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.     

Tomato Fruit and Seed Colonization by Clavibacter michiganensis subsp. michiganensis through External and Internal Routes

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis, causal agent of bacterial wilt and canker of tomato, is an economically devastating pathogen that inflicts considerable damage throughout all major tomato-producing regions. Annual outbreaks continue to occur in New York, where C. michiganensis subsp. michiganensis spreads via infected transplants, trellising stakes, tools, and/or soil. Globally, new outbreaks can be accompanied by the introduction of contaminated seed stock; however, the route of seed infection, especially the role of fruit lesions, remains undefined. In order to investigate the modes of seed infection, New York C. michiganensis subsp. michiganensis field strains were stably transformed with a gene encoding enhanced green fluorescent protein (eGFP). A constitutively eGFP-expressing virulent C. michiganensis subsp. michiganensis isolate, GCMM-22, was used to demonstrate that C. michiganensis subsp. michiganensis could not only access seeds systemically through the xylem but also externally through tomato fruit lesions, which harbored high intra- and intercellular populations. Active movement and expansion of bacteria into the fruit mesocarp and nearby xylem vessels followed, once the fruits began to ripen. These results highlight the ability of C. michiganensis subsp. michiganensis to invade tomato fruits and seeds through multiple entry routes.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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。     

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.     

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.     

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.     

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。     

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.     

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.