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     

The mechanism of resistance to sterol 14α demethylation inhibitors in a mutant (Erg 40) of Ustilago maydis

Resistance to DMI fungicides is a problem in both agriculture and medicine. Several mechanisms of resistance exist, but, as yet, few have been characterised in field resistant strains of plant pathogens. One approach to evaluating the role of mutations in the sterol 14α demethylase (14DM) target site requires cloning this gene and confirming its identity by complementation in an appropriate mutant. The azole‐resistant mutant, Erg 40, of Ustilago maydis which is totally blocked at the 14α demethylation step in sterol biosynthesis seems to be suitable for such expression studies. Transformation of Erg 40 with a plasmid containing the yeast 14α demethylase (CYP51A1) gene removed the block in sterol biosynthesis and generated azole‐sensitive transformants. Detailed analysis of these transformants failed to detect the presence of the yeast gene and suggested, instead, that changes in sterol biosynthesis resulted simply from the transformation protocol and not from the incorporation of extracellular DNA. Subsequent sequence analysis has revealed a mutation in the 14α demethylase gene of Erg 40. The results suggest that azole resistance in Erg 40 is not simply controlled by this mutation but involves some additional regulatory function, and consequently Erg 40 is not suitable for complementation studies with CYP51A1 genes. © 2000 Society of Chemical Industry     

Effects of sterol biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi

The fungicides miconazole, fenarimol, and etaconazole block ergosterol biosynthesis in fungi by inhibiting sterol 14α-demethylation, which is mediated by a cytochrome P-450 enzyme. The sensitivity of cytochrome P-450-dependent hydroxylation or demethylation of several substrates to these fungicides and similar compounds was compared to that of fungal growth and sterol 14α-demethylation. Demethylation of p-chloro-N-methylaniline (PCMA) by sporidia of Ustilago maydis and 11α-hydroxylation of progesterone by Aspergillus nidulans were relatively insensitive to these compounds and to metyrapone. The ability of a sterol 14α-demethylation-deficient mutant to demethylate PCMA indicates that this substrate is not demethylated by the sterol 14α-demethylation system of U. maydis. The 14α-hydroxylation of progesterone by cells of Curvularia lunata was quite sensitive to the three fungicides, and also to metyrapone and isopropylphenylimidazole. This system was less sensitive to the three fungicides than sterol 14α-demethylation, but was appreciably more sensitive than PCMA demethylation. A study of progesterone 14α-hydroxylation in cell-free preparations of C. lunata showed the reaction to be inhibited by CO, and to be competitively inhibited by low concentrations of miconazole. These data suggest that the primary action of sterol biosynthesis-inhibiting (SBI) fungicides is competitive inhibition of sterol/steroid-type cytochrome P-450 enzymes rather than interference with the function of sterol carrier proteins or enzyme-modulating phospholipids.     

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.     

Relationship between chemical structure and biological activity of triazole fungicides against Botrytis cinerea

The inhibitory activity of commercial and experimental triazole fungicides on the target enzyme, sterol 14α-demethylase (P450_14DM), was studied in a cell-free sterol synthesis assay of Botrytis cinerea Pers. ex Fr. In order to assess structure-activity relationships, the inhibitory activities of the compounds on radial growth of the fungus were tested as well. The EC₅₀ values (concentrations of fungicide inhibiting radial growth of B. cinerea on PDA by 50%) of all triazoles tested ranged between 10^?8 and 10^?5 m. IC₅₀ values (concentrations of fungicide inhibiting incorporation of [2-¹⁴C]mevalonate into C4-desmethyl sterols by 50%) generally ranged between 10^?9 and 10^?7 M and correlated with inhibition of radial mycelial growth. However, differences in IC₅₀ values did not reflect quantitatively the observed differences in EC₅₀ values, since the ratio between EC₅₀ and IC₅₀ increased with decreasing fungitoxicity. For a limited number of compounds the correlation between intrinsic inhibitory activity and fungitoxicity was low. Both in-vitro tests were used to investigate structure-activity relationships for stereoisomers of cyproconazole, SSF-109 and tebucona-zole. Fungitoxicity and the potency to inhibit cell-free C4-desmethyl sterol synthesis correlated for all stereoisomers tested. Mixtures of isomers of tebucona-zole or cyproconazole were slightly less active than the most potent isomer. The high activity of several commercial triazoles in both experiments implies that poor field performance of triazole fungicides against B. cinerea is due neither to insensitivity of the P450_14DM nor to low in-vitro sensitivity of the fungus.     

Inhibition of sterol biosynthesis in cell-free extracts of Botrytis cinerea by prochloraz and prochloraz analogues

The sensitivity of cytochrome-P450-dependent sterol 14α-demethylase (P450_14DM) to prochloraz and several prochloraz analogues was studied in a cell-free assay of Botrytis cinerea Pers. ex Fr. The EC₅₀ values (concentrations which inhibited radial growth of B. cinerea by 50%) of the compounds tested ranged from 3.3 × 10 ?8 to 1.7 × 10 ?5 M. The IV₅₀ values (concentrations which inhibited cell-free C₄-demethyl sterol synthesis by 50%) in cell-free assays of B. cinerea ranged from 2.6 × 10 ?9 to 4.4 × 10 ?7 M. Ranking compounds in terms of their relative inhibitory potencies showed quite similar trends to the order of fungitoxicity, but the IC₅₀ values did not quantitatively reflect the differences in toxicity. Therefore, the differential inhibition of cell-free P450_14DM activity by these compounds cannot fully account for their differences in activity towards B. cinerea. Additional mechanisms must be involved. The compounds tested were generally more potent in the B. cinerea assay than in similar assays developed for Penicillium italicum Wehmer and, in particular, Saccharomyces cerevisiae Meyen. This correlated with the relatively higher activity of most test compounds to B. cinerea. Results suggest that the cell-free assay of B. cinerea is more useful to evaluate candidate fungicides as inhibitors of sterol 14α-demethyiase activity than similar assays from model organisms. The present study confirms that the affinity of prochloraz analogues for P450_14DM depends on the nature of the N-1 substituent of the imidazole and the azole ring. It was also found that addition of an amino group at C-2 of the imidazole moiety of prochloraz gave a compound (6) which inhibited 4, 4-demethyl sterol biosynthesis in B. cinerea at a different site from the P450_14DM. This was confirmed by the observation that laboratory-generated triadimenol-resistant isolates of B. cinerea showed reduced sensitivity to triadimenol and prochloraz, but not to compound 6.     

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 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.     

New Cases of Negative Cross-resistance between Fungicides,Including Sterol Biosynthesis Inhibitors

A survey of fungicide resistance in Mycosphaerella graminicola and Tapesia acuformis, two major pathogens of winter wheat in France, respectively responsible for speckled leaf blotch and eyespot, led to the characterization of two types of resistant strains to sterol 14α-demethylation inhibitors (DMIs). Most of the strains of M. graminicola collected in France in 1997–1998 were resistant to all DMIs, and only in a few strains was the resistance to several triazoles associated with increased susceptibility to pyrimidine derivatives (i.e., fenarimol, nuarimol) and triflumizole. On the other hand, in T. acuformis the most prevalent strains were those which exhibited negative-cross resistance between DMIs. In both fungi such a phenomenon could be related to changes in cytochrome P450 sterol 14α-demethylase, the target site of these fungicides. For Botryotinia fuckeliana, the causal agent of grey mould, the extensive monitoring conducted in French vineyards before the marketing of fenhexamid revealed the presence of highly resistant strains to this promising botryticide (only in tests involving mycelial growth measurements). Negative cross-resistance to edifenphos and several sterol biosynthesis inhibitors, such as prochloraz and fenpropimorph, was observed in fenhexamid resistant strains. Synergism of the antifungal action of fenhexamid by cytochrome P450 inhibitors, such as the DMI fungicides, was only recorded in fenhexamid resistant strains. These data and those previously obtained with edifenphos resistant strains of Magnaporthe grisea (rice blast pathogen) suggest that in fenhexamid resistant strains of B. fuckeliana the same cytochrome P450 monooxygenase could be involved in detoxification of fenhexamid and activation of edifenphos. Received 6 September 1999/ Accepted in revised form 13 September 1999     

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.     

Heterologous expression of the P450 sterol 14α-demethylase gene from Monilinia fructicola reduces sensitivity to some but not all DMI fungicides

The cytochrome P450 sterol 14α-demethylase gene (MfCYP51) from Monilinia fructicola (G. Wint.) Honey was cloned and sequenced. The gene was 1680 bp in length (including introns) and was predicted to have two introns of 54 and 57 bp. The nucleotide sequence was 82.1, 53.4, 47.1, 45.1, and 33.6% and the amino acid sequence was 89.7, 76.1, 76.1, 71.8, and 66.9% identical to the CYP51 genes from Botrytis cinerea, Tapesia yallundae, T. acuformis, Erysiphe graminis, and Uncinula necator, respectively. Expression of MfCYP51 in PDR5::TN5 deficient Saccharomyces cerevisiae resulted in reduced sensitivity of the yeast transformants to myclobutanil but not to propiconazole, fenbuconazole or tebuconazole. A wildtype population of 33 M. fructicola isolates was significantly less sensitive to myclobutanil than to propiconazole, fenbuconazole, and tebuconazole. The sensitivity of the isolates to myclobutanil and the three other DMI fungicides included in this study was correlated positively, suggesting a similar or identical mode of action. The low sensitivity in M. fructicola wildtype isolates to myclobutanil could result from a less effective binding potential of the fungicide to the 14α-demethylase.     

Phase 1 and 2 Metabolism in Freshly Isolated Hepatocytes and Subcellular Fractions from Rat,Mouse, Chicken and Ox Livers

In toxicological studies hepatocytes offer an excellent alternative to whole-animal experiments, provided their metabolic competence has been established. We have compared Phase 1 and 2 metabolism in rat, mouse, chicken and ox liver microsomes and cytosol with freshly isolated hepatocytes. The relative amounts of total cytochrome P450 in microsomes and hepatocytes were equivalent. Rat liver had the highest P450 content while chicken liver had the lowest content (148·2(±75·7) and 20·6(±11·5) pmol mg^-1 hepatocellular protein, respectively). The metabolism of testosterone was assessed to determine selective cytochrome P450 isoenzyme activities. Only two metabolite products were common to all four species, namely 6β-hydroxytestosterone (6β-OHT) and androstenedione (ASD), which co-eluted with 6-dehydrotestosterone (6DHT). 16α-OHT was present in all incubations except for ox microsomes. The rate of metabolism of testosterone was generally lower in microsomes than hepatocytes, with the exception of the ox, but the pattern and quantity of metabolite formation was similar. The quantity of total products formed was 15- to 27-fold higher in rat and mouse livers than in chicken or ox. The major product formed in freshly isolated hepatocytes from mice and chickens was ASD/6DHT which accounted for 60% and 76% of the total metabolites, respectively. ASD/6DHT formation accounted for only 33% and 17% of the total metabolites formed by rat and ox hepatocytes, respectively. 2α-OHT production occurred in rat and mouse hepatocytes (14% of the total metabolites in rat and 7% in mouse hepatocytes) but was lacking in chicken or ox cells. The stability of P450 isoforms in culture was species-dependent. Rat and mouse hepatocyte cultures lost 54% and 31% of their initial P450 content after 72 h, while there was no loss in chicken hepatocytes over the same period. There was a good correlation between the relative glutathione S-transferase (GST) activities in cytosol and freshly isolated hepatocytes. Mouse liver exhibited highest GST activity (664·2(±203·5)) compared with rat, chicken or ox (320·4(±64·0), 341·5(±13·9) and 256·3(±109·9) nmol min^-1 mg^-1 cytosolic protein, respectively). © 1997 SCI.     

Strong resistance to the fungicide fenhexamid entails a fitness cost in Botrytis cinerea, as shown by comparisons of isogenic strains

BACKGROUND: Fenhexamid, a sterol biosynthesis inhibitor effective against Botrytis, inhibits the 3‐ketoreductase (Erg27) involved in C‐4 demethylation. Several fenhexamid‐resistant phenotypes have been detected in Botrytis cinerea populations from French vineyards. The field isolates with the highest resistance levels display amino acid changes in Erg27 (F412S, F412I or F412V). RESULTS: Fenhexamid‐resistant mutants were generated by site‐directed mutagenesis of the erg27 gene in a sensitive recipient strain to overcome the impact of different genetic backgrounds. The wild‐type erg27 allele was replaced by the three mutated alleles (erg27^F412S/I/V) by homologous recombination. These isogenic strains were shown to be fenhexamid‐resistant and were used to quantify the impact of F412 mutations on fungal fitness. Several parameters, including radial growth, the production of sclerotia and conidia, freezing resistance and aggressiveness, were quantified in laboratory conditions. Analysis of variance demonstrated significant differences between the mutant and parental strains for some characters. In particular, the mutants grew more slowly than the wild‐type strain and displayed variations in the production of sclerotia and conidia with temperature and susceptibility to freezing. CONCLUSIONS: The results highlight a moderate but significant impact of F412 mutations on the survival capacity of B. cinerea strains displaying high levels of resistance to fenhexamid in laboratory conditions, potentially limiting their dispersal and persistence, particularly in terms of overwintering, in field conditions. Copyright © 2011 Society of Chemical Industry     

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.     

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.     

Specific effects of tridemorph on sterol biosynthesis in Ustilago maydis

In an attempt to indicate the site of action of tridemorph in ergosterol biosynthesis by Ustilago maydis the nature of the sterol intermediates accumulating in treated cells was studied. At low growth-inhibiting concentrations of the toxicant (10 and 15 μg/ml) decline of ergosterol content during 6 hr of incubation was accompanied by an accumulation of various sterol intermediates of which ergosta-8,14-dien-3β-ol appeared to be the major sterol. After isolation of this intermediate its identity was further confirmed by direct comparisonof its ultraviolet, glc, and mass spectrum with those of an authentic synthesized sample of ergosta-8,14-dien-3β-ol (ignosterol). Results indicate that toxicity of tridemorph is caused by a specific inhibitive effect on the enzyme Δ¹⁴-reductase which is responsible for $\text{14}\text{15}$ double-bond reduction in sterol biosynthesis.     

Effects of triazole inhibitors of sterol biosynthesis on the free and esterified sterol composition of celery cell suspension cultures

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.     

Absorption,translocation, and metabolism of ethalfluralin and trifluralin in Solanum spp

Seedlings of Solanum scabrum Mill. and Solanum ptycanthum Dun. were treated with [¹⁴C]ethalfluralin (N-ethyl-α,α,α-trifluoro-N-(methylallyl)-2,6-dinitro-p-toluidine) and [¹⁴C]trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) supplied in nutrient solution to determine the basis for differences in response by these two species to these two herbicides. Plants of S. scabrum absorbed more [¹⁴C]ethalfluralin and [¹⁴C]trifluralin than plants of S. ptycanthum. During the first 24 h, S. scabrum seedlings, but not S. ptycanthum seedlings absorbed more [¹⁴C]ethalfluralin than did plants treated with [¹⁴C]trifluralin. More [¹⁴C]ethalfluralin than [¹⁴C]trifluralin was found in the shoots of plants of both species. Seventy-two hours after treatment with [¹⁴C]herbicides, the conversion to water-soluble metabolites was greater for [¹⁴C]ethalfluralin than for [¹⁴C]trifluralin. In the shoots of plants from both species an average of nearly 55% of the ¹⁴C recovered was found in the water-soluble fraction following [¹⁴C]ethalfluralin treatment whereas an average of only 40% was found in the water-soluble fraction following [¹⁴C]trifluralin treatment.     

A comparison of the potency of some fungicides as inhibitors of sterol 14-demethylation

An enzymatic assay system has been developed to measure the relative potency of fungicides such as triadimefon, triarimol, triforine, and buthiobate as inhibitors of sterol 14-demethylation. The enzyme preparation used is the 8000g supernatant derived from a homogenate of an aerobically adapted, anaerobically grown, high sterol strain of Saccharomyces cerevisiae. After incubation of the enzyme with [2-¹⁴C]mevalonic acid and the fungicide the ratio, radioactivity in 4,4-dimethyl sterols/radioactivity in 4-demethyl sterols is determined. The higher this ratio is, the more efficient is the fungicide as an inhibitor of fungal sterol 14-demethylation. The ratio has been determined for a number of commercial fungicides and two series of triazole compounds. A similar assay system based on the 10,000g supernatant from a rat liver homogenate was also tested but gave an inaccurate assessment of the relative potency of fungicides as inhibitors of fungal sterol 14-demethylation.     

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.