Detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds using immunomagnetic separation

The use of pathogen-free plant material is the main strategy for controlling bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis. However, detection and isolation of this pathogen from seeds before field or greenhouse cultivation is difficult when the bacterium is at low concentration and associated microbiota are present. Immunomagnetic separation (IMS), based on the use of immunomagnetic beads (IMBs) coated with specific antibodies, was used to capture C. michiganensis subsp. michiganensis cells, allowing removal of non-target bacteria from samples before plating on non-selective medium. Different concentrations of IMBs and of two antisera were tested, showing that IMS with 10(6)IMBs/ml coated with a polyclonal antiserum at 1/3200 dilution recovered more than 50% of target cells from initial inocula of 10(3) to 10(0)CFU/ml. Threshold detection was lower than 10CFU/ml even in seed extracts containing seed debris and high populations of non-target bacteria. The IMS permitted C. michiganensis subsp. michiganensis isolation from naturally infected seeds with higher sensitivity and faster than direct isolation on the semiselective medium currently used and could become a simple viable system for routinely testing tomato seed lots in phytosanitary diagnostic laboratories.     

The tomato pathogen Clavibacter michiganensis ssp. michiganensis: producer of several antimicrobial substances

AIM: To purify and analyse antimicrobial substances produced by the tomato pathogen Clavibacter michiganensis ssp. michiganensis (Cmm), with potential application in control of Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot of potato. METHODS AND RESULTS: After selection of a suitable producer and indicator strain, antimicrobial compounds were isolated using chromatographic techniques. The resulting preparations were analysed with respect to heat and protease sensitivity, amino acid composition, amino acid sequence and mass. Using this procedure we discovered one post-translationally modified 2145 Da peptide bacteriocin, one 14 kDa antimicrobial protein as well as low molecular weight (<1000 Da) antimicrobial compounds, putatively belonging to the tunicamycin family. CONCLUSIONS: Clavibacter michiganensis ssp. michiganensis produces various antibacterial substances that are active against Cms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the first attempt to characterize antimicrobial substances from Cmm at the molecular level. This is an important step towards investigation of the possible use of these compounds to control the potato ring rot pathogen.     

Purification, characterization, and gene sequence of michiganin A, an actagardine-like lantibiotic produced by the tomato pathogen Clavibacter michiganensis subsp. michiganensis

Members of the actinomycete genus Clavibacter are known to produce antimicrobial compounds, but so far none of these compounds has been purified and characterized. We have isolated an antimicrobial peptide, michiganin A, from the tomato pathogen Clavibacter michiganensis subsp. michiganensis, using ammonium sulfate precipitation followed by cation-exchange and reversed-phase chromatography steps. Upon chemical derivatization of putative dehydrated amino acids and lanthionine bridges by alkaline ethanethiol, Edman degradation yielded sequence information that proved to be sufficient for cloning of the gene by a genome-walking strategy. The mature unmodified peptide consists of 21 amino acids, SSSGWLCTLTIECGTIICACR. All of the threonine residues undergo dehydration, and three of them interact with cysteines via thioether bonds to form methyllanthionine bridges. Michiganin A resembles actagardine, a type B lantibiotic with a known three-dimensional structure, produced by Actinoplanes liguriae, which is a filamentous actinomycete. The DNA sequence of the gene showed that the michiganin A precursor contains an unusual putative signal peptide with no similarity to well-known secretion signals and only very limited similarity to the (only two) available leader peptides of other type B lantibiotics. Michiganin A inhibits the growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of ring rot of potatoes, with MICs in the low nanomolar range. Thus, michiganin A may have some potential in biological control of potato ring rot.     

Induction of resistance to Clavibacter michiganensis subsp. michiganensis

Tomato plants pre-inoculated with the avirulent strain NCPPB 3123 of Clavibacter michiganensis subsp. michiganensis (Cmm) were protected largely against challenge infection by virulent strains of Cmm. Effectiveness of this protective effect was mainly dependent on the inoculation sites, the bacterial cell concentration used for pre- and challenge inoculations, and the time interval between both inoculations. This defence reaction was systemic and stable throughout the whole growing season. Resistance can also be induced by pre-inoculation of heat-killed bacteria or application of isolated EPS of the strain 3123. Strain 3123 spreads out in tomato plants in the same manner as virulent Cmm isolates, but its colonization of tomato fruits and seeds was substantially lower. Papillary to spherical electron dense particles were observed at the tonoplast in parenchyma cells of the vascular system of tomato plants inoculated with the strain 3123. Numerous investigations carried out to examine the ability of 3123 to induce resistance in other host/pathogen-systems showed that it was only specific for tomato/Cmm.     

Evaluation of the efficacy of immunomagnetic separation for the detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds

Aims: To evaluate the effectiveness of the optimized immunomagnetic separation (IMS)‐plating protocol in relation to other culture, serological and molecular techniques currently used for Clavibacter michiganensis subsp. michiganensis in seed‐testing laboratories. Methods and results: Bacterial suspensions, tomato seed extracts spiked with the pathogen and naturally infected seeds were IMS‐plated for the detection of C. m. subsp. michiganensis. These results were compared with plating on general (YPGA) and semiselective (mSCM) media, double‐antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA), immunofluorescent assay (IF) or polymerase chain reaction (PCR). Different seed lots and pathogen strains were also tested. IMS‐plating allowed the detection of less than 10 CFU ml^?1 of pathogen in all assayed samples. The mSCM medium provided positive results for 10 CFU ml^?1 in naturally infected seeds, but up to 14 days was necessary for the typical colonies of the target to be come visible. By serological techniques, 10³ and up to 10⁴ CFU ml^?1 were detected by IF and ELISA, respectively. DNA extraction was required to obtain positive results by PCR in seed extracts containing 10³ CFU ml^?1 or more. Conclusions: Among the evaluated methods, IMS‐plating provided the best results regarding sensitivity and specificity for C. m. subsp. michiganensis detection, allowing the recovery of viable bacteria from seed extracts. Significance and impact of the study: IMS‐plating increases isolation rates of C. m. subsp. michiganensis and could improve standard protocols currently used for routine analysis.     

Analysis of the interaction of Clavibacter michiganensis subsp. michiganensis with its host plant tomato by genome-wide expression profiling

Genome-wide expression profiles of the phytopathogenic actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm) strain NCPPB382 were analyzed using a 70mer oligonucleotide microarray. Cmm causes bacterial wilt and canker of tomato, a systemic disease leading to substantial economic losses worldwide. Global gene expression was monitored in vitro after long- and short-term incubation with tomato homogenate to simulate conditions in planta and in vivo ten days after inoculation of tomatoes. Surprisingly, both in the presence of tomato homogenate and in planta known virulence genes (celA, chpC, ppaA/C) were down-regulated indicating that the encoded extracellular enzymes are dispensable in late infection stages where plant tissue has already been heavily destroyed. In contrast, some genes of the tomA-region which are involved in sugar metabolism showed an enhanced RNA-level after permanent growth in supplemented medium. Therefore, these genes may be important for utilization of plant derived nutrients. In the plant Cmm exhibited an expression profile completely different from that in vitro. Especially, the strong expression of genes of the wco-cluster (extracellular polysaccharide II), 10 genes encoding surface or pilus assembly proteins, and CMM_2382, coding for a putative perforin suggest a possible role of these genes in the plant-pathogenic interaction.     

Introduction and recovery of Clavibacter michiganensis subsp. michiganensis from agricultural soil

Twelve phytopathogenic Clavibacter michiganensis subsp. michiganensis strains were introduced into non-sterile agricultural loam soil at an inoculum density of about log. 6.0 cfu g^–1 dry weight soil. The soil samples were incubated at 22°C under a 12h light, 12h dark cycle and the population densities followed over a 30-day period by plating subsamples of serial dilutions of soil on Brain Heart Infusion agar amended with 0.5% (w/v) yeast extract and 30 g mL^–1 nalidixic acid. In 5 soil samples C. michiganensis cfu were not detected after 30 days incubation. Initially, C. michiganensis cfu accounted for about 90% of the cfu recovered but decreased to less than 10% after 30 days. These results suggested that some C. michiganensis strains survive in this particular soil, while other strains exhibit poor survival and/or may be difficult to detect when present in low numbers.     

Expression of putative virulence factors in the potato pathogen Clavibacter michiganensis subsp. sepedonicus during infection

The Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus is the causal agent of bacterial wilt and ring rot of potato. So far, only two proteins have been shown to be essential for virulence, namely a plasmid-encoded cellulase CelA and a hypersensitive response-inducing protein. We have examined the relative expression of CelA and eight putative virulence factors during infection of potato and in liquid culture, using quantitative real-time PCR. The examined putative virulence genes were celB, a cellulase-encoding gene and genes encoding a pectate lyase, a xylanase and five homologues of the Clavibacter michiganensis subsp. michiganensis pathogenicity factor Pat-1 thought to encode a serine protease. Six of the nine assayed genes were up-regulated during infection of potato, including celA, celB, the xylanase gene, and two of the pat genes. The pectate lyase gene showed only slightly elevated expression, whereas three of the five examined pat genes were down-regulated during infection in potato. Interestingly, the two up-regulated pat genes showed a noticeable sequence difference compared to the three down-regulated pat genes. These results reveal several new proteins that are likely to be involved in Clavibacter michiganensis subsp. sepedonicus pathogenicity.     

A family of serine proteases of Clavibacter michiganensis subsp. michiganensis: chpC plays a role in colonization of the host plant tomato

Genes for seven putative serine proteases (ChpA–ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis ( Cmm ) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm . The genes chpA , chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant.     

Evidence for plasmid-encoded virulence factors in the phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382.

The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt, harbors two plasmids pCM1 (27.5 kb) and pCM2 (72 kb). After curing of the plasmids, bacterial derivatives were still proficient in the ability to colonize the host plant and in the production of exopolysaccharides but exhibited a reduced virulence. When one of the two plasmids is lost, there is a significant delay in the development of wilting symptoms after infection and a plasmid-free derivative is not able to induce disease symptoms. By cloning of restriction fragments of both plasmids in the plasmid-free strain CMM100, two DNA fragments which restored the virulent phenotype were identified. Further analysis suggested that a fragment of plasmid pCM1 encodes an endocellulase which is involved in the expression of the pathogenic phenotype.     

Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp. michiganensis in transgenic tomato (Solanum lycopersicum)

Two tomato proteins were evaluated by over-expression in transgenic tomato for their ability to confer resistance to Clavibacter michiganensis subsp. michiganensis (Cmm). Snakin-2 (SN2) is a cysteine-rich peptide with broad-spectrum antimicrobial activity in vitro while extensin-like protein (ELP) is a major cell-wall hydroxyproline-rich glycoprotein linked with plant response to pathogen attack and wounding. Tomato plants, cultivar Mountain Fresh, were transformed via Agrobacterium tumefaciens harboring a binary vector for expression of the full-length SN2 gene or ELP cDNA under the regulation of the CaMV 35S promoter. Molecular characterization of PCR-positive putative T₀ transgenic plants by Northern analysis revealed constitutive over-expression of SN2 and ELP mRNA. Junction fragment analysis by Southern blot showed that three of the four SN2 over-expressing T₀ lines had single copies of complete T-DNAs while the other line had two complete T-DNA copies. All four ELP over-expressing T₀ lines had a single copy T-DNA insertion. Semi-quantitative RT-PCR analysis of T₁ plants revealed constitutive over-expression of SN2 and ELP. Transgenic lines that accumulated high levels of SN2 or ELP mRNA showed enhanced tolerance to Cmm resulting in a significant delay in the development of wilt symptoms and a reduction in the size of canker lesions compared to non-transformed control plants. Furthermore, in transgenic lines over-expressing SN2 or ELP bacterial populations were significantly lower (100–10,000-fold) than in non-transformed control plants. These results demonstrate that SN2 and ELP over-expression limits Cmm invasiveness suggesting potential in vivo antibacterial activity and possible biotechnological application for these two defense proteins.     

Linear plasmid in the genome of Clavibacter michiganensis subsp. sepedonicus

Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen Clavibacter michiganensis subsp. sepedonicus revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.     

Identification of a tomatinase in the tomato-pathogenic actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382

The insertion site of a transposon mutant of Clavibacter michiganensis subsp. michiganensis NCPPB382 was cloned and found to be located in the gene tomA encoding a member of the glycosyl hydrolase family 10. The intact gene was obtained from a cosmid library of C. michiganensis subsp. michiganensis. The deduced protein TomA (543 amino acids, 58 kDa) contains a predicted signal peptide and two domains, the N-terminal catalytic domain and a C-terminal fibronectin III-like domain. The closest well-characterized relatives of TomA were tomatinases from fungi involved in the detoxification of the tomato saponin alpha-tomatine which acts as a growth inhibitor. Growth inhibition of C. michiganensis subsp. michiganensis by alpha-tomatine was stronger in the tomA mutants than in the wild type. Tomatinase activity assayed by deglycosylation of alpha-tomatine to tomatidine was demonstrated in concentrated culture supernatants of C. michiganensis subsp. michiganensis. No activity was found with the tomA mutants. However, neither the transposon mutant nor a second mutant constructed by gene disruption was affected in virulence on the tomato cv. Moneymaker.     

New Type of Antimicrobial Protein Produced by the Plant Pathogen Clavibacter michiganensis subsp. michiganensis

It has previously been shown that the tomato pathogen Clavibacter michiganensis subsp. michiganensis secretes a 14-kDa protein, C. michiganensis subsp. michiganensis AMP-I (CmmAMP-I), that inhibits growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato. Using sequences obtained from tryptic fragments, we have identified the gene encoding CmmAMP-I and we have recombinantly produced the protein with an N-terminal intein tag. The gene sequence showed that CmmAMP-I contains a typical N-terminal signal peptide for Sec-dependent secretion. The recombinant protein was highly active, with 50% growth inhibition (IC₅₀) of approximately 10 pmol, but was not toxic to potato leaves or tubers. CmmAMP-I does not resemble any known protein and thus represents a completely new type of bacteriocin. Due to its high antimicrobial activity and its very narrow inhibitory spectrum, CmmAMP-1 may be of interest in combating potato ring rot disease.     

Bioluminescence Imaging of Clavibacter michiganensis subsp. michiganensis Infection of Tomato Seeds and Plants

Clavibacter michiganensis subsp. michiganensis is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement of C. michiganensis subsp. michiganensis in grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on the C. michiganensis subsp. michiganensis movement in tomato seed and grafted plants, we isolated a bioluminescent C. michiganensis subsp. michiganensis strain using the modified Tn1409 containing a promoterless lux reporter. A total of 19 bioluminescent C. michiganensis subsp. michiganensis mutants were obtained. All mutants tested induced a hypersensitive response in Mirabilis jalapa and caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed that C. michiganensis subsp. michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescent C. michiganensis subsp. michiganensis to help better elucidate the C. michiganensis subsp. michiganensis-tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation of C. michiganensis subsp. nebraskensis with Tn1409::lux. Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the agriculturally important C. michiganensis.Clavibacter michiganensis subsp. michiganensis is a Gram-positive, aerobic bacterium that belongs to a group of plant-pathogenic actinomycetes (37). Infections by C. michiganensis subsp. michiganensis cause bacterial canker and wilt in tomato, which is considered one of the most destructive and economically significant diseases of this crop. Severe epidemics can cause up to 80% yield loss, mainly due to wilting and death of plants and lesions on fruit. Bacterial canker was first discovered in Michigan greenhouses in 1909 and has now been reported to occur in most tomato production areas around the world (11, 40).Plant wounds facilitate but are not required for infection by C. michiganensis subsp. michiganensis, which invades the xylem vessels and causes vascular disease with high titers (10⁹ bacteria/g of plant tissue) (2, 29), impairing water transport and leading to plant wilting, canker stem lesions, and death (17, 23). Alternatively, asymptomatic infections can be induced by C. michiganensis subsp. michiganensis during late stages of plant development, resulting in the production of contaminated seeds, a major source of outbreaks of C. michiganensis subsp. michiganensis infections in tomato production (13, 34). Traditional bacterial-disease management measures, such as applications of antibiotics and copper bactericides, have not been successful against this disease, and canker-resistant tomato cultivars are not available. As a result, C. michiganensis subsp. michiganensis has been included under international quarantine regulation (10, 11). Consequently, seed testing and maintaining pathogen-free seeds and transplants is currently the most appropriate approach to minimize the spread of disease (23). However, even a low C. michiganensis subsp. michiganensis transmission rate (0.01%) from seed to seedling can cause a disease epidemic under favorable conditions (5). Due to overcrowding of seedlings during transplant production, the pathogen can easily spread through splashing of irrigation water and leaf contact. Despite its apparent significance in C. michiganensis subsp. michiganensis epidemiology, the mechanism of seed-to-seedling transmission of C. michiganensis subsp. michiganensis is not well understood.Another critical point for disease spread is the grafting process, which is now a common practice for the majority of plants used in production greenhouses. Desirable tomato cultivars (scions) are grafted onto rootstocks that provide greater vigor, longevity, or, in some cases, disease resistance (26). Grafting requires cutting both rootstock and scion, providing a quick way for C. michiganensis subsp. michiganensis to spread from plant to plant. However, grafting is a relatively recent innovation in tomato production, and little is known about how grafting affects the dynamics of C. michiganensis subsp. michiganensis infection. Developing adequate control measures for C. michiganensis subsp. michiganensis is complicated by the complexity of genetic manipulation of Gram-positive bacteria, which impairs analysis and characterization of pathogenesis mechanisms (23). Consequently, there is a need to develop molecular techniques that would allow a better understanding of C. michiganensis subsp. michiganensis infections.One method of interest is using engineered bioluminescent bacteria to monitor plant-pathogen interactions in real time. By exploiting natural light-emitting reactions that are encoded by the luxCDABE genes, bioluminescent bacteria have been used to assess gene expression and to monitor the internalization and distribution of bacteria in hosts (3, 6, 7, 8, 9, 12, 15, 24, 31, 35, 36). In particular, bioluminescent phytopathogenic Xanthomonas campestris pathovars and Pseudomonas spp. have been used to track bacterial movement and distribution in host plants (7, 8, 15, 31, 36), as well as to assess host susceptibility quantitatively (15). Likewise, the lux genes have also been transferred to beneficial bacteria, such as Rhizobium leguminosarum and Pseudomonas spp. to visualize colonization patterns in rhizospheres (3, 9).The genes that carry the function of light emission are luxAB, which express luciferase enzymes that catalyze the bioluminescent reaction, while luxCDE encode the enzymes required for biosynthesis of a fatty aldehyde substrate necessary for the reaction (28, 39). Bioluminescence involves an intracellular oxidation of the reduced form of flavin mononucleotide and the fatty aldehyde by luciferase in the presence of molecular oxygen; therefore, bacterial bioluminescence also requires oxygen, a source of energy (38). Cells that express the lux operon spontaneously emit photons that can be captured by a sensitive charge-coupled-device (CCD) camera, enabling imaging and visualization of bacterial cells (22). Luciferase activity depends on the metabolic integrity of the cell, while the number of photons emitted correlates with the biomass of living bacteria (12, 31). Furthermore, since the half-life of luciferase binding to its substrate is several seconds (28), captured light events reflect processes in real time and are not artifacts of accumulated signals. Consequently, live imaging of bioluminescence provides a sensitive means of visualizing bacterial colonization and invasion of hosts and allows real-time representation and examination of pathogen-plant interactions (24, 36).Very little information is available about the mechanisms of C. michiganensis subsp. michiganensis pathogenesis and its colonization of seeds and subsequent transmission to seedlings. This is largely attributable to a lack of tools and difficulties in genetically manipulating this Gram-positive bacterium (30). However, recent development of an insertion sequence element IS1409 (Tn1409)-based efficient transposon mutagenesis system for C. michiganensis subsp. michiganensis has increased our knowledge of the pathogenesis of tomato canker (16, 25). To better understand the dynamics of seed-to-seedling transmission of C. michiganensis subsp. michiganensis, as well as movement of C. michiganensis subsp. michiganensis in grafted plants, we constructed a bioluminescent C. michiganensis subsp. michiganensis strain using the Tn1409 transposon mutagenesis system. Our results demonstrated the utility of using a bioluminescent C. michiganensis subsp. michiganensis strain as a novel approach to elucidate the interaction of plants with this economically important pathogen.     

Factors Affecting Survival of Clavibacter michiganensis subsp. sepedonicus in Water

The survival of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, was studied in water, to assess the risks for dissemination of Cms via surface water and infection of potato crops by irrigation. Cms was able to survive for a maximum period of 7 days in non‐sterile surface water at 10°C, a period during which Cms can be transported over long distances, but will also be strongly diluted. It is concluded that contamination of surface water with Cms can pose a threat on potato production only if aquatic host plants can multiply Cms in high densities. Survival of a fluidal and non‐mucoid strain was also studied in sterile ditch water and simulated ‘drainage water’, in sterile MilliQ water, in tap water, in physiological salt and in artificial xylem fluid. In addition, the influence of temperature and low oxygen conditions on persistence of Cms in some of these diluents was studied. A maximum survival period of 35 days was found for Cms in sterile tap water at 20°C, independent of the strain used. In the other diluents survival periods ranged between 0 and 21 days. Relatively poor survival was found in MilliQ water and artificial xylem fluid. Low temperatures of 4°C do not favour survival as it does in soil. Oxygen depletion affected survival detrimentally. Survival periods determined by agar dilution plating and a direct viable counting method, based on the use of indicators for esterase activity and membrane integrity were similar. Therefore, it was concluded that under the experimental conditions studied, Cms did not form cells in a viable but non‐culturable state.     

Genetic diversity and population structure of Clavibacter michiganensis subsp. nebraskensis

Clavibacter michiganensis subsp. nebraskensis (CMN) is a gram-positive bacterium and an incitant of Goss's bacterial wilt and leaf blight or "leaf freckles" in corn. A population structure of a wide temporal and geographic collection of CMN strains (n = 131), originating between 1969 and 2009, was determined using amplified fragment length polymorphism (AFLP) analysis and repetitive DNA sequence-based BOX-PCR. Analysis of the composite data set of AFLP and BOX-PCR fingerprints revealed two groups with a 60% cutoff similarity: a major group A (n = 118 strains) and a minor group B (n = 13 strains). The clustering in both groups was not correlated with strain pathogenicity. Group A contained two clusters, A1 (n = 78) and A2 (n = 40), with a linkage of 75%. Group A strains did not show any correlation with historical, geographical, morphological, or physiological properties of the strains. Group B was very heterogeneous and eight out of nine clusters were represented by a single strain. The mean similarity between clusters in group B varied from 13% to 63%. All strains in group B were isolated after 1999. The percentage of group B strains among all strains isolated after 1999 (n = 69) was 18.8%. Implications of the findings are discussed.