Resistance to Acetolactate Synthase (ALS) Inhibitors in the United States: History, Occurrence, Detection, and Management

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1 Resistance to Acetolactate Synthase (ALS) Inhibitors in the United States: History, Occurrence, Detection, and Management Dale L. Shaner* Abstract: Acetolactate synthase (ALS) inhibitors are highly potent herbicides that include the sulf onylureas, imidazolinones, triazolopyrimidine sulfonamides and pyrimidyl salicylates. Currently there are ALS-inhibiting herbicides for use in all major crops including maize, soybeans, rice, and cereals. These herbicides kill plants by inhibiting the first enzyme in the biosynthetic pathway of the branched chain amino acids. Resistance to ALS inhibitors can be conferred by single mutations at multiple sites within the ALS gene. The first case of resistance to ALS inhibitors was discovered in 1987 in a wild lettuce (Lactuca serriola) population in Idaho, U.S. Resistance to ALS inhibitors in the U.S. is now found in populations of 26 species in all of the major crops. In all the cases studied, the mechanism of resistance has been due to selection of an altered form of the ALS enzyme. Detection of resistance to ALS inhibitors can be done through whole plant screening, in vitro and in vivo enzyme assays. ALS resistance can be managed through the by using ALS inhibitors in an integrated system which utilizes other herbicides as well as mechanical, cultural and biological weed control methods. Key Words: acetohydroxyacid synthase, *Charman of Hebicide Resistance Acting Committee, American Cyanamid Co., P. O. Box 400, Princeton, NJ , USA (Accepted October 9, 1999) imidazolinone, sulfonylurea, triazolopyrimidine, acetolactate synthase inhibitors Introduction In the early to mid 1980s acetolactate synthase inhibitors (ALS) were introduced into the marketplace16). These new herbicides controlled a broad spectrum of broadleaf weeds and grasses at rates ranging from 10 to 200g/ha. They also were safe to nontarget organisms including mammals, fish, birds and insects. One reason for the low toxicity of these new classes of herbicides to non-plant organisms was their site of action. All of these herbicides kill plants by inhibiting acetolactate synthase (ALS, also known as acetohydroxyacid synthase), the first enzyme in the branched chain amino acid pathway. Thus, these herbicides do not affect mammals, fish, birds or insects because this target does not exists in these organisms16). Classes of ALS Inhibitors There are several different chemical classes of ALS inhibitors from which commercial herbicides have been developed (Figure 1). They include the imidazolinones, sulf onylureas, triazolopyrimidine sulfonamides, and pyrimidinylsalicylates16). Currently there are over 30 commercial ALS inhibitors on the market throughout the world13). ALS inhibitors are very important in terms of the world herbicide market, accounting for approximately 17.5% of the total herbicide market in ).

2 Development of Resistance to ALS Inhibitors Discovery of Resistant Weeds ALS inhibiting herbicides have become widely used in many different crops because of their high potency, low cost, low toxicity and flexibility of use. One of the consequences of the high potency of these herbicides is that they can place high selective pressure on weed populations. The first ALS inhibitor to be introduced into the market was chlorsulfuron (2-chloro-N-[[ (4-methoxy-6-methyl-1, 3, 5-trazin-2-yl) amino] carbonyl] benzenesulf onamide) which was registered in 1982 in the U. S. for use in cereals2). Chlorsulfuron is a highly effective, long residual herbicide that was quickly adopted by the wheat growers in the northwestern U. S. The first recorded case of resistance to an ALS inhibitor under field conditions occurred in 1987 when a farmer in Idaho found that he was no longer controlling patches of wild lettuce (Lactuca serriola) in fields that had received continuous applications of chlorsulfuron for 5 years13). In 1988 and 1989 chlorsulfuron resistant populations of kochia (Kochia scoparia) and Russian thistle (Salsola iberica) were found throughout the cereal growing area of the U. S. where chlorsulfuron was the only herbicide that had been used in these fields for 3 to 5 years13). In 1992 populations of smallf lower umbrella sedge (Cyperus difformis) and California arrowhead (Sagittaria montevidensis) with resistance to bensulfuron (2- [[[[[(4, 6-dimethoxy-2-pyrimidinyl) amino] carbonyl] amino] sulfonyl] methyl] benzoic acid), a widely used sulfonylurea in rice, were discovered in Calif ornia13). In Mississippi a population of cocklebur (Xanthium strumarium) that had received two application of imazaquin (2-[4, 5-dihydro- 4-methyl-4-(1-methylethyl)-5-oxo-1Himidazol-2-yl]-3-quinolinecarboxylic acid) per year for a 4 year period was no longer controlled by this herbicide13). This was the first confirmed case of resistance to the imidazolinones in the U.S. At present there are over 26 species in which resistant biotypes have been selected to a number of different ALS inhibitors in the U.S.8) Distirbution of ALS Inhibitor Resistance in U. S. Resistance to ALS inhibitors in the U. S. has been confirmed in 30 states in wheat, corn, soybeans, rice, and along railroads and roadsides8,3). In some cases, the resistance can be fairly widely dispersed. In a survey for sulf onylureas resistance in kochia populations in Idaho, Montana and Colorado researchers found that 50% of the populations surveyed contained resistant biotypes10 Many of these populations grew along roadsides as well as in fields. Since kochia is a tumble weed that can disperse its seed over long distances, it is unclear whether sulf onylurea resistance was due to in situ selection or if it spread from trucks or from resistant populations in wheat fields. Mechanisms Altered ALS Enzyme of Resistance

3 In all of the populations where the mechanism of resistance has been determined, the resistance is due to the selection of an altered ALS enzyme that is no longer sensitive to the herbicides. The pattern of cross resistance between the ALS inhibitors is variable. In chlorsulfuron-resistant biotypes of kochia, there are varying levels of cross resistance to other sulfonylureas and triazolopyrimidine sulf onamides13,18 The cross resistance to imidazolinones is also variab1e14,16,17) Fitness of Resistant Biotypes ALS inhibitor resistant biotypes appear to be as fit as susceptible wild types. Seeds of the resistant (R) biotypes of wild lettuce germinated more rapidly than susceptible (S) biotypes in a study conducted by Mallory- Smith et al9). Germination studies on R and S biotypes of kochia from Kansas and Montana showed that the R seed germinated more rapidly at cool temperatures compared to S biotypes4), although the germination at higher temperatures was unaffected. Analysis of the pool sizes of the branched chain amino acids in the two biotypes showed that the R biotypes had higher levels of valine, leucine, and isoleucine4). However, the relationship between these differences in amino acid pool sizes and germination is unknown. Growth and competition studies on R and S biotypes of wild lettuce under greenhouse conditions showed that the S biotypes produced 31% more biomass than the R biotypes but the relative competitive ability of the two biotypes was similar9>. R and S biotypes of kochia were also equally competitive. R and S biotypes of kochia and wild lettuce produced similar amounts of seed, and the seed longevity of R and S biotypes of wild lettuce were similar under field conditions. Thus, there appears to be little loss of fitness associated with ALS resistance13). At the enzyme level, the catalytic efficiency of ALS appears to be unaffected by resistance to these inhibitors. The Km for pyruvate in ALS from resistant kochia, Russian thistle, chickweed (Stellaria media) and perennial ryegrass (Lolium perenne) ranged from 1.7 to 4.8 mm, which is close to Km values reported for susceptible plant ALS13). There have been reports that the feed back regulation of resistant ALS by valine and leucine is altered in some biotypes but not in others. Subramanian et al18) found that ALS isolated from resistant cotton and tobacco was less sensitive to feedback inhibition by valine and leucine but Singh et al16) found no change in the feedback sensitivity of ALS isolated from imidazolinone resistant corn lines. Since resistant biotypes appear to be as fit as wild type, altered feedback regulation appears to have little consequence in the competency of ALS in the whole plant, although it could account for the change in levels of branched chain amino acids in the seed of sulfonylurea-resistant kochia biotypes. Genetics of ALS Inhibitor Resistance Research on the genetics of ALS resistance shows that resistance is inherited as a single, semidominant trait13,16) There are at least 10 different sites within 3 conserved regions of the ALS gene where mutations result in a resistant enzyme5). However, it appears that most of the mutations occur in one of four sites (Figure 2) 13). The most common site, particularly for biotypes selected by a sulf onylurea is at a proline site where a mutation that replaces proline with any other amino acid gives resistance. In a study conducted by Guttieri et al7), they found that in 8 populations of ALS resistant kochia studied, 6 of the populations contained a mutation at this proline site. Cross resistance studies have shown that mutations at the Pro site result primarily in resistance to sulfonylureas

4 Fig. 2. Depiction of mutation sites in ALS gene for resistance to ALS inhibitors. Bolded letters show mutation site. Numbering system is for Arabidopsis thaliana (Devine and Eberlein, 1998). and triazolopyrimidines13). Another site that has been found in resistant weed populations is in another region of the ALS gene where a mutation that changes a tryptophan to leucine, the plant becomes broadly cross resistant to all ALS inhibitors13). Detection of Resistance to ALS Inhibitors The most definitive and least ambiguous method for determining if a weed biotype is resistant to an ALS inhibitor is through whole plant assays. In such assays, plants from the test population are grown from seed and compared to a known susceptible population. It is important in this type of testing to collect seed in the appropriate manner from the test population. One should 1) collect seeds when the majority are mature; 2) Collect over an area of at least 100m by 50m; 3) Collect high quality seed; 4) Air dry the seed as soon as possible; and 5) Clean samples to remove poor quality seed. It is important during the test to always include a reference susceptible population and to include enough replications to allow statistical analysis. It is also best to use a range of rates to obtain a dose response curve rather than rely on a single dose assay. If a single dose is used, be sure to use a dose high enough to adequately discriminate between true resistance and potential escapes. This dose can only be arrived at through extensive testing of known populations. Enzyme assays Most, but not all, resistance to ALS inhibitors is due to an alteration at the site of action. Thus, one can often use an in vitro enzyme assay to determine if a plant population is resistant to an ALS inhibitor or not. This type of assay has advantages over whole plant screening in terms of time and space.

5 However, such assays have to be done carefully in order to avoid artifacts. The source of the plant material from which the enzyme is extracted is critical to the success of in vitro enzyme assays. Young, rapidly growing tissue is the best source. It is difficult, if not impossible, to use plant material that has been shipped from the field to a laboratory. It is best if the plant material is from young seedlings grown from seeds of the test population. It is critical for the tester to have experience in extracting and running an in vitro ALS assay in order to get reliable results. In running the assay following standard protocols, one should include an extract from a susceptible control. In addition, one should include a positive control, such as valine plus leucine in order to eliminate presence of non-als activity, thus avoiding artifacts. the Modified ALS assay Another method exists that allows one to measure ALS activity in a semi-in vivo manner6). In this assay, one treats the test plants with an inhibitor of ketoacid reducto isomerase (KARI), which causes an accumulation of acetolactate in plant tissue. If one compares a plant is treated with a KARI inhibitor alone with a plant treated with the KARI inhibitor plus an ALS inhibitor one can determine if the ALS inhibitor is working or not. Acetolactate can be easily extracted from plant material in water. Tissue treated with a KARI inhibitor alone for 6-24 hours will have large amount of acetolactate while a susceptible plant treated with a KARI inhibitor plus an ALS inhibitor will not. A resistant plant will accumulate the same or similar levels of acetolactate in the presence or absence of the ALS inhibitor. This assay can be used in the field with the appropriate equipment and can provide a relatively rapid way to determine if a plant population is resistant or not. However, the choice of plant material that is tested is critical for success and appropriate controls must be used. Management of Resistance to ALS Inhibitors In almost all cases ALS inhibitor-resistant populations have been selected where these inhibitors were the only herbicides used to control weeds. Resistance has not been selected where the ALS inhibitors are part of an integrated program that includes other herbicides combined with mechanical, cultural and biological control methods. Common Factors Associated with Resistance Selection Most cases of resistance to ALS inhibitors have occurred in fields in which these herbicides have been used on a continuous basis for 5 to 8 years with little or no use of other herbicides with a different mechanism of action. In addition, the first weeds in which resistance has developed are those that are the most susceptible to the ALS inhibitor. In many cases these are also weeds that are prolific seed producers and the seeds have a relatively short half life in the soil. Resistance occurs in these types of weeds because they are under the most intensive selection pressure Management by the ALS inhibitor. Recommendation First, and foremost, an ALS inhibiting herbicide should not be used alone as the sole means of weed control in any field over a long period of time. Instead, these herbicides should be part of an integrated weed management program that includes other weed control methods. If ALS inhibitors are used in combinations with other herbicides it is important that the two herbicides control the same spectrum of weeds. It does not do any good from a resistance management perspective to mix herbicides that control different

6 Table 1. Herbicide modes of action used in continuous soybeans Table 2. Herbicide modes of action used in maize/soybean rotation spectrums of weeds. In addition one should tailor the herbicide program to the weed spectrum in a field and use the minimum amount of herbicide necessary to control the weeds, but avoid using herbicides in excess. Finally one should use certified crop seed and clean equipment when moving from one field to another to prevent spreading resistant weed seed or plant material. Multi-year Management Program One important thing to note in developing a *Trademark of American Cyanamid Company. weed management program is that this is a multiyear approach. It is important that farmers record what herbicides they are using year to year. American Cyanamid developed a program called SAMOATM* (Second Active Mechanism of Action) to aid in managing resistance to imidazolinones.15) The main objective of this program was to ensure that farmers used the imidazolinones in an integrated system with other herbicides. In two contrasting scenarios, a herbicide program can depend on the same mechanism of action over a two year program (Table 1)

7 or one can control the same spectrum of weeds utilizing multiple mechanisms of action (Table 2). By utilizing this type of program, farmers can evaluate their herbicide program and determine the risk of selecting for resistance to ALS inhibitors. References 1) Anderson, P. C. and M. Georgeson Herbicide-tolerant mutants of corn. Genome 31, ) Beyer, E. M., M. J. Duffy, J. V. Hay, and D. D. Schlueter Sulfonyureas, In "Herbicides: Chemistry, Degradation, and Mode of Action." Ed. by P. C. Kearney. and D. D. Kaufman, Marcel Dekker, Inc., New York, pp ) Chaleff, R. S. and T. B. Ray Herbicideresistant mutants from tobacco cell cultures. Science 223, ) Dyer, W. E., P. W. Chee, and P. K. Fay Rapid germination of sulfonylurea-resistant Kochia scoparia L. accessions is associated with elevated seed levels of branched chain amino acids. Weed Sci. 41, ) Falco, S. C., and K. S. Dumas Genetic analysis of mutants of Saccharomyces cerevisae resistant to the herbicide sulf ometuron methyl. Genetics. 109, ) Gerwick, B. C., L.C. Mireles and R. J. Eilers Rapid diagnosis of ALS/AHAS-resistant weeds. Weed Technol. 7, ) Guttieri, M. J., C. V. Eberlein, and D. C. Thill Diverse mutations in the acetolactate synthase gene confer chlorsulfuron resistance in kochia (Kochia scoparia) biotypes. Weed Sci. 43, ) Heap, I International Survey of Herbicide Resistant Weeds. Online. Internet. 1 September Available 9) Mallory-Smith, C. A., D. C. Thill, M. Alcocer- Ruthling, and C. Thompson Growth comparisons of sulf onylruea resistant and susceptible biotypes, Proceedings of the First International Weed Control Congress, 2, ) Mallory-Smith, C. A., D. C. Thill, and G. P. Stalling Survey and gene flow in acetolactate synthase resistant kochia and Russian thistle. Brighton Crop Prot. Conf.-Weeds 2, ) Matthew, P., F. Mathiesen, F. Galloway, J. McDougall Agrochemical Service, Wood Mackenzie Consultants, Ltd. London, pp ) Saari, L. L., A prognosis for discovering new herbicide sites of action. In "Pesticide Chemistry and Bioscience: The Food-Environment Challenge" Ed. by G. T. Brooks and T. R. Robers. Royal Society of Chemistry, Cambridge, pp ) Saari, L. L., J. C. Cotterman, and D. C. Thill Resistance to acetolactate synthase inhibiting herbicides. In "Herbicide Resistance in Plants: Biology and Biochemistry." Ed. by S. B. Powles and J. A. M. Holtum, Lewis Publishing. Boca Raton, FL pp ) Schmitzer, P. R., R. J. Eilers, and C. Cseke Lack of cross-resistance of imazaquin-resistant Xanthium strumarium acetolactate synthase to flumetsulam and chlorimuron. Plant Physiol. 103, ) Shaner, D. L., Ferst, D. A., and Retzinger, E. J SAMOA: One company's approach to herbicide-resistant weed management. Pesticide Sci. 51, ) Shaner, D. L., and B. K. Singh Acetohydroxyacid synthase inhibitors. In "Herbicide Activity, Toxicology, Biochemistry, and Molecular Biology" Ed. By R. M. Roe, J. D. Burton, and R. J. Kuhr, IOS Press, Amsterdam, pp ) Sprague, C. L Common cocklebur (Xanthium strumarium) resistance to selected ALSinhibiting herbicides. WSSA Abstracts 36, ) Subramanian, M. V., V, Loney-Gallant, J. Dias, and L. Mireles Acetolactate synthase inhibiting herbicides bind to the regulatory site. Plant Physiol. 96,