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.     

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.     

Sequence variation in ribosomal DNA and in the nuclear <Emphasis Type="Italic">hsp</Emphasis>90 gene of <Emphasis Type="Italic">Pratylenchus penetrans</Emphasis> (Nematoda: Pratylenchidae) populations and phylogenetic analysis

Tomato bacterial canker and wilt disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is among one of the major bacterial diseases associated with tomato (Solanum lycopersicum L.) in the western Mediterranean region of Turkey. A total of 118 Cmm isolates were obtained from the petiole and the main vein of leaves of different cultivars of diseased tomato plants, and these isolates were cultured in semiselective medium (mSCM). The identity of Cmm isolates was confirmed through gas chromatography-fatty acid methyl-esters (GC-FAME) analysis and polymerase chain reaction (PCR) using the primers, CMM5 and CMM6. The fatty acid analysis of all the Turkish isolates yielded major components that included anteisoheptadeconic acid (a15:0), palmitic acid (i16:0) and anteisoheptadeconic acid (a17:0); the analysis detected and categorized all the isolates into 10 different FAME groups. Among repetitive element sequence PCR (rep-PCR) analysis, Box primer yielded the most reproducible genomic profiles with band sizes that ranged from ~200 bp to 2 kb. The isolates were also separated into 12 groups by pulsed-field gel electrophoresis (PFGE) after digesting the total genomic DNA with SpeI, a rare cutting enzyme. The genome sizes of the different strains of Cmm were also determined after running unrestricted total genomic DNA, which yielded average values between 3.0 and 3.5 MB. All the Cmm isolates had pCM1 and pCM2 plasmids. This is the first report on the detailed characterization of the Cmm population in Turkey.     

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.     

Differentiation of Clavibacter michiganensis subsp. michiganensis from seed-borne saprophytes using ELISA, Biolog and 16S rDNA sequencing

Specificity of a monoclonal antibody (MAb), Cmm1, to geographically diverse strains of the seed-borne tomato pathogen, Clavibacter michiganensis subsp. michiganensis (Cmm), was assessed and the MAb was tested for its usefulness as a tool to separate the pathogen from saprophytes in naturally infested tomato seed. Of the 236 international Cmm strains tested, 99% reacted with MAb Cmm1. MAb Cmm1 was also strongly reactive with an additional 32 strains isolated from seed that were later identified as Cmm by the Biolog MicroLog™ microbial identification system (Biolog, Inc., Hayward, CA) and 16S rDNA sequence analysis. It correctly differentiated these strains from 12 MAb Cmm1-negative seed strains that possessed similar colony morphology but were later identified as other Gram-positive genera and species. The specificity of MAb Cmm1 to the pathogen and the near universality of the MAb Cmm1-reactive antigen among diverse Cmm strains make this antibody a useful detection and identification tool. The finding that a large proportion of the Cmm strains associated with naturally infested tomato seed were putatively hypovirulent or non-virulent indicates that such populations cannot be ignored and points to a need for studies to determine their significance in host-pathogen interactions.     

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.     

Pathogenicity of plant and soil isolates of <Emphasis Type="Italic">Phytophthora parasitica</Emphasis> on tomato and pepper

Crown and root rot of tomato and sweet pepper can be caused by Phytophthora parasitica. In this work, 23 P. parasitica isolates from diseased pepper or tomato plants as well as 54 isolates from 23 monocrop tomato soils (from Spain and Chile) and one from a pepper soil were studied for their host–pathogen response. Results show significant host specificity for the isolates from tomato plants and tomato soils (63 of 64 isolates were unable to cause disease in pepper). None of the pepper plant/soil isolates showed pathogenicity on tomato, and only four of 14 reproduced their pathogenicity on pepper. Only one tomato isolate was pathogenic to both Solanaceae species. Two different inoculation protocols were evaluated (substrate irrigation and stem cutting). All isolates which expressed pathogenicity when stem inoculated also did it when root inoculated, but not vice-versa. Therefore, the recommended test protocol for tomato and pepper breeding programmes is that based on root inoculation by irrigation.     

Effect of phytotoxic compounds produced by Clavibacter michiganensis subsp. michiganensis on resistant and susceptible tomato plants

A phytotoxic fraction of high molecular weight was isolated from the culture filtrate ofClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker of tomato, and partly purified. This high molecular weight fraction consists of sugars and a minor protein moiety and is therefore probably of similar nature to that of the toxin fromC. michiganensis subsp.michiganensis reported earlier in literature.The high molecular weight fraction was albe to induce wilting, the predominant symptom of the disease, as shown in a bioassay with tomato cuttings. However, this wilting reaction turned out to be non-specific in the bioassay, since (partially) resistant and susceptible genotypes responded similarly. No correlation could be found between the degree of virulence of fiveC. michiganensis subsp.michiganensis strains and the amount of the phytotoxic high molecular weight fraction produced in vitro.As the isolated high molecular weight fraction showed a phytotoxic effect on tomato plants it is worthwhile to test its potential for use as a selective agent in in vitro selection.Samenvatting Een fytotoxische fractie werd geïsoleerd uit cultuurfiltraat vanClavibacter michiganensis subsp.michiganensis, de veroorzaker van de bacterieverwelkingsziekte bij tomaat. Een eerste karakterisering toonde aan dat deze toxische fractie hoog-moleculaire component(en) bevat, bestaande uit polysacchariden en een gering percentage eiwit. Dit is in overeenstemming met toxines vanC. michiganensis subsp.michiganensis die al eerder beschreven zijn.Deze hoogmoleculaire toxische fractie was in staat verwelking te induceren van stengeltoppen van verschillendeLycopersicon esculentum enL. peruvianum genotypen in een bioassay. Gewichtsverandering van de stengeltoppen, uitgedrukt als percentage ten opzichte van het begingewicht, werd gebruikt als parameter voor verwelking. De toxische fractie reageerde niet-specifiek in de bioassay, want er werd geen verschil gevonden in respons van (partieel) resistente en gevoelige genotypen. Er bleek geen correlatie te zijn tussen de mate van virulentie van verschillende isolaten vanC. michiganensis subsp.michiganensis en de hoeveelheid van de toxische fractie geproduceerd in vitro.Het mogelijke gebruik van deze hoogmoleculaire toxische fractie als selectief agens bij in vitro selectie zal nader onderzocht worden.     

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.     

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.     

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.     

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.     

Incidence and pathogenicity of races and isolates of Verticillium dahliae in Crete, southern Greece

Classification of 32 Verticillium dahliae isolates originating from 19 plant species in eight different botanical families to races and determination of host range pathogenicity were carried out. The physiological races of isolates were identified using the two differential tomato cultivars ??Belladonna?? (susceptible to both races 1 and 2 of V. dahliae) and ??Ace 55VF?? (resistant to race 1, susceptible to race 2 of V. dahliae). Among these isolates, 14 were race 2 (43.8%), 12 race 1 (37.5%) and six nonpathogenic (18.7%) on tomato. The host range pathogenicity of isolates was determined using four differential hosts (eggplant, turnip, tomato (Ve ^? ) and sweet pepper). Among isolates, five were pathogenic to both eggplant and turnip (15.6%), 21 to eggplant, turnip and tomato (65.6%), five to eggplant, turnip, tomato and sweet pepper (15.6%) and one was pathogenic to eggplant, turnip and sweet pepper (3.2%). The pathogenicity of isolates on the aforementioned five hosts was investigated on the basis of external symptoms and by calculating the relative areas under disease progress curves (relative AUDPC). Results showed that eggplant was the most susceptible, followed by turnip and tomato cv. Belladonna, while sweet pepper and tomato cv. Ace 55VF were less susceptible to all the isolates used. The pathogenicity of isolates varied from highly to mildly virulent on eggplant and turnip while on Belladonna, Ace 55VF and sweet pepper it varied from highly virulent to nonpathogenic. Belladonna exhibited a similar level of susceptibility to races 1 and 2 of V. dahliae, but was more susceptible than Ace 55VF to race 2. Interestingly, the isolates originating from eggplant were clearly more virulent than those originating from tomato and black nightshade on all solanaceous plants tested.     

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.     

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.     

Pathogenicity and RAPD analysis of Phytophthora nicotianae pathogenic to pepper in Tunisia

Nine isolates of Phtophthora nicotianae were isolated from infected pepper plants. Their pathogenicity was studied in Capsicum annuum in comparison with P. nicotianae isolates from tomato and tobacco. The pathogenicity test showed that pepper isolates of P. nicotianae are adapted to their host. Banding patterns obtained by RAPD analysis with six oligonucleotide primers revealed polymorphism that grouped the isolates independently of the plant host. The polygenic dendrogram showed that pepper isolates were more similar to tomato isolates than to tobacco isolates. The RAPD bands of 1300 and 1500 bp, detected with primers OPD-01 and OPD-10, respectively, appeared specific to the most pathogenic pepper isolates. The OPK-08-1950 seems specific to the isolates of P. nicotianae from tomato. These results suggest that host specified might occur in P. nicotianae and that may be due to interspecific hybridization events resulting in novel pathogenic behavior.     

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.     

Inhibitory activities of venom alkaloids of Red Imported Fire Ant against Clavibacter michiganensis subsp. michiganensis in vitro and the application of piperidine alkaloids to manage symptom development of bacterial canker on tomato in the greenhouse

Tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (CMM) is a highly destructive disease that has caused major economic losses in tomato production worldwide. In seeking disease management alternatives, the inhibitory activity of alkaloids extracted from the Red Imported Fire Ant was studied in the laboratory and the greenhouse. Piperidine and piperideine alkaloids each significantly inhibited CMM growth on nutrient agar plates. The inhibitory activity of piperidine alkaloids was stable at 4 ^° C and 22 ^° C for 12 weeks and at 54 ^° C for 4 weeks. The growth of CMM was negatively correlated with the concentration of piperidine alkaloids in nutrient broth. In the greenhouse, piperidine alkaloids also significantly reduced the symptom development on two tomato cultivars, Better Boy and DRK7018F1. This is the first demonstration that piperidine and piperideine alkaloids from the Red Imported Fire Ant are highly inhibitory against a plant-pathogenic bacterium, viz. CMM. Piperidine alkaloids could provide satisfactory management of CMM bacterial canker on tomato seedlings in the greenhouse. Our findings may lead to the development of a new group of bactericides.     

Survival of Clavibacter michiganensis subsp. michiganensis in tomato debris under greenhouse conditions

Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis, is one of the most important diseases of tomato worldwide. Once the pathogen has been introduced into an area, i.e. by contaminated seeds or transplants, it survives mainly on host debris. In different geographic areas the survival time of the pathogen in crop residues under field conditions has been very variable, ranging from 2 months in Morocco to 2 years in Iowa (USA). This study took place in the horticultural belt of Buenos Aires – La Plata, Argentina, where greenhouse production prevails, and monoculture with two production cycles per year is a common practice. The aim was to determine the survival time of this pathogen in plant residues left on the soil surface or buried. During three consecutive years, by the end of both production cycles in July (winter) and December (summer), above‐ (stem, petiole) and belowground (root) tissues were placed into nylon netting bags and left on the soil surface or buried at 10 cm depth. The pathogen population was regularly quantified by dilution plating on semiselective medium. In host debris left on the soil surface, bacteria survived 120–260 days for crop production cycles that ended in winter and 45–75 days for those that ended in summer. In stems or roots buried in winter, this period was 45–75 days. It is concluded that host debris, including roots, might be an important primary inoculum source of the pathogen in greenhouses.     

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.