Blackleg potential of potato seed: determination of tuber contamination by Erwinia carotovora subsp. Atroseptica by immunofluorescence colony staining and stock and tuber sampling

Two methods to determine numbers of the blackleg pathogen, Erwinia carotovora subsp. atroseptica, in tuber peel extract were compared; (1) growth and cavity formation on crystal violet pectate (CVP) medium (Perombelon, Lumb and Hyman, 1987) and (2) immunofluorescent colony (IFC) staining (Van Vuurde and Roozen, 1990) using an antiserum against the bacterium conjugated with fluorescein isothiocyanate. Detection, identification and quantification of the bacterium based on the differential effect of temperature on growth in the CVP method were severely restricted and in some cases could not be done at low peel extract dilutions containing > 10(6) saprophytic bacteria ml(-1) and > 10(3) cells ml(-1) of E. carotovora subsp. carotovora. In contrast, although recovery was c. 50% relative to growth of E.c. atroseptica alone on nutrient agar, numbers of the bacteria could be determined by the IFC method regardless of numbers of saprophytic bacteria and E.c. carotovora present. Moreover, the tedium of counting colonies on a UV microscope could be avoided by automation using an imaging system on photograph him negatives of the microscope fields. Readily accessible tubers from the top layer of one tonne boxes in commercial stores were c. 10 times less contaminated than those from the middle of the boxes. For the two methods of peel extract preparation examined, the estimated sample size needed with an allowable error of log(10)10 E.c. atroseptica cells ml(-1) extract with 95% confidence, was c. five tubers per box and 14 boxes for extract prepared from individual tubers and c. three lots of 10 tubers per box and 10 boxes for extract from 10 pooled tubers. A blackleg potential index for seed stocks was proposed based on the summation of the weighted number of individually tested tubers in different classes of contamination level.