Resistance to auxin mimics and EPSPS and ALS inhibitor herbicides in dicotyledonous weeds. Resistance mechanisms

Actualmente el control químico de las malas hierbas es la principal herramienta utilizada en el mundo dentro y fuera de los cultivos. Desde la introducción de los herbicidas se ha sometido a una gran cantidad de malas hierbas a una selección constate que ha dado como resultado la rápida evolución de la resistencia a los herbicidas. La ciencia de las malas hierbas ha establecido y sigue desarrollando protocolos para conocer y dilucidar los mecanismos que confieren resistencia a uno o varios herbicidas y especies de malas hierbas. La resistencia a herbicidas se divide en: resistencia dentro del sitio de acción (TSR) y resistencia fuera del sitio de acción (NTSR). La resistencia dentro del sitio de acción se atribuye generalmente a mutaciones en el gen que codifica la enzima objetivo del herbicida que provoca una disminución de su afinidad con el herbicida. La resistencia fuera del sitio de acción es el producto de la evolución más avanzada de las malas hierbas ya este impide que el herbicida llegue adecuadamente a su sitio de acción. Dentro de este grupo encontramos la absorción y translocación reducida, el secuestro vacuolar y el metabolismo de los herbicidas. Las malas hierbas pueden tener un solo mecanismo de resistencia o acumular varios mecanismos que se traduce en resistencia múltiple a varios herbicidas y que representan un gran desafío para la sostenibilidad de los herbicidas en la agricultura. En este trabajo de investigación se da a conocer el primer caso de resistencia a glifosato en Amaranthus palmeri que involucra exclusivamente mecanismos NTSR como la absorción y translocación diferenciada entre poblaciones. También se encontró resistencia a 2,4-D en seis especies dicotiledóneas donde el metabolismo mejorado y la translocación reducida son los responsables de la resistencia a este imitador de auxinas. Por otro lado, se estudió una población de Conyza bonariensis, donde por primera vez en el mundo se informa de la aparición de resistencia múltiple a herbicidas inhibidores de la aceto lactato sintasa (ALS) y la 5-enolpiruvylshikimato-3-fosfato sintasa (EPSPS), desviadores de electrones del fotosistema I (PSI), inhibidores del fotosistema II (PSII) y herbicidas imitadores de auxinas. Conocer y estudiar los mecanismos de resistencia a fondo es imprescindible para comprender la resistencia y construir soluciones que permitan desarrollar sistemas agrícolas sostenibles. El manejo integrado de malas hierbas (IWM) debe jugar un papel importante para disminuir esa presión de selección al que las malas hierban se sometido.Chemical control of weeds is currently the main tool used in the world inside and outside of crops. Since the introduction of herbicides, large number of weeds have been subjected to constant selection pressureresulting to the rapid evolution of herbicide resistance. Weed science has established and continues to develop protocols to test and know the mechanisms that confer resistance for various herbicides and weed species. Herbicide resistance is classified into target site resistance (TSR) and non-target site resistance (NTSR). Target site resistance is generally attributed to mutations in the gene encoding the herbicide target enzyme that cause a decrease in its affinity for the herbicide. Non target site resistance is the product of the more advanced evolution and prevents the herbicide from reaching its site of action correctly. Within this group are reduced absorption and translocation, vacuolar sequestration and metabolism of herbicides. Weeds have a single resistance mechanism or accumulate several mechanisms that result in multiple resistance to several herbicides and that represent a major challenge to the sustainability of herbicides in agriculture. In this research work we report the first case of glyphosate resistance in Amaranthus palmeri involving exclusively NTSR mechanisms such as differential absorption and translocation between populations. Resistance to 2,4-D was also found in six dicot species in which enhanced metabolism and reduced translocation are responsible for resistance to this auxin mimic. On the other hand, a population of Conyza bonariensis was studied, where for the first time in the world multiple resistance to acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor herbicides, photosystem I electron diverters, photosystem II inhibitors and auxin mimic herbicides. Knowledge and study of resistance mechanisms is essential to understand resistance and build solutions for the development of sustainable agricultural systems. Integrated weed management (IWM) has an important role to play in reducing the selection pressure to which weeds have been subjected

Resistance to Fomesafen, Imazamox and Glyphosate in Euphorbia heterophylla from Brazil

Euphorbia heterophylla is a species of weed that was previously controlled by fomesafen, imazamox and glyphosate, but continued use of these herbicides has selected resistant populations from the Rio Grande do Sul (Brazil). One resistant (R) strain and one susceptible (S) strain to fomesafen, imazamox and glyphosate were compared, the latter by recurrent selection. Dose-response tests showed multiple resistance to these herbicides. The required imazamox concentration to inhibit ALS by 50% was approximately 16 times greater in the R population than in the S population. Based on the EPSPS activity results, the R population was 10 fold less sensitive to glyphosate than the S counterpart. In addition, basal EPSPS activity from R plants was 3.3 fold higher than the level detected on S plants. The Proto IX assays showed high resistance to fomesafen in the R population that accumulated less Proto IX than the S population. Malathion assays showed the participation of CytP450 in fomesafen resistance, but a molecular mechanism could also be involved. To our knowledge, this is the first characterisation of multiple resistance to these three groups of herbicides in E. heterophylla in the world

Multiple Resistance to Glyphosate and 2,4-D in Carduus acanthoides L. from Argentina and Alternative Control Solutions

Carduus acanthoides L. is an invasive species native to Europe and distributed in other parts of the world, including North and South America. In Cordoba, Argentina, control failures of this species have been reported in Roundup Ready (RR) soybean crops where glyphosate and 2,4-D have frequently been applied, although there are no confirmed reports worldwide of resistance to glyphosate and 2,4-D in this species. Dose–response tests showed multiple-resistance to both active principles. The resistant population (R) had LD50 values of 1854.27 and 1577.18 g ae ha−1 (grams of acid equivalent per hectare), while the susceptible (S) population had LD50 values of 195.56 and 111.78 g ae ha−1 for glyphosate and 2,4-D, respectively. Low accumulations of shikimic acid (glyphosate) and ethylene (2,4-D) at different doses in the R population compared to the S population support the results observed in the dose–response curves. No significant differences in leaf retention were observed for glyphosate and 2,4-D in the R and S populations. However, the use of adjuvants increased the retention capacity of herbicides in both populations. Ten alternative herbicides with seven different action mechanisms (MOAs) were evaluated and the most effective active principles were dicamba, bromoxynil, atrazine, tembotrione, flazasulfuron, glufosinate, and paraquat. These findings are the first evidence of glyphosate and 2,4 D resistance in C. acanthoides

Glyphosate Resistance Confirmation and Field Management of Red Brome (Bromus rubens L.) in Perennial Crops Grown in Southern Spain

The excessive use of the herbicide glyphosate on annual and perennial crops grown in Southern Spain has caused an increase in resistant weed populations. Bromus rubens has begun to spread through olive and almond cultivars due to low glyphosate control over these species, whereas previously it had been well controlled with field dose (1080 g ae ha−1). Characterization using Simple Sequence Repeat (SSR) markers confirmed the presence of B. rubens collected in Andalusia. A rapid shikimic acid accumulation screening showed 17 resistant (R) populations with values between 300 and 700 µg shikimate g−1 fresh weight and three susceptible (S) populations with values between 1200 and 1700 µg shikimate g−1 fresh weight. In dose–response experiments the GR50 values agreed with previous results and the resistance factors (RFs: GR50 R/GR50 S (Br1)) were between 4.35 (Br9) and 7.61 (Br19). Foliar retention assays shown no differences in glyphosate retention in both R and S populations. The tests carried out in a resistant field (Br10) demonstrated the control efficacy of pre-emergence herbicides since flazasulfuron in the tank mix with glyphosate had up to 80% control 15 to 120 days after application (DAA) and grass weed postemergence herbicides, such as propaquizafop + glyphosate and quizalofop + glyphosate, had up to 90% control 15 to 90 DAA. Results confirm the first scientific report of glyphosate-resistant B. rubens worldwide; however, the use of herbicides with another mode of action (MOA) is the best tool for integrated weed management

An Asp376Glu substitution in ALS gene and enhanced metabolism confers high tribenuron-methyl resistance in Sinapis alba

Acetolactate synthase (ALS) inhibiting herbicides (group 2) have been widely applied for the last 20 years to control Sinapis alba in cereal crops from southern Spain. In 2008, a tribenuron-methyl (TM) resistant (R) S. alba population was first reported in a cereal field in Malaga (southern Spain). In 2018, three suspected R S. alba populations (R1, R2 and R3) to TM were collected from three different fields in Granada (southern Spain, 100 km away from Malaga). The present work aims to confirm the putative resistance of these populations to TM and explore their resistance mechanisms. Dose–response assays showed that the R1, R2 and R3 populations ranging between 57.4, 44.4 and 57.1 times more resistance to TM than the susceptible population (S). A mutation in the ALS gene (Asp376Glu) was detected in the Rs S. alba populations. 14C-metabolism studies show that metabolites and TM were changing significantly faster in the R than in the S plants. Alternative chemical control trials showed that 2,4-D and MCPA (auxin mimics), glyphosate (enolpyruvyl shikimate phosphate synthase,EPSPS, inhibitor-group 9), metribuzin (PSII inhibitors/Serine 264 Binders, -group 5) and mesotrione (hydroxyphenyl pyruvate dioxygenase, HPPD, inhibitor-group 27) presented a high control of the four populations of S. alba tested, both S and R. Based on these results, it is the first case described where the Asp376Glu mutation and P450-mediated metabolism participates in resistance to TM in S. alba. Comparing these results with those found in the S. alba population in Malaga in 2008, where the resistance was TSR type (Pro197Ser), we can suggest that despite the geographical proximity (over 100 km), the resistance in these cases was due to different evolutionary events

Different Mutations Providing Target Site Resistance to ALS- and ACCase-Inhibiting Herbicides in Echinochloa spp. from Rice Fields

Echinochloa spp. is one of the most invasive weeds in rice fields worldwide. Acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) inhibiting herbicides are two of the most widely used rice herbicides. However, overuse has led to the resistance evolution of Echinochloa spp. to penoxsulam (ALS-inhibitor) and cyhalofop-methyl (ACCase-inhibitor). In this work, 137 different Echinochloa spp. populations were collected in different rice fields in Extremadura (western Spain) where lack of control was detected. Target-site based resistance (by sequencing ALS and ACCase gene) and characterization of Echinochloa species at the molecular level (based on PCR-RFLP analyses) were carried out in those populations. Most of the populations studied (111 of 137) belong to the E. oryzicola/E. oryzoides group. Three-point mutations were identified in ALS genes: Pro197Ser, Pro197Thr, and Ser653Asn, the first being the most frequent substitution in resistant plants. In the ACCase gene, the Ile1781Leu substitution was found. In both ALS and ACCase sequencing, evidence of heterozygosity was also observed. To assess whether cross-resistance patterns differed between mutations, two populations belonging to the E. oryzicola/E. oryzoides group had its most frequent mutations (Pro197Ser, population ech3-14 and Ile1781Leu, population ech114-10) chosen to be carried out in a dose-response assay. It was confirmed that Pro197Ser conferred resistance to triazolopyrimidine, imidazolinone, sulfonylurea, and pyrimidinyl benzoate families. On the other hand, the Ile1781Leu change gave resistance to aryloxyphenoxypropionate and cyclohexanedione families. Of the authorized herbicides in rice in Spain, more that 80% belong to these families. It is therefore important that farmers carry out an integrated control system that combines both chemical and non-chemical tools

Can Control of Glyphosate Susceptible and Resistant Conyza sumatrensis Populations Be Dependent on the Herbicide Formulation or Adjuvants?

In this work, we studied the effect of three glyphosate formulations (isopropylamine, ammonium and potassium salts) and two non-ionic adjuvants on the resistance response of two resistant (R1, R2) and one susceptible population of the highly invasive Asteraceae, Conyza sumatrensis, from Southern France vineyards. Only in R1, an amino acid substitution (Pro106Thr) was found in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The two adjuvants, in a similar fashion, significantly reduced GR50 values for every population and glyphosate formulation. Without adjuvants, glyphosate as potassium salt was the only formulation able to significantly reduce the GR50 values of every population. For every population, the two adjuvants improved, indistinguishably, leaf retention of the herbicidal solution and the potassium salt formulation led to the highest retention, both with and without the adjuvant added. Uptake responses paralleled those of retention and adjuvant addition was more effective in increasing foliar uptake of the lower performing formulations (isopropylamine and ammonium salts). The allocation pattern of glyphosate among plant compartments was only dependent on population, with R2 retaining most glyphosate in the treated leaf, clearly suggesting the occurrence of a Non-Target Site Resistance (NTSR) mechanism. Results indicate that control of weed populations possessing NTSR mechanisms of resistance to glyphosate may be improved through adequate selection of formulation and adjuvant use

Resistance mechanisms to 2,4-D in six different dicotyledonous weeds around the world

2,4-D resistance is increasing around the world due to both transgenic crops and resistance to other herbicides. The objective of the this study was to characterize the currently unknown mechanisms of 2,4-D resistance in five weed species from around the globe: Amaranthus hybridus (Argentina), Conyza canadensis (Hungary), Conyza sumatrensis (France), Hirschfeldia incana (Argentina) and Parthenium hysterophorus (Dominican Republic), using Papaver rhoeas (Spain) as a standard resistant (R) species. Dose-response trials using malathion and absorption, translocation and metabolism experiments were performed to unravel the resistance mechanisms. R plants produced at least 3-folds less ethylene than susceptible plants, confirming the resistance to 2,4-D, together with resistance factors >4. A. hybridus, P. hysterophorus and P. rhoeas showed both reduced translocation and enhanced metabolism. In the two Conyza sps., the only resistance mechanism found was enhanced metabolism. Malathion synergized with 2,4-D in all these species, indicating the role of cytochrome P450 in the herbicide degradation. In H. incana, reduced translocation was the only contributing mechanism to resistance. Among the six dicotyledonous weed species investigated, there was a differential contribution to 2,4-D resistance of enhanced metabolism and reduced translocation. Thus, extrapolating 2,4-D resistance mechanisms from one weed species to another is very risky, if even related.EEA BordenaveFil: Palma Bautista, Candelario. Universidad de Córdoba. Departamento de Química Agrícola y Edafología; EspañaFil: Rojano Delgado, Antonia María. Universidad de Córdoba. Departamento de Química Agrícola y Edafología; EspañaFil: Dellaferrera, Ignacio Miguel. Universidad Nacional del Litoral. Facultad de Ciencias Agrarias; ArgentinaFil: Dellaferrera, Ignacio Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); ArgentinaFil: Rosario, Jesús M. Universidad de Córdoba. Departamento de Química Agrícola y Edafología; EspañaFil: Vigna, Mario Raúl. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bordenave; ArgentinaFil: Torra, Joel. Universidad de Lleida. Departamento de Horticultura y Fruticultura. Agrotecnio; EspañaFil: de Prado, Rafael. Universidad de Córdoba. Departamento de Química Agrícola y Edafología; Españ

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