EPSPS Gene Amplification in Glyphosate-Resistant Italian Ryegrass

Subscriber access provided by HKU Libraries

Article

EPSPS Gene Amplification in Glyphosate-Resistant Italian Ryegrass (Lolium perenne ssp. multiflorum) Populations from Arkansas, USA Reiofeli A. Salas, Robert C. Scott, Franck E. Dayan, and Nilda R. Burgos J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00018 • Publication Date (Web): 11 Mar 2015 Downloaded from http://pubs.acs.org on May 8, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35

Journal of Agricultural and Food Chemistry

EPSPS Gene Amplification in Glyphosate-Resistant Italian Ryegrass (Lolium perenne ssp. multiflorum) Populations from Arkansas, USA Reiofeli A. Salas†, Robert C. Scott §, Franck E. Dayan#, and Nilda R. Burgos†*

†

Department of Crop, Soil, and Environmental Sciences, Fayetteville, Arkansas 72704, United States §

University of Arkansas Extension, P. O. Box 357 Lonoke, Arkansas 72086, Unites States

#

USDA-ARS Natural Products Utilization Research Unit, Thad Cochran Research Center, P. O. Box 1848, University, Mississippi 38677, United States *To whom correspondence should be addressed: Telephone: +1-479-575-3984. Fax: +1-479575-3975. E-mail: [email protected]

1

ACS Paragon Plus Environment


1

ABSTRACT: Glyphosate-resistant Italian ryegrass was detected in Arkansas, USA in 2007. In

2

2014, 45 populations were confirmed resistant in eight counties across the state. The level of

3

resistance and resistance mechanisms in six populations were studied to assess the severity of the

4

problem and identify alternative management approaches. Dose-response bioassays, glyphosate

5

absorption and translocation experiments, herbicide target (EPSPS) gene sequence analysis, and

6

gene amplification assays were conducted. The dose causing 50% growth reduction (GR50) was 7

7

to 19 times higher for the resistant population than the susceptible standard. Uptake and

8

translocation of 14C-glyphosate was similar in resistant and susceptible plants and no mutation in

9

the EPSPS gene known to be associated with resistance to glyphosate was detected. Resistant

10

plants contained 11-fold to >100-fold more copies of the EPSPS gene than the susceptible plants,

11

while the susceptible plants had only one copy of EPSPS. Plants surviving the recommended

12

dose of glyphosate contained at least 10 copies. The EPSPS copy number was positively related

13

to glyphosate resistance level (r=80). Therefore, resistance to glyphosate in these populations is

14

due to multiplication of the target site. Resistance mechanisms could be location-specific.

15

Suppressing the mechanism for gene amplification may overcome resistance.

16 17

KEYWORDS: gene amplification, glyphosate resistance, 5-enolpyruvylshikimate-3-phosphate

18

synthase (EPSPS), Italian ryegrass (Lolium perenne ssp. multiflorum)

19

2


Page 2 of 35

Page 3 of 35


20 21

INTRODUCTION Glyphosate is the world’s most important and widely used herbicide for postemergence

22

control of weeds.1-3 It is a potent inhibitor of the plastidic enzyme 5-enolpyruvylshikimate-3-

23

phosphate synthase (EPSPS) (EC 2.5.1.19), which catalyzes the reaction of shikimate-3-

24

phosphate and phosphoenolpyruvate to form 5-enolpyruvylshikimate-3-phosphate.4 Inhibition of

25

EPSPS by glyphosate results in the accumulation of shikimic acid and depletion of essential

26

aromatic acids, leading to plant death. When commercialized in 1974, glyphosate was mainly

27

used for total vegetation control because it is a nonselective, nonresidual, and environmentally

28

benign herbicide.5 Glyphosate usage dramatically increased in the past two decades following

29

the introduction of glyphosate-resistant crops in 1996.6 This expanded the use of glyphosate into

30

millions of crop hectares. Glyphosate-resistant crops accounted for a large majority of canola,

31

corn, cotton and soybean grown in 2011 in the United States.7 The massive adoption of

32

transgenic glyphosate-resistant crops caused excessive reliance on glyphosate for weed control

33

across vast areas.8

34

After three decades of glyphosate use, weed species have evolved resistance to

35

glyphosate. Resistance to glyphosate has evolved most often in the genetically diverse and

36

resistance-prone genera Conyza and Lolium, in situations with persistent, intense glyphosate

37

selection pressure.8 The first case of resistance to glyphosate was reported in a rigid ryegrass

38

(Lolium rigidum) population exposed to two to three glyphosate applications per year for 15

39

years.9 Today resistance to glyphosate occurs in 31 weed species around the world.10

40

Lolium species, particularly L. rigidum (rigid ryegrass), L. perenne (perennial ryegrass),

41

and L. perenne ssp. multiflorum (Italian ryegrass) are self-incompatible and can freely cross-

42

pollinate.11 They have a high propensity to evolve resistance to herbicides.11 So far, resistance 3



43

has evolved to six and ten different herbicide modes of action in Italian ryegrass and rigid

44

ryegrass, respectively.10 Today, rigid ryegrass ranks in the top 10 most important herbicide-

45

resistant species.10

46

Weed resistance to glyphosate results from a number of mechanisms. Reduced herbicide

47

translocation and target site (EPSPS) mutation have been the most common mechanisms in

48

glyphosate-resistant weeds.12 Impaired translocation mechanism has been reported in Lolium

49

spp.,13-16 horseweed (Conyza canadensis),17-19 and johnsongrass (Sorghum halepense).20,21 This

50

mechanism of resistance provide between 3- and 12-fold resistance to glyphosate.11 Target site

51

mutation, involving a proline to serine, alanine, threonine or leucine substitution at position 106

52

of the EPSPS in goosegrass (Eleusine indica)22-26 and Lolium species27-30 have been reported to

53

partially confer resistance to glyphosate. Substitutions of Pro182Thr and Tyr310Cys in the EPSPS

54

gene were recently reported in glyphosate-resistant sourgrass (Digitaria insularis).31 The level of

55

resistance due to these target site mutations is relatively low, ranging from 2- to 4-fold.32

56

Two other glyphosate resistance mechanisms have been reported more recently.

57

Horseweed33 and Lolium species34 reduced the amount of glyphosate that reaches the target site

58

by rapidly sequestering glyphosate into the vacuole. This mechanism has conferred 14-fold

59

resistance to glyphosate.34 High level of resistance to glyphosate in Palmer amaranth

60

(Amaranthus palmeri) and Italian ryegrass results from EPSPS gene ampliﬁcation.35, 36 EPSPS

61

gene amplification is heritable and correlates with glyphosate resistance in the F2 population.35

62

How rare these resistance mechanisms are is not yet known. The case with Palmer amaranth

63

indicates gene amplification could confer high levels of resistance.

64 65

Starting in the mid-2000, some Italian ryegrass populations have been surviving the spring vegetation desiccation treatments in Arkansas, USA about the same period it was reported 4


Page 4 of 35

Page 5 of 35


66

in Mississippi.16, 37 The severity of escapes and reduction in weed control has been escalating.

67

Lolium is the major weed problem in wheat in the Southern USA. This is also a problem in corn

68

and cotton as these crops are planted in the spring when Italian ryegrass is actively growing. Off-

69

season application of glyphosate has historically been effective in reducing Lolium infestation. It

70

also has already evolved resistance to acetyl-CoA carboxylase (ACCase), and acetolactate

71

synthase (ALS) herbicides used in wheat.38-43 The evolution of resistance to glyphosate has

72

compromised preplant weed management options as well and has increased the risk of economic

73

losses in crop production. The objectives of this study were to determine the level of resistance

74

to glyphosate in the Arkansas Lolium populations and investigate the mechanisms by which

75

selected populations survive a previously lethal dose of glyphosate.

76

MATERIALS AND METHODS

77

Plant Materials. Mature panicles from suspected glyphosate-resistant Italian ryegrass were

78

collected in Desha County, Arkansas in 2009 and 2010. Des05, Des09, and D8 populations were

79

collected from cotton fields; D4 and Des13 were from fallow fields; Des14 and Des15

80

populations were from soybean fields. Seeds were grown in the greenhouse maintained at 24/18

81

°C day/night temperatures with a 12-h photoperiod. Plants were watered daily and fertilized with

82

Miracle-Gro, a water-soluble all-purpose plant food containing 15–30–15% NPK, every 2 weeks.

83

Seedlings at three-leaf stage were sprayed with a discriminating dose of 870 g ae ha-1 glyphosate.

84

The surviving plants were grown to maturity for seed increase, and seeds from all plants in the

85

same population were bulked at harvest. Populations grown for seed increase were placed either

86

in the greenhouse (8 m apart, with other species in between as physical barriers) or outdoors (25

87

m apart). Multiple plants from one population were grouped together to cross-pollinate. Seeds

88

generated were used for the subsequent experiments. A susceptible population (98-3) that was 5



Page 6 of 35

89

not exposed to glyphosate selection was used as the susceptible standard (reference) in all

90

experiments.

91

Experiment 1. Population Dose-Response Bioassay. Seeds were planted into flats (25 x 25 x 5

92

cm) filled with Sunshine Mix LC1 soil (Sun Gro Horticulture Canada Ltd., Vancouver, British

93

Columbia, Canada). Flats were equally divided in two greenhouses because of space limitations;

94

one was maintained at 24/18 ºC and the other at 30/25 °C day/night temperatures at 12-h

95

photoperiod. Following emergence, plants were thinned into 15 seedlings per flat. Three- to four-

96

leaf seedlings (98-3, Des05, Des14, D4, D8 and D13) were treated with 8 doses of glyphosate

97

from 0 to 13920 g ae ha-1, which corresponds to 0 to 16 times the commercial dose of 870 g ae

98

ha-1. Treatments for 98-3 included a nontreated check and 11 doses of glyphosate from 13 to

99

3480 g ae ha-1 corresponding to 1/64 to 4 times the commercial dose of glyphosate. MON 78623

100

(58% v/w potassium salt of N-(phosphonomethyl)glycine; Monsanto Co., St. Louis, MO) was

101

applied with 0.25% nonionic surfactant (NIS). Glyphosate treatments were applied using a

102

laboratory sprayer equipped with a flat fan spray nozzle (TeeJet spray nozzles, Spraying Systems

103

Co., Wheaton, IL) delivering 187 L ha-1. The experiment was conducted in a randomized

104

complete block design with two replications. Each replication consisted of one tray (50 x 25 x 5

105

cm) accommodating two flats and placed in two greenhouses by replication.

106

The number of survivors was recorded at 28 days after treatment (28 DAT). Plants were

107

cut at the soil surface, stored in a dryer for 3 days, and the dry weight recorded. Data were

108

expressed as percentage of biomass reduction relative to the nontreated control. Regression

109

analysis was conducted using SAS JMP v. 10. The % biomass reduction and % mortality were

110

fitted to nonlinear, sigmoid, three-parameter logistic regression model defined by (Equation 1),

111

Y = c/[1 + Exp(-a*(X-b))] 6


[1]

Page 7 of 35


112

where Y is the biomass reduction or mortality expressed as a percentage of the nontreated

113

control; a is the growth rate; b is the inflection point; c is the asymptote; and X is the glyphosate

114

dose. The dose needed to kill 50% (LD50) of the population or cause 50% biomass reduction

115

(GR50) was calculated from the above equation.

116

Experiment 2. Single-plant dose-response bioassay. To determine the relationship of the

117

EPSPS copy number in glyphosate-resistant plants and resistance level, dose-response assays

118

were conducted using clones of 14 total individual plants from Des05, Des09, Des13, Des14,

119

Des15, and D8 (Table 1). Enough clones were propagated for each plant to accommodate the

120

dose range tested and calculate the resistance factor of each plant. Four plants from the

121

susceptible standard 98-3 were used as reference. The dose-response assay was conducted in the

122

greenhouse, maintained at 24/18 ºC at 12-h photoperiod. To accomplish this, tillers (adventitious

123

shoots at the base of grasses) of each plant were separated and planted in 15-cm pots to obtain 24

124

clones per plant that were sprayed with eight herbicide doses in three replicates. Resistant plants

125

were treated with glyphosate at 0, 218, 435, 870, 1740, 3480, 6960, and 13920 g ae ha-1, using a

126

laboratory sprayer calibrated to deliver 187 L ha-1. The susceptible plants were sprayed with

127

seven doses of glyphosate at 0, 54, 109, 218, 435, 653, 870, and 1740 g ae/ha-1. Glyphosate was

128

applied with 0.25% NIS. The overall effects of glyphosate such as chlorosis, stunting and

129

desiccation were visually assessed at 28 DAT relative to the nontreated control, using a scale of

130

0 to 100 where 0 = no visible injury and 100 = complete death. Visible injury was regressed

131

against glyphosate dose and modeled with a sigmoid, three-parameter, logistic function in SAS

132

JMP ver.11 (Eqn. 1). The amount of glyphosate needed to incur 50% injury (GR50) was obtained

133

from the regression equation.

7



134

Experiment 3. Absorption and Translocation of Glyphosate. Seeds from Des05, Des14, and

135

98-3 populations (Table 1) were planted in 2.5-cm pots in the greenhouse maintained at 24/18 ºC

136

day/night temperatures at 12-h photoperiod. Three weeks from emergence, when seedlings were

137

7.5- to 10-cm tall, formulated glyphosate (MON 78623) containing 0.25% NIS (Kinetic HV,

138

Helena Chemical Company, Memphis, TN, 38119) was sprayed at 870 g ae ha-1 in 187 L ha-1

139

spray volume and then spotted with 4-µL of herbicide solution containing 1.776 kBq 14C-

140

glyphosate. Plants were harvested at 24 and 48 h after treatment (HAT) and sectioned into four

141

parts: treated leaf, above treated leaf, below treated leaf, and roots. To remove nonabsorbed

142

glyphosate, the treated 5-cm portion of the treated leaf was rinsed for 15 s with 1 ml of a

143

methanol:water (1:1 v/v) solution containing 0.25% v/v NIS. The rinsate was collected in a 20-

144

ml scintillation vial, mixed with 10 ml of scintillation cocktail, and radio-assayed by liquid

145

scintillation spectroscopy (LSS) (Packard Tri-Carb 2100TR Liquid Scintillation Spectrometer,

146

Packard Instrument Co., 220 Warrenville Rd., Downers Grove, IL 60515) to determine the

147

amount of nonabsorbed 14C. After rinsing the treated leaf and dissection, all plant parts were

148

dried for 48 h at 50 ºC. Individual plant parts were oxidized (Biological Oxidizer OX500, R.J.

149

Harvey Instrument Corporation, 11 Jane St., Tappan, NY 10983) and the released CO2 was

150

trapped in 15 ml of scintillation cocktail and radio-assayed using LSS. Absorbed glyphosate was

151

calculated by dividing the amount of 14C recovered from the oxidized plant parts by the sum of

152

the radioactivity contained in the leaf wash and that recovered from the oxidized plant parts. The

153

distribution of 14C-glyphosate in plant tissues was expressed as a percentage of absorbed

154

radioactivity. The experiments were arranged in a completely randomized block design with four

155

replicates. In the absorption experiment, a factorial scheme with two factors, (population and

156

harvest time) was tested by ANOVA. The translocation experiment which had three factors

8


Page 8 of 35

Page 9 of 35


157

(population, plant section, and harvest time) was also analyzed by ANOVA in SAS JMP Pro

158

v.11.

159

Experiment 4. EPSPS Gene Sequencing. Two populations were randomly chosen for EPSPS

160

gene sequencing (Table 1). Seeds from Des05 and Des14 populations were planted in 4.5-cm

161

pots filled with Sunshine Mix LC potting soil. Tillers of 12 plants from Des05 and 13 from

162

Des14 were divided and transplanted into two pots. One set of clones was cut to 8 cm, allowed to

163

regrow to 12 cm, then sprayed with glyphosate at 870 g ae ha-1. Clones that survived at 28 DAT

164

were classified as resistant (R); otherwise they were susceptible (S). Clones of all 13 plants from

165

Des14 population survived while only 7 clones from Des05 population remained alive, 28 DAT.

166

The corresponding nontreated clones of verified R plants were used for sequencing of the EPSPS

167

gene.

168

Young leaf tissues of 7 and 13 confirmed R plants from Des05 and Des14 populations,

169

respectively, were harvested and stored at -80°C for RNA extraction. In addition, leaf tissues of 5

170

and 10 S plants, respectively, from Des05 and 98-3 populations also were harvested. Leaf tissues

171

were ground into fine powder in liquid nitrogen using mortar and pestle. Total RNA was

172

extracted using PureLink RNA Mini kit (Life Technologies, Carlsbad, California 92005). First-

173

strand complementary DNA (cDNA) was synthesized using Oligo(dT)20 supplied in the Improm-

174

II Reverse Transcription System first-strand cDNA synthesis kit (Promega, Madison, WI, USA).

175

Forward primer LPM2F (5’- TSCAGCCCATCARGGAGATCT-3’) designed by Perez-Jones et

176

al. (2005)44 and reverse primer LPM2R1 (5’- CTAGTTCTTCAC GAAGGTGCTTA-3’)

177

designed by Salas et al. (2012)36 were used to amplify the EPSPS gene. The primer pair

178

amplified a 915-bp fragment of the EPSPS region encompassing codon 106 where the known

179

resistance-conferring point mutation occurs. 9



180

The polymerase chain reaction (PCR) was performed in a 25-µL reaction mixture

181

consisting of 4 µL cDNA, 0 .4 µM of both forward and reverse primers, 12.5 µL of Taq2x

182

master mix (New England Biolabs Inc., Ipswich, MA, USA) and nuclease-free water. The

183

reaction mixture was loaded in a thermal cycler (PTC-200, MJ Research, Inc., MA) programmed

184

for the following temperature profile: initial denaturation at 94 °C for 3 min, 35 cycles of 94 °C

185

for 30 s; annealing at 57.5 °C for 30 s; elongation at 72 °C for 90 s, and final extension at 72 °C

186

for 10 min. PCR products were cleaned using Wizard SV Gel and PCR Clean-Up System

187

(Promega, Madison, WI, USA) before sequencing. The resulting DNA sequences were cleaned,

188

aligned using the EPSPS sequence of Italian ryegrass as reference, and analyzed for

189

polymorphisms using Sequencher v.5 and Bioedit v.7 softwares.

190

Experiment 5. EPSPS Copy Number Determination. Leaf tissues from 10 confirmed R plants

191

of selected populations (Des05, Des14, and D8) and 10 plants of the S population 98-3 (Table 1)

192

were harvested and stored at -80 ºC. Plants used for whole-plant dose-response assays,

193

glyphosate translocation studies, and gene sequence analysis were among those analyzed for

194

gene copy number. Genomic DNA was extracted using hexadecyltrimethylammonium bromide

195

(CTAB) method45 following the modification of Sales et al.46 Approximately 100 mg of leaf

196

tissue from each plant was ground to a fine powder in liquid nitrogen, transferred to a 1.5-mL

197

centrifuge tube, and suspended in 500 ml of CTAB extraction buffer (100 mM Tris-HCl [pH

198

8.0], 20 mM ethylenediaminetetra-acetic acid [EDTA] [pH 8.0], 2 M NaCl, 2% CTAB, 2%

199

polyvinylpyrrolidone-40, 1 mM phenanthroline, and 0.3% β-mercaptoethanol). The aqueous

200

extracts were incubated in a water bath at 55 ºC for 40 min, treated with RNAse solution, and

201

extracted with an equal volume of phenol:chloroform:isoamyl alcohol solution (25:24:1). Total

202

nucleic acids were precipitated from the supernatant by addition of an equal volume of 10


Page 10 of 35

Page 11 of 35


203

isopropanol. The DNA pellet was washed with 500 µL of absolute ethanol, dried in a vacufuge

204

for 5 min, and resuspended in 30 mL of Tris-EDTA buffer (10 mM Tris-HCl [pH 8.0], 1 mM

205

EDTA). Genomic DNA was quantified using a NanoDrop spectrophotometer model ND-1000

206

(Thermo Scientific, Wilmington DE) and checked for quality by agarose gel electrophoresis.

207

Quantitative real-time PCR was used to measure the genomic copy number of EPSPS

208

relative to cinnamoyl-CoA reductase (CCR) genomic copy number in Italian ryegrass. Primer

209

sets and qPCR conditions were described previously.36 Triplicate genomic DNA templates (10

210

ng) were amplified in a 25-µL reaction mixture using Bio-Rad iQ SYBR Green Supermix by the

211

following thermoprofile on a Bio-Rad CFX96 Real-Time System PCR machine: 10 min at 94°C,

212

40 cycles of 94°C for 15 s and 60°C for 1 min then increasing the temperature by 0.5°C every 5 s

213

to generate the product melt-curve. Data was analyzed using CFX manager software (version

214

1.5). Primer efficiency and slope were 101.8 % and −3.279 for EPSPS and were 98.64% and

215

−3.355 for CCR. Negative controls did not have amplification products. Relative quantification

216

of EPSPS was calculated as ∆Ct = (Ct, CCR – Ct, EPSPS) according to the method described by

217

Gaines et al.35 Increase in EPSPS copy number was expressed as 2∆Ct. Each sample was run in

218

three technical replicates to calculate the mean and standard error of the increase in EPSPS copy

219

number. Results were expressed as fold increase in EPSPS copy number relative to CCR.

220

RESULTS AND DISCUSSION

221

Population Dose-Response Bioassay. Dose-response bioassay confirmed resistance of Des05,

222

Des13, Des14, D5, and D8 Italian ryegrass populations to glyphosate. The glyphosate dose that

223

caused 50% growth reduction (GR50) of the S population (98-3) was 101 g ae ha-1 glyphosate

224

while those of R populations ranged from 726 to 1264 g ae ha-1 glyphosate (Table 2 and Figure

11



225

1). Resistant populations Des05, Des14, Des13, D8, and D4, respectively, were 7, 8, 9, 13 and 19

226

times less sensitive to glyphosate than the S population based on the R/S ratio calculated from

227

GR50 values. The recommended field dose of glyphosate is 870 g ae ha-1; thus the R populations

228

required 0.8 to 1.5 times the normal field dose of glyphosate to reduce aboveground biomass or

229

ryegrass by 50%. The herbicide dose that caused 50% mortality (LD50) of the 98-3 population

230

was 184 g ae ha-1, whereas those of the R populations ranged from 1524 to 2719 g ae ha-1 (Table

231

2 and Figure 2). Based on LD50 values, Des13, Des05, Des14, D8, and D4 populations had 8, 9,

232

9, 12, and 15-fold nine-fold resistance relative to the 98-3 population (Table 2). These resistance

233

levels are similar to the resistance levels calculated from the GR50 values. More than 1.8 to 3.1

234

times the commercial dose of glyphosate was needed to kill 50% of the resistant populations.

235

Thus, growers would have to apply at least double these LD50 amounts to achieve 100% control

236

in the corresponding fields; however, applying twice the normal dose is not recommended in

237

commercial practice. The use of a higher dose also increases selection pressure and will

238

accelerate the evolution of resistant populations.47

239

The survival rate of populations is indicated by the LD50 values, which allows prediction

240

of seed deposits into the soil seed bank or potential patch expansion of the resistant plants.48 The

241

GR50 values may differ slightly from the LD50 values, but together, these provide a better picture

242

of population response to glyphosate. These inform us on the proportion of plants that are

243

expected to survive a glyphosate application and how healthy these remaining plants are. The

244

GR50 and LD50 values indicate that D4 and D8 populations are more resistant than Des05, Des14,

245

and D13. The full dose of glyphosate at 870 g ae ha-1 is no longer sufficient to control these five

246

R populations. The failure of glyphosate to control Italian ryegrass calls for alternative weed

247

management approaches to mitigate the evolution of resistance.37 The use of other herbicides, or

12


Page 12 of 35

Page 13 of 35


248

addition of other herbicides to glyphosate, are needed for complete weed control during field

249

preparations prior to planting the crop. Resistance to glyphosate in these populations was lower

250

than that of the first glyphosate-resistance population previously tested (Des03), which showed

251

23-fold resistance to glyphosate and required 3,880 g ae ha-1 glyphosate for 50% biomass

252

reduction.37 Nevertheless, the proportion of plants that escape glyphosate application in these

253

populations already cause ecological concerns (of continuing resistance evolution) and economic

254

detriment either due to yield loss from competition or additional costs for controlling escapes.

255 256

Uptake and Translocation of Glyphosate. Glyphosate is a potent herbicide because of its

257

ability to translocate in the plant to the apical meristems, root, and underground reproductive

258

organs of perennial plants via xylem and phloem.49 It is possible that changes in the translocation

259

pattern of glyphosate could endow resistance in plants. Glyphosate absorption was almost 60%

260

in both S and R plants (Table 3). This result was similar to what was reported for glyphosate-R

261

and -S Italian ryegrass from Mississippi16 but differs from those in Chile50 where R and S plants

262

absorbed >90% of 14C-glyphosate at 48 HAT. On average 40% and 56% of applied glyphosate

263

was absorbed by R and S plants at 24 and 48 HAT, respectively, and this response was not

264

significantly different between R (Des05 and Des14) and S populations (P > 0.05).

265

Radioactivity recovered from the treated leaf represented glyphosate loaded into the leaf,

266

but not translocated. The quantity of the 14C glyphosate recovered from the treated leaf at 48

267

HAT was not different between R (65 to 68% of absorbed) and S (71% of absorbed) plants

268

(Table 3). Translocation of 14C glyphosate into the roots and below the treated leaf was low,

269

ranging from only 11% to 19%; the radioactivity accumulated above the treated leaf was nil (1

270

to 3% of absorbed). The proportion of 14C-glyphosate recovered above the treated leaf, below the 13



271

treated leaf, and in the roots increased between 24 and 48 HAT; however, no significant

272

difference was detected between R and S populations in any plant sections at any harvest time.

273

These results were similar to what was reported on Lolium populations from Australia 51 and

274

California,52 where the distribution patterns of 14C-glyphosate did not differ between resistant

275

and susceptible plants. On the contrary, glyphosate-resistant Italian ryegrass populations from

276

Mississippi,16 Oregon,14 and Chile50 showed reduced translocation of glyphosate. Among the

277

Arkansas populations, however, resistance to glyphosate was not due to differences in uptake

278

and translocation.

279 280

Partial EPSPS Gene Sequencing. A 915-bp PCR fragment of the EPSPS gene was amplified

281

from cDNA of the R and S Italian ryegrass plants. This fragment encompassed amino acid

282

positions 77 to 381 in the 444 amino acid-long, mature EPSPS. The sequenced region included

283

the domain where point mutations are known to confer resistance to glyphosate, e.g. in corn at

284

Pro106 ,22-30 Gly101,53 and Thr102.54. Mutations at Pro182 and Tyr310 were also observed in

285

glyphosate-resistant Digitaria insularis31 although the impact of these mutations on EPSPS

286

enzyme activity has not been verified. Some nucleotide polymorphisms were detected in our

287

recent research on Italian ryegrass; however, none was associated with resistance to glyphosate

288

(data not shown). A mutation of Gly162Ser was detected in one resistant Des14 plant, but this

289

mutation was also found in a susceptible plant from Des05. Comparison of the EPSPS sequence

290

between glyphosate-R and -S plants revealed both synonymous and nonsynonymous

291

polymorphisms, but there were no amino acid changes in the catalytic sites that are known to

292

confer resistance to glyphosate (data not shown). Therefore, mutations in the EPSPS gene known

14


Page 14 of 35

Page 15 of 35


293

to endow resistance to glyphosate are not present in Des05 and Des14 Italian ryegrass

294

populations nor in the highly resistant population Des03.37

295

The absence of point mutations in the EPSPS gene that are exclusive to the R plants and

296

the absence of other mutations previously associated with resistance to glyphosate indicates that

297

target-site alteration is not the resistance mechanism in Des05 and Des14 populations.

298

Considering that target-site mutation was also not found in the highly glyphosate-resistant

299

population Des03,36 it can be deduced that target-site mutation is not the mechanism of

300

resistance among these tested glyphosate-resistant populations in Arkansas. It could also be that

301

target-site mutation is a rare resistance mechanism among Arkansas Italian ryegrass populations

302

for reasons not yet known. The rarity of target-site mutation as resistance mechanism to

303

glyphosate is associated with the strong conservation of the catalytic site where glyphosate

304

binds.32 Glyphosate interacts with 17 invariant amino acids in the active site of the EPSPS

305

protein55 and mimics the transition state in the enol transfer reaction.56 Because the active site of

306

the EPSPS protein is highly conserved, any mutation at this site is deleterious and causes

307

significant fitness penalty.56 Single-site mutation at Thr97 to Ile or Pro101 to Ser 54 or Gly96 to

308

Ala53 in E. coli impairs the binding of glyphosate but at the same time reduces affinity for the

309

substrate phosphoenolpyruvate. Deleterious mutations of critical catalytic sites are not exclusive

310

to EPSPS. Mutation in the psbA gene which confers resistance to triazine herbicide results in

311

reduced agroecological fitness.57 On the other hand, some mutations endowing target site–based

312

resistance to ACCase, and ALS herbicides have little or no fitness costs.12 Studies comparing

313

glyphosate-resistant goosegrass with Pro106Ser mutation versus susceptible population show

314

some differences, but it is not yet evident whether there are any fitness costs associated with

315

EPSPS–binding site-based resistance.58, 59 Sammons et al.32 reported that glyphosate has a very

15



316

low risk for target-site resistance and this holds true to date. Thus, we are starting to find

317

glyphosate-resistant populations that display resistance mechanisms other than target-site

318

mutation.

319 320

EPSPS Genomic Copy Number Relative to CCR. The degree of EPSPS gene amplification

321

differed greatly among the resistant plants within each population indicating high intrapopulation

322

genetic variability. Genomic EPSPS copy numbers relative to CCR was 1 for S plants (n =10),

323

whereas the relative EPSPS copy numbers for R plants (n = 30) were higher, ranging from 11 to

324

151 (Table 4, Figure 3). The EPSPS copy number in Des05, Des14, and D8 resistant plants

325

ranged from 11 to 121, 24 to 97, and 18 to 151, respectively. Italian ryegrass is an outcrossing

326

species; 60 thus, a high degree of genetic diversity would be expected within a population. A

327

broad range of EPSPS copy numbers also was detected in glyphosate-resistant Palmer amaranth

328

which is an obligate outcrossing species.61 Individual plants that were subjected to both dose-

329

response assay and EPSPS copy number analysis showed that the increase in copy number

330

strongly correlated with the level of resistance to glyphosate (r=0.80) (Figure 4). Plants with

331

higher GR50 values had higher EPSPS copy number (Figure 4). Other studies in Italian ryegrass36

332

and Palmer amaranth62, 63 populations also reported positive correlation between EPSPS copy

333

number with level of resistance to glyphosate. The observation that plants with higher resistance

334

levels to glyphosate had higher copies of EPSPS suggests that additional EPSPS gene copies

335

have additive effects in conferring resistance to glyphosate .61 Our data suggest that >10 EPSPS

336

copies are necessary to survive the recommended field dose of glyphosate. Gaines et al.61

337

reported that between 30 to 50 EPSPS genomic copies enabled Amaranthus palmeri to withstand

338

the toxic effects of glyphosate. The EPSPS enzyme activity was not determined in this study;

16


Page 16 of 35

Page 17 of 35


339

however, previous research61, 36 revealed that increased EPSPS copy number resulted in elevated

340

EPSPS activity. Not all of the amplified genes may translate to increased level of protein (e.g.

341

EPSPS) expression because transcriptional, post-transcriptional, and translational regulatory

342

elements also play a crucial role in gene expression.64 Salas et al.36 observed that Italian ryegrass

343

with similar EPSPS copy number did not show the same level of resistance. This could be a

344

manifestation of some gene copies not resulting in a protein product; or, the involvement of other

345

resistance mechanisms.

346

Amplification of the native, glyphosate-sensitive form of EPSPS enzymes had conferred

347

resistance to glyphosate in chicory (Cichorium intybus), rock harlequin (Capnoides

348

sempervirens), soybean (Glycine max), alfalfa (Medicago sativa), and tobacco (Nicotiana

349

tabacum) in plant tissue culture with glyphosate selection.65 Resistance to glyphosate in alfalfa,

350

soybean, and tobacco from progressive selection in plant cell cultures is attributed to

351

amplification of the EPSPS gene within the genome.66 In addition, a glyphosate-tolerant wild

352

carrot (Daucus carota) cell line generated by stepwise selection with glyphosate contained a 4-

353

to 25-fold increase in EPSPS.67 Similarly, a glyphosate-resistant petunia (Petunia hybrida) cell

354

line contained a 20-fold increase in EPSPS gene copies.68 Amplification of the EPSPS gene in

355

Palmer amaranth from Georgia, USA was recently reported by Gaines et al.35 in which genomes

356

of glyphosate-resistant plants contained 5-fold to >160-fold more copies of the EPSPS gene

357

resulting in a 40-fold EPSPS overexpression. This Palmer amaranth population showed 6- to 8-

358

fold resistance to glyphosate at the population level.69Although the EPSPS enzyme activity was

359

not investigated in our study, various studies indicated that EPSPS gene amplification results in

360

increased EPSPS enzyme activity in glyphosate-resistant plants.65-67 Gene amplification can

361

produce abundant supply of EPSPS enzymes that are able to counteract the loss of metabolic

17



362

function of enzyme molecules that are inhibited by glyphosate.70 This affords the plant continued

363

synthesis of aromatic acids for normal physiological function in the presence of glyphosate.

364

Given the lethal consequence of mutations in the binding site of the EPSPS gene, the

365

selected glyphosate-resistant plants harbor other mechanisms of survival, such as EPSPS gene

366

amplification. Gene duplication serves as a mechanism of adaptation to a changing environment

367

such as environmental stresses.71, 72 Intense glyphosate usage as a selector will favor survival of

368

plants with elevated copies of the glyphosate target gene EPSPS.36 Amplification of EPSPS

369

provides a certain level of glyphosate resistance in plants;65 however, the stability EPSPS gene

370

amplification and the contribution of each additional copy is not clearly understood. EPSPS gene

371

amplification in Palmer amaranth is heritable35 but the manner by which it is inherited is

372

complex. Inheritance of EPSPS amplification in Palmer amaranth from North Carolina and

373

Georgia was consistent with polygenic inheritance.63, 73 However, studies on plant cell culture

374

revealed that gene amplification varied considerably for different cell cultures.65 Stable resistance

375

was achieved with chicory,74 tomato,75 and tobacco,76 but resistance to glyphosate was slowly

376

reduced or lost entirely when glyphosate selection was removed in Madagascar periwinkle

377

(Catharanthus roseus)77 and wild carrot cell cultures.78 In the absence of glyphosate selection

378

pressure, resistance may be reduced, suggesting a fitness penalty for cells containing amplified

379

genes.65 However, the massive amplification of EPSPS gene in glyphosate-resistant Palmer

380

amaranth did not cause any fitness cost.79 Other than endowing resistance to glyphosate, no

381

physiological advantage has been documented thus far as a consequence of EPSPS gene

382

amplification.

383

In conclusion, the resistance to glyphosate in all Italian ryegrass populations analyzed is

384

conferred by amplification of the EPSPS gene. This is the primary mechanism for resistance to

18


Page 18 of 35

Page 19 of 35


385

glyphosate among Italian ryegrass populations in Arkansas. EPSPS copy number correlated

386

positively with resistance level to glyphosate. The mechanism of EPSPS gene amplification and

387

the nature of its heritability are not yet known. Information on the mechanism of amplification,

388

stability and genetic inheritance of copy number, and fitness penalty that may be associated with

389

EPSPS gene amplification is necessary to fully understand the novel mechanism of resistance to

390

glyphosate due to EPSPS gene amplification in Italian ryegrass.

391 392

ABBREVIATIONS USED

393

EPSPS , 5-enolpyruvylshikimate-3-phosphate synthase; ACCase, acetyl-CoA carboxylase; ALS,

394

acetolactate synthase; NIS, nonionic surfactant; DAT, days after treatment; LSS, liquid

395

scintillation spectroscopy; HAT, hours after treatment; R, glyphosate-resistant; S, glyphosate-

396

susceptible; CCR, cinnamoyl-CoA reductase; CTAB, hexadecyltrimethylammonium bromide;

397

PCR, polymerase chain reaction.

398

ACKNOWLEDGMENT

399

The authors thank James Dickson for providing plant materials for the ryegrass germplasm

400

collection. We also thank Seth Bernard Abugho, George Botha, Leopoldo Estorninos, Shilpa

401

Singh, Vijay Singh, and Te Ming Tseng for their assistance in tissue collection and establishing

402

greenhouse experiments.

403 404

19



405

REFERENCES

406 407

(1) Franz, J.; Mao, M.; Sikorski, J. Glyphosate: A unique global herbicide; American Chemical Society: Washington, DC, 1997; p 653.

408 409

(2) Duke, S. O.; Powles, S. B. Glyphosate: a once-in-a-century herbicide. Pest Manag. Sci. 2008, 64, 319-325.

410 411

(3) Powles, S. B.; Yu, W. Evolution in action: Plants resistance to herbicides. Annu. Rev. Plant Biol. 2010, 61, 317-347.

412 413 414

(4) Steinrücken, H. C.; Amrhein, N. The herbicide glyphosate is a potent inhibitor of 5enolpyruvyl-shikimic acid-3-phosphate synthase. Biochem. Biophys. Res. Commun. 1980, 94, 1207–1212.

415 416 417

(5) Dyer, W. E. Resistance to glyphosate. In Herbicide Resistance in Plants: Biology and Biochemistry; Powles, S. B.; Holtum, J. A. M., Eds.; Lewis Publishers: Boca Raton, FL, 1994; pp 229-237.

418 419

(6) Woodburn, A. Glyphosate: Production, pricing and use worldwide. Pest Manag. Sci. 2000, 56, 309-312.

420 421 422

(7) Department of Agriculture, Economic Research Service (USDA ERS), Adoption of genetically engineered crops in the US [dataset]. Available athttp://www.ers.usda.gov/Data/BiotechCrops/ (accessed October 12, 2011).

423 424

(8) Powles, S. B. Evolved glyphosate-resistant weeds around the world: lessons to be learnt. Pest Manag. Sci. 2008, 64, 360−365.

425 426

(9) Powles, S. B.; Lorraine-Colwill, D. F.; Dellow, J. J.; Preston, C. Evolved resistance to glyphosate in rigid ryegrass (Lolium rigidum) in Australia. Weed Sci. 1998, 16, 604–607.

427 428

(10) Heap, I. International Survey of Herbicide Resistant Weeds, available at http://www.weedscience.org (accessed December 26, 2014).

429 430 431

(11) Preston, C.; Wakelin, A. M.; Dolman, F. C.; Bostamam, Y.; Boutsalis, P. A decade of glyphosate-resistant Lolium around the world: mechanisms, genes, fitness, and agronomic management. Weed Sci. 2009, 57, 435−441.

432 433

(12) Powles, S. B.; Preston C. Evolved glyphosate resistance in plants: Biochemical and genetic basis of resistance. Weed Tech. 2006, 20, 282-289.

434 435 436

(13) Lorraine-Colwill, D. F.; Powles, S. B.; Hawkes, T. R.; Hollinshead, P. H.l.; Warner, S. A. J.; Preston, C. Investigations into the mechanism of glyphosate resistance in Lolium rigidum. Pestic. Biochem. Physiol. 2003, 74, 62–72.

437 438

(14) Perez-Jones, A.; Park, K. W.; Polge, N.; Colquhoun, J.; Mallory-Smith, C. A. Investigating the mechanism of glyphosate resistance in Lolium multiflorum. Planta 2007, 226, 395-404.

439 440

(15) Yu, Q.; Cairns, A.; Powles, S.; Glyphosate, paraquat and ACCase multiple herbicide resistance evolved in a Lolium rigidum biotype. Planta 2007, 225, 499-513.

20


Page 20 of 35

Page 21 of 35


441 442 443

(16) Nandula, V. K.; Reddy, K. N.; Poston, D. H.; Rimando, A. M.; Duke, S. O. Glyphosate tolerance mechanism in Italian ryegrass (Lolium multiflorum) from Mississippi. Weed Sci. 2008, 56, 344-349.

444 445 446

(17) Feng, P. C.; Tran, M.; Chiu, T.; Sammons, R. D.; Heck, G. R.; Jacob, C. A. Investigations into glyphosate-resistant Conyza canadensis: retention, uptake, translocation, and metabolism. Weed Sci. 2004, 52, 498–505.

447 448

(18) Koger, C. H.; Reddy, K. N. Role of absorption and translocation in the mechanism of glyphosate resistance in horseweed (Conyza canadensis). Weed Sci. 2005, 53, 84–89.

449 450 451

(19) Dinelli, G.; Marotti, I.; Bonetti, A.; Minelli, M.; Catizone, P.; Barnes, J. Physiological and molecular insight on the mechanisms of resistance to glyphosate in Conyza canadensis (L.) cronq. biotypes. Pest. Biochem. Physiol. 2006, 86, 30–41.

452 453 454

(20) Riar, D. S.; Norsworthy, J. K.; Johnson, D.B.; Scott, R. C.; Bagavathiannan, M. Glyphosate resistance mechanism in a johnsongrass (Sorghum halepense) biotype from Arkansas. Weed Sci. 2011, 59, 299-304.

455 456 457

(21) Vila-Aiub, M. M.; Balbi, M. C.; Distéfano, A. J.; Fernández, L.; Hopp, E.; Yu, Q.; Powles, S. B. Glyphosate resistance in perennial Sorghum halepense (johnsongrass), endowed by reduced glyphosate translocation and leaf uptake. Pest Manag. Sci. 2011, 68, 430-436.

458 459 460

(22) Baerson, S. R.; Rodriguez, D. J.; Tran, M.; Feng, Y.; Biest, N. A.; Dill, G. M. Glyphosateresistant goosegrass: Identification of a mutation in the target enzyme 5enolpyruvylshikimate-3-phosphate synthase. Plant Physiol. 2002, 129, 1265–1275.

461 462 463

(23) Chong, J. L.; Wickneswari, R.; Ismail, B. S.; Salmijah, S. Nucleotide variability in the 5enolpyruvylshikimate-3-phosphate synthase gene from Eleusine indica (L.) Gaertn. Pak. J. Biol. Sci. 2008, 11, 476–479.

464 465 466

(24) Ng, C. H.; Wickneswari, R.; Salmijah, S.; Teng, Y. T.; Ismail, B. S. Gene polymorphisms in glyphosate-resistant and –susceptible biotypes of Eleusine indica from Malaysia. Weed Res. 2003, 43, 108–115.

467 468 469

(25) Yuan, C.; Hsieh, Y.; Chiang, M. Glyphosate-resistant goosegrass in Taiwan: cloning of target enzyme (EPSPS) and molecular assay of field populations. Plant Prot. Bull. 2005, 47, 251–261.

470 471 472 473

(26) Kaundun, S. S.; Zelaya, I. A.; Dale, R. P.; Lycett, A. J.; Carter, P.; Sharples, K. R.; McIndoe, E. Importance of the P106S target-site mutation in conferring resistance to glyphosate in a goosegrass (Eleusine indica) population from the Philippines. Weed Sci. 2008, 56, 637-646.

474 475 476 477

(27) Jasieniuk, M.; Ahmad, R.; Sherwood, A.; Firestone, J.; Perez-Jones, A.; Lanini, W.; Mallory-Smith, C. A.; Stednick, Z. Glyphosate-resistant Italian ryegrass (Lolium multiflorum) in California: distribution, response to glyphosate and molecular evidence for an altered target enzyme. Weed Sci. 2008, 56, 496–502.

478 479

(28) Simarmata, M.; Penner, D. The basis for glyphosate resistance in rigid ryegrass (Lolium rigidum) from California. Weed Sci. 2008, 56, 181–188.

480 481

(29) Wakelin, A. M.; Preston, C. A target-site mutation is present in a glyphosate resistant Lolium rigidum population. Weed Res. 2006, 46, 432–440. 21



482 483 484 485

(30) Kaundun, S. S.; Dale, R. P.; Zelaya, I. A.; Dinelli, G.; Marotti, I.; McIndoe, E.; Cairns, A. A novel P106L mutation in EPSPS and an unknown mechanism(s) act additively to confer resistance to glyphosate in a South African Lolium rigidum population. J. Agric. Food Chem. 2011, 59, 3227-3233.

486 487 488

(31) De Carvalho, L. B.; Cruz-Hipolito, H. E.; Rojano-Delgado, A. M.; de Prado, R.; GilHumanes, J.; Barro, F.; de Castro, M. D. L. Pool of resistance mechanisms to glyphosate in Digitaria insularis. J. Agric. Food Chem. 2012, 60, 615-622.

489 490 491

(32) Sammons, R. D.; Heering, D. C.; Dinicola, N.; Glick, H.; Elmore, G. A. Sustainability and stewardship of glyphosate and glyphosate-resistant crops. Weed Technol. 2007, 21, 347– 354.

492 493

(33) Ge, X.; d’Avignon, D. A.; Ackerman, J. J.; Sammons, R. D. Rapid vacuolar sequestration: the horseweed glyphosate resistance mechanism. Pest Manag. Sci. 2010, 66, 345−348.

494 495 496 497

(34) Ge, X.; d’Avignon, D. A.; Ackerman, J. J. H.; Collavo, A.; Sattin, M.; Ostrander, E. L.; Hall, E. L.L.; Sammons, R. D.; Preston, C. Vacuolar glyphosate-sequestration correlates with glyphosate resistance in ryegrass (Lolium spp.) from Australia, South America and Europe: a 31P-NMR investigation. J. Agric. Food Chem. 2012, 60, 1243-1250.

498 499 500 501 502

(35) Gaines, T. A.; Zhang, W.; Wang, D.; Bukun, B.; Chrisholm, S. T.; Shaner, D. L.; Nissen, S. J.; Patzoldt, W. L.; Tranel, P. J.; Culpepper, A. S.; Grey, T. L.; Webster, T. M.; Vencill, W. K.; Sammons, R. D.; Jiang, J.; Preston, C.; Leach, J. E.; Westra, P. Gene amplification confers resistance in Amaranthus palmeri. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10291034.

503 504 505

(36) Salas, R. A.; Dayan, F. E.; Pan, Z.; Watson, S. B.; Dickson, J. W.; Scott. R. C.; Burgos, N. R. EPSPS gene amplification in glyphosate-resistant Italian ryegrass (Lolium perenne ssp. multiflorum) from Arkansas. Pest Manag. Sci. 2012, 68, 1223-1230.

506 507 508

(37) Dickson, J. W.; Scott, R. C.; Burgos, N. R.; Salas, R. A.; Smith, K. L. Confirmation of glyphosate-resistant Italian ryegrass (Lolium perenne ssp. multiflorum) in Arkansas. Weed Technol. 2011, 25, 674-679.

509 510

(38) Kuk, Y. I.; Burgos, N. R.; Talbert, R. E. Cross- and multiple resistance of diclofop-resistant Lolium spp. Weed Sci. 2000, 48, 412–419.

511 512

(39) Kuk, Y. I.; N. R. Burgos. Cross-resistance profile of mesosulfuron-methyl resistant Italian ryegrass in the southern United States. Pest Manag. Sci. 2007, 63, 349–357.

513 514 515

(40) Kuk, Y. I.; Burgos, N. R.; Scott, R. C., Resistance profile of diclofop-resistant Italian ryegrass (Lolium multiflorum) to ACCase- and ALS-inhibiting herbicides in Arkansas, USA. Weed Sci. 2008, 56, 614-623.

516 517 518

(41) Chandi, A.; York, A. C.; Jordan, D. L.; Beam, J. B. Resistance to acetolactate synthase and acetyl Co-A carboxylase inhibitors in North Carolina Italian ryegrass (Lolium perenne). Weed Technol. 2011, 25, 659-666.

519 520

(42) Ellis, A. T.; Morgan, G. D.; Mueller, T. C. Mesosulfuron-resistant Italian ryegrass (Lolium multiflorum) biotype from Texas. Weed Technol. 2008, 22, 431–434.

22


Page 22 of 35

Page 23 of 35


521 522 523

(43) Ellis, A. T., Steckel, L. E.; Main, C. L.; de Melo, M. S. C.; West, D. R.; Mueller, T. C. A survey for diclofop-methyl resistance in Italian ryegrass from Tennessee and how to manage resistance in wheat. Weed Technol. 2010, 24, 303-309.

524 525 526

(44) Perez-Jones, A.; Park, K. W.; Colquhoun, J.; Mallory-Smith, C.; Shaner, D., Identification of glyphosate-resistant Italian ryegrass (Lolium multiflorum) in Oregon. Weed Sci. 2005, 53, 775-779.

527 528

(45) Doyle, J. J.; Doyle, J. L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 1987, 19, 11–15.

529 530 531

(46) Sales, M. A.; Shivrain, V. K.; Burgos, N. B.; Kuk, Y. I. Amino acid substitutions in the acetolactate synthase gene of red rice (Oryza sativa) confer resistance to imazethapyr. Weed Sci. 2008, 56, 485-489.

532 533 534

(47) Vargas, L.; Ulguim, A. R.; Agostinetto, D.; Magro, T. D.; Thuermer, L., Low level resistance of goosegrass (Eleusine indica) to glyphosate in Rio Grande do Sul-BraziL. Planta Daninha 2013, 31, 677-686.

535 536 537

(48) (Burgos, N. R.; Tranel, P. J.; Streibig, J. C.; Davis, V. M.; Shaner, D.; Norsworthy, J. K.; Ritz, C., Review: Confirmation of resistance to herbicides and evaluation of resistance levels. Weed Sci. 2013, 61, 4-20.

538 539

(49) Shaner, D. L. Role of translocation as a mechanism of resistance to glyphosate. Weed Sci. 2009, 57, 118-123.

540 541 542

(50) Michitte, P.; De Prado, R.; Espinoza, N.; Ruiz-Santaella, J. P.; Gauvrit, C. Mechanisms of resistance to glyphosate in a ryegrass (Lolium multiflorum) biotype from Chile. Weed Sci. 2007, 55, 435–440.

543 544

(51) Feng, P. C. C.; Pratley, J. E.; Bohn, J. Resistance to glyphosate in Lolium rigidum. II. Uptake, translocation, and metabolism. Weed Sci. 1999, 47, 412–415.

545 546

(52) Simarmata, M.; Kaufmann,J. E.; Penner, D. Potential basis of glyphosate resistance in California rigid ryegrass (Lolium rigidum). Weed Sci. 2003, 51, 678–682.

547 548 549

(53) Eschenburg, S.; Healy, M. L.; Priestman, M. A.; Lushington, G. H.; Schonburnn, E. How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli. Planta 2006, 216, 129-135.

550 551 552 553

(54) Funke, T.; Yang, Y.; Han, H.; Healy-Fried, M.; Olesen, S. Structural basis of glyphosate resistance resulting from the double mutation Thr97 → Ile and Pro101 → Ser in 5enolpyruvylshikimate-3-phosphate synthase from Escherichia coli. J. Biol. Chem. 2009, 284, 9854–9860.

554 555 556

(55) Schonbrunn, E.; Eschenburg, S.; Shuttleworth, W. A.; Schloss, J. V.; Amrhein, N.; Evans, J. N. S.; Kabsch, W. Interaction of the herbicide glyphosate with its target enzyme EPSP synthase in atomic detail. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1376–1380.

557 558 559

(56) Mizyed, S.; Wright, J. E. I.; Byczynski, B.; Berti, P. Identification of the catalytic residues of AroA (enolpyruvylshikimate 3-phosphate synthase) using partitioning analysis. Biochemistry 2003, 42, 6986–6995.

23



560 561 562

(57) Holt, J. S.; Thill, D. C. Growth and productivity of resistant plants. In Powles, S. B.; Holtum, J.A.M., Eds; Herbicide Resistance in Plants: Biology and Biochemistry. Boca Raton, FL: 1994; Lewis. pp. 299–316.

563 564 565

(58) Ismail, B. S.; Chuah, T. S.; Salmijah, S.; Teng, Y. T.; Schumacher, R. W. Germination and seedling emergence of glyphosate-resistant and susceptible biotypes of goosegrass [Eleusine indica (L) Gaertn.]. Weed Biol. Manag. 2002, 2,177–185.

566 567 568

(59) Lee, L. J. Glyphosate resistant goosegrass (Eleusine indica) in Malaysia and some of its morphological differences. Proc. 17th Asian-Pacific Weed Sci. Soc. Conf. Bangkok: 1999; pp 90–95.

569 570

(60) Terrell, E. E. A taxonomic revision of the genus Lolium. Technical Bulletin 1392. US Department of Agriculture, US Government Printing Office: Washington DC, 1968; p. 2

571 572 573

(61) Gaines, T. A.; Shaner, D. L.; Ward, S. M.; Leach, J. E.; Preston, C.; Westra, P. Mechanism of resistance of evolved glyphosate-resistant Palmer amaranth (Amaranthus palmeri). J. Agric. Food Chem. 2011, 59, 5886-5889.

574 575 576

(62) Ribeiro, D. N.; Pan, Z.; Duke, S. O.; Nandula, V. K.; Baldwin, B. S.; Shaw, D. R.; Dayan, F. E., Involvement of facultative apomixis in inheritance of EPSPS gene amplification in glyphosate-resistant Amaranthus palmeri. Planta 2014, 239, 199-212.

577 578 579

(63) Chandi, A.; Milla-Lewis, S. R.; Giacomini, D.; Westra, P.; Preston, C.; Jordan, D. L. Inheritance of evolved glyphosate resistance in a North Carolina Palmer amaranth (Amaranthus palmeri) biotype. Intl. J. Agron. 2012, DOI:10.1155/2012/176108.

580 581

(64) Vogel, C.; Marcotte, E. M., Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nature Rev. Genet. 2012, 13, 227-232.

582 583

(65) Pline-Srnic, W. Physiological mechanisms of glyphosate resistance. Weed Technol. 2006, 20, 290-300.

584 585 586

(66) Widholm, J. M.; Chinnala, A. R.; Ryu, J.; Song, H.; Eggetta, T.; Brothertona. J. E. 2001. Glyphosate selection of gene amplification in suspension cultures of 3 plant species. Physiol. Plant. 2001, 112, 540–545.

587 588 589

(67) Suh, H.; Hepburn, A. G.; Kriz, A. L.; Widholm, J. M. Structure of the amplified 5enolpyruvylshikimate-3-phosphate synthase gene in glyphosate resistant carrot cells. Plant Mol. Biol. 1993, 22, 195–205.

590 591 592

(68) Steinrucken, H. C.; Schulz, A.; Amrhein, N.; Porter, C. A.; Fraley, R. T. Overproduction of 5-enolpyruvyl-shikimate 3-phosphate synthase in a glyphosate-tolerant Petunia hybrida cell line. Arch. Biochem. Biophys. 1986, 244, 169–178.

593 594 595

(69) Culpepper, A. S.; Grey, T. L.; Vencill, W. K.; Kichler, J. M.; Webster, T. M.; Brown, S. M.; York, A. C.; Davis, J. W.; Hanna, W. W. Glyphosate-resistant Palmer amaranth (Amaranthus palmeri) confirmed in Georgia. Weed Sci. 2006, 54, 620–626.

596 597

(70) Powles, S. B. Gene amplification delivers glyphosate-resistant weed evolution. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 955-956.

598 599

(71) Zou, C.; Lehti-Shiu, M. D.; Thomashow, M.; Shiu, S-H. Evolution of stress-regulated gene expression in duplicate genes of Arabidopsis thaliana. PLoS Genet. 2009, 5, e1000581. 24


Page 24 of 35

Page 25 of 35


600 601

(72) Kondrashov, F. A. Gene duplication as a mechanism of genomic adaptation to a changing environment. Proc. R. Soc. B. 2012, 279, 5048-5057.

602 603 604

(73) Giacomini, D. A.; Westra, P.; Ward, S. M.; Sammons, R. D. The inheritance of amplified EPSPS gene copies in Palmer amaranth (Amaranthus palmeri). Weed Sci. Soc. Am. Proc. 2013, 53, 311.

605 606

(74) Sellin, C.; Forlani, G.; Dubois, J.; Nielsen, E.; Vasseur, J. Glyphosate tolerance in Cichorium intybus var magdebourg. Plant Sci. 1992, 85, 223-231.

607 608 609

(75) Smith, C. M.; Pratt, D.; Thompson, G. A. Increased 5-enolpyruvylshikimic acid 3-phosphate synthase activity in a glyphosate-tolerant variant strain of tomato cells. Plant Cell Rep. 1986, 5, 298-301.

610 611 612

(76) Goldsbrough, P. B.; Hatch, E. M.; Huang, B.; Kosinski, W. G.; Dyer, W. E.; Herrmann, K. M.; Weller, S. C. Gene amplification in glyphosate tolerant tobacco cellS. Plant Sci. 1990, 72, 53-62.

613 614

(77) Cresswell, R. C.; Fowler, M. W.; Scragg, A. H. Glyphosate-tolerance in Catharanthus roseus. Plant Sci. 1988, 54, 55-63.

615 616 617

(78) Murata, M.; Ryu, J. H.; Caretto, S.; Rao, D.; Song, H. S.; Widholm, J. M., Stability and culture medium limitations of gene amplification in glyphosate resistant carrot cell lines. J. Plant Physiol. 1998, 152, 112-117.

618 619 620

(79) Vila-Aiub, M. M.; Goh, S. S.: Gaines, T. A.; Han, H.; Busi, R.; Yu, Q.; Powles, S. B. No fitness cost of glyphosate resistance endowed by massive EPSPS gene amplification in Amaranthus palmeri. Planta 2014, 239, 793-801.

621

(80)

622 623 624 625 626 627 628 629 630 631

FUNDING SOURCES This research was supported by the Arkansas Wheat Research and Promotion Board.

FIGURE CAPTIONS

632

Figure 1. Shoot biomass reduction of selected Italian ryegrass populations, 28 d after treatment.

633

Error bars are standard errors of the mean. Des05, Des14, and D8 had an estimated 50%

634

biomass reduction (GR50) of 726, 831, and 1264 g ae ha-1 glyphosate. The susceptible

635

standard 98-3 had an estimated GR50 of 101 g ae ha-1 glyphosate.

25



636 637

Figure 2. Mortality of selected glyphosate-resistant and –susceptible Italian ryegrass

638

populations, 28 d after treatment. Error bars are standard errors of the mean. Des05, Des14,

639

and D8 populations had an estimated LD50 of 1702, 1587, and 2245 g ae ha-1 glyphosate. The

640

susceptible 98-3 population had an estimated LD50 of 184 g ae ha-1 glyphosate. LD50 is the

641

amount of herbicide that kills 50% of the population.

642 643

Figure 3. EPSPS relative genomic copy number in glyphosate-resistant and -susceptible L.

644

perenne ssp. multiflorum plants. Relative copy number of EPSPS in resistant populations

645

(D8, Des05, and Des14) ranged from 11 to 151 (n=30), whereas the susceptible standard (98-

646

3) contained a single copy (n=10). Values are averages of 10 plants per population, with

647

three technical replicates. Vertical bars represent the standard error of the mean.

648 649

Figure 4. Relationship between the amount of glyphosate needed to incur 50% injury (GR50) and

650

the relative EPSPS genomic copy number (r=0.80). Susceptible plants (98-3) had the lowest

651

GR50 and EPSPS copy number.

652 653

26


Page 26 of 35

Page 27 of 35


654

Table 1. Summary of Lolium perenne ssp. multiflorum plants used for each

655

experiment. Plants used for each experiment

Population

Whole-plant dose-response bioassay (Expt. 2)

Absorption and translocation (Expt. 3)

No. of plants cloneda

656

EPSPS gene sequencing (Expt. 4)b

EPSPS copy number analysis (Expt. 5)

Cloned plants from Expt. 2 plus others

Des05

3

Des09

1

1

Des13

2

2

Des14

4

Des15

3

3

D8

1

10

98-3

4

Total

18

a

6

6

6

7 R, 5 S

13 R

10 S

10

10

10 46

Twenty-four clones of each plant were used to apply 8 doses (0, 218, 435, 870,

657

1740, 3480, 6960, and 13920 g ae ha-1) for confirmed R plants and 7 doses (0,

658

54, 109, 218, 435, 653, 870, and 1740 g ae/ha-1) for S plants.

659

b

R = resistant; S = susceptible

660 661

27



662 663 664

Table 2. GR50 and LD50 values of glyphosate-resistant and -susceptible L. perenne ssp. multiflorum populations, Arkansas, USA. Population

GR50

R/Sa

g ae ha-1 Des05c

726 (629, 823)d

LD50

R/Sb

g ae ha-1 7

1702 (1419, 1986) e

9

831 (771, 892)

8

1587 (1385, 1788)

9

D4c

1908 (1485, 2332)

19

2719 (2107, 3329)

15

D8c

1264 (1031, 1496)

13

2245 (1916, 2575)

12

D13c

917 (795, 1039)

9

1524 (1200, 1847)

8

98-3d

101 (91, 111)

-

184 (161, 207)

-

Des14c

665 666 667 668 669 670 671

Page 28 of 35

a

Resistance level (R/S) calculated using the GR50 of the resistant population relative to the susceptible standard. b Resistance level (R/S) calculated using the LD50 of the resistant population relative to the susceptible standard. c Glyphosate-resistant population. d Glyphosate-susceptible population e Lower 95%, Upper 95%

28


Page 29 of 35


Table 3. 14C glyphosate absorption and distribution in various plant tissues of resistant and susceptible Lolium perenne ssp. multiflorum populations from Arkansas, USA. 14

14

C-glyphosate distributiona

C-glyphosate

Population

absorptiona 24 HATb

48 HAT

Treated leaf 24 HAT

% of applied

48 HAT

Above treated leaf 24 HAT

48 HAT

Below treated leaf 24 HAT

48 HAT

Roots 24 HAT

48 HAT

----------------------------------- % of absorbed -------------------------------------

Des05c

38

51

80

65

1

2

11

17

8

15

Des14c

44

59

79

68

1

1

12

19

8

12

98-3d

37

57

77

71

1

3

14

16

8

11

29

a

Values are the average of four plants. HAT, hours after treatment. c Resistant population. d Susceptible population.

b



Page 30 of 35

Table 4. Summary statistics of the relative EPSPS copy number in glyphosate-resistant L. perenne ssp. multiflorum populations, Arkansas, USA. EPSPS:CCR copy numbera Population

Mean

Median

Standard deviation

Coefficient of variation

Minimum

Maximum

Des05

45

39

33

73

11

122

Des14

48

48

23

47

24

97

D8

80

84

47

59

18

151

1

1

98-3 a

0.2

20

Values are average of 10 plants.

30 ACS Paragon Plus Environment

0.8

1.4

Page 31 of 35


31

Biomass reduction (% of the untreated)

120 98-3 Des05

100

Des14 D8

80

60

40

20

0 10

100

1000

10000

Glyphosate dose (g ae ha-1)

Figure 1. Shoot biomass reduction of selected L. perenne ssp. multiflorum populations, 28 d after treatment. Error bars are standard errors of the mean. Des05, Des14, and D8 had an estimated 50% biomass reduction (GR50) of 726, 831, and 1264 g ae ha-1 glyphosate. The susceptible 98-3 population had an estimated GR50 of 101 g ae ha-1 glyphosate.



120 98-3 Des05 Des14

100

D8

32

Mortality (% )

80

60

40

20

0 10

100

1000

10000

Glyphosate dose (g ae ha-1)

Figure 2. Mortality of selected glyphosate-resistant and –susceptible L. perenne ssp. multiflorum populations, 28 d after treatment. Error bars are standard errors of the mean. Des05, Des14, and D8 populations had an estimated LD50 of 1702, 1587, and 2245 g ae ha-1 glyphosate. The susceptible 98-3 population had an estimated LD50 of 184 g ae ha-1 glyphosate. LD50 is the amount of herbicide that kills 50% of the population.


Page 32 of 35

Page 33 of 35


33

EPSPS:CCR relative copy number

120

100

80

60

40

20

0 98-3

Des05

Des14

D8

Italian ryegrass population

Figure 3. EPSPS relative genomic copy number in glyphosate-resistant and -susceptible L. perenne ssp. multiflorum plants. Relative copy number of EPSPS in resistant populations (D8, Des05, and Des14) ranged from 11 to 151 (n=30), whereas the susceptible standard (98-3) contained a single copy (n=10). Values are averages of 10 plants per population, with three technical replicates. Vertical bars represent the standard error of the mean.


34

EPSPS:CCR relative genomic copy number


Page 34 of 35

70 98-3 Des15 Des13 Des09 Des05 Des14 D8

60 50 40 30 20 10 0

0

200

400

600

800

1000

1200

1400

GR50 (g ae ha-1) Figure 4. Relationship between the amount of glyphosate needed to incur 50% injury (GR50) and the relative EPSPS genomic copy number (r=0.80). Susceptible plants (98-3) had the lowest GR50 and EPSPS copy number.


Page 35 of 35


TOC GRAPHIC

35 ACS Paragon Plus Environment

EPSPS Gene Amplification in Glyphosate-Resistant Italian Ryegrass

Recommend Documents