RESUMO
Italian ryegrass (Lolium multiflorum L.) is an invasive species widely spread in croplands worldwide. The intensive use of glyphosate has resulted in the selection of resistance to this herbicide in Italian ryegrass. This work characterized the response to glyphosate of Italian ryegrass populations from the South and Southwest regions of Paraná, Brazil. A total of 44 Italian ryegrass populations were collected in farming areas, and were classified for glyphosate resistance with 75% of populations resistant to gloyphosate. Of these, 3 resistant (VT05AR, MR20AR and RN01AR) and three susceptible (VT07AS, MR05AS and RN01AS) of these populations were selected to determine the resistance level and the involvement of the target site mechanisms for glyphosate resistance. Susceptible populations GR50 ranged from 165.66 to 218.17 g.e.a. ha-1 and resistant populations from 569.37 to 925.94, providing RI ranging from 2.88 and 4.70. No mutation in EPSPS was observed in the populations, however, in two (MR20AR and RN02AR) of the three resistant populations, an increase in the number of copies of the EPSPs gene (11 to 57×) was detected. The number of copies showed a positive correlation with the gene expression (R2 = 0.86) and with the GR50 of the populations (R2 = 0.81). The increase in EPSPS gene copies contributes to glyphosate resistance in Italian ryegrass populations from Brazil.
Assuntos
Herbicidas , Lolium , Glifosato , Lolium/genética , Lolium/metabolismo , Glicina/farmacologia , Glicina/metabolismo , Brasil , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Herbicidas/metabolismo , 3-Fosfoshikimato 1-Carboxiviniltransferase/genéticaRESUMO
The characterization of the mechanisms conferring resistance to herbicides in weeds is essential for developing effective management programs. This study was focused on characterizing the resistance level and the main mechanisms that confer resistance to glyphosate in a resistant (R) Steinchisma laxum population collected in a Colombian rice field in 2020. The R population exhibited 11.2 times higher resistance compared to a susceptible (S) population. Non-target site resistance (NTSR) mechanisms that reduced absorption and impaired translocation and glyphosate metabolism were not involved in the resistance to glyphosate in the R population. Evaluating the target site resistance mechanisms by means of enzymatic activity assays and EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene sequencing, the mutation Pro106Ser was found in R plants of S. laxum. These findings are crucial for managing the spread of S. laxum resistance in Colombia. To effectively control S. laxum in the future, it is imperative that farmers use herbicides with different mechanisms of action in addition to glyphosate and adopt Integrate Management Programs to control weeds in rice fields of the central valleys of Colombia.
RESUMO
While some genetically modified (GM) plants have been targeted to confer tolerance to abiotic stressors, transgenes are impacted by abiotic stressors, causing adverse effects on plant physiology and yield. However, routine safety analyses do not assess the response of GM plants under different environmental stress conditions. In the context of climate change, the combination of abiotic stressors is a reality in agroecosystems. Therefore, the aim of this study was to analyze the metabolic cost by assessing the proteomic profiles of GM soybean varieties under glyphosate spraying and water deficit conditions compared to their non-transgenic conventional counterparts. We found evidence of cumulative adverse effects that resulted in the reduction of enzymes involved in carbohydrate metabolism, along with the expression of amino acids and nitrogen metabolic enzymes. Ribosomal metabolism was significantly enriched, particularly the protein families associated with ribosomal complexes L5 and L18. The interaction network map showed that the affected module representing the ribosome pathway interacts strongly with other important proteins, such as the chloro-plastic gamma ATP synthase subunit. Combined, these findings provide clear evidence for increasing the metabolic costs of GM soybean plants in response to the accumulation of stress factors. First, alterations in the ribosome pathway indicate that the GM plant itself carries a metabolic burden associated with the biosynthesis of proteins as effects of genetic transformation. GM plants also showed an imbalance in energy demand and production under controlled conditions, which was increased under drought conditions. Identifying the consequences of altered metabolism related to the interaction between plant transgene stress responses allows us to understand the possible effects on the ecology and evolution of plants in the medium and long term and the potential interactions with other organisms when these organisms are released in the environment.
RESUMO
At present, appearance of herbicide resistant weeds is not new because repeated herbicide treatments per agricultural year/cycle are usual in both perennial and annual crops worldwide. Characterizing resistance mechanisms implied in each herbicide resistant weed is the best tool and the basis to develop integrated weed management (IWM) strategies. The main resistance mechanisms which confer low sensibility to glyphosate in a previously confirmed glyphosate-resistant Chloris radiata population (ChrR), occurring in Colombian rice fields, were characterized. Pure line selection by clone plants showed high resistance levels in ChrR. Comparing with GR50 and LD50 values, ChrR was 9.6 and 10.8 times more resistant with respect to a representative susceptible population (ChrS). The nontarget site mechanisms reduced glyphosate absorption and translocation did not contribute to the glyphosate resistance of the ChrR population. However, enzyme activity assays and DNA sequencing demonstrated that at least one target-site resistance mechanism is involved in such resistance. All ten ChrR plants tested had the amino acid substitution Pro-106-Ser. The results may be crucial to decrease the resistance distribution of C. radiata in Colombia by implementing IWM programs. The change in weed control strategies in rice fields from Colombia must include herbicides with different mode of action from glyphosate and non chemical methods to preserve the useful life of glyphosate longer for weed control in the country.
Assuntos
Herbicidas , Oryza , Colômbia , Glicina/análogos & derivados , Glicina/toxicidade , Resistência a Herbicidas/genética , Herbicidas/toxicidade , Oryza/genética , GlifosatoRESUMO
Bromus catharticus Vahl. has been used as a valuable forage crop, but it has also been noted as a weed of winter crops and an invader in several countries. In Argentina, a putative glyphosate-resistant population of B. catharticus was identified as a consequence of the lack of effective control with glyphosate in the pre-sowing of wheat. Plant survival and shikimate accumulation analysis demonstrated a lower glyphosate-sensitivity of this population in comparison to a susceptible B. catharticus population. The resistant population was 4-fold more resistant to glyphosate than its susceptible counterpart. There was no evidence of target-site mechanisms of glyphosate resistance or an enhanced capacity to metabolize glyphosate in the resistant population. However, the resistant plants showed a lower foliar retention of glyphosate (138.34 µl solution g-1 dry weight vs. 390.79 µl solution g-1 dry weight), a reduced absorption of 14C-glyphosate (54.18 vs. 73.56%) and lower translocation of 14C-glyphosate from the labeled leaf (27.70 vs. 62.36%). As a result, susceptible plants accumulated a 4.1-fold higher concentration of 14C-glyphosate in the roots compared to resistant plants. The current work describes the first worldwide case of glyphosate resistance in B. catharticus. A reduced foliar retention of herbicide, a differential rate of glyphosate entry into leaves and an altered glyphosate translocation pattern would be the most likely mechanisms of glyphosate exclusion.
RESUMO
Glyphosate can generate positive effects on turfgrass maintenance as a form of growth control by decreasing the expenses associated with mowing. However, there is little information about the effects of this herbicide on turfgrasses. This study aimed to evaluate the response of bermudagrass and zoysiagrass to the herbicide glyphosate as a growth regulator. Two studies were performed in a greenhouse and repeated at different times. The treatments involved application of glyphosate at 10 different rates (0, 5.625, 11.25, 22.5, 45, 90, 180, 360, 720, and 1.440 g ae ha-1) with four replicates. Evaluations of green cover by digital analysis, injury, and plant height were performed at 7, 14, 21, and 28 days after application, and shoot dry matter of clippings was determined for the last evaluation period. Bermudagrass and zoysiagrass presented variedtolerance to glyphosate toxicity. Overall, the digital analysis showed that green content was negatively influenced by the increase in visual injury caused by glyphosate application. Moreover, increasing the glyphosate rate decreased plant height and shoot dry matter in both turfgrasses. Glyphosate application rates up to 45 g ae ha-1 for bermudagrass and 90 g ae ha-1 for zoysiagrass decreased plant growth without affecting the factors analyzed in this study.
Assuntos
Glicina/análogos & derivados , Reguladores de Crescimento de Plantas/farmacologia , Poaceae/efeitos dos fármacos , Poaceae/crescimento & desenvolvimento , Brasil , Cynodon/efeitos dos fármacos , Cynodon/crescimento & desenvolvimento , Glicina/farmacologia , Glicina/toxicidade , Herbicidas/farmacologia , Herbicidas/toxicidade , Reguladores de Crescimento de Plantas/toxicidade , Folhas de Planta/efeitos dos fármacos , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/crescimento & desenvolvimento , Especificidade da Espécie , GlifosatoRESUMO
Shikimic acid (SA) has witnessed a strong increase in recent years due to the increasing demand of the pharmaceutical and cosmetic industry. The SA is used as a precursor for the synthesis of oseltamivir phosphate (Tamiflu®), a potent viral inhibitor and is extracted from the plant Illicium verum Hook which has a limited availability. This article proposed the use of Urochloa plantaginea (Link.) webster and glyphosate, as an alternative source of SA. U. plantaginea plants with 3 - 4 tillers and 4 - 6 leaves were harvest at 3, 6, 9 and 12 days after application (DAT) of low rates of glyphosate. Samples were dried, extracted, analyzed by HPLC and LC-MS/MS. The maximum SA concentrations were observed at 6 days after glyphosate at 36 g.a.e.ha-1 was applied in plants of U. plantaginea with 4 to 6 leaves. The capability of this annual gramineae to produce elevated SA levels throughout the entire biomass affords its potential for a greater yield on a per hectare basis.(AU)
O interesse pelo ácido chiquímico (SA) tem apresentado um forte incremento nos últimos anos devido à crescente demanda da indústria farmacêutica e cosmética. O SA é utilizado como um precursor para a síntese do fosfato de oseltamivir (Tamiflu®), um potente inibidor viral. Este ácido é extraído principalmente da planta Illicium verum Hook. A disponibilidade desta planta é um fator limitante para o crescimento do mercado no futuro próximo. Este artigo propõe Urochloa plantaginea (Link.) webster tratada com sub doses de glifosato, como uma fonte alternativa de SA. Plantas de U. plantaginea com 3 - 4 perfilhos e 4 a 6 folhas foram tratadas com subdoses de glifosato e coletadas aos 0, 3, 6, 9 e 12 dias após sua aplicação (DAT). As amostras foram secas, extraídas e analisadas por HPLC e confirmadas por LC-MS/MS. As concentrações máximas de SA foram observadas aos seis dias após aplicação do glifosato a 36 g.e.a.ha-1 em plantas de U. plantaginea com 4 - 6 folhas. A capacidade anual dessa gramínea para produzir níveis elevados de SA em toda a biomassa, pode ser uma fonte economicamente viável de SA.(AU)
Assuntos
Ácido Chiquímico , Herbicidas/administração & dosagemRESUMO
With the advent of glyphosate-resistant soybean, postemergence applications of the herbicide have become routine. In this way, problems related to the selectivity of glyphosate have been shown to be increasingly intense, due to the use of increased dosages and applications of the herbicide at unrecommended moments. In order to evaluate the agronomic performance of soybean plants subjected to the postemergence application of glyphosate both in isolation and together with Crop+ at different stages and dosage levels, a randomized block design experiment was carried out with two adjacent checks and four repetitions. The treatments consisted of: glyphosate (1296 g·ha-1); glyphosate + Crop+ (1296 + 0.25 g or L·ha-1); glyphosate + Crop+ (1296 + 0.5 g or L·ha-1); glyphosate (2592 g·ha-1); glyphosate + Crop+ (2592 + 0.25 g or L·ha-1); glyphosate + Crop+ (2592 + 0.5 g or L·ha-1); 2 × glyphosate (2 × 1296 g·ha-1); 2 × glyphosate + Crop+ (2 × 1296 + 0.25 g or L·ha-1); 2 × glyphosate + Crop+ (2 × 1296 + 0.5 g or L·ha-1) and Crop+ (0.5 g·ha-1). The phytotoxicity, soil plant analysis development (SPAD) index, height, stand, mass of 100 grains, and yield of the soybean plants were evaluated. The use of Crop+ on soybean plants led to an increase in the SPAD index; the use of Crop+ in association with glyphosate made possible the attenuation of visible symptoms of damage, preventing reductions in crop yield.(AU)
Com o advento da soja resistente ao glifosato, aplicações em pós-emergência da cultura tornaram-se corriqueiras. Dessa forma, problemas relacionados à seletividade desse herbicida mostraram-se cada vez mais intensos, devido ao aumento da dose utilizada e à aplicação em momentos não recomendados. Para avaliar o desempenho agronômico da soja submetida à aplicação de glifosato isolado em pós-emergência e em associação ao Crop+ em diferentes estádios e doses, um experimento foi realizado em delineamento de blocos ao acaso, em esquema de testemunhas duplas adjacentes, adotando-se quatro repetições. Os tratamentos foram constituídos por: glifosato (1296 g·ha-1); glifosato + Crop+ (1296 + 0,25 g ou L·ha-1); glifosato + Crop+ (1296 + 0,5 g ou L·ha-1);glifosato (2592 g·ha-1); glifosato + Crop+ (2592 + 0,25 g ou L·ha-1);glifosato + Crop+ (2592 + 0,5 g ou L·ha-1); 2 × glifosato (2 × 1296 g·ha-1); 2 × glifosato + Crop+ (2 × 1296 + 0,25 g ou L·ha-1); 2 × glifosato + Crop+ (2 × 1296 + 0,5 g ou L·ha-1) e Crop+ (0,5 g·ha-1). Foram avaliados fitointoxicação das plantas de soja, índice SPAD (soil plant analysis development), altura, estande, massa de cem grãos e produtividade. Nos resultados, a utilização de Crop+ em plantas de soja proporcionou o aumento no índice SPAD e, quando o Crop+ foi associado ao glifosato, possibilitou a atenuação dos sintomas visuais de injúrias, prevenindo reduções na produtividade da cultura.(AU)
Assuntos
Glycine max , Resistência a Herbicidas , Solo , Pragas da Agricultura , HerbicidasRESUMO
With the advent of glyphosate-resistant soybean, postemergence applications of the herbicide have become routine. In this way, problems related to the selectivity of glyphosate have been shown to be increasingly intense, due to the use of increased dosages and applications of the herbicide at unrecommended moments. In order to evaluate the agronomic performance of soybean plants subjected to the postemergence application of glyphosate both in isolation and together with Crop+ at different stages and dosage levels, a randomized block design experiment was carried out with two adjacent checks and four repetitions. The treatments consisted of: glyphosate (1296 g·ha-1); glyphosate + Crop+ (1296 + 0.25 g or L·ha-1); glyphosate + Crop+ (1296 + 0.5 g or L·ha-1); glyphosate (2592 g·ha-1); glyphosate + Crop+ (2592 + 0.25 g or L·ha-1); glyphosate + Crop+ (2592 + 0.5 g or L·ha-1); 2 × glyphosate (2 × 1296 g·ha-1); 2 × glyphosate + Crop+ (2 × 1296 + 0.25 g or L·ha-1); 2 × glyphosate + Crop+ (2 × 1296 + 0.5 g or L·ha-1) and Crop+ (0.5 g·ha-1). The phytotoxicity, soil plant analysis development (SPAD) index, height, stand, mass of 100 grains, and yield of the soybean plants were evaluated. The use of Crop+ on soybean plants led to an increase in the SPAD index; the use of Crop+ in association with glyphosate made possible the attenuation of visible symptoms of damage, preventing reductions in crop yield.(AU)
Com o advento da soja resistente ao glifosato, aplicações em pós-emergência da cultura tornaram-se corriqueiras. Dessa forma, problemas relacionados à seletividade desse herbicida mostraram-se cada vez mais intensos, devido ao aumento da dose utilizada e à aplicação em momentos não recomendados. Para avaliar o desempenho agronômico da soja submetida à aplicação de glifosato isolado em pós-emergência e em associação ao Crop+ em diferentes estádios e doses, um experimento foi realizado em delineamento de blocos ao acaso, em esquema de testemunhas duplas adjacentes, adotando-se quatro repetições. Os tratamentos foram constituídos por: glifosato (1296 g·ha-1); glifosato + Crop+ (1296 + 0,25 g ou L·ha-1); glifosato + Crop+ (1296 + 0,5 g ou L·ha-1);glifosato (2592 g·ha-1); glifosato + Crop+ (2592 + 0,25 g ou L·ha-1);glifosato + Crop+ (2592 + 0,5 g ou L·ha-1); 2 × glifosato (2 × 1296 g·ha-1); 2 × glifosato + Crop+ (2 × 1296 + 0,25 g ou L·ha-1); 2 × glifosato + Crop+ (2 × 1296 + 0,5 g ou L·ha-1) e Crop+ (0,5 g·ha-1). Foram avaliados fitointoxicação das plantas de soja, índice SPAD (soil plant analysis development), altura, estande, massa de cem grãos e produtividade. Nos resultados, a utilização de Crop+ em plantas de soja proporcionou o aumento no índice SPAD e, quando o Crop+ foi associado ao glifosato, possibilitou a atenuação dos sintomas visuais de injúrias, prevenindo reduções na produtividade da cultura.(AU)
Assuntos
Glycine max , Resistência a Herbicidas , Solo , Pragas da Agricultura , HerbicidasRESUMO
ABSTRACT: Shikimic acid (SA) has witnessed a strong increase in recent years due to the increasing demand of the pharmaceutical and cosmetic industry. The SA is used as a precursor for the synthesis of oseltamivir phosphate (Tamiflu®), a potent viral inhibitor and is extracted from the plant Illicium verum Hook which has a limited availability. This article proposed the use of Urochloa plantaginea (Link.) webster and glyphosate, as an alternative source of SA. U. plantaginea plants with 3 - 4 tillers and 4 - 6 leaves were harvest at 3, 6, 9 and 12 days after application (DAT) of low rates of glyphosate. Samples were dried, extracted, analyzed by HPLC and LC-MS/MS. The maximum SA concentrations were observed at 6 days after glyphosate at 36 g.a.e.ha-1 was applied in plants of U. plantaginea with 4 to 6 leaves. The capability of this annual gramineae to produce elevated SA levels throughout the entire biomass affords its potential for a greater yield on a per hectare basis.
RESUMO: O interesse pelo ácido chiquímico (SA) tem apresentado um forte incremento nos últimos anos devido à crescente demanda da indústria farmacêutica e cosmética. O SA é utilizado como um precursor para a síntese do fosfato de oseltamivir (Tamiflu®), um potente inibidor viral. Este ácido é extraído principalmente da planta Illicium verum Hook. A disponibilidade desta planta é um fator limitante para o crescimento do mercado no futuro próximo. Este artigo propõe Urochloa plantaginea (Link.) webster tratada com sub doses de glifosato, como uma fonte alternativa de SA. Plantas de U. plantaginea com 3 - 4 perfilhos e 4 a 6 folhas foram tratadas com subdoses de glifosato e coletadas aos 0, 3, 6, 9 e 12 dias após sua aplicação (DAT). As amostras foram secas, extraídas e analisadas por HPLC e confirmadas por LC-MS/MS. As concentrações máximas de SA foram observadas aos seis dias após aplicação do glifosato a 36 g.e.a.ha-1 em plantas de U. plantaginea com 4 - 6 folhas. A capacidade anual dessa gramínea para produzir níveis elevados de SA em toda a biomassa, pode ser uma fonte economicamente viável de SA.
RESUMO
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.
RESUMO
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.
Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Amaranthus/efeitos dos fármacos , Amaranthus/genética , Substituição de Aminoácidos , Glicina/análogos & derivados , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Argentina , Relação Dose-Resposta a Droga , Duplicação Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glicina/metabolismo , Glicina/farmacologia , Mutação/efeitos dos fármacos , Fosfatos/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/genética , Alinhamento de Sequência , Análise de Sequência de Proteína , Ácido Chiquímico/metabolismo , Glycine max , GlifosatoRESUMO
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.
Assuntos
Amaranthus/metabolismo , Glycine max/efeitos dos fármacos , Glicina/análogos & derivados , Herbicidas/metabolismo , Plantas Daninhas/metabolismo , 3-Fosfoshikimato 1-Carboxiviniltransferase/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase/metabolismo , Amaranthus/efeitos dos fármacos , Argentina , Transporte Biológico , Glicina/metabolismo , Glicina/farmacologia , Resistência a Herbicidas , Herbicidas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Daninhas/efeitos dos fármacos , Glycine max/crescimento & desenvolvimento , GlifosatoRESUMO
BACKGROUND: The evolution of herbicide-resistant weeds is one of the most important concerns of global agriculture. Amaranthus hybridus L. is a competitive weed for summer crops in South America. In this article, we intend to unravel the molecular mechanisms by which an A. hybridus population from Argentina has become resistant to extraordinarily high levels of glyphosate. RESULTS: The glyphosate-resistant population (A) exhibited particularly high parameters of resistance (GR50 = 20 900 g ai ha-1 , Rf = 314), with all plants completing a normal life cycle even after 32X dose application. No shikimic acid accumulation was detected in the resistant plants at any of the glyphosate concentrations tested. Molecular and genetic analyses revealed a novel triple substitution (TAP-IVS: T102I, A103V, and P106S) in the 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) enzyme of population A and an incipient increase on the epsps relative copy number but without effects on the epsps transcription levels. The novel mechanism was prevalent, with 48% and 52% of the individuals being homozygous and heterozygous for the triple substitution, respectively. In silico conformational studies revealed that TAP-IVS triple substitution would generate an EPSPS with a functional active site but with an increased restriction to glyphosate binding. CONCLUSION: The prevalence of the TAP-IVS triple substitution as the sole mechanism detected in the highly glyphosate resistant population suggests the evolution of a new glyphosate resistance mechanism arising in A. hybridus. This is the first report of a naturally occurring EPSPS triple substitution and the first glyphosate target-site resistance mechanism described in A. hybridus. © 2018 Society of Chemical Industry.
Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase/genética , Amaranthus/efeitos dos fármacos , Amaranthus/genética , Substituição de Aminoácidos , Glicina/análogos & derivados , Resistência a Herbicidas/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase/química , Amaranthus/enzimologia , Sequência de Aminoácidos , Argentina , Sequência de Bases , Relação Dose-Resposta a Droga , Glicina/farmacologia , Mutação , GlifosatoRESUMO
The constant presence of genetically modified (GM) soybean in conventional seed lots has become a growing problem for international seed trade. In this context, seed companies have prompted the development of routine tests for accurate genetically modified soybean seeds detection. In this study, a quantitative PCR-based method was standardized in order to detect and quantify mixtures of seeds (i.e. certified seed) or GM grains (i.e. seeds came from field) into samples of non-GM soybean, in a way that soybean lots can be assessed within the standards established by legislation. The method involved the use of p35S-f2/petu-r1 primers targeting CP-4 enolpyruvylshikimate-3-phosphate synthase (cp4-epsps) gene (i.e. that confers herbicide tolerance in Roundup ReadyTM (RR)) for real-time PCR detection and quantification through mericon Quant GMO Detection Assay. The results revealed the method efficiency to detect and quantify the presence of even one soybean seed in batch used for routine evaluation of GM seeds. In addition, it was possible to detect of up to 0.1% of transgenic DNA relative to the soybean grains content. Thus, the sensitive GMO quantitative approach described in this study will provide support in supervising activities, and facilitate the process and control of GM soybean.
A constante presença da soja geneticamente modificada (GM) em lotes de sementes convencionais têm se tornado um grande problema para o comércio internacional de sementes. Neste contexto, as empresas de sementes estão em busca de testes de rotina extremamente precisos para a detecção de sementes de soja geneticamente modificadas. Neste estudo, um método baseado em PCR quantitativo foi padronizado para detectar e quantificar misturas de sementes (i.e. sementes certificadas) ou grãos geneticamente modificados (i.e. sementes oriundas do campo) dentro de lotes de soja não transgênica, de um modo que os lotes de soja possam ser avaliados dentro dos parâmetros estabelecidos pela legislação. O método envolveu o uso dos iniciadores p35S-f2/petu-r1 alvejando o gene CP-4 5-nolpiruvil-shikimato-3-fosfato sintase (cp4-epsps) (i.e. que confere a tolerância ao herbicida Roundup Ready® (RR)) para detecção e quantificação em PCR de tempo real via Ensaio de detecção Mericon Quant GMO. Os resultados revelaram um método eficiente para detectar e quantificar a presença de até mesmo uma única semente de soja no lote usado para a avaliação de rotina de sementes geneticamente modificadas. Adicionalmente, foi possível detectar até 0,1% de DNA transgênico relativo ao conteúdo de grãos de soja. Dessa forma, uma abordagem quantitativa sensível à soja geneticamente modificada foi descrita nesse estudo e poderá fornecer suporte em atividades de supervisão, além de facilitar o processo de controle da soja geneticamente modificada.
Assuntos
Sementes , Glycine max , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , HerbicidasRESUMO
The "second generation" of glyphosate-tolerant soybean (RR®2 soybean) was developed through a different technique of insertion of the glyphosate-insensitive EPSPs gene. Information on the selectivity of glyphosate, alone or in combination, in RR2 soybean is lacking. This study evaluated the effects of glyphosate, isolated or in associations, applied at post-emergence (V4), at agronomic performance and seed quality of soybean cultivar NS 6700 IPRO (RR2). The experimental design was randomized block with four replications and seven treatments, conducted in the field for two growing seasons. The treatments consisted of glyphosate herbicide, alone or in combination with clethodim, cloransulam, chlorimuron, lactofen and fluazifop, besides the control without application. Analysis was performed for crop injury, Soil and Plant Analyzer Development (SPAD) index, as well as variables related to agronomic performance (height, number of pods per plant, yield and 1,000-seed weight) and seed quality (vigor, germination, abnormal seedlings and dead seeds). An additional test was conducted with the same cultivar and treatments in a greenhouse in a completely randomized design with four replications. The herbicides did not affect agronomic performance and seed quality of RR2 soybean. Thus, the soybean cultivar NS 6700 IPRO (RR2) was tolerant to glyphosate, alone or combined with other herbicides applied in post-emergence (V4).(AU)
A "segunda geração" de soja tolerante ao glifosato (soja RR®2) foi desenvolvida por meio de uma diferente inserção do gene EPSPs insensível ao referido herbicida. Informações sobre a seletividade de glifosato, isolado ou em associação, são faltantes em soja RR2. Este estudo avaliou os efeitos do glifosato, isolado ou em associações, aplicado em pós-emergência (V4), no desempenho agronômico e na qualidade de sementes do cultivar de soja NS 6700 IPRO (RR2). Adotou-se o delineamento experimental de blocos ao acaso com quatro repetições e sete tratamentos, realizados no campo em duas safras. Os tratamentos consistiram no herbicida glifosato, isolado ou em associação com clethodim, cloransulam, chlorimuron, lactofen e fluazifop, além da testemunha sem aplicação. Foi realizada a avaliação dos sintomas de injúria e índice SPAD (Soil and Plant Analyzer Development), bem como de variáveis relacionadas ao desempenho agronômico (altura, número de vagens por planta, produtividade e massa de 1.000 sementes) e à qualidade das sementes (vigor, germinação, plântulas anormais e sementes mortas). Experimento complementar foi realizado com o mesmo cultivar e tratamentos em casa de vegetação em delineamento inteiramente casualizado com quatro repetições. Os herbicidas não afetaram o desempenho agronômico e a qualidade de sementes da soja RR2. Assim, o cultivar NS 6700 IPRO (RR2) foi tolerante ao glifosato, isolado ou em associação com outros herbicidas aplicados em pós-emergência (V4).(AU)
Assuntos
Glycine max , Resistência a Herbicidas , SementesRESUMO
The "second generation" of glyphosate-tolerant soybean (RR®2 soybean) was developed through a different technique of insertion of the glyphosate-insensitive EPSPs gene. Information on the selectivity of glyphosate, alone or in combination, in RR2 soybean is lacking. This study evaluated the effects of glyphosate, isolated or in associations, applied at post-emergence (V4), at agronomic performance and seed quality of soybean cultivar NS 6700 IPRO (RR2). The experimental design was randomized block with four replications and seven treatments, conducted in the field for two growing seasons. The treatments consisted of glyphosate herbicide, alone or in combination with clethodim, cloransulam, chlorimuron, lactofen and fluazifop, besides the control without application. Analysis was performed for crop injury, Soil and Plant Analyzer Development (SPAD) index, as well as variables related to agronomic performance (height, number of pods per plant, yield and 1,000-seed weight) and seed quality (vigor, germination, abnormal seedlings and dead seeds). An additional test was conducted with the same cultivar and treatments in a greenhouse in a completely randomized design with four replications. The herbicides did not affect agronomic performance and seed quality of RR2 soybean. Thus, the soybean cultivar NS 6700 IPRO (RR2) was tolerant to glyphosate, alone or combined with other herbicides applied in post-emergence (V4).(AU)
A "segunda geração" de soja tolerante ao glifosato (soja RR®2) foi desenvolvida por meio de uma diferente inserção do gene EPSPs insensível ao referido herbicida. Informações sobre a seletividade de glifosato, isolado ou em associação, são faltantes em soja RR2. Este estudo avaliou os efeitos do glifosato, isolado ou em associações, aplicado em pós-emergência (V4), no desempenho agronômico e na qualidade de sementes do cultivar de soja NS 6700 IPRO (RR2). Adotou-se o delineamento experimental de blocos ao acaso com quatro repetições e sete tratamentos, realizados no campo em duas safras. Os tratamentos consistiram no herbicida glifosato, isolado ou em associação com clethodim, cloransulam, chlorimuron, lactofen e fluazifop, além da testemunha sem aplicação. Foi realizada a avaliação dos sintomas de injúria e índice SPAD (Soil and Plant Analyzer Development), bem como de variáveis relacionadas ao desempenho agronômico (altura, número de vagens por planta, produtividade e massa de 1.000 sementes) e à qualidade das sementes (vigor, germinação, plântulas anormais e sementes mortas). Experimento complementar foi realizado com o mesmo cultivar e tratamentos em casa de vegetação em delineamento inteiramente casualizado com quatro repetições. Os herbicidas não afetaram o desempenho agronômico e a qualidade de sementes da soja RR2. Assim, o cultivar NS 6700 IPRO (RR2) foi tolerante ao glifosato, isolado ou em associação com outros herbicidas aplicados em pós-emergência (V4).(AU)
Assuntos
Glycine max , Resistência a Herbicidas , SementesRESUMO
In 2014 hairy beggarticks (Bidens pilosa L.) has been identified as being glyphosate-resistant in citrus orchards from Mexico. The target and non-target site mechanisms involved in the response to glyphosate of two resistant populations (R1 and R2) and one susceptible (S) were studied. Experiments of dose-response, shikimic acid accumulation, uptake-translocation, enzyme activity and 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene sequencing were carried out in each population. The R1 and R2 populations were 20.4 and 2.8-fold less glyphosate sensitive, respectively, than the S population. The resistant populations showed a lesser shikimic acid accumulation than the S population. In the latter one, 24.9% of 14C-glyphosate was translocated to the roots at 96 h after treatment; in the R1 and R2 populations only 12.9 and 15.5%, respectively, was translocated. Qualitative results confirmed the reduced 14C-glyphosate translocation in the resistant populations. The EPSPS enzyme activity of the S population was 128.4 and 8.5-fold higher than the R1 and R2 populations of glyphosate-treated plants, respectively. A single (Pro-106-Ser), and a double (Thr-102-Ile followed by Pro-106-Ser) mutations were identified in the EPSPS2 gene conferred high resistance in R1 population. Target-site mutations associated with a reduced translocation were responsible for the higher glyphosate resistance in the R1 population. The low-intermediate resistance of the R2 population was mediated by reduced translocation. This is the first glyphosate resistance case confirmed in hairy beggarticks in the world.
RESUMO
Glyphosate-resistant (GR) canola contains two transgenes that impart resistance to the herbicide glyphosate: (1) the microbial glyphosate oxidase gene (gox) encoding the glyphosate oxidase enzyme (GOX) that metabolizes glyphosate to aminomethylphosphonic acid (AMPA) and (2) cp4 that encodes a GR form of the glyphosate target enzyme 5-enolpyruvylshikimic acid-3-phosphate synthase. The objectives of this research were to determine the phytotoxicity of AMPA to canola, the relative metabolism of glyphosate to AMPA in GR and conventional non-GR (NGR) canola, and AMPA pool sizes in glyphosate-treated GR canola. AMPA applied at 1.0 kg ha(-1) was not phytotoxic to GR or NGR. At this AMPA application rate, NGR canola accumulated a higher concentration of AMPA in its tissues than GR canola. At rates of 1 and 3.33 kg ae ha(-1) of glyphosate, GR canola growth was stimulated. This stimulatory effect is similar to that of much lower doses of glyphosate on NGR canola. Both shikimate and AMPA accumulated in tissues of these glyphosate-treated plants. In a separate experiment in which young GR and NGR canola plants were treated with non-phytotoxic levels of [(14)C]-glyphosate, very little glyphosate was metabolized in NGR plants, whereas most of the glyphosate was metabolized to AMPA in GR plants at 7 days after application. Untreated leaves of GR plants accumulated only metabolites (mostly AMPA) of glyphosate, indicating that GOX activity is very high in the youngest leaves. These data indicate that more glyphosate is transformed to AMPA rapidly in GR canola and that the accumulated AMPA is not toxic to the canola plant.
Assuntos
Brassica napus/efeitos dos fármacos , Glicina/análogos & derivados , Resistência a Herbicidas , Herbicidas/farmacologia , Organofosfonatos/farmacologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Brassica napus/genética , Brassica napus/metabolismo , Glicina/farmacologia , Isoxazóis , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tetrazóis , GlifosatoRESUMO
For the prospective immunorecognition of 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) as a biomarker protein expressed by transgenic soybean, an extensive in silico evaluation of the referred protein was performed. The main objective of this study was the selection of a set of peptides that could function as potential immunogens for the production of novel antibodies against CP4-EPSPS protein. For this purpose, the protein was in silico cleaved with trypsin/chymotrypsin and the resultant peptides were extensively analyzed for further selection of the best candidates for antibody production. The analysis enabled the successful proposal of four peptides with potential immunogenicity for their future use as screening biomarkers of genetically modified organisms. To our knowledge, this is the first attempt to select and define potential linear epitopes for the immunization of animals and, subsequently, to generate adequate antibodies for CP4-EPSPS recognition. The present work will be followed by the synthesis of the candidate peptides to be incubated in animals for antibody generation and potential applicability for the development of an immunosensor for CP4-EPSPS detection.