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Nov 18, 2016 - 3-phosphate synthase (EPSPS) was not different in the S, R8 and R14 ... Alcántara-de la Cruz et al., 2016b); multiple EPSPS gene copy.
ORIGINAL RESEARCH published: 18 November 2016 doi: 10.3389/fpls.2016.01742

First Resistance Mechanisms Characterization in Glyphosate-Resistant Leptochloa virgata Ricardo Alcántara-de la Cruz 1† , Antonia M. Rojano-Delgado 1 , María J. Giménez 2 , Hugo E. Cruz-Hipolito 3 , José A. Domínguez-Valenzuela 4*, Francisco Barro 2 and Rafael De Prado 1 Edited by: Pankaj Kumar Arora, Mahatma Jyotiba Phule Rohilkhand University, India Reviewed by: Karl Kunert, University of Pretoria, South Africa Leonardo Bianco De Carvalho, São Paulo State University, Brazil *Correspondence: José A. Domínguez-Valenzuela [email protected] † Present address: Ricardo Alcántara-de la Cruz, Department of Agricultural Parasitology, Chapingo Autonomous University, Texcoco, Mexico

Specialty section: This article was submitted to Agroecology and Land Use Systems, a section of the journal Frontiers in Plant Science Received: 20 July 2016 Accepted: 04 November 2016 Published: 18 November 2016 Citation: Alcántara-de la Cruz R, Rojano-Delgado AM, Giménez MJ, Cruz-Hipolito HE, Domínguez-Valenzuela JA, Barro F and De Prado R (2016) First Resistance Mechanisms Characterization in Glyphosate-Resistant Leptochloa virgata. Front. Plant Sci. 7:1742. doi: 10.3389/fpls.2016.01742

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Department of Agricultural Chemistry and Edaphology, Campus of Rabanales, University of Cordoba, Cordoba, Spain, Institute for Sustainable Agriculture, Spanish National Research Council, Cordoba, Spain, 3 Bayer CropScience Mexico, Mexico City, Mexico, 4 Department of Agricultural Parasitology, Chapingo Autonomous University, Texcoco, México 2

Leptochloa virgata (L.) P. Beauv. is an annual weed common in citrus groves in the states of Puebla and Veracruz, Mexico limiting their production. Since 2010, several L. virgata populations were identified as being resistant to glyphosate, but studies of their resistance mechanisms developed by this species have been conducted. In this work, three glyphosate-resistant populations (R8, R14, and R15) collected in citrus orchards from Mexico, were used to study their resistance mechanisms comparing them to one susceptible population (S). Dose-response and shikimic acid accumulation assays confirmed the glyphosate resistance of the three resistant populations. Higher doses of up to 720 g ae ha−1 (field dose) were needed to control by 50% plants of resistant populations. The S population absorbed between 7 and 13% more 14 Cglyphosate than resistant ones, and translocated up to 32.2% of 14 C-glyphosate to the roots at 96 h after treatment (HAT). The R8, R14, and R15 populations translocated only 24.5, 26.5, and 21.9%, respectively. The enzyme activity of 5-enolpyruvyl shikimate3-phosphate synthase (EPSPS) was not different in the S, R8 and R14 populations. The R15 Population exhibited 165.9 times greater EPSPS activity. Additionally, this population showed a higher EPSPS basal activity and a substitution in the codon 106 from Proline to Serine in the EPSPS protein sequence. EPSPS gene expression in the R15 population was similar to that of S population. In conclusion, the three resistant L. virgata populations show reduced absorption and translocation of 14 C-glyphosate. Moreover, a mutation and an enhanced EPSPS basal activity at target-site level confers higher resistance to glyphosate. These results describe for the first time the glyphosate resistance mechanisms developed by resistant L. virgata populations of citrus orchards from Mexico. Keywords: 5-enolpyruvyl shikimate-3-phosphate synthase, EPSPS gene, Pro-106 substitution, reduced absorption and translocation, tropical sprangletop.

Frontiers in Plant Science | www.frontiersin.org

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Glyphosate Resistance Mechanisms of Leptochloa virgata

hypothesized that the resistance mechanisms developed by L. virgata plants could be similar to the mechanisms evolved by others glyphosate resistant species. Therefore, the objective of this work was characterize for the first time whether the glyphosate resistance developed by three resistant L. virgata populations was due either target or non-target site mechanisms, comparing them to one susceptible population (S). These populations were used as being representatives of the species in citrus cropping systems from Mexico.

INTRODUCTION Glyphosate is the most extensively used systemic foliar herbicide of broad spectrum used for over 40 years (early 1970s) to control of annual and perennial weeds (Gomes et al., 2014; Armendáriz et al., 2016). The target-site of glyphosate is the 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), an intermediate in the shikimate pathway (Gomes et al., 2014). EPSPS inhibition by glyphosate does not allow the synthesis of phenylalanine, tyrosine, and tryptophan resulting in accumulation of shikimic acid and depletion of aromatic amino acid pools (Gomes et al., 2014; Maroli et al., 2016). Therefore, glyphosate acts rapidly in reducing photosynthesis activity, carbon metabolism, mineral nutrition, and oxidative events, and to disturb plant–microorganism interactions (Gomes et al., 2014). To date 36 weed species have been reported as being resistant to glyphosate with more than 266 cases (Heap, 2016). Resistance to glyphosate could be caused by different mechanisms either in the target or non-target site (Salas et al., 2015). Both groups limiting the amount of glyphosate which reaches the EPSPS at toxic levels, or causing a loss of affinity between the EPSPS and the glyphosate (Powles, 2010). Target-site resistance (TSR) mechanisms includes amino acid substitutions in DNA sequence, represented by nucleotide changes in Thr102 and Pro-106 positions, that synthesizes the EPSPS enzyme (Sammons and Gaines, 2014; Chen et al., 2015; Yu et al., 2015; Alcántara-de la Cruz et al., 2016b); multiple EPSPS gene copy numbers resulting in a higher amplification, expression and activity of the EPSPS (Gaines et al., 2013; Vila-Aiub et al., 2014). To date, glyphosate is the only herbicide known to evolve amplification of the target-site (EPSPS) conferring resistance with field applications (Ribeiro et al., 2014). Non-target-site resistance (NTSR) mechanisms can confer unpredictable resistance to herbicides (Déyle, 2013). Some are: limited glyphosate leaf absorption (Vila-Aiub et al., 2012); reduced glyphosate translocation to meristematic zones, mainly restricted within the treated leaves (Shaner, 2009; Yanniccari et al., 2012); sequestration of glyphosate in the vacuole (Ge et al., 2012); and glyphosate degradation to non-toxic compounds (sarcosine, glyoxylate, formaldehyde and amino methyl phosphonate) (Duke, 2011) resulting in significantly less glyphosate throughout the whole plants. Weeds can evolve several herbicides resistance mechanisms, and the individuals of the same species can express different mechanism(s) (Alarcón-Reverte et al., 2015). In Leptochloa virgata (L.) P. Beauv., studies about of glyphosate efficacy by dose-response assay, genetic diversity characterization using inter simple sequence repeat (ISSR) markers of different glyphosateresistant L. virgata populations (Alcántara-de la Cruz et al., 2016c), and field trials to provide alternative herbicides for their control have been carried out (Pérez-López et al., 2014). However, no studies on the glyphosate resistance mechanisms involved in their resistance have been conducted, and currently there are no reports of others resistant populations of this species elsewhere in the world. Based on previous reports, and the lack of any knowledge on the glyphosate resistance mechanisms of L. virgata, we

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MATERIALS AND METHODS Plant Material and Growing Conditions Seeds of four L. virgata populations (S, R8, R14, and R15) collected in Persian lime groves from Veracruz, previously characterized with different levels of susceptibility to glyphosate by Alcántara-de la Cruz et al. (2016c), were used. Individuals of the L. virgata populations studied had a similar glyphosate response glyphosate within them. Glyphosate resistant R14 and R15 populations from Cuitláhuac municipality, were selected for being geographically close (18.75◦ N, 96.53◦ W), but genetically different. Also, the most glyphosate resistant population (R8) from Martínez de la Torre municipality (20.16◦ N, 97.07◦ W) was included in this study. The S population was used as control (18.79◦ N, 96.69◦ W). The seeds were sown on trays with peat saturated at field capacity. The trays were covered with a layer of plastic until emergence and placed in a growth chamber at 26/18◦ C (day/night) with a photoperiod of 16 h at 850 mmol m−2 s−1 of light density and 60% relative humidity. Germinated seedlings were individually transplanted into plastic pots in 250 mL of substrate (sand and peat 1:1). Subsequently, the pots were placed in a growth chamber under the conditions described above and watered daily.

Dose-Response Assays Plants of the four populations with 3–4 true leaves were treated with the following glyphosate rates: 0, 45, 90, 180, 360, 720, 1040, 1440, and 1800 g ae ha−1 . Glyphosate (Roundup Energy 45% w/v, Monsanto, Spain) applications were carried out in a treatment chamber (Devries Manufacturing, Hollandale, MN, USA) equipped with TeeJet 8002EVS flat fan nozzle calibrated at 200 kPa, a height of 50 cm and 200 L ha−1 of application volume. The plants were harvested at ground level 21 days after treatment (DAT) and stored separately in paper envelopes. The samples were dried in an oven (JP Selecta S.A., Barcelona, Spain) at 60◦ C for 4 days and weighed to determine the dry weight. Data were expressed as percentage of dry weight compared to the untreated control plants. The experiment was repeated twice in a completely randomized design with 10 replicates per dose.

Shikimic Acid Accumulation Samples of 50 mg (leaf disks 4 mm in diameter) of plant tissue were taken from young leaves of three plants with 3–4 true leaves of each L. virgata population according to Dayan et al. (2015). The disks were placed in 2 mL-Eppendorf tubes containing

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Glyphosate Resistance Mechanisms of Leptochloa virgata

999 µL of monoammonium phosphate (NH4 H2 PO4 10 mM, pH 4.4). Volumes of 1 µL of glyphosate solutions at different concentrations were added (0, 0.1, 1, 10, 50, 100, 200, 400, 600, and 1000 µM). The samples were incubated for 24 h in the growth chamber under growing conditions described above. Next, they were incubated at 60◦ C for 30 min. Volumes of 250 µL of HCl 1.25 N were added and incubated at 60◦ C for 15 min. Aliquots of 250 µL were transferred to new tubes adding 500 µL of periodic acid (0.25% w/v) and sodium metaperiodate (0.25 % w/v) in a 1:1 ratio. The samples were incubated at room temperature (25◦ C) during 90 min, and next, 500 µL of a mix of sodium hydroxide (NaOH 0.6 N) plus sodium sulfite (Na2 SO3 0.22 N) in a 1:1 ratio was added and mixed. The experiment was arranged in a completely randomized design with three technical replications by sample of each population for each glyphosate concentration, and the study was repeated twice. A standard curve was done using known concentrations of shikimate. The absorbance of samples was measured at 382 nm in a spectrophotometer (DU-640, Beckman Coulter Inc., Fullerton, CA, USA). The absorbance values were converted into mg of shikimic acid per g of fresh weight.

in roots + kBq from washes)/kBq total applied] × 100. The average total recovery of 14 C-glyphosate applied was >96% to the S, R8, and R14 populations, and