A dicamba resistance‐endowing IAA16 mutation leads to significant vegetative growth defects and impaired competitiveness in kochia (Bassia scoparia)^†

Abstract

BACKGROUND

Precise quantification of the fitness cost of synthetic auxin resistance has been impeded by lack of knowledge about the genetic basis of resistance in weeds. Recent elucidation of a resistance‐endowing IAA16 mutation (G73N) in the key weed species kochia (Bassia scoparia), allows detailed characterization of the contribution of resistance alleles to weed fitness, both in the presence and absence of herbicides. Different G73N genotypes from a segregating resistant parental line (9425) were characterized for cross‐resistance to dicamba, 2,4‐d and fluroxypyr, and changes on stem/leaf morphology and plant architecture. Plant competitiveness and dominance of the fitness effects was quantified through measuring biomass and seed production of three F₂ lines in two runs of glasshouse replacement series studies.

RESULTS

G73N confers robust resistance to dicamba but only moderate to weak resistance to 2,4‐d and fluroxypyr. G73N mutant plants displayed significant vegetative growth defects: (i) they were 30–50% shorter, with a more tumbling style plant architecture, and (ii) they had thicker and more ovate (versus lanceolate and linear) leaf blades with lower photosynthesis efficiency, and 40–60% smaller stems with less‐developed vascular bundle systems. F₂ mutant plants had impaired plant competitiveness, which can lead to 80‐90% less biomass and seed production in the replacement series study. The pleiotropic effects of G73N were mostly semidominant (0.5) and fluctuated with the environments and traits measured.

CONCLUSION

G73N is associated with significant vegetative growth defects and reduced competitiveness in synthetic auxin‐resistant kochia. Management practices should target resistant kochia's high vulnerability to competition in order to effectively contain the spread of resistance.

An IAA16 mutation (G73N) confers robust resistance to dicamba but moderate to low resistance to 2,4‐d and fluroxypyr in key weed species kochia. The mutation also leads to significant leaf and stem morphological changes and diminished plant size, resulting in plants that are highly vulnerable to competition. Vegetative growth defects of G73N mutant plants suggest that weed management practices which subject resistant plants to substantial competition (e.g. cover crops) will potetially be effective.

© 2020 Society of Chemical Industry.

1. INTRODUCTION

Synthetic auxins are important herbicides that function by simulating the principal natural auxin indole‐3‐acetic acid (IAA) in plants. Plants sprayed with synthetic auxins suffer from an imbalance in auxin homeostasis and impaired auxin interactions with other hormones, leading to deregulated plant growth and eventually plant death. ¹ , ² Resistance evolution for synthetic auxins has been relatively slower than other herbicide modes of action (MOA): 41 resistant weed species have been documented after more than 70 years of use. ³ , ⁴ Owing to the lack of understanding of the MOA of synthetic auxins, the genetic bases of weed resistance were not known for decades. Multiple mechanisms including impaired herbicide translocation and altered metabolism have been reported to play a role in conferring resistance. ⁵ The first target‐site based synthetic auxin resistance mechanism was recently reported in the key weed species kochia (Bassia scoparia) to be a dominant point mutation (G73N) at IAA16, one of the transcriptional repression factor auxin/indoleacetic acid (AUX/IAA) proteins. ⁶ , ⁷

Kochia is a broadleaf summer annual weed in small grains and broadleaf crops in the US Great Plains, western US and Canada. ⁸ , ⁹ Kochia has a strong taproot system which allows it to thrive in droughty, nutrient‐deficient saline soils. ¹⁰ Kochia is a prolific seed producer capable of producing up to 30 000 seeds per plant, ¹¹ which can be dispersed efficiently through tumbling across the landscape in strong winds. ¹² , ¹³ Furthermore, kochia has a high level of within‐population genetic diversity, ¹⁴ resulting in a higher propensity to evolve herbicide resistance. Kochia has evolved resistance to four herbicide MOAs including synthetic auxins. ³ Kochia biotypes that are resistant to at least one MOA, has spread across 20 US states and Canada, ³ , ¹⁵ significantly eroding farmers' options to control them.

As a key factor for predicting resistance evolutionary trajectory, fitness costs of herbicide resistance traits have practical implications for managing resistant weeds. The fitness cost of synthetic auxin resistance has been measured in multiple species including kochia. ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ However, without knowledge of the genetic bases of resistance, it is hard to unequivocally link the pleiotropic effects with herbicide resistance alleles. ²¹ Furthermore, the resistance evolutionary trajectory is not only influenced by the presence of the fitness cost, but also by dominance of the costs. Dominance of fitness costs measures the fitness of the heterozygous (RS) individuals relative to the homozygous resistant (RR) and sensitive (SS) plants. ²² , ²³ , ²⁴ The fitness of RR plants is critical for initial spread and establishment of resistance genes, because newly arisen herbicide resistance mutations are usually in RS forms. ²⁵ Unfortunately, the dominance of the fitness costs of synthetic auxin resistance has not been quantified in weeds.

In this report, we thoroughly investigated the heritability and manifestation of the fitness costs of a synthetic auxin resistance‐endowing mutation G73N, in both parental and F₂ plants grown under different growth conditions, because it is known that fitness penalties associated with adaptive traits might fluctuate with the ecological conditions under which they manifest. ²⁶ , ²⁷ We also quantified the dominance of the fitness costs of G73N to provide insights on mitigating resistance during the early phase of resistance evolution. This very first precise measurement of the fitness costs associating with synthetic auxins, will be valuable to guide the design of integrated weed management practices, to mitigate the spread of resistant weeds and preserve the durability of synthetic auxins in kochia management.

2. MATERIALS AND METHODS

2.1. Plant materials and genotyping method

Seeds from a dicamba‐sensitive kochia wild‐type (WT) line were purchased from Herbiseed (www.herbiseed.com/). A resistant parental line (9425) and three F₂ lines were derived from a previously characterized dicamba‐resistant kochia line maintained through a single seed descent (SSD) method. ⁶ , ⁷ Generation of the parental and F₂ lines is illustrated in Fig. 1. Seeds were first sowed onto germination plug trays containing commercial potting media (Premier Pro‐Mix BX mycorrhizae, Premier Horticultural Services, Quakertown, PA), incorporated with slow release granular fertilizer (Osmocote [14‐14‐14], Scotts Company LLC, Marysville, OH) at the rate of 3600 g m⁻³ (Fig. S1 in Appendix S1). Seedlings were grown in the growth chamber set as follows: 16 h:8 h, light:dark photoperiod, 26 °C/20 °C day/night temperature; and light intensity 550 μm m⁻² s⁻¹. One small cotyledon was sampled from seedlings of 3–4 cm height and genotyped through the allelic specific Taqman® assay as described in LeClere et al. ⁷ Uniform size of genotyped seedlings was selected and transplanted for downstream studies.

Figure 1.

Schematic illustration of the generation of the parental line 9425 and F₂ lines. Parental line 9425 was derived from a previously characterized dicamba‐resistant line maintained through a single seed descent (SSD) method. Owing to lack of molecular markers at the time of the bulk cross and morphological similarity between RR and RS plants (Fig. 4), either multiple RS plants were included or self‐pollination of RS plants, could result in a segregating 9425 parental line. To generate F₂ lines, WT plants were hand‐pollinated with pollen from 9425‐RR plants, followed by self‐pollinating different F₁ crossed progenies confirmed through genotyping and dicamba spray.

2.2. Contribution of G73N to cross‐resistance to broad‐spectrum synthetic auxins

Different G73N genotypes of 9425 (RR, RS, SS) and WT lines were subjected to dose–response studies on three representative auxin herbicides. Plants were grown at a Bayer CropScience glasshouse in St Louis, MO, USA (29/26 ± 3 °C day/night temperatures, 14 h:10 h, light:dark photoperiod, supplemental lighting to maintain ≥1050 μm m⁻² s⁻¹ light intensity). Plants at 8–10 cm height were sprayed with 0, 140, 280, 560, 1120 or 2240 g ha⁻¹ of Dicamba (Clarity® BASF Corporation, Florham Park, NJ), or 0, 210, 420, 840, 1680, or 3360 g ha⁻¹ of 2,4‐d (2,4‐D amine® Winfield United, Gibbon, NE), or 0, 39.25, 78.5, 157, 314 or 628 g ha⁻¹ of fluroxypyr (Starane Ultra®, Corteva AgriScience, Wilmington, DE). Herbicides were applied using a research track sprayer equipped with a TTI spray nozzle (TeeJet® spray nozzles, SpraySmarter.com) calibrated to deliver 140 L ha⁻¹ of herbicide solution at 276 kPa, moving at 2.57 km h⁻¹. Plant visual injury was evaluated at 21 days after treatment (DAT) and monitored for another three weeks to see if survivors were able to survive and reproduce.

2.3. Plant architecture, biomass and seed production

Two glasshouse fitness studies were conducted on genotyped 9425 and WT kochia plants (Exp‐1 N = 18 per genotype; Exp‐2, N = 15 per genotype) in May and July 2019. Glasshouse growth conditions are listed in Table S1 in Appendix S1. Genotyped seedlings at 5–7 cm tall were transplanted into 4.5‐in plastic pots (10.2 cm width X 10.2 cm length X 17.8 cm height) filled with the potting mix described above. Plants were watered as needed and fertilized biweekly with Jack's Professional 20–10‐20 general purpose fertilizer (JR Peters Inc., Allentown, PA, USA) at 200 ppm N. Pots were evenly spaced to avoid any interplant shading and re‐randomized weekly.

Plant heights were measured weekly. Plant architecture traits such as changes in gravitropic set point angles (GSAs) of the 6th lateral shoot branch from the soil surface were quantified through images of plants at 10 weeks after planting (WAP) in Fiji. ²⁸ Plants were harvested individually upon natural plant senescence and dried at 40°C for 72 h and weighed for biomass. Harvested plants were threshed and screened through U.S. standard brass sieves (2 mm fb 0.5 mm, Dual Manufacturing Co., Inc., Franklin Park, IL). Seeds were cleaned through a column seed blower (Hoffman Manufacturing, Inc., Corvallis, OR) and weighed. Roots of two‐week‐old seedlings were washed gently under running water and dry weights were measured (N = 6 per genotype).

2.4. Photosynthesis efficiency, vegetative morphology and microscopy

All genotyped 9425 and WT kochia plants from the above glasshouse studies were used to study photosynthesis efficiency, and a subset of the plants were used to study leaf and stem morphology. Five‐mm round leaf punches were taken from the first fully unfolded leaf at 3 WAP, and immediately placed onto 96‐well plates filled with 200 μL leaf disc assay buffer (1 mm MES + 1% sucrose, PH6.5; M2933, 84 097, Sigma‐Aldrich Inc., St. Louis, MO). Plates were dark‐adapted for 20 min and scanned through a chlorophyll fluorescence imager (Technologica CF Imager, Technologica Ltd, Essex, UK), to collect Fv/Fm values, which measure the maximum quantum efficiency of PSII photochemistry/photosynthesis efficiency. ²⁹

Leaf blade width and length of the 1st fully unfolded leaf (N = 32) were measured at 4 WAP. Histological observations were made through hand and microtome transversal sections for stems and leaves, respectively (three to four plants per genotype at 4, 8 and 10 WAP). The stems were sectioned through the internodes at 10–12 cm and 2–4 cm above the soil surface for Exp‐1 and Exp‐2, respectively. Pieces (8 × 9 mm) of leaf blade from three leaves per plant were sampled and fixed in 4% formaldehyde (Electron Microscopy Sciences, Hatfield, PA, cat. no. 15710‐S), infiltrated for 1 h, washed three times in 10% PBS, air‐dried and then embedded in (10 × 10 × 5 mm) vinyl molds filled with OCT compound (Tissue‐Tek; Sakura Finetek USA Inc., Torrance, CA), instantly frozen with liquid N₂ and then stored at −80 °C. Transverse 45‐μm sections were made on a microtome and stained for 10 s with toluidine blue (Electron Microscopy Sciences, Hatfield, PA, cat. no. 22050). Observations were made with a standard brightfield microscope (M205 FA, Leica, Wetzlar, Germany) fitted with a digital camera (Leica M205 FA, DFC310 FX) through the Leica application suite X (LAS X) platform. Images from three sections per plant were taken and subjected to leaf thickness (250 μm from the middle vein) and stem diameter measurement in Fiji. ²⁹

2.5. Glasshouse replacement series competition studies on F₂ lines

Two glasshouse replacement series competition studies (Exp‐1 N = 480; Exp‐2, N = 320) were conducted on three F₂ lines in July and September 2016 (Fig. S2 in Appendix S1). A germination test was carried out on agarose in a growth chamber maintained at 22 °C constant temperature, 16 h:8 h, light:dark. Genotyped seedlings were transplanted into 20‐L plastic pots filled with the potting mix described above. Each pot contained eight plants of two genotypes (RR:SS or RS:SS) either as pure stands or in mixtures at ratios of 8:0 (100%), 6:2 (75%), 4:4 (50%), 2:6 (25%) and 0:8 (0%). All of the pots were randomized weekly and the plants were only watered as needed. Plants were grown in a completely randomized design, with detailed timing and glasshouse conditions listed in Table S1 in Appendix S1. Plant height, biomass and seed production data were collected as described above, except that plants were air‐dried in the glasshouse for six weeks instead of being oven‐dried.

Following replacement series indices derived from biomass and seed production data were calculated using the formulas listed in Table S2 in Appendix S1: Relative yield (RY), competitive ratio (CR), relative crowding coefficient (RCC) and aggressiveness index (AI). These replacement series indices measure the competitiveness and resources niches of different genotypes. ²⁰ , ³⁰ , ³¹ Dominance of the fitness costs was calculated using the following formula ²² :

$$h=\frac{\left|\overline{\mathit{SS}}-\overline{\mathit{RS}}\right|}{\left|\overline{\mathit{SS}}-\overline{\mathit{RR}}\right|}$$ (1)

where $\overline{\mathit{RR}},\overline{\mathit{RS}}$, $\overline{\mathit{SS}}$ represent means of plant height, biomass or seed production for F₂‐RR, ‐RS and ‐SS plants, respectively. The fitness costs are dominant when h = 1 (RS = RR), semidominant when h = 0.5 (RS = 1/2SS) and recessive when h = 0 (RS=SS).

2.6. Statistical analysis

Dose–response curves were fitted using the four‐parameter log‐logistic model ³² through the DRC package in RStudio (v3.5.1, The R Foundation for Statistical Computing, Vienna, Austria):

$$\mathrm{Y}=c+\frac{d-c}{1+\exp \left(b\left(\log \left(x\right)-\log \left(e\right)\right)\right)}$$ (2)

where Y is the predicted visual injury (%), x is the herbicide dose, the upper limit d and the lower limit c were fixed, b is the slope, and e is the GR₅₀ (50% growth reduction) for each genotype. A R/S ratio was calculated by dividing the GR₅₀ values of the resistant line by that of the sensitive line. Testing of model fit, parameters estimates and SE were all done through the DRC package.

Violin plots were generated to visualize plant height, biomass and seed production of different genotypes in rstudio. ANOVA was conducted using the linear mixed effect models in SAS (V9.4, SAS Institute, Cary, NC). For parental or F₂ line fitness measurements, genotype was the fixed effect. For the replacement series indices (RCC, CR, AI), genotype, competition as well as their interactions were the fixed effects. Detailed models and testing of the assumptions of ANOVA are described in the Appendix S1. All pairwise comparisons between the genotypes were defined within the ANOVA and tested using Student's t‐tests. Overall F‐tests for interactions between competition and genotype also were defined based on the model above. The nonparametric Wilcoxon signed rank test was used for RCC comparison owing to the violation of normality distribution. A two‐sided Student's t‐test was used to determine if CR and AI were different from 1 and 0, respectively; thus, if RY observed values deviate from their expected values at each competition level and if the dominance of resistance costs is different from 0.5 or 1 individually. For the replacement series study, simple linear regression lines were plotted in scatter plots to visualize how plant height, biomass and seed production change in response to increasing resistance frequencies.

3. RESULTS

3.1. The role of G73N in cross‐resistance to broad‐spectrum synthetic auxins

The GR₅₀ of 9425‐RR plants estimated from the dose–response curves were 2187 g, 263 g and 34 g for dicamba, 2,4‐d and fluroxypyr, respectively, which were five‐, three‐ and one‐fold, and 25‐, 11‐ and 11‐fold higher than that of ‐RS and ‐SS plants [Fig. 2(a)–(l)]. However, G73N appeared to confer robust resistance to dicamba (survival at 4×), but only moderate‐to‐weak resistance to 2,4‐d and fluroxypyr [Fig. 2(m)–(o)]. The 9425‐RR plants sprayed with recommended label rates of dicamba (560 g), 2,4‐d (840 g) and fluroxypyr (157 g) were 25%, 70% and 88% injured, respectively. Although most of the RR/RS plants that were sprayed with higher rates (≥2×) of 2,4‐d or fluroxypyr were moderately to highly stunted, the injury levels did appear to be lower than that of 9425‐SS or WT line, and those plants were able to reproduce six weeks after being sprayed (Fig. S3).

Figure 2.

Cross‐resistance of different G73N genotypes to dicamba, 2,4‐d and fluroxypyr. Plants were sprayed with different rates of dicamba (a–d, m), 2,4‐d (e–h, n), fluroxypyr (i‐l, o). Photos were taken 21 days after herbicide treatment (DAT). Dose–response curves were fitted using the four‐parameter log‐logistic model. GR₅₀ and GR₉₀ values were indicated at the top right corner of each photo.

3.2. G73N leads to diminished plant size and altered plant architecture

The fitness effects of G73N also were measured in two glasshouse experiments (Exp‐1, Exp‐2) in different G73N biotypes of parental 9425 and WT lines in the absence of herbicide. No difference was observed on germination rates and seedling mortality as were observed in F₂ lines (Fig. S4). Differences in plant height and leaf morphology were obvious and noticeable shortly after emergence [Figs 3(b), S5b and S6a). The 9425‐RR plants were consistently shorter than other biotypes throughout the whole plant growth period (Fig. S6), and were 34%, 47% and 54%, and 29%, 39% and 36% shorter than 9425‐RS, ‐SS and WT plants at 10 WAP in Exp‐1 and Exp‐2, respectively [Table 1; Figs 4 and S7]. We were able to recapitulate dwarf phenotypes in F₂ lines by introgression of the G73N mutation into the WT background (Fig. 5), indicating the mutation is associated with the phenotype. Regardless of being significantly shorter, 9425‐RR and ‐RS plants had similar GSAs as the SS plants and reduced apical dominance, resulting in a sphere shape plant architecture (Table 1; Figs 4 and S7). This unique tumbling plant form might allow the resistant plants to bounce and roll more easily in the wind to spread their seeds. The WT plants were similar to 9425‐SS plants in plant height and biomass (Figs 4, 5 and S7), but had much smaller GSAs owing to its more upright lateral branches, showing phenotypic variance among kochia populations of different geographical origins. Differences also were observed in root systems of different biotypes: 9425‐RR plants had fewer lateral roots than ‐RS and ‐SS plants, and an overall reduced root biomass of 50% (Fig. S5c; Table 1).

Figure 3.

Leaf and stem morphology of different G73N endowing resistant or susceptible genotypes. (a) Example chlorophyll fluorescence images of leaf punches showing different photosynthesis efficiency (1 = Exp‐1, 2 = Exp‐2, 3 leaf punches per genotype); (b) photos of whole plant at three weeks after planting (WAP) from Exp‐1; (c) stems of 9425‐RR, ‐RS, ‐SS and WT plants from Exp‐2 (left to right, four per genotype); (d)–(e) leaf and (f–g) are stem cross‐sections for four‐ and eight‐week‐old plants of different genotypes. In each panel from left to right are 9425‐RR, ‐RS, ‐SS and WT plants, respectively. Arrows in (e) show the distortion of leaf main veins.

Table 1.

Measurement of different plant physiological and morphological traits of different G73N genotypes from the 9425 and WT B. scoparia line

Fitness measurement	Timing	Genotype				ANOVA †
		9425‐RR	9425‐RS	9425‐SS	WT
Fv/Fm	Exp‐1	0.630 ± 0.005 (a ‡ )	0.645 ± 0.006 (a)	0.669 ± 0.004 (b)	0.675 ± 0.008 (b)	<0.0001
	Exp‐2	0.726 ± 0.005 (a)	0.739 ± 0.005 (b)	0.738 ± 0.003 (b)	0.735 ± 0.004 (ab)	<0.0001
Leaf thickness (μm)	4 WAP	339.6 ± 14.3 (a)	254.8 ± 5.9 (b)	291.0 ± 14.8 (bc)	296.6 ± 16.2 (c)	0.0007
	8 WAP	381.1 ± 15.3 (a)	266.8 ± 17.3 (b)	217.3 ± 10.4 (c)	192.1 ± 13.8 (c)	<0.001
Leaf area (mm2)	4 WAP	553.4 ± 21.9 (a)	530.2 ± 36.5 (a)	521.0 ± 40.9 (a)	615.0 ± 52.0 (a)	0.3437
Leaf length (mm)	4 WAP	52.7 ± 1.5 (a)	66.0 ± 1.6 (b)	73.4 ± 2.2 (c)	83.4 ± 1.9 (d)	<0.001
Leaf width (mm)	4 WAP	16.6 ± 0.3 (a)	14.0 ± 0.8 (b)	12.0 ± 0.4 (c)	12.0 ± 0.7 (c)	<0.001
Stem diameter (mm)	4 WAP	2.8 ± 0.1 (a)	4.8 ± 0.2 (b)	5.7 ± 0.2 (c)	7.1 ± 0.2 (d)	<0.0001
	8 WAP	4.9 ± 0.1 (a)	5.7 ± 0.1 (b)	5.6 ± 0.1 (b)	6.6 ± 0.3 (c)	<0.0001
GSA § (°)	Exp‐1	64.3 ± 1.8 (a)	66.3 ± 3.5 (a)	68.0 ± 2.3 (a)	43.6 ± 2.5 (b)	<0.0001
	Exp‐2	65.8 ± 3.0 (a)	52.9 ± 3.3 (b)	64.2 ± 2.2 (a)	36.2 ± 2.3 (c)	<0.0001
Plant height (cm)	Exp‐1	57.7 ± 3.5 (a)	87.5 ± 5 (b)	109.7 ± 6.8 (c)	125.8 ± 3.8 (c)	<0.0001
	Exp‐2	67.0 ± 2.0 (a)	94.8 ± 4.3 (b)	110.2 ± 3.0 (c)	104.5 ± 4.0 (bc)	<0.0001
Root weight (g)	2 WAP	0.009 ± 0.001 (a)	0.021 ± 0.003 (b)	0.024 ± 0.004 (b)	0.021 ± 0.004 (b)	<0.0001

^† P‐values for ANOVA for the effects of genotype on different fitness traits are listed in the far‐right column.

^‡Different letters represent significant difference among the means at the P = 0.05 level.

^§Gravitropic set point angles.

Figure 4.

Variations on plant architecture of different G73N genotypes of 9425 and WT kochia lines. Plant biotypes in each panel are (a) homozygous‐resistant (RR), (b) heterozygous‐resistant (RS), (c) homozygous‐sensitive (SS) and (d) WT. Photos were taken 10 weeks after planting. N = 15 per genotype. Scale bar = 100 cm.

Figure 5.

Violin and boxplots of key fitness traits of different G73N genotypes of 9425 and F₂ kochia lines. RR, RS and SS stand for homozygous‐resistant (RR), heterozygous‐resistant (RS) and homozygous‐sensitive (SS) plants with or without the G73N mutation. F₂ plants were from monoculture pots in the replacement series study (eight plants per pot, total N: Exp‐1, 160; Exp‐2, 128). Parental plants were grown with one plant per pot (total N: Exp‐1, 18; Exp‐2, 15).

3.3. G73N leads to significant leaf and stem morphological changes

In addition to smaller plant statue, significant physiological, morphological and histological changes of leaves and stems also were observed. The leaf blades of 9425‐RR plants were more ovate (20–40% shorter and 20–40% wider), whereas leaves of other biotypes were lanceolate to linear [Table 1; Figs 3(b) and S5(a,b)]. The leaf blades of 9425‐RR plants were 33%, 17% and 14%, and 43%, 75% and 98% thicker than 9425‐RS, ‐SS and WT plants at 4 WAP and 8 WAP, respectively (Table 1). In addition, leaves of 9425‐RR plants also were associated with 2–7% lower photosynthetic efficiency, as indicated by Fv/Fm values [Table 1; Fig. 3(a)]. The Kranz anatomy (thick‐walled mesophyll cells containing large chloroplasts, cluster around bundle‐sheath cells in a ring‐like fashion) of leaves looked largely similar among the genotypes, except that the older leaves (8 WAP) of 9425‐RR and ‐RS plants exhibited obvious disruption on the main veins [Fig. 3(d),(e)].

Furthermore, the stem diameter of 9425‐RR plants was 42%, 51% and 61%, and 12%, 11% and 24% smaller than that of 9425‐RS, ‐SS and WT plants in Exp‐1 and Exp‐2, respectively (Table 1; Fig. S5d–f). Although directionally consistent, the difference in stem diameter between Exp‐1 and Exp‐2 was expected owing to more compact spherical shapes (‘tumbling’) plants observed in Exp‐1 (Figs 4 and S7). Aligned with morphological stem diameter, the cross‐sections indicated that 9425‐RR plants had less developed vascular bundle systems. Both young (4 WAP) and matured stems (8–10 WAP) of 9425‐RR plants had fewer secondary xylem cells and interfascicular fibers, that provide the mechanical support to plant stems, compared to 9425‐SS and WT plants [Figs 3(f),(g) and S5d‐f], resulting in increased lodging propensity [Fig. 4 and S6(c)]. Interestingly, the diminished plant size and vegetative growth defects were translated into lower biomass accumulation but not seed production in both the F₂ and parental line (Fig. 5).

3.4. G73N leads to impaired competitiveness in the replacement series study

Replacement series studies on F₂ lines indicated that resistant plants were very vulnerable to intraspecies competition [Fig. 6(a); Table 2]. The RY values for the RR biotype were consistently lower than the expected values (0.25, 0.5, 0.75) for both biomass and seed production in both experiments. By contrast, the RY values of the SS biotype were mostly higher than (Exp‐1) or equal to (Exp‐2) the expected values [Fig. 6(a)]. The resulting total RY values (RYT) from the two competing biotypes were equal to or lower than the expected value of 1, indicating that the RR and SS biotypes likely share similar niches and compete fully for the limiting resource(s). Interestingly, RS plants were less competitive than SS biotypes in Exp‐1 [Fig. 6(a)], but were equally competitive with SS plants in Exp‐2, indicating that the manifestation of fitness cost might be environmentally dependent.

Figure 6.

Plant competitiveness and dominance of the resistance costs through the replacement series studies on F₂ lines. (a) Relative yield (RY) of different G73N genotypes of F₂ line across different competition levels. RY values were calculated from biomass and seed production of two experiments. Dashed lines indicate the expected hypothetical values (H₀ = 0, 0.25, 0.5, 0.75, 1) for RY, which indicates the two competing genotypes (RR versus SS or RS versus SS) are equally competitive. (b) Dominance of fitness costs.

Table 2.

Comparison of different replacement indices calculated from biomass and seed production data collected on F₂ lines

Fitness traits	Genotype	Exp‐1			Exp‐2
		Replacement indices (median/mean†)			Replacement indices (median/mean)
		RCC	CR	AI	RCC	CR	AI
Biomass	RR	0.197	3.814	0.327	0.409	1.693	0.208
	RS	0.213	3.429	0.360	1.209	1.011	−0.026
	SS	1.614			1.008
Seed production	RR	0.304	5.194	0.651	0.423	1.697	0.216
	RS	0.435	2.555	0.460	1.238	1.024	0.001
	SS	0.726			1.124

	Comparison	Fitness cost‐P‐value			Fitness cost‐P‐value
		RCC	CR > 1	AI > 0	RCC	CR > 1	AI > 0
Biomass	RR versus SS	0.112	0.020*	0.045*	0.023*	0.001*	0.001*
	RS versus SS	0.029* ,†	0.006*	0.007*	0.486	0.941	0.733
Seed production	RR versus SS	0.813	0.012*	0.001*	0.017*	0.007*	0.002*
	RS versus SS	1.000	0.006*	0.002*	0.697	0.830	0.982

^*Indicate statistical significance at P = 0.05.

^†Medians were used for RCC comparison and means were used for CR and AI comparison. RCC, relative crowding coefficient; CR, competitive ratio; AI, aggressiveness index. Formulas used to calculate the secondary replacement series indicies are listed in Table S2 in Appendix S1.

Similar results were observed on the secondary replacement series indices, as RCC values of SS plants were consistently higher than RR or RS plants in both experiments, and CR and AI values (Table 2) were significantly greater than 1 and 0, respectively. Interestingly, although competition can lead to 80–90% reduction in biomass in RR plants, they had similar reproductive ability with RS and SS plants when RR become the prominent genotypes (e.g. 75% resistance proportion or in monoculture) (Figs 5 and S8). This suggests that the reduced reproductivity was more likely to have been the result of suppression from plants with bigger sizes other than lower inherent fecundity caused by G73N.

3.5. The fitness costs of G73N is largely semidominant and fluctuate with environments

The quantification of the dominance of the fitness effects of G73N on F₂ lines appeared to vary with the fitness traits measured. Plant height and biomass of RS plants were mostly intermediate to those of RR and SS plants, showing a semidominant (h = 0.5) fitness cost [Fig. 6(b)]. Furthermore, the fitness of RS biotypes also varied among the two experiments: In Exp‐1, h was between 0.5 and 1, leaning toward a dominant fitness costs, whereas in Exp‐2, h was generally lower and even leaned toward slightly recessive at higher competition levels when the proportion of RR or RS plants was <50%; however, none of the h values significantly deviated from 0.5. This again suggests that RS plants can have limited or negligible fitness cost, depending on the growth environments.

4. DISCUSSION

Auxin is one of the main regulators of plant development and controls a wide variety of plant responses and stress adaptation. ³³ , ³⁴ , ³⁵ Due to the importance of the auxin pathway in plant growth and development, the evolution of synthetic auxin resistance through mutating critical proteins such as auxin receptors was at one point thought to be difficult, and likely associated with a high fitness penalty, if not lethality. ³⁶ The elucidation of a molecular mechanism of synthetic auxin resistance in a natural weed population, provided an unique opportunity for detailed characterization of the resistance gene's contribution to plant fitness, both in the presence and absence of synthetic auxin herbicides.

Dose–response study on cross‐resistance to dicamba, 2,4‐d and fluroxypyr at the genotype level directionally agrees with the molecular and phenotypic data reported previously, ⁷ except that we observed generally lower R/S ratios especially for fluroxypyr resistance. The difference, we believe, is mainly due to visual injury instead of fresh weight data being used to fit the dose–response models. Highly stunted plants treated with synthetic auxins could still have a substantial amount of fresh weight. Also, kochia is a facultative short‐day plant species whose plant sizes vary with growth conditions (e.g. photoperiod), which could account for some variance on the magnitude of resistance. ³⁷ , ³⁸ Therefore, we believe that visual injury combined with mortality data, as reported in the current study, is a better proxy with which to characterize resistance to synthetic auxin herbicides (Fig. S3). Our results align with the visual injury data collected by LeClere et al. ⁷ (Appendix S2), as well as the molecular and root length studies. ⁷ Nevertheless, although G73N might not be the major causative gene for fluroxypyr resistance, it might still present concerns on facilitating the evolution of full resistance to fluroxypyr. A study using sublethal rates of 2,4‐d and recurrently selecting the survivors in Palmer amaranth (Amaranthus palmeri), demonstrated a shift to resistance after three generations, ³⁹ likely resulting from the accumulation of resistance alleles with minor effects. ⁴⁰ It is likely when the herbicides are applied under suboptimal spraying conditions and/or use of sublethal rates with reduced herbicide effectiveness, resistance allele(s) with minor effects (e.g. G73N to fluroxypyr) might be selected, accumulated and gradually shift a less susceptible population towards resistance.

In the absence of herbicide selection pressure, G73N led to profound vegetative growth defects, altered overall plant architecture and reduced biomass accumulation. This is not unexpected because AUX/IAAs play pivotal roles and are expressed in many tissues and throughout plant growth and development. ⁶ , ⁴¹ It has been well‐documented in the model species Arabidopsis thaliana that AUX/IAA mutants often lead to varied aberrant phenotypes, similar to what we observed in kochia: diminished adult size, decreased shoot apical dominance, abnormal gravitropism (e.g. GSAs of lateral shoots) and fewer lateral roots. ⁴¹ , ⁴² , ⁴³ , ⁴⁴ Our results also agree with most other fitness studies in synthetic auxin resistance kochia or other weed species which reported up to 90% fitness cost. ¹⁶ , ¹⁹ , ⁴⁵ , ⁴⁶

It is worth mentioning that reduced fitness could be partially due to inbreeding depression because the 9425 parental line was maintained through SSD breeding. ⁶ , ⁴⁷ , ⁴⁸ However, for a prominently outcrossing species such as kochia, multiple generations of self‐pollination also can purge some of the strongly deleterious, nearly recessive mutations endowing inbreeding depression from the genome. ⁴⁹ , ⁵⁰ , ⁵¹ , ⁵² , ⁵³ Furthermore, the generation of F₂ lines through introgression and shuffling of G73N into an unrelated and highly outcrossing WT line (Fig. 1), might serve as a genetic rescue method that helps bring beneficial variants, minimizing inbreeding depression in the 9425 line. Lastly, we observed highly distinguishable phenotypes (e.g. plant height and architecture, biomass) (Fig. 4) associating with different genotypes of G73N with similar inbreeding levels, and no reduction on fecundity when grown in monoculture ⁷ (Fig. 5), which are all strong evidences that inbreeding depression was not a major factor that can mask the effects of G73N in our kochia populations.

Through microscopy work and chlorophyll imaging, we identified specific morphological changes in leaf, stem and overall plant architecture that might have contributed to lower vegetative fitness in kochia. First, leaves of G73N mutant plants were associated with reduced photosynthesis efficiency, although this might be transient and limited to seedling stage. Mutant kochia plants also displayed increased leaf thickness and altered leaf shape, which might affect the thermoregulation of leaves under heat stress and the ability of resistant plants to optimize light interception. ⁵⁴ , ⁵⁵ , ⁵⁶ Furthermore, G73N led to less‐developed vascular bundle systems and smaller stem diameter, resulting in spherically shaped plants with higher lodging propensity. Together these changes might compromise the ability of resistant plants to build biomass and produce seeds, especially under competition, as seen in the replacement series study on F₂ lines. Interestingly, the spherical and bush shape of RR plants might present some ecological advantages as it might facilitate the spread of resistant alleles across the landscape through tumbling.

Replacement series studies were previously conducted in other kochia populations with unknown resistance mechanisms, in which vegetative traits such as plant height, stem diameter and primary branches were measured. ²⁰ In the current study, we calculated the replacement series indexes (RY, RCC, CR, AI) based on biomass and seed production, which are considered to be better proxies of plant fitness. ⁵⁷ Our results generally agree with Kumar and Jha ²⁰ in that the resistant plants were much less competitive than the sensitive plants as RY values significantly deviated from their expected values across different growth conditions in the two experiments (Summer versus late Autumn). Furthermore, secondary replacement series indexes such as RCC, CR and AI values also supported higher competitiveness of susceptible plants. The differences in the RCC values were mostly insignificant in Exp‐1. This is likely the consequence of some negative RCC values brought by the heterogenous responses of the plants grown in monoculture and mixture, which suggests that RCC values might not be a very robust index with which to describe the interfering effects in the current study. ³¹

In this study, we also measured the fitness cost of G73N in both homozygous and heterozygous status and found that deleterious effects of G73N are trait‐dependent and might be magnified or ameliorated by different ecological conditions. ²⁶ It also indicates that employment of weed management to sustain the spread of RS plants might be challenging, and that their effectiveness might vary with environments. Previously, Roux et al. ²² quantified the dominance of the cost of 2,4‐d resistance endowed by three mutations at the same gene in Arabidopsis and reported a wide range of results from recessivity (axr1‐3) to dominance (axr2‐1) and to underdominance (aux1‐7). This indicates that each AUX/IAA mutation might be independent from each other and our current observations on G73N might not predict other naturally occurring mutations for synthetic auxin resistance. It is also possible that G73N might only be a transition step during the resistance evolutionary trajectory, which may be replaced by other resistance mutations or mechanisms with lower fitness cost, as has been found for triazine resistance in common waterhemp (Amaranthus tuberculatus). ⁵⁸ , ⁵⁹ , ⁶⁰

5. CONCLUSIONS

The current study presents the first precise and systemic measurement of the fitness costs of synthetic auxin resistance with known genetic basis, allowing us to unequivocally attribute fitness costs to a specific resistance endowing mutation G73N. We demonstrated that G73N leads to significant vegetative growth defects and impaired competitiveness, which might help explain the relative slower resistance evolution for the herbicide. These results suggest that subjecting G73N mutant kochia plants to substantial competition to restrain light, water and nutrient accessibility (e.g. through the adoption of cover crop or narrower crop row spacing), ⁶¹ , ⁶² will potentially be an effective approach to decrease the frequencies of resistant biotypes. Interestingly, even though resistant plants produced much less biomass and seeds under competition, they appeared to have similar fecundity as sensitive plants in the absence of competition. More study is imminent to determine the impact of G73N on kochia's reproductive growth, especially its unique mating system, to understand the ecological consequences of novel resistance mutations. Nevertheless, it is apparent that there is no single silver bullet to reduce the kochia problem and, thus, that growers must follow integrated weed management approaches that harness evolutionary approaches, as pointed by Neve et al. ⁶³ and Norsworthy et al., ⁶⁴ to diversify our weed control programs to delay development of synthetic auxin resistance.

AUTHOR CONTRIBUTIONS

CW, SL and RS conceived the project; CW and MP performed the research with assistance from SL and AP; KL and CW conducted statistical analysis; and CW, PW and RS wrote the manuscript with contributions from all other authors.

CONFLICT OF INTEREST

CW, MP, SL, KL and AP are employed by Bayer CropScience.

DATA AVAILABILITY

Data for the current study is available at https://doi.org/10.5061/dryad.2v6wwpzk6.

Supporting information

Appendix S1: Supporting information.

Appendix S2: Supporting information.