A convenient procedure for rapid release of protoplasts from Ustilago maydis.

Filtrates from cultures of Trichoderma harzianum, grown on a liquid mineral salts medium containing vitamin and trace element supplements, 0.5 g/l glucose and 2 g/l of dried residue of ethanol-extracted commercial mushrooms (Agaricus brunnescens), are a good source of enzymes for the release of protoplasts from the sporidia of Ustilago maydis. When concentrated 50-fold by (NH4)2SO4 precipitation, the enzymes liberated 95% of the protoplasts from the sporidia within 40 min.

Mode of action of the azasteroid antibiotic 15-aza-24 methylene-d-homocholesta-8,14-dien-3 beta-ol in Ustilago maydis.

Ustilago maydis sporidia treated with 0.1 mug of azasterol (15-aza-24-methylene-d-homocholesta-8,14-dien-3beta-ol) per ml appeared branched and vacuolated after 6 h of incubation. Sporidial multiplication, dry weight increase, and synthesis of protein, deoxyribonucleic acid, and ribonucleic acid were only slightly or moderately inhibited during the initial 3 h of incubation. An increase of free fatty acids was observed in lipid extracts of treated sporidia after incubation for 3 h or more. Ergosterol synthesis was completely inhibited within 1 h and there was a gradual decline of ergosterol content during 6 h which was accompanied by an accumulation of the sterol intermediate ergosta-8,14-dien-3beta-ol. The results indicate that toxicity of the azasterol results from specific inhibition of the reduction of the sterol C-14(15) double bond. A triarimol-tolerant strain of Cladosporium cucumerinum was tolerant to the azasterol, but an imazalil-tolerant strain of Aspergillus nidulans was not.

Effects of miconazole and dodecylimidazole on sterol biosynthesis in Ustilago maydis.

Miconazole at minimal fungitoxic concentrations inhibits ergosterol biosynthesis in sporidia of Ustilago maydis by interference with sterol C14 demethylation. The action is analogous to that of the fungicides triarimol and fenarimol. The fungicide 1-dodecylimidazole at low concentrations (0.1 to 0.25 mug/ml) inhibits sterol C14 demethylation; however, at higher concentrations (1.0 mug/ml or greater) it also inhibits 2,3-oxidosqualene cyclization and subsequent transmethylation. It is postulated that this diversity of effects of 1-dodecylimidazole results from binding of the inhibitor to sterol carrier protein(s).

Effects of Ancymidol (a Growth Retardant) and Triarimol (a Fungicide) on the Growth, Sterols, and Gibberellins of Phaseolus vulgaris (L.).

The effect of the two substituted pyrimidines, ancymidol (a growth retardant) and triarimol (a fungicide) on Phaseolus vulgaris was studied. Both compounds retarded shoot and root elongation as well as increases in fresh weight. Both compounds caused production of ethylene-like responses when given in high dosages or when applied shortly after germination. As growth retardation was shown to occur in the absence of net increase in sterol levels, neither ancymidol nor triarimol apparently retards growth by inhibiting sterol synthesis.Both ancymidol and triarimol treatment drastically reduced the amount of extractable gibberellin-like activity in beans. Ancymidol also either induced or enhanced the production of a compound which gave a negative response in the bioassay plant Oryza sativa var. Tan-ginbozu. The addition of gibberellin completely relieved the dwarfing effects of both ancymidol and triarimol in dark-grown beans. It is concluded that ancymidol and triarimol affect a gibberellin-induced growth response, probably by inhibiting gibberellin biosynthesis.

Gene mutation eliminating antimycin A-tolerant electron transport in Ustilago maydis.

A class of mutants of Ustilago maydis selected on a fungitoxic oxathiin lack of antimycin A-tolerant respiratory system which is present in wild-type cells. This system provides, directly or indirectly, for considerable resistance to antimycin A because growth of mutant cells lacking the system is much more sensitive to the antibiotic than that of the wild type. Antimycin A-sensitive O(2) uptake and growth is found in half of the progeny from crosses of mutant to wild type. All antimycin A-sensitive segregants are somewhat more resistant to oxathiins than the antimycin A-resistant segregants. The respiration of the mutant is strongly inhibited by cyanide and azide at concentrations which stimulate respiration of the wild type. Respiration of both mutant and wild type is about equally inhibited by rotenone. It appears that the mutation alters some component of the respiratory system located between the rotenone inhibition site and the antimycin A inhibition site that permits shift of electron transport to an alternate terminal oxidase when the normal electron transport pathway is blocked.