EPSPS Gene Research Articles

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.

Genetic diversity and in silico evidence of target-site mutation in the EPSPS gene in endowing glyphosate resistance in Eleusine indica (L.) from Malaysia

Glyphosate-resistant populations of Eleusine indica are widespread in several states of Malaysia. A whole-plant bioassay confirmed that eight out of the 17 populations tested were resistant to glyphosate at double the recommended rate of 2.44 kg ha−1. Screening with allele-specific PCR (AS-PCR) revealed that resistant plants contained an EPSPS gene with either the homozygous S/S-106 or the heterozygous P/S-106 alleles. All susceptible plants contained only the homozygous P/P-106 allele. In addition, DNA sequences of the full-length EPSPS gene from one susceptible (SB) and four resistant (R2, R6, R8 and R11) populations revealed an amino acid substitution of T102I in all the resistant plants, while another substitution of P381L was only found in resistant populations R6 and R11. The significance of the P381L mutation was examined by Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) and residue interaction network (RIN) analyses, which suggests the P381L mutation may contribute to resistance. Mutations at 102 and 106 occur widely in the EPSPS gene of glyphosate-resistant E. indica populations from Malaysia with the TIPS mutation. In addition, the P381L mutation could also contribute to resistance.

Enhancement of glyphosate tolerance in rice (Oryza sativa L.) through mutation induction in EPSPS (5-enolpyruvylshikimate-3-phosphate synthase)

Generation of an herbicide-tolerant cultivar could be an important goal in rice breeding program. Therefore, a direct mutagenesis approach (megaprimer technique) was employed to generate the EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) mutant version called epsps in which glycine 96 was replaced by alanine. Then both EPSPS and epsps genes were separately cloned into pET22b system following transformation of E. coli BL21. The transformed bacteria showed a higher expression level of EPSPS and epsps extending their survival in presence of high levels of glyphosate. Interestingly, the bacteria harboring pET22b:epsps could grow in the medium containing 18 mM glyphosate. Subsequently, Hashemi rice cultivar was used to generate two types of transgenic rice including plants harboring either EPSPS (35S:EPSPS) or epsps (35S:epsps). Molecular analysis was performed on T3 putative transgenic plants so as to confirm the presence and expression of transgenes. The results demonstrated that 35S:epsps is actively and stably transcribed in T3 transgenic plants and developed a considerable tolerance towards glyphosate in comparison with the 35S:EPSPS plants. When compared to non-transgenic plants, the epsps mutant plants accumulated higher levels of chlorophyll and carotenoid in association with less shikimic acid, which are considered as some indicators of glyphosate side effects. Moreover, flavonoid accumulation was accompanied by enhanced levels of alkaloids and lower levels of proline in 35S:epsps plants; indicating that shikimic acid pathway is well functioning and transgenic plants especially 35S:epsps experienced less stress. Therefore, for the first time, rice was successfully transformed by 35S:epsps resulting in generation of transgenic plants with a considerable level of glyphosate tolerance.

Cyhalofop-butyl and Glyphosate Multiple-Herbicide Resistance Evolved in an Eleusine indica Population Collected in Chinese Direct-Seeding Rice.

Eleusine indica is a typical xerophytic weed species with a cosmopolitan distribution. It is invasive and highly adaptable to diverse habitats and crops. Due to rice cropping-pattern changes, E indica has become one of the main dominant grass weeds infecting direct-seeding paddy fields. A Chinese E. indica population has evolved multiple-herbicide resistance to cyhalofop-butyl and glyphosate. In this study, the multiple-resistance profile of E. indica to these two different types of herbicides and their resistance mechanisms were investigated. Whole-plant dose-response assays indicated that the multiple-herbicide-resistant (MHR) population exhibited 10.8-fold resistance to cyhalofop-butyl and 3.1-fold resistance to glyphosate compared with the susceptible (S) population. ACCase sequencing revealed that the Asp-2078-Gly mutation was strongly associated with E. indica resistance to cyhalofop-butyl. The MHR plants accumulated less shikimic acid than S plants at 4, 6, and 8 days after glyphosate treatment. In addition, no amino acid substitution in the EPSPS gene was found in MHR plants. Further analysis revealed that the relative expression level of EPSPS in MHR plants was 6-10-fold higher than that in S plants following glyphosate treatment, indicating that EPSPS overexpression may contribute to the glyphosate resistance. Furthermore, the effectiveness of nine post-emergence herbicides against E. indica were evaluated, and one PPO inhibitor pyraclonil was identified as highly effective in controlling the S and MHR E. indica populations.

Glyphosate resistance in Eleusine indica: EPSPS overexpression and P106A mutation evolved in the same individuals

Goosegrass is one of the most widespread weeds in orchards and tea plantations in China, and glyphosate is a popular herbicide used to control it. However, high glyphosate selection pressure has led to some populations becoming resistant. The objectives of this research were to determine resistance levels and possible resistance mechanisms of goosegrass populations from several tea plantations in Zhejiang Province in China. The resistance indexes in four goosegrass populations (SH, SY, CA and CX) ranged from 4.9 to 13.4, and lower shikimate accumulation in these populations compared with a glyphosate-susceptible (GS) population confirmed their resistance to glyphosate. No mutations in the target gene EPSPS were found in populations SH and SY, however, the expression of EPSPS in these two populations was 9.3 and 29.7 times higher than that in the GS population, respectively. In the CX population, a P106S mutation in EPSPS was found in 6.7% of the individuals and another 80.0% of individuals had EPSPS amplification. In population CA, all the individuals had a P106A mutation and 86.7% of them had amplification in EPSPS. The EPSPS copy numbers ranged from 5.2 to 62.3 in these four resistant populations. There was a positive correlation between signal intensities of primary anti-EPSPS antibody and the copy number of the EPSPS protein, as indicated by immunoblot analysis. In population CA, with high-level resistance to glyphosate, both P106A mutation and amplification in EPSPS evolved in the same individuals in this population.

Production of EPSPS and bar gene double-herbicide resistant castor (Ricinus communis L.)

Castor (Ricinus communis L.) is one of the world's top ten oil crops. China is the second largest producer of castor beans, but China's castor bean acreage has been declining. A principal reason for this decline is the prevalence of weeds and the absence of herbicide resistant varieties of castor plants. In this study, we used Agrobacterium-mediated gene transfer to introduce the EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, which confers resistance to glyphosate, and the Bar (phosphinothricin N-acetyltransferase) gene, encoding resistance to glufosinate, into castor 2129 (high stem excellent recovery line) and CSR 181 (dwarf stem excellent recovery line). The transgenic plants exhibited significantly increased expression of the EPSPS and Bar genes and significantly increased resistance to glyphosate and glufosinate. Proteomic analysis showed differences in the amounts of 20 proteins in the dwarf stem transgenic plants. Nine of these proteins are involved in photosynthesis and indirectly related to the mechanism of action of the Bar gene. Eight proteins are involved in defense, repair, stress response, oxidative phosphorylation, and metabolic pathways and are indirectly related to the mechanism of action of the EPSPS gene. These results provide a foundation for the preparation of high herbicide resistant castor varieties.

EPSPS gene amplification confers resistance to glyphosate resistant populations of Hordeum glaucum Stued (northern barley grass) in South Australia.

Glyphosate failed to control two populations of Hordeum glaucum (northern barley grass) along a fence line and around stockyards near Arthurton on the Yorke Peninsula, South Australia after more than a decade of regular use of glyphosate. These were investigated to confirm resistance and to determine resistance mechanisms. Dose-response experiments confirmed resistance of these populations to glyphosate with resistance levels of 2.8-6.6-fold compared with two susceptible populations. Shikimate assays confirmed resistance to glyphosate with less shikimate accumulation in the resistant populations compared with the susceptible populations. Quantitative PCR of genomic DNA showed increased gene copy number in the resistant populations with 9-11-fold more copies of EPSPS compared with the susceptible populations, suggesting resistance is likely conferred by gene amplification. This study identified the first examples of glyphosate resistance in the grass species H. glaucum with resistance associated with EPSPS gene amplification. © 2019 Society of Chemical Industry.

Distribution Differences in the EPSPS Gene in Chromosomes Between Glyphosate-Resistant and -Susceptible Goosegrass (Eleusine indica)

Abstract Glyphosate is a popular herbicide used to control goosegrass [Eleusine indica (L.) Gaertn.], one of the most troublesome weeds in cotton fields. However, high selection pressure has led to some populations being difficult to control in cotton fields in China. In this study, levels of glyphosate resistance were quantified and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) amplification was confirmed. In addition, distribution of the EPSPS gene among the chromosomes was also investigated using fluorescence in situ hybridization (FISH) methodology. One population (AHCZ) was confirmed to be glyphosate resistant with a resistance index of 3.4 and significantly reduced shikimate accumulation compared to susceptible population. All examined individuals exhibited no mutations in the EPSPS gene in AHCZ. Expression and copy numbers of EPSPS in the AHCZ population were 5.7 and 15.4 times higher, respectively, than in the susceptible population. A positive correlation was identified between signal intensities of primary anti-EPSPS antibody and copy numbers of the EPSPS protein, as indicated by immunoblot analysis. FISH results revealed that, in mitotic metaphase chromosomes, signals were observed dispersed across two sets of homologous chromosomes in a resistant individual (copy number = 31), while in susceptible individuals, signals were only partially detected in one set of homologous chromosomes. In interphase nuclei, EPSPS signals were brightest in resistant individual compared to those that were susceptible. In conclusion, one E. indica population from a cotton field in the Anhui Province has evolved resistance to glyphosate, and EPSPS gene amplification was confirmed as the resistant mechanism.

The Draft Genome of Kochia scoparia and the Mechanism of Glyphosate Resistance via Transposon-Mediated EPSPS Tandem Gene Duplication.

Increased copy number of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene confers resistance to glyphosate, the world’s most-used herbicide. There are typically three to eight EPSPS copies arranged in tandem in glyphosate-resistant populations of the weed kochia (Kochia scoparia). Here, we report a draft genome assembly from a glyphosate-susceptible kochia individual. Additionally, we assembled the EPSPS locus from a glyphosate-resistant kochia plant by sequencing select bacterial artificial chromosomes from a kochia bacterial artificial chromosome library. Comparing the resistant and susceptible EPSPS locus allowed us to reconstruct the history of duplication in the structurally complex EPSPS locus and uncover the genes that are coduplicated with EPSPS, several of which have a corresponding change in transcription. The comparison between the susceptible and resistant assemblies revealed two dominant repeat types. Additionally, we discovered a mobile genetic element with a FHY3/FAR1-like gene predicted in its sequence that is associated with the duplicated EPSPS gene copies in the resistant line. We present a hypothetical model based on unequal crossing over that implicates this mobile element as responsible for the origin of the EPSPS gene duplication event and the evolution of herbicide resistance in this system. These findings add to our understanding of stress resistance evolution and provide an example of rapid resistance evolution to high levels of environmental stress.

A novel TIPT double mutation in EPSPS conferring glyphosate resistance in tetraploid Bidens subalternans.

Bidens subalternans (greater beggarticks) is a tetraploid and troublesome weed infesting annual crops in most tropical regions of the world. A glyphosate-resistant (GR) B. subalternans biotype was detected in a soybean field from Paraguay. A series of physiological and molecular analyses were conducted to elucidate its resistance mechanisms. The GR biotype had a high level of resistance (> 15-fold LD50 ), relative to a glyphosate-susceptible (GS) biotype. Shikimate accumulation was up to ten-fold greater for GS compared with GR. We found no differences in sensitivity when plants were treated and kept under lower (10/4 °C) or higher temperatures (25/20 °C). GS and GR had the same relative EPSPS gene copy number, and similar glyphosate absorption and translocation rates. Neither biotype metabolized glyphosate. A double amino acid substitution (TIPT - Thr102Ile and Pro106Thr) was found in only one EPSPS allele from one of the two EPSPS homoeologs present in tetraploid GR B. subalternans. This is the first report of a TIPT double mutation conferring high levels of glyphosate resistance in a weed species. The presence of both wild-type and TIPT mutant EPSPS on the polyploid genome of GR B. subalternans may offset a potential fitness cost, requiring additional research to confirm the absence of deleterious effects. © 2019 Society of Chemical Industry.

Overexpression of G10-EPSPS in soybean provides high glyphosate tolerance

Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2700 g a.e. ha−1 of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.

A necessity of introduction of the system of registration of GMO is for Ukraine

The article presents the main methodological approaches and criteria for the necessity of development and introduction of the State Registration of GMOs sources in Ukraine. The list of analytical researches that are to be carried out at the registration of the GMO source, expert evaluation of GMO identification methods, reproduction of detection methods, identification and quantification of GMOs are given. Submitted and analyzed legislative documents regulating the procedure for registration and control of GMO circulation in the countries of the European Union and Ukraine. DNDKI of veterinary preparations and fodder supplements on the tasks of the State Committee of Veterinary Medicine of Ukraine during 2010-2018 a number of normative documents were developed regarding procedures related to the use and circulation of feed, feed additives, premixes and veterinary preparations that are composed, containing or made using genetically modified sources in Ukraine: the procedure for state registration, the procedure for carrying out the state veterinary and sanitary examination of GMOs for sources of forage feed additives and veterinary preparations ativ plan state monitoring GMO sources of feed, feed additives, premixes for livestock and poultry. The procedure determines the procedure for state registration of GMOs for sources of feed, feed additives and veterinary preparations containing genetically modified organisms or obtained from their use. It is envisaged that the state register of GMO sources is conducted in the form approved by the State Service of Ukraine for Food Safety and Consumer Protection. On the basis of the analysis, a draft list of feeds, feed materials, protein-vitamin concentrates and premixes that have to be monitored for GMO content has been developed. This project list includes soy, corn, rape and products of their processing, fodder, feed mixes, feed concentrates, etc. In the SSRCI of veterinary drugs and supplements, in 2018, the “Guidelines for identifying the GM soya line (Soybean MON40-3-2) GTS 40-3-2” were developed and approved. “Methodological recommendations are intended to identify the GM soya line (Soybean MON40-3 -2) GTS 40-3-2 (Genetically modified soybean that contains – genetically modified soybean containing (cp4 epsps gene inserted to confer tolerance to herbicide glyphosate) cp4 epsps gene inserted to provide resistance to glyphosate herbicide (glyphosate) in plant material, feed and food products by polymerase chain reaction in real time (PCR-RF )”. The qualitative determination of GMOs is based on the discovery of genetically modified (GM) targeted regulatory sequences of the 35S Cauliflower Mosaic Virus (CaMV) promoter or FMV promoter and/or the NOS (T-NOS) T1 terminator of Agrobacterium tumefaciens plasmids that are introduced into the genetic constructs of GM based on the use of the multiplex polymerase chain reaction method to obtain real-time research results – PCR RF.

Evaluation of the effects of feeding glyphosate-tolerant soybeans (CP4 EPSPS) on the testis of male Sprague-Dawley rats

ABSTRACTGlyphosate tolerant soybeans represent a large portion of soybeans grown and fed to farm animals around the world. Despite their widespread use for many years, some have raised questions regarding their safety because the soybeans were genetically modified. The CP4 EPSPS gene which imparts resistance to topical application of the herbicide glyphosate was introduced into soybeans. Application of glyphosate to soybean fields will reduce weed pressure and increase soybean yield. To assess their safety on the rat reproduction system, male Sprague Dawley rats were fed either glyphosate-tolerant (GM) soybean (40-3-2) or near-isogenic, non-GM (A5403) (control) soybean meal. The processed soybean meal was added to formulated rodent diets at 20% (w/w) and fed to rats for 90 days. Some rats from the control group were separately administered mitomycin C for 40 days and served as positive controls in the sperm abnormality test. Body weights and behavior were monitored daily, serum enzymes and histologic and EM appearance of the testis, and sperm morphology were also examined. After 90 days of feeding, no adverse effects were observed in rats fed glyphosate-tolerant soybeans.

Molecular basis of natural tolerance to glyphosate in Convolvulus arvensis

Convolvulus arvensis is a troublesome weed that is naturally tolerant to glyphosate. This weed tolerates glyphosate at a rate 5.1 times higher than that of glyphosate-susceptible Calystegia hederacea. Glyphosate-treated C. arvensis plants accumulated less shikimic acid than C. hederacea plants. The overexpression of EPSPS genes from the two species in transgenic Arabidopsis thaliana resulted in similar glyphosate tolerance levels. qPCR of genomic DNA revealed that the EPSPS copy number in C. arvensis was approximately 2 times higher than that in C. hederacea. Moreover, glyphosate treatment caused a marked increase in EPSPS mRNA in C. arvensis compared to C. hederacea. GUS activity analysis showed that the promoter of CaEPSPS (CaEPSPS-P) highly improved GUS expression after glyphosate treatment, while no obvious differential GUS expression was observed in ChEPSPS-P transgenic A. thaliana in the presence or absence of glyphosate. Based on the obtained results, two coexisting mechanisms may explain the natural glyphosate tolerance in C. arvensis: (i) high EPSPS copy number and (ii) specific promoter-mediated overexpression of EPSPS after glyphosate treatment.

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.

Confirmation of herbicide resistance mutations Trp574Leu, ΔG210, and EPSPS gene amplification and control of multiple herbicide-resistant Palmer amaranth (Amaranthus palmeri) with chlorimuron-ethyl, fomesafen, and glyphosate.

Herbicide-resistant weeds, especially Palmer amaranth (Amaranthus palmeri S. Watson), are problematic in row-crop producing areas of the United States. The objectives of this study were to determine if chlorimuron-ethyl, fomesafen, and glyphosate applied separately and in mixtures control A. palmeri and confirm the presence of various genotypes surviving two- and three-way herbicide mixtures. Fifteen percent of A. palmeri treated with the three-way herbicide mixture survived. Mixing fomesafen with chlorimuron-ethyl or fomesafen with glyphosate to create a two-way mixture reduced A. palmeri survival 22 to 24% and 60 to 62% more than glyphosate and chlorimuron-ethyl alone, respectively. Previously characterized mutations associated with A. palmeri survival to chlorimuron-ethyl, fomesafen, and glyphosate Trp574Leu, a missing glycine codon at position 210 of the PPX2L gene (ΔG210), and 5-enolpyruvylshikimate-3-phosphase synthase (EPSPS) gene amplification; respectively, were present in surviving plants. However, 37% of plants treated with chlorimuron-ethyl did not contain heterozygous or homozygous alleles for the Trp574Leu mutation, suggesting alternative genotypes contributed to plant survival. All surviving A. palmeri treated with fomesafen or glyphosate possessed genotypes previously documented to confer resistance. Indiana soybean [Glycine max (L.) Merr] fields infested with A. palmeri possessed diverse genotypes and herbicide surviving plants are likely to produce seed and spread if alternative control measures are not implemented.

Effect on metabolome of the grains of transgenic rice containing insecticidal cry and glyphosate tolerance epsps genes

Genetically modified organisms (GMOs) need to be evaluated for safety before their release. Metabolome techniques provide effective methods to evaluate unintended effects. In this study, the grain metabolome of six transgenic rice lines containing secticidal cry and glyphosate tolerance epsps genes were tested by using an non-targeted metabolite profiling, and 161 and 138 metabolites were identified in grains at grain-filling stage and mature stage, respectively. The metabolic profiles of genetically modified (GM) lines and non-GM lines were all significantly different from each other at grain-filling stage. Although the levels of many metabolites were significantly changed in each transgenic line, only seven of them were simultaneously changed in all the six lines compared with those in non-GM lines at grain-filling stage. The number of significantly changed metabolites was much less at mature stage than that at grain-filling stage. Besides, none of these metabolites was simultaneously changed in all the six lines, suggesting that the metabolites of GMOs are different at different stages of GMO development. This study provides useful information about the metabolic variation between GMO and non-GMO in two development stages of rice grains. These findings suggest that non-targeted metabolite profiling can strengthen the assessment of risk based on metabolites.

First Report of Kochia (Bassia scoparia) with Cross-Resistance to Dicamba and Fluroxypyr in Western Kansas

AbstractEvolution and rapid spread of herbicide-resistant (HR) kochia has become a significant challenge for growers in the U.S. Great Plains. The main objectives of this research were to confirm and characterize the response of putative auxinic HR (Aux-HR) kochia accessions (designated as KS-4A, KS-4D, KS-4H, KS-10A, KS-10-G, and KS-10H) collected from two different corn fields near Garden City, KS, to dicamba and fluroxypyr and to determine the EPSPS gene copy number to detect whether those accessions were also resistant to glyphosate. Single-dose experiments indicated that putative Aux-HR kochia accessions had 78% to 100% and 85% to 100% survivors when treated with dicamba (560 g ae ha−1) and fluroxypyr (235 g ae ha−1), respectively. Whole-plant dicamba dose–response studies revealed that the selected Aux-HR accessions had 2.9- to 15.1- and 3.1- to 9.4-fold resistance to dicamba relative to two susceptible accessions (MT-SUS and KS-SUS). In a separate fluroxypyr dose–response experiment, the selected Aux-HR accessions also exhibited 3.8- to 7.3- and 3.0- to 8.6-fold resistance to fluroxypyr on the basis of shoot fresh and dry weight responses, respectively. The confirmed Aux-HR kochia accessions also had 3 to 13 EPSPS gene copies relative to MT-SUS and KS-SUS accessions (each with 1 EPSPS gene copy). These results suggest that the putative Aux-HR kochia accessions from Kansas had developed moderate to high levels of cross-resistance to dicamba and fluroxypyr and low to high levels of resistance to glyphosate. This is the first confirmation of kochia accessions with cross-resistance to dicamba and fluroxypyr in Kansas. Growers should use diverse kochia control programs, including the proper use of dicamba and fluroxypyr stewardship, use of cover crops, occasional tillage, diversified crop rotations, and alternative effective herbicides to prevent further evolution and spread of Aux-HR kochia on their fields.

Reduced Absorption and Impaired Translocation Endows Glyphosate Resistance in Amaranthus palmeri Harvested in Glyphosate-Resistant Soybean from Argentina.

Amaranthus palmeri S. Watson is probably the worst glyphosate-resistant (GR) weed worldwide. The EPSPS (5-enolpyruvylshikimate-3-phosphate-synthase) gene amplification has been reported as the major target-site-resistance (TSR) mechanism conferring resistance to glyphosate in this species. In this study, TSR and non-target-site-resistance (NTSR) mechanisms to glyphosate were characterized in a putative resistant A. palmeri population (GRP), harvested in a GR soybean crop from Argentina. Glyphosate resistance was confirmed for the GRP population by dose-response assays. No evidence of TSR mechanisms, as well as glyphosate metabolism, was found in this population. Moreover, a susceptible population (GSP) that absorbed about 10% more herbicide than the GRP population was evaluated at different periods after treatment. The GSP population translocated about 20% more glyphosate to the remainder of the shoots and roots at 96 h after treatment than the control, while the GRP population retained 62% of herbicide in the treated leaves. This is the first case of glyphosate resistance in A. palmeri involving exclusively NTSR mechanisms.