Induction of wheat and maize glutathione S-transferase by some herbicide safeners and their effect on enzyme activity against butachlor and terbuthylazine

The expression of glutathione S-transferase (GST) activity in wheat and maize shoots was investigated in response to treatments with the herbicide safeners benoxacor, cloquintocet-mexyl, fenchlorazole-ethyl, fenclorim, fluxofenim and oxabetrinil. These safeners significantly enhanced the GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) as a 'standard' substrate, with the exception of oxabetrinil in maize. The enhancements of GST (CDNB) activity were found to be concomitant with increases in V(max) (the reaction rate when the enzyme is fully saturated by the substrate) in wheat following cloquintocet-mexyl and fenchlorazole-ethyl treatments, and in maize following fenchlorazole-ethyl treatment. Otherwise, decreases in V(max) were observed in wheat and maize following fenclorim and fluxofenim treatments. With the exception of oxabetrinil, all the safeners significantly reduced the apparent K(M) (the substrate concentration required for 50% of maximum GST activity) of both wheat and maize GST. The V(max) and K(M) variations following safener treatments are discussed in terms of an increased expression of GST enzymes and an increased affinity for the CDNB substrate. The activity of wheat and maize GST was also assayed towards butachlor and terbuthylazine respectively; the results indicate the ability of cloquintocet-mexyl, fenchlorazole-ethyl and fluxofenim to enhance the enzyme activity in wheat and of benoxacor and fenchlorazole-ethyl to do so in maize.     

Characterisation of Multiple Glutathione Transferases Containing the GST I Subunit with Activities toward Herbicide Substrates in Maize (Zea mays)

The glutathione transferases (GSTs) of maize with activities toward chloroacetanilide herbicides are relatively well characterised, but their range of substrate specificities has not been determined in detail. GST activities toward an extensive range of chemically diverse xenobiotic substrates, including the herbicides atrazine, alachlor, metolachlor and fluorodifen, have been determined in crude and purified preparations from the roots and shoots of dark-grown maize seedlings treated with and without the herbicide-safener dichlormid. With the exception of the activity toward atrazine, specific activities were higher in the roots than in the shoots in all cases. In untreated shoots activities were in the order atrazine=alachlor=metolachlor>fluorodifen with safener–treatment selectively increasing the activity toward the chloroacetanilides and fluorodifen. In the roots the highest GST activities toward herbicides were toward the chloroacetanilides. Dichlormid treatment resulted in an increase in activities toward all four herbicides in the roots of one maize cultivar (Pioneer 3394) but only enhanced the activities toward the chloroacetanilides and fluorodifen in cultivar Artus. Using the non-herbicide 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, anion-exchange chromatography showed that the roots and shoots contained a similar range of GST isoenzymes. All of these isoenzymes were enhanced in response to safeners, though the extent of this induction was organ-dependent. GST isoenzymes containing the GST I subunit were purified from safener-treated roots by a combination of hydrophobic interaction chromatography and affinity chromatography using Orange A agarose. Three isoenzymes could then be purified following resolution by anion-exchange chromatography. The three GSTs were termed GST I/I, GST I/II and GST I/III with GST I, II and III referring to the presence of 29 kDa, 27 kDa and 26 kDa subunits respectively. This revised nomenclature for the maize GSTs was considered necessary in view of the continued discovery of new isoenzymes, such as GST I/III, composed of subunits which have been previously described. GST I/I had measurable activity toward atrazine, low activities toward the other herbicides and appreciable activities toward a range of other xenobiotic substrates. GST I/II and the novel GST I/III isoenzymes both showed high activities toward the chloroacetanilides and fluorodifen but lower activities toward the other substrates and negligible activities toward atrazine. The GST II subunit of GST I/II also had activity as a glutathione peroxidase. Our results show that the GST I subunit can form dimers with the GST III subunit in addition to the GST I and GST II subunits and that the degree of specificity toward herbicide substrates of the respective isoenzymes is greater than previously reported. Our results also suggest that the safener-inducible GST II subunit has additional activities as a glutathione peroxidase. © 1997 SCI.     

Detection and Elimination of Viruses in Callus, Somatic Embryos and Regenerated Plantlets of Grapevine

The distribution of some grapevine viruses in flower explants, embryogenic and non-embryogenic calli, single somatic embryos and plants regenerated from embryogenic cultures was investigated by RT-PCR and ELISA. Immature anthers and ovaries of the cultivars Grignolino infected by GRSPaV, GLRaV-1 and GVA, Müller-Thurgau infected by GRSPaV and GLRaV-3 and Bosco infected by GRSPaV were cultivated on media inducing indirect somatic embryogenesis. Viruses were detected both in anthers and ovaries. Four months after culture initiation 65.6% of tested calli were infected by at least one virus; high percentages of virus infection were found in calli originating from ovaries. No virus was detected in calli tested 8 months after culture initiation, as well as in single somatic embryos or in embryo-derived plantlets. Somatic embryogenesis confirmed its effectiveness in eliminating phloem-limited grapevine viruses. Regeneration of RT-PCR negative plantlets occurred even when at least a sector of the callus was still infected: the mechanism whereby somatic embryos are freed of some viruses could be related to the rapid proliferation of embryogenic cells within the callus or to the origin of the embryogenic callus from virus-free cells within the original explant.     

A plant-specific tau class glutathione S-transferase from Oryza sativa with very high activity against 1-chloro-2,4-dinitrobenzene and chloroacetanilide herbicides

A plant-specific tau class GST gene homolog was successfully cloned from an Oryza sativa cDNA library by PCR using oligonucleotide primers based on the OsGSTU4 (GenBank Accession No. AF309378) sequence. The cDNA was composed of a 720-bp open reading frame encoding 239 amino acids. The deduced amino acid sequence of this gene shared over 65% sequence identity with the sequences of the tau class TaGST28e45 and ZmGST42. Conversely, the OsGSTU4 sequence showed very low identity to the GST sequences of phi, theta and zeta classes. This gene was expressed in Escherichia coli with the pET vector system, and the gene product was purified to homogeneity using GSH-Sepharose affinity column chromatography. The expressed OsGSTU4 formed a homodimer with subunits of approximately 25.5 kDa. OsGSTU4 displayed very high activity toward 1-chloro-2,4-dinitrobenzene. The activity of the OsGSTU4 was significantly inhibited by S-hexylglutathione and hematin. Plant OsGSTU4 had a unique herbicide specificity and played an important role in the detoxification reaction against fluorodifen and chloroacetanilide herbicides.     

Physiological responses of maize and sorghum to four different safeners for metazachlor

The ability of the herbicide safeners, BAS-145138 (1-dichloroacetyl-hexahydro-3,3,8a-trimethyl-pyrrolo(1,2a)pyrimidin-6(2H)-one), dichlormid (N,N-diallyl-2,2-dichloroacetamide), flurazole (phenylmethyl ester), and MG-191 (2-dichloromelhyl-2-methyl-1,3-dioxolane) for preventing metazachlor injury to maize (Zea mays L.) and sorghum (Sorghum bicolor L.) seedlings were compared with their effects on ¹⁴C-metazachlor metabolism to a glutathione (GSH) conjugate, effects on non-protein thiol contents (mainly GSH) and effects on Glutathione S-transferase (GST) activity in these two species. Sorghum shoot growth was reduced by 41% and maize shoot growth was reduced by 54%, by metazachlor concentrations in vermiculite nutrient culture of 0·6 μM and 7·5μM, respectively. In this system, all four compounds had significant activity as safeners for metazachlor in both sorghum and maize seedlings. BAS-145138 and flurazole were the most effective safeners in maize and sorghum, respectively. In the absence of safeners, the rate of non-enzymatic conjugation of metazachlor and GSH was much greater than the enzymatic rate. However, the rate of enzymatic conjugation of metazachlor with GSH was increased by safener treatment in both maize and sorghum. Safener effectiveness was highly correlated with increases in ¹⁴C-metazachlor uptake and metabolism in both species. Safener effectiveness was more highly correlated with safener effects on GST activity in maize or sorghum when ¹⁴C-metazachlor was used as the substrate than when the non-specific CDNB (1-chloro-2,4-dinitrobenzene) was used as the substrate. Safener effectiveness was also strongly correlated with safener effects on GSH levels in sorghum, but not in maize, possibly because of the greater importance of non-enzymatic conjugation of metazachlor with GSH in sorghum as compared to maize.     

Effects of the safeners CGA 154281, oxabetrinil and fenclorim on uptake and degradation of metolachlor in corn (Zea mays L.) seedlings

Summary. The influence of three crop safeners on the uptake and degradation of ¹⁴C-metolachlor was investigated in two corn varieties. Following application of herbicide and safener together to seedling shoots the concentrations of non-metabolized ¹⁴C-metolachlor in the tissues was found to be lower in the tolerant variety LG 9 than in the susceptible variety 211A. The difference between varieties was due to differences in both uptake and degradation of ¹⁴C-metolachlor.
Following shoot application most of the radioactivity was retained in the coleoptile and the mesocotyl. Two hours after application 95% of the herbicide had been degraded in coleoptiles and mesocotyls, whereas approximately 20% of non-metabolized ¹⁴C-metolachlor was present in the enclosed developing shoot leaves. In both corn varieties the safener CGA 154281 caused a substantial lowering of tissue levels of parent ¹⁴C-metolachlor. This was primarily due to an enhanced degradation. Glutalhione- S -transfer-ase (GST) enzyme activity in shoot tissues was found to be enhanced in both varieties by CGA 154281. Oxabetrinil and fenclorim were less effective than CGA 154281 both in reducing tissue levels of non-metabolized ¹⁴C-metolachlor and in enhancing GST activity in either variety.     

磷化氢对小麦中玉米象致死效果的研究

玉米象Sitophilus zeamais(Motschulsky)的卵、幼虫以及蛹在粮食籽粒内部生活与发育,粮粒结构会影响熏蒸气体的渗透以及对隐蔽虫态的杀虫效果,了解粮粒内卵、幼虫、蛹以及成虫对磷化氢耐受能力的差异,有助于科学有效杀虫。本文测定了3个玉米象品系对磷化氢的抗药性,及在100、200、300、400和500mL/m3的磷化氢浓度下玉米象成虫及其隐蔽虫态卵、幼虫和蛹在不同熏蒸时间的死亡率。主要结果为:3个品系的玉米象对磷化氢均未产生抗性。在各磷化氢浓度下,经6h熏蒸后100mL/m3浓度下成虫的死亡率近59%,而500mL/m3浓度下死亡率在91%以上;经12h熏蒸后各品系成虫的死亡率均达100%,而其卵、幼虫和蛹死亡率均小于100%;经24h熏蒸后,各品系的卵、幼虫和蛹死亡率达100%的磷化氢浓度需要在400mL/m3及以上;经36h熏蒸后,100mL/m3浓度可完全致死玉米象BJXNSz品系的卵、幼虫和蛹,但对另外两个品系的卵、幼虫和蛹的致死率小于100%;磷化氢熏蒸48h以上的各浓度均可完全致死受试害虫。结果表明,100mL/m3以上的磷化氢浓度均可在不同时间致死玉米象各虫态,但完全致死玉米象各虫态的时间在100mL/m3浓度下需要48h,在500mL/m3浓度下需要24h,浓度升高完全致死害虫的时间缩短。玉米象卵、幼虫和蛹对磷化氢耐受力相接近且远大于成虫。     

Production of desert locust feeding deterrents from in vitro cultured neem (Azadirachta indica)

Callus was initiated from cotyledon, hypocotyl and shoot tip explants of neem seedlings (Azadirachta indica A. Juss.) that were cultured on Murashige and Skoog (MS) agar medium supplemented with MS vitamins, casein hydrolysate, indole-3-acetic acid and benzylamino purine. Shoots regenerated from hypocotyl-derived callus only. Mature 10-12-week-old callus regenerated both shoots and somatic embryos (SEm). The antifeedant activity of different types of callus, leaves and SEm was determined with larvae of the desert locust Schistocerca gregaria in a no-choice feeding bioassay. In vitro tissue-cultured neem had antifeedant properties against the desert locust. To the best of our knowledge the antifeedant activity of neem somatic embryos extracts is reported here for the first time.     

Characterization of glutathione S-transferase of the rice leaffolder moth, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae): Comparison of its properties of glutathione S-transferases from other lepidopteran insects

An enzyme that possesses the glutathione S-transferase (GST) activity was found in the rice leaffolder moth, Cnaphalocrocis medinalis. The enzyme was purified to homogeneity for the first time by ammonium sulfate fractionation and affinity chromatography. The resultant enzyme revealed a single band with a molecular mass of 24 kDa by SDS-polyacrylamide gel electrophoresis under reduced conditions. When assayed with 1-chloro-2,4-dinitrobenzene, a universal substrate for GST, the purified GST had an optimum pH at 8.0, and was fairly stable at pH 3-10 and at temperatures below 50 °C. The enzyme was also able to conjugate glutathione to 4-hydroxynonenal, a cytotoxic lipid peroxidation product. The present GST was inhibited by fenitrothion, permethrin, and deltamethrin, suggesting that the GST could be involved in metabolizing these organophosphorus and pyrethroid insecticides.     

Molecular cloning, expression, and characterization of a phi-type glutathione S-transferase from Oryza sativa

The structural gene for glutathione S-transferase in Oryza sativa was successfully cloned from a cDNA library by the polymerase chain reaction method. The deduced amino acid sequence of this gene showed 44-66% similarity to the sequences of the class phi GSTs from Arabidopsis thaliana and Zea mays. This gene was expressed in Escherichia coli with the pET vector system and the gene product was purified to homogeneity by GSH-Sepharose affinity column chromatography. The expressed OsGSTF3-3 was a homo-dimer composed of 24 kDa subunit and its pI value was approximately 7.3. The OsGSTF3-3 was retained on GSH affinity column and its K_m value for GSH was 0.28 mM. The OsGSTF3-3 displayed high activity toward 1-chloro-2,4-dinitrobenzene, a general GST substrate and also had high activities towards acetanilide herbicides, alachlor, and metolachlor. The OsGSTF3-3 was highly sensitive to inhibition by benastatin A and S-hexyl-GSH. From these results, the expressed OsGSTF3-3 is a phi class GST and seems to play an important role in the conjugation of the chloroacetanilide herbicides.     

Purification and biochemical characterization of glutathione S-transferases from three strains of Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae): Implication of insecticide resistance

Glutathione S-transferases (GSTs) catalyzing the conjugation of reduced glutathione (GSH) to a vast range of xenobiotics including insecticides were investigated in the psocid Liposcelis bostrychophila Badonnel. GSTs from susceptible and two resistant strains (DDVP-R for dichlorvos-resistant strain and PH₃-R for phosphine-resistant strain) of L. bostrychophila were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene (CDNB), in a photometric microplate assay. The specific activities of GSTs purified from two resistant strains were significantly higher than their susceptible counterpart. For the resistant strains, GSTs both showed a significantly higher affinity to the substrate GSH while a declined affinity to CDNB than those of susceptible strain. The inhibitory potential of ethacrynic acid was very effective with highest I₅₀ value (the concentration required to inhibit 50% of GSTs activity) of 1.21 μM recorded in DDVP-R. Carbosulfan also exhibited excellent inhibitory effects on purified GSTs. The N-terminus of the purified enzyme was sequenced by Edman degradation, and the alignment of first 13 amino acids of the N-terminal sequence with other insect GSTs suggested the purified protein was similar to those of Sigma class GSTs.     

Parasitization by Cotesia plutellae enhances detoxifying enzyme activity in Plutella xylostella

Insecticidal tests using diazinon showed that the mortality of Plutella xylostella larvae parasitized by Cotesia plutellae was reduced by 4.6-fold compared to that of the nonparasitized hosts. The use of chemicals with synergistic effect to insecticides in toxicity assay helps to elucidate the kind of enzyme involved in lowering insect mortality. Synergism of diethyl maleate and piperonyl butoxide with diazinon resulted to 2.4- and 1.9-fold increase, respectively, in susceptibility of parasitized larvae compared to those of nonparasitized larvae. These results indicated the possibility that the decrease in susceptibility to diazinon was due to the elevated activities of glutathione-S-transferase (GST) and cytochrome P450 monooxygenase (CYP), respectively. The GST activities in parasitized larvae were significantly higher than those of nonparasitized ones starting from three days post-parasitization until emergence of parasitoid larva. High GST activities during late parasitism could be attributed to both enzyme activities toward diazinon of parasitized P. xylostella larva itself and C. plutellae larva inside larval host. High GST activity one day after parasitization, although statistical significance was not detected, was caused by polydnavirus (PDV) and the venom of C. plutellae not by parasitoid larvae. Artificial injection of PDV plus venom demonstrated that the resulting increase in GST activity is similar to the increase brought by parasitization. High CYP activity after 3 days post-parasitization in parasitized larva was attributed mainly to the activity of parasitoid larva. Carboxylesterase activity in the parasitized host remained at a high level, while that in the nonparasitized host decreased slightly as pupation approaches. On the other hand, acetylcholinesterase activity also remained constant after parasitization until larval emergence, while that of the nonparasitized hosts decreased gradually as the host larvae approach pupation. These results were supported by inhibition tests using diazoxon in vitro.     

Resistance selection and biochemical mechanism of resistance to two Acaricides in Tetranychus cinnabarinus (Boiduval)

A Tetranychus cinnabarinus strain was collected from Chongqing, China. After 42 generations of selection with abamectin and 20 generations of selection with fenpropathrin in the laboratory, this T. cinnabarinus strain developed 8.7- and 28.7-fold resistance, respectively. Resistance to abamectin in AbR (abamectin resistant strain) and to fenpropathrin in FeR (fenpropathrin resistant strain) was partially suppressed by piperonyl butoxide (PBO), diethyl maleate (DEM) and triphenyl phosphate (TPP), inhibitors of mixed function oxidase (MFO), glutathione S-transferases (GST), and hydrolases, respectively, suggesting that these three enzyme families are important in conferring abamectin and fenpropathrin resistance in T. cinnabarinus. The major resistant mechanism to abamectin was the increasing activities of carboxylesterases (CarE), glutathione-S-transferase (GST) and mixed function oxidase (MFO), and the activity in resistant strain developed 2.7-, 3.4- and 1.4-fold contrasted to that in susceptible strain, respectively. The activity of glutathione-S-transferase (GST) in the FeR strain developed 2.8-fold when compared with the susceptible strain, which meant the resistance to fenpropathrin was related with the activity increase of glutathione-S-transferase (GST) in T. cinnabarinus. The result of the kinetic mensuration of carboxylesterases (CarE) showed that the structure of CarE in the AbR has been changed.     

Variation of acephate susceptibility and correlation with esterase and glutathione S-transferase activities in field populations of the tarnished plant bug, Lygus lineolaris

The tarnished plant bug (TPB) has increasingly become an economically important pest of cotton. Heavy dependence on insecticides, particularly organophosphates and pyrethroids, for TPB control facilitated resistance development to multiple classes of insecticides. To better understand resistance and explore ways to monitor resistance in field populations, this study examined acephate susceptibility and the activities of two major detoxification enzymes in nine field populations collected in the Delta region of Mississippi and Arkansas in 2010. Two Arkansas populations from Reed and Backgate had 3.5- and 4.3-fold resistance to acephate, as compared to a susceptible laboratory strain. Extensive planting of cotton and heavy chemical sprays is a major driving force for resistance development to acephate in Mid-south cotton growing areas. Reduced susceptibility to acephate was highly correlated with elevated esterase activities. The acephate-resistant populations from Backgate, Lula, and Reed consistently had higher (up to 5.3-fold) esterase activities than susceptible populations. Regression analysis of LC₅₀s with kinetic esterase activities revealed a significant polynomial quadratic relationship with R² up to 0.89. Glutathione S-transferase (GST) also had elevated activity in most populations, but the variations of GST activities were not significantly correlated with changes of acephate susceptibility. Finally, examination of esterase and GST inhibitors indicated that suppression rates (up to 70%) by two esterase inhibitors in 2010 were slightly lower than those detected in 2006, and ethacrynic acid (EA) inhibited GST effectively in both years. Two other GST inhibitors (sulfobromophthalein and diethyl maleate) displayed significantly lower suppression rates in 2010 than those detected in 2006, suggesting a potential genetic shift in pest populations and a necessity of continued monitoring for insecticide resistance with both bioassay and biochemical approaches. Results indicated that using major detoxification enzyme activities for resistance monitoring may provide insight into acephate resistance in field populations of TPB.     

Purification and partial characterization of a glutathione S-transferase from the two-spotted spider mite, Tetranychus urticae

Glutathione S-transferases (GSTs) are known to catalyze conjugations by facilitating the nucleophilic attack of the sulfhydryl group of endogenous reduced glutathione on electrophilic centers of a vast range of xenobiotic compounds, including insecticides and acaricides. Elevated levels of GSTs in the two-spotted spider mite, Tetranychus urticae Koch, have recently been associated with resistance to acaricides such as abamectin [Pestic. Biochem. Physiol. 72 (2002) 111]. GSTs from acaricide susceptible and resistant strains of T. urticae were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene and monochlorobimane. The inhibitory potential of azocyclotin, dicumarol, and plumbagin was low (IC₅₀ values > 100 μM), whereas ethacrynic acid was much more effective, exhibiting an IC₅₀ value of 4.5 μM. GST activity is highest in 2-4-day-old female adults and dropped considerably with progressing age. Furthermore, molecular characteristics were determined for the first time of a GST from T. urticae, such as molecular weight (SDS-PAGE) and N-terminal amino acid sequencing (Edman degradation). Glutathione-agarose affinity purified GST from T. urticae strain WI has a molecular weight of 22.1 kDa. N-terminal amino acid sequencing revealed a homogeneity of ≈50% to insect GSTs closely related to insect class I GSTs (similar to mammalian Delta class GSTs).     

Glutathione transferase activities toward herbicides used selectively in soybean

Using extracts from suspension-cultured cells of soybean (Glycine max cv. Mandarin) as a source of active enzymes, the activities of glutathione transferases (GSTs) catalysing the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and selective herbicides were determined to be in the order CDNB≫ fomesafen>metolachlor=acifluorfen>chlorimuron-ethyl. GST activities showed a thiol dependence in a substrate-specific manner. Thus, GST activities toward acifluorfen and fomesafen were greater when homoglutathione (hGSH), the endogenously occurring thiol in soybean, was used as the co-substrate rather than glutathione (GSH). Compared with GSH, hGSH addition either reduced or had no effect on GST activities toward other substrates. In the absence of enzyme, the rates of hGSH conjugation with acifluorfen, chlorimuron-ethyl and fomesafen were negligible, suggesting that rapid hGSH conjugation in soybean must be catalysed by GSTs. GST activities were subsequently determined in 14-day-old plants of soybean and a number of annual grass and broadleaf weeds. GST activities of the plants were then related to observed sensitivities to post-emergence applications of the four herbicides. When enzyme activity was expressed on a mg^-1 protein basis, all grass weeds and Abutilon theophrasticontained considerably higher GST activity toward CDNB than soybean. With fomesafen as the substrate, GST activities were determined to be in the order soybean≫Echinochloa crus-galli>Digitaria sanguinalis>Sorghum halepense=Setaria faberi with none of the broadleaf weeds showing any activity. This order related well to the observed selectivity of fomesafen, with the exception of A. theophrasti, which was partially tolerant to the herbicide. Using metolachlor as the substrate the order of the GST activities was soybean>A. theophrasti≫S. halepense>Amaranthus retroflexus>Ipomoea hederacea, with the remaining species showing no activity. GST activities toward metolachlor correlated well with the selectivity of the herbicide toward the broadleaf weeds but not toward the grass weeds. Acifluorfen and chlorimuron-ethyl were selectively active on these species, but GST activities toward these herbicides could not be detected in crude extracts from whole plants. © 1997 SCI     

几种代谢酶基因与柑橘全爪螨对双甲脒抗性的关系

为了明确羧酸酯酶(carboxylesterase,CarE)基因、谷胱甘肽S-转移酶(glutathione S-transferases,GST)基因和过氧化氢酶(catalase,CAT)基因与柑橘全爪螨Panonychus citri对双甲脒抗性的关系,通过BLAST检索,从柑橘全爪螨转录组数据库中对这3种代谢酶抗性相关基因进行鉴定,并采用RPKM法对双甲脒抗性品系和敏感品系代谢抗性相关基因进行表达差异分析,对差异较大的基因作定量PCR检测.基因差异性分析发现,抗性品系中有9条CarE基因、12条GST基因及6条CAT基因表达量发生上调,13条CarE基因、12条GST基因和3条CAT基因表达量发生下调;Pc29773nrt、Pcl7807nlg和Unigene31477为上调倍数最高的3个基因,其log2 ratio (RS/SS)分别为12.95、10.81、10.01.定量分析显示,Pc29773 nrt、Pcl7807nlg和Unigene31477的上调倍数分别为3.72、2.03和3.09,Pc29773 nrt和Unigene31477上调显著.研究表明柑橘全爪螨Pc29773nrt和Unigene31477上调与其对双甲脒的抗性相关.     

Assessment of insecticide resistance in eggs and neonate larvae of Cydia pomonella (Lepidoptera: Tortricidae)

Spanish Cydia pomonella (L.) field populations have developed resistance to several insecticide groups. Diagnostic concentrations were established as the LC₉₀ calculated on a susceptible strain (S_Spain) for five and seven insecticides and tested on eggs and neonate larvae field populations, respectively. The three most relevant enzymatic detoxification systems (mixed-function oxidases (MFO), glutathione S-tranferases (GST) and esterases (EST)) were studied for neonate larvae.In eggs, 96% of the field populations showed a significantly lower efficacy when compared with the susceptible strain (S_Spain) and the most effective insecticides were fenoxycarb and thiacloprid. In neonate larvae, a significant loss of susceptibility to the insecticides was detected. Flufenoxuron, azinphos-methyl and phosmet showed the lowest efficacy, while lambda-cyhalothrin, alpha-cypermethrin and chlorpyrifos-ethyl showed the highest. Biochemical assays showed that the most important enzymatic system involved in insecticide detoxification was MFO, with highest enzymatic activity ratios (5.1-16.6 for neonates from nine field populations). An enhanced GST and EST activities was detected in one field population, with enzymatic activity ratios of threefold and fivefold for GST and EST, respectively, when compared with the susceptible strain. The insecticide bioassays showed that the LC₉₀ used were effective as diagnostic concentrations. Measures of MFO activity alongside bioassays with insecticide diagnostic concentrations could be used as tools for monitoring insecticide resistance in neonate larvae of C. pomonella.