<figure><img src="image_1.png" /></figure>

HAL
open science

# Clavibacter michiganensis Reframed: The Story of How the Genomics Era Made a New Face for an Old Enemy

Ebrahim Osdaghi, Hamid Abachi, Marie-agnes Jacques

► To cite this version:

Ebrahim Osdaghi, Hamid Abachi, Marie-agnes Jacques. Clavibacter michiganensis Reframed: The Story of How the Genomics Era Made a New Face for an Old Enemy. Molecular Plant Pathology, 2025, 26 (5), pp.e70093. 10.1111/mpp.70093. hal-05206912

HAL Id: hal-05206912

https://hal.inrae.fr/hal-05206912v1

Submitted on 11 Aug 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

# *Clavibacter michiganensis* Reframed: The Story of How the Genomics Era Made a New Face for an Old Enemy

Ebrahim Osdaghi<fnref n="1" /> ID | Hamid Abachi<fnref n="1" /> | Marie-Agnes Jacques²

**Correspondence:** Ebrahim Osdaghi (eosdaghi@ut.ac.ir)

**Received:** 9 September 2024 | **Revised:** 20 March 2025 | **Accepted:** 30 April 2025

**Funding:** This work was supported by Iran National Science Foundation (INSF) under project no. 4038663. The work of E.O. was funded by the University of Tehran, Iran.

**Keywords:** Actinobacteria | bacterial canker of tomato | Corynebacteria | *Microbacteriaceae* | quarantine pathogen | *Solanaceae* | *Solanum lycopersicum*

## ABSTRACT

**Objective:** Bacterial wilt and canker of tomato caused by the gram-positive corynebacterial species *Clavibacter michiganensis* is an economically important disease threatening the tomato industry in both open-air and greenhouse productions around the world. The disease occurs in many countries, with a particular importance in regions characterised by high temperature and water scarcity. Management of bacterial canker has been a major problem since its original description in 1909. This is due in part to the seedborne nature of the pathogen, allowing the bacterium to be transmitted over long distances via infected seeds, as well as a lack of effective treatment to clean seeds. Detection of the pathogen from seeds is difficult due to high competition on culture media with diverse members of the seed-associated microbiota. Identification of the pathogen can also be difficult owing to the presence of different colony variants on culture media. In this review, we provide a historical perspective and an updated overview on the aetiology, epidemiology and management strategies of the bacterial canker disease. We also gathered recent molecular findings in the pathogenicity mechanisms and bioecology of *C. michiganensis* to boost management of the bacterial canker disease in the 21st century tomato industry.

**Taxonomy:** Class: *Actinobacteria*; Order: *Micrococcales*; Family: *Microbacteriaceae*; Genus: *Clavibacter*; Species: *Clavibacter michiganensis*.

**Disease Symptoms:** Interskeletal leaf chlorosis leading to necrotic areas. Canker on stems and lateral branches of the plant. Discolouration of vascular and pith tissues to dark yellow or brown. Small and early ripened fruits or discolouration of the皮centa from white to yellow in the interior part of the ripening fruits.

**Host Range:** Tomato (*Solanum lycopersicum*) is the main host of the pathogen while natural infection has also been reported on eggplant, pepper and wild nightshade plants.

**Synonyms (Historical/Non-Preferred Scientific Names):** *Aplanobacter michiganensis*; *Pseudomonas michiganense*; *Pseudomonas michiganensis*; *Bacterium michiganense*; *Phytomonas michiganensis*; *Mycobacterium michiganense*; *Erwinia michiganensis* (=michiganense); *Corynebacterium michiganense*; *Corynebacterium michiganense* pv. *michiganense*; *Corynebacterium michiganense* subsp. *michiganense*; *Clavibacter michiganensis* subsp. *michiganensis*.

**Microbiological Properties:** The bacterium produces domed, round and shiny mucoid colonies on general culture media. Colonies are usually yellow-pigmented, while pink-pigmented strains are occasionally observed. Cells are gram-positive, aerobic, non-motile, non-spore-producing curved rods (coryneform).

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2025 The Author(s). *Molecular Plant Pathology* published by British Society for Plant Pathology and John Wiley & Sons Ltd.

<footnote n="1">¹Department of Plant Protection, College of Agriculture, University of Tehran, Karaj, Iran | ²Institut Agro, INRAE, IRHS, SFR QUASAV, CIRM-CFBP, Université d'Angers, Angers, France</footnote>
**Distribution:** Present in all continents.

**Phytosanitary Categorization:** EPPO A2 List no. 50, EU 2019/2072 RNQP Annex IV. See EPPO ([https://gd.eppo.int/taxon/CORBMI/categorization](https://gd.eppo.int/taxon/CORBMI/categorization)) and CABI ([https://www.cabidigitallibrary.org/doi/10.1079/cabicompendium.15338](https://www.cabidigitallibrary.org/doi/10.1079/cabicompendium.15338)) databases for further country-specific categorisations. EPPO code: CORBMI.

# 1 | Taxonomic History of the Pathogen

In 1909, Erwin F. Smith observed a previously unreported disease on tomato in Grand Rapids, MI, United States. He named the disease 'the Grand Rapids tomato disease' and the causal agent *Bacterium michiganense*, which means a bacterium from Michigan (Smith 1910). The common name of the disease was changed to bacterial canker of tomato according to the characteristic canker symptoms on petioles and stems (Bryan 1930). Jensen (1934) noted that the tomato pathogen, along with the potato ring rot pathogen, was taxonomically similar to corynebacteria (Lehmann and Neumann 1896), thus proposed renaming the two species as *Corynebacterium michiganense* and *Corynebacterium sepedonicum*, respectively. *Corynebacterium michiganense* was applied to the tomato canker pathogen during the subsequent three decades.

During the 1970s, comprehensive chemotaxonomic investigations proposed the separation of plant-pathogenic coryneform species from the original *Corynebacterium* members (Yamada and Komagata 1970; Jones 1975). For the first time, Dye and Kemp (1977) used pathovar designation in the classification of corynebacterial plant pathogens. They proposed classifying all coryneform plant-pathogenic bacteria into four species within *Corynebacterium*, where the tomato canker pathogen, along with *Corynebacterium insidiosum*, *Corynebacterium nebraskense*, *Corynebacterium sepedonicum* and *Corynebacterium rathayi*, was designated as pathovars of *Corynebacterium michiganense*; thus, the tomato canker pathogen was named *Corynebacterium michiganense* pv. *michiganense*. A few years later, the taxonomic status of the bacterial canker pathogen was changed from pathovar to subspecies level and named *Corynebacterium michiganense* subsp. *michiganense* (Carlson and Vidaver 1982).

In 1984, based on phenotypic features, for example, peptidoglycan content, Davis and his colleagues proposed the genus *Clavibacter* to include a newly described sugarcane pathogen (*Clavibacter xyli* subsp. *xyli*), as well as the pathovars/subspecies of *Corynebacterium michiganense*. Therefore, the bacterial canker pathogen was renamed *Clavibacter michiganense* subsp. *michiganense* (Davis et al. 1984). However, under the nomenclature rules of bacterial taxonomy, the name was changed to *Clavibacter michiganensis* subsp. *michiganensis* in the subsequent years (Young et al. 1996). For the next three decades (1984–2014), all plant-pathogenic members of *Clavibacter* were classified in one complex species *C. michiganense* with five subspecies reflecting their host of isolation and pathogenicity pattern (Vidaver and Davis 1988). All tomato-associated *Clavibacter* strains were referred to as *C. michiganensis* subsp. *michiganensis* regardless of their pathogenicity, phenotypic features (e.g., colony pigmentation) and phylogenetic position (Jacques et al. 2012). The strains isolated from pepper plants were also classified as *C. michiganensis* subsp. *michiganensis* (Yim et al. 2012).

By the beginning of the genomics era, high-throughput molecular techniques and DNA sequencing were used to decipher taxonomic relationships of *Clavibacter* lineages (Zaluga et al. 2011). Molecular phylogenetic analyses revealed higher taxonomic variations within *C. michiganensis* sensu lato strains than those previously reported on the basis of phenotypic data. For instance, Yim et al. (2012) noted that *C. michiganensis* subsp. *michiganensis* strains isolated from pepper had orange-pigmented colonies with lower mucoidy than those of typical *C. michiganensis* subsp. *michiganensis* strains isolated from tomato. On the other hand, using phylogenetic analysis and polyphasic characterisation, Jacques et al. (2012) showed that nonpathogenic strains of *C. michiganensis* sensu lato originating from tomato were distinct from tomato-pathogenic strains of the subspecies. In the subsequent years, tomato-associated nonpathogenic members of *C. michiganensis* sensu lato were assigned to two new subspecies, *C. michiganensis* subsp. *californiensis* and *C. michiganensis* subsp. *chilensis* (Yasuhara-Bell and Alvarez 2015). The pepper-pathogenic members of the species were also assigned to a distinct subspecies, *C. michiganensis* subsp. *capsici* (Oh et al. 2016). Furthermore, non-pathogenic peach-pigmented *Clavibacter* strains isolated from the tomato phyllosphere were shown to be distinct from all the above-mentioned members of the genus (Osdaghi, Ansari, et al. 2018).

In 2018, reclassification of *Clavibacter* spp. into several new species was proposed based on genomic information, for example, average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) indices (Li et al. 2018). The original subspecies of *C. michiganensis* sensu lato were elevated to the species level and the tomato bacterial canker pathogen was designated as *C. michiganensis* sensu stricto. Thus, unless otherwise stated, hereafter the tomato bacterial canker pathogen will be referred to as *C. michiganensis* in this text. Furthermore, nonpathogenic tomato-associated members of *C. michiganensis* sensu lato were elevated to the species level where *C. michiganensis* subsp. *californiensis* was reclassified as *C. californiensis* and *C. michiganensis* subsp. *chilensis* was first transferred into *Clavibacter michiganensis* subsp. *phaseoli* (Osdaghi, Rahimi, et al. 2020) and then reclassified as *Clavibacter phaseoli* (Arizala et al. 2022). The pepper-pathogenic subspecies *C. michiganensis* subsp. *capsici* was also designated as *Clavibacter capsici* (Li et al. 2018). More recently, peach-pigmented nonpathogenic *Clavibacter* strains isolated from tomato were assigned to a new species as *Clavibacter lycopersici* (Osdaghi, Taghavi, et al. 2023).

In addition to the above-mentioned four tomato-associated *Clavibacter* lineages, phylogenomics and pathogenicity data showed that several hypothetical novel species could be identified within the genus, four of which were isolated from asymptomatic tomato tissues or seed lots (Osdaghi, Rahimi, et al. 2020; Yañez-Olvera et al. 2024). Indeed, the only pathogenic lineage of tomato-associated strains is *C. michiganensis* while the other nonpathogenic lineages are among the natural
<figure><img src="image_5.png" /><figcaption>**FIGURE 1** | Field symptoms of tomato bacterial canker disease caused by *Clavibacter michiganensis* on tomato foliage (A–D) and fruits (E, F). Artificially inoculated eggplant and pepper plants show canker on the site of inoculation (G, H, respectively), while tomato plants show both stem canker and leaf wilting (I).</figcaption></figure>

microbiota of tomato plants (Osdaghi, Taghavi, et al. 2018). All these taxonomic refinements raise the question of whether current detection methods are technically applicable to quarantine purposes and emphasise at the same time the need for re-evaluation of those methods for sensitivity, specificity and reproducibility (EPPO 2016). This would help plant pathology agencies and tomato seed industry inspectors to specifically target the enemy and neglect the nonpathogenic lineages with lower cost and labour (see detection and identification section).

## 2 | Disease Symptoms

Symptoms of bacterial canker disease are diverse and are influenced by the pathway of infection, plant cultivar and age, as well as environmental factors, that is, temperature, humidity and nutrition (Gleason et al. 1993; Jones, Zitter, et al. 2014; Nandi et al. 2018). Infected seeds, infected tomato seedlings and transplants do not generally show any visible symptoms of the disease until maturity or unless they are exposed to high temperature, drought and physical stresses (Dhanvantari 1989;

Gitaitis et al. 1991). Seedling symptoms start with small, white and raised spots on leaves. While infrequent, leaf margin discolouration and wilting may occur, leading to complete seedling wilt and plant death. Once transplanted in the field, the first symptoms on young plants include desiccation of the leaflet margin and overall plant wilt in severe cases of infections. Infected young seedlings become stunted and wither rapidly (Figure 1A–C). Numerous small whitish or tan pustules may appear on leaf veins, petioles and later on peduncles. However, the latter symptoms are observed only under high relative humidity (Jones, Zitter, et al. 2014).

Leaf symptoms are common in fully grown tomato plants, especially under warm and drought conditions. Leaf symptoms initially appear as interveinal, pale green, flaccid zones that quickly turn to yellow-brown necrotic areas resembling sunburn (Figure 1B–D; Lamichhane et al. 2011). Marginal pale brown necrotic areas also appear on the leaves, with a scorched appearance. Leaf margins turn brown, with a yellow border, while lower leaves wilt, often only on one side. Veins on the leaves and petioles can become dark and sunken. Hydathodes of tomato leaves serve as extremely efficient infection entry
points for epiphytic populations of the pathogen. Thus, chlorotic lesions progress at the tips of leaflet lobes about 2 weeks after inoculation of guttation droplets. Lesions expand along the leaflet margins and become necrotic (Carlton et al. 1998).

Characteristic symptoms of canker that lead to the name of the disease are observed on stems (Figure 1A). Light yellow to brown streaks or cankers appear on stems, becoming deeper and darker in cases of severe infections. As the disease progresses, infected stems split lengthwise and a pale yellow-to-reddish brown discolouration of the vascular tissue is observed (Figure 1A). Severely infected stems split, forming long brown cankers, while the upper parts of the branch begin to wilt and die. Brown streaks can be seen in the vascular system when the stem is cut open. Yellow sticky fluid may emerge from the cut stem when squeezed. In advanced infection, vascular discolouration is seen as brown streaks on the stem and petiole (Figure 1C). On cutting stems, petioles and peduncles, a creamy-white yellow or reddish-brown discolouration of vascular tissue and pith, and cavities within the pith will be evident. The pith of infected stems turns brown, granular to mealy and filled with cavities. Eventually, vascular wilting and premature death of entire plants are observed (Lamichhane et al. 2011). Symptoms on tomato fruits are rare, seen only under high relative humidity and cultivar susceptibility. Fruits present small (2–5mm), creamy-white spots with tan or brown centres called ‘bird eye spot’. Fruits may remain small and fall prematurely, or ripen unevenly. They also often show external marring and internal bleaching of vascular and surrounding tissues (Figure 1F). Fruit symptom severity is in some cases unrelated to the severity of symptoms during vascular infection, suggesting different mechanisms for colonisation of different tissues (Peritore-Galve et al. 2020, 2021).

Following artificial inoculations under greenhouse conditions (Figure 1G–I), 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 (Haghverdi et al. 2025). In stem injection inoculation, the main symptoms are canker of the stem at the point of inoculation and the upward turning of one or a few of the leaves (Figure 1J). As the systemic infection progresses, the entire leaves may wilt and shrivel. Most *C. michiganensis* strains induce stem canker and wilting of more than one leaf or the entire plant, except for some strains that cause only canker (Malliarakis et al. 2023). Inoculation of eggplant and pepper under greenhouse conditions induces stem canker at the site of inoculation and is less likely to cause leaf symptoms and overall wilting (Figure 1G,H; Haghverdi et al. 2025). Disbudding and defoliation contribute to the secondary spread of bacterial canker in commercial greenhouses (Kawaguchi et al. 2010).

Although bacterial canker symptoms are fairly diagnostic depending on the environmental conditions, plant age and disease stage, wilt symptoms can be confused with several other diseases, that is, bacterial wilt caused by *Ralstonia solanacearum*, Fusarium wilt caused by *Fusarium oxysporum* f. sp. lycopersici and Verticillium wilt caused by *Verticillium dahliae*/*V. alboatrum* (Jones et al. 1991). Both bacterial canker and bacterial wilt diseases of tomato progress much more rapidly than fundamental wilt diseases where the infected plant may be completely

wilted within a few days of observing the initial symptoms (CABI 2021). *R. solanacearum* does not cause canker and cracks on the stems, nor is bird eye spot observed on the fruits. A quick and fairly reliable field diagnosis for bacterial wilt is to submerge a piece of stem (8–10cm) containing vascular tissue in a clear glass jar containing clear water. If the plant has bacterial wilt, a white cloudy bacterial streaming can often be observed coming from the vascular tissue. On the other hand, bacterial wilt can cause a yellow to brown vascular discolouration, which is usually quite distinct from the darker red discolouration associated with Fusarium wilt. Symptoms of vascular discolouration with Verticillium wilt typically do not extend into the leaf petioles. Further, as symptoms progress, Verticillium wilt often causes a characteristic V-shaped lesion on leaflets, with the V opening toward the leaflet margins. Necrotic leaflet lesions are often surrounded by a chlorotic margin (CABI 2021).

## 3 | Host Range of the Pathogen

Nowadays, *C. michiganensis* is recognised as being a specialist plant pathogen presenting a very narrow host range. Tomato is the main host of *C. michiganensis* in both field- and greenhouse-grown crops. However, recent findings showed that small variations in the genomic content of *C. michiganensis* strains drive host specificity of the bacterium (Verma et al. 2024). Natural infections by *C. michiganensis* have infrequently been reported on pepper (*Capsicum annuum*) as well as in other solanaceous species such as eggplant (*Solanum melongena*) and the wild nightshade species *Solanum douglasii*, *Solanum nigrum* and *Solanum triflorum* (Latin et al. 1995; Osdaghi 2020). Resistance in domesticated eggplant to *C. michiganensis* involves the recognition of a secreted putative serine hydrolase, ChpG (Verma and Teper 2022). Boyaci et al. (2021) noted that three cultivated eggplant genotypes were highly susceptible to the pathogen while 31 eggplant genotypes displayed no symptoms. *C. michiganensis* strains isolated from tomato showed reduced virulence on pepper and bell pepper compared to tomato plants. Symptoms on pepper and bell pepper due to artificial inoculation of tomato strains were limited to stem canker with no wilting symptoms (Yim et al. 2012; Haghverdi et al. 2025). It is difficult to recognise if the pepper infections reported before the reclassification of *C. michiganensis* sensu lato were due to the pepper pathogen *C. capsici* or by authentic strains of *C. michiganensis*. A number of solanaceous plants are reported to be susceptible upon artificial inoculation (Osdaghi 2020). *C. michiganensis* also induces disease symptoms on *Nicotiana benthamiana* (Hwang et al. 2024). Recently, Ignatov et al. (2019) reported the pathogenicity of *C. michiganensis* on potato (*Solanum tuberosum*) under natural conditions in Russia. The *C. michiganensis* strains isolated from potato were distinct from the potato pathogen *C. sepedonicus* and were pathogenic on both tomato and potato under greenhouse conditions (Ignatov et al. 2019; Osdaghi et al. 2022).

## 4 | Economic Impact of the Disease

According to the EPPO Global Database https://gd.eppo.int/taxon/CORBMI/distribution and CABI Compendium https://www.cabidigitallibrary.org/doi/10.1079/cabicompendium.15338, bacterial canker of tomato is widespread worldwide,
except for Antarctica (Osdaghi 2020). Bacterial canker, along with bacterial spot (caused by four *Xanthomonas* groups) and bacterial wilt (caused by *R. solanacearum*) are the most important bacterial diseases of tomatoes (Osdaghi et al. 2017, 2021). Yield losses due to bacterial canker infection may reach 93% plant death. Up to 50% average fruit weight decreases have been reported in the literature due to bacterial canker infection (Chang et al. 1992b). Hausbeck et al. (2000) estimated losses of up to $300,000 in Michigan (United States) from sporadic epidemics of the disease. Yield losses and the economic impact of bacterial canker disease are correlated with plant age and phenology when infection occurs. According to Chang et al. (1992b), yield and average fruit weight of processing tomatoes (cv. Heinz1810) were related to the incidence of systemic infection 1 week before harvest. In plants infected during clipping or seedling harvest, yield decreased by 46% and average fruit weight decreased by 13 g when the highest incidence of systemic infection was 31%–83%. Thus, a 5%–7% yield decrease was estimated for each 10% increase in bacterial canker incidence (Chang et al. 1992b). Plant survival and yield in the field are severely affected when transplants have a pathogen population of > $10^8$ CFU/g of tissues. Under artificially inoculated field conditions, inoculated plants produced yields that were 51%–63% of those produced by uninoculated controls (Hausbeck et al. 2000). It has also been noted that the yield of infected plants was compensated by adjacent healthy plants (Ricker and Riedel 1993). Bacterial canker infection seems to affect fruit ripening as well. When the highest incidence of systemic infection was 31%–83%, the percentage of green fruits decreased by 41% and the percentage of ripe fruits increased by 41% (Chang et al. 1992b).

## 5 | Bacteriological Features of the Pathogen

Plant-pathogenic coryneform bacteria are well known for producing a variety of lipid-soluble carotenoid pigments on culture media (Schaad et al. 2001; Hamidizade et al. 2020; Osdaghi, Young, et al. 2020, Osdaghi et al. 2024). General characteristics of *C. michiganensis* are similar to those of other *Clavibacter* species possessing gram-positive, curved rod (coryneform), non-spore-forming cells (Davis et al. 1984). The cells are negative for anaerobic growth, levan production, pectinolytic activity on potato slices and hydrolysis of Tween 80. *C. michiganensis* is known to produce domed round shiny mucoid colonies on general and semiqu selective culture media, for example, yeast extract-peptone-glucose agar (YPGA), yeast extract-dextrose-calcium carbonate (YDC) agar (Figure 2A-F) and bacterial canker of tomato (BCT) (Schaad et al. 2001; Ftayeh et al. 2011). Before the genomics era, it was thought that the bacterial canker pathogen included phenotypically diverse strains with different colony pigmentation. For instance, Kaneshiro et al. (2006) noted that atypical tomato-associated *Clavibacter* strains produced white colonies with mucoid consistency, pink and mucoid colonies, yellow and less fluid colonies than typical (dry or sticky) and orange and sticky colonies. In some cases, only colony morphology but not pigmentation was stated in the literature, where Waleron et al. (2011) reported that the colonies of all tested *C. michiganensis* strains were mucoid.

Before the comprehensive reclassification of *C. michiganensis* sensu lato, morphological variants of *C. michiganensis* have

frequently been reported in the literature, though the correlation between colony characteristics and pathogenicity on tomato was not clarified. According to Zaluga, Van Vaerenbergh, et al. (2013) *Clavibacter* strains originating from tomato in Belgium showed differences in colony morphology. Most of the *C. michiganensis* sensu lato strains studied previously displayed typically yellow fluid colonies, while some atypical strains, for example, LMG 26808 and LMG 26809, showed more orange fluid colonies. Furthermore, strains isolated from pepper (nowadays belonging to *C. capsici*) in Korea produced orange-pigmented colonies with lower mucoid than typical *C. michiganensis* strains isolated from tomato (Yim et al. 2012). Another set of tomato-associated *Clavibacter* strains with yellow-orange and peach-pigmented round mucoid colonies that have traditionally been referred to as *C. michiganensis* were recently reclassified as *C. californiensis*, *C. phaseoli* and *C. lycopersici* (Yasuhara-Bell and Alvarez 2015; Arizala et al. 2022). Thus, the current authentic *C. michiganensis* includes mostly yellow-pigmented colonies on general culture media. Accordingly, diagnostic guidelines issued by EPPO, International Seed Testing Association (ISTA) and European Food Safety Authority (EFSA) refer to the bacterial canker pathogen as a solely yellow-pigmented bacterium (Figure 2A,D; EFSA 2014; EPPO 2016). However, a pink-pigmented tomato-pathogenic variant of the pathogen has recently been isolated from tomato seeds and leaves in southern Iran, expanding the phenotypic range of the pathogen (Figure 2B,E; Haghverdi et al. 2025). This raises the need for reconsideration in diagnostic guidelines and detection procedures to ensure precise screening of tomato seeds and plant materials in quarantine ports (see Section 11). Despite their differences in colony morphology and pigmentation, scanning electron microscopy (SEM) imagery of tomato-associated *Clavibacter* strains with different colony pigmentation indicated similar cell morphology and dimensions. All tomato-associated *Clavibacter* lineages possess curved and club-shaped rod cells, occasionally being arranged at an angle to give a V formation (Figure 2G-I).

## 6 | Biology of the Pathogen

### 6.1 | Inoculum Sources

*Clavibacter michiganensis* is a seedborne pathogen capable of being introduced into areas with no history of the disease via infected seed lots (Anwar et al. 2004). Low levels of seed lot infection (as low as 1 in 10,000 seeds) may lead to epidemic development as cultural practices can cause secondary dissemination of the pathogen (Chang et al. 1991) (Figure 3I-VIII: primary infection). Very low bacterial population size, as low as 5 CFU per seed, can give rise to infection of seedlings (Lelis et al. 2014). It has been noted that weakly virulent and avirulent *C. michiganensis* strains are also frequently isolated from seeds and plants. The bacterial canker disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning and harvesting (Xu et al. 2010).

Infected transplants, tomato debris, alternative hosts and tomato volunteers may also serve as primary or secondary inoculum sources (Chang et al. 1992a) (Figure 3IX-XII). Variations in time and routes of infection affect the range of symptoms under
<figure><img src="image_6.png" /><figcaption>**FIGURE 2** | Bacterial canker pathogen differs from the other members of *Clavibacter* in colony pigmentation. Colonies of yellow- (A) and pink-pigmented (B) strains of *Clavibacter michiganensis* on YPGA (A, B) and YDC (D, E) media compared to the tomato-nonpathogenic strains of *Clavibacter lycopersici* (C and F). Bacterial cultures in section 'F' include yellow- and pink-pigmented *C. michiganensis* (I, II), *Clavibacter tessellatus* (III), *Rathayibacter* sp. (IV), *Clavibacter lycopersici* (V) and *Clavibacter zhangzhiyongii* (VI). Scanning electron microscopy (SEM) of yellow- (G: ICMP 2550T) and pink-pigmented (H: CFBP 9078) *C. michiganensis* as well as peach-pigmented *C. lycopersici* (I: CFBP 8615T) strains showed curved and club-shaped rod cells. Yellow bars (1 µm) show image scale in G, H and I.</figcaption></figure>

natural conditions. In the case of primary infections where plants are infected at early stages of their life, the disease becomes systemic, affecting fruit quality and yield, which typically leads to plant death. Secondary infection, however, includes only foliar symptoms, such as chlorosis of leaves and may affect the quality and yield of the current crop (Nandi et al. 2018). Infected host debris, including roots, might be an important inoculum source of the pathogen for the next season in greenhouses. However, under natural field conditions, the most likely sources of primary inoculum are nightshade hosts (Moffett and Wood 1984).

## 6.2 | Pathogen Survival

Survival time of the pathogen in crop residues under field conditions is variable, ranging from 2 months in Morocco to 2 years in Iowa, United States (Fatmi and Schaad 2002). In host debris

left on the soil surface, bacteria survived 120–260 days for crop production cycles that ended in winter and 45–75 days for those that ended in summer. In stems or roots buried in winter, this period was 45–75 days (Vega and Romero 2016). Soil temperature has a greater effect on survival than moisture. When the stem pieces were buried in soil kept at constant temperature (22°C) and moisture, survival was longer but still limited to 11 months (Moffett and Wood 1984). Higher temperature treatment (45°C) can cause a significant reduction in the pathogen population even after 1 week (Zanón and Jordá 2008). Moffett and Wood (1984) noted that under a crop rotation strategy, it is extremely unlikely that inoculum for outbreaks of the disease will come from buried crop debris. Jacobs et al. (2005) evaluated the role of pigmentation in population dynamics, leaf colonisation strategies and field survival of *C. michiganensis*. Pigment-deficient *C. michiganensis* mutants were significantly reduced in UVA (320–400 nm) radiation survival in vitro and showed
<figure><img src="image_7.png" /><figcaption>**FIGURE 3** | Disease cycle of tomato bacterial canker caused by *Clavibacter michiganensis* under natural conditions. Long distance dissemination of the pathogen occurs via movement of latently infected tomato seeds and transplants (primary infection, I–VIII), while contaminated machinery, rainstorms and plant handling initiate short distance spread of the pathogen (secondary infection, IX and X). Interseasonal transmission of the pathogen is driven by volunteer tomato plants and alternative hosts (XI and XII).</figcaption></figure>

reduced field populations on peanut when compared to the wild-
type strain.

**6.3 | Viable but Non-Culturable State of the Pathogen**

In response to adverse environmental conditions, *C. michiganensis*
may enter the viable but non-culturable (VBNC) state that allows
persistence in unfavourable conditions and resuscitation once
the stress is diminished (Jiang et al. 2016). The VBNC state limits
pathogen detection by culture-based assays and subsequent resus-
citation of *C. michiganensis* cells in tomato seedlings yet poses a
risk for disease development in the field (Chen, Bai, et al. 2021).
Jiang et al. (2016) noted that CuSO₄ in oligotrophic conditions
and low pH induces the VBNC state in *C. michiganensis*, where
some of the VBNC cells retained their ability to colonise tomato

seedlings but failed to produce typical bacterial canker symptoms
by 2 months post-inoculation. Rel, an enzyme that mediates the
synthesis of the secondary messenger guanosine tetraphosphate/
pentaphosphate (ppGpp), accumulates after exposure of *C. mich-
iganensis* cells to stressful conditions, leading to cells entering the
VBNC state, which can be seen as a way of reallocating cellular
resources to counter the stressful conditions (Bai et al. 2022).

7 | Disease Cycle of Tomato Bacterial Canker

Pathogen penetration occurs mainly through trichomes
and hydathodes (Chalupowicz et al. 2017). Physical wounds
during seedling production and crop maintenance facilitate
the pathogen entry (Xu et al. 2012). Tomato flower infection
may lead to fruit infection. Seed infection occurs from xylem
colonisation, which can originate from fruit lesions (Tancos
et al. 2013). For primary infection of the seedlings, the 'window of vulnerability' ranges from transplanting to the 17- to 18-leaf stage (Figure 3II,III). Plants inoculated after the vulnerability period express disease symptoms but do not wilt or die (Sharabani, Manulis-Sasson, et al. 2013; Sharabani, Shtienberg, et al. 2013). The approximate incubation period of symptomless infected tomato seedlings is 10 days (Kawaguchi et al. 2023). Maximum disease incidence on fruits under greenhouse conditions resulted when $10^8$ CFU/mL of inoculum was sprayed over flowers twice and 3 days apart. The probability of infection of healthy tomato plants is 75% after cutting with scissors soaked in a cell suspension of $10^6$ CFU/mL (Kawaguchi et al. 2022). In stem base drop-inoculated plants, the pathogen was found to be present in low densities in roots, stems and leaves only 3 h after inoculation (Lelis et al. 2014). Bacteria multiplied rapidly in cotyledon petioles that remained after clip inoculation and they moved in the stem toward both roots and shoots, leading to decreased root development (Xu et al. 2012). A bioassay using a green fluorescent protein-labelled strain showed that the bacteria extensively colonise the lumen of xylem vessels and preferentially attach to spiral secondary wall thickening of the protoxylem (Chalupowicz et al. 2012).

Movement of *C. michiganensis* from the inoculated leaflet into the rachis is slow and erratic. Upon entrance, the bacteria first multiply within intercellular spaces lying beneath the stomata (Carlton et al. 1998). In grafted seedlings when either rootstock or scion is infected via a contaminated grafting knife, bacteria are translocated in both directions from the graft union at high inoculum doses (Xu et al. 2010). While the hydraulic radius of xylem vessels is not affected, the stem-specific and the leaf-specific conductivity are significantly reduced by inoculation. The pathogen reduces the growth and alters plant-water relations of tomato plants by reducing the stem hydraulic conductivity, as a consequence of the formation of biofilms that restrict xylem sap flow (Romero et al. 2018). Acropetal movement of the pathogen resulted in extensive systemic colonisation of the whole plant reaching the apical region after 15 days (Chalupowicz et al. 2012).

According to Huang and Tu (2001), when tomato plants in hydroponic culture were inoculated with the pathogen through wounds on the stems, the bacteria moved downward from the inoculation site to the roots and were released from the roots into the nutrient solution. Infections by epiphytic *C. michiganensis* populations can also occur under a wide range of temperatures, wetness and seedling age (Frenkel et al. 2016). Exudation through guttation leads to the formation of epiphytic populations on leaflets. The pathogen is exuded in large numbers in the guttation fluid of infected plants (Sharabani, Manulis-Sasson, et al. 2013; Sharabani, Shtienberg, et al. 2013). The temperature during the initial stages of *C. michiganensis* infection affects bacterial canker development and virulence gene expression. A highly significant correlation was found between the average temperatures during the first month after inoculation and the time taken for 50% of the plants to wilt or die (T50), where the shortest T50 mortality (70 days) was observed for an average temperature of 26°C (Sharabani et al. 2014).

Real-time investigation of germinating seeds revealed that *C. michiganensis* aggregates on hypocotyls and cotyledons

at an early stage of germination (Xu et al. 2010). The approximate concentration of *C. michiganensis* in symptomless infected plants was determined as $3 \times 10^6$ CFU/g plant tissue (Kawaguchi et al. 2022). Dutta et al. (2014) noted that the seeds of non-host plants can become infested with incompatible and null-interacting bacterial species through flower colonisation, and they can be transmitted via epiphytic colonisation of seedlings. It has been noted that weakly virulent and avirulent *C. michiganensis* strains are also frequently isolated from seed and plants. For instance, Alvarez et al. (2004) reported that 81% of the *C. michiganensis* sensu lato strains isolated from tomato seed were hypovirulent or avirulent. However, most of these strains were later reclassified as novel stand-alone species (Yasuhara-Bell and Alvarez 2015).

## 8 | Genetic Diversity and Population Structure

Various DNA fingerprinting methods have been used to reveal *C. michiganensis* population structure. The repetitive sequence-based PCR (rep-PCR) method was used in the 1990s to distinguish strains of the tomato pathogen. At least four types (A, B, C and D) were differentiated within *C. michiganensis*, and it was observed that this technique was unable to differentiate pathogenic and nonpathogenic tomato-associated *C. michiganensis* strains (Louws et al. 1998). No relationship was found between rep-PCR clustering and the year/location of strains (Wassermann et al. 2017). Based on polyphasic characterisation and phylogenetic analysis, Jacques et al. (2012) showed that *C. michiganensis* is monophyletic and is distinct from its closest taxonomic neighbours. Evolutionary genome analysis provided evidence that the tomato bacterial canker pathogen emerged after a host shift from grasses. Comparative genomics and phylogenomics analyses identified conserved loci that make *C. michiganensis* a successful pathogen during the transition between these hosts (Osdaghi, Rahimi, et al. 2020; Yañez-Olvera et al. 2024). They also noted that tomato-associated nonpathogenic *Clavibacter* strains were phylogenetically distinct from the pathogenic strains while cross-reacting with *C. michiganensis* identification tools. Multilocus sequence analysis and typing (MLSA/MLST) showed that *C. michiganensis* clonal complexes linked pathogenic strains from highly diverse geographical origins and strains isolated over long periods of time in the same location (Jacques et al. 2012). MLST of *C. michiganensis* strains isolated in Uruguay using the same MLST scheme as the study of Jacques et al. (2012) revealed novel sequence types in this country that could reflect the introduction of new strains from different origins, most likely from seed importation (Croce et al. 2016). Similar analyses divided a worldwide collection of 184 *C. michiganensis* strains into two phylogroups I and II (Ansari et al. 2019). Phylogroup I clustered all strains isolated in eastern Asia (Taiwan and China), eastern Europe (Hungary and Slovenia), South Africa and Portugal, while the strains isolated in Brazil, Italy and Spain were clustered in phylogroup II (Ansari et al. 2019). Multilocus variable-number-tandem-repeats (VNTR) analysis (MLVA) developed by Zaluga, Stragier, et al. (2013) distinguished 25 haplotypes within *C. michiganensis*. Based on MLSA and MLVA, *C. michiganensis* strains from central Chile were found to exhibit low genetic diversity, and sequence types match strains from other parts of the world (Valenzuela et al. 2018). Baysal et al. (2011) introduced
intersimple sequence repeats (ISSR) primers to characterise *C. michiganensis*.

## 9 | Genomic Features

*Clavibacter michiganensis* is the most studied and richest co-
rynebacterial plant pathogen in terms of available genomic re-
sources. By April 2024, 333 complete or draft genome sequences
designated as *Clavibacter* had been deposited in the NCBI
GenBank database. Preliminary screening showed that among
the 333 genomes, 327 strains were authentically *Clavibacter*
and 282 strains were isolated from tomato. Average nucleotide
identity (ANI)-based calculations assigned 265 strains into
*C. michiganensis*, all originating from tomato plants/seeds
(Figure 4). The remaining 17 tomato-associated *Clavibacter*
strains were scattered among other species, in some cases
standing alone as a hypothetical new species. For instance, the
following clades include tomato-associated strains: *C. insidio-
sus* (CFBP 6488), *C. phaseoli* (CFBP 8217 as well as all members
of the taxon originally described as *C. michiganensis* subsp.

*chilensis*; Yasuhara-Bell and Alvarez 2015), *C. californiensis* (CFBP 8216T, A6099, CFBP 7493), *C. lycopersici* (CFBP 8615T and CFBP 8616), *C. capsici* (RA1B). The strains CASJ009 and MX14-G9D belong to a hypothetical new species (Figure 4, Osdaghi, Rahimi, et al. 2020). The bacterial canker pathogen entered the genomics era when the complete genome sequence of the reference *C. michiganensis* strain NCPPB 382 became available (GenBank: AM711867.1; Gartemann et al. 2008). Strain NCPPB 382 has a 3298 Mb circular chromosome with high G + C content (72.6%) and 3080 putative protein-encoding sequences (CDSs), similar to those of other corynebacterial plant pathogens (Chen, Khojasteh, et al. 2021). The complete genome sequence of the type strain of *C. michiganensis* LMG 7333T is also available under the accession number NZ_MZMP01000000 (Oh et al. 2022).

10 | Pathogenicity Mechanisms

Before the establishment of genome-based studies, extracellu-
lar polysaccharide (EPS) production has been underlined as a

<figure><img src="image_8.png" /><figcaption>**FIGURE 4** | Phylotaxonomic analysis of all publicly available genome resources of *Clavibacter* spp. using pyANI v0.2.11 Python pipeline. All tomato-pathogenic strains of *Clavibacter michiganensis* clustered in a monophyletic clade (C.m) while nonpathogenic tomato-associated strains were scattered among the other *Clavibacter* lineages. Results of in silico PCR using the sequence of PCR primers for *C. michiganensis* (right side of the figure) showed that the primer pairs Cmm141F/Cmm141R, Cmm1F/Cmm1R, RhuM-F/RhuM-R, CM3/CM4, cel-578up/cel-2752low, PFC3/PFC5, pCRcel-593/pCRcel-1860, tomA-F/tomA-R, Cm_tomA_F/Cm_tomA_R, TomA-F1/TomA-F2, chpC-F/chpC-R and chpG-F/chpG-R are highly specific for *C. michiganensis*. Phylogenetic clades indicated by abbreviations include C.i: *C. insidiosus*, C.n: *C. nebraskensis*, C.p: *C. phaseoli*, C.se: *C. sepedonicus*, C.l: *C. lycopersici*, C.c: *C. californiensis*, C.z: *C. zhangzhiyongii*, C.t: *C. tessellarius*, C.ca: *C. capsici* and C.m: *C. michiganensis*. The clades designated as HNS are hypothetical new species lacking valid taxonomic description (Osdaghi, Rahimi, et al. 2020).</figcaption></figure>
determinative pathogenicity feature of *C. michiganensis*. Van den Bulk et al. (1990) noted inhibition of callus development from protoplasts of *Solanum peruvianum* by EPS of *C. michiganensis*. EPS-producing strains efficiently colonised tomato plants and induced a hypersensitive reaction (HR) when injected into leaves of the non-host *Mirabilis jalapa* (four o'clock plant). In contrast, the strains producing only small amounts of EPS in vitro were avirulent, did not propagate in tomato, and failed to induce an HR on *M. jalapa* (Eichenlaub et al. 1991). Hibberd et al. (1996) demonstrated that the ability to induce an HR in *M. jalapa* is under the control of a single dominant gene. The HR-inducing activity present in *C. michiganensis* cell supernatant is heat stable, sensitive to proteases and has an apparent molecular mass in the range of 35 to 50 kDa (Alarcon et al. 1998). The HR-inducing factor in *M. jalapa* was identified as the serine protease ChpG (Stork et al. 2008). While *C. michiganensis* grows mainly in the xylem of tomato plants, no significant reduction was found in genome size and CDSs compared to soil-inhabiting coryneform bacteria (Gartemann et al. 2008). Comparative analysis revealed that tomato seed-borne nonpathogenic strains do not possess plasmids pCM1 and pCM2. Also, they lack the majority of important virulence factors described so far for pathogenic *C. michiganensis*, such as the 129-kb low G + C content region known as a pathogenicity island, harbouring *chp/tomA*, near the chromosomal origin of replication, which is necessary for successful colonisation and pathogenicity on tomato (Załuga et al. 2014; Osdaghi, Rahimi, et al. 2020). Gartemann et al. (2008) and Chalupowicz et al. (2010) identified mutant clones of the pathogenic strain NCPPB 382 that lack the entire *chp/tomA* pathogenicity island (CMM30-18 and Cmm27), rendering them completely non-pathogenic. Meletzus et al. (1993) noted that the strain NCPPB 382 carries two plasmids (pCM1 of 27.35 kb and pCM2 of 72 kb) carrying genes involved in virulence. Similar to the 129-kb chromosomal pathogenicity island, the two circular plasmids have lower G + C contents, with pCM1 of 66.5% and pCM2 of 67.6% (Gartemann et al. 2008). After curing the two plasmids, derivatives exhibited a reduced virulence but were still proficient in the ability to colonise the host plant and in the production of EPS (Meletzus et al. 1993). Chalupowicz et al. (2017) showed that plasmid-borne genes, which have a crucial role in wilting, are not required for blister formation. When one of the two plasmids is lost, there is a significant delay in the development of wilting symptoms after infection. The plasmid-borne virulence genes are *celA* on pCM1 and *pat-1* on pCM2. The gene *celA* plays a major role in pathogenicity as it encodes an endo-β-1,4-glucanase, CelA (Jahr et al. 2000), while strains with or without *pat-1* (pCM2) are indistinguishable based on disease severity (Bella et al. 2012; Thapa et al. 2017). Burger et al. (2005) demonstrated the presence of *pat-1* homologous nucleotide sequences on the chromosome and on plasmid pCM2. The introduction of the *pat-1* region into an endophytic plasmid-free *Clavibacter* strain converted the latter strain into a virulent strain (Dreier et al. 1997). Burger et al. (2005) identified the serine protease Pat-1 in *C. michiganensis*, which led to the finding of a family of serine proteases (ChpA-ChpG) that play a role in colonisation of tomato (Stork et al. 2008). In *C. michiganensis* NCPPB 382, the genes *chpA*, *chpB* and *chpD* are pseudogenes containing frameshifts and/or in-frame stop codons. In planta, the titre of the *chpC* mutant was drastically reduced and only weak disease

symptoms were observed. However, the *chpG* mutant was not impaired in virulence. *chpC* was the first chromosomal gene with a defined role in plant–*C. michiganensis* interaction (Stork et al. 2008). Hwang et al. (2019) also found that the cellulase catalytic domain and cellulose-binding domain of CelA together were sufficient for both cellulase activity and full virulence of the bacterium. Interestingly, a *celA* orthologue from *C. sepedonicus* can restore the full virulence to the *celA* mutant of *C. michiganensis* (Hwang et al. 2019). The *celA* and *pat-1* genes were not detected in pepper-pathogenic strains recently renamed as *C. capsici* (Yim et al. 2012; Valenzuela et al. 2021). Transcriptional analysis revealed that *celA* and *pat-1* are significantly induced 12–72 h post-inoculation (hpi), whereas *chpC* and *ppaA* are highly expressed only 96 hpi. Transcription of chromosomal genes involved in cell wall degradation (*pelA1*, *celB*, *xysA* and *xysB*) is also induced at early stages of infection (Chalupowicz et al. 2010).

In subsequent years, additional genes have been identified as having a significant role in the *C. michiganensis* pathogenicity, for example, transcriptional regulators Vatr1 and Vatr2 (Savidor et al. 2012). According to Savidor et al. (2014), Vatr1 and Vatr2 regulate the expression of virulence factors, membrane and secreted proteins and signal-transducing proteins. It has been noted that Pat-1 is an immune elicitor inducing HR in *Nicotiana tabacum* (non-host), but in tomato it acts as a virulence factor (Dreier et al. 1997; Hwang et al. 2022). The bacterium expresses Pat-1 and CelA not only after host cell contact in planta but also in M9 minimal and xylem-mimicking medium (Hiery et al. 2015). Recently, Hwang et al. (2024) found that the chromosomal *cviA1* gene in *C. michiganensis* plays an important role in necrosis development in *Nicotiana benthamiana* leaves. *cviA1* encodes a 180-amino acid protein with a signal peptide at the N-terminus and two putative transmembrane domains at the C-terminus. Deletion of the signal peptide or the C-terminus, including the two putative transmembrane domains in *CviA1*, failed to restore full necrosis in the mutant. Verma et al. (2024) demonstrated that the ChpG<sup>C</sup> variant is not recognised as an immune elicitor in eggplant, whereas other ChpG variants from eggplant-nonpathogenic strains are. They identified five *chpG* allelic variants within *C. michiganensis* populations and named them as *chpG<sup>A</sup>*, *chpG<sup>B1</sup>*, *chpG<sup>B2</sup>*, *chpG<sup>C</sup>*, and *chpG<sup>D</sup>*. The *chpG<sup>C</sup>* variant was only present in the eggplant-pathogenic strains of the pathogen. A single amino acid substitution in the ChpG serine protease domain eliminates its recognition in eggplant. *chpG<sup>C</sup>* has a single T506G substitution that results in a V169G amino acid alteration. The *chpG<sup>C</sup>* also elicits attenuated HR in the non-host plant *M. jalapa*.

The availability of microarray, total RNA-sequencing (RNA-seq) and CRISPR/Cas9-mediated gene-editing techniques further clarified the gene expression profile of *C. michiganensis* during tomato infection. Flügel et al. (2012) constructed and validated a *C. michiganensis* oligonucleotide microarray and found that among 9254 tomato genes represented on the array, 122 were differentially expressed in infected plants compared to non-inoculated plants. RNA-sequencing indicated that numerous genes involved in stringent response, copper resistance and stress resistance were up-regulated, and some involved in cell division were down-regulated significantly (Bai et al. 2022).
Like Chen et al. (2022), Stevens et al. (2021) developed a suite of tools for genetic manipulation in *C. michiganensis*, including the *codA::upp* counterselection system to create markerless deletions in *Clavibacter*, an integrative plasmid and an R package for the identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination-based marker-less knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. Chen et al. (2022) detailed a highly efficient unmarked CRISPR/Cas9-mediated gene-editing system in *Clavibacter* that couples the expression of *cas9* and single-guide RNA with homology-directed repair templates and the negative selectable marker *codA::upp* within a single plasmid.

# 11 | Detection and Identification of the Pathogen

Historically, detection of *C. michiganensis* in tomato seeds and plant specimens was based on culture plating on general or semiselective media. Vidaver (1982) and Gleason et al. (1993) summarised all conventional methods for detection and diagnosis of *C. michiganensis* in the pregenomics era, which mostly included culture-based isolation and immunodiagnostic techniques. Since the early 1990s, DNA sequence-based detection methods such as specific PCR, nucleotide probes and quantitative real-time PCR have been extensively used for detection of the pathogen with higher efficiency and lower effort. The French National Laboratory for Plant Health initiated an international effort on the assessment of the methods used in laboratories for detection of *C. michiganensis* in tomato seeds, that is, dilution plating on semiselective media and immunofluorescence, including an interlaboratory study on naturally and artificially contaminated seeds by eight laboratories from six European countries (Olivier et al. 2010). A comparison of different methods was performed with reference strains to determine the applicability of the molecular tests in optimal conditions (Jacques et al. 2012). These comprehensive assays led to the preparation of the EPPO, ISTA and EFSA diagnostic guidelines (EFSA 2014; EPPO 2016).

## 11.1 | Seed Extraction

Infected seed is often considered to be the primary inoculum source and the major source for outbreaks of bacterial canker. Owing to the fact that population levels of the pathogen in/on the seed may be very low, development of an efficient, sensitive and reliable seed extraction procedure is a prerequisite for the establishment of robust quarantine programmes. Seed extraction methods that include grinding the seeds are better at detecting the pathogen than methods that use only soaking (Hadas et al. 2005). This could be due in part to the fact that grinding exposes the interior parts of the seeds to culture media or solution buffer. Seedlots infested with fewer than 58 CFU/g do not cause disease under greenhouse conditions, whereas seeds with 1000 CFU/g caused disease in 78 out of 2000 test plants (Hadas et al. 2005). For a successful seed extraction, 24 g of the seed sample (approximately 10,000 seeds) is placed in a sterile double-layered plastic bag containing 150 mL sterile phosphate-Tween buffer. The specimen is incubated at 4°C for 15 min. Then

the bag is placed with its contents in a stomacher (Lab Blender Model 400 Mark II) and blended for 15 min. The resulting suspension can be streaked onto semiselective culture media or directly used for PCR assays (Osdaghi 2020).

## 11.2 | Culture-Based Methods

Bacterial canker of tomato can be diagnosed by the field symptoms and by isolation of the causal organism on a nonselective medium or a semiselective medium followed by a pathogenicity test on a 2- to 4-leaf-stage tomato seedling (EPPO 2016). The most suitable part of tomato plants for reliable detection of the bacterium is the lower stem region (Krämer and Griesbach 1995). Semiselective media are useful for detection and isolation of *C. michiganensis* in plant health surveys and quarantine inspections. Within the past three decades, several semiselective culture media have been developed mainly on the basis of original medium NBY. The EPPO diagnostic guideline (2016) provided a detailed list of semiselective media and their ingredients for isolation of *C. michiganensis*. Modified CNS agar is a semiselective medium on which *C. michiganensis* colonies appear in 6-7 days (Vidaver and Davis 1988), while on SCM agar grey-to-black speckled colonies are formed (Fatmi and Schaad 1988). A modification of SCM agar, m-SCM agar, yields clear colonies with yellow flecks that appear 7-9 days post-incubation (Waters and Bolkan 1992). Ftayeh et al. (2011) noted that all previously published semiselective media (D2, KBT, D2ANX, SCM, mSCM, CMM1 and mCNS) gave false-negative results. Thus, they developed a new selective and highly sensitive medium for isolation of *C. michiganensis* from seeds and latently infected plants and named the new medium BCT (bacterial canker of tomato). Exclusively, BCT also supports growth of the closely related species *C. insidiosus*, *C. nebraskensis* and *C. tessellarius* (Osdaghi, Robertson, et al. 2023). On CMM1 agar, colonies of *C. michiganensis* are yellow, mucoid and convex, while on BCT agar typical colonies appear creamy to yellow in colour, convex and shining (Kaneshiro et al. 2006; Ftayeh et al. 2011). A xylem-mimicking medium (XMM) was also developed by Hiery et al. (2013) based on an apoplast medium for tomato-pathogenic xanthomonads. In contrast to the apoplast medium, XMM contains no sugars but amino acids that serve as a nitrogen and carbon source. Altogether, the BCT medium seems to be more suitable for isolation of the bacterial canker pathogen despite its complex ingredients and application of rare antibiotics.

Kaneshiro et al. (2006) noted that a large proportion of *Clavibacter* strains associated with naturally infested tomato seeds were putatively hypovirulent or nonvirulent. Upon isolation of the suspected bacterial strains, pathogenicity tests should be carried out on tomato seedlings at 24°C-27°C under 60%-80% relative humidity. Inoculation techniques include infiltrating a freshly prepared aqueous suspension of bacterial cells (10⁶ CFU/mL) with a syringe into the stem, or excising a leaflet on the first or second leaf with a pair of scissors contaminated with the inoculum (Gitaitis et al. 1989). Leaf margin curling and one-sided wilting or withering of the leaves in the vicinity of the inoculation site will occur within 2-3 weeks, which is indicative of the virulence of the corresponding strain. For artificial inoculation of tomato plants, defoliation using infected scissors and inserting a sterile dissecting needle dipped into a freshly
prepared bacterial suspension are more successful than planting
in soil containing contaminated plant debris (Kawaguchi and
Tanina 2014; Ansari et al. 2019). A paintbrush could be applied to
inoculate the surface of small fruits (Medina-Mora et al. 2001).
Disease symptoms become evident from the third day. As the
disease progresses, the bacterial population increases in planta,
reaching the highest level after 6 days (Tsitsekian et al. 2021).
The bacterial concentration can increase to over 10⁶ cells/g plant
tissue at 20 cm away from the inoculated point on the stem by
10 days after inoculation.

11.3 | DNA Sequence-Based Methods

11.3.1 | Conventional PCR

For the first time, Ghedini and Fiore (1995) used a PCR test de-
veloped by Sousa Santos et al. (1997) to detect latent *C. michigan-
ensis* infections in tomato seedlings. The sensitivity threshold
of the method was estimated around 1.1 × 10³ CFU/5 µL of stem
suspension (Sousa Santos et al. 1997). Then, specific PCR prim-
ers PSA-4/PSA-R (Table 1) were developed for the detection of
the pathogen along with the other species of the genus (at that
time subspecies of *C. michiganensis* sensu lato; Pastrik and
Rainey 1997). The applicability of the latter primer pair was im-
proved via multiplexing with tomato-specific primers NS-7-F/
NS8-R as an internal PCR control primer, to provide a reliable
method for the detection of *C. michiganensis* (Zhang et al. 2009).
Thapa et al. (2020) also developed a multiplex PCR-based di-
agnostic platform using the sequences of chromosomal genes
*rhuM* and *tomA* and an internal control to amplify both bacte-
rial and plant DNA. Several other PCR primers are available for
the specific detection of *C. michiganensis* as detailed in Table 1.
Viable cells of the pathogen can be detected using dyes such as
ethidium monoazide (Luo et al. 2008) and propidium monoazide
(Han et al. 2018) in qPCR tests.

11.3.2 | Bio-PCR

Bio-PCR is an improved derivative of conventional PCR where
bacterial cells are enriched on a general or semiselective me-
dium before DNA amplification. Advantages of bio-PCR over
conventional PCR techniques include the elimination of false
positives resulting from the presence of dead cells that may be
present in the seed, elimination of false negatives due to poten-
tial PCR inhibitors in seed extracts, increased sensitivity of de-
tection and no need for DNA extraction prior to amplification
(Schaad et al. 1995). Hadas et al. (2005) noted that in tomato
seed samples containing 5–10 infected seeds per 10,000 seeds,
*C. michiganensis* was detected by agar plating assay on three
media (CNS, mSCM and D2ANX) as well as using direct PCR
from seeds and bio-PCR. However, in samples of one infected
seed per 10,000 seeds, the pathogen could be detected only by
bio-PCR.

11.3.3 | TaqMan-PCR

Besides conventional PCRs, real-time TaqMan-based methods
are also available for the detection of the pathogen. In some

cases, the PCR primers are capable of targeting all *Clavibacter*
species in a single run. For instance, a real-time TaqMan-PCR
assay was developed for the detection, differentiation and abso-
lute quantification of *C. michiganensis* by directing the amplifi-
cation of a 223 bp DNA fragment of intergenic sequences of the
rRNA operon (ITS) (Bach et al. 2003). Then, a TaqMan probe
was developed for the specific detection of *C. michiganensis* in
symptomless tomato seeds. The pathogen can be detected in 2 h
without DNA extraction when the seed infection rate is higher
than 1% (Zhao et al. 2007). The primer pair CmmG-F
CmmG-R_WF was designed to detect the *clvG* gene sequence,
which is exclusively present in *C. michiganensis*. General
*Clavibacter* primers and a universal internal control were also
added to neutralise PCR inhibitors and false-negative results in
real-time PCRs, making a reliable triplex TaqMan qPCR. The
assay was specific for *C. michiganensis* and detected up to 10 fg
of the pathogen's DNA (Ramachandran et al. 2021). Recently,
Brochu, Dumonceaux, et al. (2023) developed a multiplex
TaqMan real-time PCR assay to detect *C. michiganensis* based
on two chromosomal virulence-related genes *rhuM* and *tomA*
(Brochu, Dumonceaux, et al. 2023). The plant internal control
*tubulin* α3 was included in each of the multiplexes to improve
the reliability of the assay.

11.3.4 | Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) assay has ad-
vantages over immunodiagnostic methods and PCR-based tech-
niques because of its specificity and isothermal nature, which
allows for easy field application. Yasuhara-Bell et al. (2013) de-
veloped a LAMP technique based on the michiganin A (*micA*)
gene, which was highly specific to the bacterial canker patho-
gen. In another study, genomic analyses showed that *clvA*, *clvF*
and *clvG* were present only in *C. michiganensis*, not in other
*Clavibacter* species nor other genera of plant-associated bacte-
ria. Thus, loop-mediated amplification of *clvA* was as effective
in identifying *C. michiganensis*-positive tomato seed and tissue
samples as the ImmunoStrip method (Yasuhara-Bell et al. 2014).
Yasuhara-Bell et al. (2015) showed that the LAMP technique
can provide a reliable real-time portable in-field assay compa-
rable to accepted standards. Dobhal et al. (2019) developed an-
other LAMP assay where the sensitivity of the method was 1 fg
DNA per reaction.

A comparative in silico analysis of available PCR primers for
their specificity to *C. michiganensis* using whole genome re-
sources of *Clavibacter* species is illustrated in Figure 4. The
primer pairs CMR16F1/CMR16R1, PSA-4/PSA-R and Clav-F/
Clav-R are generic for all *Clavibacter* species (Lee et al. 1997;
Quintero-Vásquez et al. 2013). The primer pairs Cmm141F/
Cmm141R (targeting *ferredoxin reductase*), Cmm1F/Cmm1R
(targeting *tomatinase*), RhuM-F/RhuM-R (targeting *rhuM*) and
CM3/CM4 are highly specific for *C. michiganensis*, where no in
silico DNA amplification was observed in non-*C. michiganensis*
strains even if the strain was isolated from a tomato plant (Cho
et al. 2012; Kokošková et al. 2010; Thapa et al. 2020; Sousa Santos
et al. 1997). Interestingly, PCR primers originally designed for de-
tection and amplification of pathogenicity determinant genes in
*C. michiganensis* have been shown to be entirely specific for this
species, where they could amplify the expected DNA fragment
<table><caption>TABLE 1 | Nucleotide sequences and physical parameters of primers designed for detection and identification of *Clavibacter michiganensis* using conventional PCR, TaqMan-PCR and LAMP techniques.</caption><thead><tr><th>Primer name</th><th>Sequence (5'-3')</th><th>Size of amplicon (bp)</th><th>Annealing temperature (°C)</th><th>Target</th><th>Reference</th></tr></thead><tbody><tr><td colspan="6"><strong>PCR primers for phylogenetic analyses of C. michiganensis</strong></td></tr><tr><td>fD1</td><td>AGAGTTTGATCCTGGCTCAG</td><td>1484</td><td>63</td><td>16S rDNA</td><td>Weisburg et al. (1991)</td></tr><tr><td>rP2</td><td>ACGGCTACCTTGTACGACTT</td><td></td><td></td><td></td><td></td></tr><tr><td>atpD2F</td><td>GACATCGAGTTCCCGCAC</td><td>1104</td><td>55</td><td>atpD</td><td>Jacques et al. (2012)</td></tr><tr><td>atpD2R</td><td>CGATGATCTCCTGGAGCTCCTTGT</td><td></td><td></td><td></td><td></td></tr><tr><td>dnakF</td><td>GCTCGTGCACTAGGAATCG</td><td>704</td><td>59</td><td>dnaK</td><td>Jacques et al. (2012)</td></tr><tr><td>dnakR</td><td>CTTGGCGATCTGTCGTTCGAGAC</td><td></td><td></td><td></td><td></td></tr><tr><td>2F</td><td>ACCGTCGAGTTCGACTACGA</td><td>977</td><td>57</td><td>gyrB</td><td>Richert et al. (2005)</td></tr><tr><td>6R</td><td>AGSACGATCTTGTGGTA</td><td></td><td></td><td></td><td></td></tr><tr><td>ppkF</td><td>GAGAACCTCATCCAGGCCCT</td><td>604</td><td>60</td><td>ppk</td><td>Jacques et al. (2012)</td></tr><tr><td>ppkR</td><td>CGAGCTTGCAGTGGGTCTTGAG</td><td></td><td></td><td></td><td></td></tr><tr><td>recaF</td><td>GACCGCGCTCGCACAGATCGACCG</td><td>724</td><td>63</td><td>recA</td><td>Jacques et al. (2012)</td></tr><tr><td>recaR</td><td>GCCATCTTGTTCTTGACGACCTTG</td><td></td><td></td><td></td><td></td></tr><tr><td>3Fs</td><td>GACAACTTCTACTTCAAC</td><td>447</td><td>57</td><td>rpoB</td><td>Richert et al. (2007)</td></tr><tr><td>4Rs</td><td>GTTGTTCTGGTCCATGAAC</td><td></td><td></td><td></td><td></td></tr><tr><td colspan="6"><strong>PCR primers for detection of pathogenicity determinant genes in C. michiganensis</strong></td></tr><tr><td>cel-578up</td><td>ATGGCTTCCCTACGATCC</td><td>2193</td><td>59</td><td>celA</td><td>Jahr et al. (2000)</td></tr><tr><td>cel-2752low</td><td>ACAGGGTAGAAGCGGGAGG</td><td></td><td></td><td></td><td></td></tr><tr><td>pCRcel-593</td><td>TCCTTATATGACATTTCGCC</td><td>1268</td><td>57</td><td>Catalytic domain of celA</td><td>Jahr et al. (2000)</td></tr><tr><td>pCRcel-1860</td><td>GCCACTTCGCTGATACAG</td><td></td><td></td><td></td><td></td></tr><tr><td>PFC3</td><td>GGTACGAAGTTCGAGACGAC</td><td>552</td><td>62</td><td>Cellulose binding domain of celA</td><td>Kleitman et al. (2008)</td></tr><tr><td>PFC5</td><td>TGTAGCGGTGAGTCGTGGTGA</td><td></td><td></td><td></td><td></td></tr><tr><td>tomA-F</td><td>CGAACTCGACCAGGTTCTCG</td><td>529</td><td>60</td><td>tomA</td><td>Kleitman et al. (2008)</td></tr><tr><td>tomA-R</td><td>GGTCTCACGATCGGATCC</td><td></td><td></td><td></td><td></td></tr></tbody></table>

(Continues)
<table><caption>TABLE 1 | (Continued)</caption><thead><tr><th>Primer name</th><th>Sequence (5'-3')</th><th>Size of amplicon (bp)</th><th>Annealing temperature (°C)</th><th>Target</th><th>Reference</th></tr></thead><tbody><tr><td>ppaA-F</td><td>CATGATATTGGTGGGAAAG</td><td>588</td><td>56</td><td>ppaA</td><td>Kleitman et al. (2008)</td></tr><tr><td>ppaA-R</td><td>CCCCGTCCTTTTGCAAGACC</td><td></td><td></td><td></td><td></td></tr><tr><td>chpC-F</td><td>GCTCTTGGGCTAATGGCCG</td><td>639</td><td>62</td><td>chpC</td><td>Kleitman et al. (2008)</td></tr><tr><td>chpC-R</td><td>GTCAGTTGTGGAAGATGCTG</td><td></td><td></td><td></td><td></td></tr><tr><td>chpG-F</td><td>GACAACATGACCCTGCACTG</td><td>394</td><td>62</td><td>chpG</td><td>Kleitman et al. (2008)</td></tr><tr><td>chpG-R</td><td>TCGGGGTGTAGACAAGGAAG</td><td></td><td></td><td></td><td></td></tr><tr><td>Cmm-5</td><td>GCGAATAAGCCCATATCAA</td><td>609</td><td>55</td><td>pat-1</td><td>Dreier et al. (1995)</td></tr><tr><td>Cmm-6</td><td>CGTCAGGAGGTCGCTAATA</td><td></td><td></td><td></td><td></td></tr><tr><td colspan="6"><strong>Generic PCR primers for Clavibacter spp.</strong></td></tr><tr><td>CMR16F1</td><td>GTGATGTCAGAGCTTCCTCTGGCGGAT</td><td>1425</td><td>62</td><td>Clavibacter spp.</td><td>Lee et al. (1997)</td></tr><tr><td>CMR16R1</td><td>GTACGGCTACCTTGTTACGACTTAGT</td><td></td><td></td><td></td><td></td></tr><tr><td>PSA-4</td><td>TCATTGGTCAATTCTGTCTCCC</td><td>271</td><td>58</td><td>Clavibacter spp.</td><td>Pastrik and Rainey (1999)</td></tr><tr><td>PSA-R</td><td>TACTGAGATGTTTCACTTCCCC</td><td></td><td></td><td></td><td></td></tr><tr><td>Clav-F</td><td>TGGATCACCTCCTTTCTAAG</td><td>296</td><td>55.9</td><td>rRNA region</td><td>Quintero-Vásquez et al. (2013)</td></tr><tr><td>Clav-R</td><td>CACCACCATCCACAACAGGA</td><td></td><td></td><td></td><td></td></tr><tr><td colspan="6"><strong>Specific PCR primers for detection of C. michiganensis</strong></td></tr><tr><td>ITSYG-1</td><td>CGCGTCAGGCGTCTGTT</td><td>270</td><td>60</td><td>rRNA internal transcribed spacer (ITS)</td><td>Zhao et al. (2007)</td></tr><tr><td>ITSYG-2</td><td>AGTGGACGCGAGCATC</td><td></td><td></td><td></td><td></td></tr><tr><td>Probe</td><td>TGGCGGTGGCGCTCATGG*</td><td></td><td></td><td></td><td></td></tr><tr><td>Cmm141F</td><td>CAGGCGTCCGTCGGTGAGGTGGTC</td><td>141</td><td>63</td><td>Ferredoxin reductase</td><td>Cho et al. (2012)</td></tr><tr><td>Cmm141R</td><td>GCGGGAGAGCGGTGCGGGAATG</td><td>500</td><td>69</td><td>Tomatinase</td><td>Kokošková et al. (2010)</td></tr><tr><td>Cmm1F</td><td>GACAAGCACCTCTACACCTGG</td><td></td><td></td><td></td><td></td></tr></tbody></table>

(Continues)
<table><caption>TABLE 1 | (Continued)</caption><thead><tr><th>Primer name</th><th>Sequence (5'-3')</th><th>Size of amplicon (bp)</th><th>Annealing temperature (°C)</th><th>Target</th><th>Reference</th></tr></thead><tbody><tr><td>CMM-16-23S_e_</td><td>GCACCTTCTGGGTGTGTCTG</td><td rowspan="5">140</td><td rowspan="5">Multiplex</td><td rowspan="5">16S-23S rRNA</td><td rowspan="5">Peňázová et al. (2020)</td></tr><tr><td>fwd</td><td></td></tr><tr><td>CMM-16-23S_e_</td><td>TGTGATCCACCGGAAACCG</td></tr><tr><td>rev</td><td></td></tr><tr><td>CMM TP</td><td>TCCGTCGTCCTGTTGTGGATG(HEX-BHQ1)</td></tr><tr><td>CM3</td><td>CCTCGTGAGTGCCGGGAACGTATCC</td><td rowspan="2">639</td><td rowspan="2">60</td><td rowspan="2">Chromosomal DNA</td><td rowspan="2">Sousa Santos et al. (1997)</td></tr><tr><td>CM4</td><td>CCACGGTGGTTGATGCTCGCGAGAT</td></tr><tr><td>CmmG-F_wf</td><td>CGTCGAGAACCAAGCTCATCA</td><td rowspan="2">136</td><td rowspan="2">65</td><td rowspan="2">clvG</td><td rowspan="2">Ramachandran et al. (2021)</td></tr><tr><td>CmmG-R_wf</td><td>CGAGATGACGGCGTAGTACC</td></tr></tbody></table>

Abbreviation: NS, not specified.

*The 5'-end labelled with 6-carboxyfluorescein (FAM) and the 3'-end labelled with tetramethylcarboxyrhodamine (TAMRA).

only in tomato-pathogenic strains. Thus, the primers cel-578up/cel-2752low (celA), PFC3/PFC5 and pCRcel-593/pCRcel-1860 (catalytic domain of celA), tomA-F/tomA-R, Cm_tomA_F/Cm_tomA_R and TomA-F1/TomA-F2 (tomA), chpC-F/chpC-R (chpC) and chpG-F/chpG-R (chpG) are exclusively specific for C. michiganensis. The potential applicability of the latter primer pairs for detection and identification of C. michiganensis needs to be evaluated in cross-laboratory assays.

## 11.4 | Other Techniques

Amplification and sequencing of the gyrB gene using a single primer set has sufficient resolution and specificity to identify each *Clavibacter* species (Richert et al. 2005). However, MLSA/MLST using the sequences of six housekeeping genes (*atpD*, *dnaK*, *gyrB*, *ppK*, *recA* and *rpoB*) is more useful for precise identification, taxonomic delineation, typing and phylogenetic characterisation of *C. michiganensis* populations (Jacques et al. 2012). All species of the genus generate distinct and reproducible matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry profiles, with unique and specific ion peaks for each species, which could be used as biomarkers for identification of the bacteria (Zaluga et al. 2011). Early detection of the disease is achievable using machine-learning spectral analysis (Vallejo-Pérez et al. 2021). Raman spectra were obtained from asymptomatic *C. michiganensis*-infected tomato plants as well as healthy controls with a 785 nm excitation laser micro-Raman spectrometer. The Raman spectra obtained from infected tomato leaf samples exhibited peaks associated with cellular components (carbohydrates, carotenoids, chlorophyll and phenolic compounds). Raman bands associated with triterpenoids and flavonoids compounds can be considered indicators of *C. michiganensis* infection during the asymptomatic stage (Vallejo-Pérez et al. 2021).

## 11.5 | Simultaneous Detection With Other Pathogens

Simultaneous detection of *C. michiganensis* with other tomato pathogens would save cost and effort in field surveys and quarantine assays. Multiplex PCR provides rapid and low-cost results for detection of bacterial pathogens of tomato. In some cases, however, the sensitivity of detection may be reduced in simultaneous detections. Özdemir (2009a) developed a multiplex PCR test for simultaneous detection of three seedborne tomato bacterial pathogens, *C. michiganensis*, *Pseudomonas syringae* pv. *tomato* and *Xanthomonas euvesicatoria* pv. *euvesicatoria*. Another multiplex real-time PCR method based on fluorescent TaqMan probes was developed for simultaneous detection of *C. michiganensis*, *P. syringae* pv. *tomato* and leaf spot-causing xanthomonads (Peňázová et al. 2020). Besides bacterial canker, pathovars of *X. euvesicatoria*, *P. syringae* and complex species are the main bacterial pathogens infecting tomato with leaf spot, leaf speck and blight symptoms (Osdaghi et al. 2021). Simultaneous detection of *C. michiganensis* and pepino mosaic virus in tomato seed was also reported by Johnson and Walcott (2012). A multiplex PCR assay was developed for simultaneous detection of *C. michiganensis*, *Fusarium* sp., *Leveillula taurica* and begomoviruses (Quintero-Vásquez et al. 2013). A polyprobe (poly-3)
was developed for simultaneous detection of *C. michiganensis*,
pepino mosaic virus and Mexican papita viroid in tomato plants
by non-isotopic molecular hybridisation, which was comparable
with real-time PCR results (Zamora-Macorra et al. 2015). A chip
digital PCR was also developed to identify and quantify *C. mich-
iganensis* and *Ralstonia solanacearum* at the same time (Morcia
et al. 2023).

**12** | **Management of the Disease**

Due to the lack of effective bactericides, the bacterial canker
disease is managed primarily by quarantine measures, use of
pathogen-free plant materials and sanitation. A number of non-
specific chemicals, biological control agents and semi-resistant/
tolerant cultivars are also available to keep the established dis-
eases under the economic loss threshold. Quarantine inspec-
tion and early detection of the pathogen are the most effective
approaches for bacterial canker management in areas with no
history of the diseases.

**12.1 | Field Management**

Blank et al. (2016) noted that the adoption of field manage-
ment strategies is the most influential factor on bacterial can-
ker severity. Differences in farmers' experience, differences in
agricultural practices between growers and the quality of imple-
mentation of management practices are correlated with the oc-
currence and severity of the disease. The bacterial canker agent
is capable of being epiphytic on plants. All actions allowing
contact between plant surfaces or between plant sap should be
avoided. Touching symptomless infected plants bearing gutta-
tion droplets prior to touching nearby plants spreads the patho-
gen over distances within rows of up to 22 plants (Sharabani,
Manulis-Sasson, et al. 2013; Sharabani, Shtienberg, et al. 2013).
Infection is transferred to healthy plants by cutting with con-
taminated scissors after cutting infected plants with early
symptoms or symptomless ones (Sharabani, Manulis-Sasson,
et al. 2013; Sharabani, Shtienberg, et al. 2013). As the bacterium
survives easily in water, irrigation should be operated with great
caution. The sub-irrigation system reduces, but does not prevent,
pathogen dispersal. Jones, Worobo, et al. (2014) proposed UV
light inactivation of *C. michiganensis* in unfiltered surface irri-
gation water where > 99.9% inactivation was achieved. Frenkel
et al. (2016) showed that the pathogen dispersed spatially from
root-inoculated source seedlings and colonised the leaf surfaces
of surrounding seedlings to distances of 65–75 cm.

**12.2** | **Biological Control**

While Proteobacteria are the most abundant organisms
within the endophytic communities of diseased tomato (López
et al. 2020), *Pseudomonas* spp., *Streptomyces* spp. and *Bacillus*
spp. are considered the most appealing biological control agents
for *C. michiganensis* worldwide (Aksoy et al. 2017; Benchlih
et al. 2023). Fluorescent pseudomonads isolated either from
the phyllosphere or rhizosphere of tomato have frequently
been reported as antagonistic agents against the incidence of
bacterial canker (Boudyach et al. 2001; Amkraz et al. 2010;

Lanteigne et al. 2012; Bouizgarne et al. 2023). A rhizosphere
strain of *Pseudomonas entomophila* (23S) was reported to have
a strong antagonistic activity from which two anti-*C. michigan-
ensis* compounds, C15H16N2O and C16H18N2O, were isolated
(Takishita et al. 2021). Paulin et al. (2017) demonstrated that
inoculation of tomato plants with 2,4-diacetylphloroglucinol-
and hydrogen cyanide-producing *Pseudomonas brassicace-
arum* LBUM300 could significantly reduce bacterial canker
symptoms.

The gram-positive bacteria Bacilli and Actinobacteria have also
a significant role in the biological control of *C. michiganensis*
(Utkhede and Koch 2004; Zhang et al. 2010). Calderón-de la
Sancha et al. (2022) reported the antimicrobial activity in a low-
molecular-weight protein secreted naturally by *Streptomyces
lividans* TK24 when glucose or glycerol were used as carbon
sources. Water extracts of *Bacillus* sp. strains H8-1 and K203
inhibited wilting caused by *C. michiganensis* and slowed the
pathogenic colonisation in tomato plants. The relative expres-
sions of *celA*, *celB*, *pat1* and *pelA* of *C. michiganensis* treated
with the bacterial aqueous extracts were reduced compared
to controls at 72h after treatments (Jang et al. 2022). *Bacillus*
strains 1B-23 and 1D-12, capable of producing surfactins A, B
and C, significantly reduced disease incidence in a greenhouse
setting (Laird et al. 2020).

Application of *Pseudozyma aphidis* spores on tomato plants in greenhouses significantly reduced incidence of bacterial canker disease. *P. aphidis* activates PR1a and other pathogenesis-related genes in tomato plants and can trigger an induced resistance response against *C. michiganensis* that proceeds in a salicylic-acid (SA)-independent manner (Barda et al. 2015). Exogenously applied SA suppressed bacterial growth and induced the expression of WRKY transcription factors, suggesting that some *C. michiganensis*-responsive genes are regulated by SA signaling and SA signalling activation should improve tomato immunity against *C. michiganensis* (Yokotani et al. 2021). Enzymatic activity, hydrogen peroxide formation and lignin production were significantly higher in benzothiadiazole (500ppm)-treated leaves than in those observed in the control (Tripathi et al. 2022).

Promising results have also been reported on phage therapy
of the bacterial canker disease. Application of Phage33 from
tomato fields in Turkey was effective against *C. michiganen-
sis* (Bekircan Eski and Darcan 2023). Bacteriophage CMP1
(*Siphoviridae* family) infects *C. michiganensis* specifically, en-
coding a peptidase that was shown to effectively lyse the patho-
gen specifically (Wittmann et al. 2011). The endolysin gene of
CMP1 was transferred into tomato plants by *Agrobacterium*
mediated transformation, where transgenic tomato plants did
not show disease symptoms after infection with *C. michiganen-
sis* (Wittmann et al. 2010, 2016).

Preliminary investigations revealed suppression of *C. mich-
iganensis* by plant extracts and essential oils while the appli-
cability and mass production of these biological agents on a
large scale are still questionable. Aqueous extract of *Eucalyptus
globulus* leaves inhibits the growth of *P. syringae* pv. *tomato,
X. euvesicatoria* pv. *euvesicatoria* and *C. michiganensis* (Pinto
et al. 2023). Capsaicinoids are molecules found in the fruits of
*Capsicum* species that produce their spicy taste and also possess
antibacterial and antifungal effects. A synthetic capsaicinoid
oleoresin showed an inhibitory effect against *Fusarium oxysporum* and *C. michiganensis* (Valencia-Hernandez et al. 2022).
Sonicated extracts from microalgae of the genera *Leptolyngbya* and *Scenedesmus* were evaluated for their effect on bacterial canker inhibition. Bioassays on tomato seedlings showed that root application of *Scenedesmus* extract is capable of controlling *C. michiganensis*, while foliar and root application of *Leptolyngbya* extract seems to be more related to the strengthening of the plant through the SA route (Toribio et al. 2021). Growth and oxygen consumption of *C. michiganensis* were suppressed after the addition of fragarin to cultures. Fragarin is an antibiotic that was isolated and purified from a soluble fraction of strawberry leaves. Furthermore, dissipation of the membrane potential and an increase in cell membrane permeability were observed in the presence of fragarin (Filippone et al. 2001).

**12.3 | Chemical Control**

Chemical control with cupric bactericides or streptomycin is the last defensive line in canker disease management (Lamichhane et al. 2018; Lyu et al. 2019). To prevent severe bacterial canker disease in the field, growers should initiate and sustain bactericide applications to tomato transplants in the greenhouse to suppress pathogen populations. de León et al. (2008) showed that treatments containing copper sulphate greatly reduced disease symptoms on plants, while streptomycin was less effective. Coskun and Horuz (2023) noted that foliar spray of phosphites inhibited the growth of *C. michiganensis* between 50% and 74% and raised the chlorophyll concentration of tomato leaves up to 30% in phosphite-sprayed plants. Besides traditional copper-based chemicals and antibiotics, application of novel bactericides, for example, nanoparticles, provides new tools for management of the disease (Marcelino-Pérez et al. 2021). Silver nanoparticles (AgNPs) are promising inhibitors of *C. michiganensis* (Rivas-Cáeres et al. 2018). Application of copper nanoparticles and potassium silicate was effective in reducing the severity of *C. michiganensis* (Cumplido-Nájera et al. 2019). AgNPs were prepared from an aqueous extract of fresh leaves from *Larrea tridentata*, with significant disease control being achieved 42 days post-inoculation with the pathogen (Méndez-Andrade et al. 2022). Furthermore, AgNPs produced with moringa extracts reduced canker disease by 86%. Systemic acquired resistance is suggested as an important mechanism induced by Mo-AgNPs (Mercado-Meza et al. 2023).

Pretreatment of plants with acibenzolar-S-methyl (benzo [1,2,3] thiadiazole-7-carbothioic acid-S-methyl ester, ASM; Bion 50 WG) reduced the severity of the disease as well as the growth of the bacteria in planta (Soylu et al. 2003; Baysal et al. 2003). Although in vitro growth of the bacteria was not affected by DL-β-amino butyric acid (BABA) treatment, foliage sprays of 500 µg/ mL BABA significantly suppressed disease development up to 54% by day 14 after inoculation (Baysal et al. 2005). Polygodial and nordrimenone showed promising results against *C. michiganensis*, *P. syringae* pv. *tomato*, *F. oxysporum* f. sp. *lycopersici* and *Phytophthora* spp. (Xu et al. 2015; Montenegro et al. 2018). The synthetic elicitors 2,6-dichloro-isonicotinic acid (INA) and 2,4-dichloro-6-[(E)-[(3-methoxyphenyl)imino]methyl]phenol (DPMP) enhance tomato resistance against bacterial canker

disease with different molecular mechanisms (Bektas 2021). Phenolic and flavonoid contents of medicinal plants belonging to 16 species were approved against the tomato bacterial canker agent (Amkraz et al. 2014). Ombiro et al. (2018) reported that ralhibitin E completely inhibited the growth of *C. michiganensis* and *R. solanacearum* at 10 µg/mL. Eustressic doses of cadmium (60 µg/kg of soil) induce defence mechanisms and protection against *C. michiganensis* in tomato (Valencia-Hernandez et al. 2023). Rotondo et al. (2023) introduced a proprietary blend of plant extracts as a potential option for bacterial canker management and yield enhancement in hydroponic tomato greenhouse production systems.

**12.3.1 | Antibiotics**

Applications of copper hydroxide, copper hydroxide+mancozeb, copper hydroxide+mancozeb (premixed 12 h before spraying), streptomycin and streptomycin+copper hydroxide to seedlings in the greenhouse increased the survival of inoculated transplants in the field in comparison to the control (Hausbeck et al. 2000). Resistance to streptomycin was reported in *C. michiganensis* strains isolated in Chile (Valenzuela et al. 2019). Minimum inhibitory concentration in a naturally occurring streptomycin-resistant *C. michiganensis* strain TX-0702 was 128 µg/mL (Lyu et al. 2019).

**12.3.2 | Seed Treatment**

Citric acid at 0.1 M concentration has been proved to be useful for the elimination of *C. michiganensis* from tomato seeds (Özdemir 2009b). 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 (Pradhanan and Collier 2009). Ten-minute immersion of seeds in acidified nitrite resulted in 98% of the seeds being pathogen-free. Treatment with copper hydroxide and certain strains of *Bacillus* spp. resulted in 100% pathogen-free seeds (Kasselaki et al. 2011).

**13 | Host Resistance**

**13.1 | Resistance Sources**

Development of resistant cultivars is the most sustainable approach for long-term management of the bacterial canker diseases as well as for all seedborne bacterial pathogens of the crop (Osdaghi et al. 2021; Khojasteh et al. 2024). Screening and breeding practices for disease resistance will be more applicable when they are conducted simultaneously for a set of economically important diseases. The genetic nature of disease resistance is classified as either being qualitative (simple), controlled by one or few resistance genes, or quantitative (complex), governed by multiple resistance genes or quantitative trait loci (QTLs). Meta-analysis of QTLs, which gathers QTL data from independent studies across genetic backgrounds and environments and identifies stable and reliable QTL regions, is a powerful strategy to facilitate
marker-assisted selection in plant breeding. Recently, Khojasteh et al. (2024) examined 491 QTLs previously reported for resistance to tomato diseases in 40 independent studies and 54 unique mapping populations. They identified 29 and 44 meta-QTLs for resistance to bacterial and fungal pathogens, respectively. Among the 29 meta-QTLs identified for resistance to bacterial diseases, 12, 4 and 2 were specific to bacterial spot, bacterial wilt and bacterial canker diseases, respectively. Interestingly, four meta-QTLs located on chromosomes 4 and 6 contributed to resistance to both bacterial spot and bacterial wilt diseases, while two meta-QTLs located on chromosomes 2 and 5 conferred resistance to both bacterial canker and bacterial spot diseases. Abebe et al. (2022) conducted a QTL-Seq analysis for identification of resistance loci to bacterial canker in tomato. A genomic region (37.24–41.15 Mb) associated with bacterial canker resistance on chromosome 6 (Rcm6) was found. Celik (2023) constructed a physical map for bacterial canker resistance QTLs and identified QTL-specific candidate genes. Single-marker QTL analysis suggested that at least two loci originating from *Solanum hirsutum* LA407, Rcm 2.0 on chromosome 2 and Rcm 5.1 on chromosome 5, contribute to resistance in replicated trials (Kabelka et al. 2002).

While some cherry tomato varieties are tolerant, others are highly susceptible to bacterial canker. In general, cherry tomato varieties tend to be more resistant to bacterial canker but more susceptible to bacterial spot than the fresh-market tomato (Romero et al. 2003). The pathogen causes less severe symptoms in wild tomato species (e.g., *Solanum habrochaites* LA2128, *Solanum arcanum* LA2157 and *S. arcanum* LA2172) and is impeded in spread and colonisation of the vascular system (Peritore-Galve et al. 2020). QTLs conferring tolerance in *S. arcanum* and *S. habrochaites* have been identified (Peritore-Galve et al. 2020). Using RFLP markers by means of the Kruskal-Wallis rank-sum test in *Solanum peruvianum* accession LA2157, five regions on chromosomes 1, 6, 7, 8 and 10 were identified that may be involved in *C. michiganensis* resistance (Sandbrink et al. 1995; Van Heusden et al. 1999). Significant variation in disease resistance was observed among 283 somaclones from 12 tomato cultivars (De Vries and Stephens 1997). Francis et al. (2001) noted that partial resistance to *C. michiganensis* was identified in a wild relative of cultivated tomato, *S. habrochaites* LA407.

## 13.2 | Host Responses During Tomato-C. michiganensis Interactions

Upon *C. michiganensis* inoculation, several defence responsive genes were found to be differentially expressed, of which 26 genes were in the resistant line and three were in the susceptible line (Basim et al. 2021). Advanced lines of a cross between *S. arcanum* LA2157 and *S. lycopersicum* showed that introgression lines carrying a locus of *S. arcanum* LA2157 on chromosome 7 had high levels of tolerance to *C. michiganensis*. Koseoglou, Brouwer, et al. (2023) suggested that two additional loci on chromosomes 2 and 4 together with the locus on chromosome 7 are required for tolerance to *C. michiganensis*. In RNA-seq analyses, 1788 and 540 genes were up-regulated and down-regulated upon infection with *C. michiganensis*, respectively, where genes involved in the defence response, phosphorylation and hormone signalling were over-represented. Tomato genes involved in SA and phenylalanine ammonia-lyase (PAL) pathway were also

up-regulated upon infection (Yokotani et al. 2021). Koseoglou, Hanika, et al. (2023) showed that tomato gene *SIWAT1* is a susceptibility gene to *C. michiganensis*, where inactivation of this gene leads to reduced susceptibility to the bacterium, reduced free auxin content and ethylene synthesis in tomato stems. Host-derived ethylene plays an important role in the regulation of the tomato susceptible response to *C. michiganensis* (Balaji et al. 2008). Comparative transcriptome and gene ontology analysis revealed that the wall-associated receptor kinase-like 20 (WAKL20) contributes to resistance against *C. michiganensis* infection (Deng et al. 2023).

## 13.3 | Screening for Resistance

No immunity has been found in any tomato cultivars against *C. michiganensis*. Berry et al. (1989) demonstrated that it is possible to identify plants with intermediate resistance using a dilute inoculum of a virulent strain of *C. michiganensis*. Sen et al. (2013) screened 24 different wild accessions of tomato and found several new tolerant sources: *Solanum pimpinellifolium* GI.1554, *Solanum parviflorum* LA735 and *S. parviflorum* LA2072. They also confirmed the tolerance in *S. peruvianum* LA2157, *S. peruvianum* PI127829, *S. peruvianum* LA385, *S. habrochaites* LA407 and *S. lycopersicum* IRAT L3. A new method using the PathoScreen field test kit was evaluated to localise green fluorescent protein-tagged *C. michiganensis* in planta and to quantify the pathogen based on the percentage of corrected GFP (cGFP%). The system was sensitive in detecting the GFP-tagged *C. michiganensis* in the shoots, but in the roots, a high autofluorescence masked detection and thus sensitivity of the assay (Mohd Nadzir et al. 2019). Brochu, Durivage, et al. (2023) aimed to identify an artificial inoculation method to induce bacterial canker on tomato plants in greenhouse conditions to homogenise the results of different studies. The syringe inoculation with low fertilisation was the most effective inoculation method, allowing the development of a multilevel scale that can be used to study the interaction between tomato plants and *C. michiganensis*. Excision with an infected scalpel of the first true leaf of 3-week-old seedlings, followed by applying a drop of inoculum on the wound, discriminated well between populations of partially resistant and susceptible tomato genotypes (Van den Bulk et al. 1991).

## 14 | Conclusion and Future Avenues for Research

Bacterial wilt and canker caused by *C. michiganensis* is considered a devastating disease in many tomato production regions. Since the first description of bacterial canker in 1909, dozens of studies provided a foundation of knowledge, that is, understanding of pathogen diversity, colony morphology and factors influencing the pathogen spread. Investigations highlighted the role of quarantine inspections, crop sanitation, and resistant cultivars in the management of bacterial canker disease. The use of pathogen-free high-quality seedlots is the cornerstone of disease management in the areas where the pathogen is established. However, strict quarantine rules should prevent the distribution of the pathogen into new areas with no history of the disease. Recent technological advancements in high-throughput DNA sequencing ensure that in the coming years we will integrate all
discoveries into a comprehensive understanding of the biology, spread and survival of *C. michiganensis*, as well as molecular mechanisms underlying disease development in the host plant. During the past two decades, genomics has played an increasing role in the understanding of colonisation, infection, transmission and evolution of plant-pathogenic bacteria. Comparative genomics analyses and phylogenomics provided fundamentals for elucidating the global population structure of *C. michiganensis*. The first complete genome sequence of *C. michiganensis* became available in 2008, and by early 2024, 265 *C. michiganensis* genome sequences were deposited in the NCBI public database. These whole genome resources provide opportunities to deepen the understanding of the molecular basis for the morphological variations and colony pigmentation of the pathogen. Further, with the genomics data in hand, we will be able to develop state-of-the-art genome-informed detection methods to detect seed infections with lower efforts and cost. Such knowledge could aid in mitigating the negative impacts of colony variations in *C. michiganensis* in quarantine inspections and seed health tests. This will also enable molecular breeders to develop durable broad-spectrum resistance and disease control strategies that are acceptable to the tomato industry. Further studies on plant and pathogen transcriptomics and meta-transcriptomics will initiate a deeper understanding of plant-bacterial interactions and ways of controlling plant colonisation. Finally, recent advances in our understanding of molecular host-pathogen interactions of other plant pathogens in the *Microbacteriaceae* will continue to aid the development of a more comprehensive understanding of the molecular biology of corynebacterial plant pathogens and identify research paths for the sustainable management of bacterial canker in the 21st century.

## Acknowledgements

Financial support was provided by Iran National Science Foundation (INSF) under project no. 4038663. The work of E.O. was funded by the University of Tehran, Iran. We thank Hamzeh Mafakheri and Malihe Haghverdi (Shiraz University, Iran) for their assistance in bacterial culture preparation and photography.

## Conflicts of Interest

The authors declare no conflicts of interest.

## Data Availability Statement

Data sharing is not applicable to this article as no new data were generated.

## References

Abebe, A. M., C. S. Oh, H. T. Kim, et al. 2022. "QTL-Seq Analysis for Identification of Resistance Loci to Bacterial Canker in Tomato." *Frontiers in Plant Science* 12: 809959.

Aksoy, H. M., Y. Kaya, M. Ozturk, Z. Secgin, H. Onder, and A. Okumus. 2017. "*Pseudomonas putida*–Induced Response in Phenolic Profile of Tomato Seedlings (*Solanum lycopersicum* L.) Infected by *Clavibacter michiganensis* subsp. *michiganensis*." *Biological Control* 105: 6–12.

Alarcon, C., J. Castro, F. Munoz, P. Arce-Johnson, and J. Delgado. 1998. "Protein(s) From the Gram-Positive Bacterium *Clavibacter michiganensis* subsp. *michiganensis* Induces a Hypersensitive Response in Plants." *Phytopathology* 88: 306–310.

Alvarez, A. M., W. S. Kaneshiro, and B. G. Vine. 2004. "Diversity of *Clavibacter michiganensis* subsp. *michiganensis* Populations in Tomato Seed: What Is the Significance?" *Acta Horticulturae (ISHS)* 695: 205–214.

Amkraz, N., E. H. Boudyach, H. Boubaker, B. Bouizgarne, and A. Ait Ben Aoumar. 2010. "Screening for Fluorescent Pseudomonades, Isolated From the Rhizosphere of Tomato, for Antagonistic Activity Toward *Clavibacter michiganensis* subsp. *michiganensis*." *World Journal of Microbiology and Biotechnology* 26: 1059–1065.

Amkraz, N., I. Talibi, H. Boubaker, et al. 2014. "Antioxidant Activity, Phenols and Flavonoids Contents and Antibacterial Activity of Some Moroccan Medicinal Plants Against Tomato Bacterial Canker Agent." *African Journal of Biotechnology* 13: 4515–4522.

Ansari, M., S. M. Taghavi, H. Hamzehzarghani, M. Valenzuela, M. I. Siri, and E. Osdaghi. 2019. "Multiple Introductions of Tomato Pathogen *Clavibacter michiganensis* subsp. *michiganensis* Into Iran as Revealed by a Global-Scale Phylogeographic Analysis." *Applied and Environmental Microbiology* 85: e02098-19.

Anwar, A., P. S. Van der Zouwen, S. Ilyas, and J. M. Van der Wolf. 2004. "Bacterial Canker (*Clavibacter michiganensis* subsp. *michiganensis*) of Tomato in Commercial Seed Produced in Indonesia." *Plant Disease* 88: 680.

Arizala, D., S. Dobhal, A. M. Alvarez, and M. Arif. 2022. "Elevation of *Clavibacter michiganensis* subsp. *californiensis* to Species Level as *Clavibacter californiensis* sp. nov., Merging and Re-Classification of *Clavibacter michiganensis* subsp. *chilensis* and *Clavibacter michiganensis* subsp. *phaseoli* as *Clavibacter phaseoli* sp. nov. Based on Complete Genome In Silico Analyses." *International Journal of Systematic and Evolutionary Microbiology* 72: 005427.

Bach, H. J., I. Jessen, M. Schloter, and J. C. Munch. 2003. "A TaqMan-PCR Protocol for Quantification and Differentiation of the Phytopathogenic *Clavibacter michiganensis* Subspecies." *Journal of Microbiological Methods* 52: 85–91.

Bai, K., N. Jiang, X. Chen, X. Xu, J. Li, and L. Luo. 2022. "RNA-Seq Analysis Discovers the Critical Role of Rel in ppGpp Synthesis, Pathogenicity, and the VBNC State of *Clavibacter michiganensis*." *Phytopathology* 112: 1844–1858.

Balaji, V., M. Mayrose, O. Sherf, et al. 2008. "Tomato Transcriptional Changes in Response to *Clavibacter michiganensis* subsp. *michiganensis* Reveal a Role for Ethylene in Disease Development." *Plant Physiology* 146: 1797–1809.

Barda, O., O. Shalev, S. Alster, K. Buxdorf, A. Gafni, and M. Levy. 2015. "*Pseudozyma aphidis* Induces Salicylic-Acid-Independent Resistance to *Clavibacter michiganensis* in Tomato Plants." *Plant Disease* 99: 621–626.

Basim, H., E. Basim, H. Tombuloglu, and T. Unver. 2021. "Comparative Transcriptome Analysis of Resistant and Cultivated Tomato Lines in Response to *Clavibacter michiganensis* subsp. *michiganensis*." *Genomics* 113: 2455–2467.

Baysal, Ö., Y. Z. Gürsoy, H. Örnek, and A. Duru. 2005. "Induction of Oxidants in Tomato Leaves Treated With DL-β-Amino Butyric Acid (BABA) and Infected With *Clavibacter michiganensis* ssp. *michiganensis*." *European Journal of Plant Pathology* 112: 361–369.

Baysal, Ö., F. Mercati, H. İkten, et al. 2011. "*Clavibacter michiganensis* subsp. *michiganensis*: Tracking Strains Using Their Genetic Differentiations by ISSR Markers in Southern Turkey." *Physiological and Molecular Plant Pathology* 75: 113–119.

Baysal, Ö., E. M. Soylu, and S. Soylu. 2003. "Induction of Defence-Related Enzymes and Resistance by the Plant Activator Acibenzol-S-Methyl in Tomato Seedlings Against Bacterial Canker Caused by *Clavibacter michiganensis* ssp. *michiganensis*." *Plant Pathology* 52: 747–753.

Bekircan Eski, D., and C. Darcan. 2023. "Isolation of *Clavibacter michiganensis* subsp. *michiganensis*-Specific Bacteriophages From Tomato
Fields in Turkey and Their Biocontrol Potential." *Egyptian Journal of Biological Pest Control* 33: 1-10.

Bektas, Y. 2021. "The Synthetic Elicitors 2, 6-Dichloro-Isonicotinic Acid (INA) and 2, 4-Dichloro-6-{(E)-[(3-Methoxyphenyl) Imino] Methyl} Phenol (DPMP) Enhances Tomato Resistance Against Bacterial Canker Disease With Different Molecular Mechanisms." *Physiological and Molecular Plant Pathology* 116: 101740.

Bella, P., G. Ialacci, G. Licciardello, R. La Rosa, and V. Catara. 2012. "Characterization of Atypical *Clavibacter michiganensis* subsp. *michiganensis* Populations in Greenhouse Tomatoes in Italy." *Journal of Plant Pathology* 94: 635-642.

Benchlih, S., Q. Esmaeel, K. Aberkani, et al. 2023. "Modes of Action of Biocontrol Agents and Elicitors for Sustainable Protection Against Bacterial Canker of Tomato." *Microorganisms* 11: 726.

Berry, S. Z., G. G. Madumadu, M. R. Uddin, and D. L. Coplin. 1989. "Virulence Studies and Resistance to *Clavibacter michiganensis* ssp. *michiganensis* in Tomato Germplasm." *HortScience* 24: 362-365.

Blank, L., Y. Cohen, M. Borenstein, et al. 2016. "Variables Associated With Severity of Bacterial Canker and Wilt Caused by *Clavibacter michiganensis* subsp. *michiganensis* in Tomato Greenhouses." *Phytopathology* 106: 254-261.

Boudyach, E. H., M. Fatmi, O. Akhayat, E. Benizri, and A. A. B. Aoumar. 2001. "Selection of Antagonistic Bacteria of *Clavibacter michiganensis* subsp. *michiganensis* and Evaluation of Their Efficiency Against Bacterial Canker of Tomato." *Biocontrol Science and Technology* 11: 141-149.

Bouizgarne, B., M. Bakki, A. Boutasknit, et al. 2023. "Phosphate and Potash Solubilizing Bacteria From Moroccan Phosphate Mine Showing Antagonism to Bacterial Canker Agent and Inducing Effective Tomato Growth Promotion." *Frontiers in Plant Science* 14: 970382.

Boyaci, H., A. Kabaş, Y. Aysan, and J. Prohens. 2021. "Screening of Eggplant Genotypes for Resistance to Bacterial Wilt Disease Caused by *Clavibacter michiganensis* subsp. *michiganensis*." *Plant Protection Science* 57: 112-121.

Brochu, A. S., T. Dumonceaux, R. Belanger, and E. Perez-Lopez Sr. 2023. "A New Multiplex TaqMan qPCR Targeting *Clavibacter michiganensis* Virulence-Related Genes." *bioRxiv*. https://doi.org/10.1101/2023.06.20.545733. [Preprint].

Brochu, A. S., J. Durivage, D. Torres Garcia, and E. Pérez-López. 2023. "Diet and Injection, Important Recommendations to Characterize *Clavibacter michiganensis*–Tomato Interactions." *Plant Health Progress* 24: 475-481.

Bryan, M. K. 1930. "Studies on Bacterial Canker of Tomato." *Journal of Agricultural Research* 41: 825-851.

Burger, A., I. Gräfen, J. Engemann, et al. 2005. "Identification of Homologues to the Pathogenicity Factor Pat-1, a Putative Serine Protease of *Clavibacter michiganensis* subsp. *michiganensis*." *Microbiological Research* 160: 417-427.

CABI. 2021. "*Fusarium oxysporum* f.sp. *lycopersici* (Fusarium Wilt of Tomato)." *CABI Compendium*. https://doi.org/10.1079/cabicompendium.24660.

Calderón-de la Sancha, F. J., U. Carrasco-Navarro, G. Santander, J. Barrios-González, and A. Mejía. 2022. "Novel Antimicrobial Activity of Protein Produced by Streptomyces Lividans TK24 Against the Phytopathogen *Clavibacter michiganensis*." *Archives of Microbiology* 204: 687.

Carlson, R. R., and A. K. Vidaver. 1982. "Taxonomy of Corynebacterium Plant Pathogens, Including a New Pathogen of Wheat, Based on Polyacrylamide-Gel Electrophoresis of Cellular Proteins." *International Journal of Systematic Bacteriology* 32: 315-326.

Carlton, W. M., E. J. Braun, and M. L. Gleason. 1998. "Ingress of *Clavibacter michiganensis* subsp. *michiganensis* Into Tomato Leaves Through Hydathodes." *Phytopathology* 88: 525-529.

Celik, I. 2023. "In Silico Integrated Analysis of Genomic, Transcriptomic, and Proteomic Data Reveals QTL-Specific Genes for Bacterial Canker Resistance in Tomato (Solanum lycopersicum L.)." *Current Issues in Molecular Biology* 45: 1387-1395.

Chalupowicz, L., I. Barash, M. Reuven, et al. 2017. "Differential Contribution of *Clavibacter michiganensis* ssp. *michiganensis* Virulence Factors to Systemic and Local Infection in Tomato." *Molecular Plant Pathology* 18: 336-346.

Chalupowicz, L., M. Cohen-Kandli, O. Dror, et al. 2010. "Sequential Expression of Bacterial Virulence and Plant Defense Genes During Infection of Tomato With *Clavibacter michiganensis* subsp. *michiganensis*." *Phytopathology* 100: 252-261.

Chalupowicz, L., E. M. Zellermann, M. Fluegel, et al. 2012. "Colonization and Movement of GFP-Labeled *Clavibacter michiganensis* subsp. *michiganensis* During Tomato Infection." *Phytopathology* 102: 23-31.

Chang, R. J., S. M. Ries, and J. K. Pataky. 1991. "Dissemination of *Clavibacter michiganensis* subsp. *michiganensis* by Practices Used to Produce Tomato Transplants." *Phytopathology* 81: 1276-1281.

Chang, R. J., S. M. Ries, and J. K. Pataky. 1992a. "Local Sources of *Clavibacter michiganensis* ssp. *michiganensis* in the Development of Bacterial Canker on Tomatoes." *Phytopathology* 82: 553-560.

Chang, R. J., S. M. Ries, and J. K. Pataky. 1992b. "Reductions in Yield of Processing Tomatoes and Incidence of Bacterial Canker." *Plant Disease* 76: 805-809.

Chen, G., M. Khojasteh, A. Taheri-Dehkordi, S. M. Taghavi, T. Rahimi, and E. Osdaghi. 2021. "Complete Genome Sequencing Provides Novel Insight Into the Virulence Repertories and Phylogenetic Position of Dry Beans Pathogen *Curtobacterium flaccumfaciens* pv. *flaccumfaciens*." *Phytopathology* 111: 268-280.

Chen, X., K. Bai, Q. Lyu, N. Jiang, J. Li, and L. Luo. 2021. "Role of Penicillin-Binding Proteins in the Viability, Morphology, Stress Tolerance, and Pathogenicity of *Clavibacter michiganensis*." *Phytopathology* 111: 1301-1312.

Chen, X., Q. Tan, Q. Lyu, et al. 2022. "Unmarked Gene Editing in *Clavibacter michiganensis* Using CRISPR/Cas9 and 5-Fluorocytosine Counterselection." *Molecular Plant-Microbe Interactions* 35: 4-14.

Cho, M. S., J. H. Lee, N. H. Her, et al. 2012. "A Quantitative and Direct PCR Assay for the Subspecies-Specific Detection of *Clavibacter michiganensis* subsp. *michiganensis* Based on a Ferredoxin Reductase Gene." *Journal of Microbiology* 50: 496-501.

Coskun, T. A., and S. Horuz. 2023. "Phosphites for the Management of Tomato Bacterial Canker and Stem Rot." *Journal of Plant Diseases and Protection* 130: 609-617.

Croce, V., M. J. Pianzzola, K. Durand, M. González-Arcos, M. A. Jacques, and M. I. Siri. 2016. "Multilocus Sequence Typing Reveals High Variability Among *Clavibacter michiganensis* subsp. *michiganensis* Strains Affecting Tomato Crops in Uruguay." *European Journal of Plant Pathology* 144: 1-13.

Cumplido-Nájera, C. F., S. González-Morales, H. Ortega-Ortíz, G. Cadenas-Pliego, A. Benavides-Mendoza, and A. Juárez-Maldonado. 2019. "The Application of Copper Nanoparticles and Potassium Silicate Stimulate the Tolerance to *Clavibacter michiganensis* in Tomato Plants." *Scientia Horticulturae* 245: 82-89.

Davis, M. J., A. G. Gillaspie, A. K. Vidaver, and R. W. Harris. 1984. "Clavibacter: A New Genus Containing Some Phytopathogenic Coryneform Bacteria, Including *Clavibacter xyli* subsp. *xyli* sp. nov., subsp. nov. and *Clavibacter xyli* subsp. *cynodontis* subsp. nov., Pathogens That Cause Ratoon Stunting Disease of Sugarcane and Bermudagrass Stunting Disease." *International Journal of Systematic Bacteriology* 34: 107-117.

de León, L., F. Siverio, M. M. López, and A. Rodríguez. 2008. "Comparative Efficiency of Chemical Compounds for In Vitro and
In Vivo Activity Against *Clavibacter michiganensis* subsp. *michiganensis*, the Causal Agent of Tomato Bacterial Canker." *Crop Protection* 27: 1277-1283.

De Vries, R. M., and C. T. Stephens. 1997. "Response of First Generation Tomato Somaclone Progeny to *Clavibacter michiganensis* subsp. *michiganensis*." *Plant Science* 126: 69-77.

Deng, S., Z. Li, X. Liu, W. Yang, and Y. Wang. 2023. "Comparative Transcriptome Analysis Reveals Potential Genes Conferring Resistance or Susceptibility to Bacterial Canker in Tomato." *Horticulturae* 9: 242.

Dhanvantari, B. N. 1989. "Effect of Seed Extraction Methods and Seed Treatments on Control of Tomato Bacterial Canker." *Canadian Journal of Plant Pathology* 11: 400-408.

Dobhal, S., A. Larrea-Sarmiento, A. M. Alvarez, and M. Arif. 2019. "Development of a Loop-Mediated Isothermal Amplification Assay for Specific Detection of All Known Subspecies of *Clavibacter michiganensis*." *Journal of Applied Microbiology* 126: 388-401.

Dreier, J., A. Bermpohl, and R. Eichenlaub. 1995. "Southern Hybridization and PCR for Specific Detection of Phytopathogenic *Clavibacter michiganensis* subsp. *michiganensis*." *Phytopathology* 85: 462-468.

Dreier, J., D. Meletzus, and R. Eichenlaub. 1997. "Characterization of the Plasmid Encoded Virulence Region *Pat-1* of Phytopathogenic *Clavibacter michiganensis* subsp. *michiganensis*." *Molecular Plant-Microbe Interactions* 10: 195-206.

Dutta, B., R. Gitaitis, S. Smith, and D. Langston Jr. 2014. "Interactions of Seedborne Bacterial Pathogens With Host and Non-Host Plants in Relation to Seed Infestation and Seedling Transmission." *PLoS One* 9: e99215.

Dye, D. W., and W. J. Kemp. 1977. "A Taxonomic Study of Plant Pathogenic *Corynebacterium* Species." *New Zealand Journal of Agricultural Research* 20: 563-582.

EFSA PLH Panel (EFSA Panel on Plant Health). 2014. "Scientific Opinion on the Pest Categorisation of *Clavibacter michiganensis* subsp. *michiganensis* (Smith) Davis et al." *EFSA Journal* 12: 3721.

Eichenlaub, R., A. Bermpohl, and D. Meletzus. 1991. "Genetic and Physiological Aspects of the Pathogenic Interaction of *Clavibacter michiganense* subsp. *michiganense* With the Host Plant." In *Advances in Molecular Genetics of Plant-Microbe Interactions Vol. 1: Proceedings of the 5th International Symposium on the Molecular Genetics of Plant-Microbe Interactions, Interlaken, Switzerland, September 9-14, 1990*, 99-102. Springer Netherlands.

EPPO. 2016. "PM 7/42 (3) *Clavibacter michiganensis* subsp. *michiganensis*." *EPPO Bulletin* 46: 202-225.

Fatmi, M., and N. W. Schaad. 1988. "Semiselective Agar Medium for Isolation of *Clavibacter michiganense* subsp. *michiganense* From Tomato Seed." *Phytopathology* 78: 121-126.

Fatmi, M., and N. W. Schaad. 2002. "Survival of *Clavibacter michiganensis* ssp. *michiganensis* in Infected Tomato Stems Under Natural Field Conditions in California, Ohio and Morocco." *Plant Pathology* 51: 149-154.

Filippone, M. P., J. C. Diaz-Ricci, A. P. Castagnaro, and R. N. Farías. 2001. "Effect of Fragarin on the Cytoplasmic Membrane of the Phytopathogen *Clavibacter michiganensis*." *Molecular Plant-Microbe Interactions* 14: 925-928.

Flügel, M., A. Becker, K. H. Gartemann, and R. Eichenlaub. 2012. "Analysis of the Interaction of *Clavibacter michiganensis* subsp. *michiganensis* With Its Host Plant Tomato by Genome-Wide Expression Profiling." *Journal of Biotechnology* 160: 42-54.

Francis, D. M., E. Kabelka, J. Bell, B. Franchino, and D. St. Clair. 2001. "Resistance to Bacterial Canker in Tomato (*Lycopersicon hirsutum* LA407) and Its Progeny Derived From Crosses to *L. esculentum*." *Plant Disease* 85: 1171-1176.

Frenkel, O., M. Bornestein, R. Shulhani, et al. 2016. "Secondary Spread of *Clavibacter michiganensis* subsp. *michiganensis* in Nurseries and the Conditions Leading to Infection of Tomato Seedlings." *European Journal of Plant Pathology* 144: 569-579.

Ftayeh, R. M., A. von Tiedemann, and K. W. Rudolph. 2011. "A New Selective Medium for Isolation of *Clavibacter michiganensis* subsp. *michiganensis* From Tomato Plants and Seed." *Phytopathology* 101: 1355-1364.

Gartemann, K. H., B. Abt, T. Bekel, et al. 2008. "The Genome Sequence of the Tomato-Pathogenic Actinomycete *Clavibacter michiganensis* subsp. *michiganensis* NCPPB382 Reveals a Large Island Involved in Pathogenicity." *Journal of Bacteriology* 190: 2138-2149.

Ghedini, R., and N. Fiore. 1995. "The Use of Polymerase Chain Reaction to Detect Latent Infection of *Clavibacter michiganensis* subsp. *michiganensis* in Tomato Seedlings." *EPPO Bulletin* 25: 449-454.

Gitaitis, R. D., R. W. Beaver, and B. N. Dhanvantari. 1989. "Detection of *Clavibacter michiganense* subsp. *michiganense* in Tomato Transplants." In *Detection of Bacteria in Seed and Other Planting Material*, edited by A. W. Saettler, N. W. Schaad, and D. A. Roth, 116-122. APS Press.

Gitaitis, R. D., R. W. Beaver, and A. E. Voloudakis. 1991. "Detection of *Clavibacter michiganensis* subsp. *michiganensis* in Symptomless Tomato Transplants." *Plant Disease* 75: 834-838.

Gleason, M. L., R. D. Gitaitis, and M. D. Ricker. 1993. "Recent Progress in Understanding and Controlling Bacterial Canker of Tomato in Eastern North America." *Plant Disease* 77: 1069-1076.

Hadas, R., G. Kritzman, F. Klietman, T. Gefen, and S. Manulis. 2005. "Comparison of Extraction Procedures and Determination of the Detection Threshold for *Clavibacter michiganensis* ssp. *michiganensis* in Tomato Seeds." *Plant Pathology* 54: 643-649.

Haghverdi, M., S. M. Taghavi, S. Zarei, et al. 2025. "Pink-Pigmented Variant of *Clavibacter michiganensis* Expands Phenotypic Range of Tomato Bacterial Canker Pathogen." *Phytopathology* 115: 343-353.

Hamidizade, M., S. M. Taghavi, S. J. Martins, et al. 2020. "Bacterial Brown Pit, a New Disease of Edible Mushrooms Caused by *Mycetocola* sp." *Plant Disease* 104: 1445-1454.

Han, S., N. Jiang, Q. Lv, et al. 2018. "Detection of *Clavibacter michiganensis* subsp. *michiganensis* in Viable but Nonculturable State From Tomato Seed Using Improved qPCR." *PLoS One* 13: e0196525.

Hausbeck, M. K., J. Bell, C. Medina-Mora, R. Podolsky, and D. W. Fulbright. 2000. "Effect of Bactericides on Population Sizes and Spread of *Clavibacter michiganensis* subsp. *michiganensis* on Tomatoes in the Greenhouse and on Disease Development and Crop Yield in the Field." *Phytopathology* 90: 38-44.

Hibberd, A. M., G. Piperidis, and L. D. Godwin. 1996. "The Hypersensitive Reaction to Bacterial Canker in *Mirabilis jalapa* Is Simply Inherited." *Australasian Plant Pathology* 25: 64-67.

Hiery, E., S. Adam, S. Reid, J. Hofmann, S. Sonnewald, and A. Burkovski. 2013. "Genome-Wide Transcriptome Analysis of *Clavibacter michiganensis* subsp. *michiganensis* Grown in Xylem Mimicking Medium." *Journal of Biotechnology* 168: 348-354.

Hiery, E., A. Poetsch, T. Moosbauer, B. Amin, J. Hofmann, and A. Burkovski. 2015. "A Proteomic Study of *Clavibacter michiganensis* subsp. *michiganensis* Culture Supernatants." *Proteomes* 3: 411-423.

Huang, R., and J. C. Tu. 2001. "Effects of Nutrient Solution pH on the Survival and Transmission of *Clavibacter michiganensis* ssp. *michiganensis* in Hydroponically Grown Tomatoes." *Plant Pathology* 50: 503-508.

Hwang, I. S., E. J. Oh, H. B. Lee, and C. S. Oh. 2019. "Functional Characterization of Two Cellulase Genes in the Gram-Positive Pathogenic Bacterium *Clavibacter michiganensis* for Wilting in Tomato." *Molecular Plant-Microbe Interactions* 32: 491-501.
Hwang, I. S., E. J. Oh, and C. S. Oh. 2024. "A Novel Virulence Gene, cviA1 of *Clavibacter michiganensis* for Necrosis Development in the *Nicotiana benthamiana* Plant." *Microbiological Research* 285: 127743.

Hwang, I. S., E. J. Oh, E. Song, et al. 2022. "An Apoplastic Effector Pat-1Cm of the Gram-Positive Bacterium *Clavibacter michiganensis* Acts as Both a Pathogenicity Factor and an Immunity Elicitor in Plants." *Frontiers in Plant Science* 13: 888290.

Ignatov, A. N., N. A. Spechenkova, M. Taliansky, and K. P. Kornev. 2019. "First Report of *Clavibacter michiganensis* subsp. *michiganensis* Infecting Potato in Russia." *Plant Disease* 103: 147.

Jacobs, J. L., T. L. Carroll, and G. W. Sundin. 2005. "The Role of Pigmentation, Ultraviolet Radiation Tolerance, and Leaf Colonization Strategies in the Epiphytic Survival of Phyllosphere Bacteria." *Microbial Ecology* 49: 104-113.

Jacques, M. A., K. Durand, G. Orgeur, et al. 2012. "Phylogenetic Analysis and Polyphasic Characterization of *Clavibacter michiganensis* Strains Isolated From Tomato Seeds Reveal That Nonpathogenic Strains Are Distinct From *C. michiganensis* subsp. *michiganensis*." *Applied and Environmental Microbiology* 78: 8388-8402.

Jahr, H., J. Dreier, D. Meletzus, R. Bahro, and R. Eichenlaub. 2000. "The Endo-β-1, 4-Glucanase CelA of *Clavibacter michiganensis* subsp. *michiganensis* Is a Pathogenicity Determinant Required for Induction of Bacterial Wilt of Tomato." *Molecular Plant-Microbe Interactions* 13: 703-714.

Jang, H., S. T. Kim, and M. K. Sang. 2022. "Suppressive Effect of Bioactive Extracts of *Bacillus* sp. H8-1 and *Bacillus* sp. K203 on Tomato Wilt Caused by *Clavibacter michiganensis* subsp. *michiganensis*." *Microorganisms* 10: 403.

Jensen, H. L. 1934. "Studies on Saprophytic Mycobacteria and Corynebacteria." *Proceedings of the Linnean Society of New South Wales* 59: 19-61.

Jiang, N., Q. Y. Lv, X. Xu, et al. 2016. "Induction of the Viable but Nonculturable State in *Clavibacter michiganensis* subsp. *michiganensis* and In Planta Resuscitation of the Cells on Tomato Seedlings." *Plant Pathology* 65: 826-836.

Johnson, K. L., and R. R. Walcott. 2012. "Progress Towards a Real-Time PCR Assay for the Simultaneous Detection of *Clavibacter michiganensis* subsp. *michiganensis* and Pepino Mosaic Virus in Tomato Seed." *Journal of Phytopathology* 160: 353-363.

Jones, D. 1975. "A Numerical Taxonomic Study of Coryneform and Related Bacteria." *Journal of General Microbiology* 87: 52-96.

Jones, J. B., J. P. Jones, R. E. Stall, and T. A. Zitter, eds. 1991. *Compendium of Tomato Diseases*. American Phytopathological Society.

Jones, J. B., T. A. Zitter, T. M. Momol, and S. A. Miller. 2014. *Compendium of Tomato Diseases and Pests*. APS Press.

Jones, L. A., R. W. Worobo, and C. D. Smart. 2014. "UV Light Inactivation of Human and Plant Pathogens in Unfiltered Surface Irrigation Water." *Applied and Environmental Microbiology* 80: 849-854.

Kabelka, E., B. Franchino, and D. M. Francis. 2002. "Two Loci From *Lycopersicon hirsutum* LA407 Confer Resistance to Strains of *Clavibacter michiganensis* subsp. *michiganensis*." *Phytopathology* 92: 504-510.

Kaneshiro, W. S., C. Y. Mizumoto, and A. M. Alvarez. 2006. "Differentiation of *Clavibacter michiganensis* subsp. *michiganensis* From Seed-Borne Saprophytes Using ELISA, Biolog and 16S rDNA Sequencing." *European Journal of Plant Pathology* 116: 45-56.

Kasselaki, A. M., D. Goumas, L. Tamm, J. Fuchs, J. Cooper, and C. Leifert. 2011. "Effect of Alternative Strategies for the Disinfection of Tomato Seed Infected With Bacterial Canker (*Clavibacter michiganensis* subsp. *michiganensis*)." *NJAS—Wageningen Journal of Life Sciences* 58: 145-147.

Kawaguchi, A., S. Kitabayashi, K. Inoue, and K. Tanina. 2022. "An HLD Model for Tomato Bacterial Canker Focusing on Epidemics of the Pathogen due to Cutting by Infected Scissors." *Plants* 11: 2253.

Kawaguchi, A., S. Kitabayashi, K. Inoue, and K. Tanina. 2023. "A PHLID Model for Tomato Bacterial Canker Predicting on Epidemics of the Pathogen." *Plants* 12: 2099.

Kawaguchi, A., and K. Tanina. 2014. "Genetic Groups of *Clavibacter michiganensis* subsp. *michiganensis* Identified by DNA Fingerprinting and the Effects of Inoculation Methods on Disease Development." *European Journal of Plant Pathology* 140: 399-406.

Kawaguchi, A., K. Tanina, and K. Inoue. 2010. "Molecular Typing and Spread of *Clavibacter michiganensis* subsp. *michiganensis* in Greenhouses in Japan." *Plant Pathology* 59: 76-83.

Khojasteh, M., H. Darzi Ramandi, S. M. Taghavi, et al. 2024. "Unraveling the Genetic Basis of Quantitative Resistance to Diseases in Tomato: A Meta-QTL Analysis and Mining of Transcript Profiles." *Plant Cell Reports* 43: 184.

Kleitman, F., I. Barash, A. Burger, et al. 2008. "Characterization of a *Clavibacter michiganensis* subsp. *michiganensis* Population in Israel." *European Journal of Plant Pathology* 121: 463-475.

Kokošková, B., I. Mráz, and J. Fousek. 2010. "Comparison of Specificity and Sensitivity of Immunochemical and Molecular Techniques for Determination of *Clavibacter michiganensis* subsp. *michiganensis*." *Folia Microbiologica* 55: 239-244.

Koseoglou, E., M. Brouwer, D. Mudadirwa, J. M. van der Wolf, R. G. Visser, and Y. Bai. 2023. "Identification of Two Novel Loci Underlying Tolerance to *Clavibacter michiganensis* Originating From *Solanum arcanum* LA2157." *Agronomy* 13: 953.

Koseoglou, E., K. Hanika, M. M. Mohd Nadzir, et al. 2023. "Inactivation of Tomato WAT1 Leads to Reduced Susceptibility to *Clavibacter michiganensis* Through Downregulation of Bacterial Virulence Factors." *Frontiers in Plant Science* 14: 1082094.

Krämer, I., and E. Griesbach. 1995. "Use of ELISA for Detection of *Clavibacter michiganensis* subsp. *michiganensis* in Tomato." *EPPO Bulletin* 25: 185-193.

Laird, M., D. Piccoli, B. Weselowski, et al. 2020. "Surfactin-Producing *Bacillus velezensis* 1B-23 and *Bacillus* sp. 1D-12 Protect Tomato Against Bacterial Canker Caused by *Clavibacter michiganensis* subsp. *michiganensis*." *Journal of Plant Pathology* 102: 451-458.

Lamichhane, J. R., G. M. Balestra, and L. Varvaro. 2011. "Severe Outbreak of Bacterial Canker Caused by *Clavibacter michiganensis* subsp. *michiganensis* on Tomato in Central Italy." *Plant Disease* 95: 221.

Lamichhane, J. R., E. Osdaghi, F. Behlau, J. Köhl, J. B. Jones, and J. N. Aubertot. 2018. "Thirteen Decades of Anti-Microbial Copper Compounds Applied in Agriculture. A Review." *Agronomy for Sustainable Development* 38: 28.

Lanteigne, C., V. J. Gadkar, T. Wallon, A. Novinscak, and M. Filion. 2012. "Production of DAPG and HCN by *Pseudomonas* sp. LBUM300 Contributes to the Biological Control of Bacterial Canker of Tomato." *Phytopathology* 102: 967-973.

Latin, R., I. Tikhonova, and K. Rane. 1995. "First Report of Bacterial Canker of Pepper in Indiana." *Plant Disease* 79: 860.

Lee, I. M., I. M. Bartoszyk, D. E. Gundersen, B. Mogen, and R. E. Davis. 1997. "Nested PCR for Ultrasensitive Detection of the Potato Ring Rot Bacterium, *Clavibacter michiganensis* subsp. *sepedonicus*." *Applied and Environmental Microbiology* 63: 2625-2630.

Lehmann, K. B., and R. Neumann. 1896. *Atlas und Grundriss der Bakteriologie und Lehrbuch der Speciellen Bakteriologischen Diagnostik. Teil II*. J.F. Lehmann.

Lelis, F. M. V., R. Czajkowski, R. M. de Souza, D. H. Ribeiro, and J. M. van der Wolf. 2014. "Studies on the Colonization of Axenically Grown
Tomato Plants by a GFP-Tagged Strain of *Clavibacter michiganensis*
subsp. *michiganensis.* "European Journal of Plant Pathology 139: 53–66.

Li, X., J. Tambong, K. X. Yuan, et al. 2018. "Re-Classification of
*Clavibacter michiganensis* Subspecies on the Basis of Whole-Genome
and Multi-Locus Sequence Analyses." *International Journal of
Systematic and Evolutionary Microbiology* 68: 234–240.

López, S. M., G. N. Pastorino, A. J. Fernández-González, M. E. Franco, M. Fernández-López, and P. A. Balatti. 2020. "The Endosphere Bacteriome of Diseased and Healthy Tomato Plants." *Archives of Microbiology* 202: 2629–2642.

Louws, F. J., J. Bell, C. M. Medina-Mora, et al. 1998. "Rep-PCR-Mediated Genomic Fingerprinting: A Rapid and Effective Method to Identify *Clavibacter michiganensis*." *Phytopathology* 88: 862–868.

Luo, L. X., C. Walters, H. Bolkan, X. L. Liu, and J. Q. Li. 2008.
"Quantification of Viable Cells of *Clavibacter michiganensis* subsp.
michiganensis Using a DNA Binding Dye and a Real-Time PCR Assay."
*Plant Pathology* 57: 332–337.

Lyu, Q., K. Bai, Y. Kan, et al. 2019. "Variation in Streptomycin Resistance Mechanisms in *Clavibacter michiganensis*." *Phytopathology* 109: 1849–1858.

Malliarakis, D., M. G. Pagoulatou, E. Mpalantinaki, E. Trantas, F. Ververidis, and D. E. Goumas. 2023. "Phylogenetic Diversity of *Clavibacter michiganensis* subsp. *michiganensis* Isolates Causing Bacterial Canker of Tomato in Greece." *Journal of Plant Pathology* 105: 1403–1419.

Marcelino-Pérez, G., R. Ruiz-Medrano, S. Gallardo-Hernández, and B. Xoconostle-Cázares. 2021. “Adsorption of Recombinant Human β-Defensin 2 and Two Mutants on Mesoporous Silica Nanoparticles and Its Effect Against *Clavibacter michiganensis* subsp. *michiganensis*." *Nanomaterials* 11: 2144.

Medina-Mora, C. M., M. K. Hausbeck, and D. W. Fulbright. 2001. "Bird's Eye Lesions of Tomato Fruit Produced by Aerosol and Direct Application of *Clavibacter michiganensis* subsp. *michiganensis*." *Plant Disease* 85: 88–91.

Meletzus, D., A. Bermphol, J. Dreier, and R. Eichenlaub. 1993. "Evidence for Plasmid-Encoded Virulence Factors in the Phytopathogenic Bacterium *Clavibacter michiganensis* subsp. *michiganensis* NCPPB382." *Journal of Bacteriology* 175: 2131–2136.

Méndez-Andrade, R., M. R. Vallejo-Perez, E. Loera-Alvarado, G. de los Santos-Villarreal, L. A. García-Cerda, and I. Vera-Reyes. 2022. "Efficacy of Biosynthesized Silver Nanoparticles From *Larrea tridentata* Against *Clavibacter michiganensis*." *Journal of Phytopathology* 170: 91–99.

Mercado-Meza, D. Y., R. G. Guevara-González, K. Esquivel, I. Carbajal-Valenzuela, and G. D. Avila-Quezada. 2023. "Green Silver Nanoparticles Display Protection Against *Clavibacter michiganensis* subsp. *michiganensis* in Tomato Plants (*Solanum lycopersicum* L.)." *Plant Stress* 10: 100256.

Moffett, M. L., and B. A. Wood. 1984. "Survival of *Corynebacterium michiganense* subsp. *michiganense* Within Host Debris in Soil." *Australasian Plant Pathology* 13: 1–3.

Mohd Nadzir, M. M., F. M. Vieira Lelis, B. Thapa, et al. 2019. "Development of an In Vitro Protocol to Screen *Clavibacter michiganensis* subsp. *michiganensis* Pathogenicity in Different *Solanum* Species." *Plant Pathology* 68: 42–48.

Montenegro, I., A. Madrid, M. Cuellar, et al. 2018. "Biopesticide Activity From Drimanic Compounds to Control Tomato Pathogens." *Molecules* 23: 2053.

Morcia, C., I. Piazza, R. Ghizzoni, et al. 2023. "Molecular Diagnostics in Tomato: Chip Digital PCR Assays Targeted to Identify and Quantify *Clavibacter michiganensis* subsp. *michiganensis* and *Ralstonia solanacearum* In Planta." *Horticulturae* 9: 553.

Nandi, M., J. Macdonald, P. Liu, B. Weselowski, and Z. C. Yuan. 2018.
"*Clavibacter michiganensis* ssp. *michiganensis*: Bacterial Canker of

Tomato, Molecular Interactions and Disease Management.” *Molecular Plant Pathology* 19: 2036–2050.

Oh, E. J., C. Bae, H. B. Lee, et al. 2016. "*Clavibacter michiganensis* subsp. *capsici* subsp. nov., Causing Bacterial Canker Disease in Pepper." *International Journal of Systematic and Evolutionary Microbiology* 66: 4065–4070.

Oh, E. J., I. S. Hwang, I. W. Park, and C. S. Oh. 2022. "Comparative Genome Analyses of *Clavibacter michiganensis* Type Strain LMG7333T Reveal Distinct Gene Contents in Plasmids From Other *Clavibacter* Species." *Frontiers in Microbiology* 12: 793345.

Olivier, V., A. Baloche, A. Drouin, C. Audusseau, S. Paillard, and H. Soubelet. 2010. "Internal Methods Comparison Study and Inter-Laboratory Study on *Clavibacter michiganensis* subsp. *michiganensis* in Tomato Seeds." *EPPO Bulletin* 40: 248–256.

Ombiro, G. S. O., T. Sawai, Y. Noutoshi, et al. 2018. "Specific Growth Inhibitors of *Ralstonia solanacearum*, *Xanthomonas oryzae* pv. *oryzae*, *X. campestris* pv. *campestris*, and *Clavibacter michiganensis* subsp. *michiganensis*." *Microbiological Research* 215: 29–35.

Osdaghi, E. 2020. "*Clavibacter michiganensis* (Bacterial Canker of Tomato)." *CABI Compendium*. https://doi.org/10.1079/cabicompendium.15338.

Osdaghi, E., M. Ansari, S. M. Taghavi, S. Zarei, R. Koebnik, and J. R. Lamichhane. 2018. "Pathogenicity and Phylogenetic Analysis of *Clavibacter michiganensis* Strains Associated With Tomato Plants in Iran." *Plant Pathology* 67: 957–970.

Osdaghi, E., J. B. Jones, A. Sharma, et al. 2021. "A Centenary for Bacterial Spot of Tomato and Pepper." *Molecular Plant Pathology* 22: 1500–1519.

Osdaghi, E., T. Rahimi, S. M. Taghavi, et al. 2020. "Comparative Genomics and Phylogenetic Analyses Suggest Several Novel Species Within the Genus *Clavibacter*, Including Nonpathogenic Tomato-Associated Strains." *Applied and Environmental Microbiology* 86: e02873-19.

Osdaghi, E., A. E. Robertson, T. A. Jackson-Ziems, H. Abachi, X. Li, and R. M. Harveson. 2023. "*Clavibacter nebraskensis* Causing Goss's Wilt of Maize: Five Decades of Detaining the Enemy in the New World." *Molecular Plant Pathology* 24: 675–692.

Osdaghi, E., S. M. Taghavi, M. Hamidizade, et al. 2023. "*Clavibacter lycopersici* sp. nov.: A Peach-Colored Actinobacterium Isolated From Symptomless Tomato Plant." *International Journal of Systematic and Evolutionary Microbiology* 73: 006022.

Osdaghi, E., S. M. Taghavi, M. Hamidizade, et al. 2024. "Multiphasic Investigations Imply Transfer of Orange-/Red-Pigmented Strains of the Bean Pathogen *Curtobacterium flaccumfaciens* pv. *flaccumfaciens* to a New Species as *C. aurantiacum* sp. nov., Elevation of the Poinsettia Pathogen *C. flaccumfaciens* pv. *poinsettiae* to the Species Level as *C. poinsettiae* sp. nov., and Synonymy of *C. albidum* With *C. citreum*." *Systematic and Applied Microbiology* 47: 126489.

Osdaghi, E., S. M. Taghavi, H. Hamzehzarghani, et al. 2018. "Epiphytic *Curtobacterium flaccumfaciens* Strains Isolated From Symptomless Solanaceous Vegetables Are Pathogenic on Leguminous but Not on Solanaceous Plants." *Plant Pathology* 67: 388–398.

Osdaghi, E., S. M. Taghavi, H. Hamzehzarghani, A. Fazliarab, and J. R. Lamichhane. 2017. "Monitoring the Occurrence of Tomato Bacterial Spot and Range of the Causal Agent *Xanthomonas perforans* in Iran." *Plant Pathology* 66: 990–1002.

Osdaghi, E., J. M. van der Wolf, H. Abachi, X. Li, S. H. De Boer, and C. A. Ishimaru. 2022. "Bacterial Ring Rot of Potato Caused by *Clavibacter sepedonicus*: A Successful Example of Defeating the Enemy Under International Regulations." *Molecular Plant Pathology* 23: 911–932.

Osdaghi, E., A. J. Young, and R. M. Harveson. 2020. "Bacterial Wilt of Dry Beans Caused by *Curtobacterium flaccumfaciens* pv.
*flaccumfaciens*: A New Threat From an Old Enemy.” *Molecular Plant Pathology* 21: 605–621.

Özdemir, Z. 2009a. "Development of a Multiplex PCR Assay for the Simultaneous Detection of *Clavibacter michiganensis* subsp. *michiganensis*, *Pseudomonas syringae* pv. *tomato* and *Xanthomonas axonopodis* pv. *vesicatoria* Using Pure Cultures." *Journal of Plant Pathology* 91: 495-497.

Özdemir, Z. 2009b. "Growth Inhibition of *Clavibacter michiganensis* subsp. *michiganensis* and *Pseudomonas syringae* pv. *tomato* by Olive Mill Wastewaters and Citric Acid." *Journal of Plant Pathology* 91: 221–224.

Pastrik, K. H., and F. Rainey. 1997. "Differentiation and Detection of the Subspecies of *Clavibacter michiganensis* by PCR (Polymerase Chain Reaction)-Techniques." In *Diagnosis and Identification of Plant Pathogens: Proceedings of the 4th International Symposium of the European Foundation for Plant Pathology, September 9–12, 1996, Bonn, Germany*, 193. Springer Netherlands.

Pastrik, K. H., and F. A. Rainey. 1999. "Identification and Differentiation of *Clavibacter michiganensis* Subspecies by Polymerase Chain Reaction-Based Techniques." *Journal of Phytopathology* 147: 687–693.

Paulin, M. M., A. Novinscak, C. Lanteigne, V. J. Gadkar, and M. Filion. 2017. "Interaction Between 2, 4-Diacetylphloroglucinol- and Hydrogen Cyanide-Producing *Pseudomonas brassicacearum* LBUM300 and *Clavibacter michiganensis* subsp. *michiganensis* in the Tomato Rhizosphere." *Applied and Environmental Microbiology* 83: e00073-17.

Peňázová, E., M. Dvořák, L. Ragasová, et al. 2020. "Multiplex Real-Time PCR for the Detection of *Clavibacter michiganensis* subsp. *michiganensis*, *Pseudomonas syringae* pv. *tomato* and Pathogenic *Xanthomonas* Species on Tomato Plants." *PLoS One* 15: e0227559.

Peritore-Galve, F. C., C. Miller, and C. D. Smart. 2020. "Characterizing Colonization Patterns of *Clavibacter michiganensis* During Infection of Tolerant Wild Solanum Species." *Phytopathology* 110: 574–581.

Peritore-Galve, F. C., M. A. Tancos, and C. D. Smart. 2021. "Bacterial Canker of Tomato: Revisiting a Global and Economically Damaging Seedborne Pathogen." *Plant Disease* 105: 1581–1595.

Pinto, M., C. Soares, T. Andreani, F. Fidalgo, and F. Tavares. 2023. "*Eucalyptus globulus* Leaf Aqueous Extract Differentially Inhibits the Growth of Three Bacterial Tomato Pathogens." *Plants* 12: 1727.

Pradhanang, P. M., and G. Collier. 2009. "How Effective Is Hydrochloric Acid Treatment to Control *Clavibacter michiganensis* subsp. *michiganensis* Contamination in Tomato Seed?" *Acta Horticulturae* 808: 81–85.

Quintero-Vásquez, G. A., M. L. Bazán-Tejeda, E. Martínez-Peñafiel, L. Kameyama-Kawabe, and R. M. Bermúdez-Cruz. 2013. "Multiplex PCR to Detect Four Different Tomato-Infecting Pathogens." *Folia Microbiologica* 58: 269–276.

Ramachandran, S., S. Dobhal, A. M. Alvarez, and M. Arif. 2021. "Improved Multiplex TaqMan qPCR Assay With Universal Internal Control Offers Reliable and Accurate Detection of *Clavibacter michiganensis*." *Journal of Applied Microbiology* 131: 1405–1416.

Richert, K., E. Brambilla, and E. Stackebrandt. 2005. "Development of PCR Primers Specific for the Amplification and Direct Sequencing of gyrB Genes From Microbacteria, Order Actinomycetales." *Journal of Microbiological Methods* 60: 115-123.

Richert, K., E. Brambilla, and E. Stackebrandt. 2007. "The Phylogenetic Significance of Peptidoglycan Types: Molecular Analysis of the Genera *Microbacterium* and *Aureobacterium* Based Upon Sequence Comparison of *gyrB*, *rpoB*, *recA* and *Ppk* and 16S rRNA Genes." *Systematic and Applied Microbiology* 30: 102–108.

Ricker, M., and R. Riedel. 1993. "Effect of Secondary Spread of *Clavibacter michiganensis* subsp. *michiganensis* on Yield of Northern Processing Tomatoes." *Plant Disease* 77: 364–366.

Rivas-Cáeres, R. R., J. L. Stephano-Hornedo, J. Lugo, et al. 2018. "Bactericidal Effect of Silver Nanoparticles Against Propagation of

*Clavibacter michiganensis* Infection in *Lycopersicon esculentum* Mill." *Microbial Pathogenesis* 115: 358–362.

Romero, A. M., O. S. Correa, S. Moccia, and J. G. Rivas. 2003. "Effect of *Azospirillum*-Mediated Plant Growth Promotion on the Development of Bacterial Diseases on Fresh-Market and Cherry Tomato." *Journal of Applied Microbiology* 95: 832–838.

Romero, A. M., D. Vega, R. Pizzorno, G. Cordon, and O. S. Correa. 2018. "Hydraulic and Leaf Reflectance Alterations Induced by *Clavibacter michiganensis* subsp. *michiganensis* on Tomato Plants." *European Journal of Plant Pathology* 152: 567–572.

Rotondo, F., N. Khatri, A. L. Testen, and S. A. Miller. 2023. "Evaluation of a Proprietary Plant Extract to Suppress Bacterial Canker and Improve Yield in Hydroponic Tomatoes." *Plant Health Progress* 24: 364–368.

Sandbrink, J. M., J. W. Van Ooijen, C. C. Purimahua, et al. 1995. "Localization of Genes for Bacterial Canker Resistance in *Lycopersicon peruvianum* Using RFLPs." *Theoretical and Applied Genetics* 90: 444–450.

Savidor, A., L. Chalupowicz, D. Teper, et al. 2014. "*Clavibacter michiganensis* subsp. *michiganensis* Vatr1 and Vatr2 Transcriptional Regulators Are Required for Virulence in Tomato." *Molecular Plant-Microbe Interactions* 27: 1035-1047.

Savidor, A., D. Teper, K. H. Gartemann, et al. 2012. "The *Clavibacter michiganensis* subsp. *michiganensis*–Tomato Interactome Reveals the Perception of Pathogen by the Host and Suggests Mechanisms of Infection." *Journal of Proteome Research* 11: 736–750.

Schaad, N. W., S. S. Cheong, S. Tamaki, E. Hatziloukas, and N. J. Panopoulos. 1995. "A Combined Biological and Enzymatic Amplification (BIO-PCR) Technique to Detect *Pseudomonas syringae* pv. *phaseolicola* in Bean Seed Extracts." *Phytopathology* 85: 243–246.

Schaad, N. W., J. B. Jones, and W. Chun. 2001. *Laboratory Guide for the Identification of Plant Pathogenic Bacteria*. 3rd ed. APS Press.

Sen, Y., Z. Feng, H. Vandenbroucke, J. van der Wolf, R. G. Visser, and A. W. Van Heusden. 2013. "Screening for New Sources of Resistance to *Clavibacter michiganensis* subsp. *michiganensis* (Cmm) in Tomato." *Euphytica* 190: 309–317.

Sharabani, G., S. Manulis-Sasson, M. Borenstein, et al. 2013. "The Significance of Guttation in the Secondary Spread of *Clavibacter michiganensis* subsp. *michiganensis* in Tomato Greenhouses." *Plant Pathology* 62: 578–586.

Sharabani, G., S. Manulis-Sasson, L. Chalupowicz, et al. 2014. "Temperature at the Early Stages of *Clavibacter michiganensis* subsp. *michiganensis* Infection Affects Bacterial Canker Development and Virulence Gene Expression." *Plant Pathology* 63: 1119–1129.

Sharabani, G., D. Shtienberg, M. Borenstein, et al. 2013. "Effects of Plant Age on Disease Development and Virulence of *Clavibacter michiganensis* subsp. *michiganensis* on Tomato." *Plant Pathology* 62: 1114–1122.

Smith, E. F. 1910. "A New Tomato Disease of Economic Importance." *Science* 31: 794–796.

Sousa Santos, M., L. Cruz, P. Norskov, and O. F. Rasmussen. 1997. "A Rapid and Sensitive Detection of *Clavibacter michiganensis* subsp. *michiganensis* in Tomato Seeds by Polymerase Chain Reaction." *Seed Science and Technology* 25: 581–584.

Soylu, S., Ö. Baysal, and E. M. Soylu. 2003. "Induction of Disease Resistance by the Plant Activator, Acibenzolar-S-Methyl (ASM), Against Bacterial Canker (*Clavibacter michiganensis* subsp. *michiganensis*) in Tomato Seedlings." *Plant Science* 165: 1069–1075.

Stevens, D. M., A. Tang, and G. Coaker. 2021. "A Genetic Toolkit for Investigating Clavibacter Species: Markerless Deletion, Permissive Site Identification, and an Integrative Plasmid." *Molecular Plant-Microbe Interactions* 34: 1336–1345.

Stork, I., K. H. Gartemann, A. Burger, and R. Eichenlaub. 2008. "A Family of Serine Proteases of *Clavibacter michiganensis* subsp.
*michiganensis*: *chpC* Plays a Role in Colonization of the Host Plant Tomato.” *Molecular Plant Pathology* 9: 599–608.

Takishita, Y., A. Souleimanov, C. Bourguet, et al. 2021. “Pseudomonas entomophila 23S Produces a Novel Antagonistic Compound Against *Clavibacter michiganensis* subsp. *michiganensis*, a Pathogen of Tomato Bacterial Canker.” *Applied Microbiology* 1: 60–73.

Tancos, M. A., L. Chalupowicz, I. Barash, S. Manulis-Sasson, and C. D. Smart. 2013. “Tomato Fruit and Seed Colonization by *Clavibacter michiganensis* subsp. *michiganensis* Through External and Internal Routes.” *Applied and Environmental Microbiology* 79: 6948–6957.

Thapa, S. P., M. O'Leary, M. A. Jacques, R. L. Gilbertson, and G. Coaker. 2020. “Comparative Genomics to Develop a Specific Multiplex PCR Assay for Detection of *Clavibacter michiganensis*.” *Phytopathology* 110: 556–566.

Thapa, S. P., S. Pattathil, M. G. Hahn, M. A. Jacques, R. L. Gilbertson, and G. Coaker. 2017. “Genomic Analysis of *Clavibacter michiganensis* Reveals Insight Into Virulence Strategies and Genetic Diversity of a Gram-Positive Bacterial Pathogen.” *Molecular Plant–Microbe Interactions* 30: 786–802.

Thyr, B. D., R. E. Webb, C. A. Jaworski, and T. J. Ratcliffe. 1973. “Tomato Bacterial Canker: Control by Seed Treatment.” *Plant Disease Reporter* 57: 974–977.

Toribio, A. J., M. M. Jurado, F. Suárez-Estrella, J. A. López-González, M. R. Martinez-Gallardo, and M. J. López. 2021. “Application of Sonicated Extracts of Cyanobacteria and Microalgae for the Mitigation of Bacterial Canker in Tomato Seedlings.” *Journal of Applied Phycology* 33: 3817–3829.

Tripathi, R., K. Vishunavat, R. Tewari, et al. 2022. “Defense Inducers Mediated Mitigation of Bacterial Canker in Tomato Through Alteration in Oxidative Stress Markers.” *Microorganisms* 10: 2160.

Tsitsekian, D., G. Daras, K. Karamanou, et al. 2021. “*Clavibacter michiganensis* Downregulates Photosynthesis and Modifies Monolignols Metabolism Revealing a Crosstalk With Tomato Immune Responses.” *International Journal of Molecular Sciences* 22: 8442.

Utkhede, R., and C. Koch. 2004. “Biological Treatments to Control Bacterial Canker of Greenhouse Tomatoes.” *BioControl* 49: 305–313.

Valencia-Hernandez, J. A., N. Solano-Alvarez, A. A. Feregrino-Perez, et al. 2022. “In Vitro and In Vivo Antimicrobial Activity of a Synthetic Capsaicinoid Oleoresin Against *Fusarium oxysporum*, *Phytophthora capsici*, *Clavibacter michiganensis* and *Pseudomonas syringae*.” *Journal of Plant Pathology* 104: 699–710.

Valencia-Hernandez, J. A., N. Solano-Alvarez, M. A. Rico-Rodriguez, et al. 2023. “Eustressic Dose of Cadmium in Soil Induces Defense Mechanisms and Protection Against *Clavibacter michiganensis* in Tomato (*Solanum lycopersicum* L.).” *Journal of Plant Growth Regulation* 42: 407–414.

Valenzuela, M., X. Besoain, K. Durand, et al. 2018. “*Clavibacter michiganensis* subsp. *michiganensis* Strains From Central Chile Exhibit Low Genetic Diversity and Sequence Types Match Strains in Other Parts of the World.” *Plant Pathology* 67: 1944–1954.

Valenzuela, M., M. González, A. Velásquez, et al. 2021. “Analyses of Virulence Genes of *Clavibacter michiganensis* subsp. *michiganensis* Strains Reveal Heterogeneity and Deletions That Correlate With Pathogenicity.” *Microorganisms* 9: 1530.

Valenzuela, M., V. Méndez, I. Montenegro, X. Besoain, and M. Seeger. 2019. “Streptomycin Resistance in *Clavibacter michiganensis* subsp. *michiganensis* Strains From Chile Is Related to an *rpsL* Gene Mutation.” *Plant Pathology* 68: 426–433.

Vallejo-Pérez, M. R., J. A. Sosa-Herrera, H. R. Navarro-Contreras, L. G. Álvarez-Preciado, Á. G. Rodríguez-Vázquez, and J. P. Lara-Ávila. 2021. “Raman Spectroscopy and Machine-Learning for Early Detection of Bacterial Canker of Tomato: The Asymptomatic Disease Condition.” *Plants* 10: 1542.

Van den Bulk, R. W., J. Jansen, W. H. Lindhout, and H. J. M. Löffler. 1991. “Screening of Tomato Somaclones for Resistance to Bacterial Canker (*Clavibacter michiganensis* subsp. *michiganensis*).” *Plant Breeding* 107: 190–196.

Van den Bulk, R. W., H. J. M. Löffler, and J. J. M. Dons. 1990. “Inhibition of Callus Development From Protoplasts of *Lycopersicon peruvianum* by Extracellular Polysaccharides of *Clavibacter michiganensis* subsp. *michiganensis*.” *Plant Science* 71: 105–112.

Van Heusden, A. W., M. Koornneef, R. E. Voorrips, et al. 1999. “Three QTLs From *Lycopersicon peruvianum* Confer a High Level of Resistance to *Clavibacter michiganensis* ssp. *michiganensis*.” *Theoretical and Applied Genetics* 99: 1068–1074.

Vega, D., and A. M. Romero. 2016. “Survival of *Clavibacter michiganensis* subsp. *michiganensis* in Tomato Debris Under Greenhouse Conditions.” *Plant Pathology* 65: 545–550.

Verma, R. K., V. Roman-Reyna, H. Raanan, G. Coaker, J. M. Jacobs, and D. Teper. 2024. “Allelic Variations in the *chpG* Effector Gene Within *Clavibacter michiganensis* Populations Determine Pathogen Host Range.” *PLoS Pathogens* 20: e1012380.

Verma, R. K., and D. Teper. 2022. “Immune Recognition of the Secreted Serine Protease ChpG Restricts the Host Range of *Clavibacter michiganensis* From Eggplant Varieties.” *Molecular Plant Pathology* 23: 933–946.

Vidaver, A. K. 1982. “The Plant-Pathogenic Corynebacteria.” *Annual Reviews in Microbiology* 36: 495–517.

Vidaver, A. K., and M. J. Davis. 1988. “Coryneform Plant Pathogens.” In *Laboratory Guide for Identification of Plant-Pathogenic Bacteria*, edited by N. W. Schaad, 2nd ed., 104–113. APS Press.

Waleron, M., K. Waleron, J. Kamasa, W. Przewodowski, and E. Lojkowska. 2011. “Polymorphism Analysis of Housekeeping Genes for Identification and Differentiation of *Clavibacter michiganensis* Subspecies.” *European Journal of Plant Pathology* 131: 341–354.

Wassermann, E., M. S. Montecchia, O. S. Correa, V. Damián, and A. M. Romero. 2017. “*Clavibacter michiganensis* subsp. *michiganensis* Strains Virulence and Genetic Diversity. A First Study in Argentina.” *European Journal of Plant Pathology* 149: 35–42.

Waters, C. M., and H. A. Bolkan. 1992. “An Improved Semi-Selective Medium and Method of Extraction for Detecting *Clavibacter michiganensis* subsp. *michiganensis* in Tomato Seeds.” *Phytopathology* 82: 1072.

Weisburg, W. G., S. M. Barns, D. A. Pelletier, and D. J. Lane. 1991. “16S Ribosomal DNA Amplification for Phylogenetic Study.” *Journal of Bacteriology* 173: 697–703.

Wittmann, J., C. Brancato, K. W. Berendzen, and B. Dreiseikelmann. 2016. “Development of a Tomato Plant Resistant to *Clavibacter michiganensis* Using the Endolysin Gene of Bacteriophage CMP1 as a Transgene.” *Plant Pathology* 65: 496–502.

Wittmann, J., R. Eichenlaub, and B. Dreiseikelmann. 2010. “The Endolysins of Bacteriophages CMP1 and CN77 Are Specific for the Lysis of *Clavibacter michiganensis* Strains.” *Microbiology* 156: 2366–2373.

Wittmann, J., K. H. Gartemann, R. Eichenlaub, and B. Dreiseikelmann. 2011. “Genomic and Molecular Analysis of Phage CMP1 From *Clavibacter michiganensis* Subspecies *michiganensis*.” *Bacteriophage* 1: 6–14.

Xu, X., A. Kumar, L. Deblais, et al. 2015. “Discovery of Novel Small Molecule Modulators of *Clavibacter michiganensis* subsp. *michiganensis*.” *Frontiers in Microbiology* 6: 1127.

Xu, X., S. A. Miller, F. Baysal-Gurel, K. H. Gartemann, R. Eichenlaub, and G. Rajashekara. 2010. “Bioluminescence Imaging of *Clavibacter michiganensis* subsp. *michiganensis* Infection of Tomato Seeds and Plants.” *Applied and Environmental Microbiology* 76: 3978–3988.

Xu, X., G. Rajashekara, P. A. Paul, and S. A. Miller. 2012. “Colonization of Tomato Seedlings by Bioluminescent *Clavibacter michiganensis* subsp.
*michiganensis* Under Different Humidity Regimes.” *Phytopathology* 102: 177–184.

Yamada, K., and K. Komagata. 1970. "Taxonomic Studies on Coryneform Bacteria. II. Principal Amino Acids in the Cell Wall and Their Taxonomic Significance." *Journal of General and Applied Microbiology* 16: 103–113.

Yañez-Olvera, A. G., A. G. Gómez-Díaz, N. Sélem-Mojica, et al. 2024. "A Host Shift as the Origin of Tomato Bacterial Canker Caused by *Clavibacter michiganensis*." *Microbial Genomics* 10: 001309.

Yasuhara-Bell, J., and A. M. Alvarez. 2015. "Seed-Associated Subspecies of the Genus *Clavibacter* Are Clearly Distinguishable From *Clavibacter michiganensis* subsp. *michiganensis*." *International Journal of Systematic and Evolutionary Microbiology* 65: 811–826.

Yasuhara-Bell, J., F. Baysal-Gurel, S. A. Miller, and A. M. Alvarez. 2015. "Utility of a Loop-Mediated Amplification Assay for Detection of *Clavibacter michiganensis* subsp. *michiganensis* in Seeds and Plant Tissues." *Canadian Journal of Plant Pathology* 37: 260–266.

Yasuhara-Bell, J., R. Kubota, D. M. Jenkins, and A. M. Alvarez. 2013. "Loop-Mediated Amplification of the *Clavibacter michiganensis* subsp. *michiganensis* micA Gene Is Highly Specific." *Phytopathology* 103: 1220–1226.

Yasuhara-Bell, J., G. Marrero, and A. M. Alvarez. 2014. "Genes *clvA*, *clvF* and *clvG* Are Unique to *Clavibacter michiganensis* subsp. *michiganensis* and Highly Conserved." *European Journal of Plant Pathology* 140: 655–664.

Yim, K. O., H. I. Lee, J. H. Kim, S. D. Lee, J. H. Cho, and J. S. Cha. 2012. "Characterization of Phenotypic Variants of *Clavibacter michiganensis* subsp. *michiganensis* Isolated From *Capsicum annuum*." *European Journal of Plant Pathology* 133: 559–575.

Yokotani, N., Y. Hasegawa, M. Sato, et al. 2021. "Transcriptome Analysis of *Clavibacter michiganensis* subsp. *michiganensis*-Infected Tomatoes: A Role of Salicylic Acid in the Host Response." *BMC Plant Biology* 21: 476.

Young, J. M., G. S. Saddler, Y. Takikawa, et al. 1996. "Names of Plant-Pathogenic Bacteria 1864–1995." *Review of Plant Pathology* 75: 721–763.

Zaluga, J., K. Heylen, K. Van Hoorde, et al. 2011. "*GyrB* Sequence Analysis and MALDI-TOF MS as Identification Tools for Plant-Pathogenic *Clavibacter*." *Systematic and Applied Microbiology* 34: 400–407.

Załuga, J., P. Stragier, S. Baeyen, et al. 2014. "Comparative Genome Analysis of Pathogenic and Non-Pathogenic *Clavibacter* Strains Reveals Adaptations to Their Lifestyle." *BMC Genomics* 15: 392.

Zaluga, J., P. Stragier, J. Van Vaerenbergh, M. Maes, and P. De Vos. 2013. "Multilocus Variable-Number-Tandem-Repeats Analysis (MLVA) Distinguishes a Clonal Complex of *Clavibacter michiganensis* subsp. *michiganensis* Strains Isolated From Recent Outbreaks of Bacterial Wilt and Canker in Belgium." *BMC Microbiology* 13: 126.

Zaluga, J., J. Van Vaerenbergh, P. Stragier, M. Maes, and P. De Vos. 2013. "Genetic Diversity of Non-Pathogenic *Clavibacter* Strains Isolated From Tomato Seeds." *Systematic and Applied Microbiology* 36: 426–435.

Zamora-Macorra, E. J., D. L. Ochoa-Martínez, G. Valdovinos-Ponce, et al. 2015. "Simultaneous Detection of *Clavibacter michiganensis* subsp. *michiganensis*, Pepino Mosaic Virus and Mexican Papita Viroid by Non-Radioactive Molecular Hybridization Using a Unique Polyprobe." *European Journal of Plant Pathology* 143: 779–787.

Zanón, M. J., and C. Jordá. 2008. "Eradication of *Clavibacter michiganensis* subsp. *michiganensis* by Incorporating Fresh Crop Debris Into Soil: Preliminary Evaluations Under Controlled Conditions." *Crop Protection* 27: 1511–1518.

Zhang, W., W. Yang, Q. Meng, Y. Li, and D. Liu. 2010. "Screening and Identification of Antagonistic *Streptomyces* spp. Against *Clavibacter*

*michiganensis* subsp. *michiganensis* From Tomato Rhizosphere." *Frontiers of Agriculture in China* 4: 159–164.

Zhang, Y., W. Yang, Y. Li, D. Liu, and T. Zhang. 2009. "A Multiplex PCR Method for Detection of *Clavibacter michiganensis* subsp. *michiganensis* With Co-Amplification of Its Host DNA." *Frontiers of Agriculture in China* 3: 140–145.

Zhao, W. J., H. Y. Chen, S. F. Zhu, M. X. Xia, and T. W. Tan. 2007. "One-Step Detection of *Clavibacter michiganensis* subsp. *michiganensis* in Symptomless Tomato Seeds Using a TaqMan Probe." *Journal of Plant Pathology* 89: 349–351.