Enrichment of Clavibacter michiganensis subsp michiganensis in tomato seed extracts

An enrichment procedure was developed to improve detection of Clavibacter michiganensis subsp. michiganensis (Cmm) in tomato seed extracts. In the final procedure, seeds were soaked overnight in a buffer at 4(omicron)C and homogenised. Seed extracts were then filtered, supplemented with antibiotics and incubated for three days at 25(omicron)C. In seed extracts spiked with known amounts of Cmm suspensions, a 0- to 10,000-fold increase of Cmm populations was found in the incubation buffer supplemented with but not without the selective compounds, dependent on the seed lot. The procedure also enhanced enrichment of Cmm in seeds artificially infected with low or high numbers of a GFP-tagged strain of Cmm. The presence of the fluorescent bacteria in these internally-infected seeds was confirmed with epifluorescence stereo- and confocal laser scanning microscopy. Eight naturally-infected seed lots stored for three years were used for further evaluation of the procedure. Cmm was detected in seven out of eight lots by TaqMan, although Cmm was only detected by spread-plating in four lots. Enrichment resulted in an increase in Cmm population densities in two of these four seed lots.