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Physiological and molecular insight on the mechanisms of resistance to glyphosate in Conyza canadensis (L.) Cronq. biotypes

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Physiological and molecular insight on the mechanisms of resistance to glyphosate in Conyza canadensis (L.) Cronq. biotypes
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  Physiological and molecular insight on the mechanisms of resistanceto glyphosate in  Conyza canadensis  (L.) Cronq. biotypes G. Dinelli  a,* , I. Marotti  a , A. Bonetti  a , M. Minelli  a , P. Catizone  a , J. Barnes  b a Dipartimento di Scienze e Tecnologie Agroambientali, Universita`  di Bologna, V.le Fanin, 44-40127 Bologna, Italy b Syngenta Crop Protection, Basel, Switzerland  Received 24 August 2005; accepted 3 January 2006Available online 6 March 2006 Abstract The physiological and molecular basis of glyphosate resistance in susceptible (S) and resistant (R) horseweed ( Conyza canadensis  (L.)Cronq.) populations collected from regions across the USA (Arkansas, Delaware, Ohio, Virginia, Washington) was investigated. At two-leaf stage approximately the same ED 50  values were observed for the S and R populations, while at the rosette stage the R biotypes wereapproximately three times more resistant than the S biotypes. After treatment with severe glyphosate doses (more than 1 ·  the recom-mended field rate), different morphological responses in R and S biotypes were observed. In S biotypes, the first phytotoxic effects werefound in the meristematic tissues, while in the R biotypes the first phytotoxic effects were observed in leaves. At 2 to 4 weeks after thetreatment, R plants recovered by emitting new leaves and/or new branches from the center of the rosette. A significant increase of themean number of branches per surviving R plants as a function of glyphosate-applied dose was observed. As regards the physiologicalmechanism of resistance, the main difference between R and S biotypes was the dissimilar mobility of glyphosate in the whole plant. Inthe R biotypes the herbicide was less translocated in the downward direction (from leaves to roots) and more translocated in the upwarddirection (from culm to leaves) with respect to the S biotypes. Finally, in R biotypes the relative level of EPSPS mRNA was from 1.8 to3.1 times higher than that found in S biotypes. On the basis of obtained results three factors may concur to glyphosate resistance in theinvestigated R biotypes: impaired translocation of the herbicide, increase in EPSP synthase transcript levels, and enhanced ramification.   2006 Elsevier Inc. All rights reserved. Keywords: Conyza canadensis ; Glyphosate resistance; Resistance mechanism; Uptake; Metabolism; Translocation; EPSPS mRNA levels 1. Introduction Glyphosate [ N  -(phosphonomethyl) glycine] is a nonse-lective foliar-applied herbicide that has been used for over20 years for the management of annual, perennial, andbiennial herbaceous species of grasses, sedges, and broad-leaf weeds, as well as woody brush and tree species [1].Commercialization of engineered glyphosate resistance inseveral crop species has further expanded the in-crop useof the herbicide [2]. In addition to being highly effectiveon a broad spectrum of annual and perennial weed speciescommon to many cropping systems, glyphosate has otherfavorable environmental characteristics such as strongsorption to soil and very low toxicity to mammals, birds,and fish [2]. These factors have contributed to glyphosatebeing the most widely used herbicide in the world [3].Although strong arguments have been proposed againstthe likelihood of weeds developing resistance to glyphosate[1], resistance to glyphosate has occurred. The first con-firmed case of weed resistance to glyphosate was rigid rye-grass ( Lolium rigidum ) in Australia [4]. A secondoccurrence of glyphosate-resistant rigid ryegrass in Austra-lia was confirmed soon thereafter [5]. Additional cases of rigid ryegrass resistance to glyphosate were confirmed inCalifornia in 1998 and South Africa in 2001 [6].Differences in glyphosate uptake, translocation, ormetabolism were disregarded as potential resistance mech-anisms in  L. rigidum , suggesting that resistance may be 0048-3575/$ - see front matter    2006 Elsevier Inc. All rights reserved.doi:10.1016/j.pestbp.2006.01.004 * Corresponding author. Fax: +39 051 2096241. E-mail address:  gdinelli@agrsci.unibo.it (G. Dinelli). www.elsevier.com/locate/ypest PESTICIDE Biochemistry & Physiology Pesticide Biochemistry and Physiology 86 (2006) 30–41  conferred by EPSPS overexpression, an insensitive EPSPS,or improper targeting of glyphosate to the loci of action [7,8]. More recently, the mechanism of resistance in  L. rigidum  was credited to differences in cellulartranslocation of glyphosate [9]. Since reports of the L. rigidum  biotypes, glyphosate resistance was confirmedin  Eleusine indica  [10],  Lolium multiflorum  [11],  Conyzabonariensis , and  Plantago lanceolata  [12].Of much greater concern is the occurrence of glyphos-ate-resistant horseweed ( Conyza canadensis ) in soybeancropping systems in Delaware [13] and the rapid increasein its occurrence across the eastern corn belt [6]. Herbicideresistance in a weed like horseweed is the worst-case sce-nario. Notably, horseweed is adapted to conservation till-age agro-ecosystems, is essentially autogamous but cancross-pollinate (<10%), and produces many seeds that arewind-dispersed [14,15]. Furthermore, recent research reports that the resistance trait in glyphosate-resistanthorseweed is due to an incompletely dominant single locusnuclear gene [16]. Thus, the speed of the evolution of gly-phosate resistance in horseweed and the large geographicdistribution of these populations is understood [17].As with other glyphosate-resistant weeds, the mechanis-tic basis of the resistance in horseweed has not been easy toidentify, although a mechanism has been proposed. Differ-ential retention, uptake and metabolism were ruled out aspossible mechanisms of resistance in horseweed. However,a consistent correlation of translocation differences com-paring resistant and sensitive biotypes was observed andsuggested to be the mechanism of glyphosate resistance inhorseweed [18].This paper reports on the evaluation of six  C. canadensis biotypes from the USA (four resistant and two susceptible)by determining: (a) the resistance index, (b) the amount of endogenous shikimic acid accumulated in leaf tissues fol-lowing glyphosate treatments, (c) the absorption, metabo-lism, and translocation of glyphosate, and (d) theexpression levels of EPSP transcripts before and after gly-phosate application. 2. Materials and methods  2.1. Seed source and plant growth conditionsConyza canadensis  (L.) Cronq seeds were obtained fromregions across the USA including Delaware (DE), Virginia(VA), Arkansas (AR), Washington (WA), and Ohio (OH).Six horseweed biotypes were investigated. DE and RE bio-types were from Delaware and, respectively, indicated asresistant and susceptible population by the provider [13].DE seeds were collected in a soybean field from plants sur-viving after 1.6 kg ai ha  1 applications of glyphosate. REseeds were from horseweed plants growing in a fieldcropped annually with corn, soybeans or winter wheat withvery little glyphosate usage at the University of Delaware’sResearch and Education Center. AR accession was classi-fied as glyphosate-resistant on the basis of dose–responseexperiments carried out at the University of Arkansas(Kenneth Smith, personal communication). Seed samplesof susceptible WA biotype came from a roadside in NorthWestern Washington where probably some occasional gly-phosate applications occurred over the last few years(Tim Miller, personal communication). VA biotype, col-lected in northern Virginia, was considered as putativeresistant biotype following supplier indications (HenryWilson, personal communication). OH-resistant biotypewas collected from South Western Ohio in a field thathad been continuous Roundup Ready soybean for at least6 years and continuous soybean for more than 8 years andwhere glyphosate was the only herbicide used in the field(Mark Loux, personal communication).For all six biotypes, seeds were germinated in Petri dish-es with moist filter paper under controlled environmentconditions (20   C, 12 h photoperiod, 250  l mol/pho-tons m  2 s  1 ). Seedlings in the cotyledon growth stage of each biotype were singly transplanted in 6-cm pots contain-ing a 1:1 (v/v) peat:sand sterile potting mix. Plants wereplaced in a growth chamber set at 25   C and 70% relativehumidity (RH) day and 20   C and 50% RH night condi-tions, and light was supplemented to a 12-h photoperiodwith artificial illumination at 550  l mol photons m  2 s  1 .Plants were sub-irrigated as needed.  2.2. Dose–response experiments and morphological determinations Dose–response curves of   C. canadensis  biotypes weredetermined at both two-leaf and 25- to 30-leaf (rosette)growth stage (diameter of approximately 0.5 and 10 cm,respectively). For the dose–response test at the rosettestage, plants were selected within each biotype to matchin size and growth stage. Two-leaf stage plants weresprayed with the isopropylamine salt of glyphosate(Roundup Bioflow, 360 g ai L  1 , Monsanto) at doses of 0, 25, 50, 90, 140, 180, 360, 740, and 1480 g ai ha  1 , whilerosette stage plants were sprayed at doses of 0, 180, 360,740, 1480, and 3000 g ai ha  1 (Roundup Bioflow, 360 gai L  1 , Monsanto). The sprayer was equipped with a flat-fan nozzle at a height of 50 cm with an output volumeequivalent to 185 L ha  1 . The dose of 740 g ai ha  1 approximately corresponded to 0.9 ·  the recommendedfield rate for a single application in Roundup Ready(RR) soybean. Experimental design for dose–response testswas a randomized complete block (biotype) with three rep-lications of 25 plants for each herbicide dose. Plants wereassessed 21 DAT and were scored as dead or alive. Nonlin-ear regression analysis and ANOVA were used to deter-mine the effect of glyphosate dose and growth stage onplant survival of each horseweed biotype. A sigmoidallog-logistic model [19] was used to relate number of sur-vived plants as a percent of the untreated check ( Y  ) to gly-phosate dose ( x ) according to the following formula: Y    ¼ a = 1 þ e ð  x  ED 50 Þ = b . G. Dinelli et al. / Pesticide Biochemistry and Physiology 86 (2006) 30–41  31  In this equation,  a  is the difference of the upper andlower response limits (asymptotes), ED 50  is the herbicidedose that results in a 50% reduction in living plants,and  b  is the slope of the curve around ED 50 . The resis-tance index was determined by dividing the ED 50  valueof each R or S biotype by the ED 50  of the susceptibleRE biotype.Eight weeks after the treatment of rosette stage plantswith 0, 180, 360, 740, and 1480 g ae ha  1 , the mean numberof branches (with at least four leaves) per surviving plantwas recorded on three replicates of five plants for each Rbiotypes (AR, DE, OH, and VA).  2.3. Shikimic acid accumulation studies The experiment was designed as a 2  ·  3  ·  7 factorial,replicated five times, with glyphosate dose (0, 740 gae ha  1 ), time of plant harvesting (2, 4, and 8 DAT)and plant accession (RE, WA, AR, DE, OH, VA, andRR soybean) as factors. Horseweed plants at the rosettestage (diameter of approximately 10 cm; 25–30 leaves)and RR soybean plants at 4–6 leaf stage were sprayedas previously described (Section 2.2). At the harvestingtime (2, 4, and 8 DAT) shoot tissues (leaves and culms)were placed into freezer storage (  20   C) until processedand analyzed. Shikimate extraction and analysis werecarried out according to the method proposed byMueller et al. [20]. Frozen tissue was finely ground in liquid nitrogen using a mortar pestle. After grinding,the tissue was weighted into screw-cap tubes and 1 MHCl was added at a ratio of 5 ml of HCl solution per1 g of tissue. The tubes were placed on an orbital shakerat 1500 rpm for 24 h. The shikimate concentration of samples was determined by a HPLC system (BeckmanSystem Gold, Palo Alto, CA, USA) equipped withautoinjector and photodiode array detector set at thewavelength of 215 nm. A Phenomenex (Torrence, CA,USA) Luna NH 2  100 A column (250 mm  ·  4.0 mm;5  l m particle size) was employed with an injectionvolume of 20  l l. The isocratic system used was 90:9:1acetonitrile/water/phosphoric acid at the flow rate of 1 ml min  1 . The total run time was 15 min, withshikimate retention time at 6.6 min. Shikimateconcentration ( l g g  1 fresh weight) was quantified onthe basis of a six-point (5–100 ppm) external standardcurve generated with commercial shikimate standard(Sigma–Aldrich, Milan, Italy). The method detectionlimit for shikimate was approximately 3 ppm (w/w).For each accession the relative shikimate content(shikimate concentration of treated plants divided byshikimate concentration of untreated plants) wascalculated. Data were analyzed for completely random-ized design and the whole experiment was repeated twotimes. As the effect of repetition over time was not signif-icant, data of the two runs were combined and subjectedto ANOVA. Means were separated by LSD at the 0.05level.  2.4. Absorption, metabolism, and translocation studies 2.4.1. [ 14 C]-glyphosate dose and application [ 14 C]-glyphosate (specific activity 4540 MBq g  1 ) wasdissolved in ultrapure water to give a stock solution of 1000 mg ai ml  1 (4540 kBq ml  1 ). An aliquot (830  l l;3768 kBq) of the [ 14 C]-glyphosate stock solution was mixedwith 6670  l l of glyphosate commercial formulation(Roundup Bioflow, 360 g ai L  1 , Monsanto), diluted 240times with water. The final concentration of the radiolabelsolution (0.5 kBq  l L  1 ) was 1.5  l g ai l L  1 . Treatmentswere performed applying with a micro-applicator (Dispens-er PB 6000, Hamilton, USA) two 1- l l droplets to six leavesof plants at the rosette stage. At the time of [ 14 C]-glyphos-ate treatment the horseweed plants contained 4–5 whorls of leaves. The first top whorl comprised the youngest leaves(not fully expanded), while the fourth-fifth whorl was com-posed by the oldest leaves. The six treated leaves were inthe second whorl. By imagining treated leaves arrangedon the axis as to indicate the cardinal points they wereapproximately placed in the rosette according to the North,North-East, East-South, South, South-West, and West-North directions. For absorption and metabolism studies,the two 1- l l droplets were applied in the mid of adaxial leaf surface on the opposite sites of the major vein. For trans-location study in the downward direction, the two 1- l ldroplets were applied on adaxial leaf surface (opposite sitesof the major vein) at approximately 0.5 cm from the top of the leaf. Finally, for the translocation study in the upwarddirection the application of the two 1- l l droplets was per-formed on the base of leaf petiole (i.e., insertion of the leaf on the culm). The relationship between the glyphosate doseapplied and the recommended field rate was based on theestimation reported by Feng et al. [18]. In the present inves- tigation each treated plant received 18  l g ai, which isapproximately equivalent to a sublethal rate of 0.42 kgai ha  1 (0.5 ·  the recommended field rate) at 187 L ha  1 spray volume.  2.4.2. [ 14 C]-glyphosate absorption and metabolism Thirty plants per biotypes were treated. At 2, 4, and 8DAT, 10 plants per biotype were harvested. [ 14 C]-glyphos-ate remaining on the leaf surface was removed by gentlyshaking for 30 s in 10 ml methanol–water (1:9; v/v), fol-lowed by an additional washing for 30 s in 5 ml metha-nol–water (1:9; v/v). Unabsorbed radioactivity wasquantified from washing solution by liquid scintillationspectroscopy (LLS) (1409 Liquid Scintillation Analyzer,Wallac). Plants were then dissected into leaves, culm(crown meristems and immature leaves), and root. Soil par-ticles were removed from roots by low-pressure water jet.The different plant parts were weighed, frozen in liquidnitrogen, powdered, and extracted with ultrapure water(1:4 g fresh weight ml  1 ). After centrifugation (15,000  g  ,10 min), supernatant radioactivity was determined byLLS. The supernatants were vacuum-dried and re-dis-solved in 500  l l of ultrapure water before being analyzed 32  G. Dinelli et al. / Pesticide Biochemistry and Physiology 86 (2006) 30–41  by thin layer chromatography (TLC). Plant debris con-tained in the centrifugation pellet were dried and combust-ed in a biological oxidizer (Packard 387, USA). Theunextracted radioactivity was then quantified by LLS. Gly-phosate and its main metabolite, namely aminomethyl-phosphonic acid (AMPA), were separated by TLC. Fiftymicroliters of the 500- l l samples was spotted on 250  l m sil-ica gel TLC plates (SG60 with fluorescent marker; Merck,Germany) and developed in ethanol/water 15 N NH 3 OH(1:1). Electronic autoradiography and image analysis of TLC plates were performed using a Molecular Imager(Bio-Rad, USA). Image acquisition times were 12 h. Gly-phosate and AMPA were identified by comparing their R f   values (0.25 and 0.45, respectively) with those of authen-tic commercial standards. Data were analyzed for com-pletely randomized design and subjected to ANOVA.Means were separated by LSD at the 0.05 level.Considering all biotypes and sampling times, the meanradioactivity recovery was 87 ± 5% of the applied dose,while the mean unextracted radioactivity was 4 ± 3% of the applied dose. No significant difference in recoveredand unextracted radioactivity was observed between Sand R horseweed biotypes.  2.4.3. [ 14 C]-glyphosate translocation in whole plants The herbicide translocation was investigated in twodifferent experiments, hereinafter called ‘‘translocationin the downward direction’’ and ‘‘translocation in theupward direction.’’ Except for the different applicationof the radiolabel (see Section 2.4.1; adaxial leaf surfaceand leaf petiole application for downward and upwardtranslocation, respectively), the two experiments wereidentical. For each experiment 30 plants per biotypeswere treated. At 2, 4, and 8 DAT, 10 plants per biotypewere harvested. Treated leaves were rinsed and unab-sorbed radioactivity was quantified by LLS as previous-ly described (Section 2.4.2). Plants were then dissectedinto treated leaves, untreated leaves, culm (crown meris-tems and immature leaves), and roots. The differentplant parts were placed on a glass support and coveredin a fine plastic film. Electronic autoradiography andimage analysis were performed as previously described(Section 2.4.2). After autoradiography, total extractableand unextracted  14 C in the different plant parts weredetermined by LLS, as previously described (Section2.4.2). Data were analyzed for completely randomizeddesign and each experiment was repeated twice. As theeffect of repetition over time was not significant, dataof the two runs were combined and subjected toANOVA. Means were separated by LSD at the 0.05level.Considering all biotypes and sampling times, the meanradioactivity recovery was 84 ± 7% of the applied dose,while the mean unextracted radioactivity was 5 ± 3% of the applied dose. No significant difference in recoveredand unextracted radioactivity was observed between Sand R horseweed biotypes.  2.5. Semiquantitative RT-PCR analysis to assess the levelsof EPSPS transcripts 2.5.1. RNA extraction and cDNA synthesis Before and 21 days after the treatment with threeglyphosate doses (0, 740, and 1480 g ai ha  1 ), leaf sam-ples of each horseweed biotype were collected fromthree replicates of five plants. Only tissues sampled fromplants surviving glyphosate treatments (dose–responsetests of Section 2.2) were subjected to molecular analy-ses. Total RNA was isolated from leaf samples(  100 mg) of each individual S and R plant using the‘‘Nucleospin kit’’ (Macherey–Nagel–M-Medical) accord-ing to the manufacturer’s directions. RNA was quanti-fied spectrophotometrically. For complementary DNA(cDNA) synthesis, 0.5  l g of total RNA was incubatedwith oligo(dT) primers and MMLV reverse transcriptase(M-Medical) at 37   C for 60 min according to the man-ufacturer’s instructions. Bulk cDNA, representative of the six investigated horseweed biotypes, were construct-ed by mixing equivalent volumes of cDNA samplesextracted from single individuals.  2.5.2. Standard PCR for EPSPS  EPSPS gene expression determinations were conduct-ed by the multiplex titration RT-PCR (MTRP) proce-dure described in Nebenfu¨hr and Lomax [21] withminor modifications. For PCR amplification of eachbulk cDNA sample, a series of ten 0.5-fold serial dilu-tions was constructed. The first sample in each seriescontained a constant amount (20 ng) of cDNA. Twomicroliters of cDNA was amplified with 200  l M deoxy-ribonucleotide triphosphate, 5.6 mM MgCl 2 , 0.4 and0.3  l M of each EPSP and EF primers, respectively,and 1 U of   Taq  polymerase (M-Medical). Amplificationwas as follows: 95   C for 10 min; 35 cycles at 95   C for40 s, 59.2   C for 40 s, and 72   C for 40 s; and a finalextension at 72   C for 5 min. Reactions were carriedout in a T-Gradient Thermal Cycler (Biometra). RT-PCR products were separated on 1.5% agarose gels,scanned, and saved in tagged information file format(TIFF). PCR conditions and cycling parameters wereoptimized so that the amplification products were clearlyvisible on agarose gel and that the two sets of primersused in each reaction did not compete with each other.Each set of reactions always included a no-sample neg-ative control. RNA samples were tested for the presenceof genomic DNA by using extracted RNA directly as aPCR template, prior to cDNA synthesis, under thesame PCR conditions.For treated and untreated plants of each accessions, therelative transcript levels of EPSPS gene (EPSPS mRNAabundance of horseweed biotype divided by EPSPSmRNA abundance of untreated RE biotype) were calculat-ed. The obtained data were subjected to ANOVA and pro-cessed according to the experimental design adopted fordose–response tests (Section 2.2). G. Dinelli et al. / Pesticide Biochemistry and Physiology 86 (2006) 30–41  33   2.5.3. Primer selection and concentration Primers used were synthesized by Genenco (M-Medical,Milan, Italy) and the sequence was determined using thesoftware Primer 3 (developed by S. Rozen, H.J. Skaletsky,1997) available online at http://www-genome.wi.mit.edu.The design of EPSP primers was based on the published1766 bp mRNA sequence for EPSPS2 of   C. canadensis (GenBank Accession No. AY545667) while for the internalcontrol (a housekeeping gene, translation elongation factor1- a , EF1- a ) primers construction was based on the EF1- a gene sequence (GenBank Accession No. AK221176) of  Arabidopsis thaliana . Primers were always chosen accord-ing to the following parameters: length between 15 and20 bases, optimal 15–18 bases;  T  m  comprised between 50and 55   C, optimal  T  m  52–54   C; length of amplificationproducts between 170 and 500 bp so that the two fragmentsdo not overlap on the agarose gel. To determine primerspecificity, all sequences were compared with the GenBankusing the program Blast available at the National Centerfor Biotechnology Information website (www.ncbi.nlm.nih.gov). When both primer sequences showed homologyto the same gene, different from that of interest, they werediscarded. The following primers were used: EPSPS2-F 5 0 -GTTGCGGGACAAGCA-3 0 ( T  m  50.6   C) and EPSPS2-R5 0 -AGGGCAACCACAGCAA-3 0 ( T  m  51.8   C); EF1 a -F5 0 -TTAAGGCCGAGCGTG-3 0 ( T  m  50.7), and EF1 a -R5 0 -CGAAGGGGCTTGTCTGA-3 0 ( T  m  54.8). The  T  m sindicated in brackets were calculated according to the for-mula reported by Sambrook et al. [22]. EPSPS2 and EF1 a primers yielded amplification products of 180 and 500 bp,respectively. Both PCR fragments were sequenced (Genen-co, M-Medical, Milan, Italy) and the comparison of theirnucleotide composition, by using the NCBI GenBankBLAST programs, confirmed that the 180 bp and the500 bp fragment contained part of the selected EPSPSand housekeeping genes, respectively. Furthermore, the180 bp fragment showed a high matching degree with partof the mRNA sequences of EPSPS1 (GenBank AccessionNo. AY545666) and EPSPS3 (GenBank Accession No.AY545668) of   C. canadensis . 3. Results 3.1. Dose–response experiments Response of each horseweed biotype to increasing gly-phosate dose was best fit to a sigmoidal log-logistic mod-el, with  R 2 values ranging between 0.89 and 0.98. At thetwo-leaf stage, no significant differences in the level of resistance between S and R biotypes were observed(Fig. 1A). The ED 50  values were ranging between 110and 140 g ai ha  1 . At the rosette stage for either R andS biotypes, a general increase of ED 50  values was found(Fig. 1B). For the RE and WA susceptible biotypes theED 50  values were 340 and 530 g ai ha  1 , respectively.In contrast for the R biotypes the ED 50  values wereranging between 1360 and 1610 g ai ha  1 . The R bio-types were 4.0–4.7 times more resistant than the RE sus-ceptible biotype.During dose–response tests at the herbicide doses corre-spondingto1.8 · and3.6 · therecommendedfieldratediffer-ent morphological responses of R and S biotypes wereobserved. In general, within 5–7 DAT in S biotypes the firstphytotoxiceffectsweredetectedinthemeristematictissuesof the growing points (i.e., center of the rosette) (Figs. 2A–B).These phytotoxic effects mainly consisted in a strong reduc-tion of the meristematic activities (i.e., emission of newleaves) and in a manifest change of pigmentation (fadingfromdarkgreentopalegreen).Noevidentphytotoxiceffectswere observed at the leaf level. In contrast, within 1–7 DATin R biotypes the first phytotoxic effects were exclusivelydetected at the leaf level (i.e., irreversible dehydration of leaves) (Figs. 2D–E). From 10–21 DAT the S plants died(Fig. 2C), while R plants recovered by emitting new leavesfrom the growing points (Fig. 2F). In general, in R biotypesall the treated leaves dried up and were substituted by newleavesemitted bymeristematictissuesofthecrown.Inaddi-tion, in R biotypes approximately 30–70% of survivingplantsemittednewbranches.ThemeannumberofbranchespersurvivingRplantasafunctionoftheappliedglyphosatedoseisreportedinFig.3.UntreatedplantsofthefourRbio-types exhibited no or negligible ramification. Increasing theglyphosate dose a linear trend was observed for AR andDEbiotypes,whileOHandVAbiotypesexhibitedasigmoi-dal relationship between dose applied and mean number of branches. After the treatment with 1440 g ai ha  1 in the dif-ferent horseweed accessions the mean number of branchesper surviving R plant ranged between 1.7 and 3.0. 3.2. Shikimic acid accumulation in S and R biotypes Shikimate accumulated in concentrations greater thanbackground levels after glyphosate treatment in all horse-weed biotypes. There were no significant differences( P   = 0.05) in shikimate levels between R and S biotypes 2DAT: in treated plants the content of shikimic acid wasfrom 174 to 245 times higher than that found in untreatedplants (5.7 ± 0.8 ppm in S and 4.3 ± 0.6 ppm in R)(Fig. 4A). The R and S accessions differed in the trend overtime in shikimate relative content: it decreased from 2 to 8DAT in the R biotypes, whereas it increased from 2 to 8DAT in S biotypes (Figs. 4B–C). In particular, 8 DATthe mean relative shikimate content for R and S biotypeswas 124 ± 84 and 1722 ± 182, respectively. Over the timeafter glyphosate treatment only a slight increase of shikim-ate level (2–4 times with respect to the untreated control)was observed in RR soybean, notably characterized by gly-phosate-resistant EPSPS (Figs. 4A–C). 3.3. [ 14 C]-glyphosate absorption, metabolism and translocation Over the time there were no significant differences in thefoliar uptake of R and S horseweed biotypes (data not 34  G. Dinelli et al. / Pesticide Biochemistry and Physiology 86 (2006) 30–41
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