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).     

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.     

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.     

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.     

Biochemical aspects of the fungitoxic and growth regulatory action of fenarimol and other pyrimidin-5-ylmethanols

The C-14 demethylation of the sterols, dependent on cytochrome P-450, and the C-22(23) desaturation of sterols are reactions in the ergosterol biosynthesis pathway that are regarded as primary target sites in the toxicity of fungicides of the pyrimidin-5-ylmethanol type. Currently, there is no evidence for target sites in other pathways of comparable sensitivity, although the failure of added ergosterol to reverse the fungitoxicity suggests the existence of such sites. The mitochondrial respiratory systems in Ustilago maydis and Aspergillus nidulans are insensitive to this type of fungicide and are not regarded as primary targets of fungitoxicity in these organisms. Appreciable evidence indicates that the primary targets in higher plant growth regulation are reactions, dependent on cytochrome P-450, that assist in the conversion of kaurene to kaurenoic acid in the gibberellin (GA) biosynthesis pathway. Growth retardation by high concentrations of pyrimidin-5-ylmethanols, which is not reversable by GA, apparently involves action at sites outside the GA biosynthesis pathway. The data derived from various studies of the mechanisms of fungitoxicity and growth regulation suggest that any undetected primary targets of the pyrimidin-5-ylmethanols are likely to be haem enzymes, similar to the cytochrome P-450 involved in sterol C-14 demethylation.     

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.     

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.     

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.     

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.     

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 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     

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.     

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.     

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.     

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 g 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.Imazalil remt differentieel de toename in drooggewicht van 10-uur-oude gekiemde sporen van wild-type en DMI-resistente isolaten vanPenicillium italicum in vloeistofcultures van moutextract. De EC₅₀ waarden voor groei van de verschillende isolaten lopen uiteen van 0,005 tot 0,27 g ml^–1. In afwezigheid van het fungicide is in alle isolaten ergosterol het belangrijkste sterol (meer dan 95% van het totaal). DMI-resistentie kan daarom niet in verband staan met deficiëntie van het C-14 demethyleringsenzym in de ergosterol biosynthese. Imazalilbehandeling van mycelium bij concentraties rond de EC₅₀ waarde voor groeiremming, resulteerde bij alle isolaten in een afname van het ergosterolgehalte en een gelijktijdige toename van het gehalte aan 24-methyleen-24,25-dihydrolanosterol. Er bestaat dus een nauwe correlatie tussen de imazalilconcentratie die noodzakelijk is om vergelijkbare veranderingen in sterolsamenstelling te induceren en de EC₅₀ waarde voor remming van myceliumgroei van de verschillende isolaten. De differentiële effecten van imazalil op de sterolsamenstelling van de verschillendeP. italicum isolaten kunnen worden veroorzaakt door verminderde accumulatie van het fungicide in het mycelium en door andere, nog niet geïdentificeerde resistentiemechanismen.     

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.     

Variable resistance factors of fungicides acting as sterol demethylation inhibitors

The ED₅₀ values and resistance factors of 20 fungicides that all act as inhibitors of the C-14 demethylation of 24-methylenedihydrolanosterol were determined for one wild-type and four resistant strains of Ustilago avenae. All fungicides were cross-resistant to each other; however, the resistance factors varied considerably, ranging from 50 (triadimenol) to 2·2 (miconazole). A tentative structural requirement for low resistant factors was the presence of two phenyl rings separated from each other by at least three atoms. Labeling of lipids with [¹⁴C]acetate in the absence and presence of the inhibitors and subsequent sterol analysis revealed that the variable resistance factors were not related to the presence of a second target site. In spite of reported second modes of action of fenarimol, tebuconazole or miconazole, accumulation of C-14 sterol precursors in both sensitive and resistant isolates was necessary to accomplish growth inhibition.     

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.     

Effects of terbinafine on growth, squalene, and steryl ester content of a celery cell suspension culture

The allylamine terbinafine inhibited growth of a celery (Apium graveolens) cell suspension culture (I₅₀ = 90 μM) and blocked the action of squalene epoxidase, resulting in an accumulation of squalene and a decrease in the sterol content of the cells. Celery cells were tolerant to squalene accumulation; inhibition of growth of cultures was associated with a fall in the free sterol content below about 1 μg sterol/mg dry wt of cells. At Day 14, untreated celery cells contained about 60% of the total sterol in the esterified form. However, the steryl ester pool was considerably lower in terbinafine-treated cells which may reflect an attempt to maintain the free sterol content above a threshold value. The composition of free sterols of terbinafine-treated cells was different from control cultures, suggesting that terbinafine has a second site of inhibition on the pathway to major sterols in plants.