Comparison of immunofluorescence colony-staining in media,selective isolation on pectate medium,ELISA and immunofluorescence cell staining for detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in cattle manure slurry

Various isolation and serological techniques were compared for the detection ofErwinia carotovora subsp.atroseptica (Eca) andE. chrysanthemi (Ech) in cattle manure slurry containing c. 10⁸ colony forming units (cfu) per ml. The slurry samples could be preserved at –80°C for 8 months without reduction in the number of bacteria but not at –20°C. Samples stored at –80°C were inoculated with concentrations of the target bacterium ranging from 10² to 10⁸ per ml. Only immunofluorescence colony-staining (IFC) in combination with selective media was able to detect the target organism at a concentration of 100 cells per ml. No IFC-positive colonies were found in pour plates of the non-inoculated cattle slurry. The recovery of the target bacterium from slurry inoculated with 10² cfu of Ech per ml was 64% in PT medium (containing polygalacturonic acid) and 19% in crystal violet pectate medium (CVP). Recoveries of Eca were 32% and 82%, respectively. Ech and Eca could be detected at levels of 10³ cfu per ml of slurry by isolation on CVP. Crude filtration procedures were necessary for analysis of slurry samples with immunosorbent immunofluorescence (ISIF) cell staining. The detection level of ISIF for Ech was 10⁵ cells per ml of slurry. IF-positive cells were incidentally observed in the non-inoculated slurry. Detection of Ech and Eca with ELISA was only possible in slurry inoculated with 10⁸ cells of the target bacterium per ml.Samenvatting Verschillende isolatie- en serologische methoden werden vergeleken om de doelbacteriënErwinia carotovora subsp.atroseptica (Eca) enE. chrysanthemi (Ech) aan te tonen in runderdrijfmest met een natuurlijke bacterieflora van ca 10⁸ kolonievormende eenheden(cfu) per ml. De mestmonsters konden gedurende de onderzoekperiode tenminste 8 maanden bij –80°C worden bewaard zonder dat er een afname van het aantal levende mestbacteriën werd geconstateerd, terwijl bij bewaring bij –20°C wel een afname werd gevonden. De ontdooide mestmonsters werden geïnoculeerd met de doelbacterie in concentraties tussen 10² en 10⁸ per ml. De laagste concentratie van de doelbacterie, 10² cfu per ml, kon alleen worden aangetoond met de immunofluorescentie-kleuring van bacteriekolonies (IFC) in een selectief medium. Met deze techniek was het percentage herisolatie vanuit drijfmest geïnoculeerd met 10² Ech cfu per ml respectievelijk 64% in PT-medium (bevat polygalacturonzuur) en 19% in kristalviolet pectine medium (CVP). Voor Eca bedroegen deze percentages respectievelijk 82% en 32%. In de niet-geïnoculeerde mestmonsters werden geen IFC-positieve kolonies gevonden. Via isolatie op CVP konden 10³ of meer cfu van Eca en Ech worden aangetoond. Ruwe filtratie van de mestmonsters was nodig voor het aantonen van Eca- en Ech-cellen met immunoadsorptie immunofluorescentie microscopie. De detectiedrempel lag voor deze techniek op 10⁵ bacteriecellen per ml mestmonster. In niet-geïnoculeerde mest werden incidenteel IF-positieve bacteriën gevonden. Het aantonen van Ech en Eca met ELISA was slechts mogelijk in mest geïnoculeerd met 10⁸ of meer cellen van de doelbacterie per ml.     

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.     

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).     

Development of the simultaneous detection of Ralstonia solanacearum race 3 and Clavibacter michiganensis subsp. sepedonicus in potato tubers by a multiplex real-time PCR assay

Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum (Smith) Yabuuchi et al. race 3 are the causal agents of ring-rot and brown-rot of potato respectively. These diseases represent a serious threat to potato production in temperate climates. Both bacteria are listed as A2 pests in the EPPO region and as zero-tolerance quarantine organisms in the European Union. All the detection tests developed so far were only focused on the detection of a single pathogen while the absence of both bacteria has to be certified in the seed tubers. We have therefore developed a new multiplex real-time PCR assay to simultaneously detect both bacteria in a single assay. Additionally, the reliability of this molecular diagnostic test has been improved by the simultaneous amplification of an internal control, corresponding to a potato gene co-extracted from the sample. The polyvalence and the specificity of each set of bacterial primers and probes were evaluated on more than 90 bacterial strains. The limit of detection of this triplex real-time protocol was similar to those observed with other molecular protocols previously developed for the individual detection of one of these bacteria. A concordance of 100 % was obtained in a blind test mimicking the routine application of the technology. In conclusion, this new protocol represents a straightforward and convenient method potentially adapted to primary screening of potato tubers.     

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.