Interconversion of chloroneb and 2,5-dichloro-4-methoxyphenol by soil microorganisms

The stability of the chloroneb fungicide (1,4-dichloro-2,5-dimethoxybenzene) and its principal degradation product, 2,5-dichloro-4-methoxyphenol (DCMP), was tested in the presence of individual soil microorganisms. Both the fungicide and DCMP were unaltered in liquid shake cultures of the organisms when growth was minimal or static. However, in nutrient broth where active microbial growth was supported, several organisms converted chloroneb to DCMP and/or DCMP to chloroneb. Of 23 organisms grown in the presence of 5 μg chloroneb/ml, 13 demethylated the fungicide to produce DCMP. Liquid shake cultures of Fusarium spp. were most active in this respect, converting more than 50% of the chloroneb to DCMP in 5 days. When grown in the presence of 5 μg DCMP/ml, 8 organisms, especially Trichoderma viride and Mucor ramannianus, converted up to 20% of the DCMP to chloroneb. Cephalosporium gramineum, Rhizoctonia solani, Mucor ramannianus and Fusarium fungi could both methylate and demethylate DCMP to produce chloroneb and 2,5-dichlorohydroquinone, respectively. Microbial synthesis of chloroneb from DCMP, its principal degradation product, may account in part for the relative stability and long term effectiveness of the fungicide in soil.     

Uptake and metabolism of chloroneb by Phaseolus vulgaris

Chloroneb is absorbed through the roots of young bean plants and accumulated mostly in the roots and lower stem. It is metabolized to 2,5-dichloro-4-methoxyphenol and then to the β-d-glucoside of the latter. Thiram ambient to the roots does not affect chloroneb uptake, metabolism, or transport during a 48-hr exposure to the chemicals.     

Substituted alpha-(phenylhydrazono)phenylacetonitrile derivatives. Part 2: Synthesis and biological activity of pro-pesticides

Pro-pesticides of alpha-(2,6-dichloro-4-trifluoromethylphenylhydrazono)-4-nitrophenylacetonitrile have been prepared and tested against mite and insect pests. Variations in potency and spectrum were observed depending on the choice of cleavable pro-moiety. Cleavage of the pro-moiety was demonstrated in one case by measuring the rate of increase in the uncoupling activity using a mitochondrial preparation. Irradiation experiments have demonstrated a rapid isomerisation of the planar Z isomer to the E isomer, which is reversible.     

Chlordimeform: Uncoupling activity against rat liver mitochondria

The pesticide chlordimeform [N′-(4-chloro-o-tolyl)-N,N-dimethylformamidine] at 0.04 μmoles/mg protein uncouples 50% of respiratory-chain phosphorylation of rat liver mitochondria. This uncoupling activity depends on mitochondrial protein concentration and can be reversed either by the addition of bovine serum albumin or by washing. The normal inhibition of state 3 respiration by oligomycin and atractylate is completely reversed by chlordimeform. Uncoupling concentrations of chlordimeform elicit high adenosine triphosphatase activity. This activity is blocked by the above inhibitors of mitochondrial energy-transfer reactions. Evidence is presented which shows that unprotonated chlordimeform is the form effective in uncoupling. It is concluded that chlordimeform is an uncoupling agent with a potency and site of action close to but probably not identical to that of the classical uncoupler 2,4-dinitrophenol.     

On the mode of action of 3-phenylindole towards Aspergillus niger

3-Phenylindole is an antimicrobial compound active towards many fungi and gram-positive bacteria. At 5 μg/ml it inhibits growth of Aspergillus niger. Higher concentrations (50 μg/ml) also suppress spore germination; they do not kill the fungus. Dry weight of the fungus still increases for 1 or 2 days after fungicide treatment. The toxicant has no effect on O₂ uptake even at higher concentrations (100 μg/ml). The compound markedly affects composition of the lipid fraction of A. niger inducing a decrease in phospholipid concentration with a coincident increase in free fatty acids. Sterol pattern and sterol concentration were not affected. Antifungal activity was reversed by phospholipids added to the medium. 3-Phenylindole induced a slight leakage of ³²P-labeled compounds from the treated cells under growth conditions but not under nongrowth conditions. A strain of A. niger resistant to 3-phenylindole had the same phospholipid and sterol pattern as the wild type, but the level of both components was higher (40–60%). The 3-phenylindole-resistant strain showed resistance to triarimol and pimaricin. The wild type and the resistant strain both took up 3-phenylindole quite rapidly and accumulated it in the mycelium. 3-Phenylindole possibly interferes with phospholipid function in cell membranes, although the specific site of action has not yet been elucidated.     

On the mode of action of the fungicide etridiazole

Etridiazole (5-ethoxy-3(trichloromethyl)-1,2,4-thiadiazole) is a fungicide primarily effective against fungi of the Oomycete group. After addition of the compound to the medium, growth of Mucor mucedo was impaired almost immediately. Oxygen uptake of the mycelia decreased only slightly at growth-inhibiting concentrations. Respiration of isolated mitochondria of Mucor was inhibited about 50% by high concentrations of the fungicide, while the respiratory control quotient remained constant. In contrast, rat liver mitochondria were not very sensitive to etridiazole. Etridiazole stimulates the hydrolysis of membrane-bound phospholipids to free fatty acids and lysophosphatides in isolated mitochondria of Mucor. Procaine, a well-known inhibitor of phospholipases, acts as an antidote for etridiazole in growth tests as well as in the hydrolysis of phosphatidylcholine by isolated mitochondria. Calcium ions in millimolar concentrations act like procaine. Therefore, it was assumed that the fungistatic effect of etridiazole was mainly caused by an activation of phospholipases in the mitochondrial membranes. Moreover, under the influence of etridiazole, a lipid peroxidation of membranes is observed. Tocopherol acts as an antidote. This could be the primary toxic effect in the mechanism of action of this fungicide. The enzymes involved are not yet identified.     

Effects of β-pinene,a nonsubstituted monoterpene,on rat liver mitochondria

β-Pinene uncouples oxidative phosphorylation and inhibits respiration in isolated rat liver mitochondria. The uncoupling effects are observed at lower concentrations (100 to 200 μM) than the inhibition of respiration (400 μM). At low concentrations, the effects observed could be explained by an increase of the passive permeability of the mitochondrial membrane produced by the terpene. Higher concentrations seemed to inhibit respiration through an effect on the electron transport chain. At the highest concentrations tested (600 to 1200 μM), β-pinene seemed to produce a partial resealing of the mitochondrial membrane. All effects can be explained by the interaction of β-pinene with the mitochondrial membrane. Other hydrophobic molecules tested do not show the effects of β-pinene or limonene on mitochondria.     

Biochemical mode of action of the acaricide fenazaflor

The active ingredient of ‘Lovozal’ (fenazaflor) is hydrolysed in solution to 5,6-dichloro-2-trifluoromethyl-benzimidazole (DTFB). Rat liver mitochondria were uncoupled by fenazaflor after a lag phase, while uncoupling by DTFB was immediate, and a similar situation occurred with mitochondria isolated from Tetranychus telarius L. (This is the first report of isolation of mite mitochondria.) Since both compounds ultimately have the same uncoupling activity, it is likely that fenazaflor exerts its effect only after hydrolysis to DTFB. Living mites showed a stimulation of respiration of 70% on addition of either fenazaflor or DTFB before death occurred, suggesting that the acaricidal effect of fenazaflor is due to uncoupling of oxidative phosphorylation after hydrolysis to DTFB.     

Structure-activity relationships of carboxamide fungicides and the succinic dehydrogenase complex of Cryptococcus laurentii and Ustilago maydis

The systemic fungicide, carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) and a variety of carboxamide compounds exhibit a marked specificity for Basidiomycete fungi. This unique specificity resides in the mitochondrial succinic dehydrogenase complex (SDC) of sensitive Basidiomycetes such as Ustilago maydis, the corn smut fungus. The present study examines in detail the structure-activity relationships of 93 carboxamide compounds and the SDC of two carboxin-sensitive organisms, U. maydis and a Basidiomycetous yeast, Cryptococcus laurentii. It has been possible to elucidate substantially the requirement in molecular structure needed for inhibition of the mitochondrial SDC. With few exceptions, a good correlation exists between the inhibitory activity of carboxamides towards the SDC of U. maydis and C. laurentii and the inhibition of growth of carboxamide-sensitive fungi, both in vitro and in vivo on the diseased plant. The structure-activity results were used as a basis for the synthesis of new, fungicidally-active carboxamides. The compounds found to be most active against the mycelial growth of Rhizoctonia solani were also tested on spore germination or mycelial growth of non-Basidiomycete fungi. Three carboxanilides (3-methyl-thiophene-2-carboxanilide, 3′-methyl-2-methylbenzanilide and 3′-methyl-2-ethylbenzanilide) had a fungitoxic spectrum which extended beyond Basidiomycetes. The spectrum of fungicidal activity of carboxanilides appears to be altered not only by substitution in the aniline ring, but by the nature of the ring attached to the carbonyl. No correlation was found between the inhibitory activity of oxathiins and benzanilides and their calculated partition coefficients.     

In vitro sensitivity of wheat and oat mitochondria to the selective herbicide,diclofop-methyl

Compared to diclofop-methyl (methyl 2-[4-(2′,4′-dichlorophenoxy)phenoxy]propanoate), diclofop (the demethylated derivative) was a more potent inhibitor of polarographically monitored state 3 respiration of mitochondrial preparations isolated from shoots of dark-grown wheat (Triticum aestivum L. cv. Neepawa) and oat (Avena sativa L. cv. Terra) seedlings. Wheat and oat mitochondria demonstrated essentially similar concentration-response patterns for the uncoupler-like stimulation of state 4 respiration and the inhibition of state 3 respiration by diclofop, thereby intimating that differential mitochondrial sensitivity was not a selectivity factor between these species. Diclofop suppression of unconstrained oxygen utilization elicited by the respiratory uncoupler FCCP indicated that inhibition of state 3 respiration involved interference with some site(s) on the mitochondrial electron transport chain and not with energy transfer directly. Cytochrome c oxidase activity was not affected by diclofop, but succinate- and malate-PMS oxidoreductase activities were inhibited by diclofop. Enhanced rates of passive mitochondrial swelling in isotonic KCl medium in the presence of diclofop pointed to a direct influence on the permeability properties of the inner mitochondrial membrane and indicated that membrane disruption could have been a factor in the effects elicited by diclofop on mitochondrial respiration. However, it does not appear that specific interference with mitochondrial functionality is the primary mechanism of phytotoxicity in susceptible plants.     

Effects of chlordimeform on mitochondria from eggs of Spodoptera littoralis

Mitochondria were isolated from eggs of Spodoptera littoralis. With succinate (+pyruvate) as substrate, respiratory control ratios between 1.70 and 2.54 were obtained. Uncouplers and the energy transfer inhibitor oligomycin influenced these mitochondria in the well-known manner. The uncoupling activity of chlordimeform in vitro was very weak and decreased with increasing age of the eggs. Electron transport in mitochondria from chlordimeform-treated eggs was not uncoupled. Therefore, it is concluded that the ovicidal effect of this pesticide is not due to its uncoupling effect.     

Biochemical genetics of oxidative resistance to diazinon in the house fly

The genetics and biochemistry of oxidative resistance to diazinon were investigated in a diazinon-resistant strain of the house fly, Musca domestica L. The resistant strain was crossed with a multimarker susceptible strain and substrains containing portions of the resistant strain genome were prepared. Resistance, microsomal oxidase, and cytochrome P-450 spectral characteristics were then compared in the different strains. The major gene for resistance to diazinon is semidominant and is located on chromosome II, 13 crossing over units from the recessive mutant stubby wing. Additional resistance genes occur on chromosome II and on other chromosomes as well. Resistance to diazinon was introduced into a susceptible mutant-marked strain via genetic crossing over. Increases in parathion oxidase, total and P-450-specific N- and O-demethylase activity, and resistant strain type I binding spectrum were introduced along with resistance, indicating genes controlling these parameters and resistance are either identical or closely linked. No increase in activity of cytochrome P-450 itself was introduced into the mutant strain. Additional genes controlling the amount of cytochrome P-450 and several spectral changes characteristic of the resistant strains are apparently controlled by genes located at different loci on chromosome II. Resistance factors on other chromosomes are also present, but were not characterized.     

Characterization of a dicarboximide-fungicide-resistant laboratory isolate ofMonilinia laxa

Monilinia laxa, the incitant of blossom blight in stone fruits in Israel, is sensitive to 5μg/ml of the dicarboximide fungicides vinclozolin and iprodione in the growth medium. When a large number of spores, from an isolate never exposed to these fungicides, was seeded on a medium containing 15 μg/ml iprodione, spontaneous resistant mutants appeared at 10^-5 frequency. These mutants showed cross resistance to the dicarboximide fungicides vinclozolin, procymidone, l-(3,5-dichloro-phenyl)-3-propen(2)-pyrrolidin-2,5-dion (Co 4462), l-(3,5-dichlorophenyl)-3-methoxymethyl-pyrrolidin-2,5-dion (Co 6054), and to dicloran. Growth rate on fungicide-free medium was similar to that of the parental sensitive strain but sporulation was much reduced. Growth rate on media supplemented with dicarboximide fungicides decreased gradually with increasing fungicide concentrations. The resistance has been stable for more than one year in the absence of fungicides. Artificial inoculation of cherry, apricot and plum fruits previously treated with 500 μg/ml vinclozolin, iprodione, procymidone or Co 6054, with a resistant strain, resulted in brown rot; similar treatments provided full protection of the fruits against the sensitive strain.     

Triazophos resistance mechanisms in the rice stem borer (Chilo suppressalis Walker)

A field population of the rice stem borer (Chilo suppressalis Walker) with 203.3-fold resistance to triazophos was collected. After 8-generation of continuous selection with triazophos in laboratory, resistance increased to 787.2-fold, and at the same time, the resistance to isocarbophos and methamidophos was also enhanced by 1.9- and 1.4-fold, respectively, implying some cross-resistance between triazophos and these two organophosphate insecticides. Resistance to abamectin was slightly enhanced by triazophos selection, and fipronil and methomyl decreased. Synergism experiments in vivo with TPP, PBO, and DEM were performed to gain a potential indication of roles of detoxicating enzymes in triazophos resistance. The synergism results revealed that TPP (SR, 1.92) and PBO (SR 1.63) had significant synergistic effects on triazophos in resistant rice borers. While DEM (SR 0.83) showed no effects. Assays of enzyme activity in vitro demonstrated that the resistant strain had higher activity of esterase and microsomal O-demethylase than the susceptible strain (1.20- and 1.30-fold, respectively). For glutathione S-transferase activity, no difference was found between the resistant and the susceptible strain when DCNB was used as substrate. However, 1.28-fold higher activity was observed in the resistant strain when CDNB was used. These results showed that esterase and microsomal-O-demethylase play some roles in the resistance. Some iso-enzyme of glutathione S-transferase may involve in the resistance to other insecticides, for this resistant strain was selected from a field population with multiple resistance background. Acetylcholinesterase as the triazophos target was also compared. The results revealed significant differences between the resistant and susceptible strain. The V_max and K_m of the enzyme in resistant strain was only 32 and 65% that in the susceptible strain, respectively. Inhibition tests in vitro showed that I₅₀ of triazophos on AChE of the resistant strain was 2.52-fold higher. Therefore, insensitive AChE may also involved in triazophos resistance mechanism of rice stem borer.     

Uncoupling of oxidative phosphorylation in rat liver mitochondria by the lamprey larvicide TFM (3-trifluoromethyl-4-nitrophenol)

The uncoupling concentrations which caused a 50% reduction in the ADP/0 ratios for rat liver mitochondria was found to be 4.29 nM per mg of mitochrondrial protein for TFM (3-trifluoromethyl-4-nitrophenol), and 11.15 nM for DNP (2,4-dinitrophenol). The increased oxygen consumption of the mitochondrial preparation is characteristic of uncoupling effects and need not be attributed to the action of detoxication enzyme systems. Interference or synergism due to impurities of the commercial preparation of TFM may affect its differential toxicity characteristics in the field.     

Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae)

The San Roman strain of the southern cattle tick, Boophilus microplus, collected from Mexico was previously reported to have a high level of resistance to the organophosphate acaricide coumaphos. An oxidative detoxification mechanism was suspected to contribute to coumaphos resistance in this tick strain, as coumaphos bioassay with piperonyl butoxide (PBO) on larvae of this resistant strain resulted in enhanced coumaphos toxicity, while coumaphos assays with PBO resulted in reduced toxicity of coumaphos in a susceptible reference strain. In this study, we further analyzed the mechanism of oxidative metabolic detoxification with synergist bioassays of coroxon, the toxic metabolite of coumaphos, and the mechanism of target-site insensitivity with acetylcholinesterase (AChE) inhibition kinetics assays. Bioassays of coroxon with PBO resulted in synergism of coroxon toxicity in both the San Roman and the susceptible reference strains. The synergism ratio of PBO on coroxon in the resistant strain was 4.5 times that of the susceptible strain. The results suggested that the cytP450-based metabolic detoxification existed in both resistant and susceptible strains, but its activity was significantly enhanced in the resistant strain. Comparisons of AChE activity and inhibition kinetics by coroxon in both susceptible and resistant strains revealed that the resistant San Roman strain had an insensitive AChE, with a reduced phosphorylation rate, resulting in a reduced bimolecular reaction constant. These data indicate a mechanism of coumaphos resistance in the San Roman strain that involves both insensitive AChE and enhanced cytP450-based metabolic detoxification.     

Development of two strains of <Emphasis Type="Italic">Fusarium culmorum</Emphasis> with a different aggressiveness in the soil and on the roots of barley of two genotypes

Fusarium culmorum can affect plants including cereals or develop saprophytically in the soil. It is unknown whether its saprophytic ability is different in strains with a different aggressiveness. This knowledge could be used for effective breeding of resistant cultivars. Here, we aimed to study the development of two F. culmorum strains with a different aggressiveness in the soil under barley, to compare their saprophytic ability and to reveal the influence, if any, of the host plant on the development of strains in the soil and on the roots. Saprophytic development of the strains was studied on membranes inoculated with macroconidia and placed into non-sterile soil under barley of two genotypes. The fungus was identified on the membranes and on barley roots by indirect immunofluorescent method. Both strains could develop saprophytically. The more aggressive strain (Fc538) showed a lesser saprophytic fitness than the less aggressive strain (Fc885): its mycelial density was lower and the number of chlamydospores was greater. Barley genotypes influenced the development of the fungal strains. Interestingly, conditions for saprophytic development of both strains were more favourable in the soil under barley of the resistant genotype. The more aggressive strain colonized barley of the resistant genotype more actively. Rot symptoms appeared earlier in the barley of the resistant genotype, but the number of diseased plants was greater in the barley of the susceptible genotype. The presence of a saprophytic stage in life cycle should slow down the accumulation of aggressive races in field populations of F. culmorum. Possible interactions between F. culmorum strains and barley plants are discussed.     

Pyrethroid resistance and esterase activity in three strains of the cotton bollworm, Helicoverpa armigera (Hübner)

Microplate assay technique for estimation of esterase activity in a single insect was used in combination with dose mortality bioassays to detect pyrethroid resistance in three strains of Helicoverpa armigera and to study the frequency of pyrethroid resistant individuals within the population of the same strain at two larval stages, third and fifth instar. The third and fifth instar larvae of the field strains i.e., Nagpur strain and Delhi strain that displayed high degree of resistance towards deltamethrin, had higher esterase activity compared to a susceptible laboratory strain. The frequency distribution of individuals with elevated esterase activity above 1.00 absorbance unit was correlated with the resistance level of the strains. The frequency of resistant individuals in the third instar larvae of Nagpur strain and Delhi strain were 29% and 23%, respectively compared to 4% in the susceptible strain. The resistant individuals in the resistant strains have markedly increased in the fifth instar larvae with a frequency distribution of 63% and 90% in Delhi strain and Nagpur strain, respectively, while only 14% of individuals was found to have elevated esterase activity in the susceptible strain. The results demonstrated the role of esterase in pyrethroid resistance in H. armigera. Microplate assay proved to be a rapid and reliable biochemical technique for monitoring of pyrethroid resistance in H. armigera.     

抗性甜菜夜蛾Na-K-ATP酶、Ca-ATP酶和Ca-Mg-ATP酶对高效氯氟氰菊酯的敏感性

分别测定了甜菜夜蛾Spodoptera exigua敏感和抗高效氯氟氰菊酯品系神经系统Na-K-ATP酶、Ca-ATP酶和Ca-Mg-ATP酶的活力。结果表明,敏感和抗性品系Na-K-ATP酶活力差异不显著,而抗性品系Ca-ATP酶和Ca-Mg-ATP酶活力明显低于敏感品系。在浓度为1.0×10-8~1.0×10-3 mol/L时,高效氯氟氰菊酯对敏感和抗性品系Na-K-ATP酶、Ca-ATP酶和Ca-Mg-ATP酶的活力均有抑制,并且对敏感品系的抑制作用高于对抗性品系。高效氯氟氰菊酯浓度为1.0×10-4 mol/L 时,对敏感品系Na-K-ATP酶活力的抑制率为29.6%,对抗性品系的为21.8%,对敏感品系Ca-ATP酶活力的抑制率为34.3%,对抗性品系为21.9%,对敏感品系Ca-Mg-ATP酶活力的抑制率为22.3%,对抗性品系为16.9%,存在显著差异。表明甜菜夜蛾抗性品系上述3种ATP酶对高效氯氟氰菊酯的敏感性已明显下降。     

Effect of Seed Quality and Combination Fungicide-Trichoderma spp. Seed Treatments on Pre- and Postemergence Damping-Off in Cotton

ABSTRACT Good quality seeds of cotton cultivars often escaped pre-emergence damping-off incited by Pythium spp. and Rhizopus oryzae, and they were resistant to postemergence damping-off incited by Rhizoctonia solani. Poor quality seeds, however, were highly susceptible to both phases of seedling disease and required seed treatment in order to survive. Pre-emergence damping-off incited by Pythium spp. and Rhizopus oryzae could be controlled by seed treatment with biocontrol preparations of a number of Trichoderma spp., but these treatments were much less effective in controlling postemergence disease incited by Rhizoctonia solani. Postemergence seedling disease can be controlled by fungicides, but they were much less effective in controlling the pre-emergence phase of the disease. Combination seed treatments of poor quality cotton seeds with fungicides and Trichoderma spp. preparations, followed by planting in pathogen-infested soil, indicated that this technique will control both phases of seedling disease. Seed treatment with either the fungicides or the biocontrol agents alone did not achieve this goal. The optimum combination treatment for disease control was that of chloroneb plus Trichoderma spp., followed by chloroneb plus metalaxyl (Deltacoat AD) plus T. virens strain G-6.