Oral administrations of methamidophos and TOCP have different effects on in vitro protein phosphorylation levels from subcellular fractions of hen brain

The effects of methamidophos and tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of specific brain proteins were studied in Beijing white laying hens during the early stage of delayed neurotoxicity. Phosphorylation of mitochondrial and synaptosomal proteins was assayed in vitro by using [γ-³²P]ATP as phosphate donor. Tri-o-cresyl phosphate (TOCP) administration enhanced the phosphorylation of synaptosomal proteins with molecular weight of 40 and 55 kDa by as much as 36% and 65%, respectively, and decreased the phosphorylation of mitochondrial protein (35 kDa) by 33%. A single dose of methamidophos enhanced the phosphorylation of 32-kDa synaptosomal protein by 44%; however, it had no effect on brain mitochondrial proteins. The activity of neuropathy target esterase (NTE) in dosed hens’ brain and spinal cord was assayed for the effects of methamidophos and TOCP. These results showed that methamidophos inhibited brain NTE by 41% compared with that of control after 7-day exposure, while TOCP inhibited brain NTE by 66%. Moreover, NTE activity from spinal cord in treated hens also exhibited a similar trend of activity inhibition. Together, these results suggested that methamidophos and TOCP induced changes of protein phosphorylation level from hen brain and resulted in different kinds of neurotoxicity.     

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.     

Pyrethroid insecticides: Potent,stereospecific enhancers of mouse brain sodium channel activation

Deltamethrin and NRDC 157, pyrethroid insecticides that produce different poisoning syndromes in mammals, enhanced veratridine-dependent, sodium channel-mediated ²²Na⁺ uptake in mouse brain synaptosomes. Concentrations producing half-maximal enhancement were 2.5 × 10^?8M (deltamethrin) and 2.2 × 10^?7M (NRDC 157). This effect was stereospecific: The nontoxic 1S enantiomers had no significant effect on veratridine-dependent activation. At high deltamethrin concentrations, enhancement was maximal at 5 × 10^?5?1 × 10^?4M veratridine. Pyrethroid enhancement was completely blocked by 5 × 10^?6M tetrodotoxin, and neither pyrethroid affected ²²Na⁺ uptake in the absence of veratridine at concentrations up to 1 × 10^?5M. The relative potencies of deltamethrin and NRDC 157 in the synaptosomal sodium channel assay agree well with their relative acute toxicities to mice when administered by intracerebral injection. These findings demonstrate that pyrethroids exemplifying both characteristic poisoning syndromes are potent, stereospecific modifiers of sodium channel function in mammalian brain.     

The effect of piperonyl butoxide and triorthocresyl phosphate on the activity and metabolism of altosid (isopropyl 11-methoxy, 3,7,11-trimethyldodeca-2,4-dienoate) in Tenebrio molitor L. and Oncopeltus fasciatus (dallas)

Altosid (I) was found to have an ED₅₀ of 0.026 μg/g in T. molitor and 5.0 μg/g in O. fasciatus. The juvenilizing activity of I in T. molitor was increased by co-treatment with piperonyl butoxide (PB) and triorthocresyl phosphate (TOCP). The activity of I in O. fasciatus, was considerably decreased by PB and TOCP indicating the blocking of an activation reaction. The presumptive metabolities of I, isopropyl 11-hydroxy-3,7,11-trimethyldodeca-2,4-dienoate, (II), 11-methoxy-3,7,11-trimethyldodeca-2,4-dienoic acid (III), and 11-hydroxy-3,7,11-trimethyldodeca-2,4-dienoic acid (IV) were tested for juvenilizing activity in O. fasciatus and II was found to be four times more active than I. IV was more active than III but less active than I. Co-treatment of II with PB or TOCP did not decrease activity, indicating that II is a true activation product. Uptake studies with ¹⁴C-I showed that the difference in activity between the two insects was not due to differences in uptake. Forty-eight hours after treatment 30% of I was lost from the cuticle by evaporation. Metabolic studies showed that PB inhibited the formation of II and TOCP inhibited the formation of III in T. molitor while in O. fasciatus both synergists inhibited the formation of II.     

Cholinergic properties of pinched-off synaptic nerve endings from the central nervous system of the cockroach Periplaneta americana

The preparation and cholinergic properties of a subcellular fraction, enriched in pinched-off nerve-endings (synaptosomes) from the central nervous system of the cockroach Periplaneta americana, are described. The endings retained their cytoplasmic components, as shown by the presence of marker enzymes and by ultrastructural examination. A carrier-mediated, high-affinity uptake of [³H] choline into the synaptosomes was demonstrated, and this uptake was saturable, depended on a Na⁺-gradient, and was inhibited by hemicholinium-3. It had an apparent K_m value of 0.6 (±0.1) μM, and a V_max of 20.5 (±2.5) pmol min^?1 per mg of protein. The high-affinity [³H]choline uptake was associated with the synthesis of [³H]phosphocholine and [³H]O-acetylcholine. The rate of [³H]choline uptake in synaptosomes was increased by DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane] at 100 nM concentration, and this increase was inhibited by tetrodotoxin, while neostigmine appeared to be a potent inhibitor (I₅₀ = 10 pM) of the DDT-activated uptake of [³H]choline. The site of action of the insecticides was specifically on the pre-synaptic nerve terminals because the synaptosomes preparation did not retain the post-synaptic membrane of the original nerve-endings. Cockroach synaptosomes provided a useful in-vitro preparation for studying the effects of insecticides on the pre-synaptic nerve endings.     

Action of deltamethrin on N-type (Cav2.2) voltage-sensitive calcium channels in rat brain

Isolated presynaptic nerve terminals prepared from whole rat brain were used to evaluate the action of deltamethrin on voltage-sensitive calcium channels by measuring calcium influx and endogenous glutamate release. Deltamethrin-enhanced K⁺-stimulated calcium influx and subsequent Ca²⁺-dependent glutamate release. The effect of deltamethrin was concentration-dependent, stereospecific, blocked by ω-conotoxin MVIIC but unaltered in the presence of tetrodotoxin. These results suggest that N-type voltage-sensitive calcium channels are a site of action at the presynaptic nerve terminal. Electrophysiological studies were carried out using rat brain Ca_v2.2 and β₃ subunits coexpressed in Xenopus oocytes to validate such action. Deltamethrin reduced barium peak current in a concentraion-dependent and stereospecific manner, increased the rate of activation, and prolonged the inactivation rate of this channel. These experiments support the conclusion that N-type voltage-sensitive calcium channel operation is altered by deltamethrin.     

Calcium entry blockers inhibit contractures induced by the molluscicide Frescon in Lymnaea stagnalis smooth and cross-striated muscles

The molluscicide Frescon induces irreversible contracture in smooth and cross-striated muscles of the freshwater snail Lymnaea stagnalis. This contracture can be inhibited or reversed by elevated Mg²⁺ and reduced Ca²⁺ concentrations, by the heavy metals Ni²⁺, Co²⁺, and Mn²⁺, by the rare earth La³⁺, and by the calcium channel blocker D-600. The removal of extracellular Ca²⁺ also prevents the expression of Frescon action. These results are consistent with the hypothesis that Frescon causes an increase in the Ca²⁺ permeability of the sarcolemma.     

Action of cismethrin and deltamethrin on functional attributes of isolated presynaptic nerve terminals from rat brain

Isolated presynaptic nerve terminals (synaptosomes) prepared from rat brain were used to evaluate the actions of a tremor (T)-syndrome (cismethrin) and a choreoathetosis-salivation (CS)-syndrome (deltamethrin) pyrethroid on the functional attributes of synaptosomes by measuring calcium influx and endogenous neurotransmitter (l-glutamate) release with fluorescent assays. Both cismethrin and deltamethrin stimulated calcium influx, however, only deltamethrin enhanced Ca²⁺-dependent neurotransmitter release and its action was stereospecific, concentration-dependent, stimulated by depolarization, unaltered by tetrodotoxin, and blocked by ω-conotoxin GVIA. Our results delineate a separate action of deltamethrin on presynaptic nerve terminals from that elicited by cismethrin and implicate Ca_v2.2 calcium channels as target sites for deltamethrin that is consistent with the observed in vivo release of neurotransmitter at the onset of convulsive symptom caused by CS-syndrome pyrethroids. This information will allow a more complete understanding of the molecular and cellular nature of pyrethroid-induced neurotoxicity and expands our knowledge of the structure–activity relationships of pyrethroids in regards to their action on voltage-sensitive calcium channels.     

Frequency detection of pyrethroid resistance allele in Anopheles sinensis populations by real-time PCR amplification of specific allele (rtPASA)

To investigate the level of pyrethroid resistance in Anopheles sinensis Wiedemann 1828 (Diptera: Culicidae), a major malaria vector in Korea, we cloned and sequenced the IIS4-6 transmembrane segments of the sodium channel gene that encompass the most widely known kdr mutation sites. Sequence analysis revealed the presence of the major Leu-Phe mutation and a minor Leu-Cys mutation at the same position in permethrin-resistant field populations of An. sinensis. To establish a routine method for monitoring resistance, we developed a simple and accurate real-time PCR amplification of specific allele (rtPASA) protocol for the estimation of resistance allele frequencies on a population basis. The kdr allele frequency of a field population predicted by the rtPASA method (60.8%) agreed well with that determined by individual genotyping (61.7%), demonstrating the reliability and accuracy of rtPASA in predicting resistance allele frequency. Using the rtPASA method, the kdr allele frequencies in several field populations of An. sinensis were determined to range from 25.0 to 96.6%, suggestive of widespread pyrethroid resistance in Korea.     

Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors

γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [³H]4′-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [³H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [³H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [³H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [³H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [³H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B_1a, were potent inhibitors of [³H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides in insect GABARs.     

<Emphasis Type="Italic">Fusarium</Emphasis> elicitor-dependent calcium influx and associated ros generation in tomato is independent of cell death

Fusarium oxysporum f. sp. lycopersici elicitor (EFOL-2) treatment induces cytosolic influx of calcium in Fusarium-resistant tomato suspension culture. The calcium signature was found to be biphasic, which is characteristic of recognition of oligosaccharides in the elicitor preparation. Further, several lines of evidence such as, (i) attainment of saturation level of the [Ca²⁺]_cyt at a definite extra-cellular calcium concentration (ii) prominent reduction in EFOL-2-induced influx in [Ca²⁺]_cyt on treatment with the calcium channel blockers verapamil and diltiazem and (iii) establishment of a refractory stage of [Ca²⁺]_cyt level upon repeated stimulation by EFOL-2, is indicative of receptor-mediated activation of the calcium channel for cytosolic elevation. In addition, inhibition of EFOL-2-induced [Ca²⁺]_cyt increase by protein kinase inhibitor staurosporine and wortmannin indicate phosphorylation is a regulatory event of calcium influx. Additionally, monitoring of cell death on EFOL-2 treatment indicated that the degree of ROS generation is not capable of inducing cell death. Inhibition of ROS generation on two separate occasions such as, calcium-free media and on treatment with inhibitors causing calcium channel occlusion revealed ROS generation as a successive event of calcium influx.     

The action of pyrethroids on the voltage-sensitive calcium channel of Paramecium tetraurelia

Calcium regulation is an important event in synaptic transmission and neuronal function, which is governed by a very intricate signal transduction system which is not completely understood. Using a variety of pharmacological assays, we have characterized the action of deltamethrin on the ciliary voltage-sensitive calcium channel and on phospholipase C activity of Paramecium tetraurelia Sonneborn, an organism that does not possess a voltage-sensitive sodium channel. In fura-2 fluorometric assays, which examined whole cells and ciliary membrane vesicles enriched with calcium channels, deltamethrin stimulated Ca²⁺ uptake. We also determined that the phospholipase C activity of the ciliary membrane vesicles is regulated by the βγ-subunit from heterotrimeric G-proteins. Subsequent treatment with deltamethrin resulted in a substantial and highly significant increase in phospholipase C activity. These results provide evidence that the molecular mode of action of pyrethroids on the voltage-sensitive calcium channel is distinct from the action of this insecticide on the voltage-sensitive sodium channel and may be dependent, in part, upon an interaction with the βγ-subunit of heterotrimeric G-protein.     

Biochemical effects of acute phoxim administration on antioxidant system and acetylcholinesterase in Oxya chinensis (Thunberg) (Orthoptera: Acrididae)

The study was undertaken to evaluate the effects of different concentrations of phoxim on acetylcholinesterase (AChE) and esterase (EST) activities, and antioxidant system after topical application to Oxya chinensis. The results showed that phoxim inhibited AChE activity, and did not cause significant changes in the EST activity and the levels of malondialdehyde (MDA) and reduced glutathione (GSH). After phoxim administration, superoxide (SOD) and catalase (CAT) activities showed a biphasic response with an initial increase followed by a decline in their activities. Glutathione reductase (GR) and glutathione peroxidase (GPx) activities were inhibited in comparison with the control. Glutathione S-transferase (GST) activity showed irregular changes. Its activity increased significantly at the concentrations of 0.06 and 0.12 μg/μL and decreased at the concentrations of 0.09 and 0.24 μg/μL compared with the control. Changes in SOD, CAT, GST, GPx, and GR activities indicated that phoxim caused oxidative damage in O. chinensis. However, no significant changes in MDA content suggested that these enzymes played important roles in scavenging the oxidative free radicals induced by phoxim in O. chinensis. The formation of oxygen free radicals might be a factor in the toxicity of phoxim.     

Differential response of plant cell membranes to some vinyl organophosphorus insecticides

The response of plant cell membranes to vinyl organophosphorus insecticides was studied by determining the release of intracellular materials as a measure of membrane permeability and the uptake of [1-¹⁴C]-α-aminoisobutyric acid as a measure of active transport. A pretreatment with chlorfenvinphos (2-chloro-1-(2,4-dichlorophenyl)-vinyl diethyl phosphate) at 0.4 mM or higher concentrations increased the leakage of cell contents from the tissues of pea, corn, and beet, but two other vinyl organophosphorus insecticides, tetrachlorvinphos (2-chloro-1-(2,4,5-trichlorophenyl)-vinyl diethyl phosphate) and phosphamidon (2-chloro-2-diethyl carbamoyl-1-methyl vinyl dimethyl phosphate), had no effect. Simultaneous addition of phospholipids, β-sitosterol, or Ca²⁺ inhibited in varying degrees the chlorfenvinphos-induced permeability, suggesting that the leakage of cell contents might be due to alteration in membrane structure.Chlorfenvinphos or tetrachlorvinphos at 0.1 mM or higher concentrations inhibited the uptake of α-aminoisobutyric acid. The degree of inhibition varied with different plant species. The inhibition was competitive and was not prevented by phospholipids. However, Ca²⁺ and other divalent cations were stimulatory to the uptake of α-aminoisobutyric acid, either in the presence or absence of chlorfenvinphos. Chlorfenvinphos also inhibited plant growth in tobacco callus and pea stem assays.From the differences in effective concentration, structural requirement, and interaction with phospholipids, it is suggested that chlorfenvinphos affected the membrane permeability and active transport by different mechanisms. These effects probably underlie its inhibitory action on plant growth.     

Inhibition of P-glycoprotein ATPase and its transport function of Helicoverpa armigera by morin, quercetin and phloroglucinol

Flavonoids (morin, quercetin and phloroglucinol) were tested for their ability to modulate the function of P-glycoprotein ATPase of the insecticide resistant pest Helicoverpa armigera (Ha-Pgp). Flavonoids in the presence of ethylparaoxon or cypermethrin significantly reduced both larval weight as well as survival rate 40-50%. Morin and quercetin inhibited the activity of Ha-Pgp ATPase by 80-90%, whereas phloroglucinol inhibited ATPase activity by 40% at 100 μM concentration. These flavonoids inhibited the verapamil, ethylparaoxon and cypermethrin-stimulated Ha-Pgp ATPase activity. Morin, quercetin and phloroglucinol binding were quantitated by quenching of the intrinsic Trp fluorescence of purified Ha-Pgp ATPase. Drug transport was monitored in proteoliposomes containing Ha-Pgp ATPase using the high affinity fluorescent substrate tetramethylrosamine (TMR) in real time. Addition of the morin and quercetin mediated the collapse of the TMR concentration gradient generated by Ha-Pgp ATPase. The inhibition studies on Ha-Pgp ATPase activity may contribute towards understanding new strategies of the pest to overcome insecticide resistance.     

Effect of pyridazinone herbicides on lipid metabolism in groundnut (Arachis hypogaea) leaves

The effect of five substituted pyridazinones (pyrazon, San 133-410H, San 9774, norflurazon, and San 6706) on lipid metabolism in groundnut (Arachis hypogaea) leaves was investigated under nonphotosynthetic conditions. In experiments with leaf disks, the uptake of [1-¹⁴C]acetate, [³²P]orthophosphate, and [³⁵S]sulfate was significantly inhibited by these herbicides and the magnitude of inhibition varied, depending on the substituents. When the incorporation of these precursors into lipids was measured and expressed as percentage of total uptake, no effect was observed in the case of [1-¹⁴C]acetate but there was significant inhibition in the incorporation of the other two precursors, suggesting that pyridazinones interfere with the metabolism of the phospholipids and the sulfolipid. None of these compounds affected the uptake of [methyl-¹⁴C]choline but all inhibited its incorporation into phosphatidylcholine indicating that phosphatidylcholine metabolism is vulnerable to pyridazinones. The fatty acid synthetase of isolated chloroplasts assayed in the absence of light was inhibited 20–50% by the pyridazinones at 0.1–0.5 mM concentrations. San 9774 showed the most potent inhibition. In addition, the pyridazinone herbicides significantly inhibited sn-glycerol-3-phosphate acyltransferase(s) in both chloroplast and microsomal fractions but showed no effect on phosphatidic acid phosphatase. The magnitude of inhibition of fatty acid synthetase and acyltransferase(s) is related to the nature of the substituent groups on the herbicide. Trifluorophenyl substitution at position 2 or amino substitution at position 5 of the pyridazinone molecule caused the maximum inhibitory effect.     

Competition between a thiocarbamate herbicide and herbicide protectants at the level of uptake into maize cells in culture

The rapid interactions between the herbicide S-ethyl dipropyl thiocarbamate (EPTC) and the structurally similar herbicide protectant N,N-diallyl 2,2-dichloroacetamide (DDCA) at the level of herbicide uptake were examined in maize cell cultures. When the two compounds were given simultaneously, DDCA inhibited uptake of [¹⁴C]EPTC into maize cells measured for 30 min. A Lineweaver-Burk plot indicated this inhibition to be competitive. N,N-Diallyl 2-chloroacetamide (CDAA), a compound similar in structure to DDCA, inhibited uptake to a lesser extent. Other protectants having no similarity in structure to either DDCA or EPTC had no inhibitory effect on the uptake of EPTC. The data suggest that competition between DDCA and EPTC for a site of uptake may be related to their similarity in chemical structure. Experiments with metabolic inhibitors suggested that uptake of EPTC is not via an active transport mechanism. We suggest that competition for uptake between EPTC and DDCA may represent the first step in a complex series of interactions between the herbicide and its protectant that contributes to the protection of maize from herbicide injury.     

Studies on the mechanism of action of cymoxanil in Phytophthora infestans

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

Calcium/calmodulin-dependent signaling for prepenetration development in <Emphasis Type="Italic">Cochliobolus miyabeanus</Emphasis> infecting rice

Cochliobolus miyabeanus forms a specialized infection structure, an appressorium, to infect rice. Contacting a hard surface induces appressorium formation in C. miyabeanus, while the hydrophobicity of the substratum does not affect this morphogenic infection event. To determine whether the calcium/calmodulin-dependent signaling system is involved in prepenetration morphogenesis in C. miyabeanus, the effects of a calcium chelator (ethylene glycol tetraacetic acid; EGTA), phospholipase C inhibitor (neomycin), intracellular calcium channel blocker (TMB-8), calmodulin antagonists (chlorpromazine, phenoxybenzamine, and W-7), and calcineurin inhibitor (cyclosporin A) on morphogenesis and infection were examined. Addition of Ca²⁺ and the calcium ionophore A23187 did not affect conidial germination, while the number of appressoria decreased with higher concentrations. EGTA inhibited conidial germination and appressorium formation. The calcium channel blocker did not affect appressorium formation at any concentration; however, calmodulin antagonists and the calcineurin inhibitor specifically reduced appressorium formation at the micromolar level. One of the calmodulin antagonists, W-7, also inhibited accumulation of mRNA of the calmodulin gene within germinating conidia and/or appressorium-forming germ tubes. Thus, biochemical processes controlled by the calcium/calmodulin signaling system seem to be involved in the induction of prepenetration morphogenesis on rice.