Plant Materials and Herbicide

The weed species that were used are listed in Table 1. Partial E. japonica populations were collected from major rice-producing areas in China with regional representation, where the application of penoxsulam (Clipper 25 OD) at the recommended dose (15 to 30 g ai ha⁻¹) has failed to control this weed since 2015. The rest of E. japonica populations were collected from fallow fields where penoxsulam had never been applied. According to the pretest (data not shown), none of the E. japonica populations were sensitive to penoxsulam. Therefore, a sensitive E. crus-galli population JLGY-2019-S, whose herbicide target protein gene homology was as high as 91% compared with E. japonica, was selected as a control in all biological and molecular studies (Wang et al. Reference Wang, Li, Lv, Lou and Dong2013, Reference Wang, Li, Lv, Zhu, Lou and Dong2014; Yu et al. Reference Yu, Friesen, Zhang and Powles2004). All seeds were collected by hand, air-dried in the shade, and stored in paper bags at 4 C until use. Penoxsulam oil dispersion (Dow AgroSciences, Nantong, Jiangsu, China, 226000) of 25 g L⁻¹ was used.

Table 1. Sensitivity of 29 Eragrostis japonica populations and one Echinochloa crus-galli population to penoxsulam.

^a GR₅₀ refers to the effective dose of herbicide causing 50% inhibition of fresh weight and is indicated as grams of active ingredient per hectare (g ai ha⁻¹). Data are the means of two experiments.

^b RI is the relative tolerance index: ratio of GR₅₀ values relative to the susceptible E. crus-galli population (JLGY-2019-S). The recommended field dose of penoxsulam is 15–30 g ai ha⁻¹.

^c Populations with no prior herbicide exposure.

Whole-Plant Dose–Response Experiment with Penoxsulam

Greenhouse experiments were conducted to evaluate the penoxsulam sensitivity of E. japonica populations and an E. crus-galli population. Twenty seeds from each population were sown in plastic pots (9-cm diameter by 10-cm height) filled with a 2:1 (w/w) mixture of sand and pH 5.6 organic matter and grown in incubators at 30 /25 C (light/dark temperature) with a 12-h light/12-h dark cycle, a light intensity of 8,000 lux, and 85% relative humidity. The seedlings were thinned to 15 plants per pot before herbicide treatment. At the 3- to 4-leaf stage, penoxsulam was applied using a laboratory sprayer (machine model: 3WP-2000, Nanjing Research Institute for Agricultural Mechanization, National Ministry of Agriculture of China, Nanjing, China) equipped with a flat-fan nozzle delivering 280 L ha⁻¹ at 230 kPa. Based on a preliminary experiment (data not shown), penoxsulam was applied at 0, 15, 30, 60, 120, and 240 g ha⁻¹ to E. japonica and at 0, 0.94, 1.88, 3.75, 7.5, and 15 g ha⁻¹ to E. crus-galli. The treated plants were returned to the incubators and cultured as described earlier. The fresh aboveground biomass was determined after 3 wk and expressed as a percentage of the untreated control (Feng et al. Reference Feng, Gao, Zhang, Dong and Li2016; Gao et al. Reference Gao, Yu, Pan, Wu and Dong2017). This experiment was conducted twice using a completely randomized design with four replicates.

ALS Activity Assay In Vitro

Plant materials for subsequent molecular experiments from a sensitive E. crus-galli population (JLGY-2019-S) and the most tolerant population of E. japonica (JHHY-2019-2) were prepared based on the results of the dose–response bioassay with penoxsulam (for convenience, the population name will be used hereafter). The response of ALS to penoxsulam was determined using crude enzyme extracts. The soil type and growth conditions were the same as those described earlier. Seedlings at the 3- to 4-leaf stage from JHHY-2019-2 and JLGY-2019-S were used for in vitro assays of ALS activity as described by Yu et al. (Reference Yu, Friesen, Zhang and Powles2004), with slight modifications as follow: 4 g of leaf blades without the petiole were harvested from each population, powdered in liquid nitrogen, and suspended in 4.5 ml of enzyme extraction buffer (10 mM sodium pyruvate, 1 mM MgCl₂, 1 mM thiamine pyrophosphate, and 10 μM flavin adenine dinucleotide [FAD] in 100 mM potassium phosphate buffer [pH 7.5]) to prepare crude enzyme extracts. The protein concentration extracted from the leaf blades was measured according to Bradford (Reference Bradford1976) using bovine serum albumin (BSA) as a standard. Using enzyme assay buffer (100 mM potassium phosphate buffer [pH 7.5], 200 mM sodium pyruvate, 20 mM MgCl₂, 2 mM thiamine pyrophosphate, 20 μM FAD, and 1 mM dithiothreitol [DTT]), the concentration of the protein extracts was normalized to 0.30 mg ml⁻¹. Each dark reaction contained 100 μl of protein extract and 100 μl of ALS inhibitor, which was achieved using a series of concentrations of penoxsulam at 0.001, 0.01, 0.1, 1, 10, 100, 1,000, and 10,000 μM. A no-herbicide treatment was included for comparison (replacing penoxsulam with potassium phosphate buffer). Acetolactate was formed by incubating the mixtures at 37 C for 60 min. The reaction was stopped by the addition of 8 μl of 6 N H₂SO₄. The mixture was held at 60 C for 30 min to convert acetolactate to acetoin until the addition of 100 μl of 0.55% (w/w) creatine solution and 100 μl of 5.5% (v/v) α-naphthol in 5 N NaOH. Acetoin solution of a certain concentration was made with pure acetoin and H₂O, then an equal gradient dilution was performed, and in order to exclude the influence of creatine and α-naphthol, 100 μl of 0.55% (w/w) creatine solution and 100 μl of 5.5% (v/v) α-naphthol in 5 N NaOH were also added. Under the same total volume conditions as the above reaction solution, an acetoin standard curve was made with the acetoin concentration as the abscissa and the optical density at 530 nm (OD530) value as the ordinate. ALS activity was monitored colorimetrically (530 nm) on a microplate photometer (Thermo Fisher, Shanghai, China, 200000) by measuring acetoin production. According to the concentration of acetoin generated (convert OD530 to acetoin concentration based on the acetoin standard curve), ALS activity at a series of concentrations of penoxsulam was expressed as a percentage of the no-herbicide control treatment. The assay was performed twice using independent enzyme extractions, with three replicates per herbicide concentration.

ALS Gene Cloning and Sequencing

Young shoot tissues obtained from individual plants at the 3- to 4-leaf stage were used for DNA extraction using a Plant Genomic DNA kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. Primers (Table 2) were designed using Primer Premier v. 5.0 to amplify DNA fragments encompassing all previously identified resistance mutation sites in the ALS gene. ALS gene fragments of the susceptible population JLGY-2019-S were amplified on the basis of previous study (Fang et al. Reference Fang, Liu, Zhang, Li and Dong2019a). As information regarding the ALS gene of E. japonica was not available, three pairs of primers intended to amplify regions of the ALS gene in JHHY-2019-2 were designed based on the nucleotide sequences of ALS enzymes from the following species (respective GenBank nucleotide accession numbers included in parentheses): blackgrass (Alopecurus myosuroides Huds.) (AJ437300.2), E. crus-galli (MH013497), E. crus-galli var. crus-galli (LC006061.1), E. crus-galli var. formosensis (LC006063.1), rice barnyardgrass [Echinochloa phyllopogon (Stapf) Koso-Pol.] (AB636580.1), cheatgrass (Bromus tectorum L.) (AF488771), and Italian ryegrass [Lolium perenne L. ssp. multiflorum (Lam.) Husnot] (AF310684) (National Center for Biotechnology Information, Bethesda, MD, http://www.ncbi.nlm.nih.gov).

Table 2. Primers used to amplify the acetolactate synthase (ALS) gene fragments of Echinochloa crus-galli and Eragrostis japonica.

^a Amino acid sequence positions in the ALS fragment refer to the full-length sequence of ALS from Arabidopsis thaliana (GenBank accession no. NM_114714).

A polymerase chain reaction (PCR) was performed as described by Xu et al. (Reference Xu, Zhu, Wang, Li and Dong2013). The PCR products were purified using a TaKaRa MiniBEST agarose gel DNA extraction kit (TaKaRa Biotechnology, Dalian, China) and then cloned into a pMD19-T vector (TaKaRa Biotechnology). Plasmids containing the fragment insertion were bidirectionally sequenced using GenScript Biotechnology (Nanjing, China). Ten plants each of JHHY-2019-2 and JLGY-2019-S were selected for gene cloning. At least 12 transformed clones from each plant were sequenced to obtain ALS gene sequences. The sequences were aligned and compared using the BioEdit Sequence Alignment Editor v. 7.2.5 (Tom Hall, Carlsbad, CA, USA). The Basic Local Alignment Search Tool (BLAST) procedure within the NCBI database was used to verify the accuracy of the obtained sequences.

Determination of ALS Expression by RT-qPCR

Real-time quantitative PCR (RT-qPCR) was used to measure the expression level of ALS gene relative to the housekeeping gene β-actin in JHHY-2019-2 and JLGY-2019-S. Plants were cultivated and treated with 7.5 g ha⁻¹ penoxsulam, as previously described. Leaf tissue (0.1 g per population per time point) was harvested at 0, 1, 3, 5, and 7 d after treatment and flash-frozen in liquid nitrogen. RNA was extracted using an RNA Simple Total RNA Kit (Tiangen Biotech) according to the manufacturer’s instructions. After RNA extraction, cDNAs were synthesized using HiScript II Q RT SuperMix for qPCR (+ gDNA wiper; Vazyme Biotech, Nanjing, China). Based on the ALS nucleotide sequences of JHHY-2019-2 and JLGY-2019-S obtained earlier, RT-qPCR primers (Table 3) were designed at the highly conserved region. The β-actin gene was selected as the endogenous housekeeping gene for RT-qPCR (Fang et al. Reference Fang, Yang, Zhao, Chen and Dong2022; Li et al. Reference Li, Wu, Cai, Wang, Zhao and Wu2013), using its primers (Table 3). All primers were assessed for specific PCR amplification, and no PCR products were observed in the negative controls. RT-qPCR was performed according to a previously reported program (Fang et al. Reference Fang, Zhang, Liu, Yan, Li and Dong2019b). Fold changes in gene expression were calculated using the 2^−ΔΔCt method (Livak and Schmittgen Reference Livak and Schmittgen2001). To exclude the influence of inherent differences between JHHY-2019-2 and JLGY-2019-S, ALS expression was displayed based on the Ct values relative to each population at 0 d (without penoxsulam treatment) (Fang et al. Reference Fang, He, Liu, Li and Dong2020, Reference Fang, Yang, Zhao, Chen and Dong2022). Three biological replicates were used, and the experiments were repeated at least twice.

Table 3. Information for primers used in the real-time quantitative PCR (RT-qPCR) study.

Synergistic Effect of Penoxsulam and Metabolic Inhibitors on Weed Growth

Seeds from JHHY-2019-2 and JLGY-2019-S were cultured as described earlier. At the 3- to 4-leaf stage, weeds were treated with three CytP450 inhibitors (1-aminobenzotriazole [ABT], PBO, and malathion), one GST inhibitor (4-chloro-7-nitro-2,1,3-benzoxadiazole [NBD-Cl]), penoxsulam, ABT with penoxsulam, PBO with penoxsulam, malathion with penoxsulam, and NBD-Cl with penoxsulam. A pretest showed that these metabolic inhibitors had no adverse effects on seedling growth (data not shown). The applied doses and methods for ABT (1,000 g ai ha⁻¹) (Zhang et al. Reference Zhang, Wu, Xu, Gao, Zhang and Dong2017), PBO (4,200 g ai ha⁻¹) (Wang et al. Reference Wang, Li, Lv, Lou and Dong2013), malathion (1,000 g ai ha⁻¹) (Wang et al. Reference Wang, Li, Lv, Lou and Dong2013), and NBD-Cl (270 g ai ha⁻¹) (Ma et al. Reference Ma, Evans and Riechers2016) have been previously reported. Malathion, ABT, and PBO were first applied 1 h before herbicide application, and NBD-Cl was first applied 48 h before herbicide application. Subsequently, penoxsulam was administered to JHHY-2019-2 and JLGY-2019-S at the doses described in the whole-plant dose–response experiment with penoxsulam. Fresh aboveground biomass was determined after 3 wk and expressed as a percentage of the no-herbicide treatment. The experiments were conducted twice using a completely randomized design with four replicates.

Determination of Metabolic Enzymes Activity In Vivo

Based on the results of the dose–response bioassay with metabolic inhibitors, the activities of NADPH-dependent cytochrome P450 reductase toward penoxsulam in JHHY-2019-2 and JLGY-2019-S were determined in vivo according to previous studies (Wang et al. Reference Wang, Li, Lv, Lou and Dong2013; Zimmerlin and Durst Reference Zimmerlin and Durst1990). The seeds were planted in a plastic basin (5-cm radius, 10-cm height) filled with quartz sand, and the basin was wrapped with foil paper to ensure complete darkness, placed in a no-light incubator with a relative humidity of 85% at 25/30 C for 12 h/12 h, watered (no light exposure), and cultured for approximately 10 d. At the 2-leaf stage, a laboratory sprayer equipped with a flat-fan nozzle (machine model: 3WP-2000, Nanjing Research Institute for Agricultural Mechanization), delivering 280 L ha⁻¹ at 230 kPa and 291 mm s⁻¹ with a 30-ml carrier volume, was used to spray the stems and leaves; the dose of penoxsulam sprayed was one-fourth the recommended label rate in the field: 7.5 g ha⁻¹. At 1 d after spraying, the aboveground parts of weeds were cut, 2 g was taken for each treatment, and untreated weeds were used as controls. After harvest, the aboveground tissue was soaked in 10 mM Na₂S₂O₄, dried with absorbent paper, placed in liquid nitrogen, and stored at −80 C until use. The activity of CytP450 declines as the age of seedling tissues increases (Frear et al. Reference Frear, Swanson and Thalacker1991). Consequently, for comparative purposes, all excised shoot tissues needed to be of the same or similar physiological age.

Aboveground tissue was then ground with liquid nitrogen; 0.10 g polyvinylpolypyrrolidone (PVPP) was added before grinding. The finely ground powder was transferred to a new precooled mortar, and 6.0 ml enzyme extraction buffer was added (0.10 M pH 7.5 phosphoric acid buffer, containing 10% glycerol [v/v], 1 g L⁻¹ BSA, 5 mM DTT, 1 mM Li₂CO₃). After being ground evenly, it was kept at 4 C for 5 min and then filtered through gauze. Then, the liquid was transferred into a 10-ml centrifuge tube. After centrifugation at 10,000 × g for 20 min at 4 C, the supernatant was taken and added to a new centrifuge tube. After centrifugation at 100,000 × g for 80 min at 4 C, the supernatant was discarded and the precipitate was retained. The precipitate was resuspended in 1ml buffer (0.1 M pH 8.0 phosphoric acid buffer, containing 10% glycerol and 1.5 mM β-mercaptoethanol) and then stored in a refrigerator at −70 C for later use. All the above operations were carried out under temperatures of 0 to 4 C (Mcfadden et al. Reference Mcfadden, Frear and Mansager1989; Yun et al. Reference Yun, Yogo, Miura, Yamasue and Fischer2005).

The activity of NADPH-dependent cytochrome P450 reductase was determined by a modification of the method used by Feng et al. (Reference Feng, Houseman and Downe1992) and Crankshaw et al. (Reference Crankshaw, Hetnarski and Wilkinson1979). The reaction mix contained 50 μl NADPH (1.5 mg ml⁻¹), 1.5 ml of 0.1 M sodium phosphate buffer (pH 7.8, containing 50 μl cytochrome C [5 mg ml⁻¹] and 50 µl microsomal crude enzyme solution) and was mixed immediately to start the reaction. The reaction without enzyme solution was used as a control. The change value of OD550 at 550 nm was recorded for 180 s at room temperature. The amount of cytochrome C reduced was calculated based on OD550; the millimolar extinction coefficient of 21.1 mmol L⁻¹ cm⁻¹ and the protein content of the enzyme solution were calculated according to Feng et al. (Reference Feng, Houseman and Downe1992). It can be used as a standard to measure the activity of NADPH-dependent cytochrome P450 reductase. The experiments were conducted twice with three replicates per treatment.

Data Analysis

After preliminary analysis, all data were subjected to a t-test analysis using SPSS v. 21.0 (SPSS, Chicago, IL, USA). The results showed no significant differences between assay repetitions (t-test, P > 0.05). The data from the whole-plant dose–response experiment with penoxsulam, ALS activity assay in vitro, and synergistic effect of penoxsulam and metabolic inhibitors on weed growth after preliminary analysis (percentage of the no-herbicide treatment) were then pooled and fitted to the four-parameter nonlinear log-logistic regression model (Equation 1) using SigmaPlot v. 14.0 (Systat Software, Chicago, IL, USA) (Cai et al. Reference Cai, Chen, Wang, Gao, Xiang and Dong2022; Fang et al. Reference Fang, Zhang, Liu, Yan, Li and Dong2019b; Liu et al. Reference Liu, Fang, He, Li and Dong2019; Seefeldt et al. Reference Seefeldt, Jensen and Fuerst1995):

(1) $$y = c + \left( {d - c} \right)/\left\{ {1 + {\rm{exp}}\left[ {b\left( {{\rm{log}}x - {\rm{log}}{x_0}} \right)} \right]} \right\}$$

where y denotes fresh weight of aboveground tissue, expressed as a percentage of the untreated control at herbicide dose x; b is the slope; c is the lower limit; d is the upper limit; and x ₀ is the effective dose of herbicide causing 50% inhibition of fresh weight (GR₅₀) with or without metabolic inhibitors or the herbicide dose causing 50% inhibition of ALS activity (IC₅₀). Relative tolerance indices (RIs) were calculated by dividing the GR₅₀ of the tolerant population by that of the susceptible population. Significant differences in the GR₅₀ values and IC₅₀ values, the expression levels, and the activities of NADPH-dependent cytochrome P450 reductase were also subjected to a t-test analysis using SPSS v. 21.0.