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 car otovora subsp. atroseptica, in tuber peel extract were compared; (1) g rowth and cavity formation on crystal violet pectate (CVP) medium (Per ombelon, 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, ident ification and quantification of the bacterium based on the differentia l effect of temperature on growth in the CVP method were severely rest ricted and in some cases could not be done at low peel extract dilutio ns containing > 10(6) saprophytic bacteria ml(-1) and > 10(3) cells ml (-1) of E. carotovora subsp. carotovora. In contrast, although recover y 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 re gardless of numbers of saprophytic bacteria and E.c. carotovora presen t. Moreover, the tedium of counting colonies on a UV microscope could be avoided by automation using an imaging system on photograph him neg atives of the microscope fields. Readily accessible tubers from the to p 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 meth ods of peel extract preparation examined, the estimated sample size ne eded 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 t ubers per box and 10 boxes for extract from 10 pooled tubers. A blackl eg potential index for seed stocks was proposed based on the summation of the weighted number of individually tested tubers in different cla sses of contamination level.