Insecticidal Activity and HPLC Correlation of Thuringiensin from Fermentation and Two-phase Aqueous Separation Processes

Thuringiensin produced by Bacillus thuringiensis subsp. darmstadiensis was further subjected to a two-phase aqueous separation system. A modified HPLC method and a test for quantitative pathogenicity using the house fly Musca domestica were used for analysis of thuringiensin. Within a realistic range of dosages, more effect was observed in the pupal stage than in the larval stage. The percentage effective control rate (ECR) was calculated by (100-percentage emergence); malformed and non-reproductive adults were considered as emerged. Pupal mortality, pupal weight, and ECR after feeding the three-day-old larvae were the measured response criteria for bioassay. The EC₅₀ of thuringiensin for pupae mortality was 1·64 μg ml^-1 diet, and 0·83 μg ml^-1 for mortality of adults. Insecticidal activity of the broth increased with fermentation time-course from 9th to 21st hour. The bioassay curve constructed with three-hour sampling interval during the fermentation course had good correlation to thuringiensin content as determined by the HPLC method. In the two-phase aqueous separation system, a maximum of 96·7% ECR was achieved with the bottom salt layer, compared to a value of 46·7% with the upper PEG layer. These results suggest that thuringiensin, prepared through a fermentation and recovery process, is suitable for pest control. © 1997 SCI.     

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.     

Resistance to pyraclostrobin, boscalid and multiple resistance to Pristine® (pyraclostrobin + boscalid) fungicide in Alternaria alternata causing alternaria late blight of pistachios in California

Pristine® (pyraclostrobin + boscalid) is a fungicide registered for the control of alternaria late blight in pistachio. A total of 95 isolates of Alternaria alternata collected from orchards with and without a prior history of Pristine® sprays were tested for their sensitivity towards pyraclostrobin, boscalid and Pristine® in conidial germination assays. The EC₅₀ values for 35 isolates from orchards without Pristine® sprays ranged from 0·09 to 3·14 µg mL^?1 and < 0·01 to 2·04 µg mL^?1 for boscalid and Pristine®, respectively. For pyraclostrobin, 27 isolates had EC₅₀ < 0·01 µg mL^?1 and six had low resistance (mean EC₅₀ value = 4·71 µg mL^?1). Only one isolate was resistant to all three fungicides tested, with EC₅₀ > 100 µg mL^?1. Among 59 isolates from the orchard with a history of Pristine® sprays, 56 were resistant to pyraclostrobin; only two were sensitive (EC₅₀ < 0·01 µg mL^?1) and one was weakly resistant (EC₅₀ = 10 µg mL^?1). For the majority of these isolates EC₅₀ values ranged from 0·06 to 4·22 µg mL^?1 for boscalid and from 0·22 to 7·74 µg mL^?1 for Pristine®. However, seven isolates resistant to pyraclostrobin were also highly resistant to boscalid and Pristine® and remained pathogenic on pistachio treated with Pristine®. Whereas strobilurin resistance is a common occurrence in Alternaria of pistachio, this is the first report of resistance to boscalid in field isolates of phytopathogenic fungi. No cross resistance between pyraclostrobin and boscalid was detected, suggesting that Pristine® resistance appears as a case of multiple resistance.     

Structural modifications increase the insecticidal activity of ryanodine

The toxicity of ryanodine ( 1 ) and 9,21-didehydroryanodine ( 2 ) (the principal active ingredients of the botanical insecticide ryania) to adult female house flies (Musca domestica L.) is attributable to binding to the ryanodine receptor (ryr) and thereby disrupting the Ca²⁺-release channel. These ryanoids, assayed in house flies with piperonyl butoxide (PBO) to suppress cytochrome P450-dependent detoxification, give injected KD₅₀ values of 0·07–0·11 μg g^-1, injected LD₅₀ values of 0·39–0·45 μg g^-1 and topical LD₅₀ values of 12– 50 μg g^-1. They inhibit the [³H]ryanodine binding site of house fly and rabbit muscle with IC₅₀ values of 3–10 nM . This study examines the effect of structure on potency, with 15 variants of the cyclohexane substituents, two 4,6-cyclic boron and two methylated derivatives, and four modifications of the isopropyl and ester substituents. The most effective compound examined was 10-deoxy- 2 ( 3 ) which was more potent than 2 by 2–4-fold on injection and 29-fold applied topically following PBO (LD₅₀ 0·41 μg g^-1). Additional high-potency compounds were 10-oxo- 1 and the cyclohexane variants with lactam, 21-nor-9-oxo and 21-nor-10-deoxy substituents. Other modifications usually reduced toxicity. The injected knockdown potency of the ester ryanoids was generally related to their effectiveness in competing with [³H]ryanodine at the ryr of rabbit skeletal muscle. Two non-ester ryanoids, ryanodol and 9,21-didehydroryanodol, were found to be more toxic than predicted from their potency at the ryr and may therefore act in a different manner such as at a K⁺ channel, as suggested by Usherwood and Vais. Clearly ryanoids are challenging prototypes for a potential new generation of insecticides. © 1997 SCI.     

Differential responses of germinating Venturia inaequalis conidia to kresoxim-methyl

A new class of agricultural fungicides derived from strobilurins act as respiration inhibitors by binding to mitochondrial cytochrome b. The effects of the strobilurin, kresoxim-methyl, on conidia germination, mycelial growth and the protection of apple leaves from scab development were investigated for two isolates of Venturia inaequalis randomly selected from a culture collection. Inhibition of mycelial growth required relatively high doses of kresoxim-methyl (ED₅₀=1 μg ml^-1) for both isolates. In comparison, germination of conidia was highly sensitive for one of the isolates (ED₅₀=0·005 μg ml^-1), while the level of inhibition achieved for the second isolate was 60-fold less (ED₅₀=0·3 μg ml^-1). As deduced from identical sequences of cytochrome b cDNAs prepared from both isolates, the different responses of germinating conidia to kresoxim-methyl were not caused by differences in the sequence of cytochrome b as the target site for strobilurin action. Strong synergistic effects of salicylhydroxamic acid on kresoxim-methyl inhibitory potency suggested that the reduced kresoxim-methyl sensitivity observed for germinating conidia was caused by interference of the alternative respiratory pathway with inhibitor action. However, this mechanism of target site circumvention in germinating conidia had no adverse effects on the protection of apple leaves from scab infection by kresoxim-methyl. © 1998 Society of Chemical Industry     

Baseline and differential sensitivity of Peronophythora litchii (lychee downy blight) to three carboxylic acid amide fungicides

Downy blight, caused by Peronophythora litchii, is an important disease of lychee (litchi) plants in China. The in vitro sensitivities of various asexual stages of P. litchii to the three carboxylic acid amide (CAA) fungicides dimethomorph, flumorph and pyrimorph were studied with four single‐sporangium isolates. None of the three fungicides affected zoospore discharge from sporangia, but they strongly inhibited mycelial growth (mean EC₅₀ values of 0·075, 0·258 and 0·115 mg L^?1, respectively); sporangial production (mean EC₅₀ values of 0·085, 0·315 and 0·150 mg L^?1, respectively); germination of cystospores (mean EC₅₀ values of 0·140, 0·150 and 0·645 mg L^?1, respectively); and germination of sporangia (mean EC₅₀ values of 0·203, 0·5 and 0·743 mg L^?1, respectively). As mycelial growth was the most sensitive stage to dimethomorph and pyrimorph, it was chosen to test baseline sensitivities to the three fungicides. In 2007, from 131 isolates collected in Fujian, Guangdong and Guangxi provinces, 127, 116 and 113 isolates were used to establish baseline sensitivity for dimethomorph, flumorph and pyrimorph respectively. Isolates from different provinces exhibited similar baseline sensitivity to the same fungicide. Baseline sensitivities to dimethomorph, flumorph and pyrimorph were distributed as unimodal curves, with mean EC₅₀ values of 0·082 (± 0·01), 0·282 (± 0·047), and 0·115 (± 0·032) mg L^?1, respectively. This information will serve as a baseline for tracking future changes in sensitivities of P. litchii populations to these three CAA fungicides.     

Comparative testing of several juvenile hormone analogues in two species of locusts,Locusta migratoria migratorioides and Schistocerca gregaria

Effective treatment with juvenile hormone analogues (JHAs) of early penultimate or early last-instar locust hoppers induces a supernumerary ‘extra’ nymphal instar. These ‘extra’ nymphs, also termed ‘adultoids’, die in the course of, or shortly after, an ‘extra’ moult. Less effective treatment results in imperfect adults with crumpled twisted wings which presumably limit their flight and migratory abilities. Extremely effective treatment leads to death in the next moult. Comparing dose-response relations of (7S)-methoprene, fenoxycarb, pyriproxyfen and a new JHA, R70-1 (ethyl cis-N-{2-[4-(2-hydroxycyclohept-1-ylmethyl)phenoxy]ethyl}carbamate), we revealed that route of administration, instar of the recipient hopper, and species may alter over 1000-fold the ED₅₀ for the same JHA. Locusta migratoria migratorioides is much more susceptible to JHAs than Schistocerca gregaria. The lowest ED₅₀ found to induce adultoids and subsequent death in the ‘extra’ moult was 0·12 μg pyriproxyfen injected in olive oil to early penultimate instar hoppers of L. m. migratorioides (about 0·5 μg g^-1 fresh weight). R70-1 was more active than pyriproxyfen following the more practical topical application to early last-instar hoppers of L. m. migratorioides, 5·9 μg and 46 μg per hopper, respectively (about 10 μg g^-1 and 78 μg g^-1 fresh weight). The high susceptibility of last-instar L. m. migratorioides nymphs to topically applied R70-1 is promising from the practical standpoint. ©1997 SCI     

Distribution and molecular characterization of Corynespora cassiicola isolates resistant to boscalid

A total of 618 isolates of corynespora leaf spot fungus (Corynespora cassiicola) collected from 24 commercial cucumber greenhouses in 12 cities in Ibaraki Prefecture, Japan, were tested for their sensitivity to boscalid. Boscalid‐resistant isolates were detected in 17 out of 19 greenhouses with a history of use of this fungicide and detection frequencies of the resistant isolates exceeded 47% in nine greenhouses. Frequencies of very highly resistant (VHR) isolates with 50% effective concentration (EC₅₀) values of boscalid exceeding 30 μg mL^?1 were higher than those of moderately resistant (MR) isolates with EC₅₀ ranging from 2·0 to 5·9 μg mL^?1 in 11 greenhouses. Additionally, highly resistant (HR) isolates with EC₅₀ from 8·9 to 10·7 μg mL^?1 were first detected. Furthermore, molecular characterization of genes encoding succinate dehydrogenase (SDH) subunits (SdhA, SdhB, SdhC and SdhD) was carried out to elucidate the amino acid substitution responsible for the resistance to boscalid. All 23 VHR isolates had the same mutation from CAC to TAC in the SdhB gene leading to the substitution of histidine with tyrosine at amino acid position 278 (B‐H278Y). At the same position, the substitution to arginine conferred by a mutation to CGC (B‐H278R) was detected in all four HR isolates. Some MR isolates showed a substitution from serine to proline at position 73 in SdhC (C‐S73P), from serine to proline or from glycine to valine at position 89 (D‐S89P) and 109 (D‐G109V), respectively, in SdhD. There was no common mutation in SDH genes of all MR isolates.     

Disposition kinetics of cypermethrin and fenvalerate in black bengal lactating goats

Disposition kinetics of cypermethrin and fenvalerate were investigated in lactating black Bengal goats following single dose intravenous administration at 57 and 45 mg kg^?1 respectively. The maximum and minimum blood concentrations of cypermethrin were 18.49 (±3.17) and 0.06 (±0.002) μg ml^?1, while the corresponding values for fenvalerate were 14.58 (±2.37) and 0.04 (±0.005) μg ml^?1 respectively. Both cypermethrin and fenvalerate remained present in blood for 36 h. The mean t1/2β) and Vd_area values were 5.56 (±0.28) h and 10.38 (±2.20) litre kg^?1 for cypermethrin and 5.66 (±0.35) h and 11.31 (±2.20) litre kg^?1 respectively for fenvalerate. Both cypermethrin and fenvalerate persisted in goat milk for 36 h. The t1/2β) and AUC values of fenvalerate were 7.37 (±1.84) h and 122.38 (±11.65) μg h ml^?1 whilst the corresponding values for cypermethrin were 6.66 (±1.54) h and 99.48 (±7.81) μg h ml^?1 in milk respectively.     

Mefenoxam sensitivity and fitness analysis of Phytophthora nicotianae isolates from nurseries in Virginia, USA

Mefenoxam is one of the most commonly used fungicides for managing diseases caused by Phytophthora spp. on ornamentals. The objectives of this study were to determine whether Phytophthora nicotianae, a destructive pathogen of numerous herbaceous annual and perennial plant species in nurseries, has developed resistance to mefenoxam, and to evaluate the fitness of mefenoxam‐resistant isolates. Ninety‐five isolates of P. nicotianae were screened for sensitivity to mefenoxam on 20% clarified V8 agar at 100 a.i. µg mL^?1. Twenty‐five isolates were highly resistant to this compound with EC₅₀ values ranging from 235·2 to 466·3 µg mL^?1 and four were intermediately resistant with EC₅₀ values ranging from 1·6 to 2·9 µg mL^?1. Sixty‐six isolates were sensitive with EC₅₀ values less than 0·04 µg mL^?1. Nine resistant and seven sensitive isolates were tested for mefenoxam sensitivity on Pelargonium × hortorum cv. White Orbit. Mefenoxam provided good protection of pelargonium seedlings from colonization by sensitive isolates, but not by any resistant isolates. Four resistant and four sensitive isolates were compared for fitness components and their relative competitive ability on Lupinus Russell Hybrids in the absence of mefenoxam. Resistant isolates outcompeted sensitive ones within 3 to 6 sporulation cycles on lupin seedlings, regardless of their initial proportions in mixed zoospore inoculum. Resistant isolates exhibited greater infection rate and higher sporulation ability than sensitive ones when they were applied separately onto lupins. These results suggest that fungicide resistance may pose a serious challenge to the continued effectiveness of mefenoxam as a control option for nursery growers.     

Effect of atrazine on growth, production of aflatoxin, and fatty acid and sterol biosynthesis by Aspergillus spp.

The effects of atrazine were studied on growth, production of aflatoxin, and fatty acid and sterol biosynthesis by four isolates of Aspergillus in vitro. There was little effect of atrazine on Aspergillus spp. at concentrations up to 20 μg ml^?1 but at 40 μg ml^?1 or above, growth, production of aflatoxin, and fatty acid and sterol biosynthesis were remarkably reduced. Palmitic, stearic and linoleic acid synthesis were inhibited in three of the isolates tested at 60 μg ml^?1. At 100 μg ml^?1, except ergosterol, the cholesterol and 5, 7-ergostadienol synthesis was totally inhibited in all isolates. Effet de l'atrazine sur la croissance, la production d'aflatoxine, et la biosynthèse d'acides gras et de stérols par Aspergillus spp. Chez quatre isolats d'Aspergillus, les effets de l'atrazine sur la croissance, la production d'aflatoxine, et la biosynthèse d'acides gras et de stérols ont étéétudiés in vitro. Jusqu'à des concentrations de 20 μg ml^?1, l'atrazine n'a eu que peu d'effets, mais à 40 μg ml^?1 et au-dessus, la croissance, la production d'aflatoxine, et la biosynthèse d'acides gras et de stérols ont été nettement réduites. Les synthèses d'acides palmitique, stéarique et linoléique ont été inhibées chez trois des isolats, à 60 μg ml^?1. A 100 μg ml^?1, mis à part l'ergostérol, les synthèses de cholestérol et de 5, 7-ergostanediol ont été totalement inhibées chez tous les isolats. Die Wirkung von Atrazin aufdas Wachslum, die Bildung von Aflatoxin und die Fettsäuren- und Sterol-Biosynthese von Aspergillus spp. Bei vier Isolaten von Aspergillus wurde in vitro die Wirkung auf das Wachstum sowie auf die Bildung von Aflatoxin, Fettsäuren und Sterolen untersucht. Bei Atrazin-Konzentrationen bis zu 20 μg ml^?1 war keine Wirkung zu beobachten, aber ab 40 μg ml^?1 wurden das Wachstum und die Bildung von Aflatoxin, Fettsäuren und Sterolen deutlich herabgesetzt. Bei 60 μg ml^?1 war bei drei Isolaten die Bildung von Palmitin-, Stearin- und Linolensäure gehemmt. Bei 100 μg ml^?1 war bei alien Isolaten die Bildung von Cholesterol und 5, 7-Ergostadienol, aber nicht Ergosterol, unterbunden.     

The effectiveness of eleven sterol biosynthesis-inhibiting fungicides against the take-all fungus,Gaeumannomyces graminis var. tritici,in relation to their physical properties

Eleven sterol biosynthesis-inhibiting fungicides were compared in experiments to determine the physico-chemical properties required for most effective control of take-all by soil treatment. All were active in agar culture against an isolate of the pathogen which causes take-all, Gaeumannomyces grammis var. tritici, with prochloraz being the most toxic (EC₅₀ 0.02 μg ml^?1) and PP 969 the least (EC₅₀ 0.44 μg ml^?1). Penconazole and PP 969 had vapour activity against the fungus in further bioassays on agar. In soil in pots, the most strongly lipophilic compound, buthiobate, was ineffective against take-all in wheat; triadimenol was most effective and, like flutriafol, nuarimol and PP 969, retained some effectiveness after 12 weeks in soil. PP 969, unlike penconazole or nuarimol, was effective in soil treated unevenly by mixing the fungicides in layers. PP 969 is relatively polar, and it is suggested that this property, allowing redistribution in soil water rather than as vapour, outweighed its poor intrinsic toxicity. The ideal soil-treatment fungicide should therefore be polar and also have good intrinsic activity and moderate persistence. None of the compounds tested had all these properties.     

Quantitative bioassays for determining residues and availability to plants of sulphonylurea herbicides

A bioassay procedure for quantitative determination of sulphonylurea herbicides is described. Turnips (Brassica rapa) were found very suitable as test plants and gave results within 10 days. Six sulphonylurea compounds were investigated for their activity in three widely differing soils. The potential availability to plants was calculated from the dose-response curves of vermiculite (non-sorptive substrate) and the corresponding ED₅₀-values of the soils. The dose-response relationship (logistic curve) was described by a computer model by a position parameter, the slope of the curve and the minimum and maximum fresh weights of plants. The limit of quantitative detection in the range of ED₃₀ in vermiculite was 0·06 μg 1^?1 for sulfometuron and 1·03 μg 1^?1 for DPX-L5300, methy12-([4-methoxy-6-methyl-1,3,5-triazin-2-yl (methyl)carbamoyl]-sulphamoyl) benzoate. Results with turnips showed that sulfometuron was the most active compound in all substrates (ED₅₀ in vermiculite 0·12 μg 1^?1) followed by chlorsulfuron, metsulfuron-methyl, triasulfuron, DPX-M6316, methyl 3-([(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbamoyl]-aminosulphaphamoyl)-2-thiophenecarboxylate, and DPX-L5300 which had ED₅₀ or 1·98 μg 1^?1, The Horotiu sandy loam soil showed the highest ED₅₀-values and the lowest plant availability for all compounds compared to the other soils. Probit and logistic evaluation methods for deriving dose-response relationships are compared and their applicability is discussed.     

An Insecticidal and Acaricidal Polysulfide Metabolite from the Roots of Petiveria alliacea

The present study reports on the insecticidal and acaricidal potentials of dibenzyltrisulfide (DBTS) isolated from the roots of Petiveria alliacea L. using thin layer and high performance liquid chromatography. The 96-h LD₅₀ value (μg per tick) obtained for adult Boophilus microplus (Canestrini) topically treated with DBTS was 0·920. The LD₅₀ values obtained for three commercial acaricides dimethoate, lindane and carbaryl were 4·6, 9·3 and 6·9 μg per tick respectively. The IOD₅₀ and IHD₅₀ (concentrations inhibiting egg laying and hatching by 50% respectively) in μg per tick doses for DBTS were 0·22 and 0·24 respectively. The 24-h LD₅₀ dose (μg per insect) obtained for DBTS on adult Cylas formicarius elegantulus (Summer) was 0·193 μg per insect. The vapour from a stock solution of 5 g litre^-1 of DBTS was highly toxic to adult Hypothenemus hampei Ferr. inside coffee berries, inflicting 89% mortality within 24 h. © 1997 SCI.     

Deposition of aerially applied tebufenozide (RH5992) on balsam fir (Abies balsamea) and its control of spruce budworm (Choristoneura fumiferana [Clem.])

A field trial was conducted in 1994 to determine the foliar deposit of tebufenozide (RH5992), applied aerially, and its efficacy against spruce budworm, Choristoneura fumiferana (Clem.). A commercial 240 g litre^-1 formulation of the insecticide (Mimic 240LV) was mixed with water, dyed with a tracer dye (Rhodamine WT) and sprayed with a light fixed-wing aircraft. Six application strategies were tested. Five used 70 g AI ha^-1 in a spray volume of 1 or 2 litre^-1 ha^-1 with single or double applications; the sixth was an unsprayed control. Results show that the spectra of the spray applications were, with one exception, fairly uniform. Volume and number median diameters ranged from 100 to 130 μm and 27 to 72 μm, respectively. Mean number of drops cm^-2 on Kromekote cards were <2·0 for strategies where either 1 or 2 litre ha^-1 were sprayed. Nevertheless no one strategy produced droplet densities that were significantly different (P<0·05) from the other strategies. Tebufenozide recovered from foliage averaged 2·5 to 5·9 μg g foliage^-1 when 1 litre ha^-1 was sprayed and 5·8 to 6·8 μg g foliage^-1 after 2 litre ha^-1 were sprayed. When a single application was the strategy used, the mean number of droplets cm^-2 and μg tebufenozide g foliage^-1 ranged from 1·2 to 1·4 and 2·5 to 5·9, respectively. With double applications, the same response parameters ranged from 0·3 to 1·9 and 2·5 to 6·8, respectively. Budworm population reductions (%) and the number of larvae that survived tebufenozide treatments were significantly different (P<0·05) from the controls. After strategies that used 1 litre spray ha^-1, mean percentage population reductions ranged from 61·4 to 93·6 whereas populations were reduced by 85·6 to 98·3% when 2 litre ha^-1 were sprayed. After double applications the mean percentage population reductions ranged from 93·6 to 98·3, but single application strategies resulted in mean reductions of 61 to 86%. Mean population reductions in the controls were 61%. The mean number of larvae per branch that survived spray strategies of 1 litre ha^-1 ranged from 1·3 to 7·4, and from 0·4 to 1·3 when 2 litre ha^-1 was the spray volume. In the controls an average of 10·2 larvae survived. With one exception, mean percentage defoliation in the treated areas was also significantly less (P<0·05) than that in the control. Mean defoliation in trees sprayed at 1 litre spray ha^-1 ranged from 40 to 62·8% whereas those treated at 2 litre ha^-1 had mean defoliation levels from 31·5 to 62·8%. In contrast, average defoliation in the controls was 92·1%. When a single application was the spray strategy, mean defoliation ranged from 31·5 to 62·8%. These data imply that a double application of tebufenozide at 70 g in 2 litre ha^-1 was the most efficacious strategy. However, analyses of the data also show that the primary influence on deposits and defoliation was interactions between number of applications and spray. Nevertheless the two independent variables acted without significant interactions when influencing percentage reductions of spruce budworm populations. © 1998 SCI     

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.     

Alternative Respiration: a Biochemical Mechanism of Resistance to Azoxystrobin (ICIA 5504) in Septoria tritici

The mechanism of resistance to ICIA 5504 (azoxystrobin) in a Septoria tritici mutant raised in the laboratory has been investigated. This mutant was approximately 10 times less sensitive than the wild-type strain in in-vitro tests towards spore germination or fungal growth. Glucose oxidation in whole cells was inhibited in the wild type (80% inhibition at 0·1 μg ml^-1), whereas in the resistant mutant, oxygen uptake was stimulated (50% stimulation at 1·0 μg ml^-1). Respiration of the wild-type strain was inhibited by antimycin A and cyanide but not that of the mutant. These results indicate the existence of an efficient alternative respiratory pathway in the mutant, which was inhibited by the addition of 2 mM salicylhydroxamate (SHAM). Using mitochondria, antimycin A and ICIA 5504 did not completely inhibit NADH oxidation in either strain. Addition of SHAM inhibited part of the antimycin- and ICIA 5504-insensitive oxygen uptake only in mutant mitochondria. For complete inhibition of oxygen reduction, SHAM and cyanide need to be present. Thus, three systems of electron transfer from exogenous NADH to oxygen are present in S. tritici mitochondria: the cytochrome pathway which is sensitive to ICIA 5504 and antimycin A inhibition in both strains, the system of NADH-cytochrome c reductase which bypasses the methoxyacrylate inhibition at the cytochrome bc₁ complex, and the alternative oxidase which is inhibited by SHAM, and which is partially functioning only in mitochondria isolated from the ICIA 5504-resistant mutant. When the S. tritici isolates were tested for their in-vivo sensitivity to ICIA 5504 on wheat, the resistant strain was controlled better than the wild type. This indicates that the decreased ATP formation by the alternative pathway of respiration was inadequate for efficient parasitic growth on the host. © 1997 SCI.     

Presence of Muscarinic Acetylcholine Receptors in the Cattle Tick Boophilus microplus and in Epithelial Tissue Culture Cells of Chironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [³H]quinuclidinyl benzilate (QNB) with high affinity (1·2±(0·13) nM ; B_max 22·5 pmol mg protein⁻¹) were detected that do not bind nicotinic compounds specifically. The estimated IC₅₀ values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [³H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC₅₀ values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM , 0·8(±0·2) μM , 2·8(±0·6) μM and 78(±1·9) μM , respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [³H]QNB were found. Muscarinic antagonists selectively displaced [³H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC₅₀ 2·13(±1·02) μM ) among different subtype-specific mAChR antagonists (4-DAMP had IC₅₀ 49·9(±9·13) μM and methoctramine had IC₅₀ 121(±14·2) μM ) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4·8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC₅₀ 71·3(±19·6) μM ) and carbachol (IC₅₀ 253(±87·1) μM ) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.     

Nimbolide is the Principal Cytotoxic Component of Neem-Seed Insecticide Preparations

Several neem-seed extracts, some used for preparing commercial azadirachtin-containing insecticides, are cytotoxic to N1E-115 murine neuroblastoma cells with IC₅₀ values of 20–200 μg extract ml⁻¹ culture medium. Bioassay-directed fractionation by reversed-phase HPLC shows that the toxicity to N1E-115 cells is associated primarily with a single minor component identified by isolation and NMR and MS as nimbolide with an IC₅₀ of 1·5 μg ml⁻¹ (3·2 μM ). The difference in quantity of nimbolide in seven neem extract sources generally correlates with their overall cytotoxicity. Three other limonoids (epoxyazadiradione, salannin and possibly deacetylsalannin) but not azadirachtin, nimbin and deacetylnimbin contribute in small part to the cytotoxicity. Reconstituted neem extract with only nimbolide removed is less cytotoxic than the original extract. It therefore appears that nimbolide is the principal cytotoxic component of the neem extracts examined and that such minor constituents may warrant consideration in safety evaluations.     

Detection of resistance to sterol demethylation-inhibiting (DMI) fungicides in<Emphasis Type="Italic">Cercospora beticola</Emphasis> and efficacy of control of resistant and sensitive strains with flutriafol

Single-lesion isolates ofCercospora beticola (n=150) were collected in 1998 from sugar beet fields in the area of Serres, N. Greece. In this area, sterol demethylation-inhibiting (DMI) fungicides have been used for almost 20 years to control sugar beet leaf spot. The sensitivity of these isolates to the DMI fungicides flutriafol and difenoconazole (EC₅₀ values) was determined on the basis of inhibition of mycelial growth at several fungicide concentrations. The relative growth (RG) of isolates was correlated at all tested concentrations with the respective EC₅₀ values, indicating that RG provides a reliable estimate for the sensitivity of the isolates. The highest correlation coefficients were obtained for concentrations of 1 μg ml⁻¹ flutriafol and of 0.05 μg ml⁻¹ difenoconazole, respectively. Consequently, they are proposed for monitoring of DMI sensitivity inC. beticola populations, as single discriminatory concentrations in a simplified test method. Based on the RG values at the discriminatory concentration of 1 μg ml⁻¹ flutriafol,C. beticola isolates were classified as either resistant or sensitive. The efficacy of flutriafol, applied at the commercially recommended dose, in controlling Cercospora leaf spot was examined in field experiments conducted during 1999 and 2000. Disease incidence in plots artificially inoculated with resistant isolates and treated with flutriafol was significantly higher than in similar plots inoculated with sensitive strains. These results suggest that poor disease control after application of flutriafol may be based on the presence of resistant strains within the pathogen population in northern Greece. This emphasizes the risk of the development of practical resistance if there is increased frequency of such strains within the population. http://www.phytoparasitica.org posting July 13, 2003.