Analogy in the mode of action of fluotrimazole and clotrimazole in Ustilago avenae

Fluotrimazole [BUE 0620; 1-(3-trifluoromethyltriphenyl) 1,2,4-triazole] (20 μg/ml of nutrient solution) and clotrimazole [Bay b 5097; bisphenyl(2-chlorophenyl)-1-imidazolyl methane] (5 μg/ml) did not inhibit dry weight increase and only slightly reduced multiplication of sporidia of Ustilago avenae during the first doubling period (about 4 hr). After 8 hr, both fluotrimazole and clotrimazole more strongly inhibited sporidia multiplication than dry weight increase. As a consequence of treatment with both fungicides the usually single-celled sporidia appear swollen, multicellular, and branched. Both chemicals at a concentration range of 5–100 μg/ml did not affect oxidation of glucose. The effect of fluotrimazole and clotrimazole on protein, DNA, and RNA synthesis was similar to that on dry weight. Following a 6-hr incubation period total lipid synthesis was quantitatively unaffected by both chemicals. As the analysis of major fatty acids of total lipids revealed fluotrimazole substantially induced the synthesis of 20:4 carbon fatty acids, while in clotrimazole-treated sporidia the pattern of fatty acids did not differ from that of control sporidia. Fluotrimazole and clotrimazole produced a higher quantity of free fatty acids in sporidia of U. avenae. Gas-liquid chromatographic analysis of sterol fractions in treated and control sporidia (6 hr) indicated that both fluotrimazole and clotrimazole seriously inhibited ergosterol biosynthesis and concomitantly caused an accumulation of immediate ergosterol precursors which represent C-4-methyl and 4,4-dimethyl sterols. Incorporation of [¹⁴C]acetate for 2 hr into various lipid fractions of sporidia of U. avenae also revealed that radioactivity in C-4-desmethyl sterols in both fluotrimazole- and clotrimazole-treated sporidia was drastically reduced, while the radioactivity of C-4-methyl and 4,4-dimethyl sterols distinctly increased. The data suggest that fluotrimazole and clotrimazole are specific inhibitors of the oxidative demethylation of the C-14-methyl group during ergosterol biosynthesis in U. avenae.     

Inhibition of ergosterol biosynthesis by triadimenol in Ustilago avenae

In Ustilago avenae sporidia, following the first doubling period of about 4 h, triadimenol (2 μg ml^?1) affected sporidial multiplication more severely than other growth processes; daughter cells failed to separate from the parent sporidia resulting in chains of interconnected cells. Triadimenol incubated with the fungus for 8 h interfered neither with respiration nor with protein and nucleic acid synthesis but after 6 h the toxicant had induced a higher content of free fatty acids. Triadimenol markedly altered, both quantitatively and qualitatively, the sterols in sporidia of U. avenae. Incorporation of [¹⁴C]acetate (in the form of sodium acetate) into lipid fractions for a period of 2 h revealed that the toxicant powerfully inhibited the synthesis of the 4-demethyl sterol fraction (predominantly ergosterol), whilst the 4,4-dimethyl sterol fraction rapidly accumulated. This was confirmed by g.1.c. analysis of the sterols after 6 and 8 h incubation which showed that the amount of ergosterol, the major sterol in untreated sporidia, was diminished while simultaneously 4,4-dimethyl, 4-methyl and 14-methyl sterols increased. The accumulation of 14-methyl sterols suggests that triadimenol acts as a potent inhibitor of one of the metabolic steps involved in the demethylation at the 14-position during ergosterol biosynthesis.     

On the antifungal mode of action of tridemorph

Tridemorph (2,6-dimethyl-N-tridecylmorpholine) was active against representative of nearly all taxonomic groups of fungi; gram-positive bacteria were also sensitive although gram-negative were not. Tridemorph, 3–10 μg/ml, inhibited the multiplication of sporidia of Ustilago maydis more strongly than the increase of dry weight. The treated sporidia appeared swollen, multicellular, and sometimes branched. Unsaturated lipophilic compounds like α-tocopherol and trilinolein alleviated the toxicity of tridemorph to Botrytis allii and U. maydis. Protein and RNA syntheses were inhibited slightly. DNA synthesis was rather strongly affected already after 2 hr. Lipid synthesis was first inhibited but later stimulated. At an early stage (2 hr) treated cells differed already from control cells by a higher content of free fatty acids. Tridemorph also inhibited sterol biosynthesis. The antimicrobial spectrum, the characteristic morphology of treated cells of U. maydis, the observations on cross-resistance, the alleviating effect of unsaturated lipophilic compounds, and the alterations in neutral lipid pattern suggest strong similarity of the mode of action of tridemorph with that of the known inhibitors of sterol biosynthesis.     

Inhibition of ergosterol biosynthesis in Ustilago maydis by the fungicide 1-[2-(2, 4-dichlorophenyl)-4-ethyl-1, 3-dioxolan-2-ylmethyl]-1H-1, 2, 4-triazole

Inhibition of sporidial multiplication in cultures of Ustilago maydis by 1-[2-(2, 4-dichlorophenyl)-4-ethyl-1, 3-dioxolan-2-ylmethyl]-1H-1, 2, 4-triazole^a (CGA-64251), at concentrations of 0.1, 1.0 and 5.0 μg ml^?1, increased from about 15% during the first 4 h, to 58–70% during the subsequent 4 to 12-h period. Sporidia became swollen and highly branched in the presence of the fungicide. Total lipid content as a percentage of the dry weight was not affected after exposure of the sporidia to the fungicide at 0.1 or 5 μg ml^?1 for 4 h, but synthesis of ergosterol and other demethyl-sterols was inhibited by 87–92%. Large quantities of methyl-sterol precursors of ergosterol and of free fatty acids accumulated in the treated sporidia. Fungitoxicity of CGA-64251 is attributed to inhibition of ergosterol biosynthesis at the stage of sterol C-14 demethylation.     

A mutant of Ustilago maydis deficient in sterol C-14 demethylation: Characteristics and sensitivity to inhibitors of ergosterol biosynthesis

An ergosterol-deficient mutant of Ustilago maydis was compared to the wild type in regard to morphology, growth rate, lipid content, and sensitivity to ergosterol biosynthetic inhibitors. Morphology of mutant sporidia is abnormal and resembles that of fenarimol-treated wild-type sporidia. Doubling time of mutant sporidia is 6.3 hr compared to 2.5 hr for the wild type. The mutant produces 24-methylenedihydrolanosterol, obtusifoliol, and 14α-methylfecosterol; ergosterol is absent. The sterols of the mutant are the same as those which accumulate in wild-type sporidia treated with the sterol C-14 demethylation inhibitors fenarimol, etaconazole, and miconazole. The level of free fatty acids is higher in the mutant than in wild-type cells. Growth of mutant sporidia is not inhibited by fenarimol, etaconazole, and miconazole, or by the sterol Δ¹⁴-reductase inhibitor azasterol A25822B at low concentrations which inhibit growth of wild-type sporidia. The residual growth rate of wild-type sporidia treated with low concentrations of the sterol C-14 demethylation inhibitors is about the same as that of untreated mutant sporidia. Therefore, the mutant would not be recognized as resistant in a wild-type population. The mutant is deficient in sterol C-14 demethylation and is similar in all properties studied to wild-type sporidia treated with sterol C-14 demethylation inhibitors. These findings support the contention that inhibition of sterol C-14 demethylation in U. maydis is the primary mode of toxicity of fenarimol, etaconazole, and miconazole. A secondary mode of toxicity is evident for miconazole and etaconazole at higher concentrations but is doubtful for fenarimol.     

Mode of action of triarimol in Ustilago maydis

Triarimol (2 μg/ml) strongly inhibited multiplication of Ustilago maydis sporidia after one doubling, but growth continued and sporidia became abnormally large, branched and multicellular. Oxidation of glucose or acetate was not affected, and only slight limitations occurred in DNA, RNA and protein syntheses. The toxicant did not inhibit triglyceride synthesis but markedly increased the quantity and altered the quality of free fatty acids. Incorporation of [¹⁴C]acetate into ergosterol and an unidentified sterol was inhibited more than 90%, but incorporation into two other unidentified sterols was almost unaffected. Inhibition in the sterol biosynthetic pathway at a point preceeding ergosterol is regarded as a primary site of triarimol action in U. maydis.     

Ultrastructure and sterol composition of laboratory strains of Ustilago avenae resistant to triazole fungicides

The fine structure and sterol composition of wild-type and triazole-resistant laboratory strains of Ustilago avenae was investigated by electron microscopic and biochemical methods. The growth rate of the mutants was only slightly affected by a fungicide (triadimefon) concentration of about 0.1 mg/ml, whereas the wild-type cells were completely inhibited. Biochemically the sterol composition of wild-type and triazole-resistant strains did not differ. In freeze-fracture electron microscopy no ultrastructural differences were observed between the different untreated strains (wild and resistant). Filipin labeling allowed the localization of ergosterol in the plasmalemma (PF and EF). Generally, wild-type samples and mutants exhibited a clear pattern of filipin-sterol (FS-) complexes. These results are in accord with the biochemical experiments. Neither a modification of the sterol composition nor an altered localization of sterols seemed to be the prime cause of resistance in U. avenae mutants. Alternative explanations for the resistance mechanism are discussed.     

Specific effects of tridemorph on sterol biosynthesis in Ustilago maydis

In an attempt to indicate the site of action of tridemorph in ergosterol biosynthesis by Ustilago maydis the nature of the sterol intermediates accumulating in treated cells was studied. At low growth-inhibiting concentrations of the toxicant (10 and 15 μg/ml) decline of ergosterol content during 6 hr of incubation was accompanied by an accumulation of various sterol intermediates of which ergosta-8,14-dien-3β-ol appeared to be the major sterol. After isolation of this intermediate its identity was further confirmed by direct comparisonof its ultraviolet, glc, and mass spectrum with those of an authentic synthesized sample of ergosta-8,14-dien-3β-ol (ignosterol). Results indicate that toxicity of tridemorph is caused by a specific inhibitive effect on the enzyme Δ¹⁴-reductase which is responsible for $\text{14}\text{15}$ double-bond reduction in sterol biosynthesis.     

Effects of Sterol Biosynthesis Inhibitor Fungicides on Growth and Sterol Composition of Ustilago maydis,Botrytis cinerea and Pyrenophora teres

The effects of the sterol biosynthesis inhibitor (SBI) fungicides fenarimol, fenpropimorph, imazalil, prochloraz, propiconazole and triadimenol on growth and sterol composition of Ustilago maydis, Botrytis cinerea and Pyrenophora teres, grown from spores or sporidia in liquid culture, were determined. Growth of U. maydis was only slightly inhibited by SBI fungicides at concentrations which caused considerable changes in both sterol content and composition. Conversely, in B. cinerea and P. teres, growth was strongly inhibited under conditions where ergosterol was still the predominant sterol, suggesting that, in these two fungi, growth may be more sensitive to SBI fungicides than overall sterol production. Demethylase inhibitor fungicides behaved as a homogeneous group in their effects on growth and on sterol profiles of the three fungi studied.     

Mode of action of the fungicide flusilazole in ustilago maydis

Flusilazole is a potent inhibitor of Ustilago maydis sporidial growth (I₅₀= 20 μg liter⁻¹). Incorporation of [¹⁴C]acetate into ergosterol of growing sporidia is inhibited 50% by 0.5 μg liter⁻¹of the fungicide. Inhibition of ergosterol biosynthesis is concomitant with the accumulation of the precursors eburicol, obtusifoliol and 14α-methylfecosterol. A novel cell-free assay has been developed to measure the 14α-demethylation of [³H]dihydrolanosterol. Flusilazole inhibits the cell-free demethylation with an I₅₀of 15 μg liter⁻¹. These data provide strong evidence that the mode of action of flusilazole is by inhibiting ergosterol biosynthesis through direct inhibition of the 14α-demethylation of ergosterol precursors.     

Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi

The fungicides miconazole, fenarimol, and etaconazole block ergosterol biosynthesis in fungi by inhibiting sterol 14α-demethylation, which is mediated by a cytochrome P-450 enzyme. The sensitivity of cytochrome P-450-dependent hydroxylation or demethylation of several substrates to these fungicides and similar compounds was compared to that of fungal growth and sterol 14α-demethylation. Demethylation of p-chloro-N-methylaniline (PCMA) by sporidia of Ustilago maydis and 11α-hydroxylation of progesterone by Aspergillus nidulans were relatively insensitive to these compounds and to metyrapone. The ability of a sterol 14α-demethylation-deficient mutant to demethylate PCMA indicates that this substrate is not demethylated by the sterol 14α-demethylation system of U. maydis. The 14α-hydroxylation of progesterone by cells of Curvularia lunata was quite sensitive to the three fungicides, and also to metyrapone and isopropylphenylimidazole. This system was less sensitive to the three fungicides than sterol 14α-demethylation, but was appreciably more sensitive than PCMA demethylation. A study of progesterone 14α-hydroxylation in cell-free preparations of C. lunata showed the reaction to be inhibited by CO, and to be competitively inhibited by low concentrations of miconazole. These data suggest that the primary action of sterol biosynthesis-inhibiting (SBI) fungicides is competitive inhibition of sterol/steroid-type cytochrome P-450 enzymes rather than interference with the function of sterol carrier proteins or enzyme-modulating phospholipids.     

Effect of the fungicide pyrifenox on sterol biosynthesis in Ustilago maydis

Pyrifenox, a new pyridine derivative, proved to be an inhibitor of ergosterol biosynthesis, blocking the pathway at the C-14 demethylation step in Ustilago maydis (CD.) Cor da. In treated sporidia the incorporation of [1-¹⁴C]acetic acid into ergosterol and squalene was reduced and the incorporation into sterols which retain the C-14 methyl group, mainly 24-methylenedihydrolanosterol and obtusifoliol, was increased. In addition, treatment with pyrifenox markedly reduced the incorporation into sterol esters. It is possible that the methylated sterols may be unsuitable substrates for the esterification enzyme.     

Antifungal mode of action of triforine

Rapidly growing mycelia of Aspergillus fumigatus treated with 10 μg/ml triforine (N,N′-bis-(1-formamido-2,2,2-trichloroethyl)-piperazine) showed little or no inhibition in dry weight increase prior to 2 h. By 2.5–3 h, triforine inhibited dry weight increase by 85%. The effects of triforine on protein, DNA, and RNA syntheses corresponded to the effect on dry weight increase both in time of onset and magnitude. Neither glucose nor acetate oxidation were inhibited by triforine.Ergosterol synthesis was almost completely inhibited by triforine even in the first hour after treatment. Inhibition of ergosterol synthesis was accompanied by an accumulation of the ergosterol precursors 24-methylenedihydrolanosterol, obtusifoliol, and 14α-methyl-Δ^{8, 24 (28)}-ergostadienol. Mycelia treated with 5 μg/ml of triarimol (α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidinemethanol) also accumulated the same sterols as well as a fourth sterol believed to be Δ^{5, 7}-ergostadienol.Identification of 4,4-dimethyl-Δ^{8, 24 (28)}-ergostadienol in untreated mycelia indicates that the C-14 methyl group is the first methyl group removed in the biosynthesis of ergosterol by A. fumigatus. The lack of detectable quantities of 4,4-dimethyl-Δ^{8, 24 (28)}-ergostadienol in triforine or triarimol-treated mycelia and the accumulation of C-14 methylated sterols in treated mycelia suggests that both fungicides inhibit sterol C-14 demethylation. The accumulation of Δ^{5, 7}-ergostadienol in triarimol-treated mycelia further implies that triarimol also inhibits the introduction of the sterol C-22(23) double bond.Two strains of Cladosporium cucumerinum tolerant to triforine and triarimol were also tolerant to the fungicide S-1358 (N-3-pyridyl-S-n-butyl-S′-p-t-butylbenzyl imidodithiocarbonate).     

Freeze-fracture electron microscopy of the plasmalemma of Ustilago avenae treated with systemic fungicides: A quantitative analysis of the intramembrane particles

The interaction of the systemic fungicides triadimefon, nuarimol, and imazalil-nitrate with the plasmalemma of Ustilago avenae was studied using freeze-fracture electron microscopy. These antifungal agents produced alterations in the plasmalemma, such as hemispherical pits and protrusions, deformations of invaginations, and formation of craters or similar structures on the membrane fracture faces. The ultrastructural changes in the plasmalemma were more pronounced after triadimefon and nuarimol treatment than after imazalil-nitrate treatment. Intramembrane particles (IMPs) were aggregated, resulting in particle-free areas which showed some exocytotic activity. A quantitative analysis was performed to determine the topographical distribution of IMPs on the extraplasmatic and plasmatic fracture faces of the plasmalemma. The evaluation revealed statistically significant differences in the membrane structure of untreated and fungicide-treated sporidia.     

Fungitoxicity and growth regulation involving aspects of lipid biosynthesis

Triarimol and triforine inhibit ergosterol biosynthesis in fungi and cause accumulation of free fatty acids, 24-methylenedihydrolanosterol, obtusifoliol and 14α-methyl-δ^8,24(28)-ergostadienol. Triparanol also inhibits ergosterol synthesis and causes accumulation of free fatty acids, but not of the latter 3 sterols. Triparanol appears to inhibit prior to lanosterol in the sterol biosynthetic pathway of Ustilago maydis and at unidentified sites subsequent to lanosterol which lead to the accumulation of a sterol which migrates with desmethylsterols on TLC plates. Quantitative abnormalities in sterols and free fatty acids in U. maydis are not produced by the fungicides carbendazim, chloroneb, carboxin and cycloheximide. A deficiency in nitrogen leads to a marked increase in triglycerides, but a normal distribution pattern for other lipids.Inhibition of oxidative demethylation of the sterol 14α-methyl group is probably the prime mechanism of inhibition of ergosterol biosynthesis by triarimol. Rates of formation of obtusifoliol and 14α-methyl-δ^8,24(28)-ergostadienol in triarimol-treated U. maydis cells suggest that C-4 demethylation occurs along an abnormal pathway which operates effectively only at high substrate concentrations. The growth retardant action of triarimol and ancymidol in higher plants most likely results from inhibition of a reaction in the gibberellin biosynthetic pathway analogous to sterol C-14 demethylation.Free fatty acid accumulation in U. maydis cells treated with inhibitors of sterol synthesis are derived mainly from polar lipid degradation and from de novo synthesis as a consequence of the disproportionality between fatty acid synthesis and utilization. The free fatty acids may play a significant role in the lethality of these inhibitors in this organism.     

Hexagonal clustering of intramembrane particles in the plasmalemma of Ustilago avenae after fungicide treatment

The plasmalemma of sporidia of Ustilago avenae has been investigated by means of the freezeetching technique after treatment with the fungicide, triadimenol (5 μg ml^?1, 10 and 17 hr). The control samples are characterized in the exponential and stationary growth phases by homogeneous plasmatic fracture (PF) and extraplasmatic fracture (EF) faces with a random intramembrane particle (IMP) distribution. Treatment with triadimenol induces clusters of similar size IMPs in hexagonal arrangement. In between the flat clusters the plasmalemma is significantly deformed, showing hemispherical pits and protrusions on both membrane fracture halves. In the stationary growth phase no hexagonal clustering of the IMPs is observed under the described conditions.     

Resistance to fungicides which inhibit ergosterol biosynthesis in laboratory strains of Botrytis cinerea and Ustilago maydis

The strains of Botrytis cinerea or Ustilago maydis selected on fenarimol, triarimol, or triadimefon were also resistant to the other inhibitors of sterol C-14 demethylation; the sterol composition of the strains was normal. Among the isolates of U. maydis resistant to dodemorph, fenpropidin, fenpropimorph and tridemorph, some were resistant to the 15-azasteroid A 25822B and did not contain ergosterol. The other strains remained sensitive to A 25822B and had a normal sterol composition. All the resistant isolates and the wild-type were inhibited to the same extent by nystatin and pimaricin.     

Resistance in mutagen-induced mutants of Ustilago maydis to fungicides which inhibit ergosterol biosynthesis

Resistance to a number of inhibitors of sterol C-14 demethylation, (clotrimazole, imazalil, miconazole, fenarimol, nuarimol and triadimefon), as well as resistance to inhibitors of sterol C-14(15) double bond reduction, (tridemorph and fenpropi-morph), was readily induced in Ustilago maydis. Resistant mutants were obtained after mutagenic treatment by ultraviolet irradiation, or by treatment with 1-methyl-3-nitro-1-nitrosoguanidine, of sporidia of the wild-type strain, followed by selection in the presence of the toxicant. The level of resistance of these mutants varied appreciably. Although not always reciprocal, cross-resistance to fungicides which inhibit ergosterol biosynthesis (EBIs) appeared to be present in most cases. Several of the U. maydis mutants which were resistant to inhibitors of sterol C-14 demethylation lacked cross-resistance to tridemorph and fenpropimorph, or displayed increased sensitivity to fenpropimorph (negatively correlated cross-resistance). Cross-resistance between EBIs and the antimicrobial agents climbazole and lombazole was also established. It is suggested that fungal mutants that possess a resistance mechanism based on a deficiency in sterol C-14 demethylation or sterol C-14(15) double bond reduction, have a greatly reduced chance of survival.     

Effects of triazoles on fungi: IV. Growth and lipids of Cercospora arachidicola and Cercosporidium personatum

The effects of propiconazole (a sterol C-14 demethylation inhibitor) on the growth and lipid content of Cercospora arachidicola and Cercosporidium personatum were examined in vitro using gravimetric, chromatographic, and colorimetric techniques. The lipid content and composition of both species were very similar. C_16:0, C_18:1, and C_18:2 were the principal fatty acids of the major acyl lipids, ergosterol (ergosta-5,7,22-trienol) was the principal sterol, and free fatty acids comprised a large portion (ca. 30%) of lipid. Cercospora and Cercosporidium were both very sensitive to the inhibitor; 0.10 to 0.15 μg propiconzole/ml was required for an average of approximately 50% growth inhibition among isolates on a mycelial dry weight basis. Changes in lipid composition were similar in both species grown in media containing the inhibitor. The total sterol content was twofold higher than that in the corresponding controls, which was due to the accumulation of ergosterol precursors (e.g., 24-methylene dihydrolanosterol). The free fatty acid content of treated mycelia was lower than that of the controls, and the degree of unsaturation of the lipids was higher, particularly in phosphatidylcholine. Also, the ratio of saturated to unsaturated fatty acids was less in the polar lipid of inhibitor-treated mycelium than in controls.     

Resistance to Sterol Demethylation Inhibitors in Ustilago maydis. III. Cross-Resistance Patterns and Sterol Analyses

Two spontaneous triadimefon-resistant mutants of Ustilago maydis, 151ar/1 and 151ar/3, were investigated with regard to their extent of cross-resistance and their sterol composition to elicit indications about the specificity of the present resistance mechanisms. Testing resistance to various sterol biosynthesis inhibitors and toxicants with different modes of action, it could be demonstrated that, in the mutant 151ar/1, cross-resistance was limited to the sterol demethylation inhibitors (DMIs), whereas, in strain 151ar/3, resistance included most sterol biosynthesis inhibitors studied (DMIs, morpholines, piperidines, allylamines) as well as the unrelated compounds vinclozolin and cycloheximide. Sterol analyses showed that both mutants contained ergosterol as the main sterol component. In comparison with the sensitive reference strain, the mutant 151ar/1 had a slightly elevated content of C-14 methyl sterols, whereas in strain 151ar/3 the amount of ergosterol was increased. Triadimefon caused an accumulation of C-14 methyl sterols and a decrease in ergosterol content in the sensitive strain and the mutant 151ar/1, whereas the other strain 151ar/3 remained unaffected. The results indicate that several resistance mechanisms are probably operating in the two mutants.