Aajm. Franken et al., DETECTION OF CLAVIBACTER-MICHIGANENSIS SSP MICHIGANENSIS IN TOMATO SEEDS BY IMMUNOFLUORESCENCE MICROSCOPY AND DILUTION PLATING, Netherlands journal of plant pathology, 99(3), 1993, pp. 125-137
A method for detecting Clavibacter michiganensis ssp. michiganensis in
tomato seeds was evaluated. The method is based on rapid screening of
tomato seed lots using indirect immunofluorescence staining (IF), fol
lowed by dilution plating of IF positive seed lots. Different polyclon
al antisera, prepared against C. michiganensis ssp. michiganensis were
tested for their specificity using IF. All strains of C. michiganensi
s ssp. michiganensis tested reacted with the polyclonal antisera. Two
of nine saprophytic isolates from tomato seeds were positive with the
antisera as well as with the control normal serum, but cells of these
isolates were distinct in shape from cells of C. michiganensis ssp. mi
chiganensis. For extraction of the pathogen from the seed, seeds were
either blended with a stomacher or soaked at 4-6-degrees-C. The stomac
her method yielded more fluorescent cells in IF than 24 h soaking of s
eed samples. However, soaking of seeds for 48 h generally yielded less
saprophytes and overall higher numbers of C. michiganensis ssp. michi
ganensis colonies in dilution plating when compared to blending by a s
tomacher. SCM medium was generally more selective than KBT and modifie
d CNS medium. However, the efficacy of the medium was dependent on the
seed lot and/or extraction method used. Confirmation of suspected col
onies with YDC (yeast-dextrose-carbonate medium), IF and a pathogenici
ty test on tomato seedlings proved to be highly reliable (P > 0.95). F
or routine testing of seed lots it is recommended to screen tomato see
d lots after soaking seeds for 24 h at 4-6-degrees-C with IF, followed
by plating of IF-positive seed lots on modified CNS and SCM after soa
king seeds for an additional 24 h.