Nested PCR for ultrasensitive detection of the potato ring rot bacterium, Clavibacter michiganensis subsp. sepedonicus.

Oligonucleotide primers derived from sequences of the 16S rRNA gene (CMR16F1, CMR16R1, CMR16F2, and CMR16R2) and insertion element IS1121 of Clavibacter michiganensis subsp. sepedonicus (CMSIF1, CMSIR1, CMSIF2, and CMISR2) were used in nested PCR to detect the potato ring rot bacterium C. michiganensis subsp. sepedonicus. Nested PCR with primer pair CMSIF1-CMSIR1 followed by primer pair CMSIF2-CMSIR2 specifically detected C. michiganensis subsp. sepedonicus, while nested PCR with CMR16F1-CMR16R1 followed by CMR16F2-CMR16R2 detected C. michiganensis subsp. sepedonicus and the other C. michiganensis subspecies. In the latter case, C. michiganensis subsp. sepedonicus can be differentiated from the other subspecies by restriction fragment length polymorphism (RFLP) analyses of the nested PCR products (16S rDNA sequences). The nested PCR assays developed in this work allow ultrasensitive detection of very low titers of C. michiganensis subsp. sepedonicus which may be present in symptomiess potato plants or tubers and which cannot be readily detected by direct PCR (single PCR amplification). RFLP analysis of PCR products provides for an unambiguous confirmation of the identify of C. michiganensis subsp. sepedonicus.     

A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies

Real-time TaqMan-PCR assays were developed for detection, differentiation and absolute quantification of the pathogenic subspecies of Clavibacter michiganensis (Cm) in one single PCR run. The designed primer pair, targeting intergenic sequences of the rRNA operon (ITS) common in all subspecies, was suitable for the amplification of the expected 223-nt DNA fragments of all subspecies. Closely related bacteria were completely discriminated, except of Rathayibacter iranicus, from which weak PCR product bands appeared on agarose gel after 35 PCR cycles. Sufficient specificity of PCR detection was reached by introduction of the additional subspecies specific probes used in TaqMan-PCR. Only Cm species were detected and there was clear differentiation among the subspecies C. michiganensis sepedonicus (Cms), C. michiganensis michiganensis (Cmm), C. michiganensis nebraskensis (Cmn), C. michiganensis insidiosus (Cmi) and C. michiganensis tessellarius (Cmt). The TaqMan assays were optimized to enable a simultaneous quantification of each subspecies. Validity is shown by comparison with cell counts.     

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.     

A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 x 10(6) transformants per microg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per pg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.     

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.     

Clavibacter michiganensis subsp. michiganensis: first steps in the understanding of virulence of a Gram-positive phytopathogenic bacterium

Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete. It infects tomato, spreads through the xylem and causes bacterial wilt and canker. The wild-type strain NCPPB382 carries two plasmids, pCM1 and pCM2. The cured plasmid-free derivative CMM100 is still able to colonize tomato, but no disease symptoms develop indicating that all genes required for successful infection, establishment and growth in the plant reside on the chromosome. Both plasmids carry one virulence factor, a gene encoding a cellulase, CelA in case of pCM1 and a putative serine protease Pat-1 on pCM2. These genes can independently convert the non-virulent strain CMM100 into a pathogen causing wilt on tomatoes. Currently, genome projects for Cmm and the closely related potato-pathogen C. michiganensis subsp. sepedonicus have been initiated. The data from the genome project shall give clues on further genes involved in plant-microbe interaction that can be tested experimentally. Especially, identification of genes related to host-specificity through genome comparison of the two subspecies might be possible.     

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.     

玉米内州萎蔫病菌免疫学检测方法的建立

以玉米内州萎蔫病菌单抗(4H4和4G12)为基础,纯化抗体后,进行亚类鉴定、效价及特异性测定。比较间接ELISA和双单抗夹心ELISA(DAS-ELISA)的检测灵敏度,并应用于玉米种子中萎蔫病菌的检测。结果表明,两株单克隆抗体(0.4g/L)效价均可达1:256000,亚类鉴定结果分别为IgG2a和IgG2b,轻链均为K链。与供试的16株非目标细菌均无交叉反应。DAS-ELISA对萎蔫病菌种子悬浮液的检测灵敏度为1.0×109CFU/L,在此基础上建立了灵敏、特异的玉米内州萎蔫病菌双单抗DAS-ELISA检测方法。     

Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels.     

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.     

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.     

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.     

番茄溃疡病菌拮抗菌株Z-L-22的鉴定及其活性物质

摘要：【目的】鉴定一株对番茄溃疡病病原菌—密执安棒形杆菌密执安亚种(Clavibacter michiganensis subsp. michiganensis,Cmm)具有强拮抗作用的放线菌菌株Z-L-22,并分析其代谢产物,为开发新的生物活性物质奠定基础。【方法】根据菌株Z-L-22的形态特征、培养特征、生理生化特征、细胞壁组分和16S rDNA序列对菌株Z-L-22的进行了鉴定。通过薄层层析、纸层析和特征性鉴别试验对活性物质进行分离、回收和鉴定。并利用抗生素合成基因保守区域设计的引物用对基因组DNA进行PCR扩增。【结果】菌株Z-L-22属于链霉菌属,各特征与西唐链霉菌(Streptomyces setonii)相似。获得了2个主要活性成分,均为放线菌素类抗生素。利用放线菌素类抗生素合成酶保守引物在该菌基因组中扩增到770 bp的相关基因片段。【结论】活性菌株Z-L-22鉴定为西唐链霉菌,命名为西唐链霉菌Z-L-22。该菌产生的抗生活性物质为放线菌素类抗生素,本研究为开发该菌株奠定基础。     

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.     

Purification and characterization of a collagenolytic enzyme produced by Rathayibacter sp. strains isolated from cultures of Clavibacter michiganensis subsp. michiganensis

We show in this work that collagenolytic Rathayibacter sp. are isolated with phytopathogenic Clavibacter michiganensis subsp. michiganensis strains. The Rathayibacter strains isolated all produced collagenases. One of these collagenases (from the strain 1715) was purified by ammonium sulphate precipitation, DEAE cellulose and Sephadex G 200 chromatography. Characterization of the enzyme showed that it is a true collagenase which is able to degrade both native collagen, gelatin and probably other proteins from plants sharing sequence homologies with collagen.     

Lateral flow immunoassay for rapid detection of potato ring rot caused by Clavibacter michiganensis subsp. sepedonicus

A lateral flow immunoassay for the rapid detection of Clavibacter michiganensis subsp. sepedonicus bacteria causing potato ring rot was developed. Multimembrane composites (test strips) containing polyclonal antibodies against the bacteria and gold nanoparticle-antibody conjugates were used for the analysis. The test strips are suitable for the analysis of potato tuber and leaf extracts within 10 min; the detection limit of bacteria is 4 × 10⁵ cells/mL. No cross-reactivity with strains of Clavibacter michiganensis subsp. michiganensis, Pectobacterium carotovorum subsp. carotovorum and saprophytes of healthy potato plants was detected. The results of analysis of 26 potato samples by the developed tests were compared with those obtained by the PCR method and using the commercial enzyme immunoassay kits. The results of lateral flow immunoassay were confirmed in 96.2% of cases, which supports the high correlation with other analytical approaches. The developed immunoassay may be considered as a promising means of phytosanitary control.     

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.     

A Simple and Fast Extraction Procedure to Obtain Amplifyable DNA from Ralstonia (Pseudomonas) Solanacearum and Clavibacter michiganensis ssp. sepedonicus Inoculated Potato Tuber Extracts and Naturally Infected Tubers to Conduct a Polymerase Chain Reaction (PCR)

A modified NaOH alkaline boiling procedure using a mixture of lysozyme and proteases combined with minimized TRIS/HCl/BSA buffer volume was applied to extract amplifyable DNA from the two quarantine bacteria Ralstonia (Pseudomonas) solanacearum and Clavibacter michiganensis ssp. sepedonicus artificially added to potato tuber extracts of a low and a high starch potato variety. A PCR detection threshold of 10⁴−10⁵ colony forming units per ml extract of each quarantine bacterium was obtained by using the two potato varieties, the high starch potato variety resulting in a lower detection threshold.
Similar sensitivities could be obtained from potato tubers naturally infected with both quarantine bacteria. When comparing a published DNA extraction procedure suitable for Clavibacter michiganensis ssp. sepedonicus with the alkaline extraction the latter is much faster and simpler with similar detection thresholds and representing an inexpensive method to obtain suitable template DNA for routine PCR tests.     

Phylogenetic Analysis and Polyphasic Characterization of Clavibacter michiganensis Strains Isolated from Tomato Seeds Reveal that Nonpathogenic Strains Are Distinct from C. michiganensis subsp. michiganensis

The genus Clavibacter comprises one species and five subspecies of plant-pathogenic bacteria, four of which are classified as quarantine organisms due to the high economic threat they pose. Clavibacter michiganensis subsp. michiganensis is one of the most important pathogens of tomato, but the recommended diagnostic tools are not satisfactory due to false-negative and/or -positive results. To provide a robust analysis of the genetic relatedness among a worldwide collection of C. michiganensis subsp. michiganensis strains, relatives (strains from the four other C. michiganensis subspecies), and nonpathogenic Clavibacter-like strains isolated from tomato, we performed multilocus sequence-based analysis and typing (MLSA and MLST) based on six housekeeping genes (atpD, dnaK, gyrB, ppK, recA, and rpoB). We compared this “framework” with phenotypic and genotypic characteristics such as pathogenicity on tomato, reaction to two antisera by immunofluorescence and to five PCR identification tests, and the presence of four genes encoding the main C. michiganensis subsp. michiganensis pathogenicity determinants. We showed that C. michiganensis subsp. michiganensis is monophyletic and is distinct from its closest taxonomic neighbors. The nonpathogenic Clavibacter-like strains were identified as C. michiganensis using 16S rRNA gene sequencing. These strains, while cross-reacting with C. michiganensis subsp. michiganensis identification tools, are phylogenetically distinct from the pathogenic strains but belong to the C. michiganensis clade. C. michiganensis subsp. michiganensis clonal complexes linked strains from highly diverse geographical origins and also strains isolated over long periods of time in the same location. This illustrates the importance of seed transmission in the worldwide dispersion of this pathogen and its survival and adaptation abilities in a new environment once introduced.     

Proteomic analysis of resistance mediated by Rcm 2.0 and Rcm 5.1, two loci controlling resistance to bacterial canker of tomato

Two quantitative trait loci from Lycopersicon hirsutum, Rcm 2.0 and Rcm 5.1, control resistance to Clavibacter michiganensis subsp. michiganensis, the causal agent of bacterial canker of tomato. Lines containing Rcm 2.0 and Rcm 5.1 and a susceptible control line were compared at 72 and 144 h postinoculation, using 2-dimensional gel electrophoresis to identify proteins regulated in response to C. michiganensis subsp. michiganensis infection. A total of 47 proteins were subjected to tandem mass spectrometry. Database queries with resulting spectra identified tomato genes for 26 proteins. The remaining 21 proteins were either identified in other species or possessed no homology to known proteins. Spectra were interpreted to deduce peptide amino acid sequences that were then used to query publicly available data. This approach identified tomato genes or expressed sequence tags for 44 of the proteins analyzed. Three superoxide dismutase (SOD) enzymes were differentially regulated among genotypes, and patterns of hydrogen peroxide accumulation were genotype- and tissue-specific, indicating a role for oxidative stress in response to C. michiganensis subsp. michiganensis. Steady-state mRNA and protein levels for SOD, thioredoxin M-type, S-adenosylhomocysteine hydrolase, and pathogenesis-related proteins demonstrated similar patterns of differential regulation. Lines containing Rcm 2.0 and Rcm 5.1 accumulate different proteins and steady-state mRNAs in response to inoculation, suggesting that the two loci may confer resistance through distinct mechanisms.