noted that the environmental temperature at the early stages of C. michiganensis infection affects bacterial canker development and virulence gene expression (Sharabani et al., 2014). Young plants have been shown to be more susceptible to canker (van Vaerenbergh and Chauveau, 1985). 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).

Plant wounds facilitate but are not required for infection by C. michiganensis, which invades the xylem vessels and causes vascular disease with high titres impairing water transport and leading to plant wilting, canker stem lesions and death (Xu et al., 2010).

Tancos et al. (2013) have demonstrated that C. michiganensis could not only access seeds systemically through the xylem but also externally through tomato fruit lesions. Active movement and expansion of bacteria into the fruit mesocarp and nearby xylem vessels followed once the fruits began to ripen. 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).

Means of Movement and Dispersal

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C. michiganensis is a seedborne pathogen that has spread through international tomato seed trade.

Seedborne Aspects

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Incidence

The incidence of infection of tomato seeds by C. 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 indicates that a small percentage of seed infection may be deep-seated within the seed tissue (Dhanvantari and Brown, 1993).

Seeds that are contaminated externally with C. michiganensis through contact with other sources of the bacterium, can also serve as the initial source of inoculum for systemic infections (Nandi et al., 2018).

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).

Pathogen Transmission

Studies on transmission of seedborne C. 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 1000 c.f.u./g seed, finding a high correlation between c.f.u./g seed and disease incidence. Concentrations of C. 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 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 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. 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 (Kleitman 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 Treatment

Seed treatments for the eradication of C. 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, 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 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

Standard seed health testing is an essential tool for the control of C. michiganensis and is important for its regulation and control through phytosanitary certification and quarantine programmes in the domestic and international seed trade (de León et al., 2011). Traditionally, tomato seed testing for C. 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 methods

Serological techniques used to detect and identify C. michiganensis include agglutination tests, indirect immunofluorescence (IF) (van Vaerenbergh and Chauveau, 1987; Franken et al., 1993), immunofluorescence colony staining (IFC) (Veena and van Vuurde, 2002), enzyme-linked immunosorbent assays (ELISA) (Krämer 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, 2008). Polyclonal antibodies (PAbs) are commercially available for IF, ELISA, agglutination tests or immunostrips, for example, from Loewe, Neogen or Plant Research International. False positive results were observed when a collection of pathogenic and non-pathogenic tomato-associated C. michiganensis strains were subjected to the serological tests (Jacques et al., 2012).

Monoclonal antibodies (MAbs) have shown higher titre and specificity against C. 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 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 3 days at room temperature before IF (Olivier et al., 2010).

PCR-based methods

Different pairs of primers have been specifically designed for C. 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). However, it is 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 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 developed for the simultaneous detection of C. michiganensis together with other seedborne pathogens, using previously published primers and either conventional (Özdemİr, 2009) or real-time PCR (Johnson and Walcott, 2012). Further, simultaneous detection of C. michiganensis, Pepino mosaic virus and Mexican papita viroid by non-radioactive molecular hybridization using a unique polyprobe was reported by Zamora-Macorra et al. (2015). Recently, Thapa et al. (2020) have report the use of comparative genomics of 37 diverse Clavibacter strains to identify DNA sequences that are specific for C. michiganensis. A multiplex PCR-based diagnostic platform using two C. michiganensis chromosomal genes (rhuM and tomA) and an internal control amplifying both bacterial and plant DNA (16s ribosomal RNA) was developed. The multiplex PCR assay specifically detected C. michiganensis strains from a panel of 110 additional bacteria, including other Clavibacter spp. and bacterial pathogens of tomato.

Standard protocols for tomato seed testing

For C. 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 (EPPO, 2016) and they are available on the ISHI Webpage: https://worldseed.org/our-work/phytosanitary-matters/seed-health/ishi-veg-protocols/ and EPPO Webpage: https://www.eppo.int/RESOURCES/eppo_standards/pm7_diagnostics, 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. Recently, Fatmi et al. (2017) have provided a detail-oriented seed health testing procedure for detection of C. michiganensis in tomato seed lots. Furthermore, it has been noted that the viable but non-culturable (VBNC) state of C. michiganensis may result in the underestimation or false negative detection of the pathogen. Hence, Han et al. (2018) have developed a detection protocol for C. michiganensis in VBNC state from tomato seed using improved qPCR. The bacteria that are in the VBNC state can recover their culturability when returned to favourable conditions. Induction of the VBNC state in C. michiganensis using CuSO4 and low pH was reported by Jiang et al. (2016). It has also been concluded that because copper-based chemicals and low pH conditions are used for management of the disease, induction of the VBNC state in C. michiganensis cells on tomato seedlings may limit pathogen detection by culture-based assays.

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 (Krämer 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.

Media preparation

Semi-selective medium for C. 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 - 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 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 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 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 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 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 by IF staining (Schaad, 1978)

1. Grow suspected colonies and a known strain of C. 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 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 (Krämer 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 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 by pathogenicity test

1. Grow suspected colonies on YDC for 24-48 h.

2. Sterile scissors dipped in inoculum of C. 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 Causes

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CauseNotesLong DistanceLocalReferences
Crop production Yes Yes
Food Yes Yes
Garden waste disposal Yes
Horticulture Yes
Live food or feed trade Yes Yes
Nursery trade Yes Yes
Research Yes Yes
Seed trade Yes Yes

Pathway Vectors

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VectorNotesLong DistanceLocalReferences
Clothing, footwear and possessionsTransfer of seeds or seedlings. Yes Yes
Land vehiclesRisk of soil on wheels. Yes Yes
MailTransfer of seeds. Yes
Containers and packaging - non-wood Yes
Debris and waste associated with human activities Yes
Floating vegetation and debris Yes
Soil, sand and gravel Yes
Water Yes
Wind Yes

Plant Trade

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Plant parts liable to carry the pest in trade/transportPest stagesBorne internallyBorne externallyVisibility of pest or symptoms
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 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
Bark
Wood

Impact Summary

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CategoryImpact
Crop production Negative
Economic/livelihood Negative

Impact

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Since the first report of the disease in the USA in 1910, bacterial canker has spread throughout the world and causes serious losses to both greenhouse and field tomato crops either by killing the young plants or reducing marketable yields. Reduction in yield may be associated with direct plant loss, reduced numbers of fruit or fruit size. Field experiments have recorded yield losses of 20% or more in Ontario, Canada (Dhanvantari, 1989; Dhanvantari and Brown, 1993); 20-30% in France (Rat et al., 1991); 46% in Illinois, USA (Chang et al., 1992c); and a ten-fold yield reduction after plant loss in Queensland, Australia (Dullahide et al., 1983).

Bacterial canker is of high economic importance in countries with a fast-growing tomato industry, e.g. Iran and Turkey (Ansari et al., 2019). Among the top-ten tomato producing countries, yield losses due to severe outbreaks of bacterial canker disease were recorded in the USA, Turkey and Iran. Widespread occurrence of the disease was also reported in European countries, e.g. Italy and Belgium, as well as in South America, e.g. Chile and Uruguay (de León et al., 2011). 

Risk and Impact Factors

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Invasiveness
  • Invasive in its native range
  • Proved invasive outside its native range
  • Highly adaptable to different environments
  • Tolerant of shade
  • Highly mobile locally
  • Fast growing
  • Reproduces asexually
  • Has high genetic variability
Impact outcomes
  • Host damage
  • Negatively impacts agriculture
  • Damages animal/plant products
  • Negatively impacts trade/international relations
Impact mechanisms
  • Pathogenic
Likelihood of entry/control
  • Highly likely to be transported internationally accidentally
  • Highly likely to be transported internationally illegally
  • Difficult to identify/detect as a commodity contaminant
  • Difficult to identify/detect in the field
  • Difficult/costly to control

Uses List

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General

  • Laboratory use
  • Research model

Diagnosis

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Isolation of the causal organism can be attempted on nutrient dextrose agar or yeast peptone glucose agar (Lelliott and Stead, 1987). C. michiganensis develops slow-growing, smooth, shining, round, yellow colonies with entire margins. Although it has traditionally been believed that white, pink, red and orange mutants of the pathogen do occur (Hayward and Waterson, 1964), recent comprehensive complete genome sequence-based investigations showed that the non-yellow-pigmented strains could not be considered C. michiganensis, rather they belong to several hypothetical novel species within the species (Osdaghi et al., 2020). 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 grey-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. New semi-selective media, as well as an improvement of SCM medium (Koenraadt et al., 2009), have been reported, such as CMM1 agar (Kaneshiro et al., 2006) on which colonies of C. michiganensis are yellow, mucoid and convex, and BCT agar (Ftayeh et al., 2011) on which typical colonies appear creamy to yellow in colour, 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 Steekelenburg, 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 series of protocols for diagnosis of C. michiganensis in symptomatic plants (EPPO, 2005, 2013, 2016; Olivier et al., 2010). 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 seedlings. 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 of the preparation of plant tissue suspensions, dilution plating, media, rapid tests, identification of presumptive isolates and pathogenicity test, can be found in EPPO (2016).

Detection and Inspection

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Many seed testing methods have been investigated. Semi-selective media are now routinely used to isolate the pathogen from seed extracts or from plant tissue. The pathogen is not consistently isolated from the vascular tissue of tomato plants or from cankers on leaf petioles. More consistent results are obtained when isolations are made from fruit spots or discoloured vascular tissue within the fruit. The section of tissue is suspended in sterile distilled water for 30 min or macerated in a few drops of sterile distilled water. A loopful of the liquid is then spread on agar plates. 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; Krämer 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.

De León et al. (2006) have compared immunomagnetic separation (IMS)-plating protocol with plating on general (YPGA) and semi-selective (mSCM) media, DAS-ELISA, immunofluorescent assay (IF) and conventional PCR in the isolation and accurate detection of the bacterial canker pathogen. Among the evaluated methods, IMS-plating provided the best results regarding sensitivity and specificity for C. michiganensis detection, allowing the recovery of viable bacteria from seed extracts. Yasuhara-Bell et al. (2013) have developed a diagnostic micA-based LAMP method to detect C. michiganensis. Furthermore, by using a loop-mediated amplification of clvA gene and PCR of clvA, clvF and clvG genes, Yasuhara-Bell et al. (2014), showed that these genes are present only in C. michiganensis and not in other Clavibacter species as well as other genera of plant-associated bacteria. Recently, Dobhal et al. (2019) developed another sensitive, specific and robust LAMP assay for detection of all known species of Clavibacter

Prevention and Control

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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).

Chemical treatment of tomato seedlings reduced population sizes and spread of C. michiganensis among tomato seedlings in the greenhouse and impacted subsequent plant development and yield in the field. Applications of copper hydroxide, copper hydroxide/mancozeb, copper hydroxide/mancozeb (premixed 12 h before spraying), streptomycin, or streptomycin/copper hydroxide to seedlings in the greenhouse prevented development of severe disease symptoms in the field such as plant and fruit stunting and yield loss (Hausbeck et al., 2000). However, extensive resistance in plant pathogenic bacteria against copper-based chemicals (Lamichhane et al., 2018) as well as streptomycin (Lyu et al., 2019) raise alarms on the efficacy of chemical treatments in the management of the bacterial canker of tomato.

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 (van Steekelenburg, 1985; Gardner et al., 1990; Poysa, 1993) but have not yet been introduced into commercial cultivars. Recently, Wittmann et al. (2016) developed a tomato plant resistant to C. michiganensis using the endolysin gene of bacteriophage CMP1 as a transgene and the transgenic tomato plants did not show disease symptoms after infection with C. michiganensis.

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.

Bacillus velezensis strain 1B-23 and Bacillus sp. strain 1D-12 have been approved for their biocontrol abilities against C. michiganensis (Laird et al., 2020).

References

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Distribution References

Șahİn F, Uslu H, Kotan R, Donmez M F, 2002. Bacterial canker, caused by Clavibacter michiganensis ssp. michiganensis, on tomatoes in eastern Anatolia region of Turkey. Plant Pathology. 51 (3), 399. DOI:10.1046/j.1365-3059.2002.00715.x

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Ayesha Bibi, Musharaf Ahmad, Shaukat Hussain, 2018. Prevalence of (Clavibacter michiganensis subsp. michiganensis) causal organism of bacterial canker in weed species in tomato fields of north west Pakistan. Sarhad Journal of Agriculture. 34 (1), 123-129. DOI:10.17582/journal.sja/2018/34.1.123.129

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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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08/11/20 Updated by:

Ebrahim Osdaghi, Department of Plant Protection, University of Tehran, Iran

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