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

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

Effect of propiconazole on growth and sterol biosynthesis by Sclerotium rolfsii

Germination of sclerotia ofSclerotium rolfsii on agar nutrient medium was delayed or slightly inhibited by concentrations of propiconazole between 0.4 and 4.0 μg ml^?1, but was strongly inhibited by 8 μg ml^?1 and completely inhibited by 16 μg ml^?1. On the other hand, growth of hyphae from the germinated sclerotia was strongly inhibited by propiconazole at 1 μg ml^?1 or greater. Hyphal growth from agar discs on agar medium was about 8 times less sensitive than hyphal growth from the sclerotia or from hyphal inoculum in liquid media. Propiconazole at 0.25 and 1.0 μg ml^?1 strongly inhibited ergosterol biosynthesis, but this was not associated with large accumulations of C-14 methyl sterols. The ratio of eburicol to ergosterol in hyphae grown in the presence of 0.25 μg ml^?1 propiconazole for 16, 30 or 45 h was 0.11, 0.13 and 0.04, respectively, for the three intervals while for hyphae grown in the presence of 1 μg ml^?1, the ratios were 0.29, 0.36 and 0.30, respectively, for the same intervals. In view of a ratio of 23.5 for¹⁴C-acetate incorporation into the two sterols during the initial 6 h growth period in the presence of propiconazole, it is believed that the lack of large accumulation of C-14 methyl sterols is due to the feedback inhibition by eburicol or to cell lysis when the content of ergosterol becomes too low in the actively growing cells.     

Sensitivity ofFusarium graminearum to fungicide JS399-19:In vitro determination of baseline sensitivity and the risk of developing fungicide resistance

The baseline sensitivity ofFusarium graminearum Schwade [teleomorph =Gibberella zeae (Schweinitz) Petch] to the fungicide JS399-19 (development code no.) [2-cyano-3-amino-3-phenylacrylic acetate] and the assessment of risk to JS399-19 resistancein vitro are presented. The mean EC₅₀ values for JS399-19 inhibiting mycelial growth of three populations of wild-typeF. graminearum isolates were 0.102±0.048, 0.113±0.035 and 0.110±0.036 μg ml⁻¹, respectively. Through UV irradiation and selection for resistance to the fungicide, we obtained a total of 76 resistant mutants derived from five wild-type isolates ofF. graminearum with an average frequency of 1.71 × 10⁻⁷% and 3.5%, respectively. These mutants could be divided into three categories of resistant phenotypes with low (LR), moderate (MR) and high (HR) level of resistance, determined by the EC₅₀ values of 1.5–15.0 μg ml⁻¹, 15.1–75.0 μg ml⁻¹ and more than 75.0 μg ml⁻¹, respectively. There was no positive cross-resistance between JS399-19 and fungicides belonging to other chemical classes, such as benzimidazoles, ergosterol biosynthesis inhibitors and strobilurins, suggesting that JS399-19 presumably has a new biochemical mode of action. Although the resistant mutants appeared to have comparable pathogenicity to their wild-type parental isolates, they showed decreased mycelial growth on potato-sucrose-agar plates and decreased sporulation capacity in mung bean broth. Nevertheless, most of the resistant mutants possessed fitness levels comparable to their parents and had MR or HR levels of resistance. As these studies yielded a high frequency of laboratory resistance inF. graminearum, appropriate precautions against resistance development in natural populations should be taken into account. http://www.phytoparasitica.org posting August 7, 2008.     

Insecticidal activity and mode of action of 2,4‐diaminopyrimidines

Three 2,4‐diaminopyrimidines were tested against several insect species. They were active against lepidopteran pests with LC₅₀ values <3 mg liter⁻¹ for most species tested. They were also active against two‐spotted spider mite, Tetranychus urticae, (LC₅₀ 10–40 mg liter⁻¹). Folinate, but not hypoxanthine or thymidine was found to be an effective rescue agent, requiring a concentration of 100 mg liter⁻¹ diet to rescue half of the intoxicated larvae. The results confirm dihydrofolate reductase to be the site of action for these insecticides and are consistent with the mode of action of folinate rescue in mammals. © 2000 Society of Chemical Industry     

Inhibition of ergosterol biosynthesis in Ustilago maydis by the fungicide 1-[2-(2, 4-dichlorophenyl)-4-ethyl-1, 3-dioxolan-2-ylmethyl]-1H-1, 2, 4-triazole

Inhibition of sporidial multiplication in cultures of Ustilago maydis by 1-[2-(2, 4-dichlorophenyl)-4-ethyl-1, 3-dioxolan-2-ylmethyl]-1H-1, 2, 4-triazole^a (CGA-64251), at concentrations of 0.1, 1.0 and 5.0 μg ml^?1, increased from about 15% during the first 4 h, to 58–70% during the subsequent 4 to 12-h period. Sporidia became swollen and highly branched in the presence of the fungicide. Total lipid content as a percentage of the dry weight was not affected after exposure of the sporidia to the fungicide at 0.1 or 5 μg ml^?1 for 4 h, but synthesis of ergosterol and other demethyl-sterols was inhibited by 87–92%. Large quantities of methyl-sterol precursors of ergosterol and of free fatty acids accumulated in the treated sporidia. Fungitoxicity of CGA-64251 is attributed to inhibition of ergosterol biosynthesis at the stage of sterol C-14 demethylation.     

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.     

Acute toxicity of the bird repellent,methyl anthranilate,to fry of Salmo salar,Oncorhynus mykiss,Ictalurus punctatus and Lepomis macrochirus

Several laboratory and field studies have shown methyl anthranilate to be an effective, non-toxic and non-lethal bird repellent, with application potential for protecting crops, seeds, turf and fish stocks from bird damage. Furthermore, methyl anthranilate can be added to liquids for the purposes of protecting migratory birds, e.g. addition to waste water associated with mining and to standing water pools at airports. Mammalian toxicity data are favorable. Methyl anthranilate is used as a fragrance and food flavoring and is GRAS listed by the US Food and Drug Administration. Despite the favorable outlook for methyl anthranilate's use as a safe repellent, no data exist on its environmental fate and effects. We have tested the acute toxicity of methyl anthranilate in a static system against the fry of four species of fish. The LC₅₀ at 24 h for Atlantic salmon (Salmo salar L.) was 32.3 mg liter^?1, with the no observable effect limit at 6 mg liter^?1. The LC₅₀ at 24 h for rainbow trout (Oncorhynus mykiss Richardson) was 23.5 mg liter^?1, with the no observable effect limit at 5 mg liter^?1. The LC₅₀ at 24 h for channel catfish (Ictalurus punctatus Raf.) was estimated to be 20.1 mg liter^?1, with the no observable effect limit at 7 mg liter^?1. The LC₅₀ at 24 h for bluegill sunfish (Lepomis macrochirus Raf.) was estimated to be 19.8 mg liter^?1, with the no observable effect limit at 7 mg liter^?1..     

Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 μg ml^?1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.     

Effect of fenpropimorph and imazalil on sterol biosynthesis in penicillium italicum

Fenpropimorph was found to be highly active against Penicillium italicum (EC₅₀ 0.01/μg ml^?1). Conidia of P. italicum, treated with low concentrations of fenpropimorph, swelled in size and showed distorted germ tubes. During the initial stages of mycelial growth, fenpropimorph had little or no effect on the dry weight increase, which became strongly inhibited within 24 h after addition of the toxicant (0.05, 0.1 and 0.2 μg ml^?1). Irregular deposition of β–1,3 and β–1, 4 polysaccharides, probably chitin, was observed after treatment with fenpropimorph or imazalil. Fenpropimorph (0.05 and 0.2 μ ml^?1) caused the accumulation of a major demethyl-sterol that was different from ergosterol. It was identified as ergosta-8, 14, 24(28)-trien-3β-ol by mass, infrared, ultraviolet and proton nuclear magnetic resonance, spectrometric procedures. At both concentrations, the accumulation was already detected after incubation for 2 h. In contrast, imazalil (0.1 μg ml^?1) caused the accumulation of several methyl- and dimethyl-sterols which were tentatively identified as eburicol (24-methylene-24, 25-dihydrolanosterol), 4, 14α-dimethylergosta-8, 24(28)-dien-3-one, 14α-methylergosta-8, 24(28)-dien-3-one and obtusifoliol (4, 14α-dimethylergosta-8, 24(28)-dien-3α-ol). The accumulation of ergosta-8, 14,24(28)-trien-3β-ol indicates inhibition of the Δ¹⁴-reductase in P. italicum in a similar manner to that found previously in Ustilago maydis.     

Aerobic soil metabolism of metsulfuron-methyl

A laboratory study was conducted to determine the degradation rates and identify major metabolites of the herbicide metsulfuron-methyl in sterile and non-sterile aerobic soils in the dark at 20°C. Both [phenyl-U-¹⁴C]- and [triazine-2-¹⁴C]metsulfuron-methyl were used. The soil was treated with [¹⁴C]metsulfuron-methyl (0.1 mg kg⁻¹) and incubated in flow-through systems for one year. The degradation rate constants, DT₅₀, and DT₉₀ were obtained based on the first-order and biphasic models. The DT₅₀ (time required for 50% of applied chemical to degrade) for metsulfuron-methyl, estimated using a biphasic model, was approximately 10 days (9–11 days, 95% confidence limits) in the non-sterile soil and 20 days (12–32 days, 95% confidence limits) in the sterile soil. One-year cumulative carbon dioxide accounted for approximately 48% and 23% of the applied radioactivity in the [phenyl-U-¹⁴C] and [triazine-2-¹⁴C]metsulfuron-methyl systems, respectively. Seven metabolites were identified by HPLC or LC/MS with synthetic standards. The degradation pathways included O-demethylation, cleavage of the sulfonylurea bridge, and triazine ring opening. The triazine ring-opened products were methyl 2-[[[[[[[(acetylamino)carbohyl]amino]carbonyl]amino] carbonyl]-amino]sulfonyl]benzoate in the sterile soil and methyl 2-[[[[[amino[(aminocarbonyl)imino]methyl] amino]carbonyl]amino]sulfonyl]benzoate in the non-sterile soil, indicating that different pathways were operable. © 1999 Society of Chemical Industry     

Comparative antifungal effect and mode of action of tetraconazole on Ustilago maydis

Tetraconazole, a new, recently introduced antifungal triazole, has been assayed in parallel with a number of standard analogues on various sensitive strains of Ustilago maydis. The values of EC₅₀ and EC₉₀ tetraconazole concentrations, determined on strain ATCC 14826 in agar, were 0.5 × 10⁻⁶ and 3.5 × 10⁻⁵ , respectively, in reasonable agreement with those needed to inhibit by 50% and 90%, respectively, the ergosterol biosynthesis in broth cultures. Squalene and 12 sterols have been extracted from the latter, characterized and quantified. Accumulation of 14α-methylsterols and reduction of ergosterol and other late precursors are consistent with the inhibition of 14α-demethylase caused by the title compound.     

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).     

Effects of imazalil on sterol composition of sensitive and DMI-resistant isolates of Penicillium italicum

Imazalil differentially inhibited dry weight increase of 10-hour-old germlings of wild-type and DMI-resistant isolates ofPenicillium italicum in liquid malt cultures. EC₅₀ values ranged from 0.005 to 0.27 g ml^–1. In all isolates ergosterol constituted the major sterol (over 95% of total sterols) in the absence of the fungicide. Therefore, DMI-resistance cannot be associated to a deficiency of the C-14 demethylation enzyme in the ergosterol biosynthetic pathway. Imazalil treatment at concentrations around EC₅₀ values for inhibition of mycelial growth resulted in a decrease in ergosterol content and a simultaneous increase in 24-methylene-24,25-dihydrolanosterol content in all isolates. A correlation existed between the imazalil concentration necessary to induce such changes in sterol composition and the EC₅₀ values for inhibition of mycelial growth of the different isolates. The reason for the differential effects of imazalil on sterol composition in the variousP. italicum isolates may be due to decreased accumulation of the fungicide in the mycelium and to other yet non-identified mechanisms of resistance.Imazalil remt differentieel de toename in drooggewicht van 10-uur-oude gekiemde sporen van wild-type en DMI-resistente isolaten vanPenicillium italicum in vloeistofcultures van moutextract. De EC₅₀ waarden voor groei van de verschillende isolaten lopen uiteen van 0,005 tot 0,27 g ml^–1. In afwezigheid van het fungicide is in alle isolaten ergosterol het belangrijkste sterol (meer dan 95% van het totaal). DMI-resistentie kan daarom niet in verband staan met deficiëntie van het C-14 demethyleringsenzym in de ergosterol biosynthese. Imazalilbehandeling van mycelium bij concentraties rond de EC₅₀ waarde voor groeiremming, resulteerde bij alle isolaten in een afname van het ergosterolgehalte en een gelijktijdige toename van het gehalte aan 24-methyleen-24,25-dihydrolanosterol. Er bestaat dus een nauwe correlatie tussen de imazalilconcentratie die noodzakelijk is om vergelijkbare veranderingen in sterolsamenstelling te induceren en de EC₅₀ waarde voor remming van myceliumgroei van de verschillende isolaten. De differentiële effecten van imazalil op de sterolsamenstelling van de verschillendeP. italicum isolaten kunnen worden veroorzaakt door verminderde accumulatie van het fungicide in het mycelium en door andere, nog niet geïdentificeerde resistentiemechanismen.     

Antifungal mode of action of imazalil

Imazalil had no effect on the initial growth of mycelia of Penicillium italicum (for 10 hr) or Aspergillus nidulans (for 2 hr). In P. italicum during this period neither respiration nor cell permeability was affected, but uptake of [³²P]phosphate, [¹⁴C]leucine, or [¹⁴C]uridine was partially inhibited. The initial (5 hr) inhibition of substrate uptake coincided with a 50% reduction in ergosterol content. Within 0.5 hr, incorporation of [¹⁴C]acetate into C-4-desmethyl sterols was strongly inhibited in mycelia of A. nidulans treated with 0.5 μg/ml of imazalil. However, radioactivity in C-4-methyl and dimethyl sterols exceeded that of control cultures. Concentrations of imazalil as low as 0.005 μg/ml caused short-term (1 hr) declines of incorporation into desmethyl sterols and increases into the C-4-methyl and dimethyl sterols. Incorporation into phospholipids, triglycerides, and free fatty acids was not affected. These data suggest that the primary antifungal action of imazalil is inhibition of demethylation in the biosynthesis of ergosterol.     

Assessment of two biotypes of Solanum ptycanthum that differ in resistance levels to imazamox

Glasshouse and laboratory experiments were conducted on acetolactate synthase (ALS) homozygous resistant Solanum ptycanthum biotypes from Illinois (IL‐R) and Indiana (IN‐R), and homozygous susceptible biotypes from Illinois (IL‐S) and Indiana (IN‐S). Genetic similarity of biotypes was assessed by random amplified polymorphic DNA (RAPD) markers, which determined that the Illinois biotypes are more similar to each other than to the IN‐R biotype. ALS enzyme activity from the IL‐R and IN‐R biotypes had I₅₀ values of 362 and 352 μM imazamox respectively. Dose–response experiments using three‐ to four‐leaf‐stage plants of the IL‐R and IN‐R biotypes had GR₅₀ values of 242 and 69 g ae ha⁻¹ imazamox respectively. Whole‐plant and ALS enzyme results are different than previously reported values in the literature, which was attributed in the current study to the original IN‐R population having individuals that were segregating for ALS resistance. Metabolism studies showed no difference in percentage [¹⁴C]imazamox remaining between the IL‐R and IN‐R biotypes up to 72 h after treatment. The IL‐S biotype metabolised [¹⁴C]imazamox approximately two times faster than the IL‐R and IN‐R biotypes and this trait was heritable. Response of F₃ plants containing homozygous ALS‐resistant alleles from the IL‐R biotype in a genetic background of 50% Illinois and 50% Indiana biotypes suggests that genetic factors other than an altered target site or metabolism may also contribute to the magnitude of resistance at the whole‐plant level in resistant biotypes.     

Protoporphyrinogen oxidase-inhibiting activity of the new,wheat-selective isoindoldione herbicide,cinidon-ethyl

Cinidon-ethyl (BAS 615H) is a new herbicide of isoindoldione structure which selectively controls a wide spectrum of broadleaf weeds in cereals. The uptake, translocation, metabolism and mode of action of cinidon-ethyl were investigated in Galium aparine L, Solanum nigrum L and the tolerant crop species wheat (Triticum aestivum L). When plants at the second-leaf stage were foliarly treated with cinidon-ethyl equivalent to a field rate of 50 g ha⁻¹ for 48 h, the light requirement for phytotoxicity and the symptoms of plant damage in the weed species, including rapid chlorophyll bleaching, desiccation and necrosis of the green tissues, were identical to those of inhibitors of porphyrin synthesis, such as acifluorfen-methyl. The selectivity of cinidon-ethyl between wheat and the weed species has been quantified as approximately 500-fold. Cinidon-ethyl strongly inhibited protoporphyrinogen oxidase (Protox) activity in vitro, with I₅₀ values of approximately 1 nM for the enzyme isolated from the weed species and from wheat. However, subsequent effects of herbicide action, with accumulation of protoporphyrin IX, light-dependent formation of 1-aminocyclopropane-1-carboxylic acid-derived ethylene, ethane evolution and desiccation of the green tissue, were induced by cinidon-ethyl only in the weed species. After foliar application of [¹⁴C] cinidon-ethyl, the herbicide, due to its lipophilic nature, was rapidly adsorbed by the epicuticular wax layer of the leaf surface before it penetrated into the leaf tissue more slowly. No significant differences between foliar and root absorption and translocation of the herbicide by S nigrum, G aparine and wheat were found. After foliar or root application of [¹⁴C]- cinidon-ethyl, translocation of ¹⁴C into untreated plant parts was minimal, as demonstrated by combustion analysis and autoradiography. Metabolism of [¹⁴C]cinidon-ethyl via its E-isomer and acid to further metabolites was more rapid in wheat than in S nigrum and G aparine. After 32 h of foliar treatment with 50 g ha⁻¹ of the [¹⁴C]-herbicide, approximately 47%, 36%, and 12% of the absorbed radioactivity, respectively, were found as unchanged parent or its biologically low active E-isomer and acid in the leaf tissue of G aparine, S nigrum and wheat. In conclusion, cinidon-ethyl is a Protox-inhibiting, peroxidizing herbicide which is effective through contact action in the green tissue of sensitive weed species. It is suggested that a more rapid metabolism, coupled with moderate leaf absorption, contribute to the tolerance of wheat to cinidon-ethyl. © 1999 Society of Chemical Industry     

Lethal and sublethal effects of the neem insecticide formulation, ‘Margosan-O’, on the pea aphid

The effects of ‘Margosan-O’ (MO) on the pea aphid, Acyrthosiphon pisum (Harris), were determined. MO significantly reduced population increase of A. pisum in a concentration-dependent manner. At a concentration equivalent to 100 mg litre^?1 of azadirachtin, population increase was c. 3.5 times lower than the control. In more detailed studies, MO significantly reduced the number of molts, longevity, and fecundity of A. pisum that had been reared on treated broad bean. Viciafaba L., plants. MO also reduced the longevity and fecundity of young adult A. pisum exposed to MO-treated broad bean. MO was slow-acting against A. pisum. Mortality caused by MO stabilised seven days after newborn A. pisum were exposed to treated broad bean and 10 days for adults. The seven day LC₅₀ for individuals exposed from birth was 27.50 mg azadirachtin liter^?1 while the 10 day LC₅₀ for adults was 53.32 mg liter^?1. Contrary to previous studies suggesting that neem insecticides are not contact toxicants, we found that MO applied topically to adult A. pisum caused effects similar to those found in individuals that fed upon treated plants. However, MO was slower-acting when applied topically. Mortality in adult A. pisum caused by topically applied MO stabilised 17 days after treatment with a resultant LD₅₀of 2.91 μg azadirachtin g^?1.     

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

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

Phytotoxicity of m-phenoxybenzamides: Inhibition of cell-free phytoene desaturation

Inhibition of carotenoid biosynthesis by herbicidal m-phenoxybenzamide derivatives has been investigated in a cell-free carotenogenic system from Aphanocapsa. Their target is the phytoenedesaturase reaction. Double-reciprocal plots of β-carotene biosynthesis (from ¹⁴C-labeled geranylgeranyl pyrophosphate) showed that 3-(2,5-dimethylphenoxy)-N-ethylbenzamide was a noncompetitive inhibitor of the phytoene-desaturase complex. The K_i value for cell-free inhibition of β-carotene formation was almost identical to the I₅₀ value of intact cells. Furthermore, the influence of certain substituents on herbicidal activity has been investigated. Inhibition increased with the length of the unbranched N-alkyl chains. In addition, substituents at the phenoxy group with higher lipophilicities showed greater inhibitory activities. The presence of a phenoxy or trifluoromethyl moiety at position 3 is essential.     

Binding Sites for Ca2+-Channel Effectors and Ryanodine in Periplaneta americana—Possible Targets for New Insecticides

The calcium channel and the ‘calcium release channel’ of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0·1 and 300 μg ml⁻¹. Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) [³H]isradipine (PN 200-110), the phenyl-alkylamine [³H]verapamil and the alkaloid [³H]ryanodine. Preliminary binding studies with the benzothiazepine [³H]diltiazem suggest a low-affinity binding site with a IC₅₀ value of 3·3 μM . All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 μM . EGTA at 10 μM abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca²⁺ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 μM EGTA. With respect to high Ca²⁺ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respectively, at 5 mM Ca²⁺. Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K_D values of 0·95 nM (B_max=550 fmol mg⁻¹ protein) and 0·75 nM (B_max=213 fmol mg⁻¹ protein) were determined respectively. A lower-affinity binding site was identified in binding studies with verapamil (K_D=7·4 nM and B_max=27 fmol mg⁻¹ protein). [³H]isradipine displacement studies with several dihydropyridines revealed the following ranking of affinity: nitrendipine>isradipine>Bay K8664≪nicardipine. Displacement of [³H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by (±)verapamil, nor-methylverapamil and R(+)verapamil.