Precise quantification of the fitness cost of synthetic auxins resistance has been impeded by lack of knowledge for the genetic basis of resistance in weeds. Recent elucidation of a resistance endowing IAA16 mutation (G73N) in a 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. 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: 1) being 30-50% shorter with a more tumbling style plant architecture; 2) 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 produced up to 90% less biomass and seeds in the replacement series study. The pleiotropic effects of G73N was mostly semi-dominant (0.5) and fluctuated with the environments and traits measured. 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 to effectively contain the spread of resistance.

1. Plant materials

The current study aimed at measruing the fitness cost of  a resistance endowing IAA16 mutation (G73N) in a key weed species kochia (Bassia scoparia). Plants with and without the G73N can result in three different genotypes: homozygous-, heterozygous-resistant (RR and RS) and homozygous sensitive (SS) plants. The pleiotrophic effects of G73N mutation on kochia's fitness was measured in two genetic backgrounds: a segregating parental line 9425 and three F2 lines (F2-a, F2-b, F2-c).  Parental line 9425 was derived from a previously charactered dicamba resistant kochia line (9425) from Nebraska and segreates in the following frequencies: 40-45% RR or RS and 10-15% SS plants.  Another parental line (WT) was purchased from Herbiseed and is sensitive to synthetic auxins. 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. F2 lines segragate in the following frequencies: 25% RR, 50% RS, 25% SS. Details of the parental line 9425 and F2 lines were listed in Fig. 1 in https://doi.org/10.1002/ps.6080.

2. Dose response studies that chracterized the cross resistance to three synthetic auxins herbicides in parental line 9425 and WT

Different G73N genotypes from the segregating resistant parental line (9425-RR, -RS, -SS) and WT were characterized for cross resistance to dicamba, 2,4-D and fluroxypyr, and changes on stem/leaf morphology and plant architecture.  Plants at 8-10cm height were sprayed with 0, 140, 280, 560, 1120, 2240 g/ha  of  Dicamba  (Clarity® BASF Corporation, NJ, US), or 0, 210, 420, 840, 1680, 3360g/ha of 2,4-D (2,4-D amine® Winfield United, NE, US), or 0, 39.25, 78.5, 157, 314, 628 g/ha of fluroxypyr (Starane Ultra^®, Corteva AgriScience, DE, US). Plant visual injury was evaluated at 21 days after treatment (DAT) and monitored for another 3 weeks to see if survivors were able to survive and reproduce.

3. Measurement of fitness traits in different G73N genotypes in parental lines 9425 and WT 

Two glasshouse fitness studies were conducted on genotyped 9425 (9425-RR, -RS, -SS) 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 1. 

3.1 Plant architecture, biomass and seed production

5 to 7-cm tall genotyped seedlings were transplanted into 4.5in plastic pots with one plant per pot. Plants were watered as needed and fertilized biweekly. 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. Plants were harvested individually upon natural plant senescence and dried at 40°C for 72h and weighed for biomass. Harvested plants were threshed and screened through U.S. standard brass sieves (2mm fb 0.5mm, Dual Manufacturing Co., Inc. IL, US). Seeds were cleaned through a column seed blower (Hoffman Manufacturing, Inc, OR, US) and weighed. Roots of 2-weeks old seedlings were washed gently under running water and dry weights were measured (N=6 per genotype).

3.2 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 3WAP, and immediately placed onto 96-well plates filled with 200µL of leaf disc assay buffer (1 mM MES + 1% sucrose). Plates were dark adapted for 20min 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.

Leaf blade width and length of the 1^st fully unfolded leaf (N=32) were measured at 4WAP. Histological observations were made through hand and microtome transversal sections for stems and leaves, respectively (3-4 plants per genotype at 4, 8 and 10WAP). The stems were sectioned through the internodes at 10-12cm and 2-4cm above the soil surface for Exp-1 and Exp-2, respectively. 8mm x 9mm leaf blade from three leaves per plant were sampled and fixed in 4% formaldehyde, infiltrated for 1h, washed three times in 10% PBS, air-dried, and then embedded in (10mm x 10mm x 5mm) vinyl molds filled with OCT compound (Tissue-Tek; Sakura Finetek USA), instantly frozen with liquid nitrogen and then stored at -80°C. Transverse sections of 45µm were made on a microtome and stained for 10s with toluidine blue. Observations were made with a standard brightfield microscope (Leica, model: M205 FA, Wetzlar, DEU) fitted with a digital camera (Leica, model: M205 FA, DFC310 FX) through Leica application suite X (LAS X) platform. Images from three sections per plant were taken and subjected to leaf thickness (250µm from the middle vein) and stem diameter measurement in Fiji.

4. Glasshouse replacement series competition studies on F₂ lines

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 (Exp-1 N=480; Exp-2, N=320) in July and September 2016. Glasshouse growth conditions are listed in Table 1. A germination test was carried out on agarose in a growth chamber maintained at 22 °C constant temperature, 16h/8h day/night. Genotyped seedlings were transplanted into 20L 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 the pots were randomized weekly and the plants were only watered as needed. Plants were grown in a completely randomized design. Plant height, biomass and seed production data were collected.

Following replacement series indices derived from biomass and seed production data were calculated using the formulas listed in the Table 2: 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.

Dominance of the fitness costs based on biomass and seed production data was calculated using the following formula:

h=        (1)

where ,  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).

Table 1 Greenhouse settings and growth conditions for the two studies on F₂ and parental kochia (Bassia scoparia) line (9425). The supplemental lights were set to be on for 14h throughout the experimental period.

^a Data from Time and Date AS, https://www.timeanddate.com/sun/usa/st-louis?month=10&year=2016

                                                    https://www.timeanddate.com/sun/usa/st-louis?month=10&year=2019

^b Indicating the months during which experiments were conducted in.

^c For F₂ lines, Exp-1 was from 7/20/2016 to 11/20/2016 and Exp-2 from 9/1/2016 to 11/30/2016. For parental line 9425, Exp-1 was from 5/22/2019 to 9/15/2019 and Exp-2 was from 7/15/2019 to 10/15/2019.

Table 2 Summary of the formulas used for the calculation of replacement series indices

^† In the formula, P₁ and P₂ are the proportions of susceptible (S) and resistant(R) plants in the mixture (RR vs SS or RS vs SS) respectively, P₁+P₂=1, S_mix, R_mix are the mean yields (biomass, seed production) of S and R plants grown in mixture and S_mono and R_mono are yields of S and R plants grown in monoculture. Expected values for RY(S) are 1 (R:S=0:8), 0.75 (R:S=2:6), 0.5 (R:S=4:4), 0.25 (R:S=6:2) and 0 (R:S=8:0); ^‡ R biotypes refer to either RR or RS; ^§Criteria to determine if there is a fitness cost (R < S) or not (R ≥ S). CR, RCC, AI index were only calculated for the equal mixture proportion (R:S = 4:4).

Figures and tables correspond to https://doi.org/10.1002/ps.6080:

Data Set-1: Fig. 2 Parental Line Dose Response Study Visual Injury and Mortality Data

Date Set-2: Fig. 5 Parental and F2  Line Fitness Violin Plots ( Plant height, Biomass, Seed production)

Date Set-3: Fig. 6 F2 Replacement Series Index RY and Dominance of the fitness cost (h)

Date Set-4: Fig. S2, S6, S8 Data (Germination, plant height over time, plant height, biomass, seed acorss different competition levels)

Date Set-5: Table 1 Parental 9425 Line Fitness Measurement

Date Set-6:  Table 2 F2 Line Replacement Series Indexes RCC, AI, CR