Academic literature on the topic 'EPSPS target-site mutation'

AbstractThe evolution of resistance to multiple herbicides in Palmer amaranth is a major challenge for its management. In this study, a Palmer amaranth population from Hutchinson, Kansas (HMR), was characterized for resistance to inhibitors of photosystem II (PSII) (e.g., atrazine), acetolactate synthase (ALS) (e.g., chlorsulfuron), and EPSP synthase (EPSPS) (e.g., glyphosate), and this resistance was investigated. About 100 HMR plants were treated with field-recommended doses (1×) of atrazine, chlorsulfuron, and glyphosate, separately along with Hutchinson multiple-herbicide (atrazine, chlorsulfuron, and glyphosate)–susceptible (HMS) Palmer amaranth as control. The mechanism of resistance to these herbicides was investigated by sequencing or amplifying the psbA, ALS, and EPSPS genes, the molecular targets of atrazine, chlorsulfuron, and glyphosate, respectively. Fifty-two percent of plants survived a 1× (2,240 g ai ha−1) atrazine application with no known psbA gene mutation, indicating the predominance of a non–target site resistance mechanism to this herbicide. Forty-two percent of plants survived a 1× (18 g ai ha−1) dose of chlorsulfuron with proline197serine, proline197threonine, proline197alanine, and proline197asparagine, or tryptophan574leucine mutations in the ALS gene. About 40% of the plants survived a 1× (840 g ae ha−1) dose of glyphosate with no known mutations in the EPSPS gene. Quantitative PCR results revealed increased EPSPS copy number (50 to 140) as the mechanism of glyphosate resistance in the survivors. The most important finding of this study was the evolution of resistance to at least two sites of action (SOAs) (~50% of plants) and to all three herbicides due to target site as well as non–target site mechanisms. The high incidence of individual plants with resistance to multiple SOAs poses a challenge for effective management of this weed.

The Dongting and Poyang Lakes are the important rice growing areas, and the Bohai Rim and Loess Plateau are the main producing areas of apples in China, where glyphosate has been used continuously to control weeds including Eleusine. indica for many years. In this study, the resistance levels and target-site based resistance (TSR) mechanisms to glyphosate in E. indica populations, which were collected from above areas were investigated. A total of 35 out of 50 (70%) E. indica populations have evolved resistance to glyphosate with resistance index (RI) of 2.01~10.43. The glyphosate-resistant (GR) E. indica accumulated less shikimic acid than glyphosate-susceptible (GS) populations, when treated by 1.0 mg/L, 10 mg/L or 100 mg/L glyphosate. There was no mutation at Thr102 and Pro106 in 5-enolpyruvate shikimate-3-phosphate synthase (EPSPS), which endowed glyphosate resistance in E. indica and other weed species. A Pro-381-Leu was found in EPSPS in GR populations. In contrast, the expression level of EPSPS gene was highly correlated with glyphosate resistance in E. indica with a Pearson coefficient of 0.73. These indicate that the glyphosate resistance in aforementioned E. indica populations was mainly caused by the overexpression of EPSPS, not by amino acid mutation in EPSPS.

Sweet summer grass is a problematic weed in the central Queensland region of Australia. This study found glyphosate resistance in two biotypes (R1 and R2) of sweet summer grass. The level of resistance in these biotypes was greater than 8-fold. The glyphosate dose required to reduce dry matter by 50% (GR50) for the resistant populations varied from 1993 to 2100 g ha−1. A novel glyphosate resistance double point mutation in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene was identified for the first time in sweet summer grass. Multiple mutations, including multiple amino acid changes at the glyphosate target site, as well as mutations involving two nucleotide changes at a single amino acid codon, were observed. Both resistant biotypes exhibited a nucleotide change of CAA to ACA in codon 106, which predicts an amino acid change of proline to a threonine (Pro-106-Thr). In addition, the R1 biotype also possessed a mutation at codon 100, where a nucleotide substitution of T for G occurred (GCT to TCT), resulting in a substitution of serine for alanine (Ala-100-Ser). Understanding the molecular mechanism of glyphosate resistance will help to design effective management strategies to control invasive weeds.

Glyphosate (N-phosphonomethyl-glycine) is the world’s most widely used broad spectrum, post-emergence herbicide. It inhibits the chloroplast-targeted enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), a component of the plant and microorganism-specific shikimate pathway and a key catalyst in the production of aromatic amino acids. Variants of EPSPS that are not inhibited by glyphosate due to particular amino acid alterations in the active site of the enzyme are known. Some of these variants have been identified in weed species that have developed resistance to glyphosate because of the strong selective pressure of continuous, heavy glyphosate use. We have used TILLING (Targeting Induced Local Lesions in Genomes), a non-transgenic, target-selected, reverse genetics, mutation breeding technique, and conventional genetic crosses, to identify and combine, through two rounds of mutagenesis, wheat lines having both T102I and P106S (so-called TIPS enzyme) mutations in both the A and the D sub-genome homoeologous copies of the wheat EPSPS gene. The combined effects of the T102I and P106S mutations are known from previous work in multiple species to minimize the binding of the herbicide while maintaining the affinity of the catalytic site for its native substrates. These novel wheat lines exhibit substantial tolerance to commercially relevant levels of glyphosate.

Few studies on herbicide resistance report data to establish unambiguously the correlation between genotype and phenotype. Here we report on the importance of the EPSPS prolyl106point mutation to serine (P106S) in conferring resistance to glyphosate in a goosegrass population from Davao, Mindanao Island, the Philippines (Davao). Initial rate-response studies showed clear survivors within the Davao population at glyphosate rates that completely controlled the standard sensitive goosegrass population (STD1). Assessment of potential resistance mechanisms identified the presence of P106S mutant individuals in the Davao population. Polymerase chain reaction (PCR) amplification of specific alleles (PASA) analysis established that the mixed-resistant Davao population was comprised of 39.1% homozygous proline wild-type (PP106), 3.3% heterozygous serine mutant (PS106), and 57.6% homozygous serine mutant (SS106) genotypes. Further rate-response studies on plants with a predetermined genotype estimated the Davao SS106 individuals to be approximately 2-fold more resistant to glyphosate compared to Davao PP106 individuals. Extensive analysis at different goosegrass growth stages and glyphosate rates established strong correlation (P < 0.001) between presence of P106S in EPSPS and the resistant phenotype. Importantly, no differences in the pattern of absorbed or translocated14C–glyphosate were observed between PP106 and SS106 Davao genotypes or Davao and STD1 individuals, suggesting that glyphosate resistance in the Davao population was attributable to an altered target site mechanism. This study demonstrates that whilst P106S in EPSPS confers a moderate resistance level to glyphosate, the mechanism is sufficient to endow glyphosate failure at the recommended field rates.

Waterhemp is an increasingly problematic weed in the U.S. Midwest, having now evolved resistances to herbicides from six different site-of-action groups. Glyphosate-resistant waterhemp in the Midwest is especially concerning given the economic importance of glyphosate in corn and soybean production. Amplification of the target-site gene, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was found to be the mechanism of glyphosate resistance in Palmer amaranth, a species closely related to waterhemp. Here, the relationship between glyphosate resistance and EPSPS gene amplification in waterhemp was investigated. Glyphosate dose response studies were performed at field sites with glyphosate-resistant waterhemp in Illinois, Kansas, Kentucky, Missouri, and Nebraska, and relative EPSPS copy number of survivors was determined via quantitative real-time polymerase chain reaction (qPCR). Waterhemp control increased with increasing glyphosate rate at all locations, but no population was completely controlled even at the highest rate (3,360 g ae ha−1). EPSPS gene amplification was present in plants from four of five locations (Illinois, Kansas, Missouri, and Nebraska) and the proportion of plants with elevated copy number was generally higher in survivors from glyphosate-treated plots than in plants from the untreated control plots. Copy number magnitude varied by site, but an overall trend of increasing copy number with increasing rate was observed in populations with gene amplification, suggesting that waterhemp plants with more EPSPS copies are more resistant. Survivors from the Kentucky population did not have elevated EPSPS copy number. Instead, resistance in this population was attributed to the EPSPS Pro106Ser mutation. Results herein show a quantitative relationship between glyphosate resistance and EPSPS gene amplification in some waterhemp populations, while highlighting that other mechanisms also confer glyphosate resistance in waterhemp.

The mechanism and expression of resistance to glyphosate at different plant growing temperatures was investigated in an Amaranthus palmeri population (VM1) from a soybean field in Vicuña Mackenna, Cordoba, Argentina. Resistance was not due to reduced glyphosate translocation to the meristem or to EPSPS duplication, as reported for most US samples. In contrast, a proline 106 to serine target-site mutation acting additively with EPSPS over-expression (1.8-fold increase) was respectively a major and minor contributor to glyphosate resistance in VM1. Resistance indices based on LD50 values generated using progenies from a cross between 52 PS106 VM1 individuals were estimated at 7.1 for homozygous SS106 and 4.3 for heterozygous PS106 compared with homozygous wild PP106 plants grown at a medium temperature of 24 °C day/18 °C night. A larger proportion of wild and mutant progenies survived a single commonly employed glyphosate rate when maintained at 30 °C day/26 °C night compared with 20 °C day/16 night in a subsequent experiment. Interestingly, the P106S mutation was not identified in any of the 920 plants analysed from 115 US populations, thereby potentially reflecting the difference in A. palmeri control practices in Argentina and USA.

Six Conyza bonariensis (L.) Cronquist populations were screened in a pot experiment at the University of Pretoria’s Hatfield experimental farm to evaluate and confirm the degree of glyphosate response. Resistance factors ranged from 2.7- to 24.8-fold compared to the most susceptible biotype. Partial sequencing of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene found no mutation at the Thr102, Ala103 or Pro106 positions. EPSPS mRNA expression levels in glyphosate-resistant biotypes (Swellendam and Piketberg seed sampling sites) were comparable or lower than those in susceptible biotypes (George and Fauresmith sites). Additionally, the highest expression level was reported in the susceptible Fauresmith biotype. These results indicate that glyphosate resistance in the tested resistant biotypes is not caused by target-site mutations and EPSPS gene amplification. Leaf surface characteristics can influence the spread and subsequent absorption of glyphosate. The study established non-significant results in the amount of leaf wax and insufficient mean separations in cuticle thickness and trichome density data. Therefore, the observed differences in response of biotypes to glyphosate treatment could not be attributed conclusively to differences in the leaf morphological characteristics investigated. Results from the inheritance study were consistent with glyphosate resistance being inherited in an incompletely dominant manner when plants were treated with glyphosate herbicide at 900 g ae ha−1.

Background: The spread of herbicide-resistance Ambrosia artemisiifolia threatens not only the production of agricultural crops, but also the composition of weed communities. The reduction of their spread would positively affect the biodiversity and beneficial weed communities in the arable habitats. Detection of resistant populations would help to reduce herbicide exposure which may contribute to the development of sustainable agroecosystems. Methods: This study focuses on the application of target-site resistance (TSR) diagnostic of A. artemisiifolia caused by different herbicides. We used targeted amplicon sequencing (TAS) on Illumina Miseq platform to detect amino acid changes in herbicide target enzymes of resistant and wild-type plants. Results: 16 mutation points of four enzymes targeted by four herbicide groups, such as Photosystem II (PSII), Acetohydroxyacid synthase (AHAS), 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) and protoporphyrinogen IX oxidase (PPO) inhibitors have been identified in common ragweed populations, so far. All the 16 mutation points were analyzed and identified. Out of these, two mutations were detected in resistant biotypes. Conclusions: The applied next-generation sequencing-targeted amplicon sequencing (NGS-TAS) method on A. artemisiifolia resistant and wild-type populations enable TSR detection of large sample numbers in a single reaction. The NGS-TAS provides information about the evolved herbicide resistance that supports the integrated weed control through the reduction of herbicide exposure which may preserve ecological properties in agroecosystems.

A suspected glyphosate-resistant (R) junglerice population was collected from a glyphosate-R corn field near Durham in northern California where glyphosate had been applied at least twice a year for over 6 yr. Based on the amount of glyphosate required to reduce growth by 50% (ED50), the R population was 6.6 times more R than the susceptible (S) standard population. Based on the glyphosate concentration that inhibits EPSPS by 50% based on shikimate accumulation (I50) in leaf discs, R plants were four times more R than S plants. By 3 d after treatment with 0.42 kg ae ha−1glyphosate, the S population had accumulated approximately five times more shikimate than the R population. No differences in [14C]-glyphosate uptake and translocation were detected between R and S plants. However, partial sequencing of theEPSPSgene revealed a mutation in R plants causing a proline to serine change at EPSPS position 106 (P106S). Our results reveal the first case of a P106S target site mutation associated with glyphosate resistance in junglerice.

A planta daninha Amaranthus palmeri é nativa dos Estados Unidos, porém foi pela primeira vez relatada no Brasil no ano de 2015. Embora comprovadamente com resistência múltipla aos herbicidas inibidores da ALS e da EPSPS, até o momento não foram investigadas as bases moleculares da resistência. Além disso, por causa da recente introdução da planta daninha no país, alternativas de manejo com culturas tolerantes a herbicidas necessitam ser estudadas. Sendo assim, os objetivos desse trabalho são de caracterizar a espécie de planta daninha introduzida no país, identificar os mecanismos de resistência aos herbicidas inibidores da ALS e da EPSPS presentes no biótipo, e propor abordagens de manejo em ambientes dos novos eventos transgênicos resistentes a herbicidas. Um bioensaio utilizando marcadores genéticos foi desenvolvido para confirmar que a população coletada no estado do Mato Grosso (BR-R) é A. palmeri, e não A. tuberculatus, outra espécie dióica do gênero Amaranthus. Os resultados de experimentos de curvas de dose-resposta e acúmulo de chiquimato indicaram que a BR-R possui alto nível de resistência, com DL50 de 4.426 e 3.400 g glyphosate ha-1 no primeiro e segundo experimento, respectivamente, mais que o dobro da dose típicamente recomendada para o controle da espécie e, adicionalmente, observou se acúmulo mínimo de chiquimato a concentração de 1 mM nos tecidos das plantas tratadas com o herbicida. BR-R também foi resistente a herbicidas dos grupos químicos das sulfoniluréias e imidazolinonas. O mecanismo de resistência ao glyphosate encontrado nesta população foi a super expressão gência, através do aumento no número de cópias do gene da EPSPS no genoma da planta BR-R, entre 50 e 179 cópias adicionais. Além disso, duas substituições de aminoácidos foram observadas na sequência da ALS, W574L e S653N, conferindo resistência tanto a sulfoniluréias quanto a imidazolinonas. No experimento utilizandos os herbicidas correspondentes às culturas geneticamente modificadas com novos traits de tolerância a herbicidas observou se, de uma forma geral, que as associações de herbicidas apresentaram níveis de controle mais satisfatórios. Assim, esta pesquisa confirma a introdução de da espécie A. palmeri no Brasil, assim como a resistência múltipla aos herbicidas inibidores da EPSPS e da ALS. Seu manejo é mais eficaz através da associação de herbicidas, garantindo assim o uso racional das novas tecnologias de culturas geneticamente modificadas com tolerância a herbicidas.
Palmer Amaranth (Amaranthus palmeri) is a weed species native to the United States, but it was reported in Brazil for the first time in 2015. Despite this population being resistant to EPSPS and ALS inhibitors, the molecular basis of its multiple resistance is unknown up to date. Because of this species introduction to Brazil, alternatives of management with the new herbicide-tolerant crops technologies need to be studied. The objectives of this research are to characterize the weed species introduced to Brazil, identify the mechanisms conferring resistance to ALS and EPSPS inhibitors herbicides, and to propose management approaches in environments with the new genetically modified herbicide-tolerant crops. A genotyping bioassay using genetic markers was developed to confirm that the species collected in the state of Mato Grosso (BR-R) is indeed A. palmeri and not A. tuberculatus, another dioceous species in the Amaranthus genus. Dose-response experiments and shikimate accumulation bioassay data indicate high level of resistance, with LD50 of 4,426 and 3,400 g glyphosate ha-1 in the first and second experiments, respectively, higher than the double rate tipically recommended to control it, and minimal accumulation in BR-R with 1 mM of glyphosate in treated plants in the leaf disks assay. BR-R also was resistanto to sulfonilurea and imidazolinone herbicides. The mechanism conferring resistance to glyphosate identified in this population was gene amplification, with increased EPSPS copy number - between 50 and 179 more copies in BR-R. Besides, two target-site mutations were identified in the ALS gene sequencing, W574L and S653N, conferring resistance to sulfonilureas and imidazolinones. The weed control experiment, overal, herbicide tank mixtures achieved higher levels of control. Therefore, this research confirms the introduction of A. palmeri to Brazil, as well as its multiple resistance to EPSPS and ALS inhibitor herbicides. Its control is more efficient with herbicide mixtures, which guarantees more susteinable use of the new herbicide-tolerant crop technologies.

Chloris truncata and C. virgata, which are major weeds in cotton and grain crops in the sub-tropical region of Australia, have recently emerged as potential weeds of the future in southern Australia. Glyphosate, an inhibitor of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS), is the most widely used non-selective post-emergence herbicide globally. As a result of over-reliance on glyphosate combined with dominance of reduced tillage systems for weed control, glyphosate-resistant populations of C. truncata have already been reported in Australia. C. virgata is also considered hard to kill with glyphosate, but resistance has not been reported so far in the literature. Studies on growth, development and seed biology of C. truncata and C. virgata were conducted to better understand the biology of these emerging weed species. Under field conditions, C. truncata and C. virgata required 748-786 degree-days (Cd) and 1200 Cd respectively to progress from emergence to mature seed production. Freshly produced seeds of C. virgata were dormant for about 2 months, whereas 16-40% of seeds of C. truncata germinated within a week after maturation. Seed dormancy of C. virgata was released by the pre-treatment with 564 mM NaClO for 30 minutes. Exposure to light significantly increased germination of C. truncata seed from 0-2% in the dark to 77-84% in the light, and of C. virgata seed from 2-35% in the dark to 72- 85% in the light. Seeds of these two species could germinate over a wide temperature range (10-40oC), with maximum germination at 20-25oC for C. truncata and 15-25oC for C. virgata. The predicted base temperature for germination was 9.2-11.2oC for C. truncata and much lower 2.1-3.0oC for C. virgata. Seedling emergence of C. virgata (76% for seeds present on soil surface) was significantly reduced by burial at 1 (57%), 2 (49%) and 5 cm (9%), whereas seedling emergence of C. truncata was completely inhibited by burial of seed even at a shallow depth (0.5 cm). Under field conditions, both C. truncata and C. virgata seeds persisted in the soil for at least 11 months and seasons with below-average spring-summer rainfall increased seed persistence. Detailed studies were undertaken to identify glyphosate-resistant populations and to understand the mechanism of glyphosate resistance in C. truncata and C. virgata. Glyphosate resistance (GR) was confirmed in five populations of C. truncata and four populations of C. virgata. GR plants were 2.4 to 8.7-fold (C. truncata) and 2 to 9.7-fold (C. virgata) more resistant and accumulated less shikimate after glyphosate treatment than susceptible (S) plants. The differences in shikimate accumulation indicated that glyphosate did reach the target site but inhibited the EPSPS enzyme of each population differently. Glyphosate absorption and translocation did not differ between GR and S plants of either C. truncata or C. virgata. Two target-site EPSPS mutations (Pro-106-Leu and Pro-106-Ser) were likely to be the primary mechanism of glyphosate resistance in C. virgata but no previously known target-site mutations were identified in C. truncata. The C. virgata population with Pro-106-Leu substitution was 2.9 to 4.9-fold more resistant than those with Pro-106-Ser substitution. The primary mechanism of resistance to glyphosate in C. truncata was a combination of target-site EPSPS mutation (Glu-91-Ala) and amplification of the EPSPS gene. There were 16 to 48-fold more copies of the EPSPS gene in GR plants compared to S plants, with the number of EPSPS copies found to be variable both between and within populations.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food & Wine, 2017