Mode of action of triadimefon in Ustilago avenae

Triadimefon [1-(4-chlorophenoxy)-3,3-dimethyl-(1,2,4-triazol-1-yl)-2-butanone], 1.5–2.0 μ/ml, inhibited the multiplication of sporidia of Ustilago avenae more strongly than it did the increase of dry weight. The treated sporidia appeared swollen, multicellular, and branched. At concentrations of 1.5–100 μg of triadimefon/ml, the oxidation of glucose was not affected. Increase in dry weight and synthesis of protein, RNA, and DNA were inhibited slightly, whereas cell division was acutely arrested. After an incubation period of 9.5 hr, microscopic studies revealed that daughter cells of the treated sporidia also contained one nucleus. In sporidia treated for 6 hr with triadimefon, both the total lipid content and its composition of fatty acids were not appreciably altered. The treated cells, however, differed from control cells by a higher content of free fatty acids. Triadimefon markedly interfered in sterol biosynthesis in Ustilago avenae. Gas chromatographic (glc) analysis and [¹⁴C]acetate incorporation studies indicated that ergosterol biosynthesis was almost completely inhibited by triadimefon; on the other hand, sterol compounds representing precursors of ergosterol (probably 4,4-dimethyl and C-4-methyl sterols) accumulated in treated sporidia. As the results indicate, the inhibition of conversion of immediate sterol precursors to ergosterol may be regarded as the primary target for the action of triadimefon in Ustilago 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.     

Antifungal mode of action of imazalil

Imazalil had no effect on the initial growth of mycelia of Penicillium italicum (for 10 hr) or Aspergillus nidulans (for 2 hr). In P. italicum during this period neither respiration nor cell permeability was affected, but uptake of [³²P]phosphate, [¹⁴C]leucine, or [¹⁴C]uridine was partially inhibited. The initial (5 hr) inhibition of substrate uptake coincided with a 50% reduction in ergosterol content. Within 0.5 hr, incorporation of [¹⁴C]acetate into C-4-desmethyl sterols was strongly inhibited in mycelia of A. nidulans treated with 0.5 μg/ml of imazalil. However, radioactivity in C-4-methyl and dimethyl sterols exceeded that of control cultures. Concentrations of imazalil as low as 0.005 μg/ml caused short-term (1 hr) declines of incorporation into desmethyl sterols and increases into the C-4-methyl and dimethyl sterols. Incorporation into phospholipids, triglycerides, and free fatty acids was not affected. These data suggest that the primary antifungal action of imazalil is inhibition of demethylation in the biosynthesis of ergosterol.     

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.     

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.     

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.     

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).     

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.     

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.     

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.     

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.     

Sterol content and other characteristics of pimaricin-resistant mutants of Aspergillus nidulans

Pimaricin-resistant mutants of Aspergillus nidulans were selected on a medium containing the polyene-antibiotic. Some resistant mutants contained markedly reduced amounts of ergosterol, but others contained almost normal levels of this sterol. Most resistant mutants which lacked ergosterol had a biochemical lesion in sterol C-22 desaturation. Analysis of sterols in one of these isolates showed the presence of 5,7-ergostadienol, 5,7,24(28)-ergostatrienol, and 5,8-ergostadienol. The sterol C-14 demethylation inhibitor, fenarimol, was more toxic to this mutant than to the wild type. On the other hand, mutants inactive in sterol C-22 desaturation were resistant to oligomycin but showed wild type sensitivity to carboxin. Attempts to select sterol C-14-demethylation-deficient mutants of Aspergillus nidulans, Monilinia fructicola, and Pyricularia oryzae on polyene-containing media were unsuccessful. Apparently C-14-methyl sterols do not support growth of these filamentous fungi.     

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.     

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.     

Effect of propiconazole on growth and sterol biosynthesis by Sclerotium rolfsii

Germination of sclerotia ofSclerotium rolfsii on agar nutrient medium was delayed or slightly inhibited by concentrations of propiconazole between 0.4 and 4.0 μg ml^?1, but was strongly inhibited by 8 μg ml^?1 and completely inhibited by 16 μg ml^?1. On the other hand, growth of hyphae from the germinated sclerotia was strongly inhibited by propiconazole at 1 μg ml^?1 or greater. Hyphal growth from agar discs on agar medium was about 8 times less sensitive than hyphal growth from the sclerotia or from hyphal inoculum in liquid media. Propiconazole at 0.25 and 1.0 μg ml^?1 strongly inhibited ergosterol biosynthesis, but this was not associated with large accumulations of C-14 methyl sterols. The ratio of eburicol to ergosterol in hyphae grown in the presence of 0.25 μg ml^?1 propiconazole for 16, 30 or 45 h was 0.11, 0.13 and 0.04, respectively, for the three intervals while for hyphae grown in the presence of 1 μg ml^?1, the ratios were 0.29, 0.36 and 0.30, respectively, for the same intervals. In view of a ratio of 23.5 for¹⁴C-acetate incorporation into the two sterols during the initial 6 h growth period in the presence of propiconazole, it is believed that the lack of large accumulation of C-14 methyl sterols is due to the feedback inhibition by eburicol or to cell lysis when the content of ergosterol becomes too low in the actively growing cells.     

Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 μg ml^?1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.     

Action of fungicidal triazoles of the diclobutrazol series on ustilago maydis

Diclobutrazol [(2RS, 3RS)-1-(2, 4-dichlorophenyl)-4, 4-dimethyl-2-(1,2, 4-triazol-1-yl)pentan-3-ol] decreased the rate of growth of Ustilago maydis during the log phase. Marked changes in sterol composition were observed with a decrease in ergosterol and an increase in methyl-sterols, indicating a block in the removal of the 14-methyl group. The inhibition was of rapid onset (<4 h). Changes in other lipid constituents were minor and there was no build up of unsaturated fatty acids. The fungicidal activity resides in the (2R, 3R)-isomer, which is known to be more potent in blocking a yeast 14-demethylase enzyme than the (2S, 3S)-isomer. This relationship held in the 4-chlorophenyl homologues, which as a group were less fungicidal and less potent inhibitors than the 2, 4-dichlorophenyl compounds. The evidence presented indicates that the primary lesion caused by the fungicide was a build up of membrane sterols containing extra methyl groups; this, in combination with a loss of ergosterol, is believed to prevent proper membrane ordering and thus to cause a loss of membrane function.     

Mechanism of resistance to fenarimol in Aspergillus nidulans

Mycelial uptake of [¹⁴C]fenarimol (10 μg/ml) by 20 fenarimol-resistant mutants of Aspergillus nidulans was compared with uptake by wild-type strain 003. Uptake of the fungicide during the initial 10 min of incubation was significantly lower in all mutant strains than in the wild-type strain indicating that resistance is related with reduced uptake. Upon prolonged incubation a gradual decrease of accumulated radioactivity in the wild-type strain was observed. A few mutants displayed resistance to unrelated chemicals such as p-fluorophenylalanine or d-serine; this phenomenon appeared not to be due to a decreased uptake of the corresponding natural amino acids. Incorporation of [³H]adenine and [¹⁴C]leucine by mycelium of mutant M193 was hardly inhibited after 5 hr of incubation with the fungicide, whereas a distinct effect was found with the wild-type strain. At this time also fungitoxicity to the wild-type strain became apparent. Probably, this effect is indirectly caused by inhibition of ergosterol biosynthesis. Mycelium of mutant M193 incorporated [¹⁴C]acetate slightly less effectively than the wild-type strain. After 2 hr of incubation with this radiochemical leakage of [¹⁴C]acetate metabolites from mycelium of the mutant strain was observed. This indicates that resistance might be correlated with increased excretion of fungal metabolites, which in turn may be related with reduced fitness of fenarimol-resistant mutants.     

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.