Evidence for two distinct phosphonate-degrading enzymes (C-P lyases) in Arthrobacter sp. GLP-1

Arthrobacter sp. GLP-1 can utilize a wide range of organophosphonates as its sole source of phosphorus. The in-situ formation of sarcosine and methane from glyphosate and methanephosphonic acid respectively was studied. These two processes are differentially induced during phosphorus-deprivation. Methanephosphonic acid strongly inhibits glyphosate degradation (I₅₀ 10 M), but glyphosate has very little effect on methane generation (I₅₀ 150 mM). The pattern of inhibition by other organophosphonates and organophosphonate analogues is also very different for the two systems. Degradation of glyphosate and methanephosphonic acid therefore represent distinct processes.Abbreviations f.wt. fresh weight - MP-lyase methanephosphonate lyase     

A radiometric assay for 5-enolpyruvylshikimate-3-phosphate synthase

A new assay for 5-enolpyruvylshikimate-3-phosphate synthase is described. This enzyme of the shikimate pathway of aromatic amino acid biosynthesis generates 5-enolpyruvylshikimate 3-phosphate and orthophosphate from phosphoenolpyruvate and shikimate 3-phosphate. The shikimate pathway is present in bacteria and plants but not in mammals. The assay employs a paper-chromatographic separation of radiolabeled substrate from product. The method is specific, is sensitive to 50 pmol of product, and is suitable for use in crude extracts of bacteria. This enzyme appears to be the primary target site of the commercial herbicide glyphosate (N-phosphonomethyl glycine). A procedure for the enzymatic synthesis of [¹⁴C]shikimate 3-phosphate from the commercially available precursor [¹⁴C]shikimic acid is also described.     

The post-registration monitoring of glyphosate-treated plants using anecic earthworms

Glyphosate N-(phosphonomethyl) glycine is a widely-used herbicide in agriculture. The anecic earthworm, Lumbricus terrestris feeds and forages for surface plant materials meaning that this species has a unique and direct exposure to agrichemicals. At the recommended product rates, significantly (F_1,44 = 8.67, p = .005) higher numbers of L. terrestris middens were found in the glyphosate treated areas of an arable crop field. Laboratory feeding assays using field aged plant materials indicated that previous glyphosate treatment was a statistically significant factor affecting earthworm L. terrestris biomass (F₁,₁₂ = 5.75, p = .03). Negligible glyphosate residues were detectable, and the field aged plant materials were encrusted with fungal hyphae. This suggests that glyphosate influences the colonisation of plant material by a litter-fungus complex which improves the food quality to earthworms. Concentrations of epoxiconazole, a fungicide, were detected in some plant materials and may influence overall food quality to earthworms. Glyphosate treatment on fresh volunteer plant leaves (unwanted crop seedlings) was not a statistically significant factor affecting earthworm L. terrestris biomass (F₁,₆ = 0.16, p = .92). These results indicate fungal communities influence feeding behaviours, and plant materials are a direct source of agrichemicals to anecic earthworms.     

Influence of conservation reserve program mid-contract management and landscape composition on northern bobwhite in tall fescue monocultures

The United States Department of Agriculture (USDA) authorized mid-contract management (MCM) in 2004 to restore and maintain plant species composition and structural diversity in aging Conservation Reserve Program (CRP) fields for the northern bobwhite (Colinus virginianus) and other grassland-dependent wildlife. We implemented 3 USDA-approved MCM regimes (i.e., strip disking, strip glyphosate spraying, and strip glyphosate spraying in combination with legume interseeding) in 60 tall fescue (Festuca arundinaceae) CRP monocultures in south-central Illinois, USA, during 2005–2008. We hypothesized that adult bobwhite relative densities and brood presence would increase following MCM that effectively restored early successional plant communities in otherwise monotypic stands of tall fescue. We estimated annual adult bobwhite relative densities and brood presence-absence in managed and unmanaged CRP. We modeled vegetation characteristics and landscape composition to identify factors influencing adult densities and brood presence. Adult relative densities were 2-fold greater in managed fields than in unmanaged fields, and were negatively correlated with greater percentages of grass cover. Adult densities were positively correlated with greater plant species diversity, and greater percentages of bare ground and legume cover. Logistic regression and odds ratio estimates indicated that fields managed with glyphosate-interseed and glyphosate treatments were 39.6% more likely to have broods than unmanaged CRP, whereas disked fields were 10.0% more likely than unmanaged CRP. These models indicated that the probability of brood presence was greater in fields with increased percentage of bare ground, greater plant species diversity, and decreased percentage of grass and litter cover. These findings suggest that a 3-year rotation of glyphosate or glyphosate-interseed treatments can enhance habitat conditions for adult bobwhites and broods in CRP tall fescue monocultures. © 2011 The Wildlife Society.     

Circadian response of annual weeds in a natural setting to high and low application rates of four herbicides with different modes of actions

Four herbicides [glyphosate (GLYT), an amino acid synthesis inhibitor; glufosinate (GLUF), a glutamine synthetase inhibitor; fomesafen (FOME), a protoporphyrinogen oxidase inhibitor; and chlorimuron ethyl (CLIM), an acetolactate synthase inhibitor] were used to examine the influence of time of day of application on the control of a variety of annual broadleaf weeds in field studies conducted in Minnesota (five studies on GLYT and GLUF, three studies on FOME and CLIM). All herbicides were applied with an adjuvant at recommended high and low (half or quarter strength) rates every 3h between 06:00 and 24:00h local time. Visual ratings of percent weed control evaluated at 14d were analyzed by herbicide and application rate for each study and across studies for time-of-day effect by analysis of variance (ANOVA) and single cosinor. A circadian response to each herbicide was found, with greatest weed control observed between 09:00 and 18:00h. Increasing the herbicide application rate did not overcome the time-of-day effect (ANOVA: p≤0.008 for time-of-day effect for each herbicide and application rate). The least-squares fit of a 24h cosine was significant (p≤0.001) for each herbicide and application rate, with double amplitudes of 18-82% (units=% visual control) and estimated peaks (acrophases) near midday between 12:40 and 13:35h. Analysis of residuals obtained from multiple regression that included weed height, herbicide rate, temperature, and relative humidity as independent factors also found a significant time-effect by both ANOVA and cosinor for each herbicide and rate, with acrophases advancing significantly by 3 to 7h for GLYT and GLUF, but not for FOME or CLIM. These results suggest that the four herbicides, while belonging to different families with different modes of action, may reveal different peak times of efficacy when adjusting for environmental factors. Nonetheless, each displays similar circadian patterns when influenced by these factors under natural seasonal field conditions. The within-day rhythmic differences found in weed control are large enough to warrant consideration of the practical financial and environmental importance of the time-of-day that these and other herbicides are applied.     

Mutation by DNA shuffling of 5‐enolpyruvylshikimate‐3‐phosphate synthase from Malus domestica for improved glyphosate resistance

A new 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate‐tolerant plants. However, wild‐type MdEPSPS is not suitable for the development of transgenic glyphosate‐tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA shuffling on MdEPSPS, and one highly glyphosate‐resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site‐directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single‐site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate‐resistant crops.     

除草剂对蜜蜂健康影响的研究进展

除草剂是农业生产中一种被广泛使用的农药,是当前种植业重要的一环,但其长期使用也导致了自然界中广泛存在除草剂残留和污染.蜜蜂作为自然界最主要的传粉者,具有重要的经济和生态价值.近年来的研究发现,除草剂对蜜蜂有着明显的负面作用,可能会导致蜜蜂个体健康受损、蜂群损失和其在蜂产品中残留等问题.本文综述了除草剂对蜜蜂生理、行为、寿命和肠道菌群稳态的影响,旨在为进一步探索除草剂对蜜蜂危害的相关研究提供基础,并为未来除草剂的科学使用提供参考.     

Evaluating Tolerance of Herbicide and Transplantation by Cane (a Native Bamboo) for Canebrake Restoration

Canebrakes (bamboo grasslands dominated by Arundinaria spp.) were once a widespread ecosystem across the Southeastern United States, and many species of wildlife depended upon them. Early settlers replaced this system with subsistence agriculture and today few canebrakes remain. The restoration of canebrakes is critical to the recovery of several wildlife species; however, restoration is complicated because (1) seed is uncommon and often predated, (2) competition from hardwood species, including the exotic Chinese privet (Ligustrum sinesnse), often prevent cane establishment, and (3) cane depends on disturbance regimes that have been disrupted in the Southeast. We investigated the tolerance of Switch cane (Arundinaria tecta) to four commonly used herbicides that are effective at controlling privet and other hardwoods: hexazinone (Velpar‐L), glyphosate (Razor Pro), triclopyr (Garlon 3A), and imazapyr (Chopper). We also investigated the possibility of transplanting cane culms, and the factors affecting successful transplant. Cane tolerated hexazinone and triclopyr but was damaged or killed by glyphosate and imazapyr. Although many measures of weather and cane condition were not predictors of transplant success, the Keetch–Byram drought index was a strong predictor, and is available through most state forestry offices. Selective herbicides and deliberately timed transplantation may be important canebrake restoration tools.     

Using Soil Available P and Activities of Soil Dehydrogenase and Phosphatase as Indicators for Biodegradation of Organophosphorus Pesticide Methamidophos and Glyphosate

The heavy use of organophosphorus pesticides in northeastern China strongly affects the ecological functions and the quality of the soil environment. In this work, a 30-day soil incubation experiment was conducted to evaluate the potential of using soil available P and the activities of soil dehydrogenase and acid phosphatase as indicators of the application of methamidophos and glyphosate. Two kinds of unpolluted soils, phaiozem and burozem, were selected as the test soils. The higher application rate of organophosphorus pesticide to the two soils caused more release of PO₄ ^3? which finally entered the soil available P pool, suggesting that soil available P is one of the effective chemical markers for biodegradation of organophosphorus pesticides. Methamidophos exhibited a significant inhibitory effect on the activity of soil dehydrogenase. The extent of enzyme inhibition was almost positively correlated with the insecticide concentration, and the enzyme activity was gradually restored after day 15. However, its effect on soil acid phosphatase activity (stimulation or inhibition) seemed to be indefinite, and varied with the application rate, soil type, and incubation time. In the case of glyphosate, soil acid phosphatase activity was depressed significantly and the depressing extent could be a function of herbicide concentration and incubation time, but soil dehydrogenase activity showed an irregular variation with the herbicide application rate and soil type. In general, dehydrogenase activity was a good biochemical indicator for the biodegradation of methamidophos, but for glyphosate biodegradation the indicator was acid phosphatase activity.     

甘薯草甘膦抗性基因EPSPS克隆和序列分析

EPSPS基因编码5-烯醇式丙酮酰莽草酸-3-磷酸合成酶,该酶是芳香族氨基酸合成的关键酶,该基因在细菌、真菌、藻类和植物中被广泛克隆和研究。EPSPS酶是草甘膦除草剂的靶点酶,过量表达EPSPS基因可以提高作物的草甘膦抗性。该研究根据甘薯基因组数据库设计引物,以‘广薯87’为材料提取RNA,通过RT-PCR方法扩增甘薯IbEPSPS基因,测序后进行生物信息学分析和表达分析。结果表明:(1)成功克隆获得甘薯IbEPSPS基因,该基因全长CDS为1569 bp,编码522个氨基酸,其中在第98~113、173~183位氨基酸序列具有2个EPSPS的保守结构域。(2)系统进化树分析结果表明,甘薯IbEPSPS基因与三裂叶薯(Ipomoea triloba)、打碗花(Calystegia hederacea)、田旋花(Convolvulus arvensis)和牵牛(Ipomoea nil)聚在一类,其中与三裂叶薯的亲缘关系最近。(3)实时荧光定量PCR分析结果表明,甘薯IbEPSPS基因在茎、叶和茎尖表达量较高,同时受到草甘膦胁迫后IbEPSPS基因表达量提高。该研究结果为进一步探讨甘薯IbEPSPS基因的功能及甘薯对草甘膦的耐药性机制奠定了基础。