Increased activity and reduced sensitivity of glutamine synthetase in glufosinate‐resistant vetiver (Vetiveria zizanioides Nash) cells

Vetiver (Vetiveria zizanioides Nash) cells derived from an inflorescence were cultured in a modified N6 liquid medium supplemented with 10 µm 2,4‐D and 10 mm proline. Exponentially growing cell suspensions were subcultured with a selection medium containing glufosinate (ammonium dl ‐homoalanin‐4‐yl(methyl)phosphinate). The glufosinate‐resistant cells which can grow in a medium containing 5 × 10^?5 M glufosinate was selected by a stepwise selection, and its I₅₀ value was determined to be 4.2 × 10^?5 M. The growth of susceptible cells was inhibited by lower concentrations of glufosinate and its I₅₀ value was 2.5 × 10^?7 M. This indicated that the selected cells were 170‐fold resistant compared with the susceptible cells. Glutamine synthetase (GS) activity of the resistant cells was twice as high as that of the susceptible cells. The I₅₀ values of glufosinate were 3.2 × 10^?5 M and 9.0 × 10^?7 M for GS from the resistant and susceptible cells, respectively. The accumulation of ammonia caused by GS inhibition was higher in the susceptible cells. Absorption of [3,4–¹⁴C]glufosinate was not significantly different between the resistant and susceptible cells. Both cell types did not metabolize glufosinate. These results suggest that the resistance of the selected vetiver cell suspension to glufosinate is mainly due to increased GS activity and its decreased sensitivity to the herbicide.     

The glutamate dehydrogenase gene gdhA increased the resistance of tobacco to glufosinate

The gene gdhA from Escherichia coli, that encodes a NADPH‐dependent glutamate dehydrogenase (GDH), directs a novel pathway in transgenic plants that allows an increase in ammonium assimilation. Glufosinate leads to plant death by the irreversible inhibition of glutamate synthetase (GS) leading to a disruption of subsequent GS‐related processes resulting in elevated ammonium and disruption of photorespiration. Therefore, it was speculated that the gdhA‐transformed plants may exhibit a novel mechanism of resistance to glufosinate by altered activity of the GDH‐directed pathway(s) and subsequently related processes. Studies were conducted in the greenhouse to evaluate the resistance of tobacco plants containing the gdhA gene to glufosinate. Five tobacco genotype lines were investigated including a non‐transformed control line, a positive control line and three transformed lines with levels of increasing GDH activity directed by the gdhA gene. Plants transformed with the gdhA gene expressed up to six times increased level of resistance (GR₅₀) to glufosinate compared with the non‐transformed control, which is 100 times less resistant than plants transformed with the bar gene. The GDH activity among lines was highly correlated (r² = 0.9903) with the level of herbicide resistance. Thus, the use of the E. coli gdhA gene in plant transformations can provide an additional mechanism for resistance to glufosinate.     

Target-site mutation associated with glufosinate resistance in Italian ryegrass (Lolium perenne L. ssp. multiflorum)

BACKGROUND: Studies were conducted to elucidate the mechanism of glufosinate resistance in an Italian ryegrass population. RESULTS: Glufosinate rates required to reduce growth by 50% (GR₅₀) were 0.15 and 0.18 kg AI ha^?1 for two susceptible populations C1 and C2 respectively, and 0.45 kg AI ha^?1 for the resistant population MG, resulting in a resistance index of 2.8. Ammonia accumulation after glufosinate treatment was on average 1.5 times less for the resistant population than for the susceptible populations. The glufosinate concentrations (µM ) required to reduce the glutamine synthetase (GS) enzyme activity by 50% (I₅₀) were 31 and 137 for C1 and C2 respectively, and 2432 for the resistant population MG. One amino acid substitution in the plastidic GS2 gene, aspartic acid for asparagine at position 171, was identified in the resistant population. CONCLUSIONS: This is the first report of glufosinate resistance in a weed species that involves an altered target site. Copyright © 2012 Society of Chemical Industry     

The mode of action of glufosinate in algae: The role of uptake and nitrogen assimilation pathways

The purpose of this work was to investigate the in-vivo mode of action of glufosinate in comparison with that of the known glutamine synthetase inhibitor, methionine sulfoxmine, in photoautotrophic microorganisms. Using the eukaryotic green alga Chlorella fusca (Shih. & Krauss) and the cyanobacterium Anacystis nidulans (Kratz & Myers), currently developed [¹⁵N]NMR pulselabelling techniques have been employed to study the inhibition of ammonia assimilation. The results show that, while methionine sulfoximine immediately blocks glutamine synthesis, glufosinate action requires an induction process in both organisms investigated, possibly indicating the de-novo synthesis of an amino acid membrane carrier. In addition, the observed ability of C. fusca to incorporate nitrogen into glutamate under glutamine synthetase-inhibiting conditions is explicable by an anabolic function of a glutamate dehydrogenase in this organism. This might explain the large differences in observed species sensitivity to glufosinate exposure.     

Initial report of glufosinate and paraquat multiple resistance that evolved in a biotype of goosegrass (Eleusine indica) in Malaysia

Field and glasshouse studies have confirmed the presence of a glufosinate‐ and paraquat‐resistant goosegrass biotype that has infested a bitter gourd field in Air Kuning, Perak, Malaysia. Glufosinate and paraquat had been applied at least six times per year to the affected fields (originally a rubber plantation) for more than four consecutive years. Paraquat had been used since 1970 for weed control in the rubber plantation. An on‐site field trial revealed that the control of the goosegrass plants, measuring 20–35 cm in height, ranged from 20 to 35% 3 weeks after being treated with each herbicide at twice the recommended rate. Dose–response tests were conducted in the glasshouse, using seedlings at the three‐to‐four‐leaf stage that had been obtained from the plants that had received repeated exposure to these herbicides and a biotype with no history of any herbicide resistance. The comparison of the GR₅₀ (the herbicide rate that is required to reduce the shoot fresh weight by 50%) of the seedlings indicated that the resistant biotype of goosegrass is 3.4‐fold and 3.6‐fold more resistant than the susceptible biotype following treatment with glufosinate and paraquat, respectively. This study has demonstrated the world's first field‐evolved instance of multiple resistance in goosegrass to two non‐selective herbicides, glufosinate and paraquat.     

Glutamine synthetase activity and ammonium accumulation is influenced by time of glufosinate application

Time of glufosinate application impacts weed control. Although leaf angle of velvetleaf significantly contributes to the time of day (TOD) effect, it is not the sole reason for reduced efficacy. Absorption and translocation of glufosinate, glutamine synthetase (GS) activity, and ammonium accumulation were investigated as possible physiological components of the TOD effect. Absorption and translocation were not associated with a decrease in glufosinate efficacy. GS activity, ammonium accumulation, and plant biomass were influenced by the time of glufosinate application. Velvetleaf GS activity quickly diminished during the light period and remained high during the dark period following glufosinate treatment. GS activity was higher in plants treated at 10:00 pm than in those treated at 2:00 pm 12 HAT. With little GS inhibition during the dark period, ammonium accumulation occurred only during the light period. At 72 HAT, ammonium concentration was nearly 1.5-fold greater in plants treated at 2:00 pm versus those treated at 10:00 pm with 160 g/ha glufosinate. Plant biomass of plants treated with 160 g/ha glufosinate at 10:00 pm was greater than those treated at 2:00 pm at 72 HAT. Increasing the application rate from 160 to 320 g/ha removed these differences in GS activity, ammonium accumulation, and plant biomass. These results demonstrate that GS activity and ammonium accumulation are physiological processes involved in the TOD effect.     

Effectiveness and mode of action of phosphonate inhibitors of plant glutamine synthetase

BACKGROUND: Aiming at the rational design of new herbicides, the availability of the three‐dimensional structure of the target enzyme greatly enhances the optimisation of lead compounds and the design of derivatives with increased activity. Among the most widely exploited herbicide targets is glutamine synthetase. Recently, the structure of a cytosolic form of the maize enzyme has been described, making it possible to verify whether steric, electronic and hydrophobic features of a compound are in agreement with inhibitor–protein interaction geometry. RESULTS: Three series of compounds (aminophosphonates, hydroxyphosphonates and aminomethylenebisphosphonates) were evaluated as possible inhibitors of maize glutamine synthetase. Aminomethylenebisphosphonate derivatives substituted in the phenyl ring retained the inhibitory potential, whereas variations in the scaffold, i.e. the replacement of the second phosphonate moiety with a hydroxyl or an amino residue, resulted in a significant loss of activity. A kinetic characterisation showed a non‐competitive mechanism against glutamate and an uncompetitive mechanism against ATP. A docking analysis suggested the mode of bisphosphonate binding to the active site. CONCLUSION: Results made it possible to define the features required to maintain or enhance the biological activity of these compounds, which represent lead structures to be further exploited for the design of new substances endowed with herbicidal activity. Copyright © 2009 Society of Chemical Industry     

苏云金芽胞杆菌谷氨酰胺合成酶基因glnA的转录调控和过表达

氮代谢对细菌生存至关重要,谷氨酰胺和谷氨酸盐在细菌氮代谢中发挥重要作用。本文对苏云金芽胞杆菌谷氨酰胺合成酶基因glnA的转录调控及过表达进行了研究。通过构建glnR基因的缺失突变株及glnA基因启动子融合lacZ基因的表达载体,测定突变株中glnA启动子活性,发现在胞外营养充足的情况下转录因子GlnR负调控glnA基因。同时在glnR基因苏云金芽胞杆菌缺失突变株中过表达glnA基因。发现与野生型相比,过表达菌株杀虫活性得到提高。     

冬小麦冠层-大气氨交换的季节性特征及其影响因素

为探究冠层氨交换对麦田氨挥发的贡献,从农学和生理角度揭示冠层氨挥发的机理,本文以冬小麦‘小偃22’和‘郑麦366’为材料,研究了不同生育期冬小麦冠层NH_3交换动态;枯、黄叶和根茬对冠层NH_3挥发的影响;监测了不同生育期的叶片硝酸盐含量、硝酸还原酶活性、谷氨酰胺合成酶活性、质外体铵浓度及可溶性蛋白含量等氮代谢指标,考察了其与冠层氨交换的关系。结果表明:冬小麦孕穗～扬花之前冠层吸收氨,为氨汇,其对农田氨挥发的消减率可达23%～40%;孕穗～扬花之后以冠层释放氨,为氨源,其对农田氨挥发的贡献率达17%～27%。小偃22冠层氨的吸收与挥发分别较郑麦366高28%～47%和19%～68%。氮肥施用下的土壤和冠层氨交换比不施氮处理高2.82～3.19kg·hm~(-2)和0.972～1.463 kg·hm~(-2),高肥力土壤和冠层氨交换比低肥力土壤高15.18～15.55 kg·hm~(-2)和0.947～1.438 kg·hm~(-2)。枯、黄叶对冠层氨挥发的作用受品种、生育期及土壤肥力等的影响。在高肥力和施氮(N180)条件下,收获后的麦茬也是氨释放源。冠层氨挥发与土壤温度、质外体铵浓度和可溶性蛋白呈显著线性正相关,与谷氨酰胺合成酶活性和硝酸盐含量呈显著线性负相关。结论:冬小麦营养生长期冠层与大气的氨交换以吸收为主,进入灌浆期后,以释放为主,氨交换方向受到生育期及氮素代谢过程的影响,同时也受到小麦品种和土壤肥力的影响。     

艳山姜叶挥发油对赤拟谷盗的杀虫活性

为开发新型仓储防护剂,研究艳山姜Alpinia zerumbet叶子挥发油的化学成分及其对仓储害虫赤拟谷盗Tribolium castaneum的杀虫活性。通过GC-MS分析了艳山姜叶挥发油的化学成分,并通过熏蒸、触杀试验测试挥发油及单体成分的活性。艳山姜叶挥发油中鉴定出31种化合物,主要成分为邻伞花烃(14.86%)、桉油精(8.44%)、芳樟醇(8.28%)、氧化石竹烯(7.62%)、柠檬烯(7.29%)、莰烯(7.23%)、α-蒎烯(6.40%)和左旋樟脑(6.20%)。通过生物活性筛选,艳山姜叶挥发油对赤拟谷盗成虫具有触杀毒性(LD_(50)值为6.59μg/头)和熏蒸毒性(LC_(50)为5.19mg/L)。挥发油中的主要化合物莰烯、柠檬烯和桉油精对赤拟谷盗均有一定的触杀毒性,LD_(50)分别为5.13、14.97和18.83μg/头。莰烯对赤拟谷盗还显示出较强的熏蒸活性,LC_(50)为4.10mg/L。因此,艳山姜叶挥发油及其活性化合物在应用于赤拟谷盗的防治方面有潜力。     

GlnA基因对刺糖多孢菌生长发育及多杀菌素合成的影响

谷氨酰胺合成酶（GS，glutamine synthetase）是由GlnA基因编码的生物体中最古老也是最广泛存在的酶，它参与许多生物体中的氮和碳代谢，是生物体氮代谢的关键酶之一，对微生物的生命活动起着重要作用。本文研究了GlnA基因对刺糖多孢菌Saccharopolyspora spinosa的生长发育及次级代谢产物合成的影响，从刺糖多孢菌基因组中克隆了glnA 基因的部分片段，将其与穿梭载体pOJ260连接，构建敲除载体pOJ260-glnA，通过接合转移将其导入刺糖多孢菌野生菌中，获得刺糖多孢菌敲除菌株S.spinosa-△glnA；同时，利用重叠延伸PCR将glnA基因置于红霉素强启动子P_ermE下，并将融合片段P_ermE-glnA与穿梭载体pOJ260进行酶切酶连，构建过量表达载体pOJ260-P_ermE-glnA，通过接合转移将其导入刺糖多孢菌中，获得了刺糖多孢菌过量表达菌株S.spinosa-glnA。表型分析发现，glnA基因的敲除对刺糖多孢菌的菌丝形态、孢子萌发等方面都有显著的抑制作用。HPLC检测分析发现，过量表达菌株中多杀菌素产量相比原始菌株提高到170%；该研究表明glnA基因对刺糖多孢菌的生长发育与多杀菌素的合成有一定的影响，为研究其在链霉菌次级代谢过程中的作用奠定了重要基础。     

Comparison of anti-feedant and insecticidal activity of nimbin and salannin photo-oxidation products with neem (Azadirachta indica) limonoids

Photo-oxidation of the neem limonoids nimbin and salannin with UV light in the presence of oxygen gives two isomeric lactone products per limonoid, nimbinolide and isonimbinolide, and salanninolide and isosalanninolide, respectively. When compared in insect tests with the important limonoids of neem seeds, azadirachtin, nimbin and salannin, isonimbinolide and isosalanninolide show activity greater than that of nimbin or salannin and in some respects show activity approaching that of azadirachtin. The photo-oxidation products were tested for anti-feedant activity and toxicity against larvae of three species of Lepidoptera, Spodoptera littoralis (Boisd), Spodoptera frugiperda (FE Smith) and Helicoverpa armigera (Hübner) and nymphs of the locusts Schistocerca gregaria (Forsk?l) and Locusta migratoria (L).     

昆虫病原线虫共生菌BP品系杀虫毒素基因xptA2的杀虫活性分析

为进一步研究嗜线虫致病杆菌Xenorhabdus nematophila杀虫毒素基因簇中各基因的功能,从BP品系的粘粒文库中筛选出一个粘粒克隆XnBP83包含全部的xpt基因,对该粘粒克隆进一步亚克隆得到一个亚克隆菌株Sub9.0,只有一个xpt基因xptA2.以Sub9.0为研究对象,对xptA2的功能作了进一步研究.结果表明,xptA2基因的表达产物与其它xpt基因的表达产物物理混合后无明显增效作用,而将xptA2转入缺失xptA2的粘粒克隆XnBP6和XnBP203后,也同样无明显增效作用.对xptA2的序列分析发现,BP品系的xptA2预测氨基酸序列与已发表序列相比明显存在2个变异区.进一步BLAST分析发现,这2个变异区同昆虫病原线虫共生菌中与口服毒性有关的杀虫毒素蛋白具有同源性.     

Sub‐lethal effects of imidacloprid on bumblebees,Bombus terrestris (Hymenoptera: Apidae), during a laboratory feeding test

A laboratory feeding test was conducted on queenless micro‐colonies of three bumblebee workers (Bombus terrestris L) to study the effects of low doses of imidacloprid on pollen and syrup consumption, worker survival, brood size and larval development. Two doses were used: D1 = 10 µg AI kg⁻¹ in syrup and 6 µg AI kg⁻¹ in pollen; D2 was 2.5 times higher in syrup and 2.7 higher in pollen. During 85 days 27, 30 and 29 micro‐colonies were reared for control, D1 and D2 treatments respectively. Food consumption was not affected by either dose. During the 5‐day pre‐oviposition period the mean insecticide intake was 4.8 ng per day per worker in treatment D2. Both doses slightly but significantly affected worker survival rate by 10% during the first month, without any dose‐effect relationship. Brood production was significantly reduced in D1 treatment and larval ejection by workers was significantly lower in D1 and D2 than in control. No significant effect of D1 and D2 treatments on the duration of larval development was revealed. No residue could be detected in workers still alive after 85 days. It was concluded that the survival rate and reproductive capacity of B terrestris was not likely to be affected by prolonged ingestion of nectar produced by sunflower after seed‐dressing treatment with imidacloprid (Gaucho), since honey or pollen collected by honeybees foraging treated sunflower never revealed concentrations of imidacloprid higher than 10 µg kg⁻¹. © 2000 Society of Chemical Industry     

Insecticidal activity and nicotinic acetylcholine receptor binding of dinotefuran and its analogues in the housefly, Musca domestica

The insecticidal activity of dinotefuran and 23 related compounds against the housefly, Musca domestica (L) was measured by injection with metabolic inhibitors. Dinotefuran was less active than imidacloprid and clothianidin by a factor of 10 in molar concentrations. Their binding activities to the fly-head membrane preparation were measured by using [125I]alpha-bungarotoxin ([125I]alpha-BGTX) and [3H]imidacloprid ([3H]IMI) as radioligands. The activity of some selected compounds measured with [3H]IMI was 10(4)-fold higher than that measured with [125I]alpha-BGTX. With [3H]IMI as a radioligand, dinotefuran was 13-fold less active than imidacloprid. The inhibitory effect of dinotefuran on the binding of [3H]IMI to the membrane preparation was in a competitive manner. Quantitative analysis of the insecticidal activity of the test compounds with the binding activity measured with [3H]IMI showed that the higher the binding activity, the higher was the insecticidal activity.     

Residual activity of spinosad applied as a soil drench to tomato seedlings for control of Tuta absoluta

BACKGROUND

Tuta absoluta (Lepidoptera: Gelechiidae) is difficult to control by means of foliar insecticides, partly because of the endophytic feeding behavior of its larvae. The biopesticide spinosad is applied as a foliar spray for control of T. absoluta and has systemic properties when applied as a soil drench to the growing medium of tomato plants. The aims of this study were to determine the: (i) instar-dependent tolerance of larvae to spinosad; (ii) efficacy of spinosad drench application for the control of larvae; (iii) residual period of systemic activity of spinosad in leaves and fruit after drenching; and (iv) effect of spinosad drenching on tomato plant growth parameters.

RESULTS

The estimated LC₅₀ value (Lethal Concentration at which 50% of the larvae died) differed between instars. The LC₅₀ for second-instar larvae (0.41 ppm) to spinosad was significantly lower than that for third- (0.64 ppm) and fourth-instar (0.63 ppm) larvae. The LC₈₀ value (Concentration at which 80% of the larvae died) for fourth-instar larvae (2.48 ppm) was 2.6- and 1.7-fold higher than that for the second- and third-instar larvae, respectively. The spinosad concentration recorded in leaves at 25 days after treatment (DAT; 0.26 μg g⁻¹) was significantly lower than that in leaves sampled at 3, 10 and 15 DAT. High larval mortalities were, however, recorded for the duration of the experiment, which lasted 25 days (equivalent to one T. absoluta generation).

CONCLUSION

Systemic spinosad effectively controlled T. absoluta larvae over a prolonged period. However, drenching this insecticide violates the recommendation of the Insecticide Resistance Action Committee to avoid treating consecutive insect generations with the same mode of action and can therefore result in the evolution of insecticide resistance. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.