Freeze-fracture electron microscopy of the plasmalemma of Ustilago avenae treated with systemic fungicides: A quantitative analysis of the intramembrane particles

The interaction of the systemic fungicides triadimefon, nuarimol, and imazalil-nitrate with the plasmalemma of Ustilago avenae was studied using freeze-fracture electron microscopy. These antifungal agents produced alterations in the plasmalemma, such as hemispherical pits and protrusions, deformations of invaginations, and formation of craters or similar structures on the membrane fracture faces. The ultrastructural changes in the plasmalemma were more pronounced after triadimefon and nuarimol treatment than after imazalil-nitrate treatment. Intramembrane particles (IMPs) were aggregated, resulting in particle-free areas which showed some exocytotic activity. A quantitative analysis was performed to determine the topographical distribution of IMPs on the extraplasmatic and plasmatic fracture faces of the plasmalemma. The evaluation revealed statistically significant differences in the membrane structure of untreated and fungicide-treated sporidia.     

Reductive and oxidative metabolism of triazolylmethanes by two basidiomycete fungi

The metabolism of [¹⁴C]triadimefon, triadimenol, diclobutrazol, and the oxo-analogue of diclobutrazol by the basidiomycete fungi Coriolus versicolor and Rhizoctonia solani was studied. Reduction of >C = 0 to >CHOH (activation) was more important for triadimefon than oxo-diclobutrazol. Conversely, oxidation of >CHOH to >C = 0 (deactivation) occurred to a greater extent with diclobutrazol than triadimenol. Formation of primary alcohol metabolites, a possible detoxication route, occurred more with diclobutrazol and its oxo-analogue than with triadimefon and triadimenol. The implications of the reduction/oxidation processes and primary alcohol formation on fungitoxicity of the applied triazolymethane are discussed. The enantiomeric composition of metabolically produced triadimenol is discussed in relation to the in vitro activity of triadimefon supplied against each fungus.     

Metabolism of the 1,2,4-triazolylmethane fungicides,triadimefon, triadimenol,and diclobutrazol,by Aspergillus niger (van Tiegh.)

Metabolism of the triazolylmethane fungicides triadimefon, triadimenol, and diclobutrazol by Aspergillus niger was studied using a replacement culture technique and ¹⁴C substrates. Components of metabolite mixtures were characterized by TLC, GLC, radio-GC, and GC-MS analyses of the free materials and their trifluoroacetate and trimethylsilyl ether derivatives. The three compounds underwent a common metabolic change involving oxidation of C(CH₃)₃ to C(CH₃)₂CH₂OH. In this work the isopropyl analog of triadimefon, previously reported as a metabolite, was an artifact and resulted from nonbiological oxidation of the corresponding primary alcohol. The fungus also reduced triadimefon to triadimenol, giving a mixture of 1R2S, 1S2R and 1R2R, 1S2S diastereoisomers. The less fungitoxic 1R2S, 1S2R triadimenol predominated, so that this conversion may be directly associated with the relative insensitivity of A. niger to triadimefon. Implications of oxidative and reductive metabolism of these fungicides are suggested with particular reference to the differing fungitoxicities of diastereoisomers and enantiomers.     

The enantiomeric composition of triadimenol produced during metabolism of triadimefon by fungi: III. Relationship with sensitivity to triadimefon

Fifteen species of fungi, grown in shake culture in a liquid nutrient medium containing a 50:50 mixture of the 1R and 1S enantiomers of triadimefon, reduced triadimefon to triadimenol, the extent of reduction varying from <1% to >95% according to species. Each produced its own characteristic pattern of two or more of the four possible enantiomers of triadimenol and the stereoselectivity and stereospecificity of the reduction is discussed. The sensitivity of each fungus to individual enantiomers and a mixture of enantiomers of triadimefon and of triadimenol was assessed. Three broadly defined groups of fungi were found: (i) sensitive to both triadimefon and tridimenol, (ii) insensitive to both, and (iii) insensitive to triadimefon but moderately sensitive to triadimenol. In many cases it was possible to predict, at least semiquantitatively, the sensitivity of an organism to triadimefon from a knowledge of its characteristic metabolism to triadimenol and its sensitivity to the individual triadimenol enantiomers. No evidence for synergism or antagonism was found.     

Resistance to inhibitors of sterol biosynthesis in field isolates or laboratory strains of the eyespot pathogen Pseudocercosporella herpotrichoides

Strains of Pseudocercosporella herpotrichoides collected in France on winter wheat give either fast-growing mycelial colonies with regular margins or slow-growing mycelial colonies with irregular margins. Most of the fastgrowing isolates were sensitive to triadimenol (EC₅₀ below 2mg litre^?1), but some of them were resistant to this inhibitor of sterol C-14 demethylation. In contrast, all the slow-growing strains were highly resistant to triadimenol (EC₅₀ greater than 100 mg litre^?1). This resistance was also expressed in inhibition of germ-tube elongation. Positive cross-resistance was observed between most of the inhibitors of sterol C-14 demethylation, with the exception of some imidazole derivatives (clotrimazole, prochloraz). All the fast-growing strains were tolerant to fenpropimorph and fenpropidin whereas the slow-growing ones were susceptible; the reverse was true with piperalin and tridemorph. All the field isolates were inhibited to the same extent by the inhibitors of squalene-epoxidase, nafifine and terbinafine. Two types of mutant resistant to triadimenol have been induced under laboratory conditions from sensitive fast-growing strains. The most common mutants were resistant to all the inhibitors of sterol C–14 demethylation and also in some conditions to fenpropimorph, tridemorph and the inhibitors of squalene-epoxidase. The other mutants were characterised by a reduced spectrum of cross-resistance between triadimenol and the other inhibitors of sterol biosynthesis. The field isolates and laboratory mutants resistant to triadimenol and propiconazole were also resistant to each of the four enantiomers of these two fungicides.     

Inhibition of ergosterol biosynthesis by triadimenol in Ustilago avenae

In Ustilago avenae sporidia, following the first doubling period of about 4 h, triadimenol (2 μg ml^?1) affected sporidial multiplication more severely than other growth processes; daughter cells failed to separate from the parent sporidia resulting in chains of interconnected cells. Triadimenol incubated with the fungus for 8 h interfered neither with respiration nor with protein and nucleic acid synthesis but after 6 h the toxicant had induced a higher content of free fatty acids. Triadimenol markedly altered, both quantitatively and qualitatively, the sterols in sporidia of U. avenae. Incorporation of [¹⁴C]acetate (in the form of sodium acetate) into lipid fractions for a period of 2 h revealed that the toxicant powerfully inhibited the synthesis of the 4-demethyl sterol fraction (predominantly ergosterol), whilst the 4,4-dimethyl sterol fraction rapidly accumulated. This was confirmed by g.1.c. analysis of the sterols after 6 and 8 h incubation which showed that the amount of ergosterol, the major sterol in untreated sporidia, was diminished while simultaneously 4,4-dimethyl, 4-methyl and 14-methyl sterols increased. The accumulation of 14-methyl sterols suggests that triadimenol acts as a potent inhibitor of one of the metabolic steps involved in the demethylation at the 14-position during ergosterol biosynthesis.     

吲哚乙酸引导下三唑醇在大豆植株中的传导与积累研究初报

将吲哚乙酸与三唑醇发生酯化反应后生成的吲哚乙酸三唑醇酯 ,在0.5 mmol/L浓度下水培和喷雾处理6~8叶期大豆植株,同时以相同浓度的三唑醇及其与吲哚乙酸的混合物为对照,色谱法测定不同时间植株不同部位吲哚乙酸三唑醇酯和三唑醇的含量。结果表明,与对照相比,吲哚乙酸三唑醇酯在大豆中具有明显的双向传导和向根部积累的特点。喷雾处理后,在12~60 h之间内吸量和根部积累量均出现最大值。     

Sensitivity of populations ofBotrytis cinerea to triazoles,benomyl and vinclozolin

Sensitivity of field isolates (121) ofBotrytis cinerea from France (1992), Germany (1979–1992), Israel (1990) and the Netherlands (1970–1989) to the triazoles tebuconazole and triadimenol, the benzimidazole benomyl and the dicarboximide vinclozolin were tested in radial growth experiments. Resistance to benomyl (in 21 to 100% of isolates tested) and vinclozolin (in 25 to 71% of isolates tested) was common in most countries. EC₅₀s (concentrations of fungicides inhibiting radial mycelial growth ofB. cinerea on B5-agar by 50%) for tebuconazole and triadimenol ranged between 0.01–1.64 and 0.4–32.6g ml^–1, respectively, and were log-normally distributed. The variation factor (ratio between EC₅₀s of the least and most sensitive isolate tested) amounts 164 and 82 for tebuconazole and triadimenol, respectively. These values are comparable to those for azole fungicides applied in control of other pathogens. Hence, variation in sensitivity to triazoles can probably not explain limited field performance of triazoles towardsB. cinerea. Isolates from south west Germany (1992) were significantly less sensitive to tebuconazole than isolates collected earlier in Germany, Israel and the Netherlands. Such less sensitive populations may contribute to the limited field performance of DMI fungicides towardsB. cinerea. The sensitivity of isolates from south west Germany to tebuconazole was similar to that of DMI-resistant mutants generated in the laboratory. These mutants displayed stable resistance with Q-values (ratio between EC₅₀ of resistant mutant and wild type isolate) between 5 and 20. Sensitivity of field isolates and laboratory mutants to tebuconazole and triadimenol was correlated.     

Sensitivity of Erysiphe graminis f. sp. hordei to ethirimol in relation to field performance in UK1

When introduced as a seed treatment 5 years ago, triadimenol (Baytan) was more effective against Erysiphe graminis f. sp. hordei than was the alternative ethirimol (Milstem). This contrasts with the current situation on the powdery mildew-susceptible cv. Golden Promise, where ethirimol is generally better than triadimenol. In part this reflects a decline in the performance of triadimenol seed treatment, as a result perhaps of some decrease in sensitivity to this fungicide. However, laboratory assays of field isolates also revealed that negatively correlated cross-resistance between triadimenol and ethirimol might exist. In field trials the sensitivity of E. graminis to triadimenol was indeed increased after treatment with ethirimol, but triadimenol hardly altered sensitivity to ethirimol. The possible causes of these changes in fungicide sensitivity are discussed.     

Uptake of triadimenol through wheat caryopsis after application by seed treatment

The uptake of triadimenol was investigated by offering radio-labelled chemical exclusively through the caryopsis in a special experimental design with single seed grains in plexiglass holders. In totally-treated caryopses about 5% and, in partially-treated caryopses (embryo untreated), 4.5% of the applied radioactivity reached the winter wheat shoots after 21 days up to the third leaf stage. Uptake of active ingredient through the pathway pericarp-testa-endosperm-scutellum-seedling up to the third leaf stage D(13) was therefore confirmed. Furthermore, [¹⁴C] triadimenol was also translocated through the caryopses into the roots and was released to the nutrient solution.     

三唑醇对映体的反相高效液相色谱拆分热力学研究

在高效液相色谱反相条件下,利用涂敷型纤维素-三(3,5-二甲基苯基氨基甲酸酯)手性固定相(CDMPC-CSP)对三唑醇4个对映异构体进行了直接手性拆分研究。考察了不同流动相组成和柱温对三唑醇对映体的色谱保留及分离的影响。同时,利用热力学方法对三唑醇对映体与固定相之间的色谱保留和分离的热力学机理进行了探讨。结果表明:在以甲醇-水(70∶ 30,体积比)为流动相、流速0.8 mL/min、柱温15 ℃,或在以乙腈-水(30∶ 70,体积比)为流动相、流速1.0 mL/min、柱温45 ℃的条件下,三唑醇对映体可以得到较好分离。     

Effect of triadimefon and triadimenol on growth of various plant species as well as on gibberellin content and sterol metabolism in shoots of barley seedlings

The fungicides triadimefon and triadimenol markedly reduced growth of coleoptiles, primary leaves, and roots of barley seedlings when grown for 7 days in petri dishes in the dark. The addition of gibberellins (A₁, A₃, A₄, A₇, A₉) alleviated growth retardation of primary leaves and coleoptiles induced by the fungicides. While fungicide-induced growth retardation of the shoots was partly relieved by kinetin, IAA did not show an alleviating activity. Triadimefon and triadimenol also substantially retarded the elongation of shoots of tomato and cotton plants and simultaneous application of GA₃ nullified the retardation. The fungicides only slightly interfered with both α-amylase production of intact germinating barley seed and the GA₃-induced α-amylase synthesis in barley endosperm. On the other hand, extracts of triadimefon- and triadimenol-treated shoot tissue of 10- to 12-day-old barley plants contained substantially lower gibberellin-like activity than control shoots. Both compounds also interfered in sterol metabolism of shoots of barley seedlings when compared to control plants, treatment resulted in lower amounts and altered proportions of C-4,4-desmethyl sterols. While both fungicides inhibited synthesis of C-4,4-desmethyl sterol fraction, sterols possessing C-4 and C-14-methyl groups were accumulated. It is assumed that triadimefon and triadimenol interfere in gibberellin and sterol biosynthesis in barley seedlings by inhibiting oxidative demethylation reactions.     

In-vivo evaluation of adjuvants for more effective control of celery leaf-spot (Septoria apiicola) and powdery mildew (Erysiphe graminis) of wheat with fungicides

The effects of seven adjuvants (at 0, 0.5, 1.0 and 2.0 g litre^?1) on the efficacies of four fungicides al 0.5 g litre^?1 were studied in the laboratory for the control of leaf-spot in celery (caused by Septoria apiicola) and powdery mildew on winter wheat (caused by Erysiphe graminis). The most effective fungicides for controlling leaf-spot were: tebuconazole + triadimenol = flutriafol > mancozeb + oxadixyl > prochloraz. However, addition of adjuvant to the fungicides gave a modified pattern of effectiveness. The efficacy of flutriafol was strongly enhanced by addition of all adjuvants, but those of prochloraz and mancozeb+oxadixyl only partially so. The tested adjuvants were mineral oil + surfactant, a polymer/alkoxylated alkyl ether blend, an ethoxylated alkylphenol, an ethoxylated hexitan ester blend, an ethoxylated nonylphenol and an alkylpolysaccharide- based adjuvant mixture. However, the addition of adjuvants to tebuconazole + triadimenol had a negative effect. Of all the adjuvants tested, the nonylphenol ethoxylate and a mixture of mineral oil /surfactant and alkylpolysaccharides gave the highest efficacy with the fungicides, while the mineral oil/surfactant and the alkylpolysaccharides alone were less effective. There was a positive relationship between high concentrations of adjuvants and their effectiveness, but there were some exceptions. The most effective fungicides for control of powdery mildew in wheat were prochloraz, mancozeb + oxadixyl and tebuconazole + triadimenol. There was a linear relationship between the high efficacy of the fungicide and the concentration of adjuvants to control powdery mildew in wheat. The highest concentration of adjuvant (2-0 g litre^?1) gave the highest efficacy for the fungicides.     

Integrated mite management in apples in Israel: Augmentation of a beneficial mite and sensitivity of tetranychid and phytoseiid mites to pesticides

Augmentative releases of the phytoseiid miteTyphlodromus athiasae were evaluated during summer 1987 in an apple orchard designated to be maintained under an IPM (integrated pest management) regime. Evaluation for establishment and recovery 2 weeks after each release showed complete absence of the mite and high population density of the injurious tetranychid mites. It was suspected that pesticides that had been used to control key pests may have been the cause. Laboratory tests with the pesticides used showed that the insect growth regulators triflumuron and fenoxycarb and the fungicide triadimenol caused only slight mortality of the predacious mitesT. athiasae (0-12%) andPhytoseiulus persimilis (6–10%), but a highly significant reduction in fecundity. Four days after treatment a reduction of 94%, 74% and 100% in fecundity ofT. athiasae, and 2 days after treatment a reduction of 78%, 53% and 80% in fecundity ofP. persimilis was brought about by triflumuron, fenoxycarb and triadimenol, respectively. On the other hand, 4 days after treatment there was a 27%, 16% and 9% increase in fecundity of the phytophagous miteTetranychus cinnabarinus caused by triflumuron, fenoxycarb and triadimenol, respectively. Laboratory and field tests were carried out to evaluate 13 other pesticides as to their selectivity toT. athiasae. The compounds azinphos-methyl, penconazole, vamidothion and diflubenzuron were found to be relatively harmless to this beneficial mite and they replaced the materials mentioned above in the coming seasons in several orchards. This caused recovery in high density of the predacious mite, which allowed reduced application of acaricides and the achievement of integrated mite management.     

A highly mutable major gene for triadimenol resistance in Nectria haematococca var. cucurbitae

Mutants ofNectria haematococca var.cucurbitae with high resistance to triadimenol could be isolated at high frequency. Resistance of 30 such mutants studied, was due to a mutation at thetri-1 locus, that maps in linkage group I, 10 cross-over units from the mating type locus. Thetri-1 mutation has no obvious effects on growth and sporulation on media without fungicide and on virulence for squash seedlings and mature fruit. In addition, a resistant mutant killed all of artificially inoculated seedlings in the presence of triadimenol at a concentration which gave 100% protection against the original wild type strain. Thetri-1 mutants were crossresistant at various levels to triadimefon, bitertanol, and propiconazol, but not to non-triazole SBIs. Their sensitivity to three imidazole derivatives was even higher than that of the wild type strains. This is believed to be the first report of a major gene for resistance to an SBI fungicide.     

Preparations for the treatment of pruning wounds

Benodanil, triadimefon or triadimenol, prepared as solutions (30 g litre^?1) in a mixture of N-methyl-2-pyrrolidone and xylene(1+ 9 by volume), or benodanil, prepared by diluting an emulsified concentrate with water, were applied to the surface of stumps produced by removing branches from apple trees. The active ingredients penetrated in fungitoxic amounts at least 6 cm below the wound surface, except when diluted benodanil e. c. was used. No such penetration occurred when the stumps were treated with acrylic paints containing the same ingredients. Triadimefon and triadimenol persisted within the wood for at least 273 days. No treatment inhibited callus formation but none appeared to enhance it. There were more saprophytic fungi in samples taken from treated than from untreated stumps, and the number of microorganisms present in the wood decreased with distance from the wound surface. Only one basidiomycete fungus was present in wood samples taken 273 days after treatments were carried out.     

Ultrastructure and sterol composition of laboratory strains of Ustilago avenae resistant to triazole fungicides

The fine structure and sterol composition of wild-type and triazole-resistant laboratory strains of Ustilago avenae was investigated by electron microscopic and biochemical methods. The growth rate of the mutants was only slightly affected by a fungicide (triadimefon) concentration of about 0.1 mg/ml, whereas the wild-type cells were completely inhibited. Biochemically the sterol composition of wild-type and triazole-resistant strains did not differ. In freeze-fracture electron microscopy no ultrastructural differences were observed between the different untreated strains (wild and resistant). Filipin labeling allowed the localization of ergosterol in the plasmalemma (PF and EF). Generally, wild-type samples and mutants exhibited a clear pattern of filipin-sterol (FS-) complexes. These results are in accord with the biochemical experiments. Neither a modification of the sterol composition nor an altered localization of sterols seemed to be the prime cause of resistance in U. avenae mutants. Alternative explanations for the resistance mechanism are discussed.     

The enantiomeric composition of triadimenol produced during metabolism of triadimefon by fungi. Part II: Differences between fungal species

The fungi Coriolus versicolor, Cladosporium cucumerinum, Botrytis cinerea and Fusarium culmorum, in shake culture, metabolised triadimefon to triadimenol. Analyses by gas–-liquid chromatography of the triadimenol showed that the fungi produced different qualitative and quantitative combinations of the four possible enantiomers, and that these differences were reproducible. The relationship between the enantiomeric composition of the triadimenol produced and the sensitivity of the test fungi to triadimefon, is discussed in the light of published data on the differences in antifungal activity shown by these enantiomers.     

Levels of resistance of Erysiphe graminis f.sp. hordei to the systemic fungicide triadimenol

A random sample of conidiospores ofErysiphe graminis f.sp.hordei was obtained from the atmosphere above Cambridge, England, by incubating them on barley seedlings exposed on three dates in 1985. The asexual progeny of each spore was tested for its response to a range of doses of a systemic triazole fungicide, triademenol. Principal components analysis of the data revealed that the majority of isolates had a distinct level of response to triadimenol, being resistant to triadimenol at the rate apoplied to seed commercially. The remaining isolates appeared either to be sensitive to the lowest dose of triadimenol used, or to have one of two intermediate levels of resistance. There was a significant increase in the frequency of isolates with higher levels of resistance to triadimenol during 1985. This is likely to have been a response to the continuing widespread use of demethylation inhibitor (DMI) fungicides by British farmers.     

Sensitivities to DMI fungicides in populations of Podosphaera fusca in south central Spain

BACKGROUND: Cucurbit powdery mildew elicited by Podosphaera fusca (Fr.) U Braun & N Shishkoff limits crop production in Spain. Disease control is largely dependent on fungicides such as sterol demethylation inhibitors (DMIs). Fungicide resistance is an increasing problem in this pathogen. To overcome such risk, it is necessary to design rational control programmes based upon knowledge of field resistance. The aim of this study was to investigate the state of DMI sensitivity of Spanish P. fusca populations and provide tools for improved disease management. RESULTS: Using a leaf‐disc assay, sensitivity to fenarimol, myclobutanil and triadimenol of 50 isolates of P. fusca was analysed to determine discriminatory concentrations between sensitive and resistant isolates. As no clearly different groups of isolates could be identified, discriminatory concentrations were established on the basis of maximum fungicide field application rate, 100 mg L^?1 for the three fungicides tested. Subsequently, a survey of DMI resistance was carried out in different provinces located in the south central area of Spain during the cucurbit growing seasons in 2002, 2003 and 2004. Examination of a collection of 250 isolates revealed that 23% were resistant to fenarimol and 7% to triadimenol, the provinces of Almería, Badajoz and Murcia being the locations with the highest frequencies of resistance. By contrast, no resistance to myclobutanil was found. CONCLUSION: Results show that fenarimol and, to a lesser extent, triadimenol have become less efficient for controlling cucurbit powdery mildew in Spain. These are important observations that should lead to reconsideration of the current disease management programmes. Copyright © 2010 Society of Chemical Industry