White mustard (Sinapis alba) resistance to ALS-inhibiting herbicides and alternative herbicides for control in Spain

Europ. J. Agronomy 35 (2011) 57–62

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White mustard (Sinapis alba) resistance to ALS-inhibiting herbicides and alternative herbicides for control in Spain J.M. Rosario a , H. Cruz-Hipolito b , R.J. Smeda c , R. De Prado b,∗ a b c

Dominican Institute of Agriculture and Forestry Research, Rafael Augusto Sánchez #89 Ensanche Evaristo Morales. 10147 Dominican Republic Department of Agricultural Chemistry and Edaphology, University of Córdoba, Campus de Rabanales, Edificio C-3 “Marie Curie”, Ctra. Madrid km 396. 14071 Córdoba, Spain Division of Plant Sciences, University of Missouri, 204A Waters Hall. 65211 Columbia, Missouri

a r t i c l e

i n f o

Article history: Received 24 September 2010 Received in revised form 23 February 2011 Accepted 14 March 2011 Keywords: White mustard SINAL Tribenuron-methyl Cross-resistance ALS Contact angle Cuticle thickness

a b s t r a c t Sinapis alba is a competitive weed in cropping systems in southwest Spain. Over the past 10 years, reports of erratic control of S. alba with ALS-inhibiting herbicides have increased. Sixteen S. alba field accessions (AR1 to AR16 ) were collected from Malaga, in southwest Spain, and cultivated under greenhouse conditions for screening tests. AR8 and AR1 accessions were selected for in vivo and in vitro assays, while the site for the AR8 population was also used for field experiments. Results demonstrated that tribenuron-methyl reduced plant fresh weight by over 90% in the AR1 , AR2 and AR3 populations; AR4 biomass was reduced 30.7%; biomass of the AR5 to AR16 populations were only reduced from 5.7 to 13.1%. In vivo, the rate to reduce above ground plant fresh weight by 50% (ED50 ) was greater for AR8 (1.76) than AR1 (0.18), indicating a resistance factor of 9.8 for tribenuron-methyl. In vitro, a comparison of ALS enzyme activity resulted in a resistance factor of 4128 for tribenuron-methyl, and 884 (mesosulfuron) and 839 (iodosulfuron) for the other two sulfonylurea herbicides. In vivo, the AR8 biotype was cross-resistant to four of the five ALS-inhibiting herbicide groups (sulfonylurea, imidazolinone, triazolopyrimidine and sulfonylaminocarboniltriazolinone), but not to pyrimidinyloxybenzoic acid (bispyribac-sodium). The leaf contact angle for interception of tribenuron-methyl was 55.6◦ on the adaxial surface of AR8 plants compared to 17.6◦ on the leaves of AR1 plants. This resulted in significantly less tribenuron-methyl retention on R versus S (125.6 versus 166.1 ␮L g−1 dry shoot weight) plants. In field trials, tribenuron-methyl resulted in minimal control of AR8 plants (28.7%). However, tribenuron-methyl + MCPA (92.3%) and tribenuron-methyl + mecopropP (88.7), resulted in the highest white mustard control. This research confirms resistance of S. alba to tribenuron-methyl. In vitro assays suggest resistance is due to the inability of tribenuron-methyl to interact with the target site (ALS). Foliar retention and contact angle might contribute to the evolved resistance of S. alba to tribenuron-methyl. For improved control of white mustard, tribenuron-methyl combined with herbicides exhibiting different modes of action, such as MCPA or mecoprop-P, should be considered. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

1. Introduction Since herbicide resistance was first documented, there have been reported 346 resistant biotypes in 194 species (114 dicots and 80 monocots) and over 340,000 fields world-wide (Heap, 2010) infested. Among the herbicides groups based upon mode of action, acetolactate synthase (ALS) inhibitors are the more resistance-prone herbicide group, with the highest number of resistant biotypes in the last 10 years. Weeds resistant to ALS inhibitor herbicides have been selected by repeated use, with populations detected in cereals, corn/soybean rotations, rice cropping sys-

∗ Corresponding author. Tel.: +34 957218600; fax: +34 957218600. E-mail address: [email protected] (R. De Prado).

tems, highway rights-of way and forestry (Heap, 1997). Presently, 31 weed species have been confirmed resistant to herbicides in Spain. Among these, three species have evolved resistance to ALS inhibitors: Alisma plantago-aquatica and Cyperus difformis in irrigated rice and Papaver rhoeas in wheat (Duran-Prado et al., 2004; Ruiz-Santaella et al., 2004; Calha et al., 2007; Heap, 2010). ALS is a plastid enzyme (Chaudhry, 2009), and catalyzes the first common step in the biosynthesis of the branched chain amino acids leucine, isoleucine and valine (Bryan, 1991; McCourt and Duggleby, 2006). The resultant action in sensitive plants is death by starvation. Following continuous exposure within or over years, some plants will survive normally toxic doses of ALS inhibitor herbicides. One of the mechanisms underlying resistance is a decrease in the number of herbicide molecules that reach their target site (Veldhuis et al., 2000; Letouzé and Gasquez, 2003; Yu et al., 2009). Non-

1161-0301/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.eja.2011.03.002

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J.M. Rosario et al. / Europ. J. Agronomy 35 (2011) 57–62

target site resistance is based upon metabolism in some species (Christopher et al., 1991; Powles and Yu, 2010). The most frequent mechanism of resistance is a single point mutation in the gene conferring ALS (Tranel and Wright, 2002; Kuk et al., 2003a; Duran-Prado et al., 2004; Warwick et al., 2005; Christoffers et al., 2006; Tranel et al., 2008; Duggleby et al., 2008; Cruz-Hipolito et al., 2009a; Heap, 2010; Xu et al., 2010). To date, amino acid substitutions have been found at seven distinct points along the ALS gene. At some points, different base pair substitutions have contributed to unique amino acid substitutions. For example, eight amino acid substitutions have been identified for Pro 197 and three substitutions for Ser 653 (Heap, 2010). Only 7 amino acid substitutions have been reported from field-selected weed biotypes: Ala122 , Pro197 , Ala205 , Asp376 , Trp574 , Ser653 and Gly654 (Tranel and Wright, 2002). Depending upon the site of the mutation, different patterns of cross-resistance occur to the five chemical groups representing ALS inhibitors; mutations have been found within five conserved domains of the ALS gene: A–E (Gressel, 2002; Tranel and Heap, 2003; Tranel et al., 2008). ALS inhibitors have been used frequently in Spain to control weeds in rice and wheat cropping systems. Concerning wheat, S. alba is an invasive plant that is highly competitive, and is prominent in southwest Spain. Conventional control in wheat cropping systems typically involves use of tribenuron-methyl, which is also labelled for use in barley. However, a population of S. alba has recently been reported resistant to tribenuron-methyl in southwest Spain (Cruz-Hipolito et al., 2009b). The extent of resistance among S. alba populations and the mechanism(s) underlying resistance are not yet known. The objectives of this work include: (1) identify the response of 16 field-selected populations to tribenuron-methyl; (2) determine the level of cross-resistance to various ALS-inhibiting herbicides between an ALS-resistant and susceptible biotype of S. alba; (3) evaluate whether foliar retention of herbicide can explain differences in ALS-resistant and -susceptible biotypes of S. alba; (4) compare ALS enzyme activity among an ALS-resistant and -susceptible biotype of white mustard; and (5) examine alternative herbicide programs for control of white mustard resistant to tribenuron-methyl. 2. Materials and methods

chamber equipped with flat fan nozzle tips (Tee Jet 8002 EVS2 ) at 200 kPa, and calibrated to deliver 140 L ha−1 . Above-ground plant tissue was harvested 21 days after treatment (DAT), and fresh weight recorded as a percentage of untreated control plants. The experiment was arranged as a completely randomized design and conducted three times. Variance analysis and a means comparison test were conducted with Statistix 8.0 software (USDA-NRCS, 2007). 2.3. Dose–response assays Tribenuron-methyl susceptible AR1 (S) and resistant AR8 (R) S alba biotypes were characterized for response to representative ALS herbicides. Plants of each biotype in the 3-leaf growth stage were treated with the following herbicides (labelled field rate (D) in g a.i. ha−1 in parentheses): tribenuron-methyl3 (20), iodosulfuron4 (30), mesosulfuron5 (14.8), imazamox6 (60), florasulam7 (140.6), bispyribac-sodium8 (61.3) and flucarbazone9 (10). These herbicides were selected because they are representative of the five structurally diverse compounds that comprise ALS inhibitors: sulfonylurea (tribenuron-methyl, mesosulfuron, iodosulfuron); triazolopyrimidine (florasulam), imidazolinone (imazamox), sulfonylaminocarbonyltriazolinone (flucarbazone), and pyrimidinyl-thiobenzoate (bispyribac-sodium). For the AR8 biotype, each herbicide was evaluated at nine rates: D/80, D/40, D/20, D/2, D, 2D, 4D, 8D and 12D. The S biotype was treated with nine rates: D/800, D/400, D/200, D/50, D/2, D/4, D, 2D and 4D. All adjuvants were added at the recommended doses: petroleum crop oil concentrate (florasulam, iodosulfuron); non-ionic surfactant (flucarbazone, tribenuron-methyl); methylated seed oil + 2.5% urea ammonium nitrate (imazamox). Plants were harvested 21 DAT and fresh weight recorded as a percentage of the untreated control for each biotype. The experiment was arranged as a completely randomized design with ten replications; the experiment was conducted three times. The herbicide doses inhibiting plant growth by 50% (ED50 ) were estimated following the method of Cruz-Hipolito et al. (2009a). Data were fitted to a non-linear log–logistic regression model with Sigma Plot 10.010 statistical software and ED50 values for each biotype were used to calculate the resistance factor (RF): (ED50 AR8 /ED50 AR1 ). Log–logistic model equation: Y = c + (d − c)/1 + (x/g)b where Y is the fresh aboveground weight expressed as a percentage of the untreated control, c and d are coefficients corresponding to the lower and upper asymptotes, b is the response line slope, g is the herbicide dose at the point of inflection halfway between the upper and the lower asymptotes, representing the ED50 ; x (independent variable) is the herbicide dose. 2.4. Spray retention Retention of spray solutions on AR1 and AR8 plants was determined. Ten plants from each biotype at the 5-leaf growth stage were sprayed with Granstar 75 WP. The application solution also contained 100 mg/L Na-fluorescein. Once plants dried, they were cut at ground level and shook for 30 s in 50 ml of 5 mM NaOH. Spectrophotometric readings were made using plant rinsates at a wavelength of 490/510 nm. Plant tissue was then placed at 80 ◦ C for 24 h, and dry matter recorded. The experiment was arranged as a completely randomized design and conducted three times.

2.1. Plant material and growing conditions

2.5. Contact angle

All seeds of S. alba were collected from plants growing throughout Arriate, Malaga in southwest Spain. Specifically, one population (AR8 )was collected from a wheat (Tricicum aestivum) field that had been treated for five continuous years with tribenuron-methyl (6 applications over 5 years). Seed for 12 additional populations were collected from different wheat fields, one population from field bean (Vicia faba) and one population from a chick-pea (Cicer arietinum) field. Also, S. alba seed was collected from a wheat field not treated previously with tribenuron-methyl, and was considered an ALS-susceptible population (AR1 ). For all studies, seeds were pre-germinated in 9 cm plastic dishes containing two layers of Whatman1 filter paper and 5 ml of distilled water. Dishes were then incubated in darkness at 4 ◦ C for 48 h, followed by an additional 24 h at room temperature. Germinating seeds were transplanted into 10 cm diameter pots filled with soil, peat, vermiculite, and sand (3:2:2:2 v/v), and amended with a slow-release fertilizer 26-13-00 (150 g in 75 L of substrate for flowerpots). Pots were irrigated daily to field capacity. Plants were grown in greenhouse conditions at 18/14 C day/night a 16 h photoperiod with light at levels of 850 ␮mol m−2 s−1 was supplemented.

The youngest, fully expanded leaf of ten, 5- to 6-leaf plants from each R and S white mustard biotype was removed at the base of the petiole and placed in a horizontal position onto a wood slide. The leaf was treated with one, 1 ␮L droplet containing tribenuron-methyl solution (15 g ha−1 ; formulated as Granstar 75 WP) at a spray volume equal to 140 L ha−1 . Droplets were applied every 18 s, with a nonbevel Hamilton(R) pipette (25 ␮L) in the center of the adaxial surface. The droplets pattern was then observed in a horizontal microscope (Leica MZ6 1,8X-4X). Photographic images were captured with a Leica Digilux plus Supermacro Leica Digimacro 4.3, adapted to one of the oculars of the microscope. Images for digital analyses were developed digitally using the ImageJ11 program (Grangeot et al., 2006). Differences between 180◦ and the theta C value (contact angle corresponding to each analyzed

2.2. Screening test The initial response of 16 S. alba accessions (AR1 to AR16 ) to tribenuron-methyl was determined. For each accession, 10 plants in the 5–6 leaf growth stage were treated with tribenuron-methyl (Granstar 75 WG3 ) at 20 g a.i. ha−1 (recommended rates for control of white mustard). Applications were made using a laboratory spray

1 Whatman No. 5 filter paper, Fisher Scientific, P.O. Box 4829, Norcross, GA 30091, USA.

2

Spraying Systems, Co., North Avenue, Wheaton, IL 60188, USA. Granstar 75 WG formulation (750 g Kg−1 of tribenuron methyl), Du Pont De Nemours, Nambshaim-France. 4 Autumn 10 WG formulation (100 g Kg−1 of Iodosulfuron methyl sodium), Bayer Cropscience, KS, USA. 5 Osprey 4,5 WG formulation (45 g Kg−1 of mesosulfuron), Bayer Cropscience, KS, USA. 6 Beyond, 12.1% EW formulation (121 g L−1 BASF, Pasco, WA 99301, USA. 7 GF 25 WG formulation (250 g Kg−1 of florasulam), Dow AgroScineces LLC, Indianapolis, IN 46268, USA. 8 Regiment 80 WP formulation (800 g Kg−1 of Byspiribac-sodium), Valent Corporation Walnut Creek, CA, USA. 9 Everest 70 WG formulation (700 g Kg−1 of flucarbazone-sodium), Arysta LifeSciences North America, NC 27513, USA. 10 Sigma Plot 2010. Ver. 10.00. SPSS, Inc., Chicago, IL 60606, USA. 3

J.M. Rosario et al. / Europ. J. Agronomy 35 (2011) 57–62 Table 1 Herbicide treatments applied in field experiment on the AR8 biotype of S. alba in Spain.a Treatment

Rate (g a.i. ha−1 )

Timing

Untreated Tribenuron-methyl 50% Tribenuron-methyl 50% + bromoxynyl Tribenuron-methyl 50% + MCPA Tribenuron-methyl + mecoprop-P

18.75 11.25 + 168 18.75 + 450 a.e. 0.1 + 800

POST POST POST POST POST

POST, postemergence. a Assays conducted during 2008–2009.

herbicide solution droplet) were estimated with the ImageJ11 program. The design of the experiment was completely randomized and was conducted three times. An analysis of variance with data for each biotype was performed using Statistix 8.0 software (USDA-NRCS, 2007), and means separated using Fisher’s protected LSD at a P = 0.05. 2.6. ALS extraction Methodology as described by Cruz-Hipolito et al. (2009a) was followed to investigate the inhibition of ALS activity to ALS inhibitor herbicides. Using four to five leaves of different S. alba R (AR8 ) and S (AR1 ) plants at the 6–8 leaf stage, ALS was extracted from three grams of leaf tissue. Tissue was frozen and ground in liquid nitrogen using a mortar and pestle. Enzyme extract was utilized for assays with technical-grade tribenuron-methyl, iodosulfuron, mesosulfuron, florasulam, imazamox, flucarbazone and bispyribac-sodium herbicides. Enzyme activity was determined colorimetrically (520 nm) by measuring acetoin production and expressing this as a percentage of control (no herbicide). Protein concentration of the crude extract was measured using Bradford (Bradford, 1976). The herbicide concentration necessary for inhibiting the ALS activity by 50% (I50 ) was calculated. Three independent protein extractions were prepared as replicates from each population. The enzyme experiment was repeated at least twice. Data were analyzed using SigmaPlot10 version 10 software, capable of non-linear regression analysis, and fitted to a four parameter logistic model. 2.7. Field experiments In 2008 and 2009, an experiment was established under field conditions on the site where the seeds from resistant plants (AR8 ) used in this research were collected. On an ochrept inceptisol, clay loam soil, pH 7.2 and 1.4% organic matter (OM). Wheat was sown at a density of 70 kg ha−1 and depth of 3 cm on November 27 and 29 in 2008 and 2009, respectively. Urea (80 kg ha−1 ) was broadcasted 9 days before seeding, and diammonium phosphate (100 kg/ha) was drilled with the wheat. Six rates of herbicide and one untreated control (Table 1) were arranged in a randomized complete block design with three replications; plot dimensions were 2 by 5 m. Treatments were applied with a pneumatic backpack containing 11002E flat fan nozzle tips, calibrated to deliver 140 L ha−1 at 276 kPa. Commercial formulations of Granstar 50 SX (tribenuron-methyl, 50% a.i.), Granstar Combi12 (tribenuron-methyl 0.01% + mercoprop-P 73.4% a.i.), MCPA (MCPA, 40% a.i.), and bromoxinil (bromoxynil) were applied. Visual evaluations of white mustard control were made 33 DAT. Control ratings are expressed on a 0 (no effect) to 100 (plant dead) scale. S. alba aboveground biomass was harvested 78 DAT in an area of 0.25 m2 , dried for 5 days at 50 ◦ C, then weighed. Plant dry weights were averaged and converted to a percentage of the untreated control prior to data analysis. Wheat was harvested in June 2008 and 2009 from approximately 0.25 m2 in each experimental plot; grain yield was adjusted to 13.5% moisture and expressed as kilogram of wheat per hectare (kg ha−1 ). The data were subjected to combined ANOVA; means were separated using Tukey’s test at P = 0.05, using Statistix 8.0 software.

3. Results 3.1. Biotype screening assay Significant differences in response to tribenuron-methyl among the field-collected biotypes of S. alba were measured by 21 DAT (Table 2). Compared to the untreated control, reductions in fresh weight ranged from 5.7 to 91.3%. The most sensitive biotypes (fresh weight reduction of >90%) included AR1 , AR2 and AR3 ; none

11 ImageJ: Image Processing and Analysis in Java, edition 1.29, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA (URL: http://rsb.info.nih.gov). 12 Granstar Combi formulation (10 g Kg−1 of tribenuron methyl + 734 g Kg−1 of mercoprop-P), Du Pont De Nemours, Nambshaim-France.

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Table 2 Growth reduction of S. alba accessions 21 DAT following treatment with tribenuronmethyl.a Each biotype was compared to the mean value for the untreated control of that biotype; the standard error of the mean was included in the percentage value. Biotype

AR1 AR2 AR3 AR4 AR5 AR6 AR7 AR8 AR9 AR10 AR11 AR12 AR13 AR14 AR15 AR16 LSD; p = 0.05

Fresh weight reduction Grams

Percentage

11.5 14.1 19.4 06.0 02.0 01.0 02.4 01.9 02.2 01.8 01.6 01.2 00.7 01.4 01.1 01.1

90.9 ± 2.3a 90.9 ± 2.1a 91.3 ± 1.8a 13.1 ± 3.1c 13.1 ± 3.1c 11.6 ± 2.1cd 11.9 ± 2.2cd 10.9 ± 2.7de 11.1 ± 2.2d 10.2 ± 1.2de 9.0 ± 2.2ef 7.9 ± 2.8fg 7.7 ± 2.7fg 6.6 ± 1.8gh 6.4 ± 2.2gh 5.7 ± 1.8h 1.97

a AR refers to S. alba accessions collected in Arriate, Malaga, South-western Spain, 2007.

of the treated plants survived tribenuron-methyl. For AR4 , some visual injury was observed, which contributed to the reduction in fresh weight measured (30%); all plants of this biotype survived tribenuron-methyl. Little or no visible injury was evident on the AR5 to AR16 biotypes, and fresh weight reductions only ranged from 5.7 to 13.1% (Table 2). 3.2. Dose–response assays Tribenuron-methyl susceptible (AR1 ) and resistant (AR8 ) biotypes of S. alba were compared to determine cross-resistance levels to ALS-inhibiting herbicides using plant dry weight. The crossresistance of AR8 was significant to most of the representative ALS inhibitors used, ranging from 4.5 to imazamox and up to 65.9 for florasulam (Table 3). No cross-resistance to bispyribac-sodium was found; the RF (0.16) actually indicated greater sensitivity for the AR8 biotype. The ED50 for tribenuron-methyl was 1.76 and 0.18 for AR8 and AR1 , respectively, resulting in an RF of 9.8. For the other sulfonylurea herbicides, the RF was higher (mesosulfuron, 11.9; iodosulfuron, 17.5). 3.3. Spray retention and contact angle Retention of tribenuron-methyl on leaves of R S. alba was 24% lower compared to S plants (Table 4). The factor underlying reduced herbicide retention on R versus S S. alba appeared to be the contact angle for herbicide droplets. The mean surface contact angle for the adaxial surface of R plants was 3.2 times lower compared to S plants (Table 4). 3.4. ALS extraction Whole plant differences in resistance to ALS inhibitors appeared to be based upon differential inhibition of ALS enzyme. In vitro specific activity of ALS from foliar tissues of the R and S biotypes of S. alba was similar, with 340 (±19) and 366 (±24) nmol h−1 mg−1 of acetoin production, respectively (data not shown). The RF values for R versus S S. alba based upon in vitro ALS activity varied depending upon the specific group of ALS inhibitors (Table 5). The highest resistance level was observed for tribenuron-methyl (4128), but cross-resistance to the other sulfonylurea herbicides mesosulfuron (884) and iodosulfuron

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Table 3 Estimated parameters for non-linear regression equations comparing the inhibition of plant growth from R (AR8) and S (AR1) S. alba biotypes to ALS inhibitors. Comparative levels of resistance to each herbicide were also estimated. Herbicide

Regression parameters

Tribenuron-methyl Imazamox Iodosulfuron Florasulam Flucarbazone Mesosulfuron Bispyribac sodium

P value

A

C

D

b

ED50 (g a.i. ha−1 )

R2

R S R S R S R S R S R S R S

14.970 2.113 5.267 2.184 0.456 2.035 5.008 2.083 7.147 3.730 9.043 0.600 10.857 3.312

100.45 101.09 100.03 100.64 98.09 100.30 99.81 100.00 100.00 100.26 100.00 100.00 101.83 88.88

0.81 1.66 1.78 1.18 0.97 1.96 3.34 0.89 0.50 1.48 6.39 1.23 1.90 1.75

1.760 0.180 1.905 0.423 2.462 0.141 2.771 0.042 203.091 23.878 18.175 1.529 0.107 0.66

0.985 0.993 0.997 0.987 0.975 0.997 0.989 0.999 0.998 0.979 0.997 0.999 0.963 0.883

<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

RF

9.8 4.5 17.5 65.9 8.5 11.9 0.16

A = accession, C = lower limit, D = upper limit, b = Hillˇıs slope, ED50 = effective dose required for 50% reduction in plant biomass, RF = resistance Factor (ED50 R/ED50 S), R2 = 1 − (sums of squares of the regression/corrected total sums of squares). P value = probability level of significance of the non-linear model.

Table 4 Tribenuron-methyl spray retention (␮l g−1 dry matter) and droplet contact angles for R and S white mustard plants. Biotypes

R S

Spray retention* (␮l tribenuron-methyl g−1 dry matter)

3.5. Field experiments Experiments were conducted under field conditions with tribenuron-methyl alone and mixtures with herbicides possessing different modes of action (MCPA and bromoxynil). Use of tribenuron-methyl alone at 14 g a.i. ha−1 only resulted in 28.7% visual control of R S. alba (Table 6). Increasing the rate of tribenuronmethyl to 56 g a.i. ha−1 (3.7 times the labelled rate) improved the control of R S. alba to 70.3%, indicating plants are resistant at the field level. Addition of bromoxynil to tribenuron-methyl improved visual control of R plants to 66%. However, the addition of MCPA or mecoprop-P to tribenuron-methyl boosted control of R S. alba to 88.7 and 92.3%, respectively. Examining shoot dry weight of AR8 plants, the 6 herbicide treatments reduced plant biomass between 48.7 and 91.7% compared to the untreated control. The level of visual control and plant dry weight reduction were similar. Wheat yield in the untreated control was reduced by 39.9% compared to the highest yielding treatment (a mixture of iodosulfuron, mesofulfuron, mefenpyr diethyl, ioxinyl, bromoxynil, and mecoprop-P), indicating the competitiveness of S. alba in wheat (Table 6). With tribenuron-methyl at 14 g a.i. ha−1 , wheat yields were similar to the untreated control. Treatments resulting in 89% or higher visual control of S. alba also resulted in the highest wheat yields.

Contact angles*

125.6b 166.1a

55.6a 17.6b

* Both means are significantly different from based upon Fisher’s Predicted LSD at the 5% level of probability.

(839) was also high. Both sulfonylaminocarbonyltriazolinone (flucarbazone; RF = 86), and triazolopyrimidine (florasulam; RF = 24) herbicides also resulted in cross-resistance based on ALS activity. Cross-resistance to imazamox, an imidazolinone herbicide, revealed a low level of resistance (RF = 1.8). Although whole plant assays indicated R S. alba was more sensitive to bispyribac-sodium than plants from the S biotype, the RF based on ALS activity was significant, but low. Cross-resistance patterns in the R S. alba biotype showed an order from greatest to least of: tribenuronmethyl  mesosulfuron > iodosulfuron  flucarbazone  florasulam  imazamox > bispyribac-sodium (Table 5).

Table 5 Estimated parameters for non-linear regression equations comparing the inhibition of ALS from R (AR8) and S (AR1) S. alba biotypes to ALS inhibitors. Comparative levels of resistance to each herbicide were also estimated. Herbicide

A

C

D

b

I50 (nM)

Pseudo R

P value

RF

Tribenuron-methyl

R S R S R S R S R S R S R S

3.07 0.0 19.46 36.70 18.95 15.95 30.23 37.89 24.66 51.64 5.25 69.59 23.54 13.05

100.66 99.78 101.04 100.84 100.03 100 100.14 101.00 96.84 100.02 100.00 97.57 100.25 100.93

0.37 0.63 1.76 2.10 2.69 0.56 0.77 2.99 5.80 2.67 1.33 1.66 1.26 1.29

908.15 0.22 7.80 4.39 427.96 0.51 72.10 3.07 3780.37 43.87 11833.57 13.39 9.00 5.73

0.91 0.89 0.97 0.97 0.9716 0.996 0.97 0.98 0.98 0.96 0.99 0.90 0.98 0.93

<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

4128

Imazamox Iodosulfuron Florasulam Flucarbazone Mesosulfuron Bispyribac

1.8 839 24 86 884 1.6

A = accession, C = lower limit, D = upper limit, b = Hillˇıs slope, ED50 = effective dose required for 50% plant injury, RF = resistance factor (I50 R/I50 S), PseudoR = 1 − (sums of squares of the regression/corrected total sums of squares). P value = probability level of significance of the non-linear model.

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Table 6 Field evaluation of ALS herbicide activity on ALS-resistant (AR8) S. alba in Spain. Herbicide

Shoot dry weight S. alba (g m−2 )

Herbicide efficacy (%)

Wheat yield (kg ha−1 )

Untreated Tribenuron 50% Tribenuron 50% + bromoxinyl Tribenuron 50% + MCPA Tribenuron 0.1% + mecorop-P LSD (0.05)

138.5 ± 1.4a 71.0 ± 2.8b 38.5 ± 1.6c 11.5 ± 0.4d 34.5 ± 1.4c 7.2

0.0 ± 0.0d 28.7 ± 2.8c 66.0 ± 0.0b 92.3 ± 12.8a 88.7 ± 19.3a 17.8

2856.0 ± 12.0c 2924.0 ± 24.0c 3842.0 ± 50.0b 4186.0 ± 10.0a 3958.0 ± 26.0b 157.2

Means of 2008–2009 assays.

4. Discussion Screening tests indicate that several S. alba populations are resistant to tribenuron. This is based upon the similar biomass response of plants from the AR5 to AR16 biotypes (Table 2). Variable responses to tribenuron-methyl indicate that widespread resistance in S. alba has occurred; the relative short history of tribenuron-methyl usage illustrates how quickly high levels of ALS inhibitor resistance can evolve. Xu et al. (2010) confirmed high levels of resistance to tribenuron-methyl in several populations of Descurainia sophia (L.) in China, with an RF of 118. In addition to tribenuron-methyl resistance, White mustard (AR8 ) was also cross-resistant to four of five ALS inhibitor herbicide families. For a number of other species, different cross-resistance patterns to ALS inhibiting herbicides have been reported (Kuk et al., 2003a; Kuk et al., 2003b; Cruz-Hipolito et al., 2009a). Differential retention could be a determining factor regarding the quantity of herbicide absorbed into the plant leaf, and may contribute to herbicide selectivity in crop and weed species (Kogan and Perez, 2003). With a lower exposed surface area, R versus S plants would intercept less tribenuron-methyl on leaves, reducing the amount of herbicide absorbed. However, a greater than 3-fold reduction in spray retention is not likely sufficient to result in an RF of 9.8, indicating other factors are contributing to the resistance level to tribenuron-methyl. Previous studies of ALS enzyme activity in Papaver rhoeas demonstrated lower resistance levels to imidazolinones in biotypes with high resistance levels to sulfonylureas (Duran-Prado et al., 2004). Variable patterns of cross-resistance to ALS inhibitors have been identified in a number of weed species including: A. plantago-aquatica, S. mucronatus, Monochoria vaginalis, Cyperus difformis, and Amaranthus retroflexus (Kuk et al., 2003a,b; Tabacchi et al., 2004; Scarabel et al., 2005; Cruz-Hipolito et al., 2009a; Merotto et al., 2010). Similar to whole plant biomass, no crossresistance to bispyribac-sodium was measured using in vitro ALS assays. Resistance to one compound from a specific class of ALS inhibiting herbicides has not necessarily resulted in cross resistance to all members of that chemical family (Mallory-Smith et al., 1990). Biotypes selected with resistance to bensulfuron-methyl have exhibited a lower incidence of resistance to bispyribacsodium and penoxulam, relative to other ALS inhibitors (Merotto et al., 2010). This study reveals that S. alba resistance to tribenuron-methyl can be attributed to an altered herbicide target site (ALS); high levels of cross-resistance to other ALS inhibitors is likely the result of enzyme modifications affecting the binding of other chemically distinct ALS inhibitors. Both Brown and Cotterman (1994) as well as Saari et al. (1992) found that sulfonylurea use can result in very high enzyme-based resistance in weed species. Altered target sites are the principal mechanism of resistance to ALS inhibitors as reported for different weeds: Stellaria media; S. arvensis; Descurainia sophia; Alisma plantago-aquatica; Papaver rhoeas; Scirpus mucronatus; Monochoria vaginalis; Cyperus difformis; and Amaranthus retroflexus (Kudsk et al., 1995; Kuk et al., 2003a,b; Tabacchi et al., 2004; Scarabel et al., 2005; Warwick et al., 2005; Duran-Prado

et al., 2004; Christoffers et al., 2006; Cruz-Hipolito et al., 2009a; Merotto et al., 2010; Xu et al., 2010). Despite the detection of high levels of ALS inhibitor resistance in S. alba, alternative herbicides resulted in effective control (Table 6). In field experiments conducted in barley cv. Duke and wheat cv. Roblin in Alberta during 1989 and 1990, control of ALS-resistant populations was poor with sulfonylurea herbicides (chlorsulfuron, metsulfuron-methyl, triasulfuron, amidosulfuron and thifensulfuron), but good to excellent control with different herbicide mixtures (cyanazine + MCPA, linuron, metribuzin, mecoprop, bentazone, metribuzin + MCPA, linuron + MCPA, and mecoprop + bentazone) (O’Donovan et al., 1994). On the other hand, commercial application of tribenuron-methyl for control of resistant S. alba is not an economically viable strategy for wheat producers. Therefore, it is necessary to modify present chemical control strategies for S. alba. Improved management of ALS-resistant weeds involves adoption of effective herbicides, which likely retain a different mode of action (Kudsk et al., 1995). These results confirm the loss of tribenuron-methyl efficacy on R S. alba in wheat in southwest Spain. ALS herbicide may be used to control some weed species in wheat, but the presence of resistant S. alba requires that other herbicides (mecoprop-P and MCPA) be added. Acknowledgments The authors thank the technical help of Rafael Angel RoldánGómez. Part of this work has been performed at the University of Missouri (USA) and University of Cordoba (Spain) and has been co-financed by MICINN (AGL2007-60771) and DuPont Agricultural Products (France). References Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248–254. Brown, H.M., Cotterman, J.C., 1994. Recent advances in sulfonylurea herbicides. Chemistry of Plant Protection, 10. Springer-Verlag, Berlin and Heidelberg, 49–81. Bryan, J.K., 1991. Synthesis of the aspartate family and branched-chain amino acids. In: Miflin, B.F. (Ed.), The Biochemistry of Plants. A Comprehensive Treatise. Amino Acids and Derivatives, vol. 5. Academic Press, New York, pp. 403–452. Calha, I.M., Osuna, M.D., Serra, C., Moreira, I., De Prado, R., Rocha, F., 2007. Mechanism of resistance to bensulfuron methyl in Alisma plantago-aquatica biotypes from portuguese rice paddy fields. Weed Res. 47, 231–240. Chaudhry, O., 2009. Herbicide-Resistance and Weed-Resistance Management. http://www.weedscience.org/paper/Book Chapter I.pdf (accessed 15.05.09.). Christoffers, J.M., Nandula, V.K., Howatt, K.A., Wehking, T.R., 2006. Target-site resistance to acetolactate synthase inhibitors in wild mustard (Sinapis arvensis). Weed Sci. 54, 191–197. Christopher, J.T., Powles, S.B., Liljegren, D.R., Holtum, J.A.M., 1991. Cross-resistance to herbicides in annual ryegrass (Lolium rigidum). Plant Physiol. 95, 1036–1043. Cruz-Hipolito, H., Osuna, M.D., Vidal, R.A., De Prado, R., 2009a. Resistant mechanism to bensulfuron-methyl in biotypes of Scirpus mucronatus L. collected in Chilean rice fields. J. Agric. Food Chem. 57, 4273–4278. Cruz-Hipolito, H., Smeda, R.J., Ioli, G., Rojano, A., Rosario, J.M., De Prado, R., 2009b. Cross-resistance of Sinapis alba to ALS-inhibiting herbicides: first case in the world. In: XIII International Conference on Weed Biology. Dijon, France (8–10 September). Duggleby, R.G., McCourt, J.A., Guddat, L., 2008. Structure and mechanism of inhibition of plant acetohydroxyacid synthase. Plant Physiol. Biochem. 46, 309–324.

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