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     

三唑酮及其代谢产物三唑醇在橡胶树植株中的迁移富集行为

三唑酮是一种重要的橡胶树白粉病防治药剂,明确三唑酮及其代谢物三唑醇在橡胶树上的迁移富集行为有利于提高其农药的靶标利用率。本研究基于QuEChERS法,采用气相色谱-串联质谱(GC-MS/MS)技术首次建立了三唑酮和三唑醇在橡胶树植株根、茎、叶中的残留分析方法。采用水培条件下根部施药和土培条件下叶部施药的方式,分别研究了三唑酮及三唑醇在橡胶树植株中的迁移富集行为。结果表明：橡胶树植株可快速将三唑酮代谢成三唑醇,2种药剂均可从植株根部通过茎部迁移至叶部,但在植株各部位中的富集行为具有较大差异,其生物富集系数大小依次为根部>茎部>叶部,且茎部和叶部的转运系数均小于1,表明三唑酮及三唑醇很难在橡胶树植株中向上迁移,易被根部富集;在对橡胶树植株叶部施药时,三唑酮亦可快速代谢成三唑醇,2种农药在植株不同部位的叶片间迁移能力较差。该研究可为优化三唑酮等农药在橡胶树上的施用方案提供重要的数据支撑。     

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.     

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.     

Effect of low temperatures on 2,4-D behaviour in maize plants

The foliar surface of 4-leaf maize plants was found to be poorly wettable and retained 106 μl g^?1 dry matter when sprayed with a U46D (2,4-D formulation) blank. The third leaf retained 141 μl g^?1. A 7-day cold spell (17/9°C) increased retention per unit dry matter by 53% (135% on the third leaf). Cold stress lowered epicuticular wax quantity by 29% on the third leaf. Contact angles of formulated 2,4-D lay between 115 and 125° and were not significantly affected by cold stress. 2,4-D rapidly entered into maize third leaf (66% in 24 h) but migration from it was less than 1.5%. 2,4-D was readily degraded in maize (80% in 72 h). The most abundant metabolite was probably an ester conjugate; little of the hydroxy derivatives were found. Cold stress reduced 2,4-D degradation, and 72 h after treatment the amount of undegraded 2,4-D was 78% higher in cold-stressed maize plants. It was concluded that 2,4-D selectivity in maize results from low spray retention per unit dry matter and active degradation of penetrated herbicide. Cold stress affects both factors.     

Etude du stock en semences d'Echinochloa crus-galli (L.) Beauv. dans le sol des rizières du Gharb (Maroc)

Study of the seed stock of Echinochloa crus-galli (L.) Beauv. in the soil of rice-fields in Gharb (Morocco) Estimates of the numbers of viable caryopses of Echinochloa crus-galli were made on soil samples taken from twenty-six rice-fields in Gharb (Morocco). Fields were chosen according to three factors: soil type, rotation and number of years in rice. The mean value of the stock of E. crus-galli caryopses is 1285 m^?2 to 60 cm depth. Its mean contribution to the total seed stock is 7.2%. The sampling plan permitted analysis of soil effects on the rotation three rice: three alternative crops and of rotation on merja soil (this soil is characterized by the smallest stocks in rotations — three rice: three other crops and three rice crops: one other). The mean distribution of caryopses in the soil profile was 85%, 12.5% and 2.5% respectively in the 0–20, 20–40 and 40–60 cm horizons. Field emergence of seedlings of E. crus-galli. from harvest to the sowing of the following crop, represented 0.65% of the stock in the soil.     

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.     

Analysis of foliar uptake of pesticides in barley leaves: Role of epicuticular waxes and compartmentation

Uptake of pesticides into barley leaves was measured under controlled conditions. Leaves detached from plants were submerged in aqueous solutions of ¹⁴C-labelled (2,4-dichlorophenoxy)acetic acid, triadimenol, bitertanol and pentachlorophenol. Uptake was biphasic. A short (30-min) period with high rates of uptake was followed by uptake that proceeded more slowly and was steady over hours. Compartmentation of pesticides was studied by desorbing pentachlorophenol from leaves previously loaded with [¹⁴C]pentachlorophenol. From the uptake and desorption kinetics it was concluded that penetration of pesticides proceeds as follows: the compounds are first sorbed at the surface of epicuticular wax aggregates where they are in contact with the donor solutions. Solutes then diffuse through the surface wax aggregates into the cuticle. Equilibrium between donor solutions, surface wax and cuticle is established in about 30 min. After this time the amounts of solutes in these compartments no longer increase. Uptake after this time represents penetration into the leaf cells. This fraction of the pentachlorophenol is retained irreversibly, while that sorbed in wax and cutin can be desorbed again. All compounds were sorbed in cuticular waxes and partition coefficients wax/water were determined. On a mass basis only 5 to 10% of the amounts sorbed in cutin are sorbed in wax. This comparatively low solubility in wax contributes to the barrier properties of cuticular waxes. The other determinant of permeability is the very low mobility of solutes in cuticular waxes.     

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.     

ROS and 9-oxylipins are correlated with deoxynivalenol accumulation in the germinating caryopses of Triticum aestivum after Fusarium graminearum infection

Wheat germinating caryopses may represent a starting point for the Fusarium Head Blight disease; however, only few studies concern the defence repertoire of wheat caryopses against fungal challenge. The germinating caryopses of two wheat commercial varieties (Blasco and Sagittario), differentially susceptible to FHB in the field, were inoculated with F. graminearum and the redox status in the interaction milieu, oxylipin production, the expression profile of some host-defence related genes, and programmed cell death in the aleuronic layer, were analysed. In Sagittario, the redox balance was profoundly modified and 9-oxylipins accumulated during fungal contamination. In this variety, F. graminearum produced a high quantity of deoxynivalenol whilst programmed cell death, also through metacaspases activation, was enhanced in the aleuronic layer of its caryopses. In Blasco, the expression of tolerance factors such as Pathogenesis-Related-protein1, glucosyl-transferase and glutathione transferase genes was up-regulated consequent to infection. Results show that unscavenged ROS and 9-oxylipins may be related to deoxynivalenol accumulation in the germinating caryopses of wheat after F. graminearum infection.     

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.     

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。     

Hexagonal clustering of intramembrane particles in the plasmalemma of Ustilago avenae after fungicide treatment

The plasmalemma of sporidia of Ustilago avenae has been investigated by means of the freezeetching technique after treatment with the fungicide, triadimenol (5 μg ml^?1, 10 and 17 hr). The control samples are characterized in the exponential and stationary growth phases by homogeneous plasmatic fracture (PF) and extraplasmatic fracture (EF) faces with a random intramembrane particle (IMP) distribution. Treatment with triadimenol induces clusters of similar size IMPs in hexagonal arrangement. In between the flat clusters the plasmalemma is significantly deformed, showing hemispherical pits and protrusions on both membrane fracture halves. In the stationary growth phase no hexagonal clustering of the IMPs is observed under the described conditions.     

Herbicide penetration across isolated and intact leaf cuticles

Penetration of four herbicides (atrazine, diclofop-methyl, dinoseb and glyphosate-mono(isopropylammonium) salt) was followed through isolated pear leaf cuticles into agar and across intact cuticles of Argenteum (ARG) mutant pea leaves from monosized droplets (300 μ diam.) during 48 h following application. Penetration into pea leaves ranged from low for glyphosatemono(isopropylammonium) (5% of amount applied) and atrazine (14%) to rapid for dinoseb (91%) and diclofop-methyl (82%). A major proportion (90%) of the dinoseb retained in the tissues underlying the site of droplet application migrated across the cuticle and the epidermal layer of cells. In contrast, only a small proportion (13%) of the dinoseb entering the leaf was transported to the tissues surrounding the treatment zone. Corresponding values for diclofop-methyl were 61% and 4% respectively. Initially, dinoseb also penetrated rapidly through isolated pear leaf cuticles, 36% of the applied dose being recovered in an agar receiver disc 4 h after treatment, but subsequent uptake was slower. Penetration of glyphosate-mono(isopropylammonium) also decreased after 4 h whereas atrazine and diclofop-methyl diffused steadily through isolated cuticles up to 216 h after treatment. The incorporation of ‘Tween 20’ or ‘Agral 90’ (1 g litre^?1) into the formulation reduced the penetration of glyphosate-mono(isopropylammonium) through isolated pear leaf cuticles.     

Cereal leaf beetles (Oulema spp., Coleoptera: Chrysomelidae) control following various dates of wheat sowing and insecticidal treatments

In this paper, we report the sowing date and intensity of chemical control on presence of the cereal leaf beetles (CLB) on spring wheat. The CLB monitoring (Oulema melanopus L. and Oulema gallaeciana Voet.) was conducted in May–June of each year, following the announcement of the IOR-PiB in Poznań (Institute of Plant Protection, National Research Institute), that economic threshold levels in Poland of this pest is observed. For the spring sowing dates between (20 March–20 April) and late autumn sowings (21 November–7 December) larger amounts of CLB adults (1.6–1.1) in comparison to early autumn sowings (25 October–02 November) were noticed (0.4). Following the insecticidal seed treatment with imidacloprid, the occurrence of adults was 0.2–0.7 individual per stem, while the use of fungicidal treatment with triadimenol–imazalil–fuberidazol resulted in average 1.1–2.2 individual per stem. The reduction in the number of larvae per stem was 7–10 fold larger following the foliar application (pirimicarb + dimethoate + cypermethrin) compared to the seed treatment with imidacloprid. The lowest grain yield (3.83 t ha^?1) from the spring crop without any insecticide was obtained. Foliar application with blend of pirimicarb + dimethoate + cypermethrin increased the yield crop by 0.41 t ha^?1 (10.7%) and additionally by 0.78 t ha^?1 (20.4%) to compare to the fungicidal control when imidacloprid was used. Wheat seed treatment with imidacloprid and sowing date in autumn showed to be suitable and economically efficient preventive strategies for controlling the pest population.     

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.     

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.     

小麦主要病虫分期混合施药技术

研究结果表明,小麦主要病虫的发生有明显的阶段性,秋苗期主要是纹枯病和地下害虫,返青拔节期主要是纹枯病和麦蜘蛛,两者皆在3月25日前后进入侵染和为害盛期;穗期主要有白粉病、叶锈病和麦蚜,有的年份条锈病流行,它们多在5月1日前后进入盛发期。播种期、返青拔节期和穗期3期,杀虫剂与杀菌剂混合施药具有良好的药效和明显的增产作用,特别是穗期。3期混合施药的最优组合是:播种期,0.08％甲基异柳磷与0.03％三唑醇混合拌种,返青拔节期,甲基硫环磷(175.5g/ha)与三唑醇(75g/ha)混合喷雾;穗期,抗蚜威(45g/ha)与三唑醇(105g/ha)混合喷雾。3期施药经济效益显著,投入产出比1: 12.7～14.1,每ha纯收益1450.5～1797.0元。     

Neurospora crassa对三唑醇的抗药性分子机制研究

 通过紫外光诱变获得Neurospora crassa对三唑醇杀菌剂抗性突变体"Nc70"。用2.5μg/ml三唑醇处理,突变体内甲基甾醇与脱甲基甾醇比例变化幅度远远小于亲本敏感菌株"STA"。突变体对三唑醇的吸收能力与敏感菌株相似,但药剂在体细胞内积累较少。三唑醇处理N.crassa 2 h后,药剂在菌体内的含量趋向稳定,突变体内的药量较敏感菌体低56.1%,而麦角甾醇合成量则高于敏感菌株53.7%。