1) Glyphosate resistance has emerged in over 20 weed species worldwide through various mechanisms including target site mutations and increased metabolism. 2) Target site mutations in the EPSPS gene have been identified in several glyphosate resistant weed species resulting in 2-3X levels of resistance. 3) Metabolic resistance allows some weed species to break down glyphosate faster through enhanced neutralizing enzyme activity, resulting in weaker resistance.

1. Overview on
glyphosate resistant
weed mechanisms
Dr. Doug Sammons
23/3/2010
Monsanto Company

2. Glyphosate Global Resistance (17)
Weedscience.org, 2/25/2010 * Target site mutant, † Exclusion
Plantago C3-(2003), Buckhorn plantain (S. Africa)
Parthenium * C3/C4-(2004), Ragweed parthenium (Columbia ) Examples of
1-3X
Eleucine * C4-(1997), Goosegrass (Malaysia, Columbia,Taiwan) magnitude
Echinochloa C4-(2007), Barnyard grass (Australia) resistance
Euphorbia C4-(2006), Wild Poinsettia (Brasil)
Kochia C4-(2007), Tumbleweed (KS)
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) Examples of
6-8X
Ambrosia † C3-(2004), Giant ragweed (6 States)
magnitude
Ambrosia C3-(2004), Common ragweed (6 States) resistance
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)

3. Herbicide Resistance in Key Glyphosate Resistant Weeds
Europe(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
Resistant Line to
Dose Response than Survival the label Rate
Label Rate
100%
Resistant/ Label Rate
75%
~10X 16X Resistant(?)
Percent Effect
50%
Survival
25% Dry Wgt
4X
0%
0.01 0.1 1 10 100
3
Glyphosate Dose (kg/ha)

4. Weed Resistant Species in Europe
55
C1-Triazine
50
ALS
45
ACCase
40
C2 Urea
35
DNA
Species
30 ~90% Target Site Auxins
25 and Metabolism
Bipyridil.
20
Glycine
15
10
5
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Years of Use

5. Resistance Mechanisms are not Equal in
Magnitude or Probability
Target Site Mutations
• Weak to IMMUNE
• ALS, ACCase,
•Photosystem 2
•DNA
Metabolism Exclusion
• Weak Resistance • Weak Resistance; Very Rare
•ALS, ACCase, •Active (sequestration)
•Photosystem 2 •Passive (cuticle )
•DNA
•Most others Combinations Possible
11/6/2008 MONSANTO CONFIDENTIAL 5

6. EPSPS, The Lock and Key Mechanism
free
EPSP Synthase
EPSP
Phosphate
S-3-P Glyphosate
Herbicidal
Dead-End Complex
PEP
busy with
chemical CP4
catalysis
Doug Sammons
Company Confidential Resistant EPSPS, RR

7. Target Site Resistance has 4 Factors
1.) Binding Site
Conservation
Polymorphic
Conserved
2.) Herbicide 3.)Contact Amino 4.) Amino Acid
Structure Acids Function
Large More Scaffold
Small Few Function
MONSANTO
Unique =high specifity Catalytic 0
CONFIDENTIAL

8. 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).

9. Glyphosate mimics the Tetrahedral
Intermediate of the reaction.
Schönbrunn et al. 2001, PNAS 98:1376
[EPSPS:Tetrahedral Intermediate] [ EPSPS:S3P:Glyphosate ]
9 complex

10. In Planta Point mutations in EPSPS
Consensus Plant Site: G T A M R P L
102 106
Resistant Eleusine indica: G T A M R S L
Baerson et al., 2002 Plant Phys. 129:1265
Ng et al., 2004 Aust. J. Ag. Res. 55:407 G T A M R T L
Yuan et al., 2005 Plant Prot. Bul. 47:251 G T A M R A L
Resistant Lolium rigidum: G T A M R A L
Yu et al., 2007 Planta 225:499.
Wakelin & Preston, 2006 Weed Res. 46:432. G T A M R T L
Resistant Lolium multiflorum: G T A M R S L
Perez-Jones et al.,2007 Planta
Resistant Oryza sativa: G T A M R L L
Zhou et al. 2006 Plant Phys. 140:185 Directed evolution
Overall 2-3X Resistance Recorded

11. Metabolic Resistance has 3 Plant factors
1.) Time to Die Based on the 2.) Neutralizing Enz
MOA & Species
Long More
Short Less
Mechanism Seed Size
Metabolism
3.) Herbicide in the Plant
is easier for
some
Lower Rates
Higher Rates
Use Rate

12. Glyphosate Can
be Metabolized in
some Legume sp.
Vigna uniguiculata
Senna obtusifolia
Cassia occidentialis 25%
Sesbania herbacea
Desmanthus illinoensis 50%
Abutilon theophrasti
Conyza canadensis O O
H
Lolium multiflorum C N P
C OH
HO C
H H2
2 OH
O
H2N P
C OH
Reddy, K.N., A.M. Rimando, S.O. Duke, and V.K. Nandula. 2008. H2
Aminomethylphosphonic acid accumulation in plant species treated with OH
glyphosate. Journal of Agricultural and Food Chemistry 56:2125-2130.

13. Vacuole Hypothesis for Horseweed Resistance
MOA
Chloroplast Cytoplasm
pH ~7 pH ~7
Vacuole
pH 4.5-6
Spongy
Palisade Mesophyl
Cells Cells
Dr. Neela Shivakumar and Dr. Cristina Ubach, confocal microscopy

14. 31P NMR Measures Glyphosate pH
O O
-O P O-
N OH
H O
H
P
HO O-
OH
pH 7.0
G
Gcyt
Gvac
vacuole
pH 4.5

15. Xia Ge, Andre' d'Avignon, Joseph Ackerman
Chemistry Department
Washington University,
St. Louis, MO USA
11/6/2008 15

16. In vivo 31P-NMR of Horseweed treated with
WeatherMAX®.
G
Sensitive Gcyt Resistant
Source Leaves Gvac Source Leaves
Gcyt vacuole Gvac
Gcyt
G
Gvac Gcyt
G vac
vacuole
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 Sammons
Published Online: Jan 8 2010 9:54AM

17. 8C
Resistant
Horseweed
is sensitive 24 hr
when cold 0.5 0.75 1 2 4 8
acclimated 0 lb/a
25C
12 hr
2 hr
Resistance mechanism is not ON in the cold
42 DAT

18. Ryegrass also uses the Vacuole for Resistance
S332(sensitive) R332(medium)
R336 High
Glyphosate Resistant Ryegrass,
Italy
% Mortality
0.34 kg 1.6 kg 7.9 kg
ae/ha ae/ha ae/ha
Dr. Maurizio Sattin
Dr. Alberto Collavo
Consiglio Nazionale delle
Ricerche Institute of
Agro-Environmental and
Forest Biology
Legnaro, Italy
18

19. Glyphosate
Resistant
Palmer amaranth
in cotton
PNAS | January 19, 2010 | V107 | no. 3 | 1029–1034

20. EPSPS Gene Amplification as the Mechanism of
Glyphosate Resistance in Palmer amaranthus
EPSPS western blot
EPSPS genomic copy# Susceptible
vs mRNA expression Resistant
1 6 15 39 54 60 copies
EPSPS enzyme activity
0.24
0.22 1 copy
0.2 7.5 copies
0.18
(uM Pi / ug / min)
EPSPS activity
0.16 39 copies
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0.1 1 10 100 1000
glyphosate (uM)

21. Control +0.75lb
S ae/a
R
2 DAT
Glyphosate resistant Giant Ragweed,
Indiana
7 DAT
14
DAT
Dr. Steve Weller and Renae Robertson, Purdue
University

22. Glyphosate Resistance Mechanisms
Exclusion
Target Site Extracellular
Mutations • Hypersensitive, excission
• Pro106X 1
4 2 •Slow across
plasmalema
2 2
Over Expression 0
• Copy number
• Expression level Intracellular
Metabolism • Vacuole accumulation ~8
• Legumes Single gene, Dominant
• Very SLOW
Combinations Known
22

23. 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.

24. A Global Commitment To Growing Yield Sustainably®
THREE MAIN GOALS ARE AT THE HEART OF THIS EFFORT
Help farmers REDUCE by 1/3 the
DOUBLE YIELDS inputs required per
in corn, cotton & unit of output
soybeans by 2030
IMPROVE THE LIVES of
farmers around the world

25. Cold Weather
Management
Horseweed control appears to be
100 % for first 3 application dates.
This is the Jan application.
Applied another on 2/28/2010
Jan close
up
4 Fall applications to Feb Jan Dec Nov
horseweed
Bob Montgomery,
Fall 2009, West TN

26. Weed Resistance Incidents in Europe
140 C1-Triazine
(52)
ALS
120
ACCase
100 C2 Urea
auxins
Incidents
80 DNA (2)
(4)
Bipyridil.
60 (24) (13) Glycine
40
(11)
20 (4) (5)
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Years of Use

27. Target Site High-level Resistance is
Efficiently Selected
ACC’ase Resistance
fops, dims
1000
• 87% of the herbicide resistance
is like this,
very high in magnitude
Reported 100
• 5 43 casesHerbicide classes are
Common NOT controlled,
Fold dominated by thisselected
resistance IS kind of
Resistance resistance
10
•ALS
7 cases < 3X
•ACCase
Only these cases Ureas controlled
•Triazine and can be
1 0 1
Published Data for 48 Cases •Dinitroanilines
Gressel, Molecular Biology of Weed Control, Taylor & Francis, London, 2002

28. Crop and Weed Metabolism are Related
Genome Sequencing is
• Until Biotech, Crop safety revealing how similar plant
species are
required metabolism
• Genomic similarity means
weeds have the same
metabolism
2 Possibilities
1.) Reaction; Is there enough?
Bennetzen and Freeling 1997. 2.) No reaction; requires
Genome Research 7:301-306
mutation to gain function
28