Flutolanil and carboxin resistance in Coprinus cinereus conferred by a mutation in the cytochrome b560 subunit of succinate dehydrogenase complex (Complex II).

A gene that confers resistance to the systemic fungicide flutolanil was isolated from a mutant strain of the basidiomycete Coprinus cinereus. The flutolanil resistance gene was mapped to a chromosome of approximately 3.2 Mb, and a chromosome-specific cosmid library was constructed. Two cosmid clones that were able to transform a wild-type, flutolanil-sensitive, strain of C. cinereus to resistance were isolated from the library. Analysis of a subclone containing the resistance gene revealed the presence of the sdhC gene, which encodes the cytochrome b560 subunit of the succinate dehydrogenase (SDH) complex (Complex II) in the mitochondrial membrane. Comparison between the sdhC gene of a wild-type strain and that of a mutant strain revealed a single point mutation, which results in the replacement of Asn by Lys at position 80. Measurements of succinate-cytochrome c reductase activity in the transformants with mutant sdhC gene(s) suggest that flutolanil resistance of the fungus is caused by a decrease in the affinity of the SDH complex for flutolanil. This sdhC mutation also conferred cross-resistance against another systemic fungicide, carboxin, an anilide that is structurally related to flutolanil. In other organisms carboxin resistance mutations have been found in the genes sdhB and sdhD, but this is the first demonstration that a mutation in sdhC can also confer resistance. The mutant gene cloned in this work can be utilized as a dominant selectable marker in gene manipulation experiments in C. cinereus.

Rapid Discrimination of Mitochondrial DNA Type and Use of Results to Study Mitochondrial Inheritance in Pleurotus spp.

We have reported a simple and rapid method to discriminate species in the genus Pleurotus by analysis of restriction-fragment-length polymorphism of whole-cell DNA, and found that several restriction enzymes gave DNA bands useful in such discrimination, but other enzymes tested did not. In the present study, we report the reason why there were useful and useless enzymes; the effective enzymes digested rDNA into small fragments that did not interfere with the detection of DNA bands useful for discrimination. The origin of these discriminative DNA bands was found to be mitochondrial DNA when the banding profiles of whole-cell DNA, mitochondrial DNA, and nuclear DNA were compared. Consequently, our method could be used for rapid and simple identification of mitochondrial DNA type in the genus Pleurotus. The results were used to study mitochondrial inheritance, and we found that only the nucleus but not the mitochondria migrated during the mating of Pleurotus cornucopiae with P. citrinopileatus.

Flutolanil Resistance as a Genetic Marker of Coprinus cinereus Strains.

All 4 Schizophyllum commune strains among several basidiomycete species were exclusively resistant to flutolanil, which inhibits succinate dehydrogenase complex (SDC) function. Flutolanil resistant strains could be screened from monokaryotic protoplasts of sensitive Coprinus cinereus, by treating them with a cloned genomic SDC iron-sulfur protein (IP) gene from the resistant S. commune. The obtained resistance was stably transferred to the progeny.

Purification and Properties of a New Chitin-binding Antifungal CB-1 from Bacillus licheniformis.

CB-1, a new antifungal, was purified from Bacillus licheniformis. The molecular mass of CB-1 was estimated as 42 kDa by gel filtration column chromatography. CB-1 seemed to be an aggregation product from 4 kinds of peptides. CB-1 contained 10 amino acids and some fatty acids of C14-18:0 and C18:1. CB-1 irreversively bound with chitin powder.