Metabolism and cell wall incorporation of phenoxyacetic acid in soybean cell suspension culture

The metabolism of [¹⁴C]phenoxyacetic acid (POA) was studied in cell suspension culture of soybean (Glycine max). POA was metabolized to 4-HO-POA, 4-HO-POA glucoside and 4-HO-POA glycosidic ester. A large part of the 4-HO-POA glucoside and small amounts of the glycosidic ester were recovered in the medium. POA was also converted to non-extractable residues bound to cell walls. Sequential extraction of cell-wall polymers showed that non-extractable residues, partly identified with 4-HO-POA and POA, were mainly associated with hemicelluloses and lignin. Comparison of the metabolism of [carboxy-¹⁴C]- and [phenyl-¹⁴C]POA revealed some degradation of the POA side-chain, followed in all probability by the incorporation of the aromatic moiety into cell walls. However, the sturdiness of the resulting bonds prevented precise identification of these bound aromatic structures. In summary, the degradation of POA in soybean cell culture provided a good model to study the formation of non-extractable residues of pesticides. © 1999 Society of Chemical Industry     

Metabolism of [ring-2,6-14]parathion in plant cell suspension cultures of carrot (Daucus carota), purple foxglove (Digitalis purpurea), soybean,thorn apple (Datura stramonium) and wheat (Triticum aestivum)

By means of standardized procedures, the metabolism of [ring-2,6-¹⁴C]-parathion was investigated in carrot (Daucus carota L.), purple foxglove (Digitalis purpurea L.), soybean (Glycine max Merrill cv. ?Mandarin’?, and Glycine max Merrill cv. ?Harosoy 63’? cultivated on B5 and Miller media, respectively), thorn apple (Datura stramonium L.), and wheat (Triticum aestivum L.) cell suspension cultures. In the wheat and soybean (Mandarin) cells only 2.9 and 8.9%, respectively, of the applied parthion remained unmetabolized after 48 h of incubation, while 51.2, 57.9, 60.3, and 62.4% of the unchanged parent were detected in the D. purpurea, D. Stramonium, carrot and soybean (Harosoy) cultures, respectively. In all suspensions, paraoxon and 4-nitrophenol were found as phase I metabolites, thus demonstrating that plant tissues can catalyse oxidative desulfuration and dearylation of parathion. 4-Nitrophenol was also glycosylated with glucose and possibly galactose. Further, as yet unidentified, metabolites indicated that bio-transformations had also occurred at the aromatic moiety. Large amounts of non-extractable residues were detected in the wheat suspension (38.3%), while the other cultures showed a lower incorporation of ¹⁴C into insoluble cell material (0.9-9.4%). For a prospective ecotoxicological evaluation of the metabolic fate of pesticides and xenobiotics in plants in general, the differential metabolic capacity of plant cell cultures and plants should be taken into account.     

An investigation of the non-solvent-extractable residues of [14C]chlorpyrifos-methyl in stored wheat

The residues of [¹⁴C] chlorpyrifos-methyl remaining in wheat after solvent extraction accounted for 28% of the applied dose after an extended period of storage. A bioassay of these residues was carried out using Tribolium castaneum and a significant reduction in the numbers of adults reaching maturity was observed. Attempts to release the non-solvent-extractable residues using an enzymic digestion procedure were unsuccessful, as were several of the chemical solubilisation procedures investigated. A water/methanol extraction system was successful in releasing up to 86% of the radioactivity leaving 10% still non-extractable. Analysis of this solubilised residue revealed that 59% was present as the pyridinol metabolite of chlorpyrifos-methyl and 26% was a polar material composed of two main components. Bioassay tests of these metabolites revealed that the reduction in the number of insects reaching maturity could be associated with the pyridinol metabolite.     

13种杀菌剂对小麦黑胚病的防治效果

在田间测定了13种杀菌剂对小麦黑胚病的防治效果.结果表明,13种杀菌剂对小麦黑胚病均有一定的防效,防效为34.8％～67.7％.其中7.5％氟环唑乳油50 g/hm2、50％嘧菌环胺水分散粒剂 375 g/hm2、56％嘧菌·百菌清悬浮剂330 g/hm2、25％丙环唑乳油180 g/hm24个处理对小麦黑胚病有较好的防治效果,防效均达到60％以上,可在生产中推广应用.     

Determination of extractable and non-extractable radioactivity from prairie soils treated with carboxyl- and ring-labelled [14C]2,4-D

Ring- and carboxyl-labelled [¹⁴C]2,4-D were incubated under laboratory conditions, at the 2 g/g level, in a heavy clay, sandy loam, and clay loam at 85% of field capacity and 20 1C. The soils were extracted at regular intervals for 35 days with aqaeous acidic acetonitrile, and analysed for [¹⁴C]2,4-D and possible radioactive degradation products. Following solvent extraction, a portion of the soil residues were combusted in oxygen to determine unextracted radioactivity as [¹⁴C]carbon dioxide. The remaining soil residues were then treated with aqueous sodium hydroxide, and the radioactivity associated with the fulvic and humic soil components determined. In all soils there was a rapid decrease in the amounts of extractable radioacitivity, with only 5% of that applied being recoverable after 35 days. All recoverable radioactivity was attributable to [¹⁴C]2,4-D, and no [¹⁴C]-containing degradation products were observed. This loss of extractable radioactivity was accompanied by an increase in non-extractable radioactivity. Approximately 15% of the applied radioactivity, derived from carboxyl-labelled [¹⁴C]2,4-D, and 30% from the ring-labelled [¹⁴C]2,4-D was associated with the soil in a non-extractable form, after 35 days of incubation. After 35 days, less than 5% of the radioactivity from the carboxyl-labelled herbicide, and less than 10% of the ringlabelled material, was associated with the fulvic components derived from the three soils. Less than 5% of the applied radioactivities were identifiable with any of the humic acid components. It was considered that during the incubation [¹⁴C]2,4-D did not become bound or conjugated to soil components, and that non-extractable radioactivity associated with the three soil types resulted from incorporation of radioactive degradation products, such as [¹⁴C]carbon dioxide, into soil organic matter.     

The fate and distribution of chlorpropham when applied to stored potatoes as a sprout suppressant

Two varieties of potato were treated with ¹⁴C- or ³⁶Cl-radiolabelled chlorpropham to suppress sprouting, and stored under controlled ventilation conditions in the laboratory for up to 6 months at 10°C. The migration of chlorpropham into the potato tubers was studied at intervals by autoradiography and analysis of tuber extracts. Little penetration of chlorpropham beyond the peel layer occurred even after storage for 6 months. No identifiable degradation product of chlorpropham was detected in the extracts, although there was evidence of bound non-extractable residues. Volatile compounds lost by ventilation through the storage containers were collected in cold traps and analysed by liquid scintillation counting. Loss of chlorpropham by volatilisation from the tuber surface was very small.     

Metabolism of the pesticide metabolites 4-nitrophenol and 3,4-dichloroaniline in carrot (Daucus carota) cell suspension cultures

The metabolism of [ 14 C]-4-nitrophenol and [ 14 C]-3,4-dichloroaniline (the xenobiotics are degradation products of parathion and propanil, respectively) was studied in cell suspension cultures of carrot (Daucus carota L.). 4-Nitrophenol was transformed almost quantitatively to water-soluble conjugates with minor amounts of non-extractable residues. The conjugates identified were 1-(O-β-D-glucopyranosyl)-4-nitrobenzene and 1-(6′-O-malonyl-O-β-D-glucopyranosyl)-4-nitrobenzene. In addition, two unidentified metabolites were observed, possibly a disaccharide and another malonylated glycoside of 4-nitrophenol. Time-course studies demonstrated that 4-nitrophenol was rapidly taken up and conjugated; all metabolites remained associated with the cells rather than nutrient medium. 3,4-Dichloroaniline was transformed quantitatively to water-soluble conjugates and bound residues (3.6%). The water-soluble metabolites were identified as 6′-O-malonyl-N-(β-D-glucopyranosyl)-3,4-dichloroaniline, N-(β-D-glucopyranosyl)-3,4-dichloroaniline and N-malonyl-3,4-dichloroaniline. A time-course study showed that the glucosides were formed initially, then decreased, possibly due to hydrolysis. This decrease was paralleled by an increase of the main metabolite, N-malonyl-3,4-dichloroaniline, which was predominantly recovered from the medium.     

不同机制杀菌剂对小麦白粉病的敏感性及与三唑酮的交互抗性

为明确三唑酮和氟环唑、吡唑醚菌酯、啶酰菌胺、嘧菌环胺、乙嘧酚5种不同作用机制的杀菌剂对小麦白粉病的敏感性及交互抗性,采用田间小区试验和室内喷雾离体叶段法测定了不同杀菌剂对小麦白粉病的防治效果。结果表明,5种不同作用机制杀菌剂对小麦白粉病的防治效果可达90%以上,而三唑酮最高的防治效果仅为72.17%;小麦白粉病菌群体对氟环唑、吡唑醚菌酯、啶酰菌胺、嘧菌环胺、乙嘧酚的敏感性EC50分别在0.087~1.901、0.058~1.402、0.186~3.014、0.222~6.005、0.006~1.742μg/mL之间,5种不同作用机制杀菌剂的敏感性均呈连续单峰曲线,可作为小麦白粉病菌对5种不同作用机制杀菌剂的敏感基线。研究表明,三唑酮与氟环唑、吡唑醚菌酯、啶酰菌胺、嘧菌环胺、乙嘧酚之间不存在交互抗性。     

Studies on the phloroglucinol–HCl reactive material produced by squash fruit elicited with pectinase: Isolation using hydrolytic enzymes and release of p -coumaryl aldehyde by water reflux

Pathological tissues from a variety of plants turn red when treated with the histochemical reagent phloroglucinol (PG)–HCl. This induced PG–HCl reacting material has been termed both wound gum and, more recently, induced lignin or lignin-like material. We are exploring alternative approaches to better identify this induced material. In this report, we describe methods for purifying the PG–HCl reactive material from solvent-washed tissue preparations and measuring the amount of p -coumaryl aldehyde released from the purified material by boiling in water. Acorn squash fruit wall tissue was sliced, sprayed with pectinase to elicit the formation of PG–HCl reactive material and incubated up to 72 h at 27°C. Sampled tissue was washed extensively with water and organic solvents. Insoluble residues were treated with cell wall degrading enzymes to remove cell wall materials, and dimethyl sulfoxide was used to dissolve starch. Yields of residual material increased from 7.5% in the time zero samples to 29.7% after 72 h. Refluxing the purified material in water released <6 μ g p -coumaryl aldehyde g⁻¹ from preparations of tissue at time zero and 8100 μ g p -coumaryl aldehyde g⁻¹ from samples incubated for 48 h. These results suggest that the function of the material is to sequester phytoalexins, but are not consistent with the material being lignin. Published by Elsevier Science Ltd.     

QuEChERS-超高效液相色谱-串联质谱法测定双氟磺草胺和氯氟吡氧乙酸在小麦和土壤中的残留及消解动态

建立了同时测定小麦及其土壤中双氟磺草胺和氯氟吡氧乙酸残留的QuEChERS-超高效液相色谱-串联质谱方法,并采用该方法研究了低温冷藏条件下双氟磺草胺和氯氟吡氧乙酸在小麦上的储藏稳定性以及15%双氟磺草胺?氯氟吡氧乙酸悬乳剂在小麦和土壤中的最终残留及消解动态。结果表明:在添加水平为0.005~1 mg/kg范围内,双氟磺草胺和氯氟吡氧乙酸在小麦及土壤中的平均回收率在82%~108%之间,相对标准偏差在0.41%~11%之间 (n=5) ,均能满足农药残留分析的要求。在 –20℃下储藏365 d后,麦粒中双氟磺草胺和氯氟吡氧乙酸的残留量变化小于30%,符合植源性农产品中农药残留储藏稳定性试验准则要求,储藏稳定。双氟磺草胺在小麦植株和土壤中的消解半衰期分别为4.4~8.1 d和2.4~9.3 d;氯氟吡氧乙酸在小麦植株和土壤中的消解半衰期分别为7.9~10.6 d和11.8~24.8 d,即在相同的试验条件下双氟磺草胺在植株和土壤中消解速率快于氯氟吡氧乙酸的。采用推荐剂量有效成分180 g/hm2和推荐高剂量有效成分270 g/hm2的15%双氟磺草胺?氯氟吡氧乙酸悬乳剂于小麦田施药1次,在小麦收获期的麦粒中均未检出双氟磺草胺和氯氟吡氧乙酸残留。     

含苯丙氨酸和1H-茚-1-酮类氯虫苯甲酰胺类似物的合成及杀虫活性

为了开发新的杀虫化合物，以氯虫苯甲酰胺为先导，2-甲基-3-氨基苯甲酸为起始原料，经过卤化生成5-氯-3-甲基-2-氨基苯甲酸；再与3-溴-1-(3-氯吡啶-2-吡啶基)-1H-吡唑-5-甲酸进行缩合反应，生成6-氯-2-(3-溴-1-(3-氯吡啶-2-吡啶基)-1H-吡唑-5-基)-8-甲基-4H-3,1-苯并噁嗪-4-酮；最后引入苯丙氨酸片段并经环化反应，分别合成了两个系列共计23 个新化合物( 4 和 5 )，其中化合物 4 为含苯丙氨酸的氯虫苯甲酰胺类似物，化合物 5 为由化合物4环化生成的含1H-茚-1-酮片段的氯虫苯甲酰胺类似物，所有化合物的结构均通过核磁共振氢谱(1H NMR)、碳谱(13C NMR)和高分辨质谱(HRMS)的表征及确证。初步室内杀虫活性测试结果表明，多数目标化合物对黏虫Mythinma separata具有较高的杀虫活性，其中14个化合物在100 mg/L下对黏虫Mythinma separata的致死率为100%，化合物 5k ( 1H-茚-1-酮片段中取代基为羟基 ) 在4和0.8 mg/L下的致死率分别为90%和70%，其LC₅₀值为0.55 mg/L。分子对接结果表明，化合物 5k 与氯虫苯甲酰胺一样，也是作用于鱼尼汀受体(RyR)，但与靶标结合的氨基酸残基不同，这种差异可能对黏虫的抗药性治理研究有益。     

The degradation of diflubenzuron and its chief metabolites in soils. Part II: Fate of 4-chlorophenylurea

The fate of 4-chlorophenylurea in soils was studied with two preparations: one labelled with ¹⁴C in the phenyl ring and the other in the carbonyl group. The initial dose of 1 mg kg^?1 decreased to 50% in about 5 weeks in aerobic sandy clay and in about 16 weeks in anaerobic hydrosoil. Soil treatment with each of the preparations resulted in the release of [¹⁴C]carbon dioxide, pointing to decarbonylation and ring opening. The fraction of non-extractable (soil-bound) radioactivity increased during incubation. Quantities of ring-¹⁴C-labelled and carbonyl-¹⁴C-labelled bound residues differed strongly in the aerobic soil but only slightly in the anaerobic hydrosoil. It is assumed that two sorts of bound residues are formed from 4-chlorophenylurea: one is fairly stable and might consist of bound 4-chloroaniline or its transformation products, whereas the other is presumed to be a degradable derivative of 4-chlorophenylurea.     

Examination of bound (non-extractable) residues of MCPA and flamprop in wheat straw

The long term metabolism of [¹⁴C]MCPA and [¹⁴C]flamprop in wheat (Triticum aestivum) straw was found to involve incorporation of radioactivity as residues that were insoluble in acetone+water (1+1 by volume). A chemical and an enzymic solubilisation procedure were critically evaluated in attempts to release these residues for further examination. The chemical procedure resulted in complete solubilisation of all the radioactivity of both compounds in association with more than one cell wall fraction. However, routine quantitative analysis was found to be difficult for some fractions. Furthermore, the extracts did not appear to be suitable for investigation of the nature of the binding with the plant constituents. None of the enzymes employed in the enzymic procedures released significant amounts of the residues insoluble in the aqueous acetone. Despite these problems, the residues of MCPA that were insoluble in aqueous acetone were found to contain both the parent MCPA and its major metabolite 4-chloro-α-hydroxy-o-tolyloxyacetic acid.     

Investigations on the Behaviour of Fenpropimorph and its Metabolite Fenpropimorphic Acid in Soils

The fate of fenpropimorph and its metabolite fenpropimorphic acid was investigated in a silty sand soil and in a clayey silt soil. In laboratory and field experiments fenpropimorph disappeared without a lag phase. A few days after application fenpropimorphic acid was detected. Additional laboratory experiments with [¹⁴C]fenpropimorph emphasized the significance of mineralization and the formation of non-extractable residues. The determination of soil/water distribution coefficients of parent compound and metabolite yielded a higher leaching potential for fenpropimorphic acid due to its higher polarity. This was confirmed by performing a laboratory column test under worst-case conditions. Under field conditions, however, fenpropimorphic acid was detected only in the superficial soil layers (0–5 cm) of both investigation sites at very low concentrations.     

The sex-specific sulfation of the major metabolite of the novel fungicide cyprodinil in the rat

The metabolism of cyprodinil, a novel broad-spectrum fungicide, was investigated in rats. After single oral administration of 0.5 or 100 mg kg⁻¹ body weight, [phenyl-U-¹⁴C]cyprodinil was rapidly eliminated, principally in the urine. The metabolite pattern in urine exhibited a significant sex-related difference with respect to the major metabolite. Males and females both produced a dihydroxy metabolite, N-4-(hydroxyphenyl)-4-cyclopropyl-5-hydroxy-6-methylpyrimidin-2-ylamine. Female rats conjugated this metabolite with sulfate exclusively at the 5-hydroxypyrimidinyl moiety, while males formed equal amounts of the monosulfate and a disulfate conjugate. The sex dimorphism in the conjugation reaction indicates the involvement of a sex-specific sulfotransferase that catalyzed the transfer of the second sulfate group.     

细交链孢菌酮酸及其衍生物的合成与杀虫活性

为了寻找具有较高杀虫活性的特胺酸化合物，以天然活性产物细交链孢菌酮酸(TeA)作为先导化合物，利用酰基化米氏酸作为酰基化试剂，设计、合成了26个3-位不同酰基取代和5-位不同取代的含特胺酸骨架衍生物 4a~4s、5a~5g、7a 和 8a ，其中14个化合物未见文献报道，所有目标化合物的结构均经核磁共振氢谱、碳谱和高分辨质谱确证。初步杀虫活性测定结果表明，在100 μg/mL下处理72 h内，所有目标化合物对麦长管蚜Macrosiphum avenae (Fabricius)均表现出良好的杀虫活性，并具有内吸性，其中化合物 5d 和 7a 48 h致死率为100%，高于对照药剂螺虫乙酯，具有作为先导化合物进一步研究的价值。对处理后的小麦植株进行残留量测定，结果表明目标化合物 4e、5c、7a 和 8a 能被植株较好的吸收 。 该研究结果可为进一步研究具有特胺酸骨架化合物的构效关系提供参考。     

Dégradation de l'isoproturon et disponibilité de ses résidus dans le sol

Degradation if isoproturon and availability of residues in soil The availability and degradation of ¹⁴C-ring-labelled isoproturon in soil was investigated over 140 days under controlled laboratory conditions. Degradation of the active ingredient followed and 65 days later only a minor fraction (0.6%) of the parent molecule remained extractable. A demethylated-isoproturon metabolite was detectable in soil from day 15 (2.6%). The amount of ¹⁴CO₂ derived from the ¹⁴C benzene ring label and liberated over time indicated that a total of 13.6% isoproturon was mineralized during the incubation period. In parallel, the amount of ¹⁴C residue extracted from the soil by water followed by methanol or remaining within the soil—analysed by combustion—was also determined at intervals. After 140 days, 72% of the radiolabel added remained in the soil as non-extractable residue. The degradation half-life of extractable isoproturon was an estimated 14 days.     

Ultrastructural and immunocytochemical investigation of pathogen development and host responses in resistant and susceptible wheat spikes infected by Fusarium culmorum

Pathogen development and host responses in wheat spikes of resistant and susceptible cultivars infected by Fusarium culmorum causing Fusarium head blight (FHB), were investigated by means of electron microscopy as well as immunogold labelling techniques. The studies revealed similarities in the infection process and the initial spreading of the pathogen in wheat spikes between resistant and susceptible cultivars. However, the pathogen’s development was obviously more slow in the resistant cultivars as in comparison to a susceptible one. The structural defence reactions such as the formation of thick layered appositions and large papillae were essentially more pronounced in the infected host tissues of the resistant cultivars, than in the susceptible one. β -1,3-glucan was detected in the appositions and papillae. Furthermore, immunogold labelling of lignin demonstrated that there were no differences in the lignin contents of the wheat spikes between susceptible and resistant cultivars regarding the uninoculated healthy tissue, but densities of lignin in host cell walls of the infected wheat spikes differed distinctly between resistant and susceptible cultivars. The lignin content in the cell walls of the infected tissues of the susceptible wheat cultivar increased slightly, while the lignin accumulated intensely in the host cell walls of the infected wheat spikes of the resistant cultivars. These findings indicate that lignin accumulation in the infected wheat spikes may play an important role in resistance to the spreading of the pathogen in the host tissues. Immunogold labelling of the Fusarium toxin DON in the infected lemma showed the same labelling patterns in the host tissues of resistant and susceptible cultivars. However, there were distinct differences in the toxin concentration between the tissues of the susceptible and resistant cultivars. At the early stage of infection, the labelling densities for DON in resistant cultivars were significantly lower than those in the susceptible one. The present study indicates that the FHB resistant cultivars are able to develop active defence reactions during infection and spreading of the pathogen in the host tissues. The lower accumulation of the toxin DON in the tissues of the infected spikes of resistant cultivars which results from the host’s defence mechanisms may allow more intensive defence responses to the pathogen by the host.     

Biotransformation of [ring-U-14C]atrazine to dealkylated and hydroxylated metabolites in cell-suspension cultures

The biconversion of [¹⁴C]atrazine to deaikyt-ated and hydroxylated products was studied in heteroirophic cell-suspension cultures of carrot ( Daucus caroia L.), Agrostemma githago L. (corn cockle). Digitalis purpurea L. (purple foxglove), soyabean ( Giycine max L. Merr; four different cultivars). Datura stramonium L. (thorn-apple) and wheat ( Tritician aestivum L.). During 48 h of incubation, the herbicide was biotransformed by all species; turnovers yields differed considerably and were between 10.1% and 88.0% of applied ¹⁴C. Differences were also observed among the soyabean cultivars (10.1-73.5%). Hydroxy-atrazine, de-ethyl-, deisopropyl- and de-ethyt-deisopropylatrazine formed in the cultures were identified by thin-layer chromatography (tlc) (co-chromatography with reference compounds); deaikyiated metabolites were also proved by gas chromatography–mass spectrometry (gc–ms). In addition, highly polar transformation products emerged that were not identified. Portions of non-extractable residues were below 5% (one soyabean cultivar: 8.9%). Atrazine was metabolized by the cells, mainly to its dealkylated derivatives and hydroxyatrazine (totals of 9.4-54, 5%), whereas portions of highly polar products were lower (0.1-26.1%). Exceptions were A. githago (26.0 and 33.6%, respectively) and D, purpurea (4.5 and 25.2% respectively). Thus, plants generally contribute to the environmental degradation of atrazine.     

Biodegradation of the herbicide bromoxynil and its plant cell wall bound residues in an agricultural soil

The mineralization and formation of metabolites and nonextractable residues of the herbicide [¹⁴C]bromoxyniloctanoate ([¹⁴C]3,5-dibromo-4-octanoylbenzonitrile) and the corresponding agent substance [¹⁴C]bromoxynil ([¹⁴C]3,5-dibromo-4-hydroxybenzonitrile) was investigated in a soil from an agricultural site in a model experiment. The mineralization of maize cell wall bound bromoxynil residues was also investigated in the agricultural soil material. The mineralization of [¹⁴C]bromoxynil and [¹⁴C]bromoxyniloctanoate in soil within 60 days amounted up to 42 and 49%, respectively. After the experiments, 52% of the originally applied [¹⁴C]bromoxynil and 44% of the [¹⁴C]bromoxyniloctanoate formed nonextractable residues in soil. Plant cell wall bound [¹⁴C]bromoxynil residues were also mineralized to an extent of about 21% within 70 days; the main portion of 76% persisted as nonextractable residues in the soil. In bacterial enrichment cultures and in soil two polar metabolites were observed; one of it could be identified as 3,5-dibromo-4-hydroxybenzoate and the other could be described tentatively as 3,5-dibromo-4-hydroxybenzamide.