928 SHORT COMMUNICATION

First report: spotted knapweed ( stoebe) resistance to auxinic Amy R. Mangin and Linda M. Hall

Abstract: Spotted knapweed is a prohibited noxious weed that is primarily controlled with auxinic herbicides. A population collected from a managed rangeland in East Kootenay, BC, was highly resistant to both clopyralid and picloram, with R/S ratios of >25 600 and 28, respectively. This is the first report of resistance in spotted knapweed. Key words: spotted knapweed, resistance, clopyralid, aminopyralid, 2,4-D, picloram. Résumé : La centaurée maculée est une adventice nocive interdite que l’on détruit principalement avec des désherbants à auxine. Un peuplement résistant au clopyralid et au picloram (rapport entre résistants et sen- sibles respectivement supérieur à 25 600 et de 28) a été découvert sur un grand parcours aménagé d’East Kootenay, en Colombie-Britannique. Il s’agit du premier signalement d’une telle résistance chez la centaurée maculée. [Traduit par la Rédaction] Mots-clés : centaurée maculée, résistance aux herbicides, clopyralid, aminopyralid, 2,4-D, picloram.

Spotted knapweed (Centaurea stoebe subsp micranthos)isa spotted knapweed; currently-registered herbicide active prohibited noxious establishing and ingredients include 2,4-D, dicamba, picloram, clopyralid, replacing native rangeland habitat critical for sustaining aminopyralid, and aminocyclopyrachlor, all of which are wildlife and domestic animals across the northwestern synthetic auxin herbicides (Senseman 2007). Synthetic United States and southern British Columbia and Alberta auxins mimic naturally occurring hormones (Watson and Renney 1974; Sheley et al. 1998). Spotted (indole acetic acid (IAA)) and may use the same auxin-

For personal use only. knapweed is a biennial or short-lived perennial that can binding protein (ABP) sites to enter the plasma form a monoculture in poor range conditions in as little membrane. as one season (USDA 2015). Spotted knapweed is a prolific Auxins regulate gene expression of auxin responsive seed producer and survives winter as a rosette and bolts factors (ARFs) through the promotion of degradation of to produce seed in the spring. It tends not to be grazed Aux/IAA (auxin/indole acetic acid) transcription factors by animals. It thrives under a wide range of conditions viabindingtothecomplexofthetransportinhibitor and can easily withstand disturbances, all of which con- response (TIR)/auxin-signaling F-box (AFB), which are tribute to its rapid establishment and invasiveness protein components of the Skip, Cullin, F-Box (SCF) (Watson and Renney 1974; Sheley et al. 1998). (Christoffoleti et al. 2015; Mithila et al. 2011). Different Provincial laws regulate invasive species to protect types of auxinic herbicides exhibit different binding endangered habitats. Spotted knapweed is a noxious spe- affinity to the TIR complex. Uncontrolled gene expres-

Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by NDSU on 12/08/16 cies in British Columbia under the Weed Control Act sion of ARFs leads to increased synthesis of plant hor- (Government of British Columbia 2011) and a prohibited mones (abscissic acid (ABA) and ethylene), resulting in noxious species under the Alberta Weed Act excessive plant growth, epinasty, and cell death in sensi- (Government of Alberta 2010), which requires all plants tive plants (Christoffoleti et al. 2015). to be destroyed and rendered non-viable. Herbicides are Heavy reliance and repeated use of herbicides with currently the most common method of control for similar modes of action for weed control has greatly

Received 7 January 2016. Accepted 25 April 2016. A.R. Mangin and L.M. Hall. Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB T6G 2E1, Canada. Corresponding author: Linda M. Hall (email: [email protected]). Abbreviations: ABA, abscissic acid; ABP, auxin-binding protein; AFB, auxin-signaling F-box; a.i., active ingredient; ARF, auxin responsive factors; GR50, effective herbicide rate with 50% fresh weight reduction; ha, hectare; IAA, indole acetic acid; R, resistant; S, susceptible; SCF, Skip, Cullin, F-Box; TIR, transport inhibitor response. © Her Majesty the Queen in right of Canada 2016. Permission for reuse (free in most cases) can be obtained from RightsLink.

Can. J. Plant Sci. 96: 928–931 (2016) dx.doi.org/10.1139/cjps-2016-0008 Published at www.nrcresearchpress.com/cjps on 27 May 2016. Mangin and Hall 929

increased the selection of herbicide resistant weed popu- plants were nested within an individual pot, with 3–5 lations (Heap 2015). Resistance to synthetic auxins has plants per pot and each pot was a separate experimental been reported in 32 weed species worldwide, and seven unit. Aboveground fresh weight was quantified 15 d include resistance to herbicides in the picolinic acid after treatment (DAT) for each plant. Data was first fit chemical family, most of which are also resistant to to a linear mixed model in the lsmeans package of R multiple synthetic auxin chemical families (Heap 2015). (v.0.98.1091) to account for nested effects. Least-square Two Centaurea species have been reported to be resistant means for individual herbicide treatments were used to synthetic auxins. Centaurea solstitialis in Washington to create dose-response curves in the dose-response State was selected for resistance to picloram along road- curve (drc) package of R (v.0.98.1091) for each experi- sides and was later classified as cross-resistant to clopyr- ment by fitting data to a four-parameter log-logistic alid, triclopyr, dicamba, and 2,4-D (Fuerst et al. 1996; curve (Fig. 1). Dose-response curves were used to deter- Miller et al. 2001). Centaurea cyanus was observed in mine the effective dose required to reduce above- Poland to be resistant to dicamba in winter wheat fields ground biomass by 50% compared with untreated

(Heap 2015). controls (GR50). The resistance ratio (resistant/ In the fall of 2012, a putative clopyralid resistant spot- susceptible, R/S)ofGR50 values for resistant and suscep- ted knapweed biotype was identified from an exten- tible populations was calculated. sively managed rangeland in the East Kootenays of Clopyralid resistance was confirmed in the resistant

British Columbia at Mount Broadwood. A detailed herbi- spotted knapweed population with the estimated GR50 cide history was not available, but this area has received value 32 times higher than the recommended label repeated applications of clopyralid and plants have sur- rate, while the susceptible population were controlled −1 −1 vived the recommended rate of 252 g a.i. ha .During at the label rate (252 g a.i. ha ) and had a GR50 value the winter of 2013, a small replicated greenhouse experi- of 0.148 of the recommended label rate (Table 1). ment with limited seed from this site was conducted and Resistance ratios show strong cross-resistance to >95% of spotted knapweed seedlings survived a clopyr- picloram (27.85), less to aminopyralid (2.25), and no − alid rate of 600 g a.i. ha 1. Survivors were vernalized cross-resistance to 2,4-D (1.76). In the absence of herbi- and allowed to flower for a seed increase and seed was cide, there were no consistent morphological differences harvested in winter 2013 for the resistant (R) population. between susceptible and resistant biotypes. Con- Susceptible seed (S) was obtained from mature plants the siderable variation in individual plant biomass was summer of 2013 near Vernon, BC from an area with no observed in spotted knapweed treated with clopyralid known herbicide history. and picloram. Some plants were injured while others Separate dose-response experiments for clopyralid, appeared unaffected. This suggests a heterogeneous For personal use only. 2,4-D, picloram, and aminopyralid were conducted biotype, which could be further selected for herbicide to further quantify the level of herbicide resistance resistance. in the Mount Broadwood spotted knapweed popula- For aminopyralid and 2,4-D, chosen rates were higher tion. Resistant (6 seeds) and susceptible (4 seeds) spot- than those required for a 50% reduction in above ted knapweed were seeded in 4 in square pots and ground biomass, which resulted in high standard

allowed to grow for approximately 2 wk until plants error values for GR50 estimates (Table 1). Clopyralid had three true leaves. The number of plants per pot var- rates of 32 times the recommended rate did not ied from 3–5 depending on the emergence of each bio- significantly decrease aboveground biomass of the type. Biotype-specific herbicide doses were applied as resistant biotype, and higher rates would be required

fractions of the following recommended label rates: clo- to accurately estimate GR50 forthisbiotype.Limited − − pyralid (120 g a.i. ha 1), 2,4-D LV ester (560 g a.i. ha 1), amounts of susceptible seed prevented the use of Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by NDSU on 12/08/16 − picloram (250 g a.i. ha 1),andaminopyralid(120g additional doses and replication of dose-response − a.i. ha 1). Resistant biotypes were treated with 0, 1, 2, 4, experiments. 8, 16 or 32 times the recommended label rates and sus- Resistance to clopyralid has only been reported twice ceptible biotypes received 0, 0.25, 0.5, 1, 2 or 4 times rec- worldwide and both cases were detected in populations ommended label rates. Herbicides were applied using a selected for resistance to picloram (Heap 2015). Yellow moving track cabinet chamber sprayer calibrated to starthistle (Centaurea solstitialis) from Washington State − deliver 200 L ha 1 at 207 kPa using an Air Bubble Jet was reported to be resistant to picloram (5.6), clopyralid 110015 nozzle. Immediately after treatment, trays were (3.7), triclopyr (2.4), dicamba (5.1), and 2,4-D (3.2) (Miller placed in a greenhouse. Natural light was supple- et al. 2001). Resistance inheritance in this population mented with 16 h of artificial light and temperature of yellow starthistle was attributed to a single recessive was maintained at 21 °C. Pots were watered and shifted gene (Sabba et al. 2003). They suggested resistance was daily to limit positional effects. duetoanalteredauxinreceptor(AFB)thatcouldconfer Experimental design was a nested randomized com- selective resistance to picolinic acids, similar to that plete block design with 4 replicates, where individual identified in picloram-resistant Arabidopsis mutants

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Fig. 1. Dose-response curves (on logarithmic dose scale) of aboveground fresh weight per plant (g) of resistant (R) and susceptible (S) spotted knapweed biotypes when treated with (A) clopyralid, (B) picloram, (C) aminopyralid, and (D) 2,4-D.

Table 1. Estimate of GR50 and resistance ratio (R/S) values of resistant (R) and susceptible (S) spotted For personal use only. knapweed populations to clopyralid, picloram, aminopyralid, and 2,4-D in dose response experiments.

a −1 Population [GR50 (g a.i. ha )] Recommended label use Herbicide RSResistance ratio (R/S)b rate (g a.i. ha−1) Clopyralid >3840 0.15 (0.08) >25 600 120 Picloram 1293 (5855) 46.6 (14.7) 27.85 250 Aminopyralid 9.26 (6) 4.16 (4.1) 2.25 120 2,4-D 122.96 (62) 69.5 (16) 1.76 560

a −1 GR50 refers to the herbicides rate (g a.i. ha ) required for 50% aboveground fresh weight reduction compared with nontreated control. Standard errors in parentheses. b Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by NDSU on 12/08/16 Resistance ratio (R/S) was calculated by dividing the GR50 value of resistant population by that of the susceptible population.

(Walsh et al. 2006). Unlike auxinic resistance in wild resistance will limit the potential herbicidal control mustard (Hall and Romano 1995), resistant knapweed options for spotted knapweed, which if left uncon- plants showed normal growth; however, following her- trolled, may potentially affect up to 10 million ha of bicide treatment, plants showed some epinasty without western Canada, with projected forage losses of $13 mil- tissue necrosis. This may indicate a resistance mecha- lion annually (USDA 2015). nism that interferes with the complex between TIR1, AFBs or Aux/IAA (Christoffoleti et al. 2015). This is the Acknowledgements first resistant spotted knapweed population reported We would like to acknowledge Lisa Raatz for the pre- and the first case of a weed reporting a high level of liminary technical work and Don Hare, previously with clopyralid resistance. More research is needed to Dow Agrosciences, for assistance throughout this investigate possible resistance mechanisms. Auxinic project.

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References Mithila, J., Hall, J.C., Johnson, W.G., Kelley, K.B., and Christoffoleti, P.J., de Figueiredo, M.R.A., Peres, L.E.P., Riechers, D.E. 2011. Evolution of resistance to auxinic herbi- cides: historical perspectives, mechanisms of resistance, Nissen, S., and Gaines, T. 2015. Auxinic herbicides, mecha- and implications for broadleaf weed management in nisms of action, and weed resistance: a look into recent plant agronomic crops. Weed Sci. 59:445–457. doi:10.1614/ science advances. Sci. Agric. 72:356–362. doi:10.1590/ WS-D-11-00062.1. 0103-9016-2014-0360. Sabba,R.P.,Ray,I.M.,Lownds,N.,andSterling,T.M.2003. Fuerst, E.P., Sterling, T.M., Norman, M.A., Prather, T.S., Inheritance of resistance to clopyralid and picloram in yel- Irzyk,G.P.,Wu,Y.,Lownds,N.K.,andCallihan,R.H.1996. low starthistle (Centaurea solstitialis L.) is controlled by a single Physiological characterization of picloram resistance in yel- – – nuclear recessive gene. J. Hered. 94:523 527. doi:10.1093/ low starthistle. Pest. Biochem. Physiol. 56:149161. jhered/esg101. doi:10.1006/pest.1996.0069. Senseman, S.A. 2007. Herbicide handbook. 9th ed. Weed Government of Alberta. 2010. Province of Alberta Weed Control Science Society of America, Champaign, IL. Act: Alberta weed control regulation. [Online] Available: Sheley, R.L., Jacobs, J.S., and Carpinelli, M.F. 1998. Distribution, http://www.qp.alberta.ca/1266.cfm?page=2010_019.cfm& biology, and management of diffuse knapweed (Centaurea leg_type=Regs&isbncln=9780779748150 [19 Oct. 2015]. diffuse) and spotted knapweed (Centaurea maculosa). Weed Government of British Columbia. 2011. Weed Control Act: weed Tech. 12: 352–362. control regulation. [Online] Available: http://www.bclaws.ca/ USDA.2015.Speciesprofile:spottedknapweed(Centaurea Recon/document/ID/freeside/10_66_85 [12 Mar. 2016]. maculosa L.). [Online] Available: http://www.invasivespeciesinfo. Hall, J.C., and Romano, M.L. 1995. Morphological and physio- gov/plants/spotknapweed.shtml [19 Oct. 2015]. logical differences between the auxinic herbicide-susceptible Walsh,T.A.,Neal,R.,Merlo,A.O.,Honma,M.,Hicks,G.R., (S) and-resistant (R) wild mustard (Sinapis arvensis L.) biotypes. Wolff, K., Matsumura, W., and Davies, J.P. 2006. Mutations Pest. Biochem. Physiol. 52: 149–155. in an auxin receptor homolog AFB5 and in SGT1b confer re- Heap, I.M. 2015. The International Survey of Herbicide Resistant sistance to synthetic picolinate auxins and not to 2,4- Weeds. [Online] Available: http://weedscience.org/ [19 Oct. dichlorophenoxyacetic acid or indole-3-acetic acid in 2015]. Arabidopsis. Plant Physiol. 142:542–552. doi:10.1104/ Miller, T.W., Shinn, S.L., and Thill, D.C. 2001. Cross-resistance in pp.106.085969. PMID:16920877. and chemical control of auxinic herbicide-resistant yellow Watson, A., and Renney, A. 1974. The biology of Canadian starthistle (Centaurea solstitialis). Weed Tech. 15:293–299. weeds: 6. and C. maculosa.Can.J.PlantSci. doi:10.1614/0890-037X(2001)015[0293:CRIACC]2.0.CO;2. 54: 687–701. doi:10.4141/cjps74-118. For personal use only. Can. J. Plant Sci. Downloaded from www.nrcresearchpress.com by NDSU on 12/08/16

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