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Although plasmids or plasmid-like DNAs (plDNAs) have been identified recently from eukaryotes including several filamentous fungi4), only a few examples of extrachromosomal DNA have been described in phytopathogenic fungi5-7). We found mitochondrial plDNAs from three isolates of B. cinerea8). In this paper we report some properties of these plDNAs. Fifteen isolates of B. cinerea used in this study were obtained from field and kept on potato-dextrose agar containing 0.3% yeast extract at room temperature. They were grown in liquid medium containing yeast extracts (0.5%) and dextrose (1%) at 25C. For preparing mitochondrial fraction, mycelial mat was homogenized in 10mM Tris-HCl buffer (pH 7.8) containing 0.5M mannitol, 1mM EDTA, 0.1% bovine serum albumin and 1% 2-mercaptoethanol using a glasshomogenizer. The homogenate was centrifuged twice at 3,000rpm for 5min (Hitachi RPR20 rotor), and the resulting supernatant was recentrifuged at 9,000rpm for 20min. To digest nuclear DNA, the pelleted materials were resuspended in buffer containing 5mM MgCl2 and treated with DNase I (50μg/ ml) for 1hr at 4C. Mitochondria were washed twice with 50mM Tris-HCl buffer (pH 8.0) containing 0.5M sucrose and 10mM EDTA. The final pellets were lysed in 50 mM Tris-HCl buffer (pH 8.0) containing 20mM EDTA, 8% sodium lauroyl sarcosine,

Linear plasmid-like DNAs in the fungus, Botrytis cinerea.