Development of a semi-selective medium and an immunofluorescence colony-staining procedure for the detection of Clavibacter michiganensis subsp. sepedonicus in cattle manure slurry

Various compounds and basal media were tested for their suitability to create a semi-selective medium for isolation ofClavibacter michiganensis subsp.sepedonicus (Cms) from cattle manure slurry containing c. 10⁸ colony forming units (cfu) per ml.Plating efficiency of Cms in yeast glucose mineral medium (YGM) was 104% compared with yeast peptone glucose medium. Nalidixic acid, polymyxin B sulphate and the experimental disinfectant S-0208 inhibited colony growth of cattle slurry bacteria as compared with Cms in YGM. The optimal concentration of these inhibitors in combination was determined by modified agar diffusion tests and by pour plating in 24-well tissue culture plates. The semi-selective medium YGMI consisted of YGM supplemented with nalidixic acid (2 mg/l), polymyxin B sulphate (30 mg/l) and S-0208 (125 mg/l). Plating efficiency varied for Cms between 50.9 and 69.6%, for cattle slurry bacteria between 1.8 and 2.5% and for saprophytes from potato heel end extracts between 11.5 and 27.4%.Differentiation of Cms colonies from other colonies was based on their small and bluish colony morphology in pour plates and on immunofluorescence colony-staining (IFC). IFC of a pure culture of micro colonies of Cms in YGM was possible after one day incubation (colonies c. 5 cells). Green background fluorescence in the agar gels was prevented by addition of Tween 20 (0.1%) to the washing buffer and the use of 1% agar gels. IFC of macro colonies of Cms in YGMI, visible with 4x objective magnification, was possible after 4 days. The detection level of the target organism in artificially inoculated cattle slurry in YGMI based on colony morphology varied between 1.4×10³ and 2.3×10⁴ cfu per ml of cattle slurry. Miniaturized plating combined with IFC, using wells in tissue culture plates (=16 mm), proved suitable for detection, but was c. 30 times les sensitive. The recovery of Cms was negatively correlated with the number of saprophytic colonies in the agar plates (R ²=0.74).     

Detection of Erwinia carotovora subsp. atroseptica and Erwinia chrysanthemi in potato tubers using polymerase chain reaction

Soft rot erwiniae are a group of notorious plant pathogens for which currently available detection methods are inadequate. Based on the polymerase chain reaction, specific and sensitive detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in potato tubers has been achieved. The composition of the PCR primers used in two specific detection systems is based on identification of the consensus of sequences of metalloprotease-coding genes present in soft rot erwiniae. Bacterial DNA was extracted from the potato tuber matrix by differential centrifugation in order to avoid interference of potato-derived compounds with the performance of the PCR assay. The PCR assay jjerformed with the E. carotovora subsp. atroseptica specific primer set was found to be capable of distinguishing E. carotovora subsp. atroseptica from all other Erwinia species and the closely related subspecies E. carotovora subsp. carotovora. With the E. chrysanthemi specific primer set, agarose gel electrophoresis is required for unequivocal differentiation between E. chrysanthemi and other erwiniae. Combined with the efficient extraction procedure, the assay allowed specific detection of less than 10³ culturable erwiniae per tuber. The specificity and sensitivity of the assay were not reduced in the presence of a 100-fold excess of DNA from both related and unrelated bacteria. This PCR-based method for detection of erwiniae in potato tubers provides a relatively fast and sensitive alternative to routinely applied serological methods.     

Characterisation of Erwinia carotovora subspecies and detection of Erwinia carotovora subsp. atroseptica in potato plants, soil and water extracts with PCR-based methods

A PCR-RFLP test based on a pectate-lyase encoding gene permits the detection of several Erwinia carotovora subspecies, but requires complete DNA extraction. This paper reports on the suitability of a simplified PCR-RFLP protocol to characterise E. carotovora strains and on the performance of PCR, using the same primers, to detect the atroseptica subspecies in substrates of epidemiological significance. A collection of 140 strains from various hosts and geographical origins was characterised for biochemical traits and PCR-RFLPs. PCR performed on boiled bacterial suspensions yielded an amplification product of 434 bp in 109 of the 140 strains. None of the E. carotovora subsp. betavasculorum strains was amplified, even after complete DNA extraction. RFLPs of the PCR product yielded 24 groups, 3 of which were new. Twenty one groups were specific to one subspecies. Several strains biochemically similar to E. carotovora subsp. atroseptica, but growing at 37 °C, showed PCR-RFLP profiles characteristic of E. carotovora subsp. carotovora. Phenetic and cladistic analyses gave three main domains, not strictly related to hosts or geographical origins. The atroseptica (RFLP groups 1 and 2) and wasabiae (group 21) subspecies constituted one of the domains, despite clustering distantly from one another. Host specialisation and molecular homogeneity suggest a clonal structure within these subspecies. Conversely, E. carotovora subsp. odorifera, despite its limited host range and geographical distribution, and E. carotovora subsp. carotovora showed great molecular diversity, spreading respectively across five and 19 RFLP groups. These two subspecies shared RFLP groups 4, 5 and 6. The tree nodes in the phenograms showed a low robustness when bootstrapping the data matrix. PCR coupled with a 48h enrichment step in a polypectate-rich medium improved detection thresholds of E. carotovora subsp. atroseptica (1.5.10²- 1.5.10³ bacteria/ml in leaves, stems, and tuber peel extracts to 4.10⁷ bacteria/ml in wash water) relative to either immunomagnetic separation coupled with PCR or DAS-ELISA (2.10⁵ in plant samples to 2.10⁷ bacteria/ml in wash water).     

Comparison of PCR, BIO-PCR, DIA, ELISA and isolation on semiselective medium for detection of Xanthomonas albilineans, the causal agent of leaf scald of sugarcane

Polymerase chain reaction (PCR) and newly designed primers, XAF1/XAR1, were tested for selective detection of the causal agent of leaf scald of sugarcane, Xanthomonas albilineans . The efficiency and reliability of PCR were compared with dot immunobinding assay (DIA), ELISA and classical isolation techniques for detecting X. albilineans in suspensions of pure cells and extracts of field-collected stalk and leaf samples of sugarcane. In addition, classical PCR and BIO-PCR (biological amplification followed by PCR) were compared with isolation on a semiselective agar medium. Classical PCR and BIO-PCR techniques had the advantage of not requiring pathogenicity tests to confirm the identity of colonies tentatively identified as X. albilineans on modified semiselective XAM agar medium. The m-XAM medium and BIO-PCR techniques were the most sensitive; however, the former required seven days whereas the latter required only four days. The BIO-PCR technique was as sensitive as the semiselective medium technique and eliminated the need to conduct any additional tests to confirm the identification.