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

Abstract

Various compounds and basal media were tested for their suitability to create a semi-selective medium for isolation of Clavibaeter 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 YGM1 consisted of YGM supplemented with nalidixic acid (2 rag/l), polymyxin B sulphate (30 rag/l) and S-0208 (125 rag/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 4 x 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 x 103 and 2.3 x 10 4 cfu per ml of cattle slurry. Miniaturized plating combined with IFC, using wells in tissue culture plates (@ --16 ram), proved suitable for detection, but was c. 30 times less sensitive. The recovery of Cms was negatively correlated with the number of saprophytic colonies in the agar plates (R 2 = 0.74).

Selenium Nanocomposites in Natural Matrices as Potato Recovery Agent

“…Clavibacter sepedonicus ( Cms ) bacterial strain Ac-1405 that causes circular potato rot disease was obtained from the All-Russian Collection of Microorganisms (Pushchino, Moscow Region, Russia). It was grown on medium with glucose, peptone, and yeast extract (GPY) [ 38 ].…”

Selenium nanocomposites as a protection agent from potato ring rot

“…The plants were cultivated for 20 days at 26 °C and 5-6 KLC illumination. A liquid culture for Cms to investigate bacteriostatic activity with selenium nanocomposite against ring rot of potatoes was grown in the dark at 26 °C on a rocker (80 rpm) in flasks containing a GPY nutrient medium, pH 7.2 (Roozen et al, 1991). When considering the effect of NC on the plants we investigated the following biometric indicators: the growth rate of plants, growth of leaves of plants, increase in root mass of plants, the growth of the ground part of the plant, the activity of the enzyme peroxidase in plant tissues, and the content of active forms of oxygen in the tissues of plant roots.…”

Immunofluorescence colony‐staining (IFC) as a concept for bacterial detection in quality testing of plant materials and ecological research 1

“…For samples with a high background ( > 104cfu cm-2) in the plating medium and for target bacteria with a relative slow colony growth such as Clavibacter spp. (Roozen & Van Vuurde, 1991), additional dilutions are needed. This will result in a correspondingly lower detectability of the target.…”