Overview on glyphosate resistant weed mechanisms

Dr. Doug Sammons

23/3/2010

Monsanto Company

Weedscience.org, 2/25/2010

Glyphosate Global Resistance (17)* Target site mutant, † Exclusion

Examples of 1-3X

magnitude resistance

Plantago C3-(2003), Buckhorn plantain (S. Africa)

Parthenium * C3/C4-(2004), Ragweed parthenium (Columbia )

Eleucine * C4-(1997), Goosegrass (Malaysia, Columbia,Taiwan)

Echinochloa C4-(2007), Barnyard grass (Australia)

Euphorbia C4-(2006), Wild Poinsettia (Brasil)

Kochia C4-(2007), Tumbleweed (KS)

Lolium * C3-(2001), Italian Ryegrass (S. America, France , Italy, Spain, 6 States)

Examples of 6-8X

magnitude resistance

Lolium * C3-(2001), Italian Ryegrass (S. America, France , Italy, Spain, 6 States)

Lolium * † C3-(1996), Rigid Ryegrass (Aus.,4 prov., CA, S. Africa, France, Italy,Spain)

Amaranthus C4-(2005), Waterhemp (5 States)

Amaranthus* † C4-(2005), Palmer pigweed (10 States)

Sorghum † C4-(2005), Johnsongrass (Argentina, MS, AR)

Digitaria C4-(2008), Sourgrass (Brazil, Paraguay)

Ambrosia † C3-(2004), Giant ragweed (6 States)

Ambrosia C3-(2004), Common ragweed (6 States)

Conyza † C3-(2000), Horseweed (17 states, Brasil, China, Spain)

Conyza C3-(2003), Hairy fleabane (S. Africa, Spain, Brasil, Columbia, CA))Urochloa panicoides C4-(2008), Liverseedgrass (Aust- NSW)

Herbicide Resistance in Key Glyphosate Resistant WeedsEurope(115) ALS(24) ACCase(13) Photo(63) Glyp(4)

Lolium multiflorum - 3 1 1

Lolium rigidum 1 4,1 2,1 5

Bipyridilium(4)

Conyza canadensis 1,1 1 8,1 2

Conyza bonarensis 1 - 2 2

Herbicide Resistance in Key Glyphosate Resistant WeedsEurope(115) ALS(24) ACCase(13) Photo(63) Glyp(4)

Lolium multiflorum - 3 1 1

Lolium rigidum 1 4,1 2,1 5

Bipyridilium(4)

Conyza canadensis 1,1 1 8,1 2

Conyza bonarensis 1 - 2 2

Growth Reduction has a Lower Comparing Sensitive Line to the Comparing Resistant Line to

3

0%

25%

50%

75%

100%

0.01 0.1 1 10 100

Pe

rce

nt E

ffect

Glyphosate Dose (kg/ha)

Survival

Dry Wgt

~10X

Growth Reduction has a Lower Dose Response than Survival

Comparing Sensitive Line to the Label RateComparing Resistant Line to the label Rate

16X Resistant(?)

4X

Resistant/ Label Rate

25

30

35

40

45

50

55

Species

Weed Resistant Species in Europe

C1-Triazine

ALS

ACCase

C2 Urea

DNA

Auxins~90% Target Site and Metabolism

0

5

10

15

20

25

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Species

Years of Use

Auxins

Bipyridil.

Glycine

and Metabolism

Resistance Mechanisms are not Equal in Magnitude or Probability

Target Site Mutations • Weak to IMMUNE

• ALS, ACCase,

•Photosystem 2

•DNA

Combinations Possible

Metabolism• Weak Resistance

•ALS, ACCase,

•Photosystem 2

•DNA

•Most others

11/6/2008 5MONSANTO CONFIDENTIAL

•DNA

Exclusion• Weak Resistance; Very Rare

•Active (sequestration)

•Passive (cuticle )

EPSPS, The Lock and Key Mechanism

EPSP

Phosphate

EPSP Synthase

S-3-P Glyphosate

free

Company Confidential

HerbicidalDead-End Complex

PEP

CP4

Resistant EPSPS, RR

busy withchemicalcatalysis

Doug Sammons

1.) Binding Site Conservation

Polymorphic

Conserved

Target Site Resistance has 4 FactorsTarget Site Resistance has 4 Factors

MONSANTO

CONFIDENTIAL

2.) Herbicide 3.)Contact Amino 4.) Amino AcidStructure Acids Function

Large More Scaffold

Small Few Function

Unique =high specifity Catalytic 0

Conserved Amino Acids in EPSPS

Consurf: Surface-Mapping of Conservation

Glaser, F., T. Pupko, I. Paz, R. E. Bell, D. Bechor-Shental, E. Martz and N. Ben-Tal. Bioinformatics. 19:163-164 (2003).

Glyphosate mimics the Tetrahedral Intermediate of the reaction.

Schönbrunn et al. 2001, PNAS 98:1376

9

[EPSPS:Tetrahedral Intermediate] [ EPSPS:S3P:Glyphosate ]complex

In Planta Point mutations in EPSPS

G T A M R P LConsensus Plant Site:

Resistant Eleusine indica:Baerson et al., 2002 Plant Phys. 129:1265

Ng et al., 2004 Aust. J. Ag. Res. 55:407

Yuan et al., 2005 Plant Prot. Bul. 47:251

Resistant Lolium rigidum:

G T A M R S L

106102

G T A M R A L

G T A M R T L

Resistant Oryza sativa:Zhou et al. 2006 Plant Phys. 140:185 Directed evolution

Resistant Lolium rigidum:Yu et al., 2007 Planta 225:499.

Wakelin & Preston, 2006 Weed Res. 46:432.

G T A M R A L

G T A M R L L

Overall 2-3X Resistance Recorded

G T A M R T L

Resistant Lolium multiflorum:Perez-Jones et al.,2007 Planta

G T A M R S L

Metabolic Resistance has 3 Plant factors

2.) Neutralizing Enz

More

Less

Seed Size

1.) Time to Die

Long

Short

Mechanism

Based on theMOA & Species

Seed Size

3.) Herbicide in the Plant

Lower Rates

Higher Rates

Use Rate

MechanismMetabolism is easier for

some

Metabolism is easier for

some

Glyphosate Can be Metabolized in some Legume sp.

Vigna uniguiculataSenna obtusifoliaCassia occidentialisSesbania herbaceaDesmanthus illinoensis

Abutilon theophrasti

25%50%

Reddy, K.N., A.M. Rimando, S.O. Duke, and V.K. Nandula. 2008. Aminomethylphosphonic acid accumulation in plant species treated with glyphosate. Journal of Agricultural and Food Chemistry 56:2125-2130.

Reddy, K.N., A.M. Rimando, S.O. Duke, and V.K. Nandula. 2008. Aminomethylphosphonic acid accumulation in plant species treated with glyphosate. Journal of Agricultural and Food Chemistry 56:2125-2130.

POH

OH

CH2

HN

O

CH2

C

O

HO

POH

OH

CH2

H2N

O

Lolium multiflorum

Abutilon theophrastiConyza canadensis

VacuolepH 4.5-6

CytoplasmpH ~7

ChloroplastpH ~7

Vacuole Hypothesis for Horseweed Resistance MOA

Palisade

Cells

Spongy

Mesophyl

Cells

Dr. Neela Shivakumar and Dr. Cristina Ubach, confocal microscopy

31P NMR Measures Glyphosate pH

N

P-O

O

O-

O

OH

H H

PHO O-

O

OH

pH 4.5

pH 7.0

Gcyt

Gvac

vacuole

G

Xia Ge, Andre' d'Avignon, Joseph AckermanChemistry Department

Washington University,

St. Louis, MO USA

11/6/2008 15

In vivo 31P-NMR of Horseweed treated with WeatherMAX®. In vivo 31P-NMR of Horseweed treated with WeatherMAX®.

Sensitive Source Leaves

Gcyt

Gcyt

Gvac

vacuole

G

Resistant Source Leaves

Gcyt

Gvac

Gvac Gcyt

Gvac

vacuole

G

Rapid vacuolar sequestration: the horseweed glyphosate resistance mechanism, Pest Management Science, 66, p 345-348, 2010.Xia Ge, D André d'Avignon, Joseph JH Ackerman, R Douglas SammonsPublished Online: Jan 8 2010 9:54AM

0.5 0.75 1 2 4 8 0 lb/a

8CResistant Horseweed is sensitivewhen cold acclimated

24 hr

0 lb/a25C

acclimated

42 DAT

12 hr

2 hr

Resistance mechanism is not ON in the cold

Ryegrass also uses the Vacuole for Resistance

7.9 kg ae/ha

1.6 kg ae/ha

0.34 kg ae/ha

Glyphosate Resistant Ryegrass, Italy

% M

ort

ality

R336 HighS332(sensitive) R332(medium)

18

ae/haae/haae/ha

Dr. Maurizio SattinDr. Alberto CollavoConsiglio Nazionale delleRicerche Institute of Agro-Environmental and Forest Biology Legnaro, Italy

Glyphosate Resistant Palmer amaranth in cotton

PNAS | January 19, 2010 | V107 | no. 3 | 1029–1034

EPSPS Gene Amplification as the Mechanism of Glyphosate Resistance in Palmer amaranthus

Susceptible Resistant

EPSPS western blot

156 60 copies1 39 54

EPSPS genomic copy# vs mRNA expression

EPSPS enzyme activity

glyphosate (uM)

0.1 1 10 100 1000

EP

SP

S a

ctivi

ty(u

M P

i / ug

/ m

in)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

0.2

0.22

0.241 copy

7.5 copies

39 copies

ControlS R

2 DAT

7 DAT

Glyphosate resistant Giant Ragweed, Indiana

+0.75lb ae/a

7 DAT

14 DAT

Dr. Steve Weller and Renae Robertson, Purdue University

Target SiteMutations

• Pro106X

Extracellular• Hypersensitive, excission

•Slow across

plasmalema

Exclusion

Glyphosate Resistance Mechanisms

1

24

22

Metabolism

• Legumes

• Very SLOW

Over Expression• Copy number

• Expression level Intracellular• Vacuole accumulation ~8

Single gene, Dominant

Combinations Known

2

0

2

Summary

1.) Glyphosate is Unique

2.) New Mechanisms of Resistance, weaker

in magnitude, low dose selection.

3.) Mechanism studies could lead to better

management strategies.

THREE MAIN GOALS ARE AT THE HEART OF THIS EFFORT

A Global Commitment To Growing Yield Sustainably®

Help farmers

DOUBLE YIELDSin corn, cotton & soybeans by 2030

REDUCE by 1/3 the

inputs required per unit of output

IMPROVE THE LIVES of

farmers around the world

Jan close up

Horseweed control appears to be 100 % for first 3 application dates. This is the Jan application. Applied another on 2/28/2010

Cold Weather Management

NovJan DecFeb4 Fall applications to horseweed

Bob Montgomery, Fall 2009, West TN

80

100

120

140

Incidents

Weed Resistance Incidents in EuropeC1-Triazine

ALS

ACCase

C2 Urea

auxins

DNA

Bipyridil.

(52)

(2)

(4)

0

20

40

60

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Incidents

Years of Use

Bipyridil.

Glycine(24) (13)

(4)

(11)

(5)

(4)

100

1000

fops, dimsACC’ase Resistance

Reported Reported Fold

Target Site High-level Resistance is Efficiently SelectedTarget Site High-level Resistance is Efficiently Selected

43 cases NOT controlled, resistance IS selected

• 87% of the herbicide resistance is like this,

very high inmagnitude• 5 Common Herbicide classes are dominated by this kind of

1

10

0 1

7 cases < 3X

Published Data for 48 Cases

Fold Resistance

Gressel, Molecular Biology of Weed Control, Taylor & Francis, London, 2002

Only these cases can be controlled

resistance IS selecteddominated by this kind of resistance

•ALS

•ACCase

•Triazine and Ureas

•Dinitroanilines

Crop and Weed Metabolism are Related

• Until Biotech, Crop safety

required metabolism

• Genomic similarity means

weeds have the same

metabolism

Genome Sequencing is revealing how similar plant species are

28

Bennetzen and Freeling 1997. Genome Research 7:301-306

2 Possibilities

1.) Reaction; Is there enough?

2.) No reaction; requires mutation to gain function