FORMATION OF STABLE USTILAGO-HORDEI DIPLOIDS

Ustilago hordei is a fungal pathogen of barley that causes covered smu t disease. During asexual growth, this fungus reproduces as haploid un inucleate budding yeastlike cells (sporidia) that are nonpathogenic. M ating of sporidia, which is controlled by a single mating-type locus w ith two alleles, results in the formation of pathogenic dikaryotic myc elia. When sporidia of the opposite mating type with nonallelic auxotr ophic mutations were plated together on minimal medium, yeastlike colo nies grew at a frequency of 1 x 10(-6). These cultures formed mycelia on charcoal agar, confirming the presence of both mating-type genes. T hese cells had a single nucleus, as determined by fluorescence microsc opy, and protoplasts had a volume approximately twice that of protopla sts derived from haploid cells. Flow cytometry of haploid and suspecte d diploid cells treated with the fluorescent DNA stain propidium iodid e showed that the diploids had roughly twice the DNA content of haploi d cells. Pulsed field gel electrophoresis confirmed the presence in th e putative diploid cells of chromosomes with unique sizes from both pa rental haploid strains. Diploids grown under nonselective conditions f or six to 23 generations remained stable. Diploid strain W-7 was solop athogenic, i.e., capable of infecting the host when used as the sole i noculum. Sporidia isolated from teliospores from barley infected with strain W-7 contained all possible combinations of auxotrophic markers and mating-type genes. Taken together, our results confirm that stable diploids of U. hordei were formed.