Herbicide Resistance

Herbicide resistance testing 
Herbicide efficacy research and weed resistance
Herbicide resistance management strategies

Herbicide resistance is defined as ‘the evolved ability of a weed population to survive a maximum dose rate of a herbicide previously known to be lethal’.

1. Herbicide resistance is naturally acquired through plant selection.
2. It is not caused by the application of herbicides.
3. Once target-site resistance is present in a population, it will not disappear, even if the herbicide selection pressure is removed.

The following factors influence grass weed herbicide resistance:

Selection intensity

Herbicide dose (using rates lower than full recommended field rates), frequency (using herbicides from the same mode of action (MOA) for two or more consecutive years) and timing of application (spraying on older or larger plants) all increase population size and place pressure on herbicides.

Frequency of resistance present in populations

A few resistant individual plants exist naturally within populations, even before herbicide use. When a herbicide is applied, these individuals survive and set seed, while most susceptible plants die. Repeated use of the same herbicide eventually results in a larger population of resistant survivors in that field.

The biology

Outcrossing species (e.g., blackgrass, Italian ryegrass) with high seed bank populations and short soil seed bank life are likely to develop resistance rapidly, compared to predominantly self-pollinating (e.g., wild oats, bromes) species.

Weed pressure

The greater the population size, the greater the likelihood of any herbicide selecting resistant individuals within that population.

Key herbicides

In Ireland, the two most widely used post-emergence herbicide types for selective grass weed control within crops have acetyl-CoA carboxylase (ACCase)- (Group 1) or acetolactate synthase (ALS)- (Group 2) inhibiting MOA. MOA is how a herbicide interrupts the biological pathways of susceptible plants, causing immediate damage and plant death.

The ACCase herbicides control grass weeds by disrupting fatty acid synthesis, causing main shoot death at the growing point. ACCase herbicides include three chemistries: Axial, Falcon, or Stratos Ultra.

The ALS herbicides disrupt the synthesis of critical amino acids, causing stunted growth or abnormal growth at the growing point. ALS herbicides include five chemistries, of which Pacifica and Broadway Star are used for grass-weed control.

The use of ACCase/ALS herbicide chemistries is determined by the specificity of the crop and the weed species involved. There are several chemical control options for wheat, but no herbicide options to effectively control sterile brome or black grass in barley, and virtually no control options for oats (Table 1).

Furthermore, where the limited herbicides belonging to the ACCase/ALS groups are used repeatedly (and at lower than recommended rates), these factors pose a very high risk for developing herbicide cross resistance. 

Table 1. ACCase/ALS herbicide mode of action for selective control of critical grass weeds in cereals and non-cereal break crops (Product label claims)

Herbicide MOA	Trade name (examples)	Active	Weed susceptibility Wild oats	Weed susceptibility Sterile brome	Weed susceptibility Italian ryegrass	Weed susceptibility Black-grass	Crop
ACCase	Axial	Pinoxaden	x		x		Wheat & Barley
ACCase	Falcon	Propaquizafop	x	x	x	x	Oilseed rape & beans
ACCase	Stratos Ultra	Cycloxydim	x	x	x	x	Oilseed rape & beans
ACCase	Centurion Max	Clethodim				x	Winter oilseed rape
ALS	Pacifica Plus	Mesosulfuron + iodosulfuron	x	x	x	x	Winter wheat
ALS	Monolith	Mesosulfuron + propoxycarbazone	x	x	x	x	Winter wheat
ALS	Broadway Star	Pyroxsulam	x	x	x		Winter wheat

Resistance mechanisms

The primary mechanism of ACCase/ALS resistance in grass weeds is either:

1. Target-site resistance (TSR) is where a plant changes the structure of its herbicide-binding site due to mutation(s), which blocks herbicide activity.
2. Non-target-site resistance (NTSR) is where plants can degrade herbicides rapidly before reaching the binding site.

TSR is specific to Mode Of Action (MOA), whereas resistance developed through NTSR mechanisms can affect multiple herbicide chemistries. Herbicide cross-resistance to the same (ACCase) or different MOA (ACCase and ALS) via., either TSR or NTSR or sometimes both can occur within a single field population.

Symptoms of herbicide resistance

Herbicide resistance may go unnoticed initially due to low plant numbers that are resistant, and or symptoms may initially look like weed escapes from spraying or small irregular weed patches. However, herbicide-resistant plants can quickly become dominant, primarily where the same herbicides are used repeatedly. Visual symptoms of suspected resistance post herbicide application are shown in Figure 2 and include:

1. A mix of healthy and dead plants of the same species
2. Inadequate control of one species alongside other susceptible species well-controlled 

It is essential to conduct resistance testing rather than relying on your own judgment and field observations. If the weed population resists one herbicide, it does not mean it is resistant to another herbicide within the same Group.

Glasshouse testing will confirm herbicide weed resistance and the herbicide’s MOA. Predicting cross-resistance patterns is difficult in the field and creates uncertainty about herbicide use. 

Resistant annual meadow-grass plants (green) alongside herbicide controlled (yellow) and poorly controlled (green/yellow) in wheat

Herbicide resistance testing

Herbicide Resistance Testing Application form (pdf)

Prevention is better than cure is the best way to describe the approach to stop the development of grass weed herbicide resistance in arable crops. A critical prevention step in addressing the herbicide resistance threat is resistance testing. It will:

1. Confirm whether surviving weed populations are resistant or poorly controlled from sub-optimal herbicide application rates, poor spray coverage, application timings, weather, etc.
2. Determine what herbicides are effective against the weeds present in your crop. 
3. Suggest integrated methods to eliminate or control resistant populations. 
4. Allow mapping of resistance evolution and monitoring of the impact of resistant management.

For grass weeds, the project conducted controlled glasshouse screens using ACCase (e.g. Axial or Falcon) or ALS (e.g. Pacifica Plus or Broadway Star) glyphosate herbicides and pre-emergence herbicides (e.g. pendimethalin). We also conduct target-site resistance analysis where possible.

Timeline for resistance testing

• Seed cleaning, sorting individual population samples and germination testing (7 days)
• Seed dormancy breaking treatments (requires 6-7 weeks, especially for black-grass)
• Initial screening with a single rate (i.e. field label rate). Plants will be sprayed at 2-3 (black-grass, bromes, Italian ryegrass) or 3-4 (spring wild oats) leaf stages. Visual assessment for survival conducted 28 or 35 days (bromes) after spraying. (8 weeks)
• At this point, individual sensitivity results will be communicated to farmers who submitted seed samples, with information concerning less and highly effective herbicide options.

• Detailed dose-response analysis will be conducted on populations that are least sensitive to full label rate of ACCase/ALS herbicides, with dose rates from 0.25 to 8 times the full label rate to assess resistance levels. (8 weeks)
• In parallel, DNA and PCR techniques will be deployed to identify the target-site mutation(s) that contribute to resistance.

We conduct glasshouse screening for broadleaf weeds using ALS (e.g. Ally Max, Boxer, Zypar, etc.) and synthetic auxin (e.g. 2,4 D) herbicides.

Quality and quantity of seeds required for accurate resistance testing

• Seed samples must be harvested when at the ripe stage
• For smaller seeds (e.g. Italian ryegrass, lesser canary grass, black-grass) two cupful of seed is required. 
• For larger seeds (e.g. Wild oats, sterile brome) a pint glass of seed is required. 
• Ensure samples are dry and stored in a cool dry place.
• Do not mix seeds from other fields or species.

Seed submission form for resistance testing – Grass Weed Seed Collection Form (pdf)

How to collect seeds for resistance testing 

Typically testing will not begin until 4-7 weeks after seeds arrive at the laboratory because most grass weeds have a dormancy period. Different test types are used to detect resistance in the population.

The most common type is glasshouse seed testing. Different post-emergence herbicides are tested on the young plants (at 2-3 leaf stage for grass weeds or 4-6 leaf stage for broad-leaved weeds) grown from the weed seed samples and compared with known-sensitive or resistant populations. Visual assessments for plant survival are conducted 28-35 days post-spraying.

For pre-emergence herbicides, treatments are applied on unchitted or chitted seeds immediately after sowing. Detailed dose-response and molecular assays are conducted on those showing less sensitivity or resistance.

Lab petri dish seed assays are also conducted. The seeds are grown in herbicide solutions, and seedling growth will be evaluated after 14 days. For example, seedling growth in pendimethalin (pre-emergence) herbicide solutions is tested. Pendimethalin resistance is used as a proxy for NTSR in grass weeds. This quick test is not suited for all weed/herbicide types.

Weed	Herbicide tested
Blackgrass	Falcon, Stratos, Centurion, Pacifica, Glyphosate
Italian ryegrass	Axial, Falcon, Stratos, Centurion, Pacifica or Monolith, Broadway, Glyphosate
Wild oats	Axial, Falcon, Stratos, Pacifica or Monolith, Broadway
Bromes	Falcon, Stratos, Pacifica, Broadway. Glyphosate
Canary grass	Axial, Falcon, Stratos, Pacifica or Monolith, Broadway, Glyphosate
Meadow grass	Pacifica, Broadway, Centurion, Glyphosate
Rat’s tail fescue	Pacifica, Broadway, Centurion, Glyphosate
Scutch	Glyphosate, Falcon, Stratos

 

Herbicide efficacy research and weed resistance

The ECT project addressed recent reports of poor grass weed control. It also monitored and mapped herbicide-resistant weeds.

Below is a pictorial presentation of glasshouse screens conducted on difficult-to-control grass weeds collected from several problem fields nationwide since 2019.

• Initial screening with a single rate (i.e. field label rate). Plants will be sprayed at 2-3 (black-grass, bromes, Italian ryegrass) or 3-4 (spring wild oats) leaf stages. Visual assessment for survival conducted 28 or 35 days (bromes) after spraying. (8 weeks)
• At this point, individual sensitivity results will be communicated to farmers who submitted seed samples, with information concerning less and highly effective herbicide options.

• Detailed dose-response analysis will be conducted on populations that are least sensitive to full label rate of ACCase/ALS herbicides, with dose rates from 0.25 to 8 times the full label rate to assess resistance levels. (8 weeks)

Blackgrass

Except for the Cork population, which developed resistance to all herbicides from the ACCase group, the remaining four populations (Waterford, Meath, Tipperary and Dublin) exhibited multiple resistance to both ACCase and ALS groups.

Stratos Ultra was ineffective in all four resistant populations – Cork, Meath-1, Meath-2, and Waterford.

Pacifica Plus was virtually ineffective in three – Meath-1, Meath-2 and Waterford of the blackgrass populations.

One and a half times or above the recommended field rate of glyphosate was required to kill 99% of the treated plants, sensitive or either ACCase or ALS-resistant or both

Italian ryegrass

All populations from Tipperary, Meath, Dublin and Cork exhibited multiple resistance to both ACCase and ALS groups.

Broadway Star was ineffective in three populations – Dublin, Tipperary, and Wexford. At the same time, twice the recommended field rate, well above the legal limit for farmer use was required to control Meath populations effectively.

Pacifica Plus was ineffective in three populations – Dublin, Tipperary, and Wexford. At the same time, 1.5 times the recommended field rate, which is well above the legal limit for farmer use was required to achieve total control of Meath populations. 

Wild oats

Plants were sprayed at the 3-4 leaf stage. Individual wild oat plants within a population survived Falcon (sprayed at half and full rates) and Axial (half rate only). ACCase-Stratos Ultra and both the ALS herbicides were found to be very effective. 

Lesser canary grass

The weed is not listed on ACCase/ALS chemical labels. Effective control was achieved with all herbicides when applied at the recommended rate only.

Annual meadow grass

Annual meadow grass showing natural tolerance to Axial and Falcon. Despite this natural trait, Centurion Max still provides adequate control. Also shown is the control using glyphosate at dose rates between 0.15 and two times the recommended rate

Bromes

All herbicides were highly effective on all brome types, including less-sensitive ones, when used at recommended rates and at the correct growth stage (2-3 leaves).

Conclusions

1. The project confirmed resistance in blackgrass, Italian ryegrass, wild oats and annual meadow-grass
2. We have found a widespread tolerance to ALS-Pacifica among brome populations, possibly due to repetitive use of reduced field rates.
3. Resistance testing results have helped farmers plan/deploy integrated weed management (IWM) and resistance management strategies (including alternative chemistry) to eliminate or contain further spread and development of resistance.
4. Glasshouse screening has helped to establish baseline sensitivity of weed populations from which future changes can be monitored.
5. Free resistance testing service via., ECT project has significantly increased awareness of the grass weed challenge across the whole tillage sector
6. Twenty of the 123 wild oat populations collected in Wexford, Cork, Kilkenny, Tipperary and Kildare counties were resistant to at least one ACCase (Axial, Falcon or Stratos Ultra) herbicide. The primary mechanism of ACCase resistance is TSR (mutant ACCase Ile-1781 or Asp-2078), with NTSR being partial or at an early development stage.
7. Six of the 14 Italian ryegrass populations collected in Cork, Meath, Tipperary, Kilkenny and Kildare were resistant to either ACCase (Axial, Falcon or Stratos Ultra) or ALS (Pacifica or Broadway Star) herbicides or both. ALS Pro-197 mutation and ACCase NTSR predominates.
8. Thirteen of the 25 blackgrass populations collected in Meath, Cork, Waterford, Wexford and Kildare were resistant to either ACCase (Falcon or Stratos Ultra) or ALS (Pacifica) herbicides or both. ACCase Ile-1781 or ALS Pro-197 mutations were frequently associated with this resistance, with ACCase/ALS NTSR documented in a few populations.
9. No full herbicide-resistant bromes were documented from>120 brome populations (all types) collected in the southeast. However, there is a high likelihood of tolerance or creeping resistance to either ALS-Pacifica Plus or ACCase-Stratos Ultra via or both. NTSR may be widespread, which may result from the frequent use of reduced herbicide field application rates.
10. Three annual meadow grass samples collected in Dublin and Wexford were confirmed resistant to ALS (Pacifica and Broadway Star) herbicides. The primary mechanism of ALS resistance is TSR (mutant Trp-574).

Herbicide resistance management strategies

1. Keep accurate field-by-field records of weed populations and herbicide applications.
2. Use glyphosate before crop sowing, followed by residual herbicides (e.g. pendimethalin, flufenacet, prosulfocarb, etc.) to reduce the target weed population.
3. Use the recommended field rates to ensure maximum efficacy.
4. Always apply ACCase/ALS herbicides on small and actively growing weeds.
5. Avoid using ACCase/ALS herbicides as the only control option
6. If resistance is suspected (especially in fields with a long history of continuous herbicide use), do not use the same herbicide or herbicide with the same MOA.
7. Conduct resistance testing to establish i) the sensitivity status of your weed population and ii) the effective available control options.
8. Ensure surviving plants do not set and shed viable seeds to the soil seed bank using alternative herbicides, spot spraying or hand rogueing.
9. Always use chemical control jointly with cultural/non-chemical control options (e.g., crop rotation, stale seedbed technique, manipulating sowing time, etc.) and on-farm biosecurity measures (e.g., certified seed, machinery hygiene, etc.).

Effective herbicide options are available for controlling resistant or suspected less-sensitive populations.

The table below shows ACCase herbicides (Axial, Falcon, Stratos Ultra, Centurion Max) and ALS herbicides (Pacifica and Broadway Star) applied at the recommended rate. 

Based on sensitivity testing, effective herbicide options are available for controlling resistant or suspected less-sensitive populations

	Axial	Falcon	Stratos Ultra	Centurion Max	Pacifica	Broadway Star	Glyphosate	Resistance management tools
Wild Oats	R	R	R	–	S	S	S @ 1.5l/ha	Glyphosate + ALS
Bromes – all types	–	S	RS	–	R/RS/S	S	S @ 1.5L/ha	Glyphosate + Pre-emergent + Falcon/Broadway
Blackgrass	–	R	R	R	R	–	S @3L/ha	Glyphosate + Pre-emergent
Italian ryegrass	R	R	R	R	R	R	S @ 3L/ha	Glyphosate + Pre-emergent
**Annual meadow grass	NT	NT	NT	S	R	R/S	S @ 3L/ha	Glyphosate + Pre-emergent
**Rats tail fescue	NT	NT	NT	R/RS	S	S	S @ 3L/ha	Glyphosate + Pre-emergent + Centurion or ALS
*Lesser canary grass	S	S	S	S	S	S	S @ 1.5L/ha	Glyphosate + Pre-emergent + ACCase/ALS

How to minimise/stop the spread of herbicide-resistance risks 

Herbicide resistance is an irreversible process; however, you can slow its development by adopting good agricultural practices such as:

• Practicing good rotation (spring and autumn-sown crops; cereal and non-cereal break crops).
• Using as many cultural and non-chemical tactics (e.g. delayed sowing, higher than the normal seed rates, stale seedbed technique,etc.).
• Including low-risk pre-emergence residual herbicides.
• Not using ACCase/ALS herbicides as a sole weed control method in consecutive crops.
• Always use recommended label field rates of ACCase/ALS herbicides
• Always spray on small, actively growing weeds
• Correct conditions for spraying (not too cold or not too hot for herbicides to work)
• Using low-risk propyzamide (Kerb) followed by ACCase-centurion max (clethodim) where possible to reduce population size in break crops.
• Walk the crops before and after herbicide application to ensure herbicide performance.
• Practising machine hygiene before coming into fields and before moving field-to-field.
• Conducting resistance testing once every three years for certain grass weeds
• A resistance test is ‘essential’ if your field has black-grass and Italian ryegrass species populations. Our study has shown that resistance can develop in these two species even at a low population levels.

Importance of resistance testing 

The resistance profile of your weed population across the entire spectrum of herbicide options and the underlying mechanisms, whether NTSR, TSR, or both contributing, can only be determined by resistance testing (glasshouse and lab-based).

• It is important to note that because your weed population is resistant to an herbicide, it does not necessarily mean that it is resistant to all other herbicides from the same group (ACCase or ALS).

As a farmer you want to know.

• What herbicide options are still available, and how this will influence your crop rotation
• How to develop tailor-made resistance management and IWM strategies with judicious use of effective herbicides for your situation
• How to prevent the weed population from developing complete resistance
• How to avoid the spread of resistant weeds from field to field and
• Saving money and herbicide costs

Farmers share their experiences of controlling grass weeds