We investigated the mechanism of resistance to demethylation inhibitors (DMI) in Penicillium digitatum by isolating the CYP51 gene, which encodes the target enzyme (P450 _14DM ) of DMI, from three DMI-resistant and three DMI-sensitive strains. The structural genes of all six strains were identical, but in the promoter region, a unique 126-bp sequence was tandemly repeated five times in the DMI-resistant strains and was present only once in the DMI-sensitive strains. Constitutive expression of CYP51 in the resistant strains was about 100-fold higher than that in the sensitive strains. We introduced CYP51 , including the promoter region, from a DMI-resistant strain into a DMI-sensitive strain, which rendered the transformants DMI resistant and increased CYP51 expression. We also found that if the number of copies of the repeat was reduced to two, resistance and CYP51 expression also decreased. These results indicate that the 126-bp unit acts as a transcriptional enhancer and that a tandem repeat of the unit enhances CYP51 expression, resulting in DMI resistance. This is a new fungicide resistance mechanism for filamentous fungi.

The injection of bacteria (Staphylococcus aureus,Stenotrophomonas maltophilia) or true fungi (Candida albicans,Candida tropicalis) that are pathogenic to humans into the silkworm hemolymph leads to death of the larvae within 2 days. Antibiotics used for clinical purposes have therapeutic effects on silkworms infected with these pathogens. The 50% effective doses obtained by injection into the silkworm hemolymph are consistent with those reported for mice. Injection of vancomycin and kanamycin into the silkworm hemolymph was effective, but oral administration was not. Chloramphenicol, which is effective by oral administration, appeared in the silkworm hemolymph soon after injection into the midgut, whereas vancomycin did not. Isolated midgut membranes were impermeable to vancomycin. Thus, the ineffectiveness of oral administration of vancomycin to silkworms is due to a lack of intestinal absorption.