An Investigation on Strains of Clavibacter michiganensis subsp. michiganensis in North and North West of Iran

Tomato bacterial canker disease was first reported from Urmiyeh in West Azerbaijan province in Iran. The disease causes lesion (canker), wilting and dryness of infected plants, leaf and fruit spots and the decline of the whole plant. Out of 102 isolates obtained from the fields in the major tomato producing areas of understudy regions, 98 were found Gram positive, yellow‐pigmented isolates, identified as Clavibacter michiganensis subsp. michiganensis based on the morphological and biochemical characteristics described in previous studies. Among these strains, 64 were virulent and 34 showed poor virulence. A strain of Cmm (NCPPB382) was used as a check (standard) in all steps of this study. DNA fingerprinting with repetitive‐sequence‐based PCR (rep‐PCR) (BOX primer) carried out among 11 representative strains (eight strains from West Azerbaijan, two from Golestan and one as standard). The most virulent strain was chosen as representative in each location. Dendrograms were prepared using NTSYS‐pc version 2/o2e software, unweighted pair group with arithmetic average method and simple matching similarity coefficient. According to the site of cut‐off line, three groups (clusters) with 82/5% similarity and six groups with 55% similarity were separated based on biochemical and SDS‐PAGE data, and rep‐PCR reactions respectively. Low similarity among groups (55%) can be explained as high genetic diversity among the strains. One strain of west Azerbaijan and the strains of Golestan, clustered in the same group suggesting that they may have been originated from a common source. Other strains of west Azerbaijan were clustered into different groups including II, III, IV, V and VI, suggesting the possibility of occurrence of different populations in a geographical region.     

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.     

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。     

Comparison of pathogenicity of Clavibacter michiganensis subsp. insidiosus and Pseudomonas viridiflava strains on alfalfa

Hamedan province of Iran is a suitable niche for alfalfa growth but many diseases including alfalfa bacterial wilt, bacterial crown and root rot diseases cause economic crop losses. Bacterial wilt is caused by Clavibacter michiganensis subsp. insidiosus, and bacterial crown and root rot diseases are caused by Pseudomonas viridiflava. In this study, we investigated the pathogenicity of C. michiganensis subsp. insidiosus and P. viridiflava strains collected from the main alfalfa growing areas of Hamedan province. Pathogenicity of the virulent strains was tested on alfalfa and the bacterial strains caused symptoms, and data were collected about stem length, root length, wet weight and dry weight of the infected plantlets. The data about the pathogenicity of C. michiganensis subsp. insidiosus and P. viridiflava on alfalfa were compared with each other and were analysed by SAS software and Dunkan's test. Resulted data showed more pathogenicity of C. michiganensis subsp. insidiosus than P. viridiflava on alfalfa. These data also showed that both of these bacteria produced the most losses on wet weight and dry weight of alfalfa plantlets.     

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.     

Fate of Clavibacter michiganensis ssp. sepedonicus, the Causal Organism of Bacterial Ring Rot of Potato, in Weeds and Field Crops

Crops and weeds were tested for their ability to host Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot in potato. Ten crops grown in rotation with potato in Europe, namely maize, wheat, barley, oat, bush bean, broad bean, rape, pea and onion and five cultivars of sugar beet were tested by stem and root inoculation. About 6–8 weeks after inoculation, Cms could be detected in most crops except onion and sugar beet, in larger numbers in stems (10⁵–10⁶ cells/g of tissue) than in roots (≤10³ cells/g of tissue) in immunofluorescence cell‐staining (IF). Cms was successfully re‐isolated only from IF‐positive stem samples of maize, bush bean, broad bean, rape and pea, but not from roots. Twelve solanaceous weeds and 13 other weeds, most commonly found in potato fields in Europe, were tested in IF as hosts of Cms by stem and root inoculations. Only in Solanum rostratum, a weed present in northern America, Cms persisted in high numbers (10⁸ cells/g tissue) in stems and leaves, where it caused symptoms. In the other solanaceous weeds, Cms persisted at low numbers (approximately 10⁵ cells/g of tissue) in stems but less so in roots. The bacteria could be frequently re‐isolated from stem but not from root tissues. In 2 consecutive years, plants from 14 different weed species were collected from Cms‐contaminated potato field plots and tested for the presence of Cms by dilution plating or immunofluorescence colony‐staining (IFC), and by AmpliDet RNA, a nucleic acid‐based amplification method. Cms was detected in roots but not in stems of Elymus repens plants growing through rotten potato tubers, and in some Viola arvensis and Stellaria media plants, where they were detected both in stems and roots, but more frequently by AmpliDet RNA than by IFC.     

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.     

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.     

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.     

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.     

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.     

Effect of Azospirillum-mediated plant growth promotion on the development of bacterial diseases on fresh-market and cherry tomato

AIMS: Plant growth-promoting (PGP) activity of two Azospirillum strains and their effects on foliar and vascular bacterial diseases were evaluated on fresh market and cherry tomato. METHODS AND RESULTS: Tomato seeds were inoculated with A. brasilense Sp7 or Azospirillum sp. BNM-65. Four-week-old plants were challenge-inoculated with Clavibacter michiganensis subsp. michiganensis (bacterial canker) or with Xanthomonas campestris pv. vesicatoria (bacterial spot). Azospirillum-induced PGP was greater on cherry than on fresh-market tomato. Cherry tomato was more resistant to bacterial canker but more susceptible to bacterial spot than the fresh-market tomato. Canker severity was not affected by Azospirillum seed treatments. However, leaf- and plant-death were delayed on Azospirillum-treated plants compared with nontreated controls. Azospirillum increased the bacterial spot severity on cherry but not on fresh-market tomato. CONCLUSIONS: PGP was observed on both tomato genotypes, although growth effects were larger on cherry tomato. Also, Azospirillum treatments may alter tomato susceptibility to bacterial diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: The interaction between PGP rhizobacteria like Azospirillum spp., not known to induce systemic resistance, with plant pathogens distantly located is frequently overlooked. This work demonstrates the importance of this kind of evaluation.     

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.