GEORGIA DOT RESEARCH PROJECT 17-10 FINAL REPORT ENHANCING EXTENSION RECOMMENDATIONS FOR IMPROVING HERBICIDE RESISTANCE MANAGEMENT ON GEORGIA ROADSIDES OFFICE OF PERFORMANCE-BASED MANAGEMENT AND RESEARCH 600 W PEACHTREE STREET NW ATLANTA, GA 30308 1.Report No.: FHWA-GA-20-1710 2. Government Accession No.: 3. Recipient's Catalog No.: NA NA 4. Title and Subtitle: Enhancing Extension Recommendations for Improving Herbicide Resistance Management on Georgia Roadsides 7. Author(s): Patrick McCullough, Donn Shilling 9. Performing Organization Name and Address: University of Georgia 1109 Experiment Street Griffin, GA 30223 5. Report Date: June, 2020 6. Performing Organization Code: NA 8. Performing Organ. Report No.: 17-10 10. Work Unit No.: NA 11. Contract or Grant No.: 12. Sponsoring Agency Name and Address: 13. Type of Report and Period Covered: Georgia Department of Transportation Final; August, 2017- June, 2020 Office of Performance-based Management and Research 600 W Peachtree St. NW 14. Sponsoring Agency Code: NA Atlanta, GA 30308 15. Supplementary Notes: Prepared in cooperation with the U.S. Department of Transportation, Federal Highway Administration. 16. Abstract: The presence of Italian ryegrass on Georgia roadsides reduces motorist site visibility and increases maintenance costs for the DOT. Herbicide selection is limited on roadsides due to economics and the susceptibility of roadside turfgrasses to injury. Consequently, agronomists often apply the same chemistries every year without rotating herbicide modes of action. These spray programs promote the development of herbicide resistance in weed populations and may compromise the safety and sustainability of roadsides throughout Georgia. Research was conducted to evaluate the extent of herbicide resistance to glyphosate and acetolactate synthase (ALS)-inhibitors in areas it is most commonly found on Georgia roadsides. These are the two chemistries that have been primarily used for controlling ryegrass on roadsides for several decades. Twenty-eight populations were sampled on routes that had sprayed herbicides for ryegrass control in the winters of 2018 and 2019. Plants that were not controlled by the current programs were grown in the greenhouse, seed was harvested, and new plants were screened for resistance. In dose response, experiments approximately one- third of the ryegrass plants exhibited resistance or enhanced tolerance levels to glyphosate compared to known susceptible populations and the majority of other plants collected on roadsides. In hydroponic assays, approximately 20% of the ryegrass populations were resistant to ALS-inhibitor herbicides. Glyphosate-resistant ryegrass was controlled by Envoy (clethodim) and Piper (flumioxazin + pyroxasulfone) when applied alone or in combinations with Esplanade (indaziflam). The majority of the ryegrass sampled in roadsides was susceptible to glyphosate and ALS-inhibitors. Nonetheless, resistance was detected in several populations that could warrant rotation to other chemistries to help delay the spread of these biotypes on roadsides. 17. Key Words: herbicide, glyphosate, ryegrass, 18. Distribution Statement: resistance NA 19. Security Classification 20. Security 21. Number of 22. Price: (of this report): Classification (of this Pages: page): Free Unclassified Unclassified 37 GDOT Research Project No. RP 17-10 Enhancing Extension Recommendations for Improving Herbicide Resistance Management on Georgia Roadsides Final Report By Dr. Patrick McCullough Associate Professor Dr. Donn Shilling Professor Contract with Georgia Department of Transportation In cooperation with U.S. Department of Transportation Federal Highway Administration June 2020 The contents of this report reflect the views of the author(s) who is (are) responsible for the facts and the accuracy of the data presented herein. The contents do not necessarily reflect the official views or policies of the Georgia Department of Transportation or the Federal Highway Administration. This report does not constitute a standard, specification, or regulation. ii (This page intentionally left blank) iii TABLE OF CONTENTS Page LIST OF TABLES ...............................................................................................................v LIST OF FIGURES ........................................................................................................... vi LIST OF PICTURES ........................................................................................................ vii EXECUTIVE SUMMARY ............................................................................................. viii ACKNOWLEDGEMENTS ............................................................................................... ix *INTRODUCTION .............................................................................................................1 *OBJECTIVE ......................................................................................................................6 *PROCEDURES..................................................................................................................7 *FINDINGS .......................................................................................................................12 *CONCLUSIONS..............................................................................................................23 *REFERENCES ................................................................................................................26 iv LIST OF TABLES Table 1. Page Location information for annual ryegrass plants collected for herbicide screenings.....................................................................8 2. Statistics from regression analysis conducted for ryegrass plants treated with glyphosate in dose-response experiments in 2018-2019....................14 3. Statistics from regression analysis conducted for ryegrass plants treated with glyphosate in dose-response experiments in 2019-2020....................16 4. Resistance confirmations to ALS-inhibitors from hydroponic assays...........19 5. Control of glyphosate-resistant and susceptible biotypes at 28 days after treatments of various herbicides with different modes of action in the greenhouse..............................................................................21 v LIST OF FIGURES Figure Page 1. Injury of ryegrass biotypes to glyphosate in dose-response experiments, 2018-2019...............................................................................13 2. Injury of ryegrass biotypes to glyphosate in dose-response experiments, 2019-2020...............................................................................15 vi LIST OF PICTURES Picture Page 1. Italian ryegrass growing on a roadside in May 2018...............................2 2. Segregation of herbicide-resistant and susceptible biotypes after a broadcast of application of sulfonylurea herbicide .................................4 3. Establishment of new ryegrass from seed harvested off of plants collected on roadsides .............................................................................9 4. Hydroponic screening of Italian ryegrass biotypes in a greenhouse experiment ..............................................................................10 5. Ryegrass plants treated with a glyphosate in various concentrations in a dose-response experiment...............................................................................17 6. Response of glyphosate-resistant and susceptible ryegrass biotypes to herbicides in a greenhouse experiment .............................................22 vii EXECUTIVE SUMMARY The presence of Italian ryegrass on roadsides reduces site visibility for motorists and increases maintenance costs for the Georgia DOT. Herbicide applications are the most economical method to control Italian ryegrass to enhance roadside quality. Agronomists using the same herbicides every year without rotating modes of action promote the onset of herbicide resistance over time. Understanding the extent of resistance in ryegrass is critical for improving the sustainability of roadside management programs for Georgia. Research was conducted to evaluate resistance to glyphosate and acetolactate synthase (ALS)-inhibitors in areas where ryegrass is most prevalent on Georgia roadsides. These chemistries were the focus of the research because of their extensive use for controlling ryegrass on roadsides for several decades. Twenty-eight populations were sampled on routes that had sprayed herbicides for ryegrass control in the winters of 2018 and 2019. Plants that were not controlled by the current programs were grown in the greenhouse, seed was harvested, and new plants were screened for resistance. In dose response, experiments approximately one-third of the ryegrass plants exhibited resistance or enhanced tolerance levels to glyphosate compared to known susceptible populations and the majority of other plants collected on roadsides. In hydroponic assays, approximately 20% of the ryegrass populations were resistant to ALS-inhibitor herbicides. Glyphosate-resistant ryegrass was controlled by Envoy (clethodim) and Piper (flumioxazin + pyroxasulfone) when applied alone or in combinations with Esplanade (indaziflam). From this work, the majority of ryegrass sampled was susceptible to glyphosate and ALS-inhibitors. However, resistance was detected in several populations that could warrant the rotation to other herbicide modes of action to delay the spread of these biotypes on roadsides. viii ACKNOWLEDGEMENTS Special thanks to VJ Mantripragada, David Westbury, Seth Williams, Dr. Diego Gomez de Barreda, and Dr. Jialin Yu for technical support with this research. ix INTRODUCTION The presence of Italian ryegrass (Lolium multiflorum L.) on Georgia roadsides reduces motorist site visibility and increases mowing costs for the Georgia DOT (GDOT). Seed germinates from September to November when soil temperatures drop below 70 F. Ryegrass seedlings mature in fall, overwinter in a vegetative state, and resume active growth in spring. Italian ryegrass grows well under cool conditions when roadside grasses are dormant or have limited competitive growth. Plants exhibit erect growth that reaches approximately three feet in height upon maturity (Picture 1). Consequently, ryegrass may interfere with the growth of roadside grasses during spring transition and early summer. Italian ryegrass is a prolific seed producer that contributes to annual infestations. The seedhead is a long spike with at least ten alternating florets. The florets contain long awns that are not present on perennial ryegrass (Lolium perenne). Plants typically produce seedheads by March in most parts of Georgia. Seed dispersed in late spring can remain dormant in soil for years. Another key characteristic to help identify ryegrass, without seedheads, is the clasping auricles at the junction of the leaf sheath and blade. The auricle may help practitioners identify ryegrass from other weedy grasses, such as tall fescue. Mowing and Cultural Control Modifications to management programs will help reduce Italian ryegrass establishment in fall. For example, mowing before seedhead formation can suppress ryegrass growth and inhibit the production of viable seed in spring. However, this method is cost prohibitive for managing over 700,000 acres of roadside vegetation in Georgia. Modifications in cultural practices that are applicable to other cropping systems, such as 1 pastures and forages, are often not applicable for most roadside managers. Therefore, an integrated strategy consisting of timely mowing, controlling summer weeds, and promoting roadside grass competitive growth can help reduce ryegrass populations. Picture 1. Italian ryegrass growing on a roadside in May 2018. The importance of herbicides for ryegrass control Practitioners may use preemergence herbicides for preventing the establishment of ryegrass in fall. Dinitroanilines, or WSSA Group 3 herbicides, include oryzalin (Surflan, others), pendimethalin (Pendulum, others) and prodiamine (ProClipse, others). These herbicides inhibit microtubule assembly during cell division of young roots and shoots after germination that prevents ryegrass establishment (Senseman 2007). Indaziflam (Esplanade) inhibits cellulose biosynthesis and offers an alternative mode of action to DNA herbicides for ryegrass control in warm-season grasses. Applications should be timed 2 when soil temperatures drop below 70 in fall. Ideally, treatments should be applied prior to a rainfall to enhance soil incorporation and herbicide activation. However, due to the costs of applications and scheduling with DOT applications, preemergence herbicides are rarely used alone for ryegrass control and treatments are often applied with postemergence herbicides in late fall. The optimum timing for postemergence control of ryegrass on roadsides is when plants are less than 6 inches in height in early winter. Bermudagrass managers have used acetolactate synthase (ALS) inhibitors for several decades due to the selectivity and safety to roadside turf. These herbicides include Escort (metsulfuron), Oust (sulfometuron), Pastora (nicosulfuron + metsulfuron), Derigo (thiencarbazone + foramsulfuron + iodosulfuron), and Matrix (rimsulfuron). Bahiagrass managers have traditionally used Oust for postemergence ryegrass control, but most other ALS-inhibitors cause unacceptable injury. Postemergence herbicides are more effective in early winter, compared to spring timings, because of the size and maturity of plants at application. Italian ryegrass is generally susceptible to postemergence herbicides in early winter prior to the onset of freezing temperatures and before seedhead emergence. Most of these herbicides require a non-ionic surfactant at 0.25% v/v of spray solution (1 qt/100 gal) to enhance foliar uptake and spray retention. Glyphosate (Roundup, Accord, others) is a nonselective herbicide widely used on bermudagrass roadsides for ryegrass control in winter. Moderate rates of glyphosate in bermudagrass, such as 0.125 to 0.25 lb ae/acre, generally do not affect spring transition when applied in winter. However, glyphosate use in spring could cause delayed green-up and growth inhibition to bermudagrass. 3 Herbicide resistance and concerns about roadside management in Georgia A major limitation to postemergence control of ryegrass is herbicide resistance (Heap 2020). Resistance of ryegrass species to ALS-inhibitors and glyphosate has been confirmed throughout the world due to overuse of these herbicides (Feng et al. 1999; Nandula et al. 2008: Shaner 1999). Resistance develops from selection pressure caused by repeated use of the same herbicide or mode of action over years. Genetic variation among biotypes in a ryegrass population contribute to differential levels of susceptibility to herbicides through altered target-site binding or enhanced degradation (Simarmata and Penner 2008; Simarmata et al. 2003; Wiersma et al. 1989). Other resistance mechanisms for Italian ryegrass may include reduced absorption, herbicide sequestration, or overproduction of the target site enzyme (Shaner 2009; Yu et al. 2009). As susceptible biotypes are controlled by a particular herbicide over years, resistant biotypes spread in these areas. This type of selection pressure will shift a population from susceptible to resistant biotypes over years. The potential development of resistance to these herbicides in Italian ryegrass could warrant modifications to DOT spray programs that include product rotations to delay or avoid the onset of resistance. Picture 2. Segregation of herbicide-resistant and susceptible biotypes after a broadcast of application of sulfonylurea herbicide. 4 Concerns about resistance in current DOT programs for ryegrass control Glyphosate has been the primary herbicide used for ryegrass control on roadsides in Georgia for over a decade. This herbicide is nonselective and must be used during winter dormancy to minimize injury to bermudagrass and other roadside grasses. Glyphosate replaced herbicides that inhibit acetolactate synthase, or the ALS-inhibitors, due to resistance in ryegrass populations throughout the Southern U.S. While these herbicides are still in rotation for controlling other weeds, they have not been used for ryegrass control on Georgia roadsides for more than ten years. The current ryegrass control program that was adopted by GDOT several years ago includes glyphosate (Accord XRT) plus a preemergence herbicide indaziflam (Esplanade). The use of Esplanade significantly improves the residual control of annual weeds on roadside turf, especially when glyphosate is used for controlling established plants. An advantage of Esplanade over other preemergence herbicides is the potential for applications to provide early-postemergence control of seedling ryegrass. However, Esplanade does not control ryegrass that has matured and reached several inches in height. The spread of glyphosate resistance may be exacerbated when Accord XRT is applied with Esplanade from December through April due to limited efficacy of indaziflam on mature plants. The development of herbicide resistance management programs is challenging for roadside managers. Agronomists have limited resources available to manage weeds and the level of herbicide resistance on roadsides has not been extensively investigated. The location of resistant weed populations could vary significantly throughout Georgia due to regional differences in management programs. These factors could all contribute to the potential for herbicide resistance development throughout Georgia roadsides. The 5 identification of herbicide resistant ryegrass will enable agronomists to make adjustments in control programs that will prevent further spread of these populations. OBJECTIVE The objective of the proposed research was to evaluate herbicide-resistance of ryegrass on roadsides managed by GDOT and determine herbicide alternatives for programs that could control and delay the further spread of resistant populations. 6 PROCEDURES Evaluation of glyphosate resistance. Experiments were conducted to evaluate the differential tolerance levels to glyphosate for ryegrass populations collected on Georgia roadsides. Plants were collected on roadsides in late winter and spring in 2018 and 2019 that were displaying no visual signs of control from herbicide applications made to the roadsides (Table 1). Approximately fifteen plants were collected per location. Plants were placed in plastic pots with 20-cm diameters and 30-cm depths filed with potting soil and placed in a greenhouse on the UGA Griffin Campus set for approximately 25/20 C (day/night). Plants were fertilized and irrigated as needed to promote growth. For a four to six-month period after harvesting, plants were grown in the greenhouse and seed was collected from the inflorescence. Seed was then dried in a growth chamber set for 30 C for one week and seedhead material was removed using sieves to collect seeds. The seed was then scattered over plastic flats in the greenhouse filled with sand:peat moss (85:15, Picture 3). The flats were irrigated and fertilized as needed to promote establishment of the seedlings. After reaching a multi-leaf growth stage, single plants were transplanted to plastic pots with a 3.8-cm diameter and 20-cm depths filled with the aforementioned sand:peat moss soil. Plants were then fertilized and irrigated to promote growth. Once grasses reached about 10-cm height and were tillered, glyphosate (Roundup Pro) was applied at ten rates ranging 2 to 128 fl oz/acre. Treatments were applied at 20 gal/acre volume in a chamber using an air pressured sprayer equipped with a single, 8002E, flat-fan nozzle. Ryegrass control was visually evaluated after three weeks on a percent scale where 0 equaled no control and 100 equaled complete plant death. 7 Table 1. Location information for annual ryegrass plants collected for herbicide screenings. Sample Location 1 I-75 Exit 205 City Jackson, GA Codes in Collection date Figure 1 and 2 1/26/18 RG1 2 I-75 Exit 157 Macon 1/29/18 RG2 3 5700 US Hwy 41S Culloden, GA 1/29/18 RG3 4 Forsyth-Yatesville Rd Yatesville, GA 1/29/18 RG4 5 Exit 198 (N) I-75 High Falls, GA 1/29/18 RG5 6 1-85 N Fortson, GA 1/30/18 RG6 7 1-85 GA/Hwy 18 Pine Mountain, GA 1/30/18 RG7 8 I-85 N/Hwy 54 Hogansville, GA 1/30/18 RG8 9 I-85 N Moreland, GA 1/31/18 RG9 10 I-85 N Palmetto, GA 1/31/18 RG10 11 I-16 S/Sgoda Rd Macon, GA 1/31/18 RG11 12 I-16 S/GA Hwy-358 Danville, GA 1/31/18 RG12 13 I-16 W Dublin, GA 3/8/18 RG13 14 Hwy-520 Augusta, GA 3/8/18 RG14 15 I-75 Exit 64 Tifton, GA 3/25/19 RG1 16 I-75 Exit 80 Sycamore, GA 3/25/19 RG2 17 I-75 Exit 109 Vienna, GA 3/25/19 RG3 18 I-75 Exit 127 Henderson, GA 3/25/19 RG4 19 I-75 Exit 185 Forsyth, GA 3/25/19 RG5 20 I-75 Exit 205 Jackson, GA 3/25/19 RG6 21 I-20 Exit 92 Convington, GA 3/26/19 RG7 22 I-20 Exit 105 Rutledge, GA 3/26/19 RG8 23 I-20 Exit 121 Buckhead, GA 3/26/19 RG9 24 I-20 Exit 138 Union Point, GA 3/26/19 RG10 25 I-20 Exit 148 Crawfordville, GA 3/26/19 RG11 26 I-20 Exit 130 Greensboro, GA 3/26/19 RG12 - Commercial seed n/a #4 8 Picture 3. Establishment of new ryegrass from seed harvested off of plants collected on roadsides. The experimental design was a randomized complete block with five replications. Regression analysis was performed with the Linear and Nonlinear Regression Procedures in SAS. Data was plotted on figures, and regressed against the following equations: y = 0 + * 1 (1- (exp(-2*x)) where 0 is the lower asymptote, 1 is the maximum predicted response, 2 is the slope, and x is the glyphosate rate in g ai/ha. The equation was selected that described the relationship of plant response with herbicide concentrations. 9 Hydroponic assays for ALS-resistance. Ryegrass seed was planted in greenhouse flats filled with sand:peat (85:15) and irrigated as needed to promote germination. Seedlings were fertilized and irrigated in flats until developing 3 to 5 leaves. Individual plants were then removed from the flats and soil was washed from roots. Plants were then placed in a 6-L hydroponic tank filled with half-strength Hoagland nutrient solution. Fifteen holes measuring 1.5-cm each were drilled in the lids of the tanks and plants roots were placed through the holes to facilitate suspension in the nutrient solution. Fifteen plants were placed in each tank that included three biotypes with five replications. An aquarium pump provided oxygen to the solution. After one week, a sulfonylurea herbicide, flazasulfuron (Katana 25WG), was spiked in the tanks at 0 or 1 M. Plants were grown for one week after treatments and then control was visually evaluated as 0 (no response) or 1 (controlled). This methodology has shown separation of resistant grass biotypes from susceptible ones. Picture 4. Hydroponic screening of Italian ryegrass biotypes in a greenhouse experiment. 10 Evaluation of herbicide alternatives for controlling glyphosate-resistant ryegrass. A greenhouse experiment was conducted to evaluate the efficacy of glyphosate alternatives for controlling Italian ryegrass. Susceptible and resistant biotypes to glyphosate were seeded in greenhouse flats. Seed was then scattered over plastic flats in the greenhouse filled with sand:peat moss (85:15). The flats were irrigated and fertilized as needed to promote establishment of the seedlings. After reaching a multi-leaf growth stage, single plants were transplanted to plastic pots with a 3.8-cm diameter and 20-cm depths filled with the aforementioned sand:peat moss soil. Plants were then fertilized and irrigated to promote growth. Treatments were applied to 1-tiller ryegrass and included the factorial combination two Esplanade rates, 0 or 3.5 oz/acre, applied with three herbicides, Accord XRTII at 16 oz/acre, Piper at 8 oz/acre, and Envoy Plus at 9 oz/acre Piper and Envoy Plus are offer different modes of action to glyphosate and ALS-inhibitors and have shown good efficacy in previous experiments. A nontreated check was included. Treatments were applied in 40 gal/acre volume using an air pressured sprayer equipped with a single, 8002E, flat-fan nozzle. Ryegrass control was visually evaluated after four weeks on a percent scale where 0 equaled no control and 100 equaled complete plant death. The experimental design was a randomized complete block with five replications. Data were subjected to analysis of variance and means were separated with Fisher's LSD test at = 0.05. 11 FINDINGS Evaluation of glyphosate resistance. There was wide variability in the response to glyphosate in dose-response experiments from the populations collected throughout the state (Figure 1, Picture 5). The majority of the ryegrass plants collected in both years had comparable I50 levels, or glyphosate rates required to injure plants 50%, to our known susceptible biotypes (Table 2). This suggests that the majority of plants collected were susceptible to glyphosate. Three populations in the 2018 collection had I50 levels greater than most other biotypes, ranging about 3 to 5-fold higher. In 2019, five populations exhibited greater tolerance to glyphosate than the majority of the populations. Most populations identified with resistance to glyphosate are from areas off of I-75 in central Georgia and I-16 south of Macon. These areas have been repeatedly sprayed with Accord XRT, Razor Pro, and other glyphosate products during the winter for over a decade. Despite the current use of glyphosate as the key herbicide for controlling established ryegrass, we did not detect significant resistance in the majority of populations. This may have occurred from skips in spray patterns or failure to apply herbicides in these areas. Another potential limitation to collecting plants was our inability to sample in medians and shoulders on interstates. Plant collections could only be made in areas that were safe for researchers to sample such as lands adjacent to the exits. This limitation could also have prevented us in detecting resistance in plants that would be targeted for control by glyphosate in these areas. 12 Figure 1. Injury of ryegrass biotypes to glyphosate in dose-response experiments, 20182019. 13 Table 2. Statistics from regression analysis conducted for ryegrass plants treated with glyphosate in dose-response experiments in 2018-2019. Regression parametersa Populations r2 0 P I50b 95% CIc #4 0.65 3.6157 93.4369 0.0009 <0.0001 760 615-905 R2 0.82 -2.4253 99.8894 0.0015 <0.0001 500 415-585 R3 0.84 -2.9061 103.04 0.0017 <0.0001 420 365-475 R4 0.41 1.6752 47.4922 0.0007 <0.0001 >3360 >3360 R5 0.74 0.3852 104.5608 8.00E-04 <0.0001 800 615-985 R6 0.76 -0.0097 110.7863 0.0006 <0.0001 1000 730-1270 R7 0.84 -2.7064 101 0.0019 <0.0001 400 330-470 R8 0.63 -2.527 79.6453 1.60E-03 <0.0001 660 500-820 R9 0.59 -0.8615 77.8535 0.0018 <0.0001 600 380-820 R10 0.68 -1.1777 88.448 0.0013 <0.0001 700 485-915 R11 0.70 1.762 123.0075 0.0004 <0.0001 1300 940-1660 R12 0.77 -1.7973 177278.68 1.31E-07 <0.0001 2200 1990-2410 R13 0.69 -0.9384 96.9802 0.0012 <0.0001 650 430-870 R14 0.82 -2.9092 101.8823 0.0023 <0.0001 330 230-430 aData were fit to the following regression equation = y = 0 + * 1 (1- (exp(-2*x)), where 0 is the lower asymptote, 1 is the maximum predicted response, 2 is the slope, and x is the glyphosate rate in g ai/ha. bI50 = glyphosate rate required to injure ryegrass 50%. cCI = confidence interval for the calculated I50 value used to statistically separate estimates. 14 Figure 2. Injury of ryegrass biotypes to glyphosate in dose-response experiments, 20192020. 15 Table 3. Statistics from regression analysis conducted for ryegrass plants treated with glyphosate in dose-response experiments in 2019-2020. Regression parametersa Populations r2 0 I50b 95% CIc #4 0.85 6.9768 85.0818 0.0012 600 530-670 R1 0.91 3.68E+00 90.9756 0.0009 800 731-869 R2 0.71 5.8731 96.3252 0.0007 900 630-1170 R3 0.88 -0.9661 106.8294 0.0005 1300 1165-1435 R4 0.92 -1.9415 103.0912 0.0008 900 730-1070 R5 0.85 1.7789 101.0502 6.00E-04 1100 785-1415 R6 0.84 0.1312 103.9237 0.0007 950 740-1160 R7 0.78 10.8962 83.1857 0.0019 350 320-380 R8 0.74 10.1748 82.1233 3.60E-03 180 145-215 R9 0.80 5.319 86.6192 0.0026 280 260-300 R10 0.86 1.2909 96.9935 0.0009 800 640-960 R11 0.61 14.4647 83.3997 0.0014 400 290-510 R12 0.86 -0.8725 97.179 0.002 380 320-440 aData were fit to the following regression equation = y = 0 + * 1 (1- (exp(-2*x)), where 0 is the lower asymptote, 1 is the maximum predicted response, 2 is the slope, and x is the glyphosate rate in g ae/ha. bI50 = glyphosate rate required to injure ryegrass 50%. cCI = confidence interval for the calculated I50 value used to statistically separate estimates. 16 Picture 5. Ryegrass plants treated with a glyphosate in various concentrations in a doseresponse experiment. 17 Evaluation of ALS-inhibitor resistance. Plants were screened hydroponically for resistance to ALS-inhibitors by spiking a sulfonylurea, Katana (flazasulfuron), in the tanks at various concentrations. This herbicide is highly active on susceptible ryegrass biotypes and was a good indicator herbicide in pilot experiments. Form these evaluations, most ryegrass plants screened were susceptible to ALS-inhibitors. This was determined based on if the majority of the replications exhibited herbicide toxicity relative to the nontreated plants. Ryegrass with confirmed resistance to ALS-inhibitors averaged about 20% of the total populations surveyed (Table 4). These cases were scattered throughout sample sites including I-20, I-75, and I-85. There has been limited use of ALS-inhibitors for ryegrass control in the recent decade which is primarily associated with the widespread resistance throughout the southern U.S. The DOT agronomists may have limited the spread of ryegrass populations with ALS-resistance by rotating to glyphosate and indaziflam programs. The shift in control methods may have also reduced the presence of ALS-resistant ryegrass on roadsides in Georgia. The plants collected were mostly sampled after glyphosate was sprayed by applicators. This may have controlled the ryegrass plants that were resistant to ALS-inhibitors since alternative chemistries were used in these programs. The majority of our ryegrass plants sampled were susceptible to this chemistry suggesting GDOT may have potential to incorporate products like Oust (sulfometuron) or Escort (metsulfuron) back in to winter spray programs. Since resistant biotypes are present, it would be prudent to include herbicides with alternative modes of action as tank-mix partners or in sequential programs for controlling ryegrass on roadsides. 18 Table 4. Resistance confirmations to ALS-inhibitors from hydroponic assays. Population 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Location I-75 Exit 205 I-75 Exit 157 5700 US Hwy 41S Hwy-341 Exit 197 (N) I-75 185 N 185 GA/Hwy 18 I-85 N/Hwy 54 I-85 N I-85 N I-16 S/Sgoda Rd I-16 S/GA Hwy-358 I-16 W Hwy-520 I-75 Exit 64 I-75 Exit 80 I-75 Exit 109 I-75 Exit 127 I-75 Exit 185 I-75 Exit 205 I-20 Exit 92 I-20 Exit 105 I-20 Exit 121 I-20 Exit 138 I-20 Exit 148 I-20 Exit 130 ALS-resistance No No No No Yes Yes No Yes No No No No No No No No No No No No No No Yes No Yes No 19 Evaluation of herbicide alternatives for controlling glyphosate-resistant ryegrass. Esplanade alone at 3.5 oz/acre provided about 50% control of ryegrass that was resistant and susceptible to glyphosate (Table 5). When Esplanade was applied with Accord XRT (glyphosate), ryegrass was controlled 79%. The Esplanade + Accord XRT treatment provided similar control to Esplanade alone when applied to glyphosate-resistant ryegrass. The current DOT spray program consists of Esplanade with Accord which can effectively control ryegrass that is susceptible glyphosate. However, the resistant biotype did not exhibit enhanced control from Esplanade when Accord was applied in the mixture. This exemplifies the limitations to only using one postemergence herbicide mode of action for controlling ryegrass populations on roadsides. Envoy (clethodim) is an ACCase-inhibitor herbicide that effectively controls grassy weeds, including ryegrass. Envoy provided 87% and 61% control of the glyphosate susceptible and resistant biotypes in this experiment. Substituting Envoy with Esplanade improved control of both biotypes to 88% on average. Another herbicide with different modes of action to the aforementioned products, Piper (flumioxazin + pyroxasulfone), controlled both ryegrass biotypes 95% on average. There was no benefit to using Piper in combination with Esplanade or Accord for controlling ryegrass after one month. Nevertheless, the combinations could be applicable when targeting other weeds that are not controlled by these herbicides alone. 20 Table 5. Control of glyphosate-resistant and susceptible biotypes at 28 days after treatments of various herbicides with different modes of action in the greenhouse. Ryegrass control Herbicide Active Ingredient Product rate Glyphosate-susceptible Glyphosate-resistant (oz/acre) ------------------------- % ------------------------ Accord XRT glyphosate 16 fl oz 61 11 Envoy clethodim 9 fl oz 87 61 Envoy + Esplanade clethodim + indaziflam 9 + 3.5 fl oz 86 89 Esplanade indazilam 3.5 fl oz 50 56 Esplanade + Accord indazilam + glyphosate 3.5 + 16 fl oz 79 48 Piper (flumioxazin + pyroxasulfone) 8 oz 93 97 Piper + Esplanade (flumioxazin + pryoxasulfone) + indaziflam 8 oz + 3.5 fl oz 99 86 Piper + Accord (flumioxazin + pryoxasulfone) + glyphosate 8 oz + 16 fl oz 81 91 LSD0.05 21 21 Picture 6. Response of glyphosate-resistant and susceptible ryegrass biotypes to herbicides in a greenhouse experiment. 22 CONCLUSIONS Controlling problem weeds is one of the greatest challenges to maintaining safe and sustainable roadside turf. Herbicides are a fundamental component of integrated weed management programs that promote the release of desirable roadside grasses and limit the establishment of invasive weeds. Italian ryegrass is the most problematic winter weed of roadsides in Georgia that must be controlled with herbicides. Failing to control ryegrass can lead to increased mowing requirements and stand thinning as populations decline in spring. Promoting bermudagrass release and density in spring by eliminating ryegrass can also reduce the potential for new weeds to emerge when populations die out in June. Spray programs must include plans for rotating herbicides to delay the onset of resistance in Italian ryegrass. Selection pressure created by repeated use of the same herbicide over time will shift weed populations and promote the spread of resistant biotypes. Annual weeds like, Italian ryegrass, with prolific seed production are highly adaptable and prone to resistance development. Understanding the threat and spread of herbicide resistant biotypes will be important for developing programs that control established Italian ryegrass and delay the onset of resistance to current chemistries. The GDOT agronomists have been using glyphosate as the primary postemergence herbicide for controlling ryegrass throughout Georgia for over a decade. Glyphosate has been applied during winter months to maximize selectivity and reduce potential to cause severe injury to bermudagrass and other roadside species. Although glyphosate is a nonselective herbicide, it has a single site of action which is associated with most cases of herbicide resistance. Single applications of glyphosate per year for a decade without 23 rotation to other chemistries has resulted in erratic levels of control that are associated with resistance. Approximately one-third of the ryegrass plants sampled exhibited resistance or enhanced tolerance to glyphosate relative to susceptible populations. Glyphosate resistance does not appear to be a statewide problem based on the populations that we sampled in this work. However, agronomists must remain attentive that glyphosate resistance could steadily increase over time if alternative chemistries are not incorporated in spray programs. The current use of Esplanade in ryegrass control programs will help delay the spread of populations with resistance to glyphosate or other modes of action. Contractors for the DOT are currently using Esplanade with glyphosate after ryegrass has emerged. Our greenhouse experiments show that Esplanade has some postemergence activity for controlling young ryegrass plants. This is beneficial for the DOT when treatments are made in late fall as ryegrass begins to emerge. However, erratic control may result from winter treatments if the biotypes are glyphosate resistant. This is because Esplanade does not control mature ryegrass alone when glyphosate is ineffective. There are several chemistries than can be incorporated in GDOT spray programs to supplement or provide options for rotation over years. Envoy (clethodim) is a Group 1, ACCase-inhibitor, herbicide that is highly active on most grass species. It is injurious to bermudagrass in spring and must be used for ryegrass control in winter months, similar to glyphosate. Envoy would be an economical herbicide to combine with or substitute for glyphosate in current spray programs. A limitation to Envoy use is the failure to control broadleaf weeds. Therefore, mixtures with glyphosate or other modes of action will be 24 needed on roadsides if Envoy is used in winter. The Group 1 herbicides are not commonly used in DOT spray programs due to excessive injury potential to desirable grasses during the growing season. There is a high risk of injuring roadside grasses with Envoy if applications are made in late winter, but the risk levels would be comparable to winter glyphosate programs currently used by the DOT. When screening glyphosate-resistant ryegrass, Piper alone and with Esplanade also provided effective control. Piper is a newer combination herbicide for roadside management that contains flumioxazin and pyroxasulfone. These active ingredients are different modes of action than all other herbicides used on roadsides. Piper has been previously used in certain cropping systems, such as wheat, for ryegrass control due to the widespread resistance to glyphosate and ALS-inhibitors. Piper does not control mature ryegrass at labeled use rates and agronomists need to make applications when plants are no more than three inches in height. The identification of herbicide-resistant ryegrass on roadsides suggests that agronomists should modify spray programs over time when feasible. Rather than having contractors apply the same regimen every year, rotating from programs every one to two years could improve ryegrass control and delay the inevitable spread of herbicide resistance. If there is insufficient flexibility in spray programs for contractors, the risk for continued spread of ryegrass resistance to glyphosate, Esplanade, and other herbicides will increase. This could lead to increased costs for additional mowing throughout the state to maintain roadsides at acceptable heights and reduce ryegrass seed production. It may also lead to the spread of invasive weeds after ryegrass declines in summer that warrant additional resources required to maintain safe and sustainable roadsides. 25 References Feng P, Pratley J, Bohn J (1999) Resistance to glyphosate in Lolium rigidum. II. Uptake, translocation, and metabolism. Weed Sci. 47:412-415. Heap I (2020) The International Survey of Herbicide Resistant Weeds. 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