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Published online 1 September 2007
Published in Crop Sci 47:2058-2066 (2007)
© 2007 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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CROP PHYSIOLOGY & METABOLISM

Recovery of Imidazolinone-Resistant Hard Red Wheat Lines Following Imazamox Application

Bradley D. Hansona,*, Lynn Fandricha, Dale L. Shanerb, Philip Westraa and Scott J. Nissena

a Dep. of Bioagricultural Sciences and Pest Management, Colorado State Univ., Fort Collins, CO 80523
b USDA-ARS Water Management Unit, 2150 Centre Ave., Bldg. D, Suite 320, Fort Collins, CO 80526. B.D. Hanson's current address is USDA-ARS Water Management Research Unit, 9611 S. Riverbend Ave., Parlier, CA 93648. This article is a U.S. government work and is in the public domain in the USA

* Corresponding author (bhanson{at}fresno.ars.usda.gov).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Imidazolinone-resistant hard red wheat (Triticum aestivum L.) cultivars are occasionally injured by imazamox applications because a portion of the acetolactate synthase (ALS) remains susceptible to the herbicide. The growth and enzyme activity of two groups of hard red wheat near-isolines with spring or winter growth habit were examined following imazamox application. Each group of near-isolines contained a susceptible cultivar and cultivars with the imidazolinone-resistant trait on either the B or D genome. The spring wheat group also contained a line carrying both the B and D genome copies of the resistance gene. In whole plant experiments, growth of all single-gene resistant lines was delayed by both 35 and 105 g ha–1 imazamox while the two-gene line was delayed at only the highest rate. There was a herbicide rate effect on biomass accumulation but no differences among genome locations in the single-gene resistant lines or among spring vs. winter growth habit. On an ALS enzyme basis, however, there were differences among B- vs. D-genome resistance and between winter and spring growth habit. Spring wheat cultivars with the B-genome resistance had greater reductions in ALS activity compared to the D-genome cultivars, while in winter wheat, B- and D-genome lines responded similarly. Differences among genotypes existed in the recovery of ALS activity in imidazolinone wheat but other factors also likely influence the injury occasionally observed in the field.

Abbreviations: ALS, acetolactate synthase • DAT, days after treatment

Recovery of Imidazolinone-Resistant Hard Red Wheat Lines Following Imazamox Application

Bradley D. Hansona,*, Lynn Fandricha, Dale L. Shanerb, Philip Westraa and Scott J. Nissena

a Dep. of Bioagricultural Sciences and Pest Management, Colorado State Univ., Fort Collins, CO 80523
b USDA-ARS Water Management Unit, 2150 Centre Ave., Bldg. D, Suite 320, Fort Collins, CO 80526. B.D. Hanson's current address is USDA-ARS Water Management Research Unit, 9611 S. Riverbend Ave., Parlier, CA 93648. This article is a U.S. government work and is in the public domain in the USA

* Corresponding author (bhanson{at}fresno.ars.usda.gov).

Imidazolinone-resistant hard red wheat (Triticum aestivum L.) cultivars are occasionally injured by imazamox applications because a portion of the acetolactate synthase (ALS) remains susceptible to the herbicide. The growth and enzyme activity of two groups of hard red wheat near-isolines with spring or winter growth habit were examined following imazamox application. Each group of near-isolines contained a susceptible cultivar and cultivars with the imidazolinone-resistant trait on either the B or D genome. The spring wheat group also contained a line carrying both the B and D genome copies of the resistance gene. In whole plant experiments, growth of all single-gene resistant lines was delayed by both 35 and 105 g ha–1 imazamox while the two-gene line was delayed at only the highest rate. There was a herbicide rate effect on biomass accumulation but no differences among genome locations in the single-gene resistant lines or among spring vs. winter growth habit. On an ALS enzyme basis, however, there were differences among B- vs. D-genome resistance and between winter and spring growth habit. Spring wheat cultivars with the B-genome resistance had greater reductions in ALS activity compared to the D-genome cultivars, while in winter wheat, B- and D-genome lines responded similarly. Differences among genotypes existed in the recovery of ALS activity in imidazolinone wheat but other factors also likely influence the injury occasionally observed in the field.

Abbreviations: ALS, acetolactate synthase • DAT, days after treatment


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HARD RED WHEAT (Triticum aestivum L.) is grown on more than 12 million hectares in the Great Plains region of the United States and weed control is an important management consideration for wheat producers each year (USDA–National Agriculture Statistic Service, 2006). Weeds, particularly annual grasses, can be difficult to selectively control in wheat. Development of herbicide-resistant crops has resulted in new opportunities for selective weed control in many crops including wheat, and, in some cases, has simplified weed management. Herbicide-resistant crops allow for expanded use of certain broad spectrum herbicides with favorable environmental profiles (Duke, 2005; Devine, 2005; Tan et al., 2005).

Imidazolinone-resistant (Clearfield) winter wheat was commercially released in the Great Plains and Pacific Northwest regions of the USA in 2002 and 2003, respectively, and was grown on 242,000 ha in 2006 (S. Tan, personal communication, 2006). Imidazolinone herbicides inhibit acetolactate synthase (ALS, also called acetohydroxyacid synthase or AHAS, EC 4.1.3.18), the first enzyme unique to branched-chain amino acid biosynthesis (Shaner et al., 1984). Imazamox was registered for use on imidazolinone-resistant wheat because of the herbicide's broad weed spectrum, limited soil persistence, and favorable toxicological properties (BASF, 2004; Shaner et al., 1996). This technology has been widely accepted by U.S. wheat producers because of the opportunity to selectively control important annual grass weeds including jointed goatgrass (Aegilops cylindrica Host), feral rye (Secale cereale L.), Italian ryegrass (Lolium multiflorum Lam.), and the brome complex (Bromus spp.) (Geier et al., 2004; Zemetra et al., 1998).

The imidazolinone-resistant trait in wheat was isolated after seed mutagenesis and is controlled by a single gene (Newhouse et al., 1992). The original mutant (FS4) and most of the early released imidazolinone-resistant cultivars carried the resistance trait on the long arm of chromosome 6 in the D genome (renamed AhasL-D1) (Anderson et al., 2004; Pozniak and Hucl, 2004). Using mutagenesis and backcrossing programs, wheat lines with AhasL-B1 and AhasL-A1 resistance were created and multiple-genome resistant cultivars are under development (Pozniak et al., 2004a; Tan et al., 2005).

Modern bread wheat is an allohexaploid (2n = 42) with A, B, and D diploid genomes each contributing to the total ALS production (Pozniak et al., 2004a; Poehlman and Sleper, 1995). Previous research suggests that the D genome in wheat may provide more total ALS than the B genome and the A genome may likewise differ (Hanson et al., 2006). Regardless of the precise contributions of each genome to total ALS production, the susceptible portion of total ALS activity in single- and two-genome imidazolinone-resistant cultivars is reduced following treatment with imazamox (Hanson et al., 2006; Rainbolt et al., 2005). Although a temporary reduction in ALS activity usually does not result in reduced productivity of imidazolinone-resistant-winter wheat, injury from imazamox occasionally does occur. Imazamox injury on imidazolinone-resistant wheat generally consists of minor chlorosis, dark green color of developed leaves, and delayed or stunted growth (Pozniak et al., 2004b). Imidazolinone-resistant-spring wheat cultivars appear to be slightly more susceptible to injury compared to winter wheat and do not have adequate tolerance to U.S. labeled rates of imazamox (Pozniak et al., 2004b; Tan et al., 2005; Hanson et al., 2006). Wheat injury can be influenced by high imazamox rates, surfactants, application timing, and environmental conditions (Frihauf et al., 2005; Geier et al., 2004; Stougaard et al., 2004; Hanson et al., 2005b).

When a susceptible plant is treated with an ALS inhibitor, growth quickly ceases (Shaner et al., 1984; Shaner and Mallipudi, 1991). However, the relationship of ALS activity and biomass accumulation is not well understood for plants containing both resistant and susceptible forms of the enzyme (either as heterozygous diploids or as polyploids such as wheat) (Hanson et al., 2005a; Rainbolt et al., 2005). One possibility is that after an imazamox application, activity of susceptible ALS is completely stopped, while the resistant ALS remains active. Depending on the requirements for branched chain amino acids and the amount of resistant ALS in the plant, biomass accumulation may slow or stop if these products are in limited supply. As the herbicide is metabolized over time, the activity of the sensitive form of the enzyme would be expected to begin to recover and eventually return to pre-application levels. Thus, the plant growth should likewise begin to recover as full ALS activity returns. Wheat cultivars with multiple-genome resistance or resistance on a more active genome may therefore be suppressed to a lesser degree or for a shorter duration.

One factor contributing to whole plant growth recovery could be the rate at which the sensitive ALS enzyme returns to normal activity. However, very little information exists on the relative recovery of growth and ALS activity of imidazolinone-resistant wheat treated with imazamox. Therefore, the objectives of this research were to monitor the recovery of several near-isolines of hard red imidazolinone-resistant wheat with different resistance gene locations and growth habit at a whole plant and ALS-enzyme level after treatment with imazamox.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Seed for selected hard red winter wheat lines were obtained from public and private breeders. Wheat lines used in these experiments included both commercially available cultivars and advanced breeding selections. The specific wheat lines used included two sets of near isolines based on the hard red spring cultivar Gunner and on the hard red winter cultivar Wahoo (Baenziger et al., 2002). Each set included a susceptible parent, a B-genome resistant line, and a D-genome resistant line, and the Gunner group also contained an imidazolinone-resistant line with both B- and D-genome resistance. Each imidazolinone-resistant wheat line carried the same mutation resulting in a serine to asparagine substitution at amino acid residue 653 relative to the Arabidopsis thaliana consensus sequence (M. Dahmer, personal communication, 2005).

Whole Plant Growth Recovery Experiments
Four wheat seeds were planted 1 cm deep in commercial potting media (Metro-Mix 200 potting mix, Sun Gro Horticulture, Inc., Bellevue, WA) in 12 by 12 by 8 cm pots (801 True series inserts, ITML Horticultural Products, Inc., Brantford, ON). Eight pots of an individual wheat line were placed in a 25 by 50 cm flat. Plants were grown in a greenhouse under natural light conditions supplemented with 400-W sodium halide lamps to provide a 14-h daylength. Day and night temperatures were 24 ± 5 and 18 ± 3°C, respectively, and plants were irrigated with an automatic sprinkler system. At the 2.5- to 3-leaf stage, each flat containing eight pots was randomly assigned to an imazamox treatment (35 or 105 g ha–1 imazamox) or left untreated. Recommended field rates of imazamox range from 35 to 53 g ha–1 in winter wheat grown in the United States (BASF, 2004). Imazamox treatments were applied with a cabinet sprayer calibrated to deliver 145 L ha–1 and included 0.25% v/v nonionic surfactant and 2.5% v/v urea ammonium nitrate. On the day of herbicide application, wheat plants in one pot from each flat were clipped at the soil surface, fresh weight was measured, and plants were dried in a 60°C oven for 24 h for zero days after treatment (DAT) for dry weight determination. Treated and untreated plants were maintained under previously described greenhouse conditions. Twice per week (3- or 4-d intervals) a randomly selected pot was removed from each flat and the plants were harvested, dried, and weighed as previously described. A total of seven biomass harvests were performed over the experimental time course (0, 3, 7, 10, 14, 17, and 21 DAT).

Each group of wheat lines (i.e., those with a common parent), along with ‘Above’ (Haley et al., 2003) as a standard, was examined in a separate experiment. All experiments were arranged in a randomized complete block design with three replications and each experiment was repeated. Treatments were in a full factorial (wheat line x imazamox rate) arrangement and each flat was considered an experimental unit. Dry biomass data from each wheat cultivar were then converted to biomass accumulation after application by subtracting the appropriate average zero-time biomass from each sample. Biomass accumulation data were converted to a percentage of the weight of the untreated control plants 21 DAT (potential biomass) to allow direct comparisons between groups. Data were analyzed using regression techniques in proc GLM (SAS Institute, 2001) assuming a normal distribution.

In Vitro Acetolactate Synthase Dose–Response Experiments
Wheat plants were grown in 25 by 50 by 8 cm flats containing commercial potting mix (Sunshine Mix no. 1/LC 1, Bellevue, WA) in a greenhouse under the previously described conditions. Plants were fertilized twice weekly with water soluble fertilizer (Scotts Champion 21–18–18, Scotts Company, Marysville, OH) at a rate equivalent to 200 mg L–1 N. Plants at the two- to three-leaf growth stage were sprayed with 105 g ha–1 imazamox plus labeled adjuvants as previously described (treated) or left undisturbed (control). Acetolactate synthase was extracted and enzyme activity was measured on actively growing tissue collected from the base of tillering wheat plants using assay protocols adopted from Pozniak et al. (2004a) and Singh et al. (1988). Enzyme activity was quantified in treated and control plants at select time points after foliar imazamox treatment. Spring wheat plants were assayed 1, 2, 3, 7, 8, and 14 DAT, while winter wheat plants were assayed 1, 3, 7, 10, and 14 DAT. Assay points included measurements collected from repeated experiments with four replications.

Assay reactions were performed in 100-µL volumes within 96-well microtiter plates using a 1:1 ratio of crude extract supernatant to test solution (Hanson et al., 2006). Enzyme activity was measured in the presence of 0, 50, and 100 µmol L–1 imazamox. Two additional reactions were included as controls: (i) 100 µmol L–1 ACC 299,016 (an uncommercialized sulfonyl carboxamide, was provided by BASF Company, Florham Park, NJ) dissolved in acetone (inhibits both imidazolinone-resistant and -susceptible forms of ALS [Alvarado et al., 1992]), and (ii) 5% sulfuric acid diluted with distilled water (inhibits all enzyme activity). Acetolactate production was quantified indirectly by decarboxylating acetolactate to acetoin and measuring absorbance spectrophotometrically at 535 nm according to the methods of Westerfeld (1945). Before further analysis, all OD 535 values were corrected for background absorbance by subtracting the value obtained from the ACC 299,016 inhibition from each test solution.

Enzyme Resistance
Enzyme resistance was determined from plants that did not receive a foliar imazamox treatment. Resistant enzyme activity was calculated as OD 535 absorbance in the presence of 50 µmol L–1 imazamox expressed as a percentage of the no-imazamox control wells. Percent activity data were analyzed with a linear regression model designed to test the effects of wheat isoline, DAT, and an interaction term using PROC GLM (SAS Institute, 2001). Specific questions addressed by the statistical analyses were (i) is there a difference in ALS activity among the wheat isolines; and (ii) is the difference in ALS activity consistent across a several time intervals? Specific linear contrasts were performed using {alpha} = 0.05.

Total Extractable Enzyme
Extractable enzyme was determined from plants that received a foliar imazamox treatment but no in vitro imazamox. The OD 535 values from these plants were expressed as a percentage of the OD 535 values from plants that received neither foliar nor in vitro imazamox. Absorbance values were normalized by the amount of crude protein in untreated and treated plant extracts measured through a standard Bradford Protein Assay. Data were analyzed using nonlinear regression techniques (Systat Software, 2004. An exponential decay model,

Formula 1[1]
where y0 = an intercept adjustment, a and b = parameters describing the exponential decay function, x = DAT in the greenhouse, and c = a parameter describing the tailing upward slope of the function, was found to adequately fit data for all wheat isolines resistant to imazamox. Data from susceptible wheat isolines were not fit to a model because all ALS enzyme activity was inhibited by an application of 105 g ha–1 imazamox. Specific questions addressed by the statistical analyses were (i) is there a difference in extractable ALS among the wheat isolines; and (ii) is the difference in extractable ALS consistent across several time intervals?

Recovered Susceptible Enzyme
Recovery of the susceptible enzyme was determined mathematically by subtracting resistant enzyme activity from total activity. Total ALS activity at each time point was determined from plants treated with foliar imazamox but no in vitro imazamox. Resistant enzyme activity was determined from plants treated with both foliar imazamox and 50 µmol L–1 in vitro imazamox. Total and resistant enzyme activity data were adjusted for total extractable enzyme at each time point (see above). Percent susceptible enzyme activity at each time point was determined by subtracting the proportion of resistant enzyme activity from 1.00. Data were analyzed using nonlinear regression techniques (SAS Institute, 2001). Data were fit to an exponential rise model described by

Formula 2[2]
where a and b are parameters describing the asymptote and slope of the line, respectively. Nonlinear contrasts were performed using {alpha} = 0.05.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Whole Plant Growth Recovery Experiments
There were no significant interactions between treatment and experiment; therefore, data from the repeated experiments were pooled for analysis. The standard imidazolinone-resistant wheat cultivar Above responded similarly among replications and experiments and the data were not included in the analysis of the near-isolines (data not shown). Linear regression using a quadratic equation provided an adequate fit to biomass accumulation over this 21 d time course experiment (Fig. 1 ). In general, little to no biomass was accumulated by susceptible wheat lines treated with either 35 or 105 g ha–1 imazamox and regression parameters were not different from zero (Table 1 ). Single gene resistant imidazolinone-resistant wheat lines treated with either 35 or 105 g ha–1 imazamox had an initial slope estimate (DAT) that was not different from zero while the untreated plants usually had a positive initial slope. The initial slope for the two-gene resistant ‘AP604’ was reduced only by the 105 g ha–1 imazamox treatment. The change in the slope of the regression curves over time (DAT*DAT) was similar among all treated and untreated resistant wheat lines except ‘AP601’ (spring, D genome) treated with 35 g ha–1 imazamox. This treatment resulted in a significantly steeper response curve than the other single-gene resistant lines. Imazamox treatment usually affected biomass accumulation rates of single-gene resistant wheat cultivars. This effect was more pronounced with the 105 g ha–1 rate and apparently was due to an initial delay in growth (DAT) rather than change in growth rate over time (DAT*DAT). No clear differences in biomass accumulation were observed between the winter and spring wheat lines with the same resistance gene location.


Figure 1
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  		Figure 1. Biomass production of hard red wheat near-isolines after imazamox treatment in the greenhouse. Data are expressed as a percent of the untreated plants 21 d after treatment (DAT) (potential biomass). Symbols and lines represent quadratic regression predictions and 95% confidence intervals.

 

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  		Table 1. Model parameter estimates, standard errors and P values estimated by quadratic regression for untransformed biomass (g pot–1) accumulation by seven wheat cultivars in response to foliar imazamox treatment.

 
In Vitro Acetolactate Synthase Dose–Response Experiments
Enzyme Resistance
Resistance-gene location, growth habit, and gene copy number affected the activity of resistant ALS enzyme in the presence of 50 µmol L–1 imazamox (Fig. 2 ). Furthermore, a significant interaction term between isoline and DAT demonstrated that slopes differed among the lines (data not shown); however, the effect of isoline was 51 times greater than the interaction, and more than 175 times greater than the DAT effect, suggesting that the variance in absorbance could be explained adequately by isoline alone. The main effect of DAT was retained in the final model for solvency, the interaction term was dropped, and the model rerun.


Figure 2
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  		Figure 2. Imazamox-resistant acetolactate synthase (ALS) enzyme activity in hard red wheat near-isolines following an in vitro application of 50 µmol L–1 imazamox. Data are expressed as a percentage of the untreated controls. Lines are linear regression predictions and symbols are mean and standard error values from two independent experiments with eight replications.

 
Approximately 37% of the ALS enzyme in spring wheat D-genome plants was resistant to in vitro imazamox applications (Fig. 2). In contrast, 32% of the enzyme was active in spring wheat B-genome plants. A similar trend was observed in winter wheat plants with significantly less enzyme inhibited in plants that carried the resistance-gene on the D rather than the B genome. Furthermore, winter wheat plants had consistently greater enzyme activity than spring wheat plants for both B- and D-genome resistant plants (Table 2 ). Approximately 62% of the enzyme was unaffected by the herbicide application in the two-gene spring wheat (BD genomes, AP604). Comparison of single-gene wheat with a two-gene winter wheat was not possible due to a lack of available seed; however, there was greater enzyme activity for all assay points in the two-gene spring wheat than in any single-gene wheat.


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  		Table 2. Contrasts for the regression line intercept term within and among resistance-gene genome locations in herbicide-resistant and -susceptible hard red wheat near-isolines for resistant enzyme values in response to 50 µmol L–1 in vitro imazamox as a percentage of untreated controls.

 
Total Extractable Enzyme
Resistance-gene location, growth habit, and gene copy number also affected the amount of ALS enzyme extracted from plants treated with 105 g ha–1 imazamox. The foliar application initially affected the resistant and susceptible forms of the enzyme in the spring wheat BD isoline; approximately 15% more enzyme was inhibited by the foliar application compared to the 50 µmol L–1 in vitro application (Fig. 2 and 3 ). The total amount of enzyme extracted from these plants increased such that by 14 DAT the amount of extractable enzyme was similar to untreated plants (86%).


Figure 3
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  		Figure 3. Extractable acetolactate synthase (ALS) enzyme activity in herbicide-resistant and -susceptible hard red wheat near-isolines following treatment with a 105 g ha–1 imazamox. Data are expressed as a percentage of untreated plants. Lines are predicted values based on an exponential decay nonlinear regression model (Eq. [1]) while symbols are mean and standard error values from two independent experiments with eight replications.

 
The total amount of enzyme extracted from plants with single-genome resistance (B or D genome) decreased between imazamox application and three DAT and remained constant through the rest of the experiment (Fig. 3). Differences in extractable enzyme were also observed between growth habit and between gene location within the winter wheat isolines (Table 3 ). Resistant and susceptible forms of the enzyme in spring wheat containing a single copy of the herbicide resistance gene were affected by the foliar overspray. Twenty-six percent of the enzyme was extracted from B- and D-genome spring wheat plants 1 DAT, and this value dropped to 14% for the B-genome wheat and 16% for the D-genome wheat by the end of the experiment. The initial amounts of enzyme extracted from single-gene winter wheat isolines differed; 36 and 57% of the enzyme was extracted from the B- and D-genome isolines, respectively, 1 DAT. The amount of enzyme extracted from these plants 3 DAT and beyond was not different within winter wheat lines, but was consistently greater than values from spring wheat B- and D-genome isolines through the same time interval.


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  		Table 3. Model parameter estimates, standard errors, and P values ({alpha} = 0.05) estimated by an exponential linear combination model for extractable ALS enzyme in treated plants as a percent of untreated plants.

 
Recovered Susceptible Enzyme
The amount of susceptible enzyme in the two-gene spring wheat (AP604) treated with 105 g ha–1 imazamox was constant through 14 DAT. In contrast, the amount of susceptible enzyme in single resistant-gene isoline plants (AP602, AP601, Wah-002, Wah-001) changed rapidly over 2 d (Fig. 4 ), and there were no differences in responses within or among the isolines (data not shown). The amount of susceptible enzyme in B- and D-genome resistant spring wheat averaged 83% 1 DAT but there was no further change in susceptible enzyme 3 DAT. We observed also that the amount of susceptible enzyme in winter wheat D-genome plants was slower to recover between 1 and 3 DAT than the enzyme in B-genome plants (Fig. 4). Although this difference was not statistically relevant, this small biological difference, especially over a short time period may contribute to the ability of these plants to tolerate imazamox treatment. In less than 2 d, the susceptible enzyme fraction in B-genome resistant wheat reached its maximum value of 83%, but 3.5 d were required by D-genome resistant wheat to reach a similar value. This indicates that a greater portion of the total ALS activity during this critical time period is due to the resistant form of the enzyme and which likely contributes to greater tolerance to imazamox in D-genome plants.


Figure 4
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  		Figure 4. Recovery of imazamox-susceptible acetolactate synthase (ALS) enzyme activity in hard red wheat near-isolines following a 105 g ha–1 imazamox treatment. Data are expressed as a percentage of total ALS activity and are corrected for total extractable enzyme activity. Lines are predicted values based on an exponential rise nonlinear regression model (Eq. [2]) while symbols are mean and standard error values from two independent experiments with 8 replications.

 

   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The wheat plants used in the whole plant biomass accumulation experiments and the in vitro ALS assays were grown under slightly different conditions; therefore these data are not directly comparable. However, these two experiments provide some insights into the response of imidazolinone-resistant hard red wheat to imazamox. At the whole plant level, biomass accumulation was delayed in all single-gene resistant wheat lines by both imazamox treatments and in the two-gene resistant line only by the highest imazamox rate. During the short time course of this experiment, the initial delay in growth often resulted in reduced overall biomass production particularly in plants treated with 105 g ha–1 imazamox. It is difficult to predict if stunting 21 DAT observed in the greenhouse also would reduce biomass and grain yield under field conditions. In contrast to previous reports (Hanson et al., 2006; Pozniak et al., 2004a, 2004b), we did not observe marked differences between winter and spring growth habit or between B- and D-genome resistance although the two-gene wheat was injured less than either single-gene resistant spring wheat. The lack of difference between winter and spring lines in these experiments may have been due to relatively poor growth of untreated spring wheat plants caused by warmer conditions compared to previous biomass experiments

In contrast to the whole plant data, the ALS enzyme activity was strongly influenced by genome location, number of genes, and growth habit. In the absence of foliar applied imazamox, D-genome imidazolinone-resistant wheat always had higher levels of resistant ALS activity compared to B-genome cultivars and winter lines always had higher resistant ALS activity compared to spring lines. This response remained consistent across time points; however, when the plants were treated with 105 g ha–1 imazamox, the amount of extractable ALS activity was suppressed much more than predicted by the in vitro imazamox treatment effect, suggesting that more than just the susceptible form of the enzyme is inhibited. The amount of extractable enzyme reached its lowest point approximately 3 DAT and remained relatively constant through 14 DAT, while in the whole plant experiments, biomass accumulated at a reduced rate compared to the control plants after an initial lag period. It is not known if the changes in extractable ALS correspond directly to total ALS activity in treated plants or if the herbicide treatment reduces the extractability of otherwise active ALS enzyme. Additionally, the fraction of extractable ALS that was sensitive to imazamox rose to a plateau approximately 2 to 3 DAT indicating that the recovery of the sensitive ALS form is quite rapid, while the recovery of total extractable ALS is much slower.

The results from the ALS assay may have been complicated by the high imazamox rate used in the greenhouse. The 105 g ha–1 rate was chosen to ensure that whole plant symptoms were observed; however, this may have caused too much suppression of ALS for us to quantify recovery in a short time frame. Field applications of imazamox often occur during periods of relatively slow growth (fall or spring) rather than during a logarithmic growth period as in these greenhouse experiments. A greater lag time between herbicide application and rapid growth periods could allow for greater imazamox metabolism and subsequent recovery of total ALS activity.

These results demonstrate that there are differences in ALS recovery among winter and spring wheat lines, and among B, D, and two-gene resistant lines. The ALS assay data did not have the same trends in recovery as the whole plant biomass recovery experiments. Although this may be partially due to environmental conditions during the biomass experiments, these data suggest that other factors besides growth habit and resistance gene location affect wheat response to imazamox. These could include the ability of the different genotypes to metabolize the herbicide to nontoxic forms, the physiological state of the plant at application, the relative expression of the imidazolinone-resistant genes, rate of enzyme turnover, and could also include other fitness traits linked to the ALS gene. Further research is needed to determine why foliar applications of imazamox reduce ALS activity to a greater extent than in vitro imazamox and how this correlates to field response of imidazolinone-resistant hard red wheat to the herbicide.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Received for publication January 16, 2007.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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C. J. Willenborg, A. L. Brule-Babel, L. F. Friesen, and R. C. Van Acker
Response of Heterozygous and Homozygous Imidazolinone-Resistant Spring Wheat Genotypes to Imazamox
Crop Sci., November 24, 2008; 48(6): 2107 - 2114.
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Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome