Uptake and reaction to roundup ultra 360 SL in soybean seedlings

Due to the widespread and frequent use of Roundup Ultra 360 SL in crops production, the active substance glyphosate is often present (in the soil or in post-harvest remnants) and may be toxic to plants, including the non-target species. The aim of the current study was to determine the sensitivity of young soybean seedlings to glyphosate in concentrations ranging from 0 to 10 μM. It was demonstrated that the seedlings take small quantities of soil glyphosate up. More of the active substance was found in the shoots than in the roots. From the doses applied, the plant absorbs up to 4% of soil glyphosate, while over 96% remains in the soil. This suggests that only 4% of glyphosate taken up from the soil affects plant seedling development and water management. It modifies the contents of the biogenic amines cadaverine and putrescine as well as the activity of enzymes involved in their biosynthesis, i.e. ornithine decarboxylase and lysine decarboxylase. The free radical content of the roots increased with increasing herbicide doses and time of exposure. The main enzyme involved in the rapid removal of free radicals was superoxide peroxidase, activated by the herbicide treatment, while catalase was not significantly stimulated.     

Novel AroA with high tolerance to glyphosate, encoded by a gene of Pseudomonas putida 4G-1 isolated from an extremely polluted environment in China

Glyphosate has been used globally as a safe herbicide for weed control. It inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (AroA), which is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. A Pseudomonas putida strain, 4G-1, was isolated from a soil heavily contaminated by glyphosate in China. Its AroA-encoding gene (aroA) has been cloned, sequenced, and expressed in Escherichia coli. Phylogenetic analysis revealed that this AroA belongs neither to class I nor to class II AroA enzymes. When compared with E. coli AroA, 4G-1 AroA shows similar values for K(m)[PEP], K(m)[S3P], and specific enzyme activity. Moreover, 4G-1 AroA exhibits high tolerance to glyphosate, which indicates a protein with a high potential for structural and functional studies of AroA in general and its potential usage for the generation of transgenic crops resistant to the herbicide.     

Biochemical bases for a widespread tolerance of cyanobacteria to the phosphonate herbicide glyphosate

Possible non-target effects of the widely used, non-selective herbicide glyphosate were examined in six cyanobacterial strains, and the basis of their resistance was investigated. All cyanobacteria showed a remarkable tolerance to the herbicide up to millimolar levels. Two of them were found to possess an insensitive form of glyphosate target, the shikimate pathway enzyme 5-enol-pyruvyl-shikimate-3-phosphate synthase. Four strains were able to use the phosphonate as the only phosphorus source. Low uptake rates were measured only under phosphorus deprivation. Experimental evidence for glyphosate metabolism was also obtained in strains apparently unable to use the phosphonate. Results suggest that various mechanisms may concur in providing cyanobacterial strains with herbicide tolerance. The data also account for their widespread ability to metabolize the phosphonate. However, such a capability seems limited by low cell permeability to glyphosate, and is rapidly repressed when inorganic phosphate is available.     

Effect of glyphosate on the microbial activity of two Romanian soils

Glyphosate applied to soils potentially affect microbial activity. A series of field and laboratory experiments assessed the effect of this herbicide on soil microorganisms. The aim of experiments was to evaluate the effect of glyphosate application on the soil microbial community structure, function and their activity. We studied "in vitro", changes in the microbial activity of typical Chernozem and Gleysol soils, with and without applied glyphosate. The herbicide was applied at a rate of 2, respectively 4 mg kg(-1) of soil and microbial activity were measured by fluorescein diacetate (FDA) hydrolysis. We found an increase of 9 to 13% in FDA hydrolyses in the presence of glyphosate in rate of 2 mg kg (-1) compared with the same type of soil which had never received herbicide. The double quantity of glyphosate decrease soil microbial activity; the amount of hydrolyzed fluorescein is lower than the addition of 2 ppm. The greater decrease was observed in the Gleysol type where the fluorescein hydrolyzed is with 4, 85% lower than version control without glyphosate. Chemical characters of soil, influence soil biological activity when herbicide is added. In Chemozem case, rich in humus, whose predominant micro flora is represented by actinomycetes through glyphosate treatment these organisms growths of as major producers of antibiotics actinomycetes determine an inhibitory effect on eubacteria and micromycetes growth, which is highlighted by estimating a relatively small number of them. After 10 days, once with decreasing of glyphosate content in soil, decreases the number of active actinomycetes, therefore we are witnessing to a numerical growth of bacterial population. In Gleysol type the indigenous micro flora is represented by eubacteria, so when the glyphosate is added it was registered a high growth of these organisms fraction.     

5-Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive to glyphosate

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the penultimate step of the shikimate pathway, and is the target of the broad-spectrum herbicide glyphosate. Kinetic analysis of the cloned EPSPS from Staphylococcus aureus revealed that this enzyme exerts a high tolerance to glyphosate, while maintaining a high affinity for its substrate phosphoenolpyruvate. Enzymatic activity is markedly influenced by monovalent cations such as potassium or ammonium, which is due to an increase in catalytic turnover. However, insensitivity to glyphosate appears to be independent from the presence of cations. Therefore, we propose that the Staphylococcus aureus EPSPS should be classified as a class II EPSPS. This research illustrates a critical mechanism of glyphosate resistance naturally occurring in certain pathogenic bacteria.     

Responses of invertebrates to herbicide in Salix cinerea invaded wetlands: Restoration implications

The use of herbicides to control weeds, particularly large invasions, has now become an essential management tool in many ecological restoration projects. The herbicide glyphosate is routinely used to control the invasive weed, Grey Willow (Salix cinerea), within New Zealand wetlands. However, little is known about the effects of glyphosate on invertebrates. We determine the short‐term effects of glyphosate on the abundance and composition of the nontarget canopy invertebrate community in wetlands invaded by Grey Willow in New Zealand. Initially, the application of glyphosate and a surfactant showed no detectable effect on the canopy invertebrates examined in this study. However, 27 days after herbicide application, significant Grey Willow canopy loss caused dramatic decreases in the abundance of invertebrates in the glyphosate‐treated plots compared with the unsprayed plots. Invertebrates appeared to be sensitive to changes in vegetation structure, such as canopy loss. These results agree with previous studies that have shown that the negative impacts of glyphosate on invertebrate communities are related to indirect effects via habitat modification as the herbicide‐treated vegetation dies. From a terrestrial invertebrate perspective, this study suggests that the use of glyphosate herbicide is suitable for the control of invasive weeds within wetland restoration projects as it appears to have negligible impact on the canopy invertebrate assemblage.     

Molecular mechanisms underlying glyphosate resistance in bacteria

Glyphosate is a nonselective herbicide that kills weeds and other plants competing with crops. Glyphosate specifically inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase, thereby depleting the cell of EPSP serving as a precursor for biosynthesis of aromatic amino acids. Glyphosate is considered to be toxicologically safe for animals and humans. Therefore, it became the most-important herbicide in agriculture. However, its intensive application in agriculture is a serious environmental issue because it may negatively affect the biodiversity. A few years after the discovery of the mode of action of glyphosate, it has been observed that bacteria evolve glyphosate resistance by acquiring mutations in the EPSP synthase gene, rendering the encoded enzyme less sensitive to the herbicide. The identification of glyphosate-resistant EPSP synthase variants paved the way for engineering crops tolerating increased amounts of the herbicide. This review intends to summarize the molecular mechanisms underlying glyphosate resistance in bacteria. Bacteria can evolve glyphosate resistance by (i) reducing glyphosate sensitivity or elevating production of the EPSP synthase, by (ii) degrading or (iii) detoxifying glyphosate and by (iv) decreasing the uptake or increasing the export of the herbicide. The variety of glyphosate resistance mechanisms illustrates the adaptability of bacteria to anthropogenic substances due to genomic alterations.     

Isolation and characterization of endophytic bacteria from soybean (Glycine max) grown in soil treated with glyphosate herbicide

Endophytic bacteria are ubiquitous in most plant species influencing the host fitness by disease suppression, contaminant degradation, and plant growth promotion. This endophytic bacterial community may be affected by crop management such as the use of chemical compounds. For instance, application of glyphosate herbicide is common mainly due to the use of glyphosate-resistant transgenic plants. In this case, the bacterial equilibrium in plant–endophyte interaction could be shifted because some microbial groups are able to use glyphosate as a source of energy and nutrients, whereas this herbicide may be toxic to other groups. Therefore, the aim of this work was to study cultivable and noncultivable endophytic bacterial populations from soybean (Glycine max) plants cultivated in soil with and without glyphosate application (pre-planting). The cultivable endophytic bacterial community recovered from soybean leaves, stems, and roots included Acinetobacter calcoaceticus, A. junii, Burkholderiasp., B. gladioli, Enterobacter sakazaki, Klebsiella pneumoniae, Pseudomonas oryzihabitans, P. straminea, Ralstonia pickettii,and Sphingomonassp. The DGGE (Denaturing Gradient Gel Electrophoresis) analysis from soybean roots revealed some groups not observed by isolation that were exclusive for plants cultivated in soil with pre-planting glyphosate application, such as Herbaspirillum sp., and other groups in plants that were cultivated in soil without glyphosate, such as Xanthomonas sp. and Stenotrophomonas maltophilia. Furthermore, only two bacterial species were recovered from soybean plants by glyphosate enrichment isolation. They were Pseudomonas oryzihabitans and Burkholderia gladioliwhich showed different sensibility profiles to the glyphosate. These results suggest that the application at pre-planting of the glyphosate herbicide may interfere with the endophytic bacterial communitys equilibrium. This community is composed of different species with the capacity for plant growth promotion and biological control that may be affected. However, the evaluation of this treatment in plant production should be carried out by long-term experiments in field conditions.     

Mechanism of inactivation of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase by o-phthalaldehyde

In order to identify the essential reactive amino acid residues of 5-enolpyruvoylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosphate (N-phosphonomethylglycine), chemical modification studies with o-phthalaldehyde were undertaken. Incubation of the enzyme with the reagent resulted in a time-dependent loss of enzyme activity. The inactivation followed first-order and saturation kinetics with a Kinact of 25 microM and a maximum rate constant of 0.34 min-1. The inactivation was prevented by preincubation of the enzyme with the substrates shikimate 3-phosphate, 5-enolpyruvoylshikimate 3-phosphate, or by a combination of shikimate 3-phosphate plus glyphosate, but not by phosphoenolpyruvate or glyphosate alone. Absorbance and fluorescence spectra studies indicate that complete inactivation of the enzyme resulted from the formation of two isoindole derivatives per molecule of enzyme. Tryptic mapping of the enzyme modified in the absence of shikimate 3-phosphate and glyphosate resulted in the isolation of two peptides which were not found for the enzyme modified in the presence of shikimate 3-phosphate and glyphosate. Analyses of these two peptides indicate that Lys-22 and Lys-340 were the modified sites. The amino acid sequences around these residues are conserved in bacterial, fungal, as well as plant enzymes, suggesting that these regions may constitute part of the enzyme active site.     

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.     

Complex Interaction and Adsorption of Glyphosate and Lead in Soil

Glyphosate [N-(phosphonomethyl)-glycine] is a herbicide widely used in large quantities in agricultural applications. It is also known to form complexes with metal ions, although its influence on metal behavior, such as lead (Pb) in soil, is not well understood. In this study, the adsorption and co-adsorption of Pb and glyphosate were determined on two soils [a red (RS) soil, Udic Ferrisol, and a yellow-brown (YB) soil, Udic Luvisol] of distinctly different chemical characteristics at varying pH conditions. Results indicate that the adsorption of lead and glyphosate strongly depends on soil types: the RS soil, characterized by a relatively high iron/aluminum content but a low pH and organic matter content, shows a much lower adsorption capacity for Pb but a higher sorption for glyphosate than the YB soil. The co-existence of Pb and glyphosate in soils resulted in complex interactions among Pb, glyphosate, Pb-glyphosate complexes, and soil minerals. The presence of glyphosate decreased Pb adsorption on the two soils, which was attributed primarily to the formation of soluble Pb-glyphosate complexes having relatively low affinities to soil surfaces. On the other hand, addition of Pb increased the adsorption of glyphosate on both soils, which was attributed to: (1) a decreased solution pH due to the ion exchange between Pb²⁺ and H⁺ on soil surfaces; and (2) increased sorption sites where Pb was adsorbed and acted as a bridge between glyphosate and the soil. The present study illustrates that the complex interactions among glyphosate, Pb, and soil may have important implications for the mobility and bioavailability of Pb in soil and should thus be considered in future environmental risk assessments.     

Amplification of the aroA gene from Escherichia coli results in tolerance to the herbicide glyphosate.

The predominant cellular target of the herbicide glyphosate is thought to be the enzyme 5-enolpyruvylshikimate-3-phosphoric acid synthase (EPSP synthase). As a means of biologically testing this finding, we cloned a segment of DNA from Escherichia coli that encodes this enzyme. Clones carrying the gene for EPSP synthase were identified by genetic complementation. Cells that contain a multicopy plasmid carrying the EPSP synthase gene overproduce the enzyme 5- to 17-fold and exhibit at least an 8-fold increased tolerance to glyphosate. These experiments provide direct biological evidence that EPSP synthase is a major site of glyphosate action in E. coli and that, in an amplified form, it can serve as a selectable glyphosate resistance marker.     

Amplification of the aroA gene from Escherichia coli results in tolerance to the herbicide glyphosate

The predominant cellular target of the herbicide glyphosate is thought to be the enzyme 5-enolpyruvylshikimate-3-phosphoric acid synthase (EPSP synthase). As a means of biologically testing this finding, we cloned a segment of DNA from Escherichia coli that encodes this enzyme. Clones carrying the gene for EPSP synthase were identified by genetic complementation. Cells that contain a multicopy plasmid carrying the EPSP synthase gene overproduce the enzyme 5- to 17-fold and exhibit at least an 8-fold increased tolerance to glyphosate. These experiments provide direct biological evidence that EPSP synthase is a major site of glyphosate action in E. coli and that, in an amplified form, it can serve as a selectable glyphosate resistance marker.     

Bacterial glyphosate resistance conferred by overexpression of an <Emphasis Type="Italic">E. coli</Emphasis> membrane efflux transporter

Glyphosate herbicide-resistant crop plants, introduced commercially in 1994, now represent approximately 85% of the land area devoted to transgenic crops. Herbicide resistance in commercial glyphosate-resistant crops is due to expression of a variant form of a bacterial 5-enolpyruvylshikimate-3-phosphate synthase with a significantly decreased binding affinity for glyphosate at the target site of the enzyme. As a result of widespread and recurrent glyphosate use, often as the only herbicide used for weed management, increasing numbers of weedy species have evolved resistance to glyphosate. Weed resistance is most often due to changes in herbicide translocation patterns, presumed to be through the activity of an as yet unidentified membrane transporter in plants. To provide insight into glyphosate resistance mechanisms and identify a potential glyphosate transporter, we screened Escherichia coli genomic DNA for alternate sources of glyphosate resistance genes. Our search identified a single non-target gene that, when overexpressed in E. coli and Pseudomonas, confers high-level glyphosate resistance. The gene, yhhS, encodes a predicted membrane transporter of the major facilitator superfamily involved in drug efflux. We report here that an alternative mode of glyphosate resistance in E. coli is due to reduced accumulation of glyphosate in cells that overexpress this membrane transporter and discuss the implications for potential alternative resistance mechanisms in other organisms such as plants.     

Sorption and microbial degradation of glyphosate in soil suspensions

Sorption and microbial destruction of glyphosate, the active agent of the herbicide Ground Bio, in suspensions of sod-podzol and gray forest soils has been studied. According to the adsorptive values (3560 and 8200 mg/kg, respectively) and the Freundlich constants (K_f, 15.6 and 18.7, respectively), these soils had a relatively high sorption capacity as related to the herbicide. Sorbed glyphosate is represented by extractable and bound (non-extractable) fractions. After long-term incubation of sterile suspensions, the ratio of these fractions reached 2: 1 for sod-podzol soil and 1: 1 for gray forest soil. Inoculation of a native suspension of sod-podzol soil with cells of a selected strain-degrader Ochrobactum anthropi GPK 3 resulted in a 25.4% decrease in the total glyphosate content (dissolved and extractable), whereas in a noninoculated suspension, the loss did not exceed 5.5%. The potential for the use of a selected bacterial strain in the glyphosate destruction processes in soil systems is demonstrated for the first time.     

Molecular basis for the glyphosate-insensitivity of the reaction of 5-enolpyruvylshikimate 3-phosphate synthase with shikimate

The shikimate pathway enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSP synthase) has received attention in the past because it is the target of the broad-spectrum herbicide glyphosate. The natural substrate of EPSP synthase is shikimate-3-phosphate. However, this enzyme can also utilize shikimate as substrate. Remarkably, this reaction is insensitive to inhibition by glyphosate. Crystallographic analysis of EPSP synthase from Escherichia coli, in complex with shikimate/glyphosate at 1.5 Angstroms resolution, revealed that binding of shikimate induces changes around the backbone of the active site, which in turn impact the efficient binding of glyphosate. The implications from these findings with respect to the design of novel glyphosate-insensitive EPSP synthase enzymes are discussed.     

Effect of the herbicide glyphosate on glyphosate‐tolerant maize rhizobacterial communities: a comparison with pre‐emergency applied herbicide consisting of a combination of acetochlor and terbuthylazine

A comparison was made of the effect of glyphosate (Roundup^®Plus), a post‐emergency applied herbicide, and of Harness^®GTZ, a pre‐emergency applied herbicide, on the rhizobacterial communities of genetically modified NK603 glyphosate‐tolerant maize. The potential effect was monitored by direct amplification, cloning and sequencing of soil DNA encoding 16S rRNA, rhizobacterial DNA hybridization to commercially available genome‐wide microarrays from the soil bacterium Streptomyces coelicolor, and high‐throughput DNA pyrosequencing of the bacterial DNA coding for 16S rRNA hypervariable V6 region. The results obtained strongly suggest that both herbicides do in fact affect the maize rhizobacterial communities, glyphosate being, to a great extent, the environmentally less aggressive herbicide.     

Evaluation of glyphosate application in regulating the reproduction of riparian black locust (Robinia pseudoacacia L.) after clear-cutting,and the possibility of leaching into soil

Black locust (Robinia pseudoacacia L.)—an invasive alien species in riparian forests—is becoming more prevalent in many rivers of eastern Japan. Riparian black locust forests are typically cut down to maintain river-flow capacity. However, such forests often reproduce rapidly by stump sprouting and root suckering and regenerate by germination. Thus, more effective riparian forest management approaches are required. To regulate the reproduction of black locust forests after clear-cutting, we examined the regrowth-inhibiting effects of glyphosate herbicide application to stumps, in accordance with current river management protocol (i.e., winter logging operation). Further, we investigated the concentrations of glyphosate leaching into the soil at a depth of 30 cm in a riparian area of the Tenryu River. Our results showed that glyphosate application to stumps completely inhibited stump sprouting but not root suckering or seedling germination. The glyphosate concentration leaching into the soil reached a maximum (2.6 ± 0.7 mg kg^?1, mean ± standard error) on day 1 after the application, and subsequently declined to below the detection limit on day 2. Thus, the rapid degradation of glyphosate was confirmed, despite the fact that the herbicide leached into the soil after application to the stumps. The glyphosate application has limited effectiveness against root suckering and germination of riparian black locust forests after clear-cutting in winter, in accordance with the current river management protocol.