Characteristics of immunofluorescence microscopy and of dilution-plating to detect Pseudomonas syringae pv. phaseolicola in bean seed lots and for risk assessment of field incidence of halo blight

Routine laboratory testing of 710 bean seed lots from various origins forPseudomonas syringae pv. phaseolicola (Psp) with immunofluorescence microscopy (IF) showed that 27.5% of the seed lots (five subsamples of 1000 seeds tested per sample) contained two or more IF-positive cells in a total of 500 microscope fields (magnification 500×). Simultaneously performed dilution-platings of IF-positive subsamples on King's medium B confirmed presence of Psp for one-third of these IF-positive seed lots. The grey area of disagreement between both laboratory tests was studied by comparison of test data and by field trials.The number of IF-positive cells per subsample was positively correlated with isolation and identification of Psp (R=0.85). The detection level of IF was ca. 10² Psp cells per ml of undiluted subsample extract. The detection level of Psp by isolation on King's medium B was variable, being inversely related with the saprophyte to Psp ratio. The high sensitivity of IF was in part due to high percentages of dormant or dead IF-positive cells in the sample extract. Field trials over two years with 10 000 seeds per seed lot, showed disease incidence for 9 of the 22 seed lots. Of ten IF-positive lots with five positive subsamples per sample, nine were positive in the field test plot (the negative lot gave primary infection spots of Psp when used for commercial growing). By isolation, seven of these ten IF-positive lots were positive. Of the five IF-positive lots with two or less positive subsamples, isolation and field trial were both negative. Based on data on seed transmission from literature, field incidence was unlikely for these five samples in a 10 000 seeds field trial. All seven IF-negative lots were negative in the field trial. The value of IF and isolation for indexing bean seed lots for Psp is discussed.This study was carried out at the Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Binnenhaven 1, P.O. Box 16, 6700 AA Wageningen the Netherlands, to which address correspondence should be addressed.