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 triforine: A nonexistent problem?

Permanent resistance to triforine in Cladosporium cucumerinum was obtained after UV irradiation of spore suspensions and selection of resistant strains in the presence of triarimol or triforine. About 50 strains were examined for mycelial growth on malt agar, viability upon routine subculturing, growth and inhibition by triforine in thin-layer chromatographic bioassays, and virulence towards cucumber seedlings. In addition, sporulation and spore germination, as well as effects of ergosterol were investigated in a restricted number of strains. Although all strains differed considerably from each other in these characteristics, they were quite similar in a reduced overall-fitness. These triforine-resistant mutants of C. cucumerinum might, therefore, have a greatly reduced chance of survival in competition with the wild-type strain, as was actually demonstrated for two strains in greenhouse experiments with mixed-inoculations of cucumber seedlings. Such a reduced chance of survival might also explain why under practical conditions development of resistance to triforine has not been observed yet.The triforine-resistant C. cucumerinum strains showed cross-resistance to a number of systemic fungicides known or supposed to inhibit ergosterol biosynthesis, viz. Denmert, fenarimol, imazalil, triadimefon and triarimol. The practical implications and potential hazards of such cross-resistance for the use of already established fungicides are discussed.     

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.     

Association of heat shock induced resistance to disease with increased accumulation of insoluble extensin and ethylene synthesis

Cucumber seedlings (Cucumis sativus L. cv Marketer), heat-shocked for 40 s at 50 °C, acquire disease resistance to the fungal pathogen Cladosporium cucumerinum E11. and Arth. The resistance develops 15 to 21 h after heat shock treatment and is associated with stimulated ethylene production and changes in the plant cell wall. There was a two-fold increase in the production of ethylene and in its precursor 1-aminocyclopropane-1-carboxylic acid by 6 h after the heat shock. Heat shock also enhanced the accumulation of insoluble extensin, a hydroxyproline-rich glycoprotein found in cell walls of seedlings. A heat shock treatment of seedlings 24 h before inoculation with C. cucumerinum resulted in a doubling of extensin content at 72 h after the inoculation. Cell walls from heat shocked seedlings were up to 55% more resistant to degradation by enzymes from C. cucumerinum than were cell walls from unshocked seedlings, but increased lignin depositions did not appear responsible. The accumulation of extensin after heat shock and its crosslinking by peroxidase is discussed as a possible mechanism of induced resistance to C. cucumerinum.     

Metabolism of some sterol inhibitors in fungi and higher plants,with special reference to the selectivity of fungicidal action

The metabolism of triforine, chloraniformethan, the α-(pyrimidin-5-yl)benzhydryl alcohols (fenarimol, nuarimol and triarimol), the trityl-azoles (fluotrimazole and clotrimazole), and the morpholine types of sterol inhibitors is reviewed; the metabolism of the azolyl-alkane derivatives (mainly triadimefon and triadimenol) is discussed in detail. Redox and hydrolytic reactions are of primary importance. Enzymic inactivation may be one factor influencing fungicide selectivity. Metabolism is the dominant factor of selectivity if it represents the activation process, as with triadimefon. Transformations in higher plants do not differ significantly from those occurring in fungi, except that factors such as the formation of conjugates with natural compounds of plant tissues also play a role, as with triforine and triadimenol. The selectivity of fungitoxic action may be influenced by metabolism both in the host plant and the pathogen.     

Simultaneous resistance in fungi to ergosterol biosynthesis inhibitors and dicarboximides

Several strains ofAspergillus nidulans, Cladosporium cucumerinum andPenicillium italicum with known resistance to ergosterol biosynthesis inhibitors were tested for resistance to three dicarboximides. Negligible levels of resistance to iprodione and vinclozolin were observed in one out of three strains ofA. nidulans. Two out of three strains ofC. cucumerinum displayed a low resistance to iprodione, and a high resistance to procymidone and vinclozolin. The latter strains were also moderately resistant to the isoflavonoid phytoalexins medicarpin and pisatin, but sensitive to the antibiotic pimaricin. All sixP. italicum strains examined displayed wild-type sensitivity to all three dicarboximides; the two of these tested in thin-layer chromatographic bioassays proved to be resistant to pimaricin.Iprodione and vinclozolin induced energy-dependent fenarimol efflux inA. nidulans. In line with this observation, in crossed-paper strip assays iprodione and fenarimol antagonized each other in their toxicity towardsA. nidulans; towardsC. cucumerinum, on the other hand, these fungicides behaved independently.The implications and practical consequences of the phenomena observed are briefly discussed.Samenvatting Verscheidene tegen ergosterolbiosyntheseremmers resistente stammen vanAspergillus nidulans, Cladosporium cucumerinum enPenicillium italicum werden getoetst op resistentie tegen dicarboximiden. Eén der drie onderzochte stammen vanA. nidulans bezat enige resistentie tegen iprodione en vinchlozoline. Twee van de drie onderzochte stammen vanC. cucumerinum vertoonden een lage graad van resistentie tegen iprodione en een zeer hoge tegen procymidone en vinchlozoline. Ze waren ook enigermate resistent tegen de fytoalexinen medicarpine en pisatine, maar gevoelig voor het antibioticum pimaricine. Alle onderzochte stammen vanP. italicum waren voor de drie getoetste dicarboximiden even gevoelig als het wild-type; voorzover onderzocht, bleken deze stammen resistent tegen pimaricine.Iprodione en, hoewel in mindere mate, vinchlozoline induceerden energieafhankelijke efflux van fenarimol inA. nidulans. In overeenstemming hiermee antagoneerden iprodione en fenarimol elkander in hun activiteit ten opzicht vanA. nidulans. Ten opzichte vanC. cucumerinum gedroegen deze fungiciden zich onafhankelijk van elkaar.De practische consequenties van de waargenomen verschijnselen worden kort aangeduid.     

Allelism of theFcu-1 andFoc genes conferring resistance to fusarium wilt in cucumber

The inheritance of resistance toFusarium oxysporum f.sp.cucumerinum race 1 was determined in the cucumber cv. WIS-248 by analyzing segregation of F₁, F₂, and BC populations of crosses with the susceptible cv. Straight-8. Resistance was conferred by a single dominant gene. In an allelism test, it was proven that theFcu-1 gene, which confers resistance toF. oxysporum f.sp.cucumerinum races 1 and 2 in cucumber cv. SMR-18 and theFoc gene, which confers resistance toF. oxysporum f.sp.cucumerinum race 2 in cucumber cv. WIS-248, are indistinguishable.     

Metabolism of a phosphoramidate by Pyricularia oryzae in relation to tolerance and synergism by a phosphorothiolate and isoprothiolane

Metabolism of dibutyl N-methyl-N-phenylphosphoramidate (BPA) by mycelial cells of Pyricularia oryzae was studied to elucidate the mechanism of synergism and negatively correlated cross-resistance in fungicidal action between phosphoramidates and phosphorothiolate derivatives. Rapid metabolism of BPA by a wild-type strain through hydroxylation and N-demethylation was observed. The metabolism was inhibited by diisopropyl S-benzyl phosphorothiolate (IBP; Kitazin P) and by isoprothiolane (diisopropyl 1,3-dithiolan-2-ylidenemalonate; Fuji-One). This inhibition of BPA metabolism is probably the mechanism of synergistic fungicidal action between the phosphoramidate and the thiol derivatives. The metabolism was, however, not inhibited by S-1358 (S-butyl S′-p-tert-butylbenzyl N-3-pyridyldithiocarbonimidate; Denmert) or triarimol [α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidinemethanol; EL-273], both of which are considered to be inhibitors of hydroxylation of a methyl radical in ergosterol biosynthesis. The metabolism of BPA by P. oryzae was much slower when mutants selected for IBP resistance and for isoprothiolane resistance were used. This phenomenon probably explains the differential sensitivity to phosphoramidate of wild-type strains and mutants resistant to the thiol derivatives.     

Induced resistance to<Emphasis Type="Italic">Cladosporium cucumerinum</Emphasis> in cucumber by pectinases extracted from<Emphasis Type="Italic">Penicillium oxalicum</Emphasis>

Pectinases extract (PE) from the fermentation product ofPenicillium oxalicum BZH-2002 was tested for its ability to induce protection against scab caused byCladosporium cucumerinum on cucumber (Cucumis sativus L.) plants. Seedlings with one true leaf were sprayed with various concentrations of PE (10–200 units ml⁻¹) 3 days before inoculation withC. cucumerinum. Results showed that the induced local protection against the pathogen was dose-dependent when the concentrations of PE were between 20 and 120 units ml⁻¹; systemically induced resistance against the pathogen was not observed. Boiled PE had a slight effect on disease reduction. Commercial pectinases prepared fromAspergillus niger showed lower protection against scab compared with PE when they were used at the same concentration of enzyme activity. No inhibitory activity was observed on conidial germination or germ-tube growth ofC. cucumerinum. PE was further evaluated for its enhancement of defense-related enzymes. Peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) increased in cucumber seedlings after treatment with PE. PO and PPO remained at a higher level in PE-pretreated seedlings throughout the experiment period whether pathogen-inoculated or non-inoculated, whereas PAL activity began to decrease 2 days after PE treatment. http://www.phytoparasitica.org posting July 14, 2004.     

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.     

Response of transgenic cucumber expressing a rice class I chitinase gene to two fungal pathogens with different infectivities

A transgenic cucumber line (CR32) over-expressing the rice class I chitinase gene exhibited resistance to Phytophthora rot (Phytophthora nicotianae var. parasitica) but not to Fusarium wilt (Fusarium oxysporum f. sp. cucumerinum). The infection behavior of these fungi on CR32 and nontransgenic plants was examined with an optical microscope. In zoosporangia of P. nicotianae var. parasitica, the rates of germination and penetration on leaves of both CR32 and nontransgenic plants were almost equal. After infection, however, the growth of infection hyphae was markedly suppressed in CR32 compared with their growth in the nontransgenic plants. In F. oxysporum f. sp. cucumerinum, the infection hyphae localized in petiole vessels of both CR32 and nontransgenic plants, and growth did not differ in the two plants. We investigated the antifungal activity of a high-molecular-weight fraction (HF) and a low-molecular-weight fraction (LF) of crude leaf extracts from CR32 and from the nontransgenic line. CR32 HF, which included the rice chitinase, had antifungal activity only against F. oxysporum f. sp. cucumerinum. In contrast, CR32 LF, which did not have the rice chitinase, had strong antifungal activity against the two fungi. These results suggested that a low-molecular-weight antifungal substance(s) was induced in CR32 and might function as a factor of resistance to P. nicotianae var. parasitica, which has cell walls that almost never contain chitin. Because rice chitinase has already been demonstrated not to localize in vessels of CR32, the infection localization of F. oxysporum f. sp. cucumerinum in vessels might enable the fungus to avoid antifungal substance(s), resulting in Fusarium wilt in transgenic cucumber.     

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.     

Metabolism of [3H]triforine in barley grown in the field: The unbound radioactive residue

Barley was sown and grown normally in an experimental field. At the growth stage J,

1 Growth stage J: during the stem extension stage; second node of stem formed and next-to-last leaf just visible.

the aerial part of the plants was sprayed with an aqueous emulsion of a mixture of triforine and [³H]triforine (uniformly labelled in the piperazine ring) using the recommended dose rate of about 240g triforine ha^?1. The barley was harvested when ripe and the straw and grain were analysed separately. The total radioactivity concentration was 20 times higher in straw than in grain. In straw and grain, 12 and 25% respectively of the total incorporated radioactivity in each of these tissues was methanol soluble. The composition of the methanol-soluble radioactive residue was investigated and was shown to contain triforine and its metabolites which were free and unbound in barley straw and grain. No radioactive piperazine was observed, in spite of the high detection sensitivity for radioactivity. The concentrations of triforine and its identified metabolites in straw and grain respectively (mg kg^?1, relative to the fresh weight of tissue) were: triforine, 0.034 and 0.0018; N-[(2,2,2-trichloro-1-(piperazin-1-yl)ethyl]formamide), 0.009 and 0.0006; iminodiacetic acid, 0.021 and 0.001; glycine, 0.043 and 0.0033. Other radioactive water-soluble and very polar, unidentified compounds were observed, corresponding to advanced metabolic products of triforine.     

Control of Fusarium oxysporum f. sp. cucumerinum of cucumbers by soil solarization with impermeable plastics and/or reduced doses of methyl bromide*

Soil solarization is not broadly adopted as a soil deinfestation method mainly because of its long duration (4–6 weeks). We present evidence showing that the duration of solarization can be reduced to nearly half using impermeable plastics and/or low doses of methyl bromide, while still ensuring effective control of Fusarium oxysporum f. sp. cucumerinum. Chlamydospores of a pathogenic isolate of F. o. cucumerinum, formed in sterile soil, were inserted into nylon mesh envelopes and incorporated into the soil prior to treatment at 20‐ and 30‐cm soil depths. Soil treatments included untreated control, soil solarization with polyethylene or impermeable plastics (LMG), and soil solarization with polyethylene or impermeable plastics plus 20 g m^?2 methyl bromide. According to the effects on artificial inocula of F. o. cucumerinum checked at weekly intervals for 4 weeks, soil solarization with impermeable plastics was most effective in destroying pathogen populations even two weeks after soil covering.     

Experiments with some of the newer systemic fungicides on apple and marrow

In field trials against apple scab (caused by Venturia inaequalis) and powdery mildew (caused by Podosphaera leucotricha), improved control of one or other disease has been given by the newer systemic fungicides benomyl, triarimol and the thiophanates. However, in tests on apple seedlings in the greenhouse, biological evidence of translocation from individual deposits was generally limited to movement within the treated leaf. In greenhouse tests with cucumbers and marrows, using Oidium sp., there was again little evidence for movement of toxicant from a treated leaf, although effective disease control was readily obtained by root application of several compounds at low dosage. These results suggest that the systemic properties of the compounds evident when used as soil or seed treatments are of little account when they are applied as foliar sprays.     

Fungicide resistance in diseases of vegetables growing under plastic in Crete1

Serious fungicide resistance problems have been encountered in several major vegetable pathogens in glasshouses in Crete. Botrytis cinerea populations are now widely resistant to either or both benzimidazoles and dicarboximides. Pseudoperonospora cubensis very rapidly became resistant to metalaxyl, with cross resistance to other acylalanines. Didymella bryoniae has shown some benzimidazolc resistance. Sphaerotheca fuliginea, against which a great diversity of fungicides is used, has mostly not given problems. However, strains with resistance to benomyl, triforine and ditalimfos have been found in experimental plots. Ways of managing resistance in Crete are discussed.     

Inheritance and Mechanisms of Resistance to Tebuconazole, a Sterol C14-Demethylation Inhibitor, inNectria haematococca

Genetic control and mechanisms of resistance to tebuconazole, a sterol C14-demethylation inhibitor, were investigated in the phytopathogenic fungusNectria haematococca. Resistant mutants have been selected from the laboratory, following UV irradiation. They have been characterized through genetic crosses and mutations in at least three genes were found to be responsible for resistance. The genesTeb1, Teb2, andTeb3were clearly identified, a fourth gene calledTeb4could be hypothesized. Mutations at lociTeb2andTeb3induced pleiotropic effects such as reduced sporulation and growth rate, mycelium pigmentation (Teb2), or altered ascospore viability (Teb3). The resistance levels determined by mutations in the different genes were relatively low (below 10). When associated in double mutants, the additive effect was recorded. Cross-resistance toward other sterol C14-demethylation inhibitors was observed in all the resistant strains, except in theTeb4-carrying strain; moreover, for some C14-demethylation inhibitors hypersensitivity was expressed. A constitutive energy-dependent efflux seemed implicated in the mechanism of resistance for theTeb1-carrying strain and probably also for theTeb2andTeb3-carrying strains. However, theTeb4-carrying strain exhibited a kinetic of fungicide uptake similar to that of the wild-type strain. The sterol profile of theTeb4-carrying strain was similar to that of all the other resistant mutants and wild-type strains. Thus the resistance mechanism induced by mutation at theTeb4locus has not been found yet.     

Adsorption of systemic fungicides and a herbicide by some components of plant tissues,in relation to some physicochemical properties of the pesticides

Lignin, an important component of plant tissues, adsorbed five systemic fungicides and one herbicide (carbendazim, triadimefon, nuarimol, triarimol, fenarimol and fluometuron) more strongly than bovine serum albumin, cellulose, ethylcellulose or sodium polygalacturonate. Significant correlations were found between the extent of the adsorption of the pesticides on the lignins extracted from three different plant species, and the log Poet (Poet is the octan-1-ol/water partition coefficient) of the compounds. The more lipophilic fungicides triarimol and fenarimol (log Poet about 2.6) were adsorbed to the greatest extent. Fluometuron, triadimefon and nuarimol (log Poet about 2.0) were moderately adsorbed, whereas carbendazim with the lowest log Poet (1.34 at pH 5.0), was adsorbed more than expected from its Poet value. The anomaly of carbendazim is discussed; it is ascribed to it's partial protonation at pH 5.0, whereas the other pesticides were non-ionised.     

Characterisation of bound residues of [3H]triforine in the straw of barley grown in the field

Barley was grown in an experimental field and, at the growth stage J (during the stem extension stage when the second node of the stem was formed and the next-to-last leaf just visible), the aerial part of the plants was sprayed with an aqueous emulsion of a mixture of triforine and [³H]triforine (uniformly labelled in the piperazine ring) at the recommended rate of 250 g triforine ha^?1. Barley was harvested when ripe, and the straw was analysed separately. Extraction of the straw with methanol left methanol-insoluble solids containing an amount of radioactivity (the bound residue) which represented 88 % of the tritium (as with all the subsequently quoted percentages, relative to the total tritium incorporated into the straw). Methanol acidified with hydrochloric acid extracted a further 8% of the triforine-derived bound residues as radioactive iminodiacetic acid (0.4%). glycine (3.2%), serine (2.0%), ethanolamine (0.2%) and unidentified compounds (2.2%); a neutral detergent solution extracted a further 3% of the radioactive triforine-derived bound residues as unidentified compounds; these radioactive compounds, which were dissolved by the acidified methanol and by the neutral detergent solution, were thus complexed in the straw to straw constituents. An acid detergent solution extracted a further 58% of the total tritium, which was incorporated by means of σ chemical bonds into the hemicelluloses fraction. Acid hydrolysis of the hemicelluloses yielded a mixture of mono-saccharides; these were converted into a mixture of osazones (50%), recrystallised several times, and had a constant specific radioactivity, indicating that the mono-saccharides were tritiated. The plant solids (which contained 19% of the total tritium), left by the acid-detergent extraction, were processed and separated into the tritium-containing cellulose (13%) and lignin (6%) fractions. No piperazine was observed in the bound residue of straw. Biochemical pathways are suggested for the radioactive metabolites of [³H]triforine.     

Mode of action of triarimol in Ustilago maydis

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.