Effects of sterol biosynthesis inhibitor fungicides in the phytopathogenic fungus,Nectria haematococca: ergosterol depletion versus precursor or abnormal sterol accumulation as the mechanism of fungitoxicity

The relative importance of the depletion of ergosterol versus the accumulation of precursor or abnormal sterols in the mechanism of fungal growth inhibition by sterol biosynthesis inhibitor fungicides is incompletely understood. In order to investigate this problem further, the degree of inhibition of the growth of Nectria haematococca by fungicides with different enzymatic targets in the sterol biosynthetic pathway was determined and compared with their effects on the sterol profile. The sensitivity of N. haematococca was highest towards fenpropimorph, followed by tebuconazole, terbinafine, fenpropidin and tridemorph. Terbinafine, a squalene epoxidase inhibitor, induced a very large accumulation of squalene without very significant inhibition of ergosterol biosynthesis and growth. The fungus appeared able to tolerate large amounts of squalene. In the case of tebuconazole, a sterol 14α-demethylase inhibitor, it seemed that the accumulation of C4 mono- and dimethyl sterols was responsible for fungitoxicity. Fenpropimorph and fenpropidin seemed to be good inhibitors of both sterol Δ¹⁴-reductase and Δ⁸→Δ⁷-isomerase, whereas tridemorph was a better inhibitor of Δ⁸→Δ⁷-isomerase than of the Δ¹⁴-reductase. Large accumulations of Δ^8,14- or Δ⁸-sterols and correspondingly large decreases in the ergosterol content are both implicated in the fungitoxicity of these compounds in N. haematococca. © 1998 Society of Chemical Industry     

Chemical structure-activity relationships of the inhibition of sterol biosynthesis by N-substituted morpholines in higher plants

Tridemorph and fenpropimorph as well as several related N-alkyl morpholines have been tested in vitro on the cycloeucalenol-obtusifoliol isomerase, a microsomal enzyme involved in higher plant sterol biosynthesis. The results showed that N-substituted morpholines inhibit powerfully the enzyme (I₅₀ = 0.4 μM for fenpropimorph). The following important molecular parameters of the inhibition could be determined: (i) the inhibitory capacity was probably related to the presence of a positive charge on the nitrogen atom, (ii) the length of the alkyl group was critical, with a maximum activity for n = 13 carbons in the case of a linear hydrocarbon chain, (iii) the presence of bulky substituents at the proximity of the nitrogen atom led to a strong decrease of the inhibitory power, (iv) in the fenpropimorph series where a chiral center is present at C-2 of the alkyl chain, a remarkable enantiomeric selectivity of the inhibition was observed, (v) the N-oxide derivative of fenpropimorph was shown to be as active as the parent compound. The N-alkyl morpholines have been also assayed on suspension cultures of bramble cells and led to a strong accumulation of 9β, 19-cyclopropyl- and Δ⁸-sterols. This result confirmed that the cycloeucalenol-obtusifoliol isomerase was a major target of the N-substituted morpholines and suggested that the Δ⁸ → Δ⁷-sterol isomerase was also a target for these chemicals. The molecular parameters implied in the in vivo accumulation of 9β, 19-cyclopropyl sterols were very similar to those resulting from the in vitro study. The chemical structure-inhibitory activity relationship of N-alkyl morpholines was discussed with respect to their fungicidal activity which has been described in a previous study [E. H. Pommer, Pestic. Sci. 15, 285 (1984)]. The comparison revealed that the better the inhibitory capacity on the cycloeucalenol-obtusifoliol isomerase was, the higher was the fungicidal activity in vivo.     

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.     

Effect of triadimefon and triadimenol on growth of various plant species as well as on gibberellin content and sterol metabolism in shoots of barley seedlings

The fungicides triadimefon and triadimenol markedly reduced growth of coleoptiles, primary leaves, and roots of barley seedlings when grown for 7 days in petri dishes in the dark. The addition of gibberellins (A₁, A₃, A₄, A₇, A₉) alleviated growth retardation of primary leaves and coleoptiles induced by the fungicides. While fungicide-induced growth retardation of the shoots was partly relieved by kinetin, IAA did not show an alleviating activity. Triadimefon and triadimenol also substantially retarded the elongation of shoots of tomato and cotton plants and simultaneous application of GA₃ nullified the retardation. The fungicides only slightly interfered with both α-amylase production of intact germinating barley seed and the GA₃-induced α-amylase synthesis in barley endosperm. On the other hand, extracts of triadimefon- and triadimenol-treated shoot tissue of 10- to 12-day-old barley plants contained substantially lower gibberellin-like activity than control shoots. Both compounds also interfered in sterol metabolism of shoots of barley seedlings when compared to control plants, treatment resulted in lower amounts and altered proportions of C-4,4-desmethyl sterols. While both fungicides inhibited synthesis of C-4,4-desmethyl sterol fraction, sterols possessing C-4 and C-14-methyl groups were accumulated. It is assumed that triadimefon and triadimenol interfere in gibberellin and sterol biosynthesis in barley seedlings by inhibiting oxidative demethylation reactions.     

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.     

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.     

Sterol composition of isolates of Erysiphe graminis f.sp. tritici differing in sensitivity to fenpropimorph

Isolates of Erysiphe graminis f.sp. tritici with wild-type or reduced sensitivity to fenpropimorph were similar in sterol composition, viz. ergosta-5,24(28)-dienol (±90%) and episterol (±10%). Following treatment with fenpropimorph, the relative content of episterol increased in conidia of all isolates tested, while that of ergosta-5,24(28)-dienol decreased. These results suggest that fenpropimorph, under the test conditions used, does not inhibit activity of sterol Δ¹⁴-reductase or Δ⁸→Δ⁷-isomerase but probably interferes with the final part of the demethyl sterol synthesis. However, modifications in this part of the pathway are probably not responsible for the decreased sensitivity of the pathogen to the fungicide. © 1998 SCI.     

Mechanism of inhibition of sterol biosynthesis enzymes by N-substituted morpholines

Tridemorph, fenpropimorph and derivatives have been tested in vitro on Δ⁸→Δ⁷-sterol isomerase (SI) from maize seedlings. The results show that these N-substituted morpholines strongly inhibit this enzyme (I₅₀: 0.4 μM for fenpropimorph, 0.6 μM for tridemorph). In fenpropimorph, where a chiral centre is present at C-2 of the alkyl chain, good enantioselectivity was observed for enzyme inhibition. Inhibition of SI and of the cycloeucalenol-obtusifoliol isomerase (COI), another enzyme of plant sterol biosynthesis, is probably due to protonation of the N-substituted morpholines, giving a positively charged nitrogen atom, at the pH (7.4) of the enzyme assays. In order to test this hypothesis, the pH dependence of the inhibition constants has been measured. Fenpropimorph, (pK_a 7.5) which can be protonated to give a morpholinium cation and N-methylfenpropimorph, the corresponding quaternary ammonium derivative, have been tested as inhibitors in a pH range from 6 to 8.5. The I₅₀ value of fenpropimorph toward the SI increased 20 times as the pH increased from 6 to 8.5. In contrast the I₅₀ value of fenpropimorph for COI did not change significantly in this pH range. While the I₅₀ value of N-methyl fenpropimorph towards COI decreased more than 50 times as the pH increased from 6 to 8.5, its I₅₀ value for SI only varied slightly in this pH range. Taken together, these results suggest the existence of an interaction of the morpholinium cations with one or several enzyme amino acid residues of a much higher pK_a in the case of COI than SI. Moreover for maize COI, the pK_a of the amino acid residue(s) interacting with these morpholines derivatives is probably close to that of these molecules.     

Conformational analysis of the fungicide fenpropimorph by molecular mechanics calculations and NMR spectroscopy

Fenpropimorph is an inhibitor of Δ8→Δ7-isomerase and Δ14-reductase in fungi and cycloeucalenol-obtusifoliol-isomerase and Δ8→Δ7-isomerase in higher plants. A detailed conformational analysis of the most potent enantiomer (S)-fenpropimorph is described. The conformational analysis is based on three different methods: molecular mechanics calculations. NMR spectroscopy and X-ray crystallography. In solution several conformations coexist. The molecular mechanics calculations using MM2(85) show a folded conformation of the unprotonated form of fenpropimorph to be stabilized by at least 3 kcal mol^?1 relative to the next lowest energy conformer, suggesting only one conformation of fenpropimorph to be present. A corresponding stabilization of the protonated form (5.4 kcal mol^?1) is obtained. However, this stabilization is found partly to be due to attractive steric interactions and may be a phenomenon occurring only in vacuum. In the solid state, fenpropimorph exists in an L-shaped conformation resembling one of the low-energy conformers obtained with MM2(85). Based on comparison with the natural substrate, a totally extended conformation of fenpropimorph is suggested to be the biologically active conformation for interaction with the Δ8 → Δ7-isomerase in fungi.     

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.     

Resistance to inhibitors of sterol biosynthesis in field isolates or laboratory strains of the eyespot pathogen Pseudocercosporella herpotrichoides

Strains of Pseudocercosporella herpotrichoides collected in France on winter wheat give either fast-growing mycelial colonies with regular margins or slow-growing mycelial colonies with irregular margins. Most of the fastgrowing isolates were sensitive to triadimenol (EC₅₀ below 2mg litre^?1), but some of them were resistant to this inhibitor of sterol C-14 demethylation. In contrast, all the slow-growing strains were highly resistant to triadimenol (EC₅₀ greater than 100 mg litre^?1). This resistance was also expressed in inhibition of germ-tube elongation. Positive cross-resistance was observed between most of the inhibitors of sterol C-14 demethylation, with the exception of some imidazole derivatives (clotrimazole, prochloraz). All the fast-growing strains were tolerant to fenpropimorph and fenpropidin whereas the slow-growing ones were susceptible; the reverse was true with piperalin and tridemorph. All the field isolates were inhibited to the same extent by the inhibitors of squalene-epoxidase, nafifine and terbinafine. Two types of mutant resistant to triadimenol have been induced under laboratory conditions from sensitive fast-growing strains. The most common mutants were resistant to all the inhibitors of sterol C–14 demethylation and also in some conditions to fenpropimorph, tridemorph and the inhibitors of squalene-epoxidase. The other mutants were characterised by a reduced spectrum of cross-resistance between triadimenol and the other inhibitors of sterol biosynthesis. The field isolates and laboratory mutants resistant to triadimenol and propiconazole were also resistant to each of the four enantiomers of these two fungicides.     

Chemistry and biology of novel amine fungicides: Attempts to improve the antifungal activity of fenpropimorph

In spite of considerable efforts by many workers, there has been a lack of progress in the area of amine fungicides since fenpropimorph. Random synthesis of a large variety of different amine compounds, as well as intelligent structural modification of the lead structure fenpropimorph (well over 15 000 amines have been screened at BASF alone) have not led to a new market product so far. Further work has been focused on the reported mode of action of fenpropimorph, notably on the inhibition of the sterol Δ¹⁴-reductase. Although some doubt has to be cast on the hypothesis that fenpropimorph behaves as a sterol mimic, the concept of ‘high energy intermediate’ inhibitors has been employed successfully. Rational drug design of azasterol mimics has led to a number of very potent inhibitors of the sterol Δ¹⁴-reductase which also displayed high fungicidal activity in the greenhouse. Although many of these compounds are more powerful reductase inhibitors than fenpropimorph, under field conditions none showed significant advantages over this established fungicide. Most likely, fenpropimorph already exhibits the maximum fungicidal activity which can be attained by blocking the sterol Δ¹⁴-reductase. This would mean that, with the development of the ‘second generation’ amine fungicide fenpropimorph, this class of compounds has already virtually been optimized.     

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).     

The lipid compositions of two isolates of Cladosporium cucumerinum do not explain their differences in sensitivity to fungicides which inhibit sterol biosynthesis

Isolate 840905 of Cladosporium cucumerinum, when grown on agar or in liqiud medium, was sensitive to triadimenol, HWG 1608 (tebuconazole), fenpropimorph and pimaricin but relatively resistant to terbinafine. Conversely, isolate 49628 was sensitive to terbinafine but relatively resistant to the other fungicides. Changes in sterol composition following treatment with the fungicides reflected the known modes of action of each fungicide. When individual enantiomers of triadimenol were tested against isolate 840905 the order of activity in reducing mycelial growth was 1 S, 2R > 1R, 2R > 1R, 2S ≈? 1S, 2S, and this was paralleled by the depletion of ergosterol and the appearance of 14α-methyl sterols. Isolate 49628 had a greater saturated:unsaturated fatty acid ratio than did isolate 840905 but no major changes in fatty acid composition of either isolate were induced by fungicide treatment. There appears to be no obvious explanation for the differences in fungicide sensitivity of the isolates in terms of their lipid compositions.     

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.     

Novel inhibitors of sterol C-14 demethylase and Δ14 Reductase/Δ8 → Δ7 isomerase for cereal disease control

The activity of five experimental fungicides combining structural elements responsible for sterol C-14 demethylase inhibition and sterol nuclear double-bond transformations has been investigated by examining sterol accumulation in Ustilago maydis (DC.) Corda, inhibition of sterol-biosynthesis enzymes in Saccharomyces cerevisiae Meyer using a cell-free system and protective activity in cereals against Erysiphe graminis DC. and Puccinia coronata Corda. Combining the fenpropidin/fenpropimorph basic structure with a pyridine moiety offered no advantage in fungicidal activity. However, the combination of the pyrifenox structure with a morpholine moiety yielded a compound which was a good inhibitor of C-14 demethylase and Δ⁸ → Δ⁷ isomerase in vitro and gave good cereal protection.     

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.     

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.     

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.