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Single-spore strains of Verticillium albo-atrum from hop, which had previously been classified into three serotypes by immunoelectrophoretic and immunofluorescence methods, have been further characterized using a series of morphological and biochemical parameters. Serotype 1 strains were characterized by high sporulation and low polygalacturonase activity when cultured on hop root cell walls, the presence of certain protein bands on isoelectric focusing of mycelial extracts, and a characteristic dark-pigmented mycelial morphology on prune/lactose/yeast extract agar. It is suggested that the expression of serotype 1 characteristics is controlled by the previously reported hyl+ cytoplasmic factor. Serotypes 2 and 3 were both associated with low sporulation and high polygalacturonase activity and a fully hyaline fluffy mycelium, although many serotype 3 strains were only partially hyaline. Serotype 2 was associated with a characteristic pattern of mycelial proteins on isoelectric focusing gels: serotype 3 strains showed a heterogeneous series of patterns. None of the characters studied could be correlated with virulence for hop but the results do allow the selection for future studies of well-characterized strains which are extremely similar in many respects and yet still differ markedly in virulence.