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1.
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-Δ8, 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 Δ5, 7-ergostadienol.Identification of 4,4-dimethyl-Δ8, 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-Δ8, 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 Δ5, 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).  相似文献   

2.
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.  相似文献   

3.
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.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
Treatment of celery cell suspension cultures with paclobutrazol and three other triazoles resulted in decreased growth and an accumulation of 14α-methylsterols in both the free sterol and the steryl ester pools, thus indicating that the triazoles were inhibiting the action of the plant obtusifoliol 14α-demethylase system. Obtusifoliol, 14α-methylcampesta-8,24(241)-dien-3β-ol and 14α-methylcampest-8-en-3β-ol were the main 14α-methylsterols to increase in the free sterol pool. In the presence of the triazoles the steryl esters became virtually depleted of sitosterol, campesterol, stigmasterol and isofucosterol, which were replaced by 14α-methylsterols with obtusifoliol becoming the dominant esterified sterol. Treatment of the celery culture with a fourth triazole which did not have a noticeable effect on growth and caused negligible accumulation of 14α-methylsterols in the free sterol pool nevertheless produced some enrichment of the steryl esters in obtusifoliol. The results indicate that, following triazole treatment, a rapid esterification of the accumulating 14α-methylsterol intermediates occurs before they start to build up in significant amount in the free sterol pool.  相似文献   

7.
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.  相似文献   

8.
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.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
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.  相似文献   

12.
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 Δ8,14- 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  相似文献   

13.
An assay for measuring ergosterol synthesis in cell-free extracts of the filamentous plant pathogen Botrytis cinerea is described. The extracts capable of synthesizing C4-desmethyl sterols from [2- 14 C]mevalonate were derived by mechanical disruption of young conidial germlings in a Bead-Beater apparatus. The C4-desmethyl sterol fraction consisted of three distinct compounds and totalled 39% of the non-saponifiable lipids formed. Ergosterol accounted for 63% of the C4-desmethyl sterols. Only small amounts of C4-monomethyl sterols were synthesized, while C4, 4-dimethyl sterols made up 29% of the non-saponifiable lipids. The latter fraction mainly consisted of lanosterol (54%) and eburicol (28%). The cell-free system had a narrow pH optimum for synthesis of C4-desmethyl sterols of pH 7.3–7.4. Cell-free synthesis of C4-desmethyl sterols was inhibited by the imidazole fungicide imazalil, concomitant with an accumulation of eburicol. The IC50 value (concentration of fungicide which inhibited cell-free synthesis of C4-desmethyl sterols by 50%) was 9.1 × 10 ?9 M. These results are consistent with the hypothesis that imazalil is a potent inhibitor of the cytochrome P450-dependent sterol 14x-demethylase of B. cinerea. The method described may be used to screen compounds biochemically for inhibition of sterol synthesis in an agriculturally important plant pathogen.  相似文献   

14.
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.  相似文献   

15.
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.  相似文献   

16.
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.  相似文献   

17.
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. C16:0, C18:1, and C18:2 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.  相似文献   

18.
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-triazolea (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.  相似文献   

19.
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.  相似文献   

20.
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.  相似文献   

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