Level Of Glyphosate Resistance Research Articles

No fitness cost of glyphosate resistance endowed by massive EPSPS gene amplification in Amaranthus palmeri

Amplification of the EPSPS gene has been previously identified as the glyphosate resistance mechanism in many populations of Amaranthus palmeri, a major weed pest in US agriculture. Here, we evaluate the effects of EPSPS gene amplification on both the level of glyphosate resistance and fitness cost of resistance. A. palmeri individuals resistant to glyphosate by expressing a wide range of EPSPS gene copy numbers were evaluated under competitive conditions in the presence or absence of glyphosate. Survival rates to glyphosate and fitness traits of plants under intra-specific competition were assessed. Plants with higher amplification of the EPSPS gene (53-fold) showed high levels of glyphosate resistance, whereas less amplification of the EPSPS gene (21-fold) endowed a lower level of glyphosate resistance. Without glyphosate but under competitive conditions, plants exhibiting up to 76-fold EPSPS gene amplification exhibited similar height, and biomass allocation to vegetative and reproductive organs, compared to glyphosate susceptible A. palmeri plants with no amplification of the EPSPS gene. Both the additive effects of EPSPS gene amplification on the level of glyphosate resistance and the lack of associated fitness costs are key factors contributing to EPSPS gene amplification as a widespread and important glyphosate resistance mechanism likely to become much more evident in weed plant species.

Multiple Resistance of Horseweed to Glyphosate and Paraquat and Its Control with Paraquat and Metribuzin Combinations

Greenhouse and field studies were conducted in 2007 and 2008 to investigate possible multiple-resistance of horseweed to paraquat and glyphosate, and to evaluate the effect of the addition of metribuzin to paraquat on control of paraquat-resistant horseweed. Results indicated that the GR50 (herbicide dose required to cause a 50% reduction in plant growth) value for the susceptible population S102 was 0.066 kg ae/ha glyphosate, and for the resistant population MDOT was 0.78 kg/ha glyphosate. The level of glyphosate resistance for MDOT was 12-fold compared with S102. The GR50 value for the susceptible population S102 was 0.078 kg ai/ha paraquat, and for the resistant population MDOT was 0.67 kg/ha paraquat. The level of paraquat resistance for MDOT was 9-fold compared to S102, suggesting multiple-resistance to glyphosate and paraquat in the MDOT population. In field studies the addition of metribuzin to paraquat improved horseweed control.

Glyphosate resistance in Sorghum halepense and Lolium rigidum is reduced at suboptimal growing temperatures

Glyphosate resistance in populations of the C(4) perennial Sorghum halepense (Johnsongrass) and C(3) annual Lolium rigidum (rigid ryegrass) has evolved and been documented in many cropping areas around the globe. In S. halepense and in the majority of reported cases in L. rigidum the glyphosate resistance trait has been associated with a mechanism that reduces glyphosate translocation within plants. Here, the significant decrease in the glyphosate resistance level when resistant plants of S. halepense and L. rigidum are grown at suboptimal cool temperature conditions is reported. Lowering temperature from 30 to 19 °C in S. halepense and from 19 to 8 °C in L. rigidum significantly reduced both plant survival and above-ground biomass produced by glyphosate-resistant plants. Thus, glyphosate resistance parameters significantly decreased when glyphosate-treated resistant plants of both species were grown under non-optimal temperature conditions. The results suggest that the resistance mechanism against glyphosate damage is less efficient at sub-optimal [corrected] growing temperatures. It is possible to increase the control of glyphosate-resistant S. halepense and L. rigidum populations by treatment with glyphosate during growing conditions at suboptimal low temperatures. Conversely, glyphosate failure will continue to occur on glyphosate-resistant populations treated during periods of higher temperatures

Vacuolar Glyphosate-Sequestration Correlates with Glyphosate Resistance in Ryegrass (Lolium spp.) from Australia, South America, and Europe: A 31P NMR Investigation

Lolium spp., ryegrass, variants from Australia, Brazil, Chile, and Italy showing differing levels of glyphosate resistance were examined by (31)P NMR. Extents of glyphosate (i) resistance (LD(50)), (ii) inhibition of 5-enopyruvyl-shikimate-3-phosphate synthase (EPSPS) activity (IC(50)), and (iii) translocation were quantified for glyphosate-resistant (GR) and glyphosate-sensitive (GS) Lolium multiflorum Lam. variants from Chile and Brazil. For comparison, LD(50) and IC(50) data for Lolium rigidum Gaudin variants from Italy were also analyzed. All variants showed similar cellular uptake of glyphosate by (31)P NMR. All GR variants showed glyphosate sequestration within the cell vacuole, whereas there was minimal or no vacuole sequestration in the GS variants. The extent of vacuole sequestration correlated qualitatively with the level of resistance. Previous (31)P NMR studies of horseweed ( Conyza canadensis (L.) Cronquist) revealed that glyphosate sequestration imparted glyphosate resistance. Data presented herein suggest that glyphosate vacuolar sequestration is strongly contributing, if not the major contributing, resistance mechanism in ryegrass as well.

Resistance to glyphosate in Lolium rigidum selected in Italian perennial crops: bioevaluation, management and molecular bases of target‐site resistance

Collavo A & Sattin M (2011). Resistance to glyphosate in Lolium rigidum selected in Italian perennial crops: bioevaluation, management and molecular bases of target‐site resistance. Weed Research 52, 16–24.SummaryThe resistance status of two Lolium rigidum populations (IBAF code 332 and 336) collected in northern Italian vineyards and southern Italian olive groves was investigated. Glasshouse and outdoor pot experiments confirmed that both populations were resistant to glyphosate, exhibiting a resistance index of 4 (pop. 332) and 16 (pop. 336) based on plant survival. Multiple resistance to ACCase and ALS inhibitors was also investigated, and it has been demonstrated that pop. 332 has a lower susceptibility to fluazifop but could be controlled by cycloxydim and flazasulfuron, whereas the other populations tested did not survive herbicides other than glyphosate. The molecular bases of possible target‐site resistance were also investigated. Four different patterns of substitution at position 106 were discovered only in the sequences of pop. 336: (i) heterozygous proline–leucine (CCA‐CTA); (ii) heterozygous proline–serine (CCA‐TCA); (iii) heterozygous leucine–serine (CTA‐TCA); and (iv) homozygous serine–serine. The high glyphosate resistance level of pop. 336 indicated that resistance mechanism(s) other than target site are likely to be present. Integrated weed management based on rotation of herbicide mode of action, a careful use of glyphosate, as well as non‐chemical weed control, like mowing the vegetation between crop rows, should be implemented to effectively control and limit the dissemination of resistant populations.

Glyphosate-Resistant Common Waterhemp (Amaranthus rudis) Confirmed in Texas

Glyphosate-resistantAmaranthusspecies are a recognized risk to U.S. agriculture. With affected cropland exceeding 1.2 million ha, this epidemic is particularly pertinent to agricultural regions that utilize an intensive glyphosate-based management program to control weedy pests. Before 2006, Texas had no identified glyphosate-resistant populations. Two independent common waterhemp populations exhibiting poor control by glyphosate were identified in Wharton County and Fort Bend County, TX in 2006 and 2008, respectively. The objective of the present research was to characterize the level of glyphosate resistance (50% lethal dose [LD50] and 50% reduction in growth rate [GR50]) in each population. Resistance levels in four putatively glyphosate-resistant common waterhemp biotypes selected from these two populations were compared with confirmed glyphosate-resistant and -susceptible common waterhemp populations under greenhouse conditions. The LD50value for the susceptible population (736 g ae ha−1) was equivalent to the 0.9× labeled rate of glyphosate, whereas the putatively resistant lines exhibited a broad range of resistance with LD50values ranging from 3.5 to 59.7× the labeled rate of glyphosate. The GR50value for the most resistant line was 2.5-fold greater than the susceptible biotype (317 g ae ha−1of glyphosate). These results confirm the first documented case of a glyphosate-resistant weed species in Texas.

Flaxleaf fleabane (Conyza bonariensis) populations have developed glyphosate resistance in north-east Australian cropping fields

In recent years flaxleaf fleabane has become a widespread and difficult-to-control weed in no-tilled fallowed fields, where weeds are controlled by applications of glyphosate, in annual cropping systems of north-east Australia. Fifty-two populations, collected in a national survey from agricultural and non-agricultural areas, were tested in two glyphosate dose-response pot experiments. In two subsequent pot experiments, a sub-set of these populations was tested with a field rate of glyphosate when weeds of two ages were grown at different soil moistures. In the first and second experiments, most populations collected from chemical fallowed or cropped fields in north-east Australia had GR50 (estimated dose for 50% biomass reduction) values three to six times greater than the susceptible populations, indicating low levels of glyphosate resistance. Several populations from roadsides adjacent to chemical fallowed or cropped fields also had higher GR50 values, indicating movement of seeds from resistant plants. In the third experiment, weed biomass of all populations from chemical fallowed or cropped fields was 70–98% of unsprayed compared to 2–3% for the susceptible populations, irrespective of weed age or soil moisture. In the fourth experiment which treated older weeds, the response of several resistant populations to glyphosate was unaffected by differences in weed age and soil moisture, whereas the biomass of the other resistant populations was greater following spraying of older and/or moisture stressed plants compared with smaller non-stressed plants. Thus, exclusive reliance on glyphosate for fallow weed control in this region has resulted in the evolution of resistance in flaxleaf fleabane populations in a cropping system with annual non-transgenic crops. Prolific production of windborne seeds, combined with poor control associated with spraying large moisture-stressed weeds, is likely to have contributed also to flaxleaf fleabane becoming such a problem weed.

Evolution of glyphosate resistance in a Lolium rigidum population by glyphosate selection at sublethal doses

The majority of the documented cases of field-evolved herbicide-resistant weed biotypes established that single major genes confer glyphosate resistance. However, the contribution of minor genes endowing substantial plant survival at sublethal herbicide doses may be a potential complementary path to herbicide resistance evolution in weed populations under selection. Here, we subjected a number of susceptible individuals of Lolium rigidum to recurrent glyphosate selection to test the potential for sublethal glyphosate doses to additively select for glyphosate resistance. After 3-4 cycles of glyphosate selection in two distinct environments, the progenies of the initially susceptible population were shifted toward glyphosate resistance. The results indicate progressive enrichment of minor gene trait(s) contributing toward plant survival in the glyphosate-selected progenies. After three generations of selection, the estimated LD(50) values were doubled compared with the original population and up to 33% plant survival was obtained in the glyphosate-selected progeny at the recommended glyphosate label rate. This level of resistance probably was the maximum shift achievable with sublethal glyphosate dose selection in this small population. Cross-pollination was a crucial factor enabling the rapid rate of accumulation of minor glyphosate resistance gene trait(s) that are likely to be present at a relatively high frequency in a small susceptible population. The mechanistic basis of the moderate glyphosate resistance level selected by sublethal glyphosate doses remains unknown and warrants future research. Studying the main factors influencing the evolution of resistant weed populations is crucial for understanding, predicting and managing herbicide resistance.

Glyphosate-Resistant Italian Ryegrass (Lolium multiflorum) in California: Distribution, Response to Glyphosate, and Molecular Evidence for an Altered Target Enzyme

Selection by herbicides has resulted in widespread evolution of herbicide resistance in agricultural weeds. In California, resistance to glyphosate was first confirmed in rigid ryegrass in 1998. Objectives of this study were to determine the current distribution and level of glyphosate resistance in Italian ryegrass, and to assess whether resistance could be due to an altered target site. Seeds were sampled from 118 populations and seedlings were treated with glyphosate at 866 g ae ha−1. Percentage of survivors ranged from 5 to 95% in 54 populations. All plants from 64 populations died. One susceptible (S) population, four putatively resistant (R) populations, and one S accession from Oregon were used for pot dose–response experiments, shikimic acid analyses, and DNA sequencing. Seedlings were treated with glyphosate at eight rates, ranging from 108 to 13,856 g ae ha−1. Shoot biomass was evaluated 3 wk after treatment and fit to a log-logistic regression equation. On the basis of GR50(herbicide rate required to reduce growth by 50%) values, seedlings from putatively R populations were roughly two to 15 times more resistant to glyphosate than S plants. Shikimic acid accumulation was similar in all plants before glyphosate treatment, but at 4 and 7 DAT, S plants from California and Oregon accumulated approximately two and three times more shikimic acid, respectively, than R plants. Sequencing of a cDNA fragment of the EPSPS coding region revealed two different codons, both of which encode proline at amino acid position 106 in S individuals. In contrast, all R plants sequenced exhibited missense mutations at site 106. Plants from one population revealed a mutation resulting in a proline to serine substitution. Plants from three R populations exhibited a mutation corresponding to replacement of proline with alanine. Our results indicate that glyphosate resistance is widespread in Italian ryegrass populations of California, and that resistance is likely due to an altered target enzyme.

Glyphosate and Multiple Herbicide Resistance in Common Waterhemp (Amaranthus rudis)Populations from Missouri

Field and greenhouse experiments were conducted to determine the level of glyphosate resistance in common waterhemp populations from Platte County (MO1) and Holt County, Missouri (MO2), and to determine the level and distribution of resistance to glyphosate, acetolactate synthase (ALS)–inhibiting herbicides, and protoporophyrinogen oxidase (PPO)–inhibiting herbicides across the MO1 site. Results from greenhouse experiments revealed that the MO1 and MO2 waterhemp populations were 19 and 9 times more resistant to glyphosate, respectively, than a susceptible waterhemp population. In field experiments, greater than 54% of waterhemp at the MO1 site survived 1.7 kg glyphosate ae ha−1(twice the labeled rate) 6 wk after treatment. Tank-mix combinations of ALS- and PPO-inhibiting herbicides with glyphosate also failed to provide complete control of the waterhemp population at the MO1 site. Collection and screening of seed from individual female waterhemp accessions revealed multiple resistance to glyphosate, ALS-, and PPO-inhibiting herbicides across the MO1 site. All 14 waterhemp accessions collected across the MO1 site exhibited greater than 65% survival to 2× rates of glyphosate and thifensulfuron, and these accessions were spread across a 5-km2(503-ha) area. Four waterhemp accessions collected across a 0.9-km2(87-ha) area also exhibited 26 to 38% survival to 2× rates of lactofen. The results from these experiments provide evidence and confirmation of the first glyphosate-resistant waterhemp population in the United States and reveal that multiple resistance to glyphosate, ALS-, and PPO-inhibiting herbicides can occur in waterhemp.

Glyphosate-Resistant Horseweed (Conyza canadensis) in Mississippi

Survival of horseweed in several glyphosate-tolerant cotton and soybean fields treated with glyphosate at recommended rates preplant and postemergence was observed in Mississippi and Tennessee in 2001 and 2002. Plants originating from seed collected from fields where horseweed escapes occurred in 2002 were grown in the greenhouse to the 5-leaf, 13- to 15-leaf, and 25- to 30-leaf growth stages and treated with the isopropylamine salt of glyphosate at 0, 0.025, 0.05, 0.1, 0.21, 0.42, 0.84, 1.68, 3.36, 6.72, and 13.44 kg ae/ha to determine if resistance to glyphosate existed in any biotype. All biotypes exhibited an 8- to 12-fold level of resistance to glyphosate when compared with a susceptible biotype. One resistant biotype from Mississippi was two- to fourfold more resistant than other resistant biotypes. Growth stage had little effect on level of glyphosate resistance. The glyphosate rate required to reduce biomass of glyphosate-resistant horseweed by 50% (GR50) increased from 0.14 to 2.2 kg/ha as plant size increased from the 5-leaf to 25- to 30-leaf growth stage. The GR50rate for the susceptible biotype increased from 0.02 to 0.2 kg/ha glyphosate. These results demonstrate that the difficult-to-control biotypes were resistant to glyphosate, that resistant biotypes could survive glyphosate rates of up to 6.72 kg/ha, and that plant size affected both resistant and susceptible biotypes in a similar manner.

Plastid‐expressed 5‐enolpyruvylshikimate‐3‐phosphate synthase genes provide high level glyphosate tolerance in tobacco

Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.

Stability and culture medium limitations of gene amplification in glyphosate resistant carrot cell lines

The stability of resistance to the herbicide glyphosate caused by amplification of the 5-enolpyruvylshiki-mate-3-phosphate synthase (EPSPS) gene was followed in six cloned Daucus carota (carrot) cell lines and other carrot lines grown in the absence of the selection agent for periods up to about four years. The lines originated from three different carrot suspension cultures with high levels of glyphosate resistance. The cloned lines lost resistance with a half-time of about two years. The resistance was correlated with decreased levels of EPSPS enzyme activity and EPSPS gene copy number. In some, but not all cases, glyphosate resistance was correlated with increased DNA content as measured by flow cytometry. The C1 wild type cell line, which had 2.3-times as much nuclear DNA as carrot plants, also had a correspondingly higher chromosome number, about 42 versus 18. The medium containing high glyphosate (70 mmol · L−1) was much less toxic if the K+ level was decreased, indicating that growth inhibition of the most resistant lines was caused in part by the high K+ concentration of the medium. These studies show that the selected glyphosate resistance was unstable and was lost slowly when grown away from the selection agent and that high K+ concentrations limited growth of resistant lines at high glyphosate concentrations.