Genus Clavibacter Research Articles

A host shift as the origin of tomato bacterial canker caused by Clavibacter michiganensis.

The Actinomycetota (formerly Actinobacteria) genus Clavibacter includes phytopathogens with devasting effects in several crops. Clavibacter michiganensis, the causal agent of tomato bacterial canker, is the most notorious species of the genus. Yet, its origin and natural reservoirs remain elusive, and its populations show pathogenicity profiles with unpredictable plant disease outcomes. Here, we generate and analyse a decade-long genomic dataset of Clavibacter from wild and commercial tomato cultivars, providing evolutionary insights that directed phenotypic characterization. Our phylogeny situates the last common ancestor of C. michiganensis next to Clavibacter isolates from grasses rather than to the sole strain we could isolate from wild tomatoes. Pathogenicity profiling of C. michiganensis isolates, together with C. phaseoli and C. californiensis as sister taxa and the wild tomato strain, was found to be congruent with the proposed phylogenetic relationships. We then identified gene enrichment after the evolutionary event, leading to the appearance of the C. michiganesis clade, including known pathogenicity factors but also hitherto unnoticed genes with the ability to encode adaptive traits for a pathogenic lifestyle. The holistic perspective provided by our evolutionary analyses hints towards a host shift event as the origin of C. michiganensis as a tomato pathogen and the existence of pathogenic genes that remain to be characterized.

Galactofuranans and Galactomannan of Cell Walls as Chemotaxonomic Characteristics of the Genus and Species of <i>Clavibacter</i>

Two types of glycopolymers of different structures were found in the cell walls of type strains of two species of phytopathogenic bacteria, Clavibacter insidiosus VKM Ac-1402T and Clavibacter nebraskensis VKM Ac-1404T (family Microbacteriaceae, class Actinomycetes). The first type is represented by new, previously undescribed (1→6)-linked β-D-galactofuranans, which differ in the studied strains of the two species by the structure of side oligosaccharide chains (the structures are given in the text). The structure of the second glycopolymer, pyruvate-containing galactomannan, was identical in both strains. The results obtained in this work, together with those previously obtained, indicate that the presence of pyruvate-containing galactomannan and galactofuranans with an identical core structure and different oligosaccharide side substituents can be considered as a chemotaxonomic trait of the genus Clavibacter, and galactofuranans with di-, tri-, or tetrasaccharide substituents of different composition and structures can serve as chemotaxonomic markers of species. The data obtained expand our understanding of the structural diversity of natural glycopolymers and structural features of the bacteria cell walls of various taxa and may be of interest for taxonomic studies and studies aimed at elucidating the molecular mechanisms of interaction between bacteria and plant cells.

Clavibacter tessellarius causing bacterial mosaic of wheat establishes in the Old World

AbstractThe genus Clavibacter (family Microbacteriaceae) includes gram‐positive actinobacterial species infecting diverse agricultural crops such as alfalfa, maize, pepper, potato, tomato and wheat. Clavibacter tessellarius, causing bacterial mosaic of wheat, was reported for the first time in 1976 in Nebraska (United States). Since then, the pathogen was thought to be restricted to North America (Canada and the United States). While bacterial strains suspected to be C. tessellarius have occasionally been isolated from wheat samples outside North America, occurrence of the pathogen in the Old World has not yet been confirmed by the plant protection authorities. In spring 2020 (January to May) dozens of wheat fields with young plants showing leaf chlorosis and mild mosaic symptoms were surveyed and sampled in southern Iran. Among 192 surveyed wheat fields, gram‐positive, orange‐pigmented actinobacterial strains were isolated from samples of 53 fields. Altogether, 61 bacterial strains were identified as C. tessellarius based on the phenotypic features, PCR‐based molecular tests and pathogenicity assays. Multilocus sequence analysis of five housekeeping genes (atpD, dnaK, gyrB, ppk and rpoB) using 10 representative strains from diverse geographic areas in southern Iran confirmed the phylogenetic status of the strains within C. tessellarius. Our data indicate that the bacterial mosaic pathogen occurs in four southern Iranian provinces including Bushehr, Fars, Kerman and Khuzestan. Thus, occurrence of C. tessellarius outside North America is confirmed. Potential risks associated with the emergence of the pathogen in the Old World, and the need for strict quarantine measures in the area, is further discussed.

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.

The Gram-positive genus Clavibacter is currently divided into seven species (Clavibacter michiganensis, Clavibacter nebraskensis, Clavibacter capsici, Clavibacter sepedonicus, Clavibacter tessellarius, Clavibacter insidiosus and Clavibacter zhangzhiyongii) and three subspecies (C. michiganensis subsp. californiensis, C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli). Recent studies have indicated that the taxonomic rank of the subspecies must be re-evaluated. In this research, we assessed the taxonomic position of the three C. michiganensis subspecies and clarified the taxonomic nomenclature of other 75 Clavibacter strains. The complete genomes of the type strains of the three Clavibacter subspecies, the type strain of C. tessellarius and C. nebraskensis A6096 were sequenced using PacBio RSII technology. Application of whole-genome-based computational approaches such as average nucleotide identity (ANI), digital DNA-DNA hybridization, multi-locus sequence analysis of seven housekeeping genes (acnA, atpD, bipA, icdA, mtlD, recA and rpoB), a phylogenomic tree reconstructed from 1 028 core genes, and ANI-based phylogeny provided sufficient justification for raising C. michiganensis subsp. californiensis to the species level. These results led us to propose the establishment of Clavibacter californiensis sp. nov. as a species with its type strain C55T (=CFBP 8216T=ATCC BAA-2691T). Moreover, the orthologous and in silico dot plot analyses, along with the above described bioinformatic strategies, revealed a high degree of similarity between C. michiganensis subsp. chilensis and C. michiganensis subsp. phaseoli. Based on these analyses, we propose that both subspecies be combined into a single taxon and elevated to the species level as Clavibacter phaseoli sp. nov., with LPPA 982T (= CECT 8144T= LMG 27667T) as the type strain.

Isolation and identification of phosphate solubilizing bacteria from the rhizosphere of rice in organic and non-organic rice fields in Sukoharjo District, Indonesia

Abstract. Purwanti D, Setyaningsih R, Susilowati A. 2019. Isolation and identification of phosphate solubilizing bacteria from the rhizosphere of rice in organic and non-organic rice fields in Sukoharjo District, Indonesia. Bonorowo Wetlands 9: 108-115. One of the essential nutrients in the soil is phosphate. Phosphate fertilization is often inefficient because phosphate is bound and difficult for plants to absorb. The use of phosphate solubilizing bacteria (PSB) can be used as an alternative to the availability of P in the soil. This study aimed to obtain PSB isolates with high phosphate solubilization ability and identify these PSB isolates molecularly. Soil samples were taken from organic and non-organic rice (Oryza sativa L.) fields in Sukoharjo, Central Java, Indonesia, namely Grogol Village, Menuran Village, and Pondok Village. The research activity was carried out in several stages: isolation of PSB on Pikovskaya medium, characterization and testing of PSB in dissolving P, and Gram staining. PSB colonies with a phosphate solubilization index and showed different colony colors were taken, and then DNA was isolated. DNA isolation was carried out by extraction method using a DNA extraction kit. The gene encoding 16S rRNA amplified the obtained DNA samples. Sequencing was carried out using a genetic analyzer. Data analysis by comparing sequence data on the BLASTN program. Based on the isolation of rhizosphere samples from organic paddy fields, 14 isolates of PSB were obtained, and ten isolates of non-organic paddy were PSB. P13, P1, Q7, and Q8 isolates had the highest P solubilization ability with dissolution index (IP), namely 3.35, 2.13, 1.82, and 1.47. The 16S rRNA gene sequence analysis showed that isolates P1 and P13 had 99% similarity to Acinetobacter sp. ADP1 strain. The isolate Q7 had a 96% similarity with the genus Clavibacter strain NCPPB 382, ??while Q8 had a 99% similarity with Pseudomonas aeruginosa strain PAO1.

Unmarked Gene Editing in Clavibacter michiganensis Using CRISPR/Cas9 and 5-Fluorocytosine Counterselection.

Plant-pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis, which 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. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site-directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the C. michiganensis rpsL gene to generate a streptomycin-resistant mutant, and complete gene knockout, in which the deletion of the C. michiganensis celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments, the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 5-fluorocytosine at 100 μg/ml, in which an unmarked katA mutant lacking the transformation vector was recovered. Compared with conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient because it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into C. michiganensis and other Clavibacter spp.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Multiplex recombinase polymerase amplification assay developed using unique genomic regions for rapid on-site detection of genus Clavibacter and C. nebraskensis

Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg (~ 3000 copies) and 100 fg (~ 30 copies) was determined for Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1 pg (~ 300 copies) when 1 µL of host sap was added into the RPA reaction containing tenfold serially diluted genomic DNA; though no effect was observed on Clavibacter-specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA assay for any plant pathogen.

Novel galactofuranan and pyruvylated galactomannan in the cell wall of Clavibacter michiganensis subsp. michiganensis VKM Ac-1403T

The cell wall of Clavibacter michiganensis subsp. michiganensis VKM Ас-1403Т (family Microbacteriaceae, class Actinobacteria) contains two polysaccharides. The first one is neutral (1 → 6) linked galactofuranan in which every second galactofuranose residue in the main chain substituted at position 3 by side trisaccharide, β-D-GlcpNAc-(1 → 3)-α-L-Rhap-(1 → 2)-α-D-Fucp-(1 →. The second polymer is pyruvylated galactomannan with the repeating unit, →3)-α-D-Galp-(1 → 3)-α-D-[4,6-S-Pyr]-Manp-(1 → 3)-α-D-Manp-(1 →. The cell wall glycopolymer structures were established by chemical and NMR spectroscopic methods. The obtained results provide new data on the cell wall composition of plant pathogenic species of the genus Clavibacter and can promote understanding the molecular mechanisms involved in colonization and infection of plants.

Comparative Genomics of Pathogenic Clavibacter michiganensis subsp. michiganensis Strains from Chile Reveals Potential Virulence Features for Tomato Plants.

The genus Clavibacter has been associated largely with plant diseases. The aims of this study were to characterize the genomes and the virulence factors of Chilean C. michiganensis subsp. michiganensis strains VL527, MSF322 and OP3, and to define their phylogenomic positions within the species, Clavibacter michiganensis. VL527 and MSF322 genomes possess 3,396,632 and 3,399,199 bp, respectively, with a pCM2-like plasmid in strain VL527, with pCM1- and pCM2-like plasmids in strain MSF322. OP3 genome is composed of a chromosome and three plasmids (including pCM1- and pCM2-like plasmids) of 3,466,104 bp. Genomic analyses confirmed the phylogenetic relationships of the Chilean strains among C. michiganensis subsp. michiganensis and showed their low genomic diversity. Different virulence levels in tomato plants were observable. Phylogenetic analyses of the virulence factors revealed that the pelA1 gene (chp/tomA region)—that grouped Chilean strains in three distinct clusters—and proteases and hydrolases encoding genes, exclusive for each of the Chilean strains, may be involved in these observed virulence levels. Based on genomic similarity (ANIm) analyses, a proposal to combine and reclassify C. michiganensis subsp. phaseoli and subsp. chilensis at the species level, as C. phaseoli sp. nov., as well as to reclassify C. michiganensis subsp. californiensis as the species C. californiensis sp. nov. may be justified.

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

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. michiganensissensu 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. michiganensissensu 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.IMPORTANCEClavibacter 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. michiganensissensu 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. michiganensissensu 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.

Comparative Genomics to Develop a Specific Multiplex PCR Assay for Detection of Clavibacter michiganensis.

Clavibacter michiganensis is a Gram-positive bacterial pathogen that proliferates in the xylem vessels of tomato, causing bacterial wilt and canker symptoms. Accurate detection is a crucial step in confirming outbreaks of bacterial canker and developing management strategies. A major problem with existing detection methods are false-positive and -negative results. Here, we report the use of comparative genomics of 37 diverse Clavibacter strains, including 21 strains sequenced in this study, to identify specific sequences that are C. michiganensis detection targets. Genome-wide phylogenic analyses revealed additional diversity within the genus Clavibacter. Pathogenic C. michiganensis strains varied in plasmid composition, highlighting the need for detection methods based on chromosomal targets. We utilized sequences of C. michiganensis-specific loci to develop a multiplex PCR-based diagnostic platform using two C. michiganensis chromosomal genes (rhuM and tomA) and an internal control amplifying both bacterial and plant DNA (16s ribosomal RNA). The multiplex PCR assay specifically detected C. michiganensis strains from a panel of 110 additional bacteria, including other Clavibacter spp. and bacterial pathogens of tomato. The assay was adapted to detect the presence of C. michiganensis in seed and tomato plant materials with high sensitivity and specificity. In conclusion, the described method represents a robust, specific tool for detection of C. michiganensis in tomato seed and infected plants.

The role of incorrect citation of the International Code of Nomenclature of Prokaryotes and subsequent misinterpretation in causing unnecessary nomenclatural confusion.

The list that notifies names published in volume 68, part 1 of the International Journal of Systematic and Evolutionary Microbiology provides the information that a number of names of species in the genus Clavibacter that had previously been treated as names at the rank of subspecies were illegitimate because they contravene Rule 34a and Rule 50a of the International Code of Nomenclature of Prokaryotes. Rule 34a deals with combinations at the same rank and this rule does not apply to names that involve a change in rank. Rule 34a, b and c all fall under the heading new combinations and that in the case of names at the rank of species and subspecies where a change in rank is made Rule 34c applies. Rule 50a applies to names at the rank of subspecies that are elevated to species, but it is unclear why these nomenclatural changes lead to illegitimate names. Fortunately the Code is explicit in stating the role of the Notification Lists is limited to allowing orthographic corrections to be made. It is necessary to publish an interpretation of the status of these names that is consistent with the current wording of the Code.

Descriptions of Clavibacter insidiosus sp. nov. and Clavibacter tessellarius sp. nov.

To complete the valid publication of the new species names resulting from reclassification of the genus Clavibacter, we here provide descriptions of Clavibacter insidiosus sp. nov. and Clavibacter tessellarius sp. nov.

Бактериозы картофеля в Российской Федерации

Картофель поражается с заметным экономическим ущербом по меньшей мере 54 видами фитопатогенных грибов, 39 видами вирусов, 19 видами нематод, 3 видами фитоплазм и 11 видами бактерий. Бактериальные патогены вызывают болезни различной этиологии: от мокрой гнили до «зебры чипсов». Примерно 8-12 лет назад появились первые сообщения о проникновении и распространении новых возбудителей бактериозов во всех регионах России, в том числе о заболеваниях, вызываемых бактериями родов Dickeya dianthicola, D. solani и Clavibacter michiganensis subsp. michiganensis. В связи с усилением в России вредоносности бактериальных болезней картофеля, вызываемых различными группами бактерий, необходима точная идентификация патогенов, изучение путей их распространения, сохранения и разработка мер борьбы, ограничивающих распространение и обеспечивающих снижение ущерба от бактериальных болезней. Мы оценили распространение бактериальных патогенов картофеля как на основе собственных данных, так и по опубликованным сообщениям.Potato is affected at least by 54 species of phytopathogenic fungi, 39 viruses, 19 species of nematodes, 3 species of phytoplasmas, and 11 species of bacteria. Bacterial pathogens can cause diseases of different etiology: from "soft rot" to “zebra chips”. About 8-12 years ago, the increased severity of bacterial diseases in regions of Russia was reported, including diseases, caused by bacteria of genus Dickeya and Clavibacter michiganensis subsp. michiganensis. In connection with the increased harmfulness of bacterial diseases of sugar beet in Russia, caused by different groups of bacteria, we need an accurate identification of the pathogens, ways of their distribution, preservation, and search for control measures to limit the spread and reduce the damage from bacterial diseases of potato. We assessed and confirmed the distribution of potato bacterial pathogens based on own data as well as on reported incidence.

Re-classification of Clavibacter michiganensis subspecies on the basis of whole-genome and multi-locus sequence analyses.

Although the genus Clavibacter was originally proposed to accommodate all phytopathogenic coryneform bacteria containing B2γ diaminobutyrate in the peptidoglycan, reclassification of all but one species into other genera has resulted in the current monospecific status of the genus. The single species in the genus, Clavibacter michiganensis, has multiple subspecies, which are all highly host-specific plant pathogens. Whole genome analysis based on average nucleotide identity and digital DNA–DNA hybridization as well as multi-locus sequence analysis (MLSA) of seven housekeeping genes support raising each of the C. michiganensis subspecies to species status. On the basis of whole genome and MLSA data, we propose the establishment of two new species and three new combinations: Clavibacter capsici sp. nov., comb. nov. and Clavibacter tessellarius sp. nov., comb. nov., and Clavibacter insidiosus comb. nov., Clavibacter nebraskensis comb. nov. and Clavibacter sepedonicus comb. nov.

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

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 non-pathogenic 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 single-gene 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) ( = ATCC BAA-2691(T) = CFBP 8216(T)) and ZUM3936(T) ( = ATCC BAA-2690(T) = CFBP 8217(T)), respectively. Recognition of separate subspecies is essential for improved international seed testing operations.

PM 7/42 (2) Clavibacter michiganensis subsp. michiganensis

Specific scopeThis standard describes a diagnostic protocol for Clavibacter michiganensis subsp. michiganensis.Specific approval and amendmentApproved in 2004‐09.Revision adopted in 2012‐09.

GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter

The bacterial genus Clavibacter has only one species, Clavibacter michiganensis, containing five subspecies. All five are plant pathogens, among which three are recognized as quarantine pests (mentioned on the EPPO A2 list). Prevention of their introduction and epidemic outbreaks requires a reliable and accurate identification. Currently, identification of these bacteria is time consuming and often problematic, mainly because of cross-reactions with other plant-associated bacteria in immunological tests and false-negative results in PCR detection methods. Furthermore, distinguishing closely related subspecies is not straightforward. This study aimed at evaluating the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fragment of the gyrB sequence for the reliable and fast identification of the Clavibacter subspecies. Amplification and sequencing of gyrB using a single primer set had sufficient resolution and specificity to identify each subspecies based on both sequence similarities in cluster analyses and specific signatures within the sequences. All five subspecies also generated distinct and reproducible MALDI-TOF MS profiles, with unique and specific ion peaks for each subspecies, which could be used as biomarkers for identification. Results from both methods were in agreement and were able to distinguish the five Clavibacter subspecies from each other and from representatives of closely related Rathayibacter, Leifsonia or Curtobacterium species. Our study suggests that proteomic analysis using MALDI-TOF MS and gyrB sequence are powerful diagnostic tools for the accurate identification of Clavibacter plant pathogens.

Free flow and capillary isoelectric focusing of bacteria from the tomatoes plant tissues

The means of the preconcentration and preseparation of selected species or pathovars of bacteria directly from the plant tissue suspension by free flow isoelectric focusing are introduced here. After the focusing, the resulting fraction of microorganisms, native or dynamically modified by the non-ionogenic tenside on the basis of pyrenebutanoate, was separated by capillary isoelectric focusing and/or cultivated and positively identified by gas chromatographic analysis of fatty acid methyl esters. Simultaneously, capillary isoelectric focusing with UV and fluorometric detection was used for the rapid estimation of unknown isoelectric points of the examined plant pathogenic species of genus Clavibacter, Xanthomonas and Pseudomonas prior to the preconcentration and preseparation. The microorganisms were of different origin, native and/or dynamically modified by the non-ionogenic tenside.

The status of molecular biological research on the plant pathogenic genus Clavibacter

The species of the high-GC Gram-positive genus Clavibacter can be divided into three groups: (1) The subspecies within C. michiganensis; (2) the Clavibacter species causing gumming diseases; and (3) the fastidious, xylem-limited subspecies within C. xyli. All members of the taxon are plant pathogens. Basic studies have demonstrated the presence of native plasmids in various species and allowed construction of cloning vectors for a few of these species. Infection of plants by C. michiganensis and C. xyli subspecies can be difficult to detect, since they often give symptomless infections. Therefore, serological and DNA-based detection techniques have been developed to aid in disease control. Efforts to determine what factors may be important in pathogenicity have focused on extracellular polysaccharides in several of the species, but a clear role for these molecules in virulence has not been found. The development of transformation methods for several of the C. michiganensis subspecies has allowed elucidation of other pathogenicity factors, e.g., plasmid-encoded extracellular cellulase. For C. toxicus, the involvement of a phage in annual ryegrass toxicity has been determined. Of special interest is C. xyli ssp. cynodontis, which has been genetically engineered for protecting plants from insect damage. Further progress in elucidating the molecular biology of these organisms will require the development of more sophisticated molecular genetic tools.