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.     

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     

Guanidinium and amidinium fungicides: A new class of carbocation mimetic ergosterol biosynthesis inhibitors

A novel class of chemical has been designed with the aim of inhibiting the Δ¹⁴-reductase and Δ⁸-Δ⁷-isomerase enzymes in the ergosterol biosynthesis pathway in fungi. Use was made of knowledge about the mechanisms of both enzymes and the mode of action of known, fungicidal inhibitors of these enzymes. Pioneer examples have been synthesised and have been demonstrated to be potent inhibitors of ergosterol biosynthesis in Ustilago maydis (DC) Corda, acting in the same manner as the commercial fungicide fenpropimorph. They also showed excellent fungicidal activity against Erysiphe graminis DC f. sp. hordei Marchal (powdery mildew of barley) and Puccinia recondita Rob. ex Desm. (wheat leaf rust) in in-vivo glasshouse tests. Using these compounds as a starting point, systematic structural variation has been carried out. Testing of a wide range of analogues at high volume confirms the potential of this class of compound to control mildew and rust pathogens at levels comparable to those of the standards. Correlation of in-vivo and enzymatic data is good and the structure-activity relationship developed for this series of compounds closely parallels that found for the morpholine/piperidine class of fungicides, suggesting a common mode of action.     

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.     

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.     

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.     

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.     

The biochemical mechanism of action of the dinitroaniline herbicide oryzalin

Dinitroaniline herbicides such as oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfanilamide) disrupt mitosis in the meristematic cells of seedling plants by inhibiting the formation of microtubules. For further understanding of the biochemical mechanism of action of oryzalin, laboratory analyses with isolated plant tubulin must be employed. Plant tubulin from flagella of the alga Chlamydomonas was isolated and purified. This tubulin was incubated with [¹⁴C]oryzalin, and free oryzalin was separated from oryzalin bound to plant tubulin by miniature DEAE-cellulose chromatography. Scatchard analysis predicts a molar ratio of oryzalin bound to plant tubulin of 1.0 ± 0.1 when oryzalin is incubated with plant tubulin for 30 min at pH 6.9 and 25°C. The association constant for the oryzalin-tubulin complex is 2.08 ± 0.08 × 10⁵M^?1 at 25°C. The thermodynamic values for the formation of the oryzalin-tubulin complex at 25°C are ΔG^o = ?7.25 ± 0.02 kcal mol^?1, ΔH^o = 6.5 ± 0.2 kcal mol^?1, and ΔS^o = 46 ± 2 cal mol^?1 deg^?1 (mean ± standard error). Oryzalin has little or no affinity for intact microtubules, previously denatured plant tubulin, actin, bovine serum albumin, calmodulin, ferredoxin, trypsin, or urease, indicating oryzalin is specific for the biologically active conformation of plant tubulin. Oryzalin binds to plant tubulin to form a complex that may be incapable of polymerizing into microtubules.     

Effect of fenpropimorph and imazalil on sterol biosynthesis in penicillium italicum

Fenpropimorph was found to be highly active against Penicillium italicum (EC₅₀ 0.01/μg ml^?1). Conidia of P. italicum, treated with low concentrations of fenpropimorph, swelled in size and showed distorted germ tubes. During the initial stages of mycelial growth, fenpropimorph had little or no effect on the dry weight increase, which became strongly inhibited within 24 h after addition of the toxicant (0.05, 0.1 and 0.2 μg ml^?1). Irregular deposition of β–1,3 and β–1, 4 polysaccharides, probably chitin, was observed after treatment with fenpropimorph or imazalil. Fenpropimorph (0.05 and 0.2 μ ml^?1) caused the accumulation of a major demethyl-sterol that was different from ergosterol. It was identified as ergosta-8, 14, 24(28)-trien-3β-ol by mass, infrared, ultraviolet and proton nuclear magnetic resonance, spectrometric procedures. At both concentrations, the accumulation was already detected after incubation for 2 h. In contrast, imazalil (0.1 μg ml^?1) caused the accumulation of several methyl- and dimethyl-sterols which were tentatively identified as eburicol (24-methylene-24, 25-dihydrolanosterol), 4, 14α-dimethylergosta-8, 24(28)-dien-3-one, 14α-methylergosta-8, 24(28)-dien-3-one and obtusifoliol (4, 14α-dimethylergosta-8, 24(28)-dien-3α-ol). The accumulation of ergosta-8, 14,24(28)-trien-3β-ol indicates inhibition of the Δ¹⁴-reductase in P. italicum in a similar manner to that found previously in Ustilago maydis.     

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).     

Sterol metabolism in wheat treated by N-substituted morpholines

Wheat caryopses were treated with racemic fenpropimorph. As shown previously in other plant species, 9(β,19-cyclopropyl sterols were found to accumulate markedly. A distinctive feature was a remarkable accumulation of 31 -norcyclobranol, a very rare sterol in nature. A⁸-sterols were also identified. The ratio A⁸-sterols: cyclopropyl sterols was shown to depend greatly on the configuration of the methyl substituent in position 2 of fenpropimorph. Whereas cyclopropyl sterols predominated in the case of seedlings treated with the (2S)-2-methyl enantiomer, A⁸-sterols were shown to be very abundant in plants treated with the (2R)-2-methyl enantiomer. A⁸-sterols were shown also to be more abundant in leaves than in roots. Experiments were conducted to find out whether the phytotoxic response of plants to fenpropimorph could be ascribed to its action on sterol biosynthesis. From the results obtained it appears that this is not the case and thus the phytotoxic effect is probably related to a cellular target other than sterol biosynthesis.     

Fluorescence microscope studies of Ustilago maydis and Penicillium italicum after treatment with imazalil or fenpropimorph

Imazalil and fenpropimorph caused morphological changes in sporidia of Ustilago maydis and in germinating conidia of Penicillium italicum, as observed by fluorescence microscopy using an optical brightener. Sporidia of U. maydis appeared swollen, distorted, multicellular and, sometimes, branched; conidia of P. italicum swelled in size, and extension of the germ tubes was strongly inhibited. Mycelium of P. italicum, treated with fenpropimorph, showed much enlarged hyphal diameters and relatively short distances between septa. Imazalil and fenpropimorph also caused an irregular deposition of β–1,3 and β-1,4 polysaccharides, probably chitin, in U. maydis and P. italicum. The latter phenomenon is discussed in relation to the following observed effects of fungicides that inhibit ergosterol biosynthesis: differences in effect on the morphology of budding and filamentous fungi; preferential inhibition of yeast-hypha conversion in dimorphic fungi; disorganisation of septum formation in budding fungi; and inhibition of spheroplast formation from budding fungi.     

Influence of soil pH and contents of organic carbon and clay on the volatilization of [14C]fenpropimorph after application to bare soil

The behaviour of the morpholine fungicide fenpropimorph applied to soil was investigated in a laboratory chamber. The volatility and metabolism of a ¹⁴C-labelled fenpropimorph formulation (Corbel^®) was studied after application to three soils (sandy loam, loamy clay and loamy sand), simulating a four-day weather scenario in the volatilization chamber. Additional experiments were conducted under standard climatic conditions over a period of 24 h using sandy soils with different pH values. The results of the first experiments showed that most of the radioactivity applied remained in the soils as unchanged fenpropimorph four days after application. In the experiments with the sandy loam and loamy clay, less than 5% of the applied radioactivity was removed by volatilization whereas 11·4% volatilized from the surface of the loamy sand. The comparatively higher volatilization of the fungicide from the loamy sand was confirmed by the later experiments indicating that higher soil pH favoured volatilization of [¹⁴C]fenpropimorph from sandy soils. Thus 5·6% (pH 5·0), 18·9% (pH 5·8) and 28·3% (pH 6·6) of the radioactivity applied volatilized within one day after application. The overall recoveries were between 93·8% and 111·3% in these experiments. © 1998 SCI     

Rationally Designed Guanidine and Amidine Fungicides

A previous investigation established that compounds containing a guanidinium or amidinium grouping are effective inhibitors of sterol Δ⁸–Δ⁷ isomerase and/or Δ¹⁴ reductase activity in plant pathogenic fungi. A binding model for known fungicidal inhibitors of this enzyme has now been used to rationally design further guanidinium or amidinium inhibitors. Three novel classes of chemistry were investigated. The results of biochemical testing against ergosterol biosynthesis in Ustilago maydis (DC) Corda and of in-vivo testing for fungicidal activity against Erysiphe graminis DC f. sp. hordei Marchal (powdery mildew of barley), do much to support the binding model, and compounds with significant fungicidal activity have been found. © 1997 SCI.     

1(R)-(2,6-cis-dimethylmorpholino)- 3(S)-(p-tert-butylphenyl)cyclopentane: A representative of a novel,potent class of bio-rationally designed fungicides

Inhibition of the Δ⁸-Δ⁷ sterol isomerase in the ergosterol biosynthesis pathway is known to lead to fungistatic or even fungicidal effects. Our approach to the rational design of new putative inhibitors of the enzyme was based on the assumed mechanism of the enzymic transformation and in particular on the structure of the unstable carbocationic intermediate, i.e. the ‘transition-state-analogue’ principle. The synthesis and the biological properties of 1(R)-(2,6-cis- dimethylmorpholino)-3(S)-(p-tert-butylphenyl)cyclopentane, a representative of a novel, potent class of rationally designed fungicides, as well as the synthesis and biological activities of several other related compounds are described and discussed.     

Characterization of triadimefon sorption in soils using supercritical fluid (SFE) and accelerated solvent (ASE) extraction techniques

Sorption–desorption of the fungicide triadimefon in field‐moist silt loam and sandy loam soils were determined using low‐density supercritical fluid extraction (SFE). The selectivity of SFE enables extraction of triadimefon from the soil water phase only, thus allowing calculation of sorption coefficients (K_d) at field‐moist or unsaturated conditions. Triadimefon sorption was influenced by factors such as soil moisture content and temperature; sorption increased with increased moisture content up to saturation, and decreased with increased temperature. For instance, K_d values for triadimefon on the silt loam and the sandy loam soils at 40 °C and 10% water content were 1.9 and 2.5 ml g⁻¹, respectively, and at 18% water content, 3.3 and 6.4 ml g⁻¹, respectively. Isosteric heats of sorption (ΔH_i) were −42 and −7 kJ mol⁻¹ for the silt loam and sandy loam soils, respectively. Sorption–desorption was also determined using an automated accelerated solvent extraction system (ASE), in which triadimefon was extracted from silt loam soil by 0.01 M CaCl₂. Using the ASE system, which is basically a fast alternative to the batch equilibration system, gave a similar ΔH_i value (−29 kJ mol⁻¹) for the silt loam soil (K_f = 27 µg^{1 − 1/n} ml^1/n g⁻¹). In order to predict transport of pesticides through the soil profile more accurately on the basis of these data, information is needed on sorption as a function of soil water content. © 2000 Society of Chemical Industry     

Investigations on the Behaviour of Fenpropimorph and its Metabolite Fenpropimorphic Acid in Soils

The fate of fenpropimorph and its metabolite fenpropimorphic acid was investigated in a silty sand soil and in a clayey silt soil. In laboratory and field experiments fenpropimorph disappeared without a lag phase. A few days after application fenpropimorphic acid was detected. Additional laboratory experiments with [¹⁴C]fenpropimorph emphasized the significance of mineralization and the formation of non-extractable residues. The determination of soil/water distribution coefficients of parent compound and metabolite yielded a higher leaching potential for fenpropimorphic acid due to its higher polarity. This was confirmed by performing a laboratory column test under worst-case conditions. Under field conditions, however, fenpropimorphic acid was detected only in the superficial soil layers (0–5 cm) of both investigation sites at very low concentrations.     

Toxicity of fenpropimorph to fenarimol-resistant isolates of Penicillium italicum

Fenarimol-resistant isolates ofPenicillium italicum with cross-resistance to imidazole and triazole fungicides which inhibit ergosterol biosynthesis were tested for their sensitivity to fenpropimorph. Radial growth tests on PDA showed that the isolates (n=6) lacked cross-resistance to fenpropimorph or displayed enhanced sensitivity to the fungicide (negatively correlated cross-resistance). Control of blue mold decay of oranges incited by the wild-type isolate could be achieved by dipping fruits in suspensions of fenpropimorph at a concentration of 100 mg ml^–1. Decay of oranges incited by the fenarimol-resistant isolates was controlled at the same or at a lower concentration (30 mg ml^–1), thus showing that the normal or increased sensitivity to fenpropimorph is also expressed under in vivo conditions.Samenvatting Fenarimol-resistente isolaten vanPenicillium italicum met kruisresistentie tegen imidazool-en triazoolfungiciden die de ergosterolbiosynthese remmen, werden getoetst op hun gevoeligheid voor fenpropimorf. Radiale groeiproeven op PDA toonden aan dat de isolaten (n=6) geen kruisresistentie bezaten met fenpropimorf of een verhoogde gevoeligheid voor het middel vertoonden (negatief gecorreleerde kruisresistentie). Op sinaasappels konPenicillium-rot, veroorzaakt door het wild-type bestreden worden door middel van een dompelbehandeling met fenpropimorf bij een dosering van 100 g ml^–1). Bestrijding van rot veroorzaakt door fenarimil-resistente isolaten werd verkregen bij dezelfde of een lagere dosering (30 g ml^–1); aldus werd aangetoond dat de normale of verhoogde gevoeligheid voor fenpropimorf ook in vivo tot uiting komt.     

Hydrolysis of 1, 3-dichloropropene in dilute aqueous solution

Hydrolysis of [¹⁴C]-radiolabeled 1, 3-dichloropropene (1, 3-D) was studied at pH 5, 7 and 9 at 10, 20 and 30°C in sterile buffered water. The rate of hydrolysis was independent of pH at each temperature, with measured half-lives of 3-1 (±0.l), 11.3 (±0.5) and 51 (±2.3) days at 30, 20 and 10°C, respectively. The activation energy for the hydrolysis reaction was determined to be 23.9 kcal mol^?1 deg^?1. One hydrolysis product was formed during the course of the study and was identified by co-chrornatography with analytical standards, using h.p.l.c., to be 3-chloroallyl alcohol. The alcohol appeared to be stable to further hydrolytic conversion and was formed in the same cis: trans ratio as in the initial 1, 3-D starting material, indicating essentially identical rates of hydrolysis for the cis and trans isomers of 1, 3-D.     

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.