Metabolism of fungicidal cyanooximes, cymoxanil and analogues in various strains of Botrytis cinerea

BACKGROUND: The metabolism of cymoxanil [1‐(2‐cyano‐2‐methoxyiminoacetyl)‐3‐ethylurea] and fungicidal cyanooxime analogues was monitored on three phenotypes of Botrytis cinerea Pers. ex Fr. differing in their sensitivity towards cymoxanil. For this purpose, labelled [2‐¹⁴C]cymoxanil was added either to the culture medium of these strains or to its cell‐free extract. RESULTS: In the culture medium of the most sensitive strain, four main metabolites were detected. Three were isolated and identified. Cymoxanil was quickly metabolised by at least three concurrent enzymatic pathways: (i) cyclisation leading, after hydrolysis, to ethylparabanic acid, (ii) reduction giving demethoxylated cymoxanil, (iii) hydrolysis followed by reduction and then acetylation leading to N‐acetylcyanoglycine. In the cell‐free extract of the same strain, only the first and the second of these enzymatic reactions occurred. By comparing the metabolic profile of the most sensitive strain with that of the less sensitive ones, it was shown that the decrease in sensitivity to cymoxanil correlates with a reduced acetylcyanoglycine formation. Among all metabolites, only N‐acetylcyanoglycine is active against the most sensitive strain. Moreover, in a culture of this strain, two other fungicidal cyanooximes were also metabolised into this metabolite. CONCLUSION: The formation of N‐acetylcyanoglycine may play an important role in the fungitoxicity of cymoxanil and cyanooxime derivatives. Copyright © 2008 Society of Chemical Industry     

Synergism between fungicides with different mechanisms of action against acylalanine-resistant strains of Phytophthora spp.1

The authors have studied the efficacy of two systemic compounds, cymoxanil and propamocarb, used as partners of acylalanines, in limiting mycelial growth of acylalanine-sensitive and resistant strains of Phytophthora capsici and P. nicotianae var. parasitica. The fungicidal activity of the single components was compared to that of the different mixtures, at various ratios. The degree of synergy was calculated using the method of Wadley. The results obtained indicate synergistic interactions of various intensities: synergistic factors (SF) up to seven were found. The highest synergistic effects were observed in the case of resistant Phytophthora strains.     

Influence of pyrethroid ester,oxime ether,and other central linkages on insecticidal activity,hydrolytic detoxification,and physicochemical parameters

The relative potency to target and nontarget insects of ester pyrethroids and the analogous oxime ethers, and the degree of synergism of the esters with tributyl phosphorotrithioate, provide a useful guide to the importance of esterase detoxification in species specificity. These criteria indicate that esterase detoxification is a more important component of pyrethroid tolerance in Chrysopa carnea and Cryptolaemus montrouzieri larvae than in Exochomus flavipis larvae and Musca domestica adults. Studies with 3-phenoxybenzyl 2-(4-chlorophenyl)-2-cyclopropylacetate and the corresponding 2,3,4,5,6-pentafluorobenzyl ester, their oxime ether analogs, and permethrin and its thiol ester and amide analogs provide evidence that the high insecticidal activity of some ester and E-oxime ether pyrethroids, relative to that of the corresponding thiol ester and amide, is more closely associated with the dipole moment and polarizability of the central linkage and its resistance to esterase detoxification than with bond lengths or lipophilicities conferred by a specific linkage. Pentafluorobenzyl tetramethylcyclopropanecarboxylate is very effective in the vapor state for house fly control.     

Sensitivity ofPhytophthora Infestans to cymoxanil

The efficacy of cymoxanil, Pulsan (oxadixyl + mancozeb + cymoxanil, 1:7:0.4) and Sandocur-M (oxadixyl + mancozeb + cymoxanil, 1:7:2) in controlling late blight was examined in potatoes (cv. ‘Alpha’). Nine oxadixyl-sensitive and seven oxadixyl-resistant isolates ofPhytophthora infestons from six countries were tested. ED₉₀ values of cymoxanil ranged between 64 and 467 mg a.i./l. No relationship was observed between sensitivity to cymoxanil and sensitivity to oxadixyl. Isolate S6A (1305) from California was the most sensitive to cymoxanil (ED₉₀ = 64 mg/l), whereas isolate R11 from Israel was the least sensitive (ED₉₀ = 467 mg/l). Most isolates from Israel were less sensitive (ED₉₀ = 151–467 mg/l) than isolates from Switzerland, the Netherlands, Ireland, Mexico or California (ED₉₀ = 64–152 mg/l). Three-way mixtures of cymoxanil with mancozeb and oxadixyl were more inhibitory than cymoxanil alone, regardless of the isolate. Sandocur-M was, for most isolates, more effective than Pulsan. The correlation coefficient between sensitivity to cymoxanil and sensitivity to Sandocur-M was 0.67, while that between cymoxanil and Pulsan was 0.76.     

Sensitivity of European Plasmopara viticola populations to cymoxanil

Between 1995 and 1997, 278 grape downy mildew (Plasmopara viticola) populations originating from European vineyards were characterised for their sensitivity to cymoxanil in a leaf-disc assay. The sensitivity profile revealed a wide distribution, with minimum inhibitory concentrations (MIC) ranging from 10 to more than 800 mg litre⁻¹. EC₅₀ values ranged from 1 to more than 800 mg litre⁻¹ with an average of 125 mg litre⁻¹. The sensitivity distribution was stable between 1995 and 1997. Surprisingly, populations from Portugal appeared significantly more sensitive than those from France or Italy, which could not be linked to differential cymoxanil usage in these countries. P viticola populations collected outside Europe and never exposed to cymoxanil appeared significantly more sensitive than exposed European populations, with an average EC₅₀ value of 10 mg litre⁻¹. The level of sensitivity of European P viticola populations was relatively unaffected by the number of cymoxanil applications made during a season or by the number of years of cymoxanil use. No link was found between the level of sensitivity in the leaf-disc assay and the level of performance of the cymoxanil mixtures used in the fields where the populations originated. Specific field trials conducted in Italy and Portugal have shown that the performance of cymoxanil-based mixtures remained good even on populations of the grape downy mildew fungus characterised as less sensitive in the leaf-disc assay. While there are no baseline sensitivity data for pre-commercialisation P viticola populations, the results of our study suggest that a shift in sensitivity (12.5-fold) may have occurred in some areas since introduction of cymoxanil on grapes nearly 20 years ago. Because cymoxanil is never used alone, it is difficult to determine whether or not practical resistance is occurring in European vineyards. © 1999 Society of Chemical Industry     

抑制番茄早疫病菌活性化合物的 高通量筛选方法研究

以OD 630 值作为评价指标,在高通量筛选设备上建立了番茄早疫病菌Alternaria solania的高通量定量筛选方法,并对嘧菌酯、百菌清、 NFDA1 醚唑、霜脲氰、甲霜灵、代森锰锌、福美双和咪鲜胺8种常用杀菌剂对番茄早疫病菌孢子萌发和菌丝生长的抑制效果进行了评价,结果与孢子萌发法和目测筛选法所得结论基本一致。     

Synthesis of a new class of pyridine herbicide

A new class of fluoroalkylated pyridine herbicides has been discovered. The 3,5-pyridinedicarboxylates are prepared either from ethyl 4,4,4-trifluoroacetoacetate via Hantzsch dihydropyridine synthesis or from trifluoroacetonitrile via cyclocondensation and carboxylation. Hydrolysis of the ester groups, followed by derivatization of the resulting acids yields a variety of herbicidally active analogs including ester, thioester, amide, halogen, amino, alkoxy and alkylthio derivatives.     

江西省辣椒疫霉病菌的交配型及对霜脲氰敏感性的测定

由辣椒疫霉病菌Phytophthora capsici引起的辣椒疫病是辣椒生产上一种重要病害,在我国各地均有发生.辣椒疫霉是一种异宗配合的卵菌.异宗配合的疫霉菌的不同交配型菌株成对培养时,可以形成有性器官并发生基因重组,使该种病菌获得更强的生存能力、致病力和更广泛的寄主范围.因此,研究疫霉交配型在理论和实践上均具有重要意义.霜脲氰(cymoxanil)由美国杜邦公司开发,用于卵菌纲病害的防治.自投入市场使用,至今未发现在自然条件下关于疫霉菌对霜脲氰抗药性报道.     

Studies on the mechanism of action of cymoxanil in Phytophthora infestans

Colony growth and germ tube emergence of sporangia and encysted zoospores of Phytophthora infestans were highly sensitive to cymoxanil (ED₅₀ 0.5–1.5 μg/ml), whereas differentiation of sporangia and zoospore release were insensitive at concentrations up to 100 μg/ml. Treated sporangia did not show distorted germ tubes. Oxygen consumption for glucose oxidation by germinating sporangia and zoospore motility were not inhibited at concentrations up to 100 μg/ml. Cymoxanil hardly affected the uptake of radiolabeled precursors of DNA, RNA, and protein at concentrations up to 100 μg/ml. Incorporation of [¹⁴C]phenylalanine into protein was completely insensitive. RNA synthesis as measured by [³H]uridine incorporation was differentially inhibited in the various developmental stages of the fungus. Inhibition did not occur at differentiation of sporangia, whereas at cyst and sporangial germination and mycelial growth this process was inhibited 20–45% at a concentration of 100 μg cymoxanil/ml. Endogenous RNA polymerase activity of isolated nuclei was not inhibited by cymoxanil. DNA synthesis as measured by [methyl-³H]thymidine incorporation was inhibited 20–80% at the various stages of development at cymoxanil concentrations higher than 10 μg/ml. Metalaxyl, a specific inhibitor of ribosomal RNA synthesis, inhibited [³H]uridine incorporation 40–60% at all developmental stages. The data suggest that although DNA synthesis is affected more than RNA synthesis, inhibition of both biosynthetic processes is a secondary effect. The primary mode of action of cymoxanil thus remains unknown.     

Microsomal metabolism of juvenile hormone analogs in the house fly,Musca domestica L.

Of six juvenile hormone analogs of the alkyl 3,7,11-trimethyl-2,4-dodecadienate type, only the isopropyl ester was strongly morphogenic in the house fly, Musca domestica L. In vitro assays revealed that house fly microsomes contain B-esterases as well as oxidases which metabolize such analogs. However, these esterases did not hydrolyze the isopropyl ester, ZR-515. Enzymes prepared from larvae, pupae, and adults were all active and there was evidence that in the late larval stage the esterase activity was cyclic, showing a minimum in the early third instar and a maximum a few hours later. When microsomes from two susceptible and two resistant house fly strains were compared for metabolic activity against the juvenile hormone analogs, those from the resistant strains were 1.3 to 20 × higher in oxidase activity but there was no difference in esterase activity. The oxidative metabolism of two analogs ZR-515 and 512 was greatly enhanced when the flies were induced with phenobarbital but there was no enhancement in metabolism of three of the remaining analogs and only a slight enhancement of a fourth. It is concluded that the insecticidal action of ZR-515 is largely due to its stability in the presence of the house fly esterases.     

疫霉菌对霜脲氰抗性遗传及对霜脲氰和甲霜灵的交互抗性

就疫霉菌对霜脲氰抗性的产生和遗传，以及疫霉菌对霜脲氰和甲霜灵的交互抗性进行了研究。结果表明，苎麻疫霉容易对霜脲氰产生抗药性突变，但抗性在连续３代单游动孢子分离过程中逐渐丧失。大雄疫霉对霜脲氰不易产生抗性突变，恶疫霉、大雄疫霉和苎麻疫霉对甲霜灵和霜脲氰不存在交互抗性     

Characterization of acetylcholinesterases of acaricide resistant and susceptible strains of the cattle tick Boophilus microplus (Can.): II. The substrate specificity and catalytic efficiency of the critical enzyme component

Concentrations and turnover numbers have been determined for the critical acetylcholinesterase component of the organophosphorus susceptible Yeerongpilly and resistant Biarra and Ridgelands strains. The lower specific activity for the substrate acetylthiocholine in the resistant strains was shown to be due to decreased catalytic efficiency and not to any change in the titer of the enzyme. Thioester analogs of acetylthiocholine, with additional methylene groups between the carbonyl carbon and the quaternary nitrogen of the molecule and in the alkyl moiety, have been used to derive K_m and V_max values for the enzymes from each strain. An hypothesis is proposed concerning the alteration of the active site that has occurred to produce the dissimilar aberrant enzymes of the resistant strains and the implications of this hypothesis for inhibitor design are discussed.     

Effects of alkyl groups attached to the carbamoyl nitrogen atom of an N-arylcarbamoylpyrazoline on insecticidal activity

N-Alkylated analogs (C₁–C₃) of an N-arylcarbamoylpyrazoline were prepared. The introduction of these alkyl groups completely changed the crystal structure in respect of the torsion angle of the amide C–N bond of the non-alkyl compound. The introduction of methyl and ethyl groups slightly, and that of an isopropyl group markedly, decreased insecticidal activity against American cockroaches and house flies. Conformational analyses of the compounds suggested that the insecticidally active conformer of this class of compounds is in the anti-position regarding the N′–C(=O) and N-aryl bonds in which the non-alkyl compound is energetically the most stable. The most stable conformers of the alkylated compounds are in the syn-position, and these compounds would interact with target sites in the less stable anti-form. © 1999 Society of Chemical Industry     

Fungicidal activity of N-(2-cyano-2-methoximinoacetyl)amino acids and their derivatives

More than 20 N-(2-cyano-2-methoximinoacetyl)amino acids and derivatives were synthesised and tested for antifungal activity against grape downy mildew, caused by Plasmopara viticola (de Bary) Berl. & de Toni, and rape downy mildew, Peronospora parasitica Fr. Two of the compounds containing a free carboxylic acid group, a moiety which has been shown to confer phloem mobility on compounds, showed high activity, especially against P. parasitica, in protectant tests. These results indicate that there is no incompatibility between the acid function and fungicidal activity. A number of the esters showed activity comparable with that of cymoxanil in the protectant tests, and the tert-butyl esters of the methionine derivative and its sulfone were more than ten times as active as the commercial compound.     

Characterization of European Plasmopara viticola isolates with reduced sensitivity to cymoxanil

Cymoxanil has been used for over 30 years to control grape downy mildew (Plasmopara viticola) in European vineyards, prevalently in mixture with other fungicides active on this disease. In the 1990’s cases of P. viticola resistant to cymoxanil were detected using a leaf disc assay. In this study, we establish that the presence of only 1 % of resistant isolates in a P. viticola population will allow the detection of cymoxanil resistance in the leaf disc assay. A poor correlation (R?=?0.194) was observed between the leaf disc assay and a whole- plant test for 38 P. viticola field populations collected in 2004. Over 60 % of these populations were characterized as fully sensitive in a whole-plant assay compared to 10 % in the leaf disc assay. Five P. viticola field isolates resistant to cymoxanil reverted to full sensitivity after six to nine transfers to untreated vines, indicating that cymoxanil resistance in P. viticola is unstable. Two European P. viticola populations sensitive to cymoxanil became resistant when transferred 12–14 times on vines treated with cymoxanil. In contrast, two populations originating from the USA and three monozoospore isolates from France retained full sensitivity to the fungicide after 13 cycles on cymoxanil-treated plants. Whole-plant experiments were conducted in the laboratory to compare the efficiency of spray programs to delay the development of cymoxanil resistance. Whereas the continuous use of cymoxanil alone quickly selected for resistance, the mixture of cymoxanil and folpet applied either continuously or in strict or block alternation effectively prevented the development of resistance over 10 generations of the fungus. These results demonstrate that resistance to cymoxanil in P. viticola can be managed with appropriate spray programs.     

Effects of Bacillus subtilis metabolites on larval Aedes aegypti L

The culture supernatant of a strain of Bacillus subtilis isolated from soil samples killed larvae of the mosquito Aedes aegypti. The metabolites produced by B. subtilis were characterized using high performance liquid chromatography (HPLC). Mortality rate was dose-dependent for all larval instars of A. aegypti. Log probit analysis (95% confidence level) revealed an LC₅₀ of 1.73 and an LC₉₀ 3.71 μg/ml. Molecular weights/masses of B. subtilis metabolites were confirmed using SDS–PAGE analysis. B. subtilis metabolites were confirmed using HPLC analysis. We demonstrate that secondary metabolites from B. subtilis have larvicidal activity against A. aegypti and may be suitable for the control of this and other mosquito vectors of human disease. The larvae to the metabolites, significant reduction in the activities of acetylcholinesterse, α-carboxylesterase, and acid phosphatases were recorded.     

Bactericidal and fungicidal activity of Aspergillus ochraceus metabolites and some derivatives

The bactericidal and fungicidal activities of aspyrone and asperlactone, secondary metabolites of Aspergillus ochraceus, and some aspyrone derivatives were studied. In general, aspyrone exhibited better activity than asperlactone or aspyrone derivatives. The inhibition patterns of the assayed compounds were different. Helminthosporium monoceras was the tested mould most inhibited by the studied compounds. The comparative study of the activity of the different compounds showed that the epoxy group seems to be necessary for activity against some micro-organisms. © 1998 SCI     

Activity of fungicides and modulators of membrane drug transporters in field strains of Botrytis cinerea displaying multidrug resistance

In Botrytis cinerea, multidrug resistant (MDR) strains collected in French and German vineyards were tested in vitro, at the germ-tube elongation stage, towards a wide range of fungicides. Whatever the MDR phenotype, resistance was recorded to anilinopyrimidines, diethofencarb, iprodione, fludioxonil, tolnaftate and several respiratory inhibitors (e.g., penthiopyrad, pyraclostrobin). In MDR1 strains, overproducing the ABC transporter BcatrB, resistance extended to carbendazim and the uncouplers fluazinam and malonoben. In MDR2 strains, overproducing the MFS transporter BcmfsM2, resistance extended to cycloheximide, fenhexamid and sterol 14α-demethylation inhibitors (DMIs). MDR3 strains combined the overexpression of both transporters and exhibited the widest spectrum of cross resistance and the highest resistance levels. The four transport modulators, amitriptyline, chlorpromazine, diethylstilbestrol, and verapamil, known to affect some ABC transporters, were tested in B. cinerea. In our experimental conditions, the activity of several fungicides was only enhanced by verapamil. Interestingly, synergism was only recorded in MDR2 and/or MDR3 isolates treated with tolnaftate, fenhexamid, fludioxonil or pyrimethanil, suggesting that verapamil may inhibit the MFS transporter BcmfsM2. This is the first report indicating that a known modulator of ABC transporters could also block MFS transporters.     

Resistance to metalaxyl-M and cymoxanil in a dominant clonal lineage of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Huánuco,Peru, an area of continuous potato production

Fifty-eight isolates of Phytophthora infestans were evaluated in vitro and on detached leaves of potato for their sensitivities to metalaxyl-M and cymoxanil. The isolates belonged to the clonal lineage, EC-1, which is dominant on potato in Peru and Ecuador. All isolates were collected in Huánuco, Peru, an area of year-round potato production, where the potential for development of fungicide resistance is high. All isolates were resistant to metalaxyl-M, with in vitro EC₅₀ values ranging from 468.30—813.57 mg l⁻¹. In contrast, we found no evidence for resistance to cymoxanil for which in vitro EC₅₀ values ranged from 0.03—1.11 mg l⁻¹. Resistance to each fungicide was also evaluated for five isolates in a detached leaf assay in which the fungicide was sprayed on the leaf surface prior to inoculation. With metalaxyl-M, the range of EC₅₀ values was 158.85—828.29 mg l⁻¹, similar to that for the in vitro assay. For cymoxanil, EC₅₀ values ranged from 1.41 to 2.31 mg l⁻¹, which was higher than in the in vitro assay but still two orders of magnitude lower than the concentration applied by farmers in the field.     

Synergism of organophosphorus insecticides by diethyl maleate and related compounds in house flies

The toxicity of several organophosphorus and one carbamate insecticide for house flies is enhanced by simultaneous administration of diethyl maleate. Synergism factors vary from 2 to 116 and are strongly dependent on the combination of strain and insecticide studied. In general, it seems that thiono compounds are less synergized than their oxon analogs. Comparison of some analogs of maleic acid esters showed that the diethyl ester was not the most active compound. trans-Phenylbutenone was found to be a good alternative for diethyl maleate. Both diethyl maleate and trans-phenylbutenone deplete glutathione in the flies, but this depletion per se is certainly not the most important mode of action. In vitro experiments made clear that sufficient glutathione remained to permit a substantial insecticide degradation rate. Moreover, treatment of the flies with diethyl maleate 2–4 hr before the insecticide application resulted in a partial or complete disappearance of synergism in a period in which the glutathione concentration in the flies was still very low. Apart from this effect on the glutathione levels in the insect tissues it appeared from further experimental work in vitro that both compounds had a direct inhibitory effect on glutathione-dependent transferase(s) as well as on oxidative enzymes involved in insecticide degradation. The contribution of these reactions to the eventual synergism factor is discussed.