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. 2016 Oct 3:7:1492.
doi: 10.3389/fpls.2016.01492. eCollection 2016.

Target and Non-target Site Mechanisms Developed by Glyphosate-Resistant Hairy beggarticks (Bidens pilosa L.) Populations from Mexico

Ricardo Alcántara-de la Cruz  1 , Pablo T Fernández-Moreno  2 , Carmen V Ozuna  3 , Antonia M Rojano-Delgado  2 , Hugo E Cruz-Hipolito  4 , José A Domínguez-Valenzuela  5 , Francisco Barro  3 , Rafael De Prado  2
Affiliations

Target and Non-target Site Mechanisms Developed by Glyphosate-Resistant Hairy beggarticks (Bidens pilosa L.) Populations from Mexico

Ricardo Alcántara-de la Cruz et al. Front Plant Sci. .

Abstract

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.

Keywords: 5-enolpyruvyl shikimate-3-phosphate synthase; Bidens pilosa; EPSPS2; TIPS mutation; glyphosate translocation; resistance mechanisms.

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Figures

FIGURE 1
FIGURE 1
Log–logistic curves of glyphosate-susceptible and -resistant Bidens pilosa populations evaluated at 21 days after treatment (DAT). (A) Dose-response curve with respect to percentage of dry mass reduction. The equations of log–logistic curves to estimates the ED50 values are: S: Y = 0.178 + {(99.354 - 0.178)/[1 + (dose/ED50)2.134]}; R1: Y = -3.295 + {(100.56 + 3.295)/[1 + (dose/ED50)1.140]}; R2: Y = 12.121 + {(101.39 - 12.121)/[1 + (dose/ED50)1.288]}. (B) Dose-response curve with respect to percentage of survival. The equations of log–logistic curves to estimates the LD50 values are: S: Y = 0.623 + {(100.17 - 0.623)/[1 + (dose/LD50)5.396]}; R1: rates used did not permit to estimate LD50 value; R2: Y = -1.156 + {(101.47 + 1.156)/[1 + (dose/LD50)3.238]}. Vertical bars represent the standard error of the mean (n = 10).
FIGURE 2
FIGURE 2
Shikimic acid accumulation of glyphosate-susceptible and -resistant B. pilosa populations. (A) Shikimic acid accumulation after a glyphosate application at 360 g ae ha-1 at different intervals of time. (B) Shikimic acid accumulation at different glyphosate concentrations. Vertical bars represent the standard error of the mean (n = 6 technical replicates).
FIGURE 3
FIGURE 3
14C-Glyphosate uptake and translocation in glyphosate-susceptible and -resistant plants of B. pilosa populations. (A) 14C-glyphosate uptake in glyphosate-susceptible and -resistant B. pilosa plants. Different letters are statistically different at 95% probability determined by LSD test. Vertical bars represent the standard error of the mean (n = 5). (B) Digital images (upper plants) and autoradiograph images (lower plants) that show the distribution of 14C-glyphosate translocation in glyphosate-susceptible and -resistant B. pilosa plants at 96 HAT. The highest concentration of 14C-glyphosate is highlighted in red. Arrows indicate the treated leaf.
FIGURE 4
FIGURE 4
5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) activity in glyphosate-susceptible and -resistant plants of B. pilosa populations. (A) Basal EPSPS activity for glyphosate-susceptible and -resistant B. pilosa plants. Histograms represent treatment means and vertical bars SE of the mean (n = 3). (B) EPSPS enzyme activity expressed as percentage of the untreated control in leaf extracts of plants from glyphosate-susceptible and resistant B. pilosa populations. The equations of log–logistic curves to estimates the I50 values are: S: Y = 0.227 + {(99.98 - 0.227)/[1 + (dose/I50)2.619]}; R1: Y = -4.065 + {(99.92 + 4.065)/[1 + (dose/I50)1.477]}; R2: Y = -2.471 + {(99.79 + 2.471)/[1 + (dose/I50)1.108]}. Vertical bars represent the standard error of the mean (n = 3).
FIGURE 5
FIGURE 5
Partial alignment of protein sequences of EPSPS1 and EPSPS2 genes in glyphosate-susceptible and -resistant B. pilosa populations. The highlighted color indicates changes to codons from the consensus nucleotide sequence. Box includes from the 102 to 106 positions (amino acid number based on the start codon (ATG) of A. thaliana [GenBank: CAA29828.1] EPSPS sequence). The B. pilosa EPSPS cDNA sequences information can be found in GenBank with accession numbers KU984452–KU984458.
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