Synthesis of 2-amino-6-oxocyclohexenylsulfonamides and their activity against Botrytis cinerea

BACKGROUND: With the objective of exploring the fungicidal activity of 2‐oxocyclohexylsulfonamides (2), a series of novel 2‐amino‐6‐oxocyclohexenylsulfonamides (6 to 23) were synthesised, and their fungicidal activities against Botrytis cinerea Pers. were evaluated in vitro and in vivo. RESULTS: The compounds were characterised by IR, ¹H NMR and elemental analysis. Bioassay results of mycelial growth showed that compounds 6 to 23 had a moderate antifungal activity against B. cinerea. N‐(2‐methylphenyl)‐2‐(2‐methylphenylamino)‐4,4‐dimethyl‐6‐oxocyclohexenylsulfonamide (13) and N‐(2‐chlorophenyl)‐2‐(2‐chlorophenylamino)‐6‐oxocyclohexenylsulfonamide (21) showed best antifungal activities, with EC₅₀ values of 8.05 and 10.56 µg mL^?1 respectively. Commercial fungicide procymidone provided an EC₅₀ value of 0.63 µg mL^?1. The conidial germination assay showed that most of compounds 6 to 23 possessed excellent inhibition of spore germination and germ‐tube elongation of conidia of B. cinerea. For in vivo control of B. cinerea colonising cucumber leaves, the compound N‐cyclohexyl‐2‐(cyclohexylamino)‐4,4‐dimethyl‐6‐oxocyclohexenylsulfonamide (19) showed a better control effect than the commercial fungicide procymidone. CONCLUSION: The present work demonstrated that 2‐amino‐6‐oxocyclohexenylsulfonamides can be used as possible new lead compounds for further developing novel fungicides against B. cinerea. Copyright © 2011 Society of Chemical Industry     

Ovicidal,larvicidal and juvenilizing activity of a picolinephenoxyanilide against Cydia pomonella

N‐(4‐phenoxyphenyl)‐2‐pyridinecarboxamide (1) was synthesized from commercially available materials and its ovicidal and larvicidal activity against Cydia pomonella (L) was tested. The compound showed a LC₅₀ of 0.98 mg ml⁻¹ when eggs less than 24 h were sprayed using a Potter Tower, but it had no effect when eggs older than this were sprayed. The compound did not have larvicidal activity when larvae were treated with 1200 µg g⁻¹. However, the larval head capsules were smaller than those in the controls when treated at this concentration. To assess its possible juvenile‐hormone‐like activity, the compound was topically applied to young pupae of Tribolium confusum duVal, where it produced clear juvenilization effects, which were dependent on the applied dose. © 2000 Society of Chemical Industry     

Influence of environmental pH modulation on efficiency of apoplastic PR proteins during Fusarium culmorum– wheat seedling interaction

Variation of apoplastic pH by Fusarium culmorum and its influence on the production, activity and isoenzymes patterns of the pathogenesis‐related (PR) proteins β1,3‐glucanase, chitinase and peroxidase enzymes were detected in apoplastic fluids (AFs) from infected wheat seedlings. The time course in the 24–48 h interval post infection was characterized by an increase in activity and isoenzymatic differential induction of the selected PR proteins and by a concomitant rise of apoplastic pH. Chitinase attained maximum activity at pH 8·0 in the case of inoculated seedlings. Optimal β1,3‐glucanase activity in the pH range 6·0–8·0, was observed at pH 7·0. Peroxidase was strongly affected by pH, with enzyme activity having a maximum rate at pH 6·0 and thereafter rapidly declining at higher pH. Maximum peroxidase activity paralleled the appearance of the complete isoenzymatic pattern. In order to investigate the biological role of PR proteins in AFs, the in vitro antifungal activity was evaluated. In the interval 0–6 h, pH of macroconidia suspensions rose up to 7·2. AFs revealed inhibitory activity against germinating macroconidia of F. culmorum by decreasing the germination efficiency of macroconidia apical compartments, while this effect was compensated by an increased germination capacity of middle compartments. Present results suggest that during infection of wheat seedlings by F. culmorum the pH modulation favours host colonization by enhancing the activity of pectin lyase, and simultaneously inhibits the capacity of the host to oppose the pathogen by interfering with peroxidases which represent an important component of the defence arsenal.     

Structure–biological activity relationships in triterpenic saponins: the relative activity of protobassic acid and its derivatives against plant pathogenic fungi

BACKGROUND: Triterpenic saponins from Sapindus mukorossi Gaertn. and Diploknema butyracea JF Gmelin were evaluated for in vitro antifungal activity against four phytopathogenic fungi. The study of the structure–antifungal activity relationships of protobassic acid saponins was widened by including semi‐synthetic derivatives. RESULTS: Diploknema butyracea saponins exhibited significant antifungal activity against three fungi (ED₅₀ 230–455 µg mL^?1), whereas S. mukorossi saponin was effective against two fungi (ED₅₀ 181–407 µg mL^?1). The n‐butanol extract after preparative HPLC separation provided two saponins from D. butyracea saponin mixture: 3‐O‐[β‐D ‐glucopyarnosyl‐β‐D ‐glucopyranosyl]‐16‐α‐hydroxyprotobassic acid‐28‐O‐[arabinopyranosyl‐glucopyranosyl‐xylopyranosyl]‐arabinopyranoside (MI‐I), and 3‐O‐β‐D ‐glucopyranosyl‐glucopyranosyl‐glucopyranosyl‐16‐α‐hydroxyprotobassic acid‐28‐O‐[arabinopyranosyl‐xylopyranosyl‐arabinopyranosyl]‐apiofuranoside (MI‐III). The single saponin extracted from S. mukorossi saponin mixture was identified as 3‐O‐[O‐acetyl‐β‐D ‐xylopyranosyl‐β‐D ‐arabinopyranosyl‐β‐D ‐rhamnopyranosyl] hederagenin‐28‐O[β‐D ‐glucopyranosyl‐β‐D ‐glucopyranosyl‐β‐D ‐rhamnopyranosyl] ester (SM‐I). Monodesmosides resulting from the partial degradation of hederagenin and hydroxyprotobassic acid bisdesmosides exhibited significant reduction in antifungal effect. Further removal of sugar moiety yielded complete loss in activity. The antifungal activity of the triterpenic saponins was associated with their aglycone moieties, and esterification of the hydroxyl group led to change in antifungal activity. CONCLUSION: Sapindus mukorossi saponin, which is effective against Rhizoctonia bataticola (Taub.) Briton Jones and Sclerotium rolfsii Sacc., can be exploited for the development of a natural fungicide. A sugar moiety is a prerequisite for the antifungal activity of triterpenic saponin. Copyright © 2010 Society of Chemical Industry     

Effect of the ecdysone agonists,RH-2485 and tebufenozide,on the southwestern corn borer,Diatraea grandiosella

The effect of the ecdysone agonists RH-2485 (proposed name methoxyfenozide) and tebufenozide (RH-5992), was examined on eggs and larvae of the southwestern corn borer, Diatraea grandiosella Dyar. Both compounds exhibited a concentration-dependent ovicidal activity. More than 95% of eggs died when egg masses were dipped in solutions of 100 or 200 mg liter^-1 of either compound in acetone+distilled water (1+1 by volume). Although some eggs treated with 1 or 10 mg liter^-1 of the compounds hatched, the survival rate was low. Newly hatched larvae were fed for seven days on an artificial diet containing RH-2485 or tebufenozide. The LC₅₀ values were 0·049 mg kg^-1 for RH-2485 and 0·185 mg kg^-1 for tebufenozide, showing that RH-2485 was about four times more active than was tebufenozide. Although increasing the time of exposure to either compound decreased the LC₅₀ value significantly, the relative potency of RH-2485 versus tebufenozide was not changed. Newly ecdysed 4th-instar larvae fed with diets containing 0·125, 0·25 or 0·5 mg kg^-1 RH-2485 or tebufenozide ceased feeding approximately 8 h after exposure, indicating that larvae had prematurely entered a molting cycle. Larvae treated with RH-2485 ecdysed earlier and died more quickly than those treated with tebufenozide. Ingestion of sublethal concentrations of RH-2485 (0·005 and 0·01 mg kg^-1) or tebufenozide (0·03 and 0·06 mg kg^-1) retarded larval growth, and decreased pupal weight and adult emergence. Increasing exposure time to tebufenozide tended to increase the larval mortality, significantly retarded larval growth, and decreased the mean weights of male and female pupae and adult emergence. RH-2485 (0·125 and 0·25 mg kg^-1) and tebufenozide (0·25 and 0·5 mg kg^-1) were lethal to newly hatched larvae, even after diets containing these compounds were held for 20 days at 30°C under long days (16 h light: 8 h dark). Our results suggest that field trials to assess the potential of RH-2485 and tebufenozide to control D. grandiosella are warranted. © 1998 SCI     

Insecticidal properties of benzofuran-2-carboxylic acid derivatives

A series of amides and esters of substituted benzo[b]furan-2-carboxylic acids have been synthesised, and their activity against adult sweet potato weevils, Cylas formicarius elegantulus (Summer) studied. The topical insecticidal potency of these compounds was compared in acetone solution and in a mixture of piperonyl butoxide (PB) and acetone (0·05+99·95 by volume). The compounds were much more active when administered in the acetone/PB mixture, and exhibited 48-h LD₅₀ values ranging from 1·7 to 26·6 μg per insect. The most active compound, 2-(3,5-dimethylpyrazol-1-ylcarbonyl)-6-methoxy-3-methylbenzofuran, was equiactive with technical grade dimethoate (in acetone/PB) on a weight basis. © 1998 SCI     

The peach (Prunus persica) defensin PpDFN1 displays antifungal activity through specific interactions with the membrane lipids

Ppdfn1 is a defensin gene previously identified in peach (Prunus persica). The biological role of Ppdfn1 was investigated by analysing its expression profile in leaves, flowers and fruits, either inoculated with the Monilinia laxa fungal pathogen or mock‐inoculated. Ppdfn1 expression was highest in flowers and, in fruits, did not vary upon M. laxa inoculation. To characterize the PpDFN1 antifungal activity, the recombinant mature peptide was expressed in Escherichia coli and purified; recombinant PpDFN1 displays antifungal activity against Botrytis cinerea, M. laxa and Penicillium expansum, with IC₅₀ values of 15·1, 9·9 and 1·1 μg mL^?1, respectively. Treatment of fungal hyphae with FITC‐labelled PpDFN1 indicated that the peptide is not internalized by fungal hyphae, but localizes on their external cell surface. At this site, PpDFN1 is capable of membrane destabilization and permeabilization, as demonstrated by SYTOX Green fluorescence uptake by the treated mycelia. Using artificial lipid monolayers, it was shown that PpDFN1 interacts with sphingolipid‐containing membranes; however the strongest interaction occurs with monolayers composed of lipids extracted from sensitive fungi, such as P. expansum. These data suggest that the lipid composition of fungal membranes is of key relevance for defensin specificity.     

Synthesis and Structure–Activity Relationships for the Fungicidal Activity of O,O-bisaryl sec-butylphosphonates

Sixteen O,O-bisaryl sec-butylphosphonates have been synthesised by condensing sec-butylphosphonyl dichloride with substituted phenols. The compounds were tested against two phytopathogenic fungi, Rhizoctonia bataticola and Helminthosporium oryzae. The most active compound against R. bataticola is O,O-bis(3-methylphenyl) sec-butylphosphonate (ED₅₀ 29·56 mg litre^-1) and against H. oryzae, O,O-bis(2-chlorophenyl) sec-butylphosphonate and its para-chloro analogue (ED₅₀ 0·14 and 0·13 mg litre^-1 respectively). Quantitative structure–activity relationships on the fungicidal activity have been analysed by means of multiple regression analysis using physicochemical substituent parameters. Electronic parameters viz., σ and F have expressed significant variability in fungitoxicity against both the fungi, viz. R. bataticola and H. oryzae. Hydrophobic and steric parameters are also found to be important in the correlation studies. © 1997 SCI.     

Characterization and Inhibitor Studies of Chitinases from a Parasitic Blowfly (Lucilia cuprina), a Tick (Boophilus microplus), an Intestinal Nematode (Haemonchus contortus) and a Bean (Phaseolus vulgaris)

The molecular weight pattern and the stage-specific activities of chitinases from the blowfly Lucilia cuprina, the tick Boophilus microplus and the intestinal nematode Haemonchus contortus were examined. Chitinolytic enzymes could be detected in all parasite species tested, but the activity was different between the stages. Highest chitinolytic titers were found in blowfly pupae (83 kDa, 118 kDa), hatching larvae of ticks (58 kDa, 94 kDa) and nematode eggs (43 kDa). Leaves from ethylene-treated bean plants Phaseolus vulgaris expressed two basic Class I chitinases (Ia, Ib) of 34 kDa, differing in their amino acid sequences at residue 33 and 34 (Ia: glycine, proline; Ib: lysine, aspartic acid). Inhibitor studies with blowfly pupae revealed that allosamidin (IC₅₀=0·32 (±0·02) μM ) was by far the best inhibitor when compared with various amino sugar derivatives. This compound also inhibited chitinases from tick larvae (IC₅₀=0·69(±0·10) μM ) and nematode eggs (IC₅₀=0·048(±0·0045) μM ) specifically. Whereas Class Ia chitinase from bean leaves was inhibited only up to 18% by 10 μM allosamidin, it had an IC₅₀ of 1(±0·14) μM for the Ib type, which is the first plant chitinase described to be highly sensitive to allosamidin.     

Synergistic and antagonistic interactions of terpenes against Meloidogyne incognita and the nematicidal activity of essential oils from seven plants indigenous to Greece

BACKGROUND: Biorational means for phytonematode control were studied within the context of an increasingly ecofriendly pest management global approach. The nematicidal activity and the chemical composition of essential oils (EOs) isolated from seven plants grown in Greece and ten selected compounds extracted from them against second‐stage juveniles (J2) of Meloidogyne incognita (Kof. & White) Chitwood were evaluated using juvenile paralysis experiments. Additionally, synergistic and antagonistic interactions between nematicidal terpenes were studied using an effect addition model, with the comparison made at one concentration level. RESULTS: The 96 h EC₅₀ values of Foeniculum vulgare Mill., Pimpinella anisum L., Eucalyptus meliodora A Cunn ex Schauer and Pistacia terebinthus L. were 231, 269, 807 and 1116 µg mL^?1, respectively, in an immersion bioassay. Benzaldehyde (9 µg mL^?1) was the most toxic compound, followed by γ‐eudesmol (50 µg mL^?1) and estragole (180 µg mL^?1), based on 96 h EC₅₀ values. The most potent terpene pairs between which synergistic actions were found, in decreasing order, were: trans‐anethole/geraniol, trans‐anethole/eugenol, carvacrol/eugenol and geraniol/carvacrol. CONCLUSION: This is the first report on the activity of F. vulgare, P. anisum, E. meliodora and P. terebinthus, and additionally on synergistic/antagonistic nematicidal terpene interactions, against M. incognita, providing alternative methods for nematode control. Copyright © 2010 Society of Chemical Industry     

竹红菌甲素对18种植物病原菌的抑制作用

Inhibition of Hypocrellin A (HA) against 18 pathogenic fungi by mycelium growth rate in light and dark conditions was evaluated. The results showed that 50 mg·L^-1 HA exhibited antifungal activities against all of 18 pathogenic fungi under luminous intensity of 12 000 Lx. The inhibition rates of HA to Lecanosticta acicola, Fusarium graminearum, Valsa mali and Botryosphaeria dothidea were all higher than 90%. The EC₅₀ of HA to Botryosphaeria dothidea and Lecanosticta acicola was 0.60 mg·L^-1 and 0.77 mg·L^-1 respectively. However, HA showed weak and even no antifungal activity in dark condition. The study suggested that the potential of HA could be as a new kind of photoactivated biopesticide.     

Acaricidal activities of the active component of Lycopus lucidus oil and its derivatives against house dust and stored food mites (Arachnida: Acari)

BACKGROUND: Recent studies have focused on materials derived from plant extracts as mite control products against house dust and stored food mites because repeated use of synthetic acaricides had led to resistance and unwanted activities on non‐target organisms. The aim of this study was to evaluate the acaricidal activity of materials derived from Lycopus lucidus against Dermatophagoides farinae, D. pteronyssinus and Tyrophagus putrescentiae. RESULTS: The LD₅₀ values of L. lucidus oil were 2.19, 2.25 and 8.45 µg cm^?2 against D. farinae, D. pteronyssinus and T. putrescentiae. The acaricidal constituent of L. lucidus was isolated by chromatographic techniques and identified as 1‐octen‐3‐ol. In a fumigant method against D. farinae, the acaricidal activity of 1‐octen‐3‐ol (0.25 µg cm^?2) was more toxic than N,N‐diethyl‐m‐toluamide (DEET) (36.84 µg cm^?2), followed by 3,7‐dimethyl‐1‐octen‐3‐ol (0.29 µg cm^?2), 1‐octen‐3‐yl butyrate (2.32 µg cm^?2), 1‐octen‐3‐yl acetate (2.42 µg cm^?2), 3,7‐dimethyl‐1‐octene (9.34 µg cm^?2) and benzyl benzoate (10.02 µg cm^?2). In a filter paper bioassay against D. farinae, 1‐octen‐3‐ol (0.63 µg cm^?2) was more effective than DEET (20.64 µg cm^?2), followed by 3,7‐dimethyl‐1‐octen‐3‐ol (1.09 µg cm^?2). CONCLUSION: 1‐Octen‐3‐ol and 3,7‐dimethyl‐1‐octen‐3‐ol could be useful as natural agents for the management of three mite species. Copyright © 2011 Society of Chemical Industry     

Bacterial pathogens of rice in the Kingdom of Cambodia and description of a new pathogen causing a serious sheath rot disease

A study of rice diseases in Cambodia from 2005 to 2007 showed widespread occurrence of diseases caused by Acidovorax avenae subsp. avenae, Burkholderia gladioli, B. cepacia and Pantoea ananatis. This is the first report of these pathogens in Cambodia. Additionally, a pseudomonad causing a widespread disease similar to sheath brown rot (caused by Pseudomonas fuscovaginae) was isolated. The studied strains were pathogenic to rice cvs Sen Pidau and IR 66, producing similar, though slightly less severe, symptoms to those observed in the field. Based on comparative 16S rDNA gene sequence analysis, combined with cell wall fatty acid analysis and metabolic profiles, the isolated strains were allocated to the genus Pseudomonas. The novel species were differentiated from Pseudomonas fuscovaginae and P. putida by their inability to metabolize d ‐fructose, d ‐galactose, d ‐galactonic acid lactone, d ‐galacturonic acid, d ‐glucosaminic acid, d ‐glucuronic acid, p‐hydroxy phenylacetic acid, d ‐saccharic acid and urocanic acid. The major fatty acids were C_16:0, summed feature 3 (C_16:1ω7c and C_16:1ω6c) and summed feature 8 (C_18:1ω7c), representing 80% of the total. Partial 16S rRNA gene sequences (1460 bp) were identical, except for two nucleotide changes amongst the six strains. Alignment of the causal strains within type‐culture databases revealed similarities of 99·7% with Pseudomonas parafulva AJ 2129^T, 99·2% with P. fulva IAM 1592^T, 98·9% with P. plecoglossicidia FPC 951^T, and 98·1% with P. fuscovaginae MAFF 301177^T. On the basis of data from this polyphasic study, it is proposed that the unknown strains isolated from rice represent a novel species of the genus Pseudomonas.     

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.     

Laboratory Evaluation of the Novel Naturally Derived Compound Spinosad against Ceratitiscapitata

Laboratory studies were conducted to determine the effect of the naturally derived compound spinosad on Ceratitis capitata Wied. (Diptera, Tephritidae). The organophosphate fenthion was used as a standard. Direct dose-dependent mortality and reduced fecundity were observed in oral treatment of adults with spinosad. The LC₉₀ values 14 h and seven days after treatment were 19·50 and 0·49 mg litre⁻¹ respectively. Fenthion was less active (the LC₅₀ eight days after treatment was 1·17 mg litre⁻¹) and did not affect the fecundity of the fly. Adults were also very susceptible to spinosad and fenthion via residual contact. For spinosad, 100% mortality was recorded 48 h after treatment for a dose of 10 mg litre⁻¹. Spinosad was more effective than fenthion in suppressing larval development when neonate larvae were reared on treated diet supplemented with a range of concentrations from 0·02 to 0·83 mg kg⁻¹ diet. Last-instar larvae were much less susceptible to spinosad or fenthion when exposed via dipping or when they pupated in treated medium and both products had similar performance. A lack of ovicidal activity was observed in direct egg-treatments with spinosad but significant reductions from 1 mg litre⁻¹ onwards were recorded for fenthion.     

基于丁香酚的异(口恶)唑啉类化合物的设计合成及抑菌活性

为了探索新型异齅唑啉类化合物作为杀菌剂候选化合物开发的潜力,本研究以廉价易得的芳香醛类化合物为原料制备了32个氯代肟类化合物,通过其与天然产物丁香酚的1,3-偶极环加成反应和后期官能团化反应,制备了35个异齅唑啉类化合物 ( D1 ~ D32 , E1 ~ E3 ),其中34个为新化合物。所有化合物的结构均经过液相色谱-电喷雾质谱 (LC-ESI-MS)、核磁共振氢谱 (1H NMR) 及元素分析确认。初步抑菌活性测试结果表明:多数目标化合物对油菜菌核病菌、水稻纹枯病菌、番茄早疫病菌和辣椒疫霉病菌具有较好的抑菌活性,其中化合物 D26 对油菜菌核病菌的活性最高,有效抑制中浓度 (EC₅₀) 为14.3 mg/L,具有进一步研究的价值。     

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.     

Evaluation of different methods for the characterization of carrot resistance to the alternaria leaf blight pathogen (Alternaria dauci) revealed two qualitatively different resistances

Alternaria leaf blight (ALB), caused by Alternaria dauci, is one of the most damaging foliar diseases of carrot worldwide. The aim of this study was to compare different methods for evaluating levels of carrot resistance to ALB. Three techniques were investigated by comparison with a visual disease assessment control: in vivo conidial germination, a bioassay based on a drop‐inoculation method, and in planta quantification of fungal biomass by quantitative PCR (Q‐PCR). Three carrot cultivars showing different degrees of resistance to A. dauci were used, i.e. a susceptible cultivar (Presto) and two partially resistant genotypes (Texto and Bolero), challenged with an aggressive or a very aggressive isolate of A. dauci. Both partially resistant genotypes produced a higher mean number of germ tubes per conidium (up to 3·42±0·35) than the susceptible one (1·26±0·18). The drop‐inoculation results allowed one of the partially resistant genotypes (Bolero, log₁₀(S+1) = 1·34±0·13) to be distinguished from the susceptible one (1·90±0·13). By contrast, fungal growth measured by Q‐PCR clearly differentiated the two partially resistant genotypes with log₁₀(I) values of 2·77±0·13 compared to the susceptible cultivar (3·65±0·13) at 15 days post‐inoculation. This result was strongly correlated (r² = 0·91) with the disease severity index scored at the same date. Data obtained with the different assessment methods strongly suggest that the Texto and Bolero genotypes have different genetic resistance sources.