Rapid Identification of Clavibacter michiganensis Subspecies sepedonicus Using Two Primers Random Amplified Polymorphic DNA (TP-RAPD) Fingerprints

Twenty strains of Clavibacter michiganensis subsp. sepedonicus from different geographic origins and other reference strains of the same and different species, including other potato pathogens, were analysed with a new procedure named TP-RAPD that originates fingerprints of bacterial species. This procedure uses two primers to amplify the 16S rDNA gene. At 45 °C of annealing, the PCR product electrophoresed in agarose gels produced a band pattern that was different in all bacterial species studied as well as in the subspecies of C. michiganensis. All strains of C. michiganensis subsp. sepedonicus displayed the same TP-RAPD number of pattern. Unlike Gram negative bacteria, Gram positives of high G + C content, such as Clavibacter, produced low bands in TP-RAPD. By using a different set of two primers also based in the 16S rDNA sequence from Escherichia coli a more adequate amplification of Gram positives of high G + C including a greater number of bands was obtained. TP-RAPD patterns using the new set of primers described in this work is a reliable and fast method to identify C. michiganensis subsp. sepedonicus.     

Rapid Identification of Clavibacter michiganensis Subspecies Sepedonicus based on the Stable Low Molecular Weight RNA (LMW RNA) Profiles

Clavibacter michiganensis subsp. sepedonicus causes potato ring rot disease. The identification process for this bacterium is complex and long. This work demonstrates that the stable low-molecular-weight (LMW) RNA profiles allow their rapid identification. Staircase electrophoresis in polyacrylamide gels was used to analyze the LMW RNA profiles of 54 strains of C. michiganensis subsp. sepedonicus from different geographic origins. The profiles of several strains of other subspecies of C. michiganensis and other pathogens of potatoes were also analyzed. All the strains of C. michiganensis subsp. sepedonicus had the same LMW RNA profile. They had a band in class 2 of tRNA that was absent in the other subspecies of the species C. michiganensis. Also, the LMW RNA of C. michiganensis subsp. sepedonicus was different with respect to the LMW RNA profiles of other pathogens of potato. The results indicate the possible utilization of LMW RNA profiles in identification of the bacteria causing potato ring rot disease.     

Evaluation of the API 50CH and API ZYM systems for rapid characterization of Clavibacter michiganensis subsp. sepedonicus, causal agent of potato ring rot

API 50CH and API ZYM systems were used to characterize fifty-three strains of Clavibacter michiganensis subsp. sepedonicus from different geographic locations and several reference strains of the same and different species, including other potato pathogens. Clavibacter michiganensis subsp. sepedonicus strains displayed a high level of homogeneity, both in carbohydrate utilization and in enzymatic activity. Using API 50CH and API ZYM it was possible to differentiate C. michiganensis subsp. sepedonicus strains from the remaining taxa analysed in this study, which included representative strains of the other subspecies of C. michiganensis as well as other bacterial pathogens affecting potatoes. Therefore, these systems could be used as an effective method to characterize C. michiganensis subsp. sepedonicus. Such a procedure would constitute an alternative system to the conventional nutritional and physiological identification tests currently included in the official methods employed in the European Union to detect and identify this bacterium. The results obtained with the API systems agreed with the current taxonomic classification of C. michiganensis, clearly separating sepedonicus from the other subspecies belonging to this species.     

In Planta - Complementation of Clavibacter Michiganensis Subsp. sepedonicus Strains Deficient in Cellulase Production or HR Induction Restores Virulence

Clavibacter michiganensis subsp. sepedonicus, a Gram positive bacterium that causes bacterial ring rot of potato, was studied in eggplant, an alternate host, using strains that differed in phenotype. Two factors affecting virulence, the ability to induce a hypersensitive response (HR) and cellulase production, were studied. A plasmid-free isolate of C. michiganensis subsp. sepedonicus that causes HR on tobacco but is unable to produce cellulase multiplied efficiently in planta, but caused only weak symptoms. In contrast, a strain that is unable to induce HR on tobacco but produces cellulase was impaired in the ability to multiply in the host and caused no symptoms. When the two non-virulent strains were coinoculated into eggplants, typical disease symptoms developed. This enhancement was not due to formation of a new phenotype or significant increases in population density of either of the strains. Our results suggest that both cellulase production and the ability to induce HR are required for a successful infection process and disease induction by C. michiganensis subsp. sepedonicus. Our results additionally suggest that the ability to induce HR on non-host plants is required for multiplication in the host plant, whereas cellulase expression is necessary for induction of disease symptoms.     

植物病原棒形杆菌属分类最新进展

植物病原棒形杆菌属Clavibacter的分类随着研究的深入和鉴定技术水平的不断进步一直在发生着变化,之前普遍认可的观点为该属仅含有1个种,即密执安棒形杆菌C. michiganensis,种下又分为5个亚种,分别为密执安亚种C. michiganensis subsp. michiganensis、诡橘亚种C. michiganensis subsp. insidiosus、尼布拉斯加亚种C. michiganensis subsp. nebraskensis、环腐亚种C. michiganensis subsp. sepedonicus和花叶亚种C. michiganensis subsp. tessellarius。最近几年又有4个新亚种陆续被报道,分别为菜豆亚种C. michiganensis subsp. phaseoli、辣椒亚种C. michiganensis subsp. capsici、加利福尼亚亚种C. michiganensis subsp. californiensis和智利亚种C. michiganensis subsp. chilensis。随着全基因组分析和多基因分析技术在细菌分类上的应用,之前被广泛认可的4个亚种(诡橘亚种、尼布拉斯加亚种、环腐亚种和花叶亚种)及新亚种辣椒亚种也曾被建议定义为种,即C. insidiosus、C. nebraskensis、C. sepedonicus、C. tessellarius和C. capsici。为便于研究人员了解该属最新的分类研究现状,本文对植物病原棒形杆菌属的分类历史及最新分类现状进行了系统梳理,期望为该属病原菌的风险评估、检测鉴定等研究提供分类学参考。     

Evaluation of antagonistic bacteria for suppression of bacterial ring rot of potato

Bacterial strains with potential for biological control of bacterial ring rot of potato caused byClavibacter michiganensis subsp.sepedonicus were isolated from the surface of potato tubers. Eighty-eight potential biocontrol candidates, selected on the basis ofin vitro antibiosis toC. m. sepedonicus, produced inhibition zones with radii ranging from 0.5 to 16 mm on test plates. All antagonistic isolates were screened in the greenhouse for biocontrol activity on micropropagated potato plantlets root-inoculated withC. m. sepedonicus. Eight strains consistently prevented infection of plantlets but there was no significant correlation between the width of the inhibition zone in thein vitro assay and ring rot suppression in the plant bioassay. Three strains that showed a high level of biological control potential were identified as a saprophytic enteric bacterium (strain 7G), anArthrobacter sp. (strain 16C), and a soil coryneform bacterium (strain 18A). These were tested in a field plot by co-inoculating cut seed potato tubers withC. m. sepedonicus and antagonists. Strains 7G and 18A significantly increased plant stand whereas 16C decreased disease incidence. The relative number of ostensibly ring rot-free progeny tubers was generally greater when antagonists were present.     

Polymorphism analysis of housekeeping genes for identification and differentiation of <Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> subspecies

The utility of polymorphism analysis was determined for differentiation of the following subspecies of the Gram-positive plant pathogenic bacterium, Clavibacter michiganensis: C. m. subsp. michiganensis, C. m. subsp. sepedonicus, C. m. subsp. insidiosus C. m. subsp. nebraskensis, and C. m. subsp. tessellarius. Specific primers designed for amplification of the housekeeping genes recA, rpoB, and rpoD generated 827-, 1037-, and 862-bp DNA fragments, respectively. PCR products obtained from 40 C. michiganensis strains were analysed using RFLP with four restriction endonucleases, and those PCR products with specific RFLP patterns were sequenced. The genotypes discriminated after PCR–RFLP were specific for each subspecies and also allowed for differentiation of C. m. subsp. michiganensis strains. Sequence analysis of the recA, rpoB, and rpoD gene fragments also distinguished C. michiganensis subspecies and was useful for phylogenetic analysis of all subspecies. For rapid, inexpensive, and effective differentiation of the five subspecies in this research, we recommend the amplification of recA and/or rpoD gene fragments and digestion of the PCR products with the restriction endonuclease FnuDII.     

Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by Multiplex PCR with Coamplification of Host DNA

A new multiplex PCR assay was developed for the detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers. The assay combines two different tests in one reaction mixture. First, a highly specific and sensitive detection of the pathogen and second, an indicator test for successful amplification (internal PCR control), which monitors potentially false-negative PCR results, caused by inhibition of the PCR. For the simultaneous amplification of two different targets in one reaction mixture, a mix of two different primer sets was used. For the detection of C. michiganensis subsp. sepedonicus, a pathogen-specific primer set PSA-1/PSA-R was used, based on the intergenic spacer region of the 16S–23S rRNA genes of C. michiganensis subsp. sepedonicus. For the simultaneous amplification of the internal PCR control, the plant-specific primer set NS-7-F/NS-8-R was employed, permitting amplification of target sequence from plant DNA present in DNA extractions from potato core fluid. The applicability of the multiplex PCR was verified in 3500 composite samples of 200 seed potato tubers from 143 different cultivars in a survey for C. michiganensis subsp. sepedonicus by parallel testing using immunofluorescence, a bioassay in eggplant seedlings and multiplex PCR.     

Validation of four real‐time TaqMan PCRs for the detection of Ralstonia solanacearum and/or Ralstonia pseudosolanacearum and/or Clavibacter michiganensis subsp. sepedonicus in potato tubers using a statistical regression approach

A new DNA extraction method and a new multiplex real‐time TaqMan PCR test for detection of Ralstonia solanacearum, Ralstonia pseudosolanacearum and Clavibacter michiganensis subsp. sepedonicus in asymptomatic potato tubers are presented. This new multiplex PCR and three published TaqMan PCRs for detection of R. solanacearum and/or R. pseudosolanacearum and/or R. syzygii spp. and/or C. michiganensis subsp. sepedonicus were validated using linear regression analysis for estimating the Ct values and its variation at 5 × 10³ bacteria mL^?1. The three published PCRs that have been validated are Massart et al. (2014, detecting R. solanacearum and C. michiganensis subsp. sepedonicus), Weller et al. (1999, detecting R. solanacearum, R. pseudosolanacearum and R. syzygii spp.) and Gudmestad et al. (2009, detecting C. michiganensis subsp. sepedonicus). All tested PCRs were fit for purpose for their target organisms. The PCR tests have different target genes, allowing one of the sets to be used as first screening test and another as second screening test for the detection of R. solanacearum and/or R. pseudosolanacearum and/or C. michiganensis subsp. sepedonicus in asymptomatic potato tubers.     

Analysis of the toxicity of purothionins and hordothionins for plant pathogenic bacteria

Purothionins (PTHs) and hordothionins (HTHs) were purified by cation-exchange chromatography from petroleum-ether extracts of wheat and barley flour respectively. The HTHs could be separated into two fractions, HTH-1 and HTH-2. Radial diffusion assays and micro-plate broth dilution assays with a number of plant pathogenic bacteria showed that these proteins were toxic forClavibacter michiganensis subsp.michiganensis, the causal agent of bacterial canker on tomato,C. m. subsp.sepedonicus, the causal agent of ring rot on potato, andXanthomonas campestris pv.vesicatoria, the causal agent of a spot disease on tomato and pepper. Only minor differences in toxicity between PTHs and HTHs, and between HTH-1 and HTH-2, were detected. Minor differences in toxicity of these thionins were also detected for different strains of these bacteria. The use of these plant proteins for engineering bacterial disease resistance into solanaceous crops will be discussed.     

Euphresco inter‐laboratory comparison (2009–2012) on detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers: proposal to include TaqMan® real‐time PCR as a primary (core) screening test in EU/EPPO standard methods

In the European Union (EU) potato production is surveyed for Clavibacter michiganensis subsp. sepedonicus (potato ring rot) and Ralstonia solanacearum (potato brown rot) under Commission Directives 93/85/EEC with its amendment 2006/56/EC and 98/57/EEC with its amendment 2006/63/EC. A regular update of the Directives is required in view of developments in understanding of the biology of these organisms and the diagnostics recommended for their detection and identification. Three inter‐laboratory tests (ILT1, ILT2 and ILT3) were performed from 2009 to 2012 as part of a Euphresco Phytosanitary ERA‐NET project to assess performance of current official methods for C. michiganensis subsp. sepedonicus and R. solanacearum. A major aim of the ILTs was to generate data on the performance of real‐time PCR protocols to support their introduction as primary (core) screening tests for both pathogens. In ILT1, 29 laboratories from 23 countries participated, in ILT2, 23 laboratories from 18 countries and in ILT3 42 laboratories from 24 countries. Relative accuracies for real‐time PCR tests averaged 92% for R. solanacearum and 96% for C. michiganensis subsp. sepedonicus) and compared with existing primary (core) screening tests (immunofluorescence, conventional PCR, semi‐selective plating and bioassay) in terms of analytical sensitivity, analytical specificity and robustness. It was concluded that all methods tested, including real‐time PCR, can be considered as equivalent. Therefore TaqMan ^® real‐time PCR is recommended for inclusion in EU Directives and EPPO Standards as a reliable primary (core) screening method.     

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.     

Performance of real‐time PCR and immunofluorescence for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing

This paper describes a comparison study of test methods and supports the use of real‐time polymerase chain reaction (PCR) for the detection of Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum in potato tubers in routine testing. These 2 bacteria are quarantine organisms under European Union (EU) regulatory control and testing for (latent) infections of these bacteria in seed potatoes is mandatory. Real‐time PCR tests were performed on 276 routine potato tuber samples, including samples infected with either C. michiganensis subsp. sepedonicus or R. solanacearum, and the performance of these real‐time PCR tests was compared with that of immunofluorescence (IF). Real‐time PCR tests, using different primer sets and extraction and PCR protocols, proved to be sensitive and specific for the detection of C. michiganensis subsp. sepedonicus and R. solanacearum in potato tubers in routine testing, and performed at least as well as IF. Real‐time PCR is a good addition to the detection protocols as laid down in EU regulations (EU Council Directives 2006/56/EC and 2006/63/EC).     

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.     

Evaluation of a diagnostic microarray for the detection of major bacterial pathogens of potato from tuber samples

Potato can be infected with many bacterial pathogens, the detection of which is necessary in seed certification. In this study, a diagnostic microarray previously tested for specificity of probes for detecting the potato bacteria causing blackleg and soft rot (Pectobacterium atrosepticum, Pectobacterium carotovorum, and Dickeya spp.), ring rot (Clavibacter. michiganensis subsp. sepedonicus), scab (Streptomyces scabies and Streptomyces turgidiscabies) and brown rot (Ralstonia solanacearum) from pure culture was evaluated for analytical sensitivity when testing directly from tuber samples. The microarray readily detected all the bacterial species when 100 ng of the target bacterial DNA from pure culture was mixed with DNA from soil microbes and potato. However, detection was inconsistent when total DNA isolated directly from infected tubers or enriched bacterial culture was used. While the high specificity of the probes could be confirmed from the results of the DNA cocktail experiment used as a control, the study demonstrated that the level of analytical sensitivity of the microarray under the tested condition was not sufficient to detect bacteria directly from tubers. Therefore, in addition to the cost and organizational complexities, the low analytical sensitivity and limited reproducibility of the microarray are constraints for establishing the platform for routine detection of potato bacterial pathogens from tuber samples.     

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.     

Antibacterial activity of petroleum ether fraction from Laminaria japonica extracts against Clavibacter michiganensis subsp. sepedonicus

We prepared an ethanol extract from Laminaria japonica, and obtained five fractions from the extract using various solvents. We investigated the antibacterial activities of the fractions against Clavibacter michiganensis subsp. sepedonicus (Spieckermann & Kotthoff) Davis et al., the causal agent of bacterial ring rot of potato (BRR). Among all the fractions, the petroleum ether fraction showed the strongest (P?C. michiganensis subsp. sepedonicus. We analyzed the petroleum ether fraction to detect the components with antibacterial activity, and identified 24 compounds. The main constituents of the petroleum ether extract were esters (35.28 %) and acids (44.84 %). Cell membrane permeability assays, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses revealed that the petroleum ether fraction caused drastic ultrastructural changes in the cells of the pathogen. The petroleum ether fraction caused envelope disruption, cell deformation, the separation of the cell wall from the cell membrane, and the formation of vacuoles. These results clearly indicate that the petroleum ether fraction obtained from L. japonica shows strong antibacterial activity against the causal agent of BRR.     

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.     

rep-PCR-Mediated Genomic Fingerprinting: A Rapid and Effective Method to Identify Clavibacter michiganensis

ABSTRACT The genomic DNA fingerprinting technique known as repetitive-sequence-based polymerase chain reaction (rep-PCR) was evaluated as a tool to differentiate subspecies of Clavibacter michiganensis, with special emphasis on C. michiganensis subsp. michiganensis, the pathogen responsible for bacterial canker of tomato. DNA primers (REP, ERIC, and BOX), corresponding to conserved repetitive element motifs in the genomes of diverse bacterial species, were used to generate genomic fingerprints of C. michiganensis subsp. michiganensis, C. michiganensis subsp. sepedonicus, C. michiganensis subsp. nebraskensis, C. michiganensis subsp. tessellarius, and C. michiganensis subsp. insidiosum. The rep-PCR-generated patterns of DNA fragments observed after agarose gel electrophoresis support the current division of C. michiganensis into five subspecies. In addition, the rep-PCR fingerprints identified at least four types (A, B, C, and D) within C. michiganensis subsp. michiganensis based on limited DNA polymorphisms; the ability to differentiate individual strains may be of potential use in studies on the epidemiology and host-pathogen interactions of this organism. In addition, we have recovered from diseased tomato plants a relatively large number of naturally occurring avirulent C. michiganensis subsp. michiganensis strains with rep-PCR fingerprints identical to those of virulent C. michiganensis subsp. michiganensis strains.     

Risk of dissemination of Clavibacter michiganensis ssp. sepedonicus with potato waste

To estimate the risk of dissemination of Clavibacter michiganensis ssp. sepedonicus through potato residues from processing industries, the various processes and the usage of residues from plants from different processing branches were analysed with regard to the effect they can have on the pathogen. The residues were classified into different risk categories, from category 0 (no risk of dissemination) to category 4 (high risk of dissemination). Residues not heated during processing and used in agriculture, e.g., as fertilizer, were pooled in the highest risk category 4. Residues that were sanitised before use in agriculture, e.g., by composting or pasteurisation, were still classified as probably high risk (risk category 3), as no information on these treatments concerning the inactivation of the pathogen was available so far. Therefore the effect of composting and pasteurisation under varying conditions was tested on samples (ready-made compost mould) contaminated with Clavibacter michiganensis ssp. sepedonicus. Viable bacteria could be extracted after all experiments via bioassay on eggplants, and cultivated on semi-selective media from plant sap forming characteristic colonies. The viable pathogen could be extracted after composting for 6 days at maximum temperatures at 70 °C, 13 days at 55 °C and 90 min pasteurisation at 70 °C. It can be concluded that these sanitation treatments are not sufficient to inactivate Clavibacter michiganensis ssp. sepedonicus and the previous classification of treated residues in category 3 (probably high risk) could thus be confirmed.