Characterization of Clavibacter michiganensis subsp. michiganensis strains from recent outbreaks of bacterial wilt and canker in Serbia

Sixty-eight Clavibacter michiganensis subsp. michiganensis (Cmm) strains from recent outbreaks of bacterial wilt and canker in Serbia were collected from several tomato growing regions during a three-year period. The pathogen was identified based on bacteriological characteristics and pathogenicity tests and the identity of strains was confirmed by DAS ELISA and PCR amplification using primers CMM5/6 and PSA4/R. The strains showed homogeneity in biochemical and physiological properties. However, pathogenicity tests revealed differences in virulence that are presumably due to a loss of the pat-1 gene. Further strain characterization using DNA-based methods revealed a high diversity of the Serbian Cmm strains. Based on multi-locus sequence typing (MLST) analyses of five genes, Cmm strains were divided into seven groups. The pulsed-field gel electrophoresis (PFGE) pattern of a selection of strains supported the groupings based on trees of the kdpA/sdhA sequences. On the other hand, groupings made according to PFGE and MLST were not correlated to plasmid content in all cases. This study suggested that high genetic variability of the Serbian Cmm strains was detected both in MLST and PFGE analyses, and could have resulted either from new Cmm strains being introduced by seeds from different origins or as a consequence of an intraspecific hybridization process. In addition, this study proposed MLST as an efficient tool in epidemiological studies, population biology investigations and tracking the routes of transmission of pathogens. Four of the five house-keeping genes (kdpA, sdhA, ligA and gyrB) selected to characterize Cmm strains proved to be suitable for the MLST analysis. This is the first study carried out on the characterization of Cmm using MLST.     

Induction of the viable but nonculturable state in Clavibacter michiganensis subsp. michiganensis and in planta resuscitation of the cells on tomato seedlings

Bacterial canker of tomato is an economically important seedborne disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm). Copper‐based bactericides and seed treatment with hydrochloric acid are commonly used for bacterial canker management. Recent studies have shown that some bacteria can enter a viable but nonculturable (VBNC) state, and fail to form colonies on microbiological agar media. Bacteria in the VBNC state can recover their culturability when returned to favourable conditions. This study reports the induction of the VBNC state in Cmm by CuSO₄ and low pH, and resuscitation of VBNC cells on tomato seedlings. Flow cytometry using the nucleic acid dyes SYTO 9 and propidium iodide, combined with agar plating, was used to assess VBNC cell counts. It was demonstrated that CuSO₄ and low pH induced the VBNC state in Cmm and the rate of induction increased with copper ion concentration and acidity. Pathogenicity tests showed that some of the VBNC cells induced by CuSO₄ retained their ability to colonize tomato seedlings but failed to produce typical bacterial canker symptoms by 2 months post‐inoculation. This was probably due to low levels of resuscitation of VBNC Cmm cells resulting in low levels of initial inoculum. This study has improved understanding of the VBNC state of Gram‐positive phytopathogenic bacteria. Most importantly, because copper‐based chemicals and low pH conditions are used for disease management, induction of the VBNC state and subsequent resuscitation of Cmm cells on tomato seedlings may limit pathogen detection by culture‐based assays yet present a risk for disease development in the field.     

Streptomycin resistance in Clavibacter michiganensis subsp. michiganensis strains from Chile is related to an rpsL gene mutation

Streptomycin has been used for decades in Chile to control Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of tomato bacterial canker. The aim of this work was to evaluate streptomycin resistance and to analyse the presence of resistance-related genes in Cmm strains from Chile. A collection of 25 Cmm strains isolated from different localities in central Chile between 1996 and 2015 was analysed. Minimum inhibitory concentration (MIC) of streptomycin was determined. A search of streptomycin resistance-related genes was carried out in Cmm genomes, and the presence of these genes was studied in all Chilean strains using PCR and sequencing techniques. MIC results showed that four of 25 strains were highly sensitive to streptomycin, with MIC values <2 μg mL⁻¹. The remaining 21 strains possessed MIC of streptomycin ≥100 μg mL⁻¹. The strB gene, encoding an aminoglycoside 6-phosphotransferase that inactivates streptomycin, was detected in all Chilean strains, including sensitive and resistant strains. In the 21 resistant strains, a mutation in codon 43 of the rpsL gene was determined, conferring high streptomycin resistance. Interestingly, the four streptomycin-sensitive Cmm strains did not possess this mutation. This study proposes that the continuous use of streptomycin leads to emergence of resistant Cmm strains, challenging researchers to look for novel alternatives to control this plant pathogenic bacterium.     

Clonal populations of Clavibacter michiganensis subsp. michiganensis are responsible for the outbreaks of bacterial canker in greenhouse tomatoes in Italy

Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected in greenhouses from 17 farms during tomato bacterial canker outbreaks occurring between 2005 and 2008 in Sicily, were analysed by a multiphasic approach. Population studies were conducted to investigate the possible sources of inocula. Cmm strains were characterized by PCR assays targeting virulence genes, fingerprinting techniques, metabolic profiles and virulence. These strains were comparatively analysed with Cmm strains isolated in other parts of Italy over a period of 15 years. Chromosomal genes encoding virulence determinants tomA, ppaA, chpC, and the plasmid‐encoded genes pat‐1 and celA were detected by PCR in all tested strains, except for four Sicilian Cmm strains where the pat‐1 gene was not amplified. Using BOX‐PCR, Cmm strains were differentiated into 13 haplotypes and clonal populations were identified. Cmm strains isolated from different farms in 2008 showed the same BOX‐PCR haplotype. A distinct BOX‐PCR haplotype was obtained from atypical Cmm strains lacking pat‐1 and isolated in 2006/7 from three farms. Cmm strains with two different haplotypes were detected in one farm, whereas the other farms contained strains with only a single haplotype. A new fAFLP protocol based on the amplification of ApaI/MseI fragments was developed and was able to differentiate C. michiganensis subspecies. Different populations were delineated for the multiple outbreaks occurring in Sicily, whereas similar populations were recorded in other Italian regions over a period of 12 years. The results are consistent with previous studies that demonstrate that Cmm outbreaks are associated with propagation material.     

Development of an in vitro protocol to screen Clavibacter michiganensis subsp. michiganensis pathogenicity in different Solanum species

Clavibacter michiganensis subsp. michiganensis (Cmm) is a quarantine organism in Europe and in many other countries. It is one of the most severe bacterial pathogens affecting tomato. Screening tomato plants for their resistance level to Cmm requires a large amount of space under quarantine conditions and is therefore costly. This project developed a new inoculation protocol on in vitro tomato plants to facilitate a more economic and higher throughput disease screening. A new method using the PathoScreen system was tested to localize green fluorescent protein-tagged Cmm in planta and to quantify the pathogen based on the percentage of corrected GFP (cGFP%). The system was sensitive in detecting the GFP-tagged Cmm in the shoots, but in the roots a high autofluorescence masked detection and thus sensitivity of the assay. The in vitro protocol was tested on several wild relatives of tomato, which were previously screened in a greenhouse assay. The correlation between wilt symptoms in vitro and wilt symptoms in the greenhouse was overall moderate (r = 0.6462). The protocol worked well in differentiating the two parents that were used in the mapping studies. This study shows that the in vitro protocol can be efficiently used for resistance breeding in many tomato genotypes.     

Detection of Clavibacter michiganensis ssp. michiganensis in tomato seeds by immunofluorescence microscopy and dilution plating

A method for detectingClavibacter michiganensis ssp.michiganensis in tomato seeds was evaluated. The method is based on rapid screening of tomato seed lots using indirect immunofluorescence staining (IF), followed by dilution plating of IF positive seed lots. Different polyclonal antisera, prepared againstC. michiganensis ssp.michiganensis were tested for their specificity using IF. All strains ofC. michiganensis ssp.michiganensis tested reacted with the polyclonal antisera. Two of nine saprophytic isolates from tomato seeds were positive with the antisera as well as with the control normal serum, but cells of these isolates were distinct in shape from cells ofC. michiganensis ssp.michiganensis.For extraction of the pathogen from the seed, seeds were either blended with a stomacher or soaked at 4–6 °C. The stomacher method yielded more fluorescent cells in IF than 24 h soaking of seed samples. However, soaking of seeds for 48 h generally yielded less saprophytes and overall higher numbers ofC. michiganensis ssp.michiganensis colonies in dilution plating when compared to blending by a stomacher. SCM medium was generally more selective than KBT and modified CNS medium. However, the efficacy of the medium was dependent on the seed lot and/or extraction method used. Confirmation of suspected colonies with YDC (yeast-dextrose-carbonate medium), IF and a pathogenicity test on tomato seedlings proved to be highly reliable (P>0.95). For routine testing of seed lots it is recommended to screen tomato seed lost after soaking seeds for 24 h at 4–6 °C with IF, followed by plating of IF-positive seed lots on modified CNS and SCM after soaking seeds for an additional 24 h.     

Characterization of a Clavibacter michiganensis subsp. michiganensis population in Israel

Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected during the last decade from different locations in Israel, were analyzed by macrorestriction pulsed-field gel electrophoresis (PFGE). Fifty-eight strains from Israel and 18 from other sources were differentiated into 11 haplotypes with either VspI or DraI restriction enzymes. The strains from Israel formed four distinct groups among which groups A (16 strains) and B (32 strains) constituted the major clusters. These two groups originated from the Besor region, which is the main area for growing tomatoes under cover. Rep-PCR, with either ERIC or BOX primers, confirmed results obtained by PFGE. PCR with primers based on three genes – ppaA, chpC and tomA – that spanned the pathogenicity island of the reference strain NCPPB382, produced the expected products with the tested pathogenic strains. Plasmid analysis of representative strains revealed different profiles of one or two plasmids. However all the strains, including five non-pathogenic ones, reacted positively in PCR with primers based on celA gene, which resides on the plasmid pCM1 of NCPPB382. Southern hybridization of total DNA with a 3.2-kb BglII-fragment of pCM1 containing the celA gene was positive when carried out with 31 strains, but the size of the reacting band was not always the same as that of pCM1, suggesting that the plasmids carrying celA may differ in size. Comparison between the colonization rates of strain Cmm42 (group A) and of Cmm32 (group B) did not show any significant differences. The high diversity of the Cmm strains, on the one hand, and the presence of two persistent groups in the Besor region, on the other hand, suggests that the primary inoculum originated each year from residual plants in the soil rather than from infested seeds, in spite of extensive control measures taken by the growers in this area.     

Comparison of extraction procedures and determination of the detection threshold for Clavibacter michiganensis ssp. michiganensis in tomato seeds

Several seed extraction procedures, used for detection of Clavibacter michiganensis ssp. michiganensis ( Cmm ) in naturally infected and artificially infested tomato seed lots were evaluated. Extraction methods that included grinding the seeds were significantly better at detecting the pathogen in three different seed lots than methods that used only soaking. The detection threshold of Cmm in relation to seed sample size was determined by adding naturally infected seeds into samples of three different sizes. Cmm was detected by agar plating assay, on three media (CNS, mSCM, D₂ANX), and by direct PCR from seeds and Bio-PCR (bacteria cultured on agar media prior to PCR). In samples of 10 000 seeds containing one infected seed, Cmm could be detected only by Bio-PCR and in only one replicate out of five. In samples containing five or 10 infected seeds per 10 000 seeds, three of five and five of five replicates, respectively, were detected by the three detection methods. In samples of 5000 seeds, one infected seed could be detected in all five replicates only after adding a concentration step. A high correlation ( R ² = 0·9448) between artificially infested seeds and the disease incidence was found. Seed lots infested with less than 58 colony-forming units (CFU) per g did not cause disease under glasshouse conditions, whereas lots with about 1000 CFU g⁻¹ caused disease in 78 plants out of 2000.     

Studies on the colonization of axenically grown tomato plants by a GFP-tagged strain of Clavibacter michiganensis subsp. michiganensis

In this study, colonization and disease development of axenically-grown tomato plants by Clavibacter michiganensis subsp. michiganensis (Cmm), the causative agent of bacterial wilt and canker, was investigated. For this, a spontaneous rifampicin resistant strain of Cmm was tagged with a marker that expressed a green fluorescent protein (GFP) in a stable way and which possessed a similar virulence to the parental strain. In vitro plants were drop-inoculated at the stem base and the population dynamics was determined by dilution pour-plating in a selective medium. At 3 h after inoculation, Cmm was already present in low densities in roots, stems and leaves. At 16 dpi, Cmm was found throughout the entire plant in high densities of ca. 10¹⁰ cfu g^?1. Symptoms developed in the in vitro plants typical for Cmm, such as canker, wilting and growth reduction. The presence of Cmm in vascular and parenchymatic tissue of in vitro tomato plants was confirmed by epifluorescence stereo- and confocal laser scanning microscopy. This study showed that in vitro tomato plants can be effectively used for detailed studies on interactions between Cmm and its host, in particular if a GFP-tagged strain of the pathogen is used.     

The significance of guttation in the secondary spread of Clavibacter michiganensis subsp. michiganensis in tomato greenhouses

Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), can spread in commercial tomato greenhouses causing epidemics. Results of greenhouse experiments with Cmm‐contaminated tools demonstrated disease spread for only a limited distance (<4 plants) from infected plants. However, touching symptomless infected plants bearing guttation droplets prior to touching nearby plants spread the pathogen over longer distances within rows (>22 plants). The pathogen was exuded in large numbers in the guttation fluid of infected plants; its presence in the guttation fluid was not influenced by the inoculation procedures, leaf age or the volume of the guttation droplets. Population size of Cmm and the incidence of leaflets with epiphytic bacteria were significantly higher in plants placed in a guttation‐induction chamber than in those kept in a growth chamber with high humidity, suggesting exudation through guttation contributed to the formation of epiphytic populations on leaflets. This new knowledge may provide a simple and environmentally friendly means for decreasing the spread of the disease by avoiding contact with plants during periods when they bear guttation droplets.     

Virulence and pCM1 plasmid carriage are related to BOX-PCR fingerprint type in strains of Clavibacter michiganensis subsp. michiganensis that cause bacterial wilt and canker of tomato in Argentina

Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker in tomato, producing important economic losses worldwide. Its virulence has been related to several putative virulence factors present on a chromosomal pathogenicity island and on plasmids pCM1 and pCM2, in strain NCPPB382. We genotypically characterized a collection of Cmm isolates from the main greenhouse tomato-producing areas of Argentina by BOX-PCR fingerprinting and screened for the presence of genes and plasmids involved in pathogenicity by PCR. In addition, we evaluated in vitro cellulolytic activity and virulence in planta of selected strains. BOX-PCR fingerprinting clustered strains into four groups. Group II was dominant and included the most virulent strains, while Group III was the smallest and had the least virulent strains. All local strains exhibited similar cellulolytic activity. Most of the examined strains carry two plasmids of similar size to those of NCPPB382, although there were strains with one or three plasmids. By PCR amplification of repA, pCM1 was detected only in strains belonging to Group III, which includes local strains closely related to reference strain NCPPB382. All analysed pathogenicity genes were widespread among strains, and so in strains belonging to Groups I and II, celA found on pCM1 in NCPPB382 could be found in the chromosome or in plasmids other than pCM1. This study contributes to a better understanding of the diversity of Cmm genetic profiles and virulence of strains present in Argentina. Such information could be useful for the selection of strains for screening of host resistance and development of resistant tomato varieties.     

Characterization of phenotypic variants of Clavibacter michiganensis subsp. michiganensis isolated from Capsicum annuum

Phenotypic variants of Clavibacter michiganensis subsp. michiganensis (Cmm) were isolated from pepper fields and from pepper seeds during quarantine inspections. All strains isolated from pepper (pepper isolates) produced orange-coloured colonies with lower mucoidy than typical Cmm strains isolated from tomato (tomato isolates). However, the results of ELISA, fatty acid analysis, 16S rDNA sequencing, and PCR analysis showed that all pepper isolates were similar enough to be identified as Cmm. In addition to phenotypic variations, the pepper isolates showed different pathogenic and genetic characteristics from tomato isolates from the USA, Europe, or other countries. They could be clearly distinguished in terms of pathogenicity, as they showed increased pathogenicity to pepper but reduced pathogenicity to tomato. Tomato isolates caused strong wilting and canker in tomato, but caused only canker and no wilting in pepper and bell pepper. However, pepper isolates caused no wilting, even in tomato, and only caused canker in the three host plants. In addition, compared to tomato isolates, pepper isolates showed increased colonization efficiency and caused a greater reduction in shoot dry weight in pepper. Pepper and tomato isolates could be separated into two groups according to host origin on the basis of 16S rDNA and ITS sequence analysis. They also showed different rep-PCR genomic fingerprints. All pepper isolates showed higher cellulase activity than tomato isolates on M9CMC plates. However, two plasmid-borne virulence genes of Cmm, pat-1, and celA, were not detected in any pepper isolates by PCR. Furthermore, PCR for pathogenicity-related genes located on a pathogenicity island (PAI) revealed that all tomato isolates were positive for these genes, whereas the pepper isolates did not show any PCR products for the chpC, chpG, ppaA, or tomA genes. Therefore, we suggest that the pepper isolates may represent a separate Cmm population that has evolved within the limits of this host.     

Application of Trichoderma harzianum Strain KABOFT4 for Management of Tomato Bacterial Wilt Under Greenhouse Conditions

Clavibacter michiganensis subsp. michiganensis is a very important pathogen that causes bacterial wilt of tomato (BWT). Biological control of plant diseases is a critical tool for protecting the environment from chemical pollution. Twenty-five isolates of the genus Trichoderma were obtained from a healthy tomato root. Of the 25 isolates, KABOFT4 showed highly antagonistic activity that controlled the growth of C. michiganensis subsp. michiganensis (Cmm7) under in vitro conditions. The 5.8S ribosomal RNA gene and internal transcribed spacer identified the isolate as Trichoderma harzianum KABOFT4. The effect of this isolate as a soil drench and/or foliar application on bacterial wilt under greenhouse conditions was studied. The germination percentage of tomato seed treated with KABOFT4 increased by 36.7% compared to infected seed treated with only the pathogen Cmm7. Under greenhouse conditions, tomato seedlings treated with KABOFT4 as a soil drench, foliar and soil treatment, and foliar treatment had a 61.3, 26.7, and 40% reduced disease severity relative to the infected control, respectively. All treatments had a positive effect on tomato plants that presented as greater vegetative growth and accumulation of dry matter. The best fresh and dry weight was recorded when plants were treated with KABOFT4 as a soil and foliar application. Tomato plants treated with KABOFT4 also had increased total phenol and flavonoid contents in inoculated and non-inoculated plants compared to untreated plants. Under greenhouse conditions, T. harzianum strains can be used as an environmentally friendly way to manage the most economically important tomato disease. The results showed that a native endophytic strain of T. harzianum was a potent biocontrol agent against C. michiganensis subsp. michiganensis. Application of this strain to tomatoes in the greenhouse resulted in a decrease in disease severity and an increase in crop biomass.

     

番茄溃疡病菌PCR快速检测技术

番茄溃疡病是一种严重危害番茄生产的细菌性病害，许多国家将其列为检疫性病害。利用ITS通用引物扩增了番茄溃疡病菌（Clavibacter michiganensis subsp．michiganensis）的ITS序列，并进行克隆测序。根据序列比较结果设计了引物BT1和BT2，该引物特异性好，能专一扩增出268bp电泳条带，而马铃薯环腐病菌等不同亚种、不同属的细菌及健康的番茄材料均无扩增条带。从接种但未显症番茄苗叶片及人工模拟染菌种子上提取总DNA，以此为模板均能稳定地扩增出特异性目的条带。该方法直接对种子或植株进行检测，不需进行病原菌分离培养，快速简便，适用于出入境检验检疫及种苗健康检测领域。     

Internal methods comparison study and inter‐laboratory study on Clavibacter michiganensis subsp. michiganensis in tomato seeds1

Bacterial canker of tomato is a disease caused by Clavibacter michiganensis subsp. michiganensis, a quarantine bacterium, the spread of which has not been completely controlled in spite of the phytosanitary measures taken within the EPPO region. Since 2008 the French National Laboratory for Plant Health (LNPV) has been working on the assessment of the methods used in laboratories to detect the presence of Clavibacter michiganensis subsp. michiganensis in tomato seeds i.e. dilution plating on semi‐selective media and immunofluorescence. In the 1st stage of the assessment, a methods comparison study was performed with reference strains to determine the performance criteria of the tests in optimal conditions. In the 2nd stage, an inter‐laboratory study on naturally and artificially contaminated seeds was performed with 8 laboratories from 6 European countries. This study demonstrated the strengths and weaknesses of the tests currently in use. Two laboratories took the opportunity the collaborative study offered to evaluate alternative tests: BIO‐PCR and IMS‐plating. These could offer interesting alternatives to optimise the detection procedure for Clavibacter michiganensis subsp. michiganensis on tomato seeds.     

Effects of plant age on disease development and virulence of Clavibacter michiganensis subsp. michiganensis on tomato

The effect of plant age at the time of inoculation on the severity of bacterial wilt and canker disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm) was examined in six greenhouse experiments. The period during which inoculations led to wilt and death of tomato plants was defined. This period, designated ‘window of vulnerability’, ranged from transplanting to the 17‐ to 18‐leaf stage. Plants inoculated after this period expressed disease symptoms but did not wilt or die. No significant changes in disease incidence were observed when leaves of different ages were inoculated. Yield accumulation was significantly reduced in plants inoculated within the window of vulnerability compared with those inoculated after this period. Expression of virulence genes, viz. celA, encoding a secreted cellulase, and the serine protease‐encoding pat‐1, chpC and ppaA, was induced during the early stages after inoculation in plants inoculated within the window of vulnerability. Differences in Cmm population between plants inoculated within and outside of this period were insignificant after the first week post‐inoculation, indicating that differences in disease severity, yield loss and expression of virulence determinants are not correlated with Cmm population level. Results suggest that implementation of precautionary measures during the window of vulnerability to avoid secondary spread of Cmm will have a season‐long effect on plant mortality and may minimize, or even prevent, yield losses.     

Clavibacter michiganensis subsp. michiganesis: Tracking strains using their genetic differentiations by ISSR markers in Southern Turkey

There is no available data published related to the dissemination of bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm) and its genetic diversity in Turkey. It is important to control new introduced inoculum sources by commercial seeds and seedlings. Pathogens were identified by morphological features and the identity was confirmed by PCR amplification using a specific primer PSA-4, PSA-R in addition to microbiological tests. ISSR markers showing high polymorphism ratios were selected and used to characterize Cmm strains. The collected strains were classified into different groups on the basis of ISSR-PCR fingerprints, which showed remarkable genetic specificity and diversity not previously identified in Cmm, suggesting that genetic differences are related to dissemination of the pathogen in the region. This is the first ever study carried out on the characterization of Cmm using ISSR. The selected ISSR primers to characterize Cmm can be used to determine genetic differences in further studies.     

Enhanced tomato resistance to bacterial canker by application of turtle oil

Pretreatment with oil of sea turtle Caretta caretta protected tomato plants against bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm). The turtle oil was ineffective in inhibiting Cmm in an agar diffusion test, suggesting a mechanism of induced resistance. Under controlled conditions in the greenhouse, turtle oil lowered the disease index and had reduced the growth of bacteria up to 50.4% by 7 days after inoculation. Applying turtle oil to the foliage of tomato plants increased per-oxidase and lipoxygenase activities. Gas chromatography analyses of turtle oil indicated the presence of polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. These fatty acids may lead to the higher activity of the enzymes and probably induced plant resistance against the pathogen.     

Temperature at the early stages of Clavibacter michiganensis subsp. michiganensis infection affects bacterial canker development and virulence gene expression

Clavibacter michiganensis subsp. michiganensis (Cmm), the causal agent of bacterial canker and wilt, causes severe economic losses in tomato net‐houses and greenhouses worldwide. In this study, seedlings which were transplanted and inoculated monthly over 2 years wilted and died earlier in the spring (21–24°C) and autumn (18–23°C) than in the winter (15–18°C) and summer (28–31°C): T₅₀ (the time taken for 50% of the plants to wilt or die) was 2 and 3–4 months after inoculation, respectively. A highly significant correlation was found between the average temperatures during the first month after inoculation and T₅₀; the shortest T₅₀ mortality (70 days) was observed for an average temperature of 26°C. Expression of virulence genes (pat‐1, celA, chpC and ppaA) by Cmm was higher in plants inoculated in the spring than in those inoculated in the summer. In another set of experiments, seedlings were inoculated and maintained in controlled‐environment growth chambers for 2 weeks. Subsequently, they were transplanted and maintained in commercial‐type greenhouses for 4–5 months. The temperatures prevailing in the first 48 h after inoculation were found to affect Cmm population size and virulence gene expression and to have season‐long effects on bacterial canker development.     

Pathogenicity and phylogenetic analysis of Clavibacter michiganensis strains associated with tomato plants in Iran

During 2013–2016, 277 tomato fields were surveyed across Iran to monitor the status of bacterial canker of tomato, caused by Clavibacter michiganensis subsp. michiganensis. Altogether, 450 plant samples were collected, both with and without symptoms, from which 35 bacterial strains were recovered. These were positive for the PCR test performed using the Clavibacter‐specific primer pair CMR16F1/CMR16R1. Based on the phylogeny of the gyrB gene sequences, 31, three and one of the 35 strains were identified as C. michiganensis, Microbacterium sp. and Agrococcus sp., respectively. The 31 strains of C. michiganensis were further identified as C. michiganensis subsp. michiganensis (23 strains), C. michiganensis subsp. tessellarius (six strains) and Clavibacter spp. (two strains). This was subsequently confirmed by multilocus sequence analysis (MLSA) of five housekeeping genes (atpD, gyrB, ppk, recA and rpoB). In pathogenicity tests, all 23 strains induced wilting symptoms on tomato plants in greenhouse conditions, while no symptoms were observed on eggplant, bell pepper and chili pepper plants. All evaluated pathogenicity determinant genes (celA, pat‐1, tomA, ppaA, chpC and chpG) were detected in 18 out of 31 C. michiganensis strains, using eight specific primer pairs. Estimation of the number of nucleotide differences, sequence similarity matrix and MLSA clustered two peach‐coloured strains (Tom495 and Tom532) separately from all nine previously described subspecies, thereby suggesting these two strains are a new subspecies of C. michiganensis. However, a detailed taxonomic study using multiphased molecular approaches is needed to delineate a formal taxonomic name for these atypical strains.