Genes clvA, clvF and clvG are unique to Clavibacter michiganensis subsp. michiganensis and highly conserved

Yasuhara-Bell, Jarred; Marrero, Glorimar; Alvarez, Anne M.

doi:10.1007/s10658-014-0495-5

Cited by 7 publications

(8 citation statements)

References 39 publications

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“…All other strains tested were negative by PCR for all three genes. These results confirm previous findings (Yasuhara-Bell & Alvarez, 2012; Yasuhara-Bell et al , 2013, 2014) that the clvA (formerly micA ) gene, and associated clvF and clvG genes are highly specific for Clavibacter michiganensis subsp. michiganensis .…”

supporting

confidence: 92%

“…Additionally, PCR primers were designed to react only with Clavibacter michiganensis subsp. michiganensis and not with the seed-associated CMB (Yasuhara-Bell et al , 2014).…”

mentioning

confidence: 95%

“…PCR amplifications were carried out using primer pairs designed to amplify dnaA (Schneider et al , 2011), partial 16S rRNA (Weisburg et al , 1991), atpD , dnaK , gyrB , ppK , recA (Jacques et al , 2012), rpoB , clvA , clvF and clvG (Yasuhara-Bell et al , 2014) gene sequences (). The rpoB primers used in this study were the reverse complements to those previously published by Jacques et al (2012).…”

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confidence: 99%

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Seed-associated subspecies of the genus Clavibacter are clearly distinguishable from Clavibacter michiganensis subsp. michiganensis

Yasuhara-Bell

Alvarez

2015

International Journal of Systematic and Evolutionary Microbiology

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The genus Clavibacter contains one recognized species, Clavibacter michiganensis. Clavibacter michiganensis is subdivided into subspecies based on host specificity and bacteriological characteristics, with Clavibacter michiganensis subsp. michiganensis causing bacterial canker of tomato. Clavibacter michiganensis subsp. michiganensis is often spread through contaminated seed leading to outbreaks of bacterial canker in tomato production areas worldwide. The frequent occurrence of non-pathogenic Clavibacter michiganensis subsp. michiganensis-like bacteria (CMB) is a concern for seed producers because Clavibacter michiganensis subsp. michiganensis is a quarantine organism and detection of a nonpathogenic variant may result in destruction of an otherwise healthy seed lot. A thorough biological and genetic characterization of these seed-associated CMB strains was performed using standard biochemical tests, cell wall analyses, metabolic profiling using Biolog, and singlegene and multilocus sequence analyses. Combined, these tests revealed two distinct populations of seed-associated members of the genus Clavibacter that differed from each other, as well as from all other described subspecies of Clavibacter michiganensis. DNA-DNA hybridization values are 70 % or higher, justifying placement into the single recognized species, C. michiganensis, but other analyses justify separate subspecies designations. Additionally, strains belonging to the genus Clavibacter isolated from pepper also represent a distinct population and warrant separate subspecies designation. On the basis of these data we propose subspecies designations for separate non-pathogenic subpopulations of Clavibacter michiganensis: Clavibacter michiganensis subsp. californiensis subsp. nov. and Clavibacter michiganensis subsp. chilensis subsp. nov. for seed-associated strains represented by C55 T (5ATCC BAA-2691 T 5CFBP 8216 T) and ZUM3936 T (5ATCC BAA-2690 T 5CFBP 8217 T), respectively. Recognition of separate subspecies is essential for improved international seed testing operations.

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supporting

confidence: 92%

“…Additionally, PCR primers were designed to react only with Clavibacter michiganensis subsp. michiganensis and not with the seed-associated CMB (Yasuhara-Bell et al , 2014).…”

mentioning

confidence: 95%

mentioning

confidence: 99%

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Seed-associated subspecies of the genus Clavibacter are clearly distinguishable from Clavibacter michiganensis subsp. michiganensis

Yasuhara-Bell

Alvarez

2015

International Journal of Systematic and Evolutionary Microbiology

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“…Furthermore, BLASTn/BLASTp-based investigation was performed to decipher whether the pathogenicity determinant genes/clusters are present in the genomes (26). Using the complete genome of C. michiganensis sensu stricto NCPPB 382, one-versus-one BLASTn/BLASTp searches were done against the sequences of the pathogenicity island (a 129-kb low-GϩC region which includes chp and tomA clusters) as well as several individual genes proposed to have effective contributions to the virulence of C. michiganensis sensu stricto (22,25,26,28,42,43). Proteins with amino acid sequence similarities higher than 50% and with a query coverage higher than 70% were considered homologs (28).…”

Section: Methodsmentioning

confidence: 99%

Comparative Genomics and Phylogenetic Analyses Suggest Several Novel Species within the Genus Clavibacter , Including Nonpathogenic Tomato-Associated Strains

Osdaghi

Rahimi

Ansari

et al. 2020

Appl Environ Microbiol

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Members of the genus Clavibacter are economically important bacterial plant pathogens infecting a set of diverse agricultural crops (e.g., alfalfa, corn, potato, tomato, and wheat). Tomato-associated Clavibacter sp. strains account for a great portion of the genetic diversity of the genus, and C. michiganensis sensu stricto (formerly C. michiganensis subsp. michiganensis), causing bacterial canker disease, is considered one of the most destructive seed-borne agents for the crop worldwide. However, current taxonomic descriptions of the genus do not reflect the existing diversity of the strains, resulting in unsatisfactory results in quarantine surveys for the pathogens. In this study, we used all the available genome sequences of Clavibacter sp. strains, including the type strains of newly described subspecies, to provide precise insight into the diversity of tomato-associated members of the genus and further clarify the taxonomic status of the strains using genotypic and phenotypic features. The results of phylogenetic analyses revealed the existence of nine hypothetical new species among the investigated strains. None of the three new subspecies (i.e., C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis, and C. michiganensis subsp. phaseoli) is included within the tomato-pathogenic C. michiganensis sensu stricto lineage. Although comparative genomics revealed the lack of chp and tomA pathogenicity determinant gene clusters in the nonpathogenic strains, a number of pathogenicity-related genes were noted to be present in all the strains regardless of their pathogenicity characteristics. Altogether, our results indicate a need for a formal taxonomic reconsideration of tomato-associated Clavibacter sp. strains to facilitate differentiation of the lineages in quarantine inspections. IMPORTANCE Clavibacter spp. are economically important bacterial plant pathogens infecting a set of diverse agricultural crops, such as alfalfa, corn, pepper, potato, tomato, and wheat. A number of plant-pathogenic members of the genus (e.g., C. michiganensis sensu stricto and C. sepedonicus, infecting tomato and potato plants, respectively) are included in the A2 (high-risk) list of quarantine pathogens by the European and Mediterranean Plant Protection Organization (EPPO). Although tomato-associated members of Clavibacter spp. account for a significant portion of the genetic diversity in the genus, only the strains belonging to C. michiganensis sensu stricto (formerly C. michiganensis subsp. michiganensis) cause bacterial canker disease of tomato and are subjected to the quarantine inspections. Hence, discrimination between the pathogenic and nonpathogenic Clavibacter sp. strains associated with tomato seeds and transplants plays a pivotal role in the accurate detection and cost-efficient management of the disease. On the other hand, detailed information on the genetic contents of different lineages of the genus would lead to the development of genome-informed specific detection techniques. In this study, we have provided an overview of the phylogenetic and genomic differences between the pathogenic and nonpathogenic tomato-associated Clavibacter sp. strains. We also noted that the taxonomic status of newly introduced subspecies of C. michiganensis (i.e., C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis, and C. michiganensis subsp. phaseoli) should be reconsidered.

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“…Probe-based TaqMan quantitative PCR (qPCR) combines the advantages of PCR with quantitative measurement of pathogen titre, thus offering a highly sensitive and specific tool for pathogen detection that can be used in routine diagnosis and enumeration of bacterial titres in a given sample (Ouyang et al 2013;Ross and Somssich 2016;Dobhal et al 2020). Although serological and molecular assays are available for detection of Cm (Nemeth and Vuurde 2006;Kaneshiro et al 2006;Sudarshana et al 2012;Yasuhara-Bell et al 2013;Yasuhara-Bell et al 2014Sueno et al 2015), these assays are time-consuming and have no internal controls to inform about the false positives. Compared to other assays, a qPCR-based diagnostic test eliminates the additional step of running agarose gels, cutting down the time required for detection (Ouyang et al 2013).…”

Section: Introductionmentioning

confidence: 99%

Improved multiplex TaqMan qPCR assay with universal internal control offers reliable and accurate detection of Clavibacter michiganensis

Ramachandran

Dobhal

Alvarez

2021

J of Applied Microbiology

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Aim: Clavibacter michiganensis (Cm) is a seed-borne plant pathogen that significantly reduces tomato production worldwide. Due to repeated outbreaks and rapid spread of the disease, seeds/transplants need to be certified free of the pathogen before planting. To this end, we developed a multiplex TaqMan qPCR assay that can accurately detect Cm in infected samples. Methods and Results: A specific region of Cm (clvG gene) was selected for primer design using comparative genomics approach. A fully synthetic universal internal control (UIC) was also designed to detect PCR inhibitors and false-negative results in qPCRs. The Cm primers can be used alone or in a triplex TaqMan qPCR assay with UIC and previously described Clavibacter primers. The assay was specific for Cm and detected up to 10 fg of Cm DNA in sensitivity and spiked assays. Addition of the UIC did not change the specificity or sensitivity of the multiplex TaqMan qPCR assay. Conclusion:The triplex TaqMan qPCR provides a specific and sensitive diagnostic assay for Cm. Significance and Impact of the Study: This assay can be used for biosecurity surveillance, routine diagnostics, estimating bacterial titres in infected material and for epidemiological studies. The UIC is fully synthetic, efficiently amplified and multiplex compatible with any other qPCR assay.

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Genes clvA, clvF and clvG are unique to Clavibacter michiganensis subsp. michiganensis and highly conserved

Cited by 7 publications

References 39 publications

Seed-associated subspecies of the genus Clavibacter are clearly distinguishable from Clavibacter michiganensis subsp. michiganensis

Seed-associated subspecies of the genus Clavibacter are clearly distinguishable from Clavibacter michiganensis subsp. michiganensis

Comparative Genomics and Phylogenetic Analyses Suggest Several Novel Species within the Genus Clavibacter , Including Nonpathogenic Tomato-Associated Strains

Improved multiplex TaqMan qPCR assay with universal internal control offers reliable and accurate detection of Clavibacter michiganensis

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