ABSTRACT Eight strains of Metschnikowia pulcherrima isolated over a 4-year period from an unmanaged orchard and selected for their biocontrol activity against blue mold (caused by Penicillium expansum) of apples were characterized phenotypically, genetically, and for their biocontrol potential against blue mold on apples. All strains grew well and only differed slightly in their growth in nutrient yeast dextrose broth medium at 1 degrees C after 216 h, but large differences occurred at 0 degrees C, with strain T5-A2 outgrowing other strains by more than 25% transmittance after 360 h. This strain was also one of the most resistant to diphenylamine (DPA), a postharvest antioxidant treatment. All strains required biotin for growth in minimum salt (MS) medium, although strain ST2-A10 grew slightly in MS medium containing riboflavin or folic acid, as did ST3-E1 in MS medium without vitamins. None of the strains produced killer toxins against an indicator strain of Saccharomyces cerevisiae. Analysis of Biolog data from YT plates for all eight strains using the MLCLUST program resulted in separation of the strains into one major cluster containing four strains and four scattered strains from which strain ST1-D10 was the most distant from all other strains. This was particularly apparent in 3-D and principle component analysis. Genetic differentiation of the eight strains using maximum parsimony analysis of nucleotide sequences from domain D1/D2 of nuclear large subunit (26S) ribosomal DNA resulted in detection of two clades. Strain ST1-D10 grouped with the type strain of M. pulcherrima but the remaining seven strains grouped separately, which might possibly represent a new species. All strains significantly reduced blue mold on mature Golden Delicious apples during 1 month of storage at 1 degrees C followed by 7 days at room temperature, but strains T5-A2 and T4-A2 were distinctly more effective under these conditions. Strain T5-A2 also was the most effective in tests on harvest mature apples treated with the lowest concentration of the antagonist and stored for 3 months at 0.5 degrees C. Populations of all eight strains increased in apple wounds by approximately 2 log units after 1 month at 1 degrees C followed by 5 days at 24 degrees C. Our results indicate that M. pulcherrima is an excellent candidate for biological control of postharvest diseases of pome fruit. The variation in phenotypic, genetic, and biocontrol characteristics among strains of M. pulcherrima isolated from the same orchard should make it possible to select antagonists with characteristics that are most desirable for postharvest application.