Glyphosate-tolerant Crops Research Articles

Development of a double-antibody sandwich ELISA for quantification of mutated EPSPS gene expression in rice

Glyphosate resistance is a critically important trait for genetically modified (GM) crops. Mutation of the rice EPSPS gene results in a high level of glyphosate resistance, presenting significant potential for the development of glyphosate-tolerant crops. The resistance of rice to glyphosate is correlated with the expression levels of resistance genes. Therefore, developing a convenient, stable, and sensitive method for quantifying the OsmEPSPS protein is crucial for the development of glyphosate-resistant crops. We developed a double-antibody sandwich quantitative ELISA (DAS-ELISA) using a specific monoclonal antibody (mAb) for OsmEPSPS capture and an HRP-conjugated anti-OsmEPSPS rabbit polyclonal antibody (pAb). The method could be used to detect OsmEPSPS within a linear range of 16–256 ng/mL with robust intra- and inter-batch duplicability (%CV values: 0.17 %–7.24 %). OsmEPSPS expression in the transgenic rice lines (54.44–445.80 μg/g) was quantified using the DAS-ELISA. Furthermore, the expression of the OsmEPSPS gene was validated through Western blotting. This study demonstrated the reliability and stability of the DAS-ELISA for OsmEPSPS detection in GM rice.

Economic implications of the loss of glyphosate and paraquat on Australian mixed enterprise farms

CONTEXTGlyphosate and paraquat are effective, affordable non-selective herbicides widely used in Australian agriculture. However, for reasons described in this study, their use is under threat and a ban on their use is a possibility. OBJECTIVEThis study describes the farm business and farming system changes resulting from a ban on use of glyphosate and paraquat in a main mixed farming region of Australia. METHODSThe bioeconomic farm model, MIDAS, is used to represent mixed enterprise farms of Western Australia. The model incorporates the suite of management strategies and tactics farmers are likely to employ in response to loss of access to the key herbicides, glyphosate and paraquat. Scenario analyses are applied to different farm types. RESULTS AND CONCLUSIONSLoss of these herbicides is estimated to cause increased costs of crop production and large declines in farm profit, if the herbicide ban does not similarly apply to other major grain exporters. Farming systems shift towards sheep production and away from cropping, increasing farm greenhouse gas emissions. Farm businesses that are more crop dominant experience the greatest declines in profit. However, if a global ban on glyphosate and paraquat applies, then Australian farm businesses will be advantaged as they rely far less on glyphosate-tolerant crops. SIGNIFICANCEDespite several tactics and investments that farmers might employ to combat the loss of these herbicides, none prevents a reduction in farm profit. For all farm types considered under all management scenarios, loss of these herbicides generates sizeable declines farm profit. The likelihood of such large declines in profit however should spur the invention and development of cost-effective means of weed control.

Environmental and economic concerns surrounding restrictions on glyphosate use in corn

Since the commercialization of transgenic glyphosate-tolerant (GT) crops in the mid-1990s, glyphosate has become the dominant herbicide to control weeds in corn, soybean, and other crops in the United States and elsewhere. However, recent public concerns over its potential carcinogenicity in humans have generated calls for glyphosate-restricting policies. Should a policy to restrict glyphosate use, such as a glyphosate tax, be implemented? The decision involves two types of tradeoffs: human health and environmental (HH-E) impacts versus market economic impacts, and the use of glyphosate versus alternative herbicides, where the alternatives potentially have more serious adverse HH-E effects. Accounting for farmers' weed management choices, we provide empirical evaluation of the HH-E welfare and market economic welfare effects of a glyphosate use restriction policy on US corn production. Under a glyphosate tax, farmers would substitute glyphosate for a combination of other herbicides. Should a 10% glyphosate tax be imposed, then the most conservative welfare estimate is a net HH-E welfare gain with a monetized value of US$6 million per annum but also a net market economic loss of US$98 million per annum in the United States, which translates into a net loss in social welfare. This result of overall welfare loss is robust to a wide range of tax rates considered, from 10 to 50%, and to multiple scenarios of glyphosate's HH-E effects, which are the primary sources of uncertainties about glyphosate's effects.

Characterization of a Glyphosate-Tolerant Enzyme from Streptomyces svecius: A Distinct Class of 5-Enolpyruvylshikimate-3-phosphate Synthases.

Natural and modified versions of the 5-enolpyruvylshikimate-3-phosphate synthase (epsps) gene have been used to confer tolerance to the broad-spectrum herbicide glyphosate in a variety of commercial crops. The most widely utilized trait was obtained from the Agrobacterium tumefaciens strain CP4 and has been commercialized in several glyphosate-tolerant crops. The EPSPS gene products are enzymes that have been divided into three classes based on sequence similarity, sensitivity to glyphosate, and steady-state catalytic parameters. Herein, we describe the informatics-guided identification and biochemical and structural characterization of a novel EPSPS from Streptomyces sviceus (DGT-28 EPSPS). The data suggest DGT-28 EPSPS and other closely related homologues exemplify a distinct new class (Class IV) of EPSPS enzymes that display intrinsic tolerance to high concentrations of glyphosate (Ki ≥ 5000 μM). We further demonstrate that dgt-28 epsps, when transformed into stable plants, provides robust (≥4× field rates) vegetative/reproductive herbicide tolerance and has utility in weed-control systems comparable to that of commercialized events.

Glyphosate-induced hormesis: impact on seedling growth and reproductive potential of common sowthistle (Sonchus oleraceus)

AbstractIn Australia, glyphosate is widely used in glyphosate-tolerant crops and fallows to control weeds such as common sowthistle (Sonchus oleraceus L.). It has been hypothesized that glyphosate at sublethal doses, as a consequence of herbicide drift, may have a stimulatory effect on S. oleraceus growth. In 2017, pot trials were conducted to evaluate the effect of low doses of glyphosate on growth and seed production of this weed at the Weed Science Screenhouse Facility at the University of Queensland, Australia. At the 4- to 5-leaf stage (3-wk-old rosette), plants were treated with low doses of glyphosate (0 [control], 5, 10, 20, 40, 80, and 800 g ae ha−1), and their responses were recorded until plant maturity. The study was repeated after completion of the first experimental run. An additional glyphosate dose (2.5 g ha−1) was added in the second run. The low doses of glyphosate (<40 g ha−1) caused a significant increase in S. oleraceus plant height and number of leaves compared with the no-glyphosate treatment. The highest stimulatory effect was observed at 5 g ha−1. At 5 g ha−1 glyphosate, S. oleraceus seed production increased by 154% and 101% in the first and second experimental runs, respectively, compared with the no-glyphosate treatment. The results of this study suggest that the sublethal doses of glyphosate produced hormetic effects on growth and seed production of S. oleraceus that changed the dynamics of weed–crop competition.

Novel aroA of Glyphosate-Tolerant Bacterium Pseudomonas sp. Strain HA-09 Isolated from Roundup-Contaminated Garden Soils in Iran.

Background: Glyphosate is a non-selective systemic herbicide with a broad spectrum of weed control that inhibits a key enzyme, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, in the shikimate pathway.Objectives: Isolation and analysis of the epsps (aroA) gene responsible for glyphosate-tolerance in bacteria from Roundup- contaminated soils was the aim of this study.Materials and Methods: Sampling was done from the soil of the gardens which were heavily contaminated by Roundup herbicide and then bacterial screening was performed in the presence of high concentrations of glyphosate. The genus of bacterium was identified via molecular methods such as 16S rRNA sequencing. The aroA gene of this bacterium (aroAHA-09) was isolated using the primers designed-upon specific regions of aroA genes available in NCBI GenBank database. The PCR product was cloned, sequenced and subcloned into pET28a as an expression vector and transferred into E. coli strain BL21(DE3). The cells were inoculated in liquid M9 minimal medium containing IPTG and different concentrations of glyphosate.Results: The genus of bacterium was identified as Pseudomonas sp. strain HA-09. The isolated aroAHA-09 gene from this bacterium was approximately 2.2 kb in size. Bioassay of E. coli expressing this gene showed high tolerance to glyphosate (up to 300 mM).Conclusion: The aroAHA-09 gene could be considered as a novel and efficient candidate for development of glyphosate-tolerant crop plants.

Isolation and functional characterization of 5-enolpyruvylshikimate 3-phosphate synthase gene from glyphosate-tolerant Pseudomonas nitroreducens strains FY43 and FY47.

5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is the primary target for the broad-spectrum herbicide, glyphosate. Improvement of EPSPS gene for high level of glyphosate tolerance is important to generate glyphosate-tolerant crops. In this study, we report the isolation and characterization of EPSPS genes of glyphosate-tolerant Pseudomonas nitroreducens strains FY43 and FY47. Both P. nitroreducens strains FY43 and FY47, which showed glyphosate tolerance up to 8.768% (518.4mM, 32× higher than field application), were isolated from soil samples collected from oil palm plantation with a long history of glyphosate application. The glyphosate tolerance property of EPSPS genes of strains FY43 and FY47 was functionally characterized by expressing the genes in Escherichia coli strain BL21(DE3). Error-prone PCR was performed to mutagenize native EPSPS gene of strains FY43 and FY47. Ten mutagenized EPSPS with amino acid changes (R21C, N265S, A329T, P71L, T258A, L184F, G292C, G292S, L35F and A242V) were generated through error-prone PCR. Both native and mutated EPSPS genes of strains FY43 and FY47 were introduced into Escherichia coli strain BL21(DE3) and transformants were selected on basal salt medium supplemented with 8.768% (518.4mM) glyphosate. Mutants with mutations (R21C, N265S, A329T, P71L, T258A, L35F, A242V, L184F and G292C) showed sensitivity to 8.768% glyphosate, whereas glyphosate tolerance for mutant with G292S mutation was not affected by the mutation.

Identification and integrated analysis of glyphosate stress-responsive microRNAs, lncRNAs, and mRNAs in rice using genome-wide high-throughput sequencing

BackgroundGlyphosate has become the most widely used herbicide in the world. Therefore, the development of new varieties of glyphosate-tolerant crops is a research focus of seed companies and researchers. The glyphosate stress-responsive genes were used for the development of genetically modified crops, while only the EPSPS gene has been used currently in the study on glyphosate-tolerance in rice. Therefore, it is essential and crucial to intensify the exploration of glyphosate stress-responsive genes, to not only acquire other glyphosate stress-responsive genes with clean intellectual property rights but also obtain non-transgenic glyphosate-tolerant rice varieties. This study is expected to elucidate the responses of miRNAs, lncRNAs, and mRNAs to glyphosate applications and the potential regulatory mechanisms in response to glyphosate stress in rice.ResultsLeaves of the non-transgenic glyphosate-tolerant germplasm CA21 sprayed with 2 mg·ml− 1 glyphosate (GLY) and CA21 plants with no spray (CK) were collected for high-throughput sequencing analysis. A total of 1197 DEGs, 131 DELs, and 52 DEMs were identified in the GLY samples in relation to CK samples. Genes were significantly enriched for various biological processes involved in detoxification of plant response to stress. A total of 385 known miRNAs from 59 miRNA families and 94 novel miRNAs were identified. Degradome analysis led to the identification of 32 target genes, of which, the squamosa promoter-binding-like protein 12 (SPL12) was identified as a target of osa-miR156a_L + 1. The lncRNA-miRNA-mRNA regulatory network consisted of osa-miR156a_L + 1, two transcripts of SPL12 (LOC_Os06g49010.3 and LOC_Os06g49010.5), and 13 lncRNAs (e.g., MSTRG.244.1 and MSTRG.16577.1).ConclusionLarge-scale expression changes in coding and noncoding RNA were observed in rice mainly due to its response to glyphosate. SPL12, osa-miR156, and lncRNAs (e.g., MSTRG.244.1 and MSTRG.16577.1) could be a novel ceRNA mechanism in response to glyphosate in rice by regulating transcription and metal ions binding. These findings provide a theoretical basis for breeding glyphosate-tolerant rice varieties and for further research on the biogenesis of glyphosate- tolerance in rice.

Weed management strategies effect on glyphosate‐tolerant maize and soybean yields and quality

AbstractWeed management (WM) is necessary to prevent crop losses through competition with weeds and maintain high yields. However, in the case of glyphosate‐tolerant (GT) crops, phytotoxic effects can occur after glyphosate‐based herbicide (GBH) applications, which could impact yields and quality. In order to assess the agronomic performance of six WM strategies on GT soybean [Glycine max (L.) Merr.] and maize (Zea mays L.), field experiments were conducted in randomized blocks replicated four times (T1: Mechanical weeding; T2: Other herbicide application [Soybean: Chlorimuron ethyl + Imazethapyr] [Corn: Saflufenacil + Dimethenamid‐P]; T3: One GBH application; T4: One GBH + other herbicide application [Soybean: Imazethapyr] [Corn: S‐metolachlor + Mesotrione]; T5: Two GBH applications; T6: Two GBH applications + other herbicide application [Soybean: Chlorimuron ethyl + Imazethapyr] [Corn: S‐metolachlor + Mesotrione]). In soybean, T1 was the least productive treatment with an average yield of 2,652 kg ha−1, while T4, T5, and T6 produced significantly higher yields (4,315, 4,646, and 4,248 kg ha−1 respectively). However, the protein content was higher in T1 (42%) than in T3‐6 (40.85, 40.55, 40.68, and 40.65%), as well as the linolenic acid content whereas the total oil content was significantly lower. For maize, there were no significant differences in yields nor in nutritional content for all treatments. These findings question the systemic usage of GBHs in GT crops. If unnecessary, GBH applications could be reduced, which would relieve the selection pressure for glyphosate‐resistant weeds, especially in the case of GT soybean and maize crop rotation.

Response of Annual Weeds to Glyphosate: Evaluation and Optimization of Application Rate Based on Fecundity-Avoidance Biomass Threshold Criterion

The increased availability and high adoption rate of glyphosate-tolerant crops have selected for several glyphosate-resistant weed species. The response of representative weed species to glyphosate was assessed to provide insights and tools for optimizing glyphosate use for economic, agronomic and environmental reasons. Anoda cristata, Chenopodium album, Digitaria sanguinalis, Eleusine indica and Portulaca oleracea were grown outdoors in pots containing commercial potting medium. An increasing dose of glyphosate was applied to these species at three growth stages. Weed response was evaluated visually compared to the nontreated control and shoot dry weights were recorded. Fecundity was also determined. Based on visual evaluations, the dose of glyphosate required to attain 90% control of the species tested exhibited an application rate margin up to 28.5-fold compared to recommended rate, denoting a potential for rate optimization. Except for A. cristata, the recommended dose of glyphosate could be reduced by 30%–60% and still achieve 90% or greater control. The order of species sensitivity, based on effective dose 50 (ED50 )values, was E. indica > C. album > D. sanguinalis > P. oleracea > A. cristata. The ratio of ED90/ED50 was constant, indicating that increasing the glyphosate dose 8.7-fold would reduce weed biomass 1.8-fold. In most cases, the fecundity-avoidance biomass threshold (i.e., the maximum allowable weed biomass for herbicide application in order to prevent weed seed production and dispersal) for glyphosate was below the ED90 value. Complimentary measures such as fecundity-avoidance biomass threshold will improve herbicide evaluation procedures and preserve the effectiveness of herbicides, including glyphosate, on sensitive species, an important issue particularly when action to reduce herbicide resistance development is highly required.

Glyphosate in livestock: feed residues and animal health1.

Glyphosate is a nonselective systemic herbicide used in agriculture since 1974. It inhibits 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, an enzyme in the shikimate pathway present in cells of plants and some microorganisms but not human or other animal cells. Glyphosate-tolerant crops have been commercialized for more than 20 yr using a transgene from a resistant bacterial EPSP synthase that renders the crops insensitive to glyphosate. Much of the forage or grain from these crops are consumed by farm animals. Glyphosate protects crop yields, lowers the cost of feed production, and reduces CO2 emissions attributable to agriculture by reducing tillage and fuel usage. Despite these benefits and even though global regulatory agencies continue to reaffirm its safety, the public hears conflicting information about glyphosate's safety. The U.S. Environmental Protection Agency determines for every agricultural chemical a maximum daily allowable human exposure (called the reference dose, RfD). The RfD is based on amounts that are 1/100th (for sensitive populations) to 1/1,000th (for children) the no observed adverse effects level (NOAEL) identified through a comprehensive battery of animal toxicology studies. Recent surveys for residues have indicated that amounts of glyphosate in food/feed are at or below established tolerances and actual intakes for humans or livestock are much lower than these conservative exposure limits. While the EPSP synthase of some bacteria is sensitive to glyphosate, in vivo or in vitro dynamic culture systems with mixed bacteria and media that resembles rumen digesta have not demonstrated an impact on microbial function from adding glyphosate. Moreover, one chemical characteristic of glyphosate cited as a reason for concern is that it is a tridentate chelating ligand for divalent and trivalent metals; however, other more potent chelators are ubiquitous in livestock diets, such as certain amino acids. Regulatory testing identifies potential hazards, but risks of these hazards need to be evaluated in the context of realistic exposures and conditions. Conclusions about safety should be based on empirical results within the limitations of model systems or experimental design. This review summarizes how pesticide residues, particularly glyphosate, in food and feed are quantified, and how their safety is determined by regulatory agencies to establish safe use levels.

Maternal glyphosate-based herbicide exposure alters antioxidant-related genes in the brain and serum metabolites of male rat offspring

In response to the rapid development of genetically engineered glyphosate-tolerant crops, the use of glyphosate-based herbicides (GBHs), in agriculture, has increased substantially. Currently, it is estimated that 747 million kg of GBHs are applied per year. Although several epidemiological studies have demonstrated that there are health risks associated with GBH exposure, the effects these chemicals have on the oxidative and inflammatory response in the brain are still unclear. In fact, alterations in these processes could contribute to the development of neurological diseases, such as Alzheimer’s disease and autism spectrum disorders. The present study exposed pregnant rats to GBH and evaluated changes in the expression of genes related to oxidnte defense and inflammation response and monitored the serum metabolome in the adult male offspring. Pregnant Wistar rats were administered distilled water or Roundup®, at either 5 and 50 mg/kg/day, (p.o.) from gestational day (GD) 18 to postnatal day (PND) 5. There was a significant increase in the gene expression levels of Neuroglobin (Ngb – oxygen storage and tissue protection) (105%, p = 0.031), Glutathione Peroxidase 1 (Gpx1 – oxidative stress) (95%, p = 0.005), Prostaglandin-Endoperoxidase Synthase 1 (Ptgs1 - inflammation) (109%, p = 0.033) and Hypoxia inducible factor 1 subunit alpha (Hif1α – oxygen sensor) (73%, p = 0.017), in the cerebellum of PND90 rats perinatally exposed to 50 mg GBH/kg/day. Moreover, both GBH-exposed groups displayed a significant decrease in the expression of Catalase (Cat – oxidative stress) (49%, p = 0.003; and 31% p = 0.050, respectively) expression, in the cortex. Serum metabolites analyses, from the same animals of each group, demonstrated that there were significant changes in the concentrations of lysophosphatidylcholine and phosphatidylcholine, which have been associated with neurodegenerative diseases. The results of the present study suggest GBH exposure during pregnancy alters the expression of genes associated with oxidant defense, inflammation and lipid metabolism. It is plausible that maternal GBH exposure could have lasting neuronal effects on the offspring later in life.

Exposure to Roundup® affects behaviour, head regeneration and reproduction of the freshwater planarian Girardia tigrina

The demand of glyphosate-based herbicides including Roundup® is rising in the tropics due to increase occurence of glyphosate-resistant weeds that require higher herbicide application rates but also because of their use associated with genetically engineered, glyphosate-tolerant crops. Consequently, there is now an excessive use of glyphosate in agricultural areas with potential adverse effects also for the surrounding aquatic environments. This study aimed to determine the sensitivity of the freshwater planarian Girardia tigrina to acute and chronic exposures of Roundup®. Planarians were exposed to a range of lethal and sub-lethal concentrations of Roundup® to determine the median lethal concentration (LC50) concerning its active ingredient glyphosate and also effects on locomotor velocity (pLMV), feeding rate, regeneration, reproductive parameters and morphological abnormalities. Regeneration endpoints included length of blastema and time for photoreceptors and auricles regeneration after decapitation, while effects on reproduction were assessed measuring fecundity (number of deposited cocoons) and fertility (number of hatchlings) over five weeks of exposure to glyphosate. The estimated 48 h LC50 of was 35.94 mg glyphosate/L. Dose dependent effects were observed for feeding, locomotion and regeneration endpoints with Lowest observed effect concentration (LOEC) values as low as 3.75 mg glyphosate/L. Chronic exposures to environmentally relevant concentrations of glyphosate significantly impaired fecundity and fertility rates of exposed planarians (median effective concentration, EC50 = 1.6 mg glyphosate/L for fecundity and fertility rates). Our results show deleterious effects of Roundup® on regeneration, behavior and reproduction of freshwater planarians and add important ecotoxicological data towards the environmental risk assessment of glyphosate-based herbicide in freshwater ecosystems.

Pesticides in agriculture

Pesticides in agriculture

Desensitizing plant EPSP synthase to glyphosate: Optimized global sequence context accommodates a glycine-to-alanine change in the active site

5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the transfer of a carboxyvinyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate and in plants is the target of the herbicide glyphosate. EPSPSs with high catalytic efficiency and insensitivity to glyphosate are of microbial origin, including the enzyme from Agrobacterium strain CP4, in which insensitivity is conferred by an active site alanine. In the sequence context of plant EPSPSs, alanine in place of glycine at the equivalent position interferes with the binding of both glyphosate and PEP. We show here that iterative optimization of maize EPSPS containing the G101A substitution yielded variants on par with CP4 in terms of catalytic activity in the presence of glyphosate. The improvement relative to G101A alone was entirely due to reduction in Km for PEP from 333 to 18 μm, versus 9.5 μm for native maize EPSPS. A large portion of the reduction in Km was conferred by two down-sizing substitutions (L97C and V332A) within 8 Å of glyphosate, which together reduced Km for PEP to 43 μm Although the original optimization was conducted with maize EPSPS, contextually homologous substitutions conferred similar properties to the EPSPSs of other crops. We also discovered a variant having the known glyphosate-desensitizing substitution P106L plus three additional ones that reduced the Km for PEP from 47 μm, observed with P106L alone, to 10.3 μm The improvements obtained with both Ala101 and Leu106 have implications regarding glyphosate-tolerant crops and weeds.

Alternative Herbicides for Control of Glyphosate-Resistant Giant Ragweed in Nebraska

Giant ragweed is an early emerging and one of the most competitive summer annual species found in many fields throughout North America. Extensive use of glyphosate in glyphosate-tolerant (GT) crops has evolved giant ragweed populations with glyphosate resistance. Field dose-response studies were conducted to determine the influence of growth stage on the level of glyphosate resistance in a suspected giant ragweed population. In addition, efficacy of alternative pre-plant, pre-emergence (PRE) and post-emergence (POST) herbicides were evaluated in corn and soybeans for glyphosate-resistant (GR) giant ragweed control. The field glyphosate dose-response studies confirmed that the suspected giant ragweed population were resistant ranging from 14- to 32-fold resistance depending on the growth stage of glyphosate application. The 10, 20 and 30 cm tall giant ragweed had 14, 17 and 32X resistance level, respectively. The dose-response studies indicated that the 10, 20, and 30 cm tall GR giant ragweed was controlled 90% with 214, 402 and 482 g ae ha-1 of dicamba, respectively, when tank-mixed with glyphosate (1060 g ae ha-1) 21 days after treatment (DAT). All evaluated pre-plant herbicides for corn provided ≥ 90% control of the GR giant ragweed at 30 DAT; among which the best control (100%) was achieved with pre-plant application of atrazine (2240 g ai ha-1), isoxaflutole (90 g ai ha-1), and premix of flumioxazin/pyroxasulfone (315 g ai ha-1). Herbicide combinations of different site of action provided greater than 90% control of the GR giant ragweed population in a PRE followed by POST herbicide program in corn and soybean, suggesting that alternative herbicide for giant ragweed control are available.

Non-destructive Determination of Shikimic Acid Concentration in Transgenic Maize Exhibiting Glyphosate Tolerance Using Chlorophyll Fluorescence and Hyperspectral Imaging.

The development of transgenic glyphosate-tolerant crops has revolutionized weed control in crops in many regions of the world. The early, non-destructive identification of superior plant phenotypes is an important stage in plant breeding programs. Here, glyphosate-tolerant transgenic maize and its parental wild-type control were studied at 2, 4, 6, and 8 days after glyphosate treatment. Visible and near-infrared hyperspectral imaging and chlorophyll fluorescence imaging techniques were applied to monitor the performance of plants. In our research, transgenic maize, which was highly tolerant to glyphosate, was phenotyped using these high-throughput non-destructive methods to validate low levels of shikimic acid accumulation and high photochemical efficiency of photosystem II as reflected by maximum quantum yield and non-photochemical quenching in response to glyphosate. For hyperspectral imaging analysis, the combination of spectroscopy and chemometric methods was used to predict shikimic acid concentration. Our results indicated that a partial least-squares regression model, built on optimal wavelengths, effectively predicted shikimic acid concentrations, with a coefficient of determination value of 0.79 for the calibration set, and 0.82 for the prediction set. Moreover, shikimic acid concentration estimates from hyperspectral images were visualized on the prediction maps by spectral features, which could help in developing a simple multispectral imaging instrument for non-destructive phenotyping. Specific physiological effects of glyphosate affected the photochemical processes of maize, which induced substantial changes in chlorophyll fluorescence characteristics. A new data-driven method, combining mean fluorescence parameters and featuring a screening approach, provided a satisfactory relationship between fluorescence parameters and shikimic acid content. The glyphosate-tolerant transgenic plants can be identified with the developed discrimination model established on important wavelengths or sensitive fluorescence parameters 6 days after glyphosate treatment. The overall results indicated that both hyperspectral imaging and chlorophyll fluorescence imaging techniques could provide useful tools for stress phenotyping in maize breeding programs and could enable the detection and evaluation of superior genotypes, such as glyphosate tolerance, with a non-destructive high-throughput technique.

Glyphosate Tolerant Soybean Response to Different Management Systems

The benefits of glyphosate tolerant crops technology are well-known, and its acceptance by farmers is undeniable. However, results of recent research indicate that, in some situations, glyphosate applied to herbicide-tolerant soybean crops may have phytotoxic effects affecting nutritional balance, photosynthesis and others biochemical process in plants. Despite the increasing information available on this subject, there are still scientific and technical issues that need to be clarified. Therefore, the present study aimed to assess the impact of applying different rates, management systems, and formulations of glyphosate to glyphosate-tolerant soybean trough different regions of Brazil in different environmental conditions. Two experiments were conducted over two crop seasons. A 2 × 2 × 5 (formulations × stage of application × doses) factorial design was used in each of them, for a total of 20 treatments with four replications. The study assessed a series of variables related to agronomic performance such as total chlorophyll and yield. The results suggest some problems associated with post-emergent use of glyphosate in tolerant soybean crop as 5% total yield reduction even without phytotoxicity symptoms dependent of season. There was not found any formulation interaction with yield decrease.

Phytotoxicity of soilborne glyphosate residues is influenced by the method of phosphorus fertiliser application

Glyphosate use has increased in recent decades with the adoption of minimum tillage techniques and the emergence of glyphosate-tolerant crop cultivars. There is evidence that glyphosate residues are common in cropping soils at sowing, but any potential impact of these residues on crop growth is not known. This study aimed to determine concentrations of soilborne glyphosate that are phytotoxic to common crop species and investigate any interactions between glyphosate residues and phosphorus (P) fertiliser. A pot study examined the impact of soilborne glyphosate residues on the growth of wheat (Triticum aestivum L.) and narrow leaf lupin (Lupinus angustifolius L) seedlings in a sandy soil with a low P buffering capacity in the absence or presence of P fertiliser applied as a liquid to the topsoil. A second pot study investigated whether the phytotoxic effects of soilborne glyphosate residues on lupin growth in the presence of P fertiliser could be mitigated by banding the P fertiliser (as superphosphate) as per standard practice by farmers. Wheat was more tolerant of glyphosate residues than lupin, with glyphosate levels up to 14.8 mg kg−1 having no effect on wheat shoot growth at 48 days after sowing (DAS), while lupin shoot growth at 48 DAS had an ED10 (i.e. a 10% yield reduction) of glyphosate at 6.4 mg kg−1 in the absence of P fertiliser. When P fertiliser was applied, wheat shoot biomass had an ED10 of 5.8 mg glyphosate kg−1 soil while lupins had an ED10 of 3.4 mg glyphosate kg−1 soil. When P fertiliser (superphosphate) was banded at sowing, lupins grown adjacent to band were unaffected by glyphosate whereas the shoot weights of lupins planted above the band were significantly reduced by 68% in soil containing 1.3 mg kg−1 glyphosate and 81% in soil containing 3.4 mg kg−1 glyphosate. Where P fertiliser was applied, growth reductions in lupin seedlings were observed at soil glyphosate concentrations that have been observed in farmers’ fields, suggesting that glyphosate residues may be problematic in sandy soils with low P buffering capacities. Banding of P fertiliser exacerbated, rather than mitigated, the phytotoxicity of glyphosate residues to lupins where P fertiliser was banded directly beneath the seed.

Split application of glyphosate in herbicide-tolerant maize provides efficient weed control and favors beneficial epigeic arthropods

Wide deployment of genetically modified herbicide-tolerant (GMHT) maize may affect the efficiency of weed control methods and impair ecosystem functioning. We examined these potential threats using glyphosate-tolerant maize NK603 (Monsanto Technology LLC, St. Louis, MO, USA) planted in 54 randomly distributed plots in 2013 and 2014. Maize was grown in three herbicide treatments combined with six tillage regimes. Conventional, single post-emergence application of a selective herbicide MaisTer (Bayer CropScience GmbH, Frankfurt am Main, Germany; hereafter as MT) was compared with two herbicide strategies used in the glyphosate-tolerant crops: split application of Roundup Rapid (Monsanto Europe S.A./N.V., Brussels, Belgium; hereafter as RRRR) and application of this herbicide mixed with the soil residual herbicide Guardian Extra (Monsanto Europe S.A./N.V.; hereafter as RRGE). MT proved unreliable, whereas RRRR and especially RRGE provided efficient weed control and affected weed performance during the season. RRRR permitted restoration of weed cover during the latter half of the cultivation season. Conventional, reduced, and conservation (mulching with Hordeum vulgare, Phacelia tanacetifolia, Sinapis alba, and Trifolium incarnatum) tillage had minor effect on the weed performance. Carabids, staphylinids, and spiders were monitored to assess environmental impact of tested weed management practices. Carabid communities were not affected by the type of tillage but responded to the herbicide treatments. The plots treated with MT harbored the highest carabid activity abundance and species richness, followed by RRRR, and then by RRGE. RRRR and RRGE treatments also reduced the rise of staphylinid abundance and species richness after harvest, while conventional tillage negatively affected staphylinids at the start of the cultivation season. Spider and carabid activity abundance was similar, but spider species richness was highest in the RRRR plots. Neither herbicides nor tillage strongly affected arthropod species evenness. Multivariate analysis showed that weed species richness was significantly correlated with the species activity abundance of all three arthropod groups; weed coverage had a similar but smaller effect but the effect of herbicides and tillage was negligible. We concluded that herbicide treatments curbed weed performance which consequently influenced associated arthropods.The RRRR herbicide treatment adequately regulates weeds and exerts restoration of weed cover later in growing season that is beneficial to the arthropods. Thus, GMHT crops have a potential to combine economic and environmental advantages for agroecosystem sustainability and can be recommended for implementation in European crop production systems.