Planta DOI 10.1007/s00425-006-0364-3 ORIGINAL ARTICLE Glyphosate, paraquat and ACCase multiple herbicide resistance evolved in a Lolium rigidum biotype Qin Yu · Andrew Cairns · Stephen Powles Received: 22 June 2006 / Accepted: 19 July 2006 © Springer-Verlag 2006 Abstract Glyphosate is the world’s most widely used biotype, indicating a co-occurrence of two distinct herbicide. A potential substitute for glyphosate in some glyphosate resistance mechanisms within the R popula- use patterns is the herbicide paraquat. Following many tion. In total, this R biotype displays at least four co- years of successful use, neither glyphosate nor paraquat existing resistance mechanisms, endowing multiple resis- could control a biotype of the widespread annual rye- tance to glyphosate, paraquat and ACCase herbicides. grass (Lolium rigidum), and here the world’s Wrst case of This alarming case in the history of herbicide resistance multiple resistance to glyphosate and paraquat is con- evolution represents a serious challenge for the sustain- Wrmed. Dose–response experiments established that the able use of the precious agrochemical resources such as glyphosate rate causing 50% mortality (LD50) for the glyphosate and paraquat. resistant (R) biotype is 14 times greater than for the sus- ceptible (S) biotype. Similarly, the paraquat LD50 for the Keywords ACCase · EPSP synthase mutation · R biotype is 32 times greater than for the S biotype. Glyphosate · Herbicide resistance · L. rigidum · Thus, based on the LD50 R/S ratio, this R biotype of L. Paraquat · Translocation rigidum is 14-fold resistant to glyphosate and 32-fold resistant to paraquat. This R biotype also has evolved resistance to the acetyl-coenzyme A carboxylase Introduction (ACCase) inhibiting herbicides. The mechanism of paraquat resistance in this biotype was determined as Glyphosate is the world’s most widely used herbicide restricted paraquat translocation. Resistance to (Baylis 2000). Glyphosate is versatile, controls a broad ACCase-inhibiting herbicides was determined as due to spectrum of annual and perennial weeds (non-selec- an insensitive ACCase. Two mechanisms endowing tive) under varied agricultural, industrial and domestic glyphosate resistance were established: Wrstly, a point situations, and has low mammalian toxicity and little mutation in the 5-enolpyruvylshikimate-3-phosphate soil activity. Glyphosate inhibits the enzyme 5-eno- synthase (EPSPS) gene, resulting in an amino acid sub- lpyruvylshikimate-3-phosphate synthase (EPSPS, EC stitution of proline to alanine at position 106; secondly, 2.5.1.19), resulting in shikimate accumulation and reduced glyphosate translocation was found in this R reduced production of aromatic amino acids (Steinrüc- ken and Amrhein 1980; Schönburnn et al. 2001). Para- quat is also a non-selective herbicide with a broad- Q. Yu · S. Powles (&) spectrum and rapid-action. Paraquat is toxic because it Western Australian Herbicide Resistance Initiative, School of Plant Biology, University of Western Australia, diverts photosynthetic electron transport to oxygen to Crawley, Perth, WA 6009, Australia produce free radicals that cause lipid peroxidation and e-mail: [email protected] membrane damage. In world cropping, glyphosate and paraquat have A. Cairns Department of Agronomy, University of Stellenbosch, helped enable minimum/zero tillage, providing produc- Matieland, 7602, South Africa tivity and soil conservation beneWts. Already high 123 Planta usage of glyphosate dramatically increased from 1995 glyphosate had been used over 25 years, paraquat for onwards with the introduction of transgenic glyphosate over 40 years, and ACCase inhibiting herbicides for resistant soybean, cotton, oilseed rape and maize. This only about 3 years. Seedlings of AFLR2 were initially innovation allowed glyphosate to be used as a selective screened by sequentially spraying glyphosate herbicide in transgenic crops (Shaner 2000). Now, (Roundup Power Max®) and paraquat (Gramoxone®) transgenic glyphosate resistant crops dominate much at commercial rates. Survivors were grown to maturity of North and South American cropping. and seeds obtained from bulk crosses within the popu- Among the attributes of glyphosate has been the lation were used in subsequent experiments (L. rigi- absence of evolved glyphosate resistance in weed spe- dum is obligate cross-pollinated). A known herbicide cies (reviewed by Dyer 1994; Bradshaw et al. 1997). susceptible (S) L. rigidum biotype (VLR1) from Aus- However, evolved glyphosate resistance, Wrst reported tralia was used as a control. Seeds stocks were main- in Lolium rigidum (Pratley et al 1996, 1999; Powles tained at Western Australian Herbicide Resistance et al. 1998), is now reported in at least eight weed spe- Initiative. Seeds of the R and S biotypes were germi- cies. (Heap website: http://www.weedscience.org; nated in 16-cm diameter plastic pots containing potting reviewed by Powles and Preston 2006). In the limited soil and seedlings (20–30 per pot) grown in a glass- biochemical studies thus far conducted on evolved house at 20/15°C day/night temperature under natural glyphosate resistant weeds, two diVerent mechanisms sunlight. Seedlings were well watered and fertilized have been shown to confer glyphosate resistance, i.e. and were herbicide treated at the 2–3 leaf stage. For mutations of the EPSPS gene, or reduced glyphosate radiolabeled herbicide translocation experiments, translocation (reviewed by Powles and Preston 2006). plants were thinned to 2–3 seedlings per pot and Biotypes of 23 weed species worldwide have evolved moved to a growth chamber with 20/15°C day/night paraquat resistance and recently we have identiWed the temperature, 12 h/12 h day/night photoperiod, and a instance of Weld-evolved paraquat resistance in L. rigi- photon Xux density of 250 mol quanta m¡2 s¡1. dum (Yu et al. 2004a). Most evidence has implicated a paraquat resistance mechanism of reduced transloca- Dose response to herbicides tion/or sequestration, although enhanced oxygen radi- cal detoxiWcation has been proposed as the mechanism In dose–response experiments, R and S plants were in a few biotypes (reviewed by Hart and DiTomaso sprayed with rates of glyphosate (0, 0.028, 0.056, 0.112, 1994; Preston 1994; Szigeti and Lehoczki 2003). 0.224, and 0.45 kg ha¡1 for S plants, 0, 0.45, 0.9, 1.8, 3.6, Despite much eVort, the precise mechanisms endowing 7.2kgha¡1 for R plants), paraquat (0, 0.0125, 0.025, paraquat resistance are only partially understood. 0.05, 0.1, and 0.2 kg ha¡1 for S plants; 0, 0.2, 0.4, 0.8, 1.6, Lolium rigidum is a very resistance prone weed spe- and 3.2kgha¡1 for R plants), diclofop-methyl (0, 0.25, cies with extensive resistance to numerous herbicides 0.5, 1, 2 and 4 kg ha¡1), Xuazifop (0, 25, 50, 100, and (Powles and Matthews 1992; Preston et al. 1996). Until 200 g ha¡1), haloxyfop (0, 13, 26, 52, 104, and 208 this study, there were no weed species known to pos- gha¡1), propaquizafop (0, 12.5, 25, 50, 100, and 200 sess multiple resistance to both glyphosate and para- gha¡1), sethoxydim (0, 25, 50, 100, 200, and 400 g ha¡1) quat. Here, we conWrm the Wrst case of Weld-evolved and tralkoxydim (0, 38, 76, 152, 304, and 608 g ha¡1). In multiple resistance to glyphosate and paraquat (plus single-dose experiments, R and S plants were sprayed resistance to acetyl-coenzyme A carboxylase at the following herbicide doses known to control S (ACCase)-inhibiting herbicides). We investigate the plants: 100gha¡1 chlorsulfuron, 100 g ha¡1 triasulfuron, resistance mechanisms in this biotype and reveal at 15 g ha¡1 sulfometuron, 20 g ha¡1 imazapyr, 72 g ha¡1 least four co-existing mechanisms endowing multiple imazethapyr, 1,000 g ha¡1 atrazine, 1,800 g ha¡1 diuron, resistance across three very diVerent herbicide modes 500 g ha¡1 triXuralin, and 500 g ha¡1 metolachlor. Her- of action. bicides were applied as commercial formulations plus adjuvant as required (either as 0.1% v/v BS1000 or 0.1% v/v Hasten) using a cabinet sprayer delivering Materials and methods 106 l ha¡1 water at a pressure of 200 kPa. Plants were returned to the glasshouse after treatment, and the Plant material mortality and shoot dry mass (oven-dried at 70°C for 2 days) were recorded 21 days after herbicide applica- Seeds of the putative resistant (R) population of L. tion. Plants were recorded as alive if they had strongly rigidum (termed AFLR2) were originally collected tillered since herbicide application. For application of from a farm in the Tulbagh Valley, South Africa, where the soil applied triXuralin and metolachlor herbicides, 123 Planta 100 seeds were placed on the soil surface, covered At harvest, whole plants were gently washed out of the with 0.5 cm of soil, watered and left for 1 day to soil, rinsed in 0.1% Triton X-100, blotted dry, pressed imbibe before herbicide treatment. After herbicide and oven-dried at 70°C for 48 h, then exposed to a treatment, 1 cm of untreated soil was placed on the phosphor imager plate (24 h) before scanning for soil surface. The number of emerged seedlings was radioactivity. The experiment was repeated at least recorded 21 days after herbicide spraying. Dose– two times for each herbicide and each harvest had 10– response experiments were repeated during April and 12 individual replicates. October in 2005 and each treatment contained 3–4 replicates. EPSPS gene sequencing Leaf uptake and translocation of herbicides The R biotype plants were sprayed with 900 g ha¡1 glyphosate at the 2–3 leaf stage and the survivors were In order to simulate commercial agricultural herbicide used for RNA preparation. Total RNA was extracted treatment, and follow the movement of the radiola- from shoot tissue of the R and S biotypes using the beled herbicides, plants at the 2 or 3 leaf stage were Plant RNeasy Mini Kit (Qiagen, Pty Ltd., Doncaster sprayed with commercial herbicides (450 g ha¡1 VIC, Australia).