Herbicide Resistance Mechanism(s) in Italian Ryegrass (Lolium perenne ssp. multiflorum) Populations in the Southern United States

Italian ryegrass is a principal weed problem in wheat production fields in the Southern US. Resistance to herbicides diclofop, mesosulfuron, and pinoxaden among ryegrass populations has been reported. Glyphosate-resistant Italian ryegrass populations were identified in Desha County, Arkansas. This research aimed to 1) determine resistance patterns to ACCase (diclofop and pinoxaden) and ALS (imazamox, mesosulfuron, and pyroxsulam) herbicides among Italian ryegrass populations from the southern US; 2) determine if cytochrome P450-mediated enhanced herbicide metabolism contributed to resistance; and 3) elucidate the resistance mechanism to glyphosate in four Arkansas populations (Des03, Des05, Des14, and D8). For objective 1, 30 accessions from problematic fields in the southern US between 2008 and 2010 were subjected to dose-response bioassays. Among the 30 accessions, 27 were resistant to both diclofop and mesosulfuron, 25 of which were also resistant to pyroxsulam. Ten Arkansas accessions collected in 2008 were resistant to diclofop, mesosulfuron, pyroxsulam, and imazamox. One accession from Georgia and three accessions from North Carolina were resistant to diclofop, mesosulfuron, pyroxsulam, and pinoxaden. For objective 2, six ryegrass populations with different resistance patterns to glyphosate, ALS- and ACCase herbicides, were treated with P450 inhibitors malathion (1000 g ai ha-1) and 1-aminobenzotriazole (100 µM ABT) before herbicide application. Malathion improved herbicide activity in some populations; but did not completely overcome resistance to any herbicide. This indicates that P450-mediated metabolism is only partially responsible for resistance in these populations. For objective 3, plants from Des03 population were analyzed for resistance level, EPSPS genetic mutation(s), EPSPS enzyme activity, and EPSPS gene copy number. The absorption and translocation of 14C-glyphosate were similar in R and S plants. The EPSPS gene in the R plants did not contain any point mutation(s) associated with glyphosate resistance. Resistance to glyphosate in Des03 is due to increased basal EPSPS enzyme activity resulting from amplification of the EPSPS gene. Follow-up experiments conducted on other glyphosate-R populations Des05, Des14, and D8 showed 11-fold to 516-fold more copies of the EPSPS gene in resistant plants than their susceptible counterparts indicating that EPSPS gene amplification also confers resistance to glyphosate in these populations

Herbicide-resistant weeds : from research and knowledge to future needs

Synthetic herbicides have been used globally to control weeds in major field crops. This has imposed a strong selection for any trait that enables plant populations to survive and reproduce in the presence of the herbicide. Herbicide resistance in weeds must be minimized because it is a major limiting factor to food security in global agriculture. This represents a huge challenge that will require great research efforts to develop control strategies as alternatives to the dominant and almost exclusive practice of weed control by herbicides. Weed scientists, plant ecologists and evolutionary biologists should join forces and work towards an improved and more integrated understanding of resistance across all scales. This approach will likely facilitate the design of innovative solutions to the global herbicide resistance challenge

Regional whole plant and molecular response of Kochia scoparia to glyphosate

2012 Fall.Includes bibliographical references.Globally, glyphosate (Roundup®) resistant weeds pose a serious challenge to modern agricultural practices that utilize glyphosate for weed control, including Roundup Ready® cropping regimes. Locally, glyphosate resistant Kochia scoparia have been identified throughout the central Great Plains, and the infested range is expanding rapidly. Glyphosate and Roundup Ready® crops form the foundation of no-till technology, which has considerably reduced water use and soil loss in arid to semi-arid regions of North America. Unfortunately, the continued spread of glyphosate-resistant K. scoparia will jeopardize the utility of glyphosate and the sustainability of no-till agricultural practices. In an effort to suppress glyphosate-resistant K. scoparia, more needs to be known about 1) the spread of resistance, 2) the level of resistance, and 3) the mechanism responsible for glyphosate resistance in K. scoparia. Suspected glyphosate-resistant K. scoparia accessions were collected from Kansas, Colorado, North Dakota, South Dakota, and Alberta. Whole plant glyphosate dose response and shikimate assays were used to confirm resistance and assess the level of resistance. Then PCR, quantitative PCR, sequencing, and immunoblotting techniques were used to determine the mechanism responsible for glyphosate resistance. Sequence of the EPSPS binding site proline confirmed that amino acid substitution at that residue was not responsible for resistance in K. scoparia. However, quantitative PCR estimates of EPSPS copy number revealed increased copy number in all glyphosate-resistant individuals —ranging from 3 to 9 EPSPS copies relative to the reference ALS gene. Furthermore, increased EPSPS copy number was correlated to increased transcript and protein abundance. Based on these finding, I confirm resistance for all tested accessions throughout the North American central Great Plains, and conclude that increased glyphosate rates will have little effect in controlling glyphosate-resistant K. scoparia. Furthermore, I suggest that EPSPS gene amplification may be the mechanism responsible for glyphosate resistance in K. scoparia, and that lower level increases in EPSPS expression (as compared to A. palmeri) are sufficient for glyphosate resistance. Moreover, this research, again, demonstrates the adaptability of plants and foreshadows the need for diversifying weed management practices

Survey for Herbicide Resistance in Palmer Amaranth and Waterhemp in Texas

The distribution of herbicide resistance of Palmer amaranth and waterhemp to glyphosate, atrazine, tembotrione, fomesafen, and dicamba are unknown in the State of Texas. A random, semi-stratified survey targeting 6 farming regions where these species would most likely be found was conducted. Seeds from the two species were collected from 15 to 20 individuals to evaluate the sensitivity of these two species to the aforementioned herbicides. A total of 125 Palmer amaranth samples arriving from four regions, High Plains, Central TX, Gulf Coast, and Rio Grande Valley, were screened with the 5 herbicides applied POST at a recommended field rate. For waterhemp, a total of 115 samples originating from the two regions, Upper Gulf Coast and Central TX, were screened with these herbicides. At 21 days after application, a visual rating of survival (yes/no) and injury (0-100%) was taken as compared to a non-treated check and known susceptible/resistant standards. Results showed that resistance to glyphosate is widespread in the High Plains and Upper Gulf Coast, in Palmer amaranth and waterhemp, respectively. An EPSPS gene copy number analysis has revealed that the injury ratings were highly and negatively correlated with gene copy numbers, suggesting that EPSPS gene amplification is an important mechanism that endows resistance in the tested populations. Palmer amaranth resistance to atrazine was the greatest in the Upper High Plains where corn-based cropping systems are predominant. Likewise, atrazine resistant waterhemp was also found widespread in the Upper Gulf Coast region where corn and grain sorghum and widely grown. No resistance was observed for tembotrione, but when ammonium sulfate was not added to the tank-mix (as recommended by the label), it revealed the regional differences in the level of sensitivity to this herbicide. Upper High Plains and the Upper Gulf Coast regions had the greatest number of Palmer amaranth and waterhemp populations, respectively that showed reduced sensitivity to tembotrione. Resistance to fomesafen and dicamba were not detected in the surveyed populations. However, at least one waterhemp population and two Palmer amaranth populations showed reduced sensitivity to reflex and dicamba, respectively. Results revealed that herbicide resistance is an emerging issue in Texas. Best management practices need to be implemented to manage existing resistant populations and also to reduce the risk of resistance evolution to future herbicide options

Mutation site identification confers glyphosate resistance in goosegrass (Eleusine indica L. Gaertn) from Jonggol, Bogor, Indonesia

Glyphosate resistance is a serious problem in weed control, especially in oil palm plantations. However, evaluation of suspected resistant weeds such as Eleusine indica L. Gaertn at the gene level is still scarce in Indonesia. Here, target-site resistance to glyphosate of the EPSPS gene was evaluated. The objective of this study was to identify glyphosate resistance caused by mutation points in goosegrass biotypes from oil palm plantation in Jonggol, West Java, Indonesia. Bio-assay analysis was carried out by planting goosegrass in pots. Glyphosate was applicated using eight-level, namely 0, 0.125x, 0.25x, 0.5x, x, 2x, 4x, and 8x, where x was the recommended dose (972 g ha-1) for three replications. Weed damage was evaluated from plant biomass and then regression analysis was performed to obtain the LD50 and resistance index. The E. indica from block V had a reduced sensitivity status with 2.343 value of resistance index, and those from Block II biotype had a sensitive status with 1.588 resistance index. Alignment of the EPSPS gene showed no mutation was observed at two target-points, indicating that increasing resistance of E. indica in the study site could be controlled by other factors. Nevertheless, E. indica of block V biotype had a resistance potential due to the highest LD50. It is necessary to evaluate further the possibility of mutation in other target-points of the EPSPS gene. Keywords: bio-assay; EPSPS; LD50; resistance index; sequencing; target-site resistanceGlyphosate resistance is a serious problem in weed control, especially in oil palm plantations. However, evaluation of suspected resistant weeds such as Eleusine indica L. Gaertn at the gene level is still scarce in Indonesia. Here, target-site resistance to glyphosate of the EPSPS gene was evaluated. The objective of this study was to identify glyphosate resistance caused by mutation points in goosegrass biotypes from oil palm plantation in Jonggol, West Java, Indonesia. Bio-assay analysis was carried out by planting goosegrass in pots. Glyphosate was applicated using eight-level, namely 0, 0.125x, 0.25x, 0.5x, x, 2x, 4x, and 8x, where x was the recommended dose (972 g ha-1) for three replications. Weed damage was evaluated from plant biomass and then regression analysis was performed to obtain the LD50 and resistance index. The E. indica from block V had a reduced sensitivity status with 2.343 value of resistance index, and those from Block II biotype had a sensitive status with 1.588 resistance index. Alignment of the EPSPS gene showed no mutation was observed at two target-points, indicating that increasing resistance of E. indica in the study site could be controlled by other factors. Nevertheless, E. indica of block V biotype had a resistance potential due to the highest LD50. It is necessary to evaluate further the possibility of mutation in other target-points of the EPSPS gene. Keywords: bio-assay; EPSPS; LD50; resistance index; sequencing; target-site resistanc

Interspecific hybridization transfers a previously unknown glyphosate resistance mechanism in Amaranthus species

A previously unknown glyphosate resistance mechanism, amplification of the 5-enolpyruvyl shikimate-3-phosphate synthase gene, was recently reported in Amaranthus palmeri. This evolved mechanism could introgress to other weedy Amaranthus species through interspecific hybridization, representing an avenue for acquisition of a novel adaptive trait. The objective of this study was to evaluate the potential for this glyphosate resistance trait to transfer via pollen from A. palmeri to five other weedy Amaranthus species (Amaranthus hybridus, Amaranthus powellii, Amaranthus retroflexus, Amaranthus spinosus, and Amaranthus tuberculatus). Field and greenhouse crosses were conducted using glyphosate-resistant male A. palmeri as pollen donors and the other Amaranthus species as pollen recipients. Hybridization between A. palmeri and A. spinosus occurred with frequencies in the field studies ranging from <0.01% to 0.4%, and 1.4% in greenhouse crosses. A majority of the A. spinosus × A. palmeri hybrids grown to flowering were monoecious and produced viable seed. Hybridization occurred in the field study between A. palmeri and A. tuberculatus (<0.2%), and between A. palmeri and A. hybridus (<0.01%). This is the first documentation of hybridization between A. palmeri and both A. spinosus and A. hybridus

The triple amino acid substitution TAP-IVS in the EPSPS gene confers high glyphosate resistance to the superweed amaranthus hybridus

The introduction of glyphosate-resistant (GR) crops revolutionized weed management; however, the improper use of this technology has selected for a wide range of weeds resistant to glyphosate, referred to as superweeds. We characterized the high glyphosate resistance level of an Amaranthus hybridus population (GRH)—a superweed collected in a GR-soybean field from Cordoba, Argentina—as well as the resistance mechanisms that govern it in comparison to a susceptible population (GSH). The GRH population was 100.6 times more resistant than the GSH population. Reduced absorption and metabolism of glyphosate, as well as gene duplication of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or its overexpression did not contribute to this resistance. However, GSH plants translocated at least 10% more 14C-glyphosate to the rest of the plant and roots than GRH plants at 9 h after treatment. In addition, a novel triple amino acid substitution from TAP (wild type, GSH) to IVS (triple mutant, GRH) was identified in the EPSPS gene of the GRH. The nucleotide substitutions consisted of ATA102, GTC103 and TCA106 instead of ACA102, GCG103, and CCA106, respectively. The hydrogen bond distances between Gly-101 and Arg-105 positions increased from 2.89 Å (wild type) to 2.93 Å (triple-mutant) according to the EPSPS structural modeling. These results support that the high level of glyphosate resistance of the GRH A. hybridus population was mainly governed by the triple mutation TAP-IVS found of the EPSPS target site, but the impaired translocation of herbicide also contributed in this resistance

Quantitative analysis of Roundup Ready soybean content in soy-derived food and animal feed by using Real-time PCR incorporated with cloned DNA fragments

Malaysia, Biosafety Bill 2006 was approved by Parliament in July 2007, and labeling legislation will be implemented soon. In this study, duplex polymerase chain reaction (PCR) was carried out to detect endogenous soybean lectin gene and exogenous cp4-epsps (5’-enolpyruvylshikimate-3-phospate synthase) gene simultaneously. Additionally, real-time PCR utilizing SYBR Green fluorescence dye were established for the quantitative analysis of Roundup Ready soybean (RRS), which is based on the two established calibration curve from cloned fragment of cp4-epsps gene and lectin gene respectively. Approximately, 39.5% (45/114) of the samples examined in this study contain RRS, animal feeds (31), processed food (13) and raw soybean (1). Additionally, 75.6% (34/45) of the positive samples were found contained RRS above 0.9%. The sensitive GMO quantitative approach described in this study enable the analysis of various samples and this will facilitate the labeling process