Overview on
glyphosate resistant
weed mechanisms

Dr. Doug Sammons
23/3/2010
Monsanto Company
Glyphosate Global Resistance (17)
    Weedscience.org, 2/25/2010         * Target site mutant,    † Exclusion



Plantago ...
Herbicide Resistance in Key Glyphosate Resistant Weeds
Europe(115)                                  ALS(24)             AC...
Weed Resistant Species in Europe
          55
                                                             C1-Triazine
   ...
Resistance Mechanisms are not Equal in
          Magnitude or Probability
Target Site    Mutations
• Weak to IMMUNE
   • A...
EPSPS, The Lock and Key Mechanism

                         free
                                                EPSP Synt...
Target Site Resistance has 4 Factors
                                        1.) Binding Site
                            ...
Conserved Amino Acids in
EPSPS




Consurf: Surface-Mapping of Conservation
Glaser, F., T. Pupko, I. Paz, R. E. Bell, D. B...
Glyphosate mimics the Tetrahedral
        Intermediate of the reaction.
                 Schönbrunn et al. 2001, PNAS 98:1...
In Planta Point mutations in EPSPS
          Consensus Plant Site:           G     T       A   M    R   P     L
          ...
Metabolic Resistance has 3 Plant factors
1.) Time to Die    Based on the 2.) Neutralizing Enz
                  MOA & Spec...
Glyphosate Can
                                                       be Metabolized in
                                  ...
Vacuole Hypothesis for Horseweed Resistance
MOA
           Chloroplast                      Cytoplasm
             pH ~7  ...
31P     NMR Measures Glyphosate pH
     O           O
-O                   P   O-


         N               OH

     H   ...
Xia Ge, Andre' d'Avignon, Joseph Ackerman
Chemistry Department
Washington University,
St. Louis, MO USA




   11/6/2008  ...
In vivo 31P-NMR of Horseweed treated with
    WeatherMAX®.
                                                     G
Sensitiv...
8C
Resistant
Horseweed
is sensitive            24 hr

when cold        0.5    0.75     1   2   4    8
acclimated     0 lb/...
Ryegrass also uses the Vacuole for Resistance
 S332(sensitive)                    R332(medium)
                           ...
Glyphosate
         Resistant
         Palmer amaranth
         in cotton



PNAS | January 19, 2010 | V107 | no. 3 | 1029...
EPSPS Gene Amplification as the Mechanism of
 Glyphosate Resistance in Palmer amaranthus
                                 ...
Control                     +0.75lb
                              S     ae/a
                                             ...
Glyphosate Resistance Mechanisms
                                                     Exclusion
Target Site               ...
Summary
1.) Glyphosate is Unique
2.) New Mechanisms of Resistance, weaker
   in magnitude, low dose selection.
3.) Mechani...
A Global Commitment To Growing Yield Sustainably®
THREE MAIN GOALS ARE AT THE HEART OF THIS EFFORT


Help farmers         ...
Cold Weather
                          Management
                            Horseweed control appears to be
            ...
Weed Resistance Incidents in Europe
            140                                                    C1-Triazine
       ...
Target Site High-level Resistance is
           Efficiently Selected
       ACC’ase Resistance
                         fo...
Crop and Weed Metabolism are Related
                                            Genome Sequencing is
• Until Biotech, Cro...
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D Sammons

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Overview on glyphosate resistant weed mechanisms

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D Sammons

  1. 1. Overview on glyphosate resistant weed mechanisms Dr. Doug Sammons 23/3/2010 Monsanto Company
  2. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 15. Xia Ge, Andre' d'Avignon, Joseph Ackerman Chemistry Department Washington University, St. Louis, MO USA 11/6/2008 15
  16. 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. 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. 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. 19. Glyphosate Resistant Palmer amaranth in cotton PNAS | January 19, 2010 | V107 | no. 3 | 1029–1034
  20. 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. 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. 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. 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. 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. 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. 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. 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. 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
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