Insecticidal activity of glufosinate through glutamine depletion in a caterpillar

The herbicide glufosinate‐ammonium (GLA) is a competitive inhibitor of glutamine synthetase (GS), an enzyme converting glutamate to glutamine in both plants and animals. Because GS is essential for ammonia detoxification in plants, GLA treatment disrupts photorespiration by causing a build‐up of ammonia and a loss of glutamine in plant tissues. This study reports that GLA applied to leaf surfaces is also toxic to 5th‐instar caterpillars of the skipper butterfly Calpodes ethlius (LD₅₀ = 400 mg kg⁻¹). After ingesting GLA, caterpillars stopped feeding and became dehydrated through a loss of rectal function. Caterpillars showed symptoms of neurotoxicity, such as proleg tremors, body convulsions and complete paralysis before death. Incubation of several tissues isolated from normal feeding‐stage caterpillars with the GS substrates glutamate and ammonium showed that GLA inhibited GS activity in vitro. Within 24 h of ingesting GLA, caterpillars had a greatly reduced glutamine content and the ammonium ion levels had more than doubled. Injection of ammonium chloride into non‐GLA‐treated caterpillars had no deleterious effect, suggesting that glutamine depletion, and not a rise in body ammonium, was the primary cause of GLA toxicity following GS inhibition. This was supported by the observation that the onset of the symptoms of GLA poisoning could be postponed by giving GLA‐fed caterpillars several subsequent daily injections of glutamine. The effective GLA dose fed to 5th‐instar caterpillars in this study was comparable to the amount that might realistically be acquired from feeding on GLA‐treated crops. © 2001 Society of Chemical Industry     

RT-PCR based detection of resistance conferred by an insensitive GS in glufosinate-resistant maize cell lines

Selection of maize (Zea mays L.) cell lines resistant to glufosinate was carried out using cell suspension cultures induced from an embryo. The cell suspension was cultured on MS liquid medium supplemented with 150 mg/l asparagine, 1 mg/l thiamine, 3 mg/l 2,4-D and 2% sucrose (pH 5.8) and treated with different concentrations of glufosinate herbicide. In vitro selection from these cell suspension cultures resulted in the identification of a maize cell line resistant to glufosinate. The resistance index of the resistant cell line was 120-fold more than the normal cells. Moreover, the resistance of the cell line remained stable for at least 6 months when kept in herbicide-free medium. Glutamine synthetase (GS) activity in the resistant cells was higher than the normal cells by about 2.30-fold at 5 days after treatment of 10⁻⁶ M glufosinate. The low sensitivity of GS in resistant cells resulted in the non-inhibition of the enzyme activity, hence, the high resistance of the cell line to the herbicide. Using RT-PCR to amplify the GS mRNA, a substitution of 12 nucleotides was observed in resistant maize cells compared to that of normal cells. Conceptual translation of this sequence shows a substitution of 10 amino acids in the GS protein sequence from Genbank database of NCBI. The GS gene sequence of the newly identified glufosinate-resistant maize cell was submitted to Genbank and was given the Accession No. AY339214. The results of this study indicate a functional role of mutation in the evolution of glufosinate-resistant plants. It also supports the view that glufosinate resistance in plants was primarily due to the alteration of GS making it less sensitive to inhibitory effect of the herbicide.     

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.     

Multiple resistance across glufosinate,glyphosate, paraquat and ACCase‐inhibiting herbicides in an Eleusine indica population

An Eleusine indica population was previously reported as the first global case of field‐evolved glufosinate resistance. This study re‐examines glufosinate resistance and investigates multiple resistance to other herbicides in the population. Dose–response experiments with glufosinate showed that the resistant population is 5‐fold and 14‐fold resistant relative to the susceptible population, based on GR₅₀ and LD₅₀ R/S ratio respectively. The selected glufosinate‐resistant subpopulation also displayed a high‐level resistance to glyphosate, with the respective GR₅₀ and LD₅₀ R/S ratios being 12‐ and 144‐fold. In addition, the subpopulation also displayed a level of resistance to paraquat and ACCase‐inhibiting herbicides fluazifop‐P‐butyl, haloxyfop‐P‐methyl and butroxydim. ACCase gene sequencing revealed that the Trp‐2027‐Cys mutation is likely responsible for resistance to the ACCase inhibitors examined. Here, we confirm glufosinate resistance and importantly, we find very high‐level glyphosate resistance, as well as resistance to paraquat and ACCase‐inhibiting herbicides. This is the first confirmed report of a weed species that evolved multiple resistance across all the three non‐selective global herbicides, glufosinate, glyphosate and paraquat.     

Preliminary findings of potentially resistant goosegrass (Eleusine indica) to glufosinate‐ammonium in Malaysia

Goosegrass (Eleusine indica), regarded as one of the world's worst weeds, is highly pernicious to cash crop‐growers in Malaysia. Following reports in 2009 that glufosinate‐ammonium failed to adequately control goosegrass populations in Kesang, Malacca, and Jerantut, Pahang, Malaysia, on‐site field trials were conducted to assess the efficacy of glufosinate‐ammonium towards goosegrass in both places. Preliminary screenings with glufosinate‐ammonium at the recommended rate of 495 g ai ha^?1 provided 82% control of the weed at a vegetable farm in Kesang, while the same rate failed to control goosegrass at an oil palm nursery in Jerantut. The ensuing greenhouse evaluations indicated that the “Kesang” biotype exhibited twofold resistance, while the “Jerantut” biotype exhibited eightfold resistance towards glufosinate‐ammonium, compared to susceptible goosegrass populations. The occurrence of glufosinate resistance in goosegrass calls for more integrated management of the weed to prevent escalating resistance and further proliferation of the weed in Malaysia.     

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.     

Metabolism of the herbicide glufosinate‐ammonium in plant cell cultures of transgenic (rhizomania‐resistant) and non‐transgenic sugarbeet (Beta vulgaris), carrot (Daucus carota), purple foxglove (Digitalis purpurea) and thorn apple (Datura stramonium)

The metabolism of the herbicide glufosinate‐ammonium was investigated in heterotrophic cell suspension and callus cultures of transgenic (bar‐gene) and non‐transgenic sugarbeet (Beta vulgaris). Similar studies were performed with suspensions of carrot (Daucus carota), purple foxglove (Digitalis purpurea) and thorn apple (Datura stramonium). ¹⁴C‐labelled chemicals were the (racemic) glufosinate, L ‐glufosinate, and D ‐glufosinate, as well as the metabolites N‐acetyl L ‐glufosinate and 3‐(hydroxymethylphosphinyl)propionic acid (MPP). Cellular absorption was generally low, but depended noticeably on plant species, substance and enantiomer. Portions of non‐extractable residues ranged from 0.1% to 1.2% of applied ¹⁴C. Amounts of soluble metabolites resulting from glufosinate or L ‐glufosinate were between 0.0% and 26.7% of absorbed ¹⁴C in non‐transgenic cultures and 28.2% and 59.9% in transgenic sugarbeet. D ‐Glufosinate, MPP and N‐acetyl L ‐glufosinate proved to be stable. The main metabolite in transgenic sugarbeet was N‐acetyl L ‐glufosinate, besides traces of MPP and 4‐(hydroxymethylphosphinyl)butanoic acid (MPB). In non‐transgenic sugarbeet, glufosinate was transformed to a limited extent to MPP and trace amounts of MPB. In carrot, D stramonium and D purpurea, MPP was also the main product; MPB was identified as a further trace metabolite in D stramonium and D purpurea. © 2001 Society of Chemical Industry     

Host‐induced gene silencing in the necrotrophic fungal pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a necrotrophic fungus that causes a devastating disease called white mould, infecting more than 450 plant species worldwide. Control of this disease with fungicides is limited, so host plant resistance is the preferred alternative for disease management. However, due to the nature of the disease, breeding programmes have had limited success. A potential alternative to developing necrotrophic fungal resistance is the use of host‐induced gene silencing (HIGS) methods, which involves host expression of dsRNA‐generating constructs directed against genes in the pathogen. In this study, the target gene chosen was chitin synthase (chs), which commands the synthesis of chitin, the polysaccharide that is a crucial structural component of the cell walls of many fungi. Tobacco plants were transformed with an interfering intron‐containing hairpin RNA construct for silencing the fungal chs gene. Seventy‐two hours after inoculation, five transgenic lines showed a reduction in disease severity ranging from 55·5 to 86·7% compared with the non‐transgenic lines. The lesion area did not show extensive progress over this time (up to 120 h). Disease resistance and silencing of the fungal chs gene was positively correlated with the presence of detectable siRNA in the transgenic lines. It was demonstrated that expression of endogenous genes from the very aggressive necrotrophic fungus S. sclerotiorum could be prevented by host induced silencing. HIGS of the fungal chitin synthase gene can generate white mould‐tolerant plants. From a biotechnological perspective, these results open new prospects for the development of transgenic plants resistant to necrotrophic fungal pathogens.     

Joint action of amino acid biosynthesis-inhibiting herbicides

The joint action of binary mixtures of the amino acid biosynthesis‐inhibiting herbicides glyphosate, glufosinate‐ammonium, metsulfuron‐methyl and imazapyr was assessed in pot experiments applying the Additive Dose Model (ADM). Plants of Sinapis arvensis or S. alba were sprayed with seven doses of the herbicides alone and binary fixed‐ratio mixtures of the four herbicides. In total, 73 binary mixtures were studied in six separate experiments. Mixtures of glyphosate and glufosinate‐ammonium were less phytotoxic than predicted by ADM whether commercial formulations or technical grade products were applied. In contrast, mixtures of glyphosate and metsulfuron‐methyl, glyphosate and imazapyr, glufosinate‐ammonium and metsulfuron‐methyl, glufosinate‐ammonium and imazapyr, and metsulfuron‐methyl and imazapyr either followed ADM or were synergistic. Synergism was observed most frequently for mixtures of glyphosate or glufosinate‐ammonium with metsulfuron‐methyl. Synergism was also more pronounced for commercial formulations of glyphosate and glufosinate‐ammonium than for the corresponding technical grade formulations, implying that synergism was caused by the presence of the formulation constituents of the commercial glyphosate and glufosinate‐ammonium formulations in the spray solution.     

Potential gene flow from transgenic rice (Oryza sativa L.) to different weedy rice (Oryza sativa f. spontanea) accessions based on reproductive compatibility

BACKGROUND: The possibility of gene flow from transgenic crops to wild relatives may be affected by reproductive capacity between them. The potential gene flow from two transgenic rice lines containing the bar gene to five accessions of weedy rice (WR1–WR5) was determined through examination of reproductive compatibility under controlled pollination. RESULTS: The pollen grain germination of two transgenic rice lines on the stigma of all weedy rice, rice pollen tube growth down the style and entry into the weedy rice ovary were similar to self‐pollination in weedy rice. However, delayed double fertilisation and embryo abortion in crosses between WR2 and Y0003 were observed. Seed sets between transgenic rice lines and weedy rice varied from 8 to 76%. Although repeated pollination increased seed set significantly, the rank of the seed set between the weedy rice accessions and rice lines was not changed. The germination rates of F₁ hybrids were similar or greater compared with respective females. All F₁ plants expressed glufosinate resistance in the presence of glufosinate selection pressure. CONCLUSIONS: The frequency of gene flow between different weedy rice accessions and transgenic herbicide‐resistant rice may differ owing to different reproductive compatibility. This result suggests that, when wild relatives are selected as experimental materials for assessing the gene flow of transgenic rice, it is necessary to address the compatibility between transgenic rice and wild relatives. Copyright © 2009 Society of Chemical Industry     

大豆凝集素基因lec-s转化烟草>增强对烟草花叶病毒的抗性

 将编码大豆凝集素的lec-s基因插入植物表达载体pBI121中,构建植物重组表达质粒pBI121:: lec-s。由根癌土壤杆菌EHA105介导的叶盘法转化烟草,获得了转基因烟草株系。PCR和RT-PCR检测证明lec-s基因已转入烟草植株中。接种烟草花叶病毒（Tobacco mosaic virus,TMV）进行抗病性试验结果表明,转基因烟草叶片上的病斑数显著减少,说明转基因烟草表现出对TMV的抗性。定量RT-PCR检测发现,接种TMV后,抗病防卫基因（PR-1a、GST1、Pal和hsr515）在转基因烟草叶片中显著上调表达。这些结果表明,大豆凝集素基因lec-s转化烟草可对TMV产生抗性,其作用机制可能在于lec-s基因参与了植物的防卫信号通路,诱导了抗病防卫基因在转基因植株体内的表达,增强了植株对TMV的系统抗性。     

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     

酵母pac1基因介导对葡萄B病毒的抗性

为明确广谱性抗病毒基因—酵母pac1基因对葡萄B病毒(Grapevine virus B,GVB)的抗性效果,通过农杆菌介导的遗传转化,将pac1基因导入西方烟37B,对转基因植株进行PCR鉴定及Southern blot分析,通过病毒摩擦接种观察症状以及实时荧光定量RT-PCR检测植株体内病毒含量,并对转基因植株抗病性进行初步鉴定。结果表明,目的基因pac1成功导入并整合至西方烟37B基因组,共获得10个转基因株系。不同株系的T1代烟草中阳性植株比例为16.7%~72.7%,表明目的基因可成功遗传到子代。接种病毒后转基因植株普遍延迟发病,但后期症状与非转基因对照相似,其中仅1个转基因株系B6具有不表现典型症状等抗性反应。接种植株病毒含量检测中,所有转基因植株均检测到病毒存在,但表现为抗病的B6株系中病毒含量显著低于非转基因对照,表明该转基因植株虽不能完全抵抗GVB侵染,但对GVB具有耐病性。     

Are herbicide‐resistant crops the answer to controlling Cuscuta?

BACKGROUND: Herbicide‐resistant crop technology could provide new management strategies for the control of parasitic plants. Three herbicide‐resistant oilseed rape (Brassica napus L.) genotypes were used to examine the response of attached Cuscuta campestris Yuncker to glyphosate, imazamox and glufosinate. Cuscata campestris was allowed to establish on all oilseed rape genotypes before herbicides were applied. RESULTS: Unattached seedlings of C. campestris, C. subinclusa Durand & Hilg. and C. gronovii Willd. were resistant to imazamox and glyphosate and sensitive to glufosinate, indicating that resistance initially discovered in C. campestris is universal to all Cuscuta species. Glufosinate applied to C. campestris attached to glufosinate‐resistant oilseed rape had little impact on the parasite, while imazamox completely inhibited C. campestris growth on the imidazolinone‐resistant host. The growth of C. campestris on glyphosate‐resistant host was initially inhibited by glyphosate, but the parasite recovered and resumed growth within 3–4 weeks. CONCLUSION: The ability of C. campestris to recover was related to the quality of interaction between the host and parasite and to the resistance mechanism of the host. The parasite was less likely to recover when it had low compatibility with the host, indicating that parasite‐resistant crops coupled with herbicide resistance could be highly effective in controlling Cuscuta. Published 2009 by John Wiley & Sons, Ltd.     

广东省稻菜轮作区中牛筋草对十种除草剂的抗性水平及靶基因序列分析

为明确广东省稻菜轮作区中牛筋草对10种常用除草剂的抗性水平及抗性分子机制,采用整株生物测定法测定广东省稻菜轮作区内8个牛筋草种群P1～P8对草甘膦、草铵膦和乙酰辅酶A羧化酶(acetyl-CoA carboxylase,ACCase)抑制剂类等10种除草剂的抗性水平,并进一步分析P1和P8种群相关靶标酶基因5-烯醇丙酮酰莽草酸-3-磷酸合酶(5-enolpyruvyl-shikimate-3-phosphate synthase,EPSPS)、谷氨酰胺合成酶(glutamine synthetase,GS)和ACCase的部分功能区序列特征。结果显示,牛筋草P1～P8种群对草甘膦抗性指数为敏感种群的5.9倍～17.7倍,其中P8种群对草甘膦的抗性水平最高;8个种群对草铵膦也产生了不同程度的抗性,抗性指数为敏感种群的2.3倍～14.2倍,其中P1种群抗性最高。牛筋草P1和P8种群均对ACCase抑制剂类除草剂精喹禾灵、氰氟草酯和噁唑酰草胺产生了交互抗性;P1种群ACCase基因在第2 041位氨基酸处发生突变,该突变在牛筋草种群中首次发现;而P8种群ACCase基因则在第2 027位氨基...     

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.     

Glyphosate resistance in common ragweed ( A mbrosia artemisiifolia L.) from Mississippi,USA

Glyphosate is one of the most commonly used broad‐spectrum herbicides over the last 40 years. Due to the widespread adoption of glyphosate‐resistant (GR) crop technology, especially corn, cotton and soybean, several weed species have evolved resistance to this herbicide. Research was conducted to confirm and characterize the magnitude and mechanism of glyphosate resistance in two GR common ragweed ( A mbrosia artemisiifolia L.) biotypes from Mississippi, USA. A glyphosate‐susceptible (GS) biotype was included for comparison. The effective glyphosate dose to reduce the growth of the treated plants by 50% for the GR1, GR2 and GS biotypes was 0.58, 0.46 and 0.11 kg ae ha^?1, respectively, indicating that the level of resistance was five and fourfold that of the GS biotype for GR1 and GR2, respectively. Studies using ¹⁴ C‐glyphosate have not indicated any difference in its absorption between the biotypes, but the GR1 and GR2 biotypes translocated more ¹⁴ C‐glyphosate, compared to the GS biotype. This difference in translocation within resistant biotypes is unique. There was no amino acid substitution at codon 106 that was detected by the 5‐enolpyruvylshikimate‐3‐phosphate synthase gene sequence analysis of the resistant and susceptible biotypes. Therefore, the mechanism of resistance to glyphosate in common ragweed biotypes from Mississippi is not related to a target site mutation or reduced absorption and/or translocation of glyphosate.