© 2002 Plant Management Network.
Confirmation of Shattercane (Sorghum bicolor) Resistance to ALS-Inhibiting Herbicides in Ohio
Traci L. Brenly-Bultemeier, Jeff Stachler, and S. Kent Harrison, Department of Horticulture & Crop Science, Ohio State University, Columbus 43210
Brenly-Bultemeier, T. L., Stachler, J., and Harrison, S. K. 2002. Confirmation of shattercane (Sorghum bicolor) resistance to ALS-inhibiting herbicides in Ohio. Online. Plant Health Progress doi:10.1094/PHP-2002-1021-01-RS.
A population of shattercane (Sorghum bicolor (L.) Moench) located in Fairfield County, Ohio, was investigated for herbicide resistance after it persisted in a field that had been treated repeatedly with herbicides that inhibit acetolactate synthase (ALS). Herbicide bioassays confirmed cross-resistance of the suspected resistant (R) population to the ALS inhibitors nicosulfuron, primisulfuron, and imazethapyr. Herbicide doses required to reduce R shattercane shoot dry weight 50% (i.e., the GR50 values) were > 35,000, > 40,000, and 34,215 g ai/ha for nicosulfuron, primisulfuron, and imazethapyr, respectively. In contrast, GR50 values for the same herbicides applied to a susceptible (S) shattercane population from an adjacent county were 0.185, 0.025, and 0.038 g/ha, respectively. The high levels of resistance exhibited by the R population suggest that the resistance mechanism is due to one or more alterations in ALS, the herbicide target site. Effective management of ALS herbicide-resistant shattercane will require an integrated strategy designed to isolate the R population and deplete its soil seed bank while minimizing herbicide selection pressure.
Shattercane is a genetically diverse, summer annual grass that infests thousands of hectares of cropland throughout the southern and central United States (3). It can attain a height of 3 m and is capable of producing over 1000 seeds per plant, some of which may remain viable in soil for 13 years (12). A weed-crop competition experiment showed that dense populations of shattercane reduced corn (Zea mays L.) grain yield 22%, which was greater than maximum yield losses caused by giant foxtail (Setaria faberi L.), common cocklebur (Xanthium strumarium L.), or common lambsquarters (Chenopodium album L.) (4). Shattercane also competes with rotational crops following corn and has caused soybean (Glycine max (L.) Merr.) yield losses ranging from 10 to 50% (8).
Shattercane is an increasing weed problem in Ohio crops, especially continuous cornfields where conventional (nontransgenic) corn hybrids are grown. There are relatively few options available for chemical control of shattercane in conventional corn, and since 1990 many producers have relied on the use of the sulfonylurea herbicides primisulfuron or nicosulfuron for selective postemergence shattercane control. The mode of action of sulfonylurea herbicides in susceptible plants is inhibition of acetolactate synthase (ALS), a key enzyme in the branched chain amino acid synthesis pathway (15). Other chemical classes of ALS inhibitors used for weed control in corn, soybean, and/or small grains include the imidazolinones, triazolopyrimidine sulfonanilides, and pyrimidinylthiobenzoates.
The widespread use of ALS-inhibiting herbicides in several major crops over the past two decades has contributed to the development of ALS-herbicide resistance in 73 weed species worldwide (9). The resistance mechanism has generally involved an altered form(s) of the ALS enzyme with decreased herbicide sensitivity (1,2,5,6,14). A shattercane biotype resistant to primisulfuron was identified in Nebraska fields that had been treated with primisulfuron or nicosulfuron for a minimum of three consecutive years (3). A later study reported that primisulfuron resistance in each of three shattercane accessions from Nebraska was due to different ALS mutations (13). Previous research suggests that shattercane populations may contain multiple inherent genotypes with resistance to ALS-inhibiting herbicides, so strong ALS-herbicide selection pressure may lead to rapid development of resistant (R) populations.
A population of shattercane located in a Fairfield County, Ohio, cornfield was suspected of having resistance to ALS-inhibiting herbicides based on poor shattercane control and a 10-year history of ALS-herbicide use. The objectives of this study were to verify herbicide resistance in the suspect population and to determine its magnitude of cross-resistance to two chemical classes of ALS-inhibiting herbicides.
Shattercane Population Sampling and Preliminary
Seed were collected from approximately 40 random plants growing in each of three central Ohio shattercane populations. Populations included the suspected R population in Fairfield County, a known ALS herbicide-susceptible (S) population with no prior history of ALS-herbicide application in Perry County, and a third population with unknown (U) susceptibility in Madison County from a field that had been treated only twice in previous years with an ALS-inhibiting herbicide (Table 1). Seed from plants within each population were mixed well and stored at 5░C and 50% relative humidity for a period of 4 months prior to the greenhouse experiments.
Table 1. Cropping and herbicide use history of Ohio fields infested with ALS herbicide-resistant (R), susceptible (S), and unknown (U) shattercane biotypes.
Before planting, shattercane seed were scarified in concentrated sulfuric acid for six minutes, then rinsed in a saturated sodium bicarbonate solution. Seed were imbibed for 12 hours and planted 2 cm deep in 500-ml plastic cups filled with a commercial potting soil. The cups were transferred to a greenhouse maintained at 24 ▒ 3░C and equipped with high-pressure sodium lights that provided approximately 220 Ámol m-2 s-1 supplemental photosynthetic photon flux for a 16-hour daily photoperiod. Shattercane seedlings were thinned to three plants per pot for the preliminary screen and one plant per pot for the dose-response bioassay and irrigated as needed.
Herbicide applications were made with a laboratory spray chamber equipped with a Teejet 8002EVS nozzle tip (Spraying Systems Co., Wheaton, IL) and calibrated to deliver a carrier volume of 187 liter/ha at 290 kPa. Before transferring plants to the greenhouse, they were moved to an area where they were spaced apart and allowed to dry to avoid cross-contamination of treated plants. All experiments were conducted in a randomized complete block design with 4 replications and repeated for confirmation of results.
Representatives from two herbicide chemical classes, the sulfonylureas and imidazolinones, were used to test shattercane populations for resistance and cross-resistance to ALS-inhibiting herbicides in a preliminary screen. Nicosulfuron (sulfonylurea), primisulfuron (sulfonylurea), and imazethapyr (imidazolinone) were applied to plants from the S, suspected R, and U shattercane populations at 70, 80, and 140 g/ha respectively. These application rates are twice (2x) those recommended by the herbicide labels. Plants were at growth stage V4 and approximately 23 cm tall at the time of herbicide application. Phytotoxicity ratings were made on a scale of 0 (no injury based on untreated controls) to 100% (complete plant death) at 14 days after treatment. The experiment was conducted twice, and a combined analysis of variance was performed using the general linear models procedure of SAS software (SAS Institute, Inc., Cary, NC) to determine experiment, herbicide, and population effects. There were no experiment by treatment interactions, so data were combined for subsequent analysis. Means were separated by Fisher's protected least significant difference test. All statistical tests were performed at a significance level of 0.05.
The preliminary screen indicated that the Perry County S and Madison County U shattercane populations were highly susceptible to nicosulfuron, primisulfuron and imazethapyr at the 2x application rate. Individual treatment data are not shown, since all herbicide treatments resulted in phytotoxicity ratings of > 98% for the S populations and < 3% for the R population (P < 0.001). Results from the preliminary screen confirmed that the Fairfield County shattercane population was strongly cross-resistant to ALS inhibitors. Responses of S and R shattercane biotypes to the three herbicides in the preliminary screen are shown in Fig. 1.
Herbicide Dose-Response Bioassay of R and S
Based on results from the preliminary screen, dose-response bioassays were conducted on the confirmed R and S shattercane populations to determine levels of cross-resistance to the three herbicides tested in the preliminary screen. Treatments were in a 2-x-3-x-8 factorial arrangement with shattercane population, herbicide, and herbicide rate as factors, respectively. Nicosulfuron, primisulfuron, and imazethapyr were each applied at a logarithmic range of rates corresponding to 0, 0.001, 0.01, 0.1, 1.0, 10, 100, and 1000 times the recommended label rates of 35, 40, and 70 g/ha, respectively. Herbicide applications were made to 25-cm-tall plants in the V5 growth stage. Two weeks after herbicide treatment, plants were harvested by cutting off the shoots at the soil surface, then oven-dried for 72 hours at 50░C and weighed.
A combined analysis of variance was performed as described earlier, and data from duplicate experiments were combined for subsequent analysis. When dose was significant, shoot dry weight means were regressed on herbicide dose and fit to a second-order logarithmic equation for the R population or a hyperbolic decay equation for the S population using SigmaPlot 7.0 statistical software (SPSS, Inc., Chicago, IL). The herbicide doses required to cause a 50% reduction in shoot biomass (GR50 values) of each shattercane population were calculated from the regression equations, and resistance ratios (GR50 of resistant population/GR50 of susceptible population) were calculated for each herbicide.
Nicosulfuron at the maximum dose of 35,000 g/ha reduced shoot dry weight of the R shattercane population only 21%, so the GR50 value was not reached within the range of doses tested (Fig. 2A). In contrast, the GR50 of nicosulfuron in the S population was 0.185 g/ha and the labeled rate of 35 g/ha nicosulfuron reduced shoot dry weight 96%. The R:S resistance ratio for nicosulfuron exceeded 189,000, indicating high differential sensitivity between the S and R population.
The R population was insensitive to primisulfuron, and its dose response was not significant across the range of doses tested (Fig. 2B). The primisulfuron GR50 in the S population was 0.025 g/ha, so the R:S resistance ratio for primisulfuron exceeded 1,600,000. The S population was more sensitive to primisulfuron than nicosulfuron, yet the R population exhibited a higher level of resistance to primisulfuron than nicosulfuron.
The R shattercane population also exhibited a high level of resistance to imazethapyr, confirming strong cross-resistance to the sulfonylurea and imidazolinone herbicides tested (Fig. 2C). Imazethapyr doses higher than 100 g/ha reduced shoot dry weights in the R population, resulting in a GR50 of 34,200 g/ha. These data indicate that shattercane's level of resistance to imazethapyr was somewhat lower than its resistance to the sulfonylurea herbicides, but the resistance level was still exceedingly high from a practical standpoint. The imazethapyr GR50 in the S population was 0.037 g/ha, thus the relative toxicity of imazethapyr to S shattercane was between that of primisulfuron and nicosulfuron. The R:S resistance ratio for imazethapyr was approximately 925,000.
These experiments confirmed the presence of ALS herbicide-resistant shattercane in central Ohio, and that the R population exhibited strong cross-resistance to herbicides from two chemical classes of ALS inhibitors. The high resistance ratios and cross-resistance observed in the dose-response experiment suggest that there may be one or more alterations in ALS that conferred herbicide resistance in the R population (13). The development of an herbicide-resistant shattercane population at the Fairfield County site was predictable based on its history (Table 1), since repeated application of herbicides with the same mode of action exerts strong selection pressure that screens for resistant biotypes (7,11).
Implications for Management
Herbicide resistance prevention and management are based on integrated weed control strategies that employ a combination of cultural, mechanical, biological, and chemical methods. In fields where ALS-resistant shattercane has been confirmed, a basic approach is to isolate the resistant population as much as possible and use a combination of available methods to reduce its size and density over time. This necessarily involves practices that will prevent future seed production by resistant plants and avoid transfer of seed to non-infested fields via farm equipment or animal manure. Crop producers should also avoid continued use of ALS-inhibiting herbicides on the R population unless they are mixed with other non-ALS inhibitor herbicides that are effective on shattercane.
Since options are limited for herbicidal control of shattercane in non-transgenic corn and other grass crops, rotation of R shattercane-infested fields to a broadleaf crop or set-aside land will offer effective non-ALS herbicide options and help speed the rate of decline of the R population. Another alternative is to use transgenic herbicide-resistant crop varieties that would permit the use of a highly effective non-ALS herbicide (e.g., glyphosate in Roundup Ready« crops). However, crop producers should maintain vigilance and be aware that over-reliance on herbicides with the same mode of action may inevitably lead to a resurgence of other herbicide-resistant populations. The goal of keeping shattercane populations low while minimizing herbicide selection pressure will ultimately require a long-term, integrated strategy that is compatible with crop producers' goals and economic constraints. Additional guidelines for herbicide resistance management are available from the Herbicide Resistance Action Committee (10).
The authors gratefully acknowledge the financial support provided by the Weed Science Society of America. Salaries and additional research support were provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, Ohio State University.
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