Distribution and Mechanism of Japanese Brome (Bromus japonicus) Resistance to ALS-Inhibiting Herbicides in China

Bromus japonicus is a common monocot weed that occurs in major winter wheat fields in the Huang–Huai–Hai region of China. Pyroxsulam is a highly efficient and safe acetolactate synthase (ALS)-inhibiting herbicide that is widely used to control common weeds in wheat fields. However, B. japonicus populations in China have evolved resistance to pyroxsulam by different mutations in the ALS gene. To understand the resistance distribution, target-site resistance mechanisms, and cross-resistance patterns, 208 B. japonicus populations were collected from eight provinces. In the resistant population screening experiment, 59 populations from six provinces showed different resistance levels to pyroxsulam compared with the susceptible population, of which 17 B. japonicus populations with moderate or high levels of resistance to pyroxsulam were mainly from the Hebei (4), Shandong (4) and Shanxi (9) Provinces. Some resistant populations were selected to investigate the target site-resistance mechanism to the ALS-inhibiting herbicide pyroxsulam. Three pairs of primers were designed to amplify the ALS sequence, which was assembled into the complete ALS sequence with a length of 1932 bp. DNA sequencing of ALS revealed that four different ALS mutations (Pro-197-Ser, Pro-197-Thr, Pro-197-Phe and Asp-376-Glu) were found in 17 moderately or highly resistant populations. Subsequently, five resistant populations, QM21-41 with Pro-197-Ser, QM20-8 with Pro-197-Thr and Pro-197-Phe, and QM21-72, QM21-76 and QM21-79 with Asp-376-Glu mutations in ALS genes, were selected to characterize their cross-resistance patterns to ALS inhibitors. The QM21-41, QM20-8, QM21-72, QM21-76 and QM21-79 populations showed broad-spectrum cross-resistance to pyroxsulam, mesosulfuron–methyl and flucarbazone–sodium. This study is the first to report evolving cross-resistance to ALS-inhibiting herbicides due to Pro-197-Phe mutations in B. japonicus.


Introduction
Bromus japonicus is an annual weed of the Poaceae family, a troublesome weed for winter wheat (Triticum aestivum) and widely distributed in the Huang-Huai-Hai Plain of China [1].B. japonicus usually germinates in early autumn and overwinters in the field as "rosettes" and restores vigorous growth in the next spring with the onset of warmer weather.It begins to flower in early May, and then seeds mature in late June or early July [1].A plant of B. japonicus growing normally can produce 8.2 tillers and 1885 seeds on average [2].B. japonicus and wheat grow at the same time and are difficult to distinguish.B. japonicus has stronger fecundity and tiller ability and may reduce yield by at least 30% in wheat fields seriously infected by B. japonicus [1].Acetolactate synthase (ALS)-inhibiting herbicides have been used for more than 15 years to control B. japonicus in wheat fields.Recently, there have been four reports of resistance to ALS inhibitors in B. japonicus [3][4][5][6].
Plants 2024, 13, 1139 2 of 12 ALS inhibitors have been commonly applied to control weeds in wheat fields because they provide broad-spectrum effective weed control and safety to crops combined with low environmental toxicity [7].The target of these herbicides is ALS, which can control weeds by inhibiting the biosynthesis of the branched-chain amino acids leucine, isoleucine and valine, ultimately resulting in weed death [8].The typical symptoms of herbicide after the application of ALS inhibitors are as follows: the top buds or new leaves of the plant die, the leaves lose their green color or turn purple, the internodes are shortened and finally the whole plant dies [9].According to their different chemical structures, ALS inhibitors mainly include five classes: sulfonylureas (SU), imidazolinones (IMI), triazolopyrimidines (TP), pyrimidinylthiobenzoates (PTB) and sulfonylamino-carbonyl-triazolinones (SCT).Unfortunately, following the frequent and extensive use of ALS inhibitors in the past few decades, 170 weed species in locations worldwide have been reported to be resistant to these herbicides [10].
Recently, ALS inhibitor-resistant B. japonicus populations has been found in Hebei Province, Shandong Province and Tianjin municipality, and the Pro-197 and Asp-376 mutations in the ALS gene are associated with resistance to ALS-inhibiting herbicides in B. japonicus [3][4][5][6].In China, control failures of B. japonicus have recently been observed in wheat fields where ALS-inhibiting herbicides are constantly used.In the present study, Plants 2024, 13, 1139 3 of 12 208 B. japonicus populations were collected from the Anhui, Hebei, Henan, Hubei, Jiangsu, Shandong, Shanxi and Shaanxi Provinces of China, where wheat is the main grain crop.This study aimed (1) to monitor the resistance situation and determine its distribution in B. japonicus in eight provinces of China; (2) to obtain the complete sequence of the ALS gene and identify target site mutations of 17 pyroxsulam-resistant B. japonicus populations; and (3) to evaluate the cross-resistance for different mutations based on whole-plant response assays with three ALS inhibitors in B. japonicus.

Resistant Population Screening and Distribution
The level and geographical distribution of 208 B. japonicus populations' resistance to pyroxsulam are shown in Figure 1 (levels and geographical distribution of pyroxsulam resistance in Bromus japonicus populations in China).The susceptible population preserved and identified by the laboratory was selected as the control, and the GR 50 value of the susceptible population was 3.0 g a.i.ha −1 .Resistant population screening assays indicated that 59 of 208 populations showed different resistance levels to pyroxsulam, and 149 populations were sensitive to pyroxsulam.Among the 59 resistant populations, 42 had low resistance levels (1 < RI ≤ 3), 9 had moderate resistance levels (3 < RI ≤ 10) and 8 had high resistance levels (RI > 10) to pyroxsulam.A total of 149 susceptible populations had no surviving plants under the recommended dose of pyroxsulam, which was 12 g a.i.ha −1 in the field.The plant survival rate of the 42 low-resistance populations was about 90%, but the plant growth was significantly inhibited at 12 g a.i.ha −1 , and there were no surviving plants at 24 g a.i.ha −1 .The plant survival rate of the nine moderately resistant populations was about 90%, but the plant growth was significantly inhibited at 24 g a.i.ha −1 , and the plant survival rate was about 40% at 96 g a.i.ha −1 .The plant survival rate of the eight highly resistant populations was about 90%, but the plant growth was significantly inhibited at 96 g a.i.ha −1 , and the plant survival rate was about 30% at 192 g a.i.ha −1 .
tion in B. japonicus in eight provinces of China; (2) to obtain the complete sequence of the ALS gene and identify target site mutations of 17 pyroxsulam-resistant B. japonicus populations; and (3) to evaluate the cross-resistance for different mutations based on wholeplant response assays with three ALS inhibitors in B. japonicus.

Resistant Population Screening and Distribution
The level and geographical distribution of 208 B. japonicus populations' resistance to pyroxsulam are shown in Figure 1 (levels and geographical distribution of pyroxsulam resistance in Bromus japonicus populations in China).The susceptible population preserved and identified by the laboratory was selected as the control, and the GR50 value of the susceptible population was 3.0 g a.i.ha −1 .Resistant population screening assays indicated that 59 of 208 populations showed different resistance levels to pyroxsulam, and 149 populations were sensitive to pyroxsulam.Among the 59 resistant populations, 42 had low resistance levels (1 < RI ≤ 3), 9 had moderate resistance levels (3 < RI ≤ 10) and 8 had high resistance levels (RI > 10) to pyroxsulam.A total of 149 susceptible populations had no surviving plants under the recommended dose of pyroxsulam, which was 12 g a.i.ha −1 in the field.The plant survival rate of the 42 low-resistance populations was about 90%, but the plant growth was significantly inhibited at 12 g a.i.ha −1 , and there were no surviving plants at 24 g a.i.ha −1 .The plant survival rate of the nine moderately resistant populations was about 90%, but the plant growth was significantly inhibited at 24 g a.i.ha −1 , and the plant survival rate was about 40% at 96 g a.i.ha −1 .The plant survival rate of the eight highly resistant populations was about 90%, but the plant growth was significantly inhibited at 96 g a.i.ha −1 , and the plant survival rate was about 30% at 192 g a.i.ha −1 .
The pyroxsulam GR50 values of 208 Bromus japonicus populations from different provinces are shown in Figure 2. A total of 99 populations were collected from Anhui, Henan, Hubei, Jiangsu and Shaanxi Provinces, and the results showed that 87.9% of the populations were at the sensitive level and 12.1% of the populations were at the lowresistance level.The moderately or highly resistant populations were mainly distributed in Hebei, Shandong and Shanxi Provinces, accounting for 15.6% of the total samples in the three provinces.There were nine moderately resistant populations in Shanxi Province, four highly resistant populations in Hebei Province and four highly resistant populations in Shandong Province.The pyroxsulam GR 50 values of 208 Bromus japonicus populations from different provinces are shown in Figure 2. A total of 99 populations were collected from Anhui, Henan, Hubei, Jiangsu and Shaanxi Provinces, and the results showed that 87.9% of the populations were at the sensitive level and 12.1% of the populations were at the lowresistance level.The moderately or highly resistant populations were mainly distributed in Hebei, Shandong and Shanxi Provinces, accounting for 15.6% of the total samples in the three provinces.There were nine moderately resistant populations in Shanxi Province, four highly resistant populations in Hebei Province and four highly resistant populations in Shandong Province.

Amplification and Sequencing of the ALS Gene Fragment
To determine whether the presence of any known target-site mutation in B. japonicus confers resistance to ALS inhibitors, targeted sequence results were acquired from the amplification products obtained from three pairs of primers.In this study, three fragments of the amplified ALS gene, with lengths of 593 bp, 1440 bp and 598 bp, were assembled into the complete ALS sequence with a length of 1932 bp.

Amplification and Sequencing of the ALS Gene Fragment
To determine whether the presence of any known target-site mutation in B. japonicus confers resistance to ALS inhibitors, targeted sequence results were acquired from the amplification products obtained from three pairs of primers.In this study, three fragments of the amplified ALS gene, with lengths of 593 bp, 1440 bp and 598 bp, were assembled into the complete ALS sequence with a length of 1932 bp.

Discussion
Three ALS inhibitors, pyroxsulam, mesosulfuron-methyl and flucarbazone-sodium, are important herbicides that can effectively control some weeds in the Poaceae family in wheat fields, including Bromus japonicus, Alopecurus aequalis and Alopecurus japonicus [28].The three ALS inhibitors in this study have been used in China for more than 10 years, which has resulted in the rapid evolution of herbicide resistance [29].Twenty-six species of weeds, including Lolium perenne ssp.multiflorum, ripgut brome (Bromus diandrus), poverty brome (Bromus sterilis) and silky windgrass (Apera spica-venti), have been found to be resistant to pyroxsulam in countries such as the United States, Australia, France and Germany [10].In the resistant population screening experiment, four, one and three populations with high resistance to pyroxsulam were from Shijiazhuang City, Hebei Province; Binzhou City, Shandong Province; and Zibo City, Shandong Province, respectively.Nine populations with moderate resistance to pyroxsulam were from Linfen City, Shanxi Province.B. japonicus populations moderately and highly resistant to pyroxsulam were detected mainly in Shanxi, Hebei and Shandong Provinces, which may be closely related to the application history of ALS inhibitors in these areas.In Hubei and Jiangsu Provinces, the number of the collected populations was small, and these populations were all sensitive to pyroxsulam, which could not explain the occurrence of

Discussion
Three ALS inhibitors, pyroxsulam, mesosulfuron-methyl and flucarbazone-sodium, are important herbicides that can effectively control some weeds in the Poaceae family in wheat fields, including Bromus japonicus, Alopecurus aequalis and Alopecurus japonicus [28].The three ALS inhibitors in this study have been used in China for more than 10 years, which has resulted in the rapid evolution of herbicide resistance [29].Twenty-six species of weeds, including Lolium perenne ssp.multiflorum, ripgut brome (Bromus diandrus), poverty brome (Bromus sterilis) and silky windgrass (Apera spica-venti), have been found to be resistant to pyroxsulam in countries such as the United States, Australia, France and Germany [10].In the resistant population screening experiment, four, one and three populations with high resistance to pyroxsulam were from Shijiazhuang City, Hebei Province; Binzhou City, Shandong Province; and Zibo City, Shandong Province, respectively.Nine populations with moderate resistance to pyroxsulam were from Linfen City, Shanxi Province.B. japonicus populations moderately and highly resistant to pyroxsulam were detected mainly in Shanxi, Hebei and Shandong Provinces, which may be closely related to the application history of ALS inhibitors in these areas.In Hubei and Jiangsu Provinces, the number of the collected populations was small, and these populations were all sensitive to pyroxsulam, which could not explain the occurrence of B. japonicus resistance in these two provinces.The B. japonicus populations collected for this study were from wheat fields across eight provinces, where farmers commonly use ALS inhibitor herbicides to control weeds.Notably, in some wheat fields with a higher prevalence of resistant populations in Hebei, Shandong and Shanxi Provinces, the use of ALS inhibitors has exceeded a decade.
Target-site mutations are often identified as the most common mechanism of resistance to ALS inhibitor herbicides in many weeds.For example, Pro-197 and Asp-376 mutations of the ALS gene conferring resistance to ALS inhibitors were found in many weed species, including Raphanus raphanistrum [25], Galium aparine [14], Descurainia sophia [30], Cyperus difformis [31] and Monochoria vaginalis [32].To date, four mutations (Pro-197-Ser, Pro-197-Thr, Pro-197-Arg and Asp-376-Glu) have been reported as being related to resistance to ALS inhibitor herbicides in B. japonicus [3][4][5][6].In the present study, four amino acid substitutions in the ALS gene from 17 resistant populations, including Pro-197-Ser/Thr/Phe and Asp-376-Glu, were identified by molecular analysis.Compared with the often reported Pro-197-Ser, Pro-197-Thr and Asp-376-Glu mutations, the Pro-197-Phe mutations reported here in B. japonicus have been reported in only two other weed species, Sisymbrium orientale [33] and Lactuca serriola [34].This is the first report of Pro-197-Phe mutations in B. japonicus.This study reveals that the resistance mechanism of 17 B. japonicus populations from Hebei, Shandong and Shanxi to pyroxsulam is due to mutations in the ALS gene, which is the gene targeted by ALS inhibitor herbicides.This finding suggests to local farmers that using ALS inhibitor herbicides may be ineffective in controlling B. japonicus.
In recent years, there have been increasing reports on the mechanisms of herbicide resistance in Bromus spp.Yanniccari et al. found that the resistance mechanism of Bromus catharticus to glyphosate is due to reduced absorption and translocation of the herbicide [35].Sen et al. discovered that the resistance mechanism of Bromus sterilis to pyroxsulam includes overexpression of the ALS gene and enhanced metabolic detoxification mediated by P450 enzymes [36].Owen et al. reported that the resistance mechanism of Bromus rigidus to ALS inhibitors is due to its own enhanced metabolic detoxification of the herbicide [37].Kumar and Jha found that the resistance mechanisms of Bromus tectorum with Ser-653-Asn have evolved resistance to ALS inhibitors [13].
To date, 27 weeds with the Pro-197-Ser mutation have evolved resistance to ALS inhibitors.Furthermore, the cross-resistance to ALS inhibitors in many weeds with the Pro-197-Ser point mutation has been previously tested, such as in Descurainia sophia [38], Sagittaria trifolia [39], Galium aparine [14] and Ludwigia prostrata [26].The level of resistance to ALS inhibitors may be different in different weeds with the same ALS mutation [40].Beckmannia syzigachne with the Pro-197-Ser mutation exhibited high resistance to all five ALS-inhibiting herbicides, while the same mutation in Alopecurus japonicus exhibited high resistance to SUs, TPs and SCTs but sensitivity to IMIs [41,42].In this study, whole-plant response experiments showed that QM21-41 with Pro-197-Ser had evolved high levels of resistance to pyroxsulam (TP), mesosulfuron-methyl (SU) and flucarbazone-sodium (SCT).The Pro-197-Thr mutation was first reported in Kochia scoparia in 1990 [43].To date, 14 weeds have evolved resistance to ALS inhibitors with the Pro-197-Thr mutation [10].Zhao et al. found that Pro-197-Thr in Alopecurus japonicus conferred low resistance levels to TPs, moderate resistance levels to SCTs and high resistance levels to SUs [42].In the present study, however, the whole-plant response experiments revealed that QM20-8 with Pro-197-Thr/Phe was highly resistant to TPs, SUs and SCTs.Weeds with the Asp-376-Glu mutation commonly exhibited cross-resistance to ALS inhibitors.Cyperus difformis with the Asp-376-Glu mutation showed moderate resistance to TP, SU and IMI [31].Raphanus raphanistrum with the Asp-376-Glu mutation showed high resistance to TP and SU herbicides but no resistance to imazapyr (IMI) [25].Li et al. reported that the Asp-376-Glu mutation was characterized in B. japonicus as conferring high-level resistance to pyroxsulam (TP), mesosulfuron-methyl (SU) and flucarbazone-sodium (SCT) [4].In this study, QM21-72, QM21-76 and QM21-79 with Asp-376-Glu mutation were resistant to pyroxsulam (TP), mesosulfuron-methyl (SU) and flucarbazone-sodium (SCT).The resistance level of the populations for Pro-197 (25.9-59.5)mutations to flucarbazone-sodium were significantly higher than that of the populations for Asp-376 (2.9-5.9).The cross-resistance pattern is complex and depends on many factors, including weed species, mutation types and specific ALS inhibitors [44].Although in many cases, the application of ALS inhibitors is still the most effective method for controlling B. japonicus, it will be necessary to implement integrated weed management to reduce the risk of resistant B. japonicus development.

Resistant Populations Screening
Mature seeds of a total of 208 B. japonicus populations from the provinces of Anhui, Hebei, Henan, Hubei, Jiangsu, Shandong, Shanxi and Shaanxi were collected in wheat fields from 2020 to 2022.A total of 208 B. japonicus populations from eight provinces were first examined to reveal the occurrence region of resistant B. japonicus populations by a whole-plant bioassay test.
The seeds were soaked in water for 48 h before sowing.Seeds of 208 populations were sown into 9 cm × 9 cm × 11 cm plastic pots containing seedling matrix.A total of twenty to 30 seeds were sown evenly in each pot and covered with a 1 cm thick layer of fine soil.These pots were randomly placed in a greenhouse with natural light and temperature conditions, where they were watered regularly and fertilized as needed.The seedlings of B. japonicus were reduced to 15 plants with uniform growth and even distribution per pot before herbicide treatment.
In the resistant population screening experiment, pyroxsulam (recommended field dose is 12 g a.i.ha −1 ) was sprayed with 0, 6, 12, 24, 48, 96 and 192 g a.i.ha −1 on 208 B. japonicus populations.Two replicate pots were made for each herbicide treatment.Pyroxsulam was applied by foliar application when the seedlings had reached the threeleaf stage.A moving-nozzle cabinet sprayer equipped with a TeeJet ® XR8002 flat-fan nozzle (Compressed Air Cabinet Sprayer ASS-4, Beijing Research Center for Information Technology in Agriculture, Beijing, China) was used, which delivered 450 L ha −1 at a pressure of 0.275 MPa.
After 21 days of herbicide treatment, the survival rate and fresh weight of the aboveground parts of each treatment were determined.The GR 50 (50% growth reduction in the aboveground biomass) was calculated by a nonlinear log-logistic regression model using SigmaPlot version 12.5.
C is the lower limit, D is the upper limit and b is the slope of the curve.Y is correspondingly expressed as the percentage of the control at herbicide dose X.The resistance index (RI) was calculated by dividing the GR 50 value of the resistant biotype by that of the susceptible biotype.According to the RI value, the resistance level was divided into four grades: sensitive level: RI ≤ 1; low resistance level: 1 < RI ≤ 3; moderate resistance level: 3 < RI ≤ 10; and high resistance level: RI > 10.

Amplification and Sequencing of the ALS Gene Fragment
The plant materials used for amplifying the ALS gene were obtained from the plants in the resistant population screening test.The leaf tissues of the susceptible population were taken from untreated plants, while the leaf tissues of the resistant population plants were obtained from plants that survived after treatment with a two-fold higher dose of pyroxsulam than the recommended dose in the field.Fifteen selected plants from each population (17 resistant and 1 susceptible) were used for DNA extraction.The results of preliminary screening of resistance showed that the plant survival rates of the 17 resistant populations were 100% after treatment with two times the recommended dose of pyroxsulam in the field.Approximately 100 mg of leaf tissue was collected from each plant and stored at −80 • C. Total genomic DNA was extracted from the leaf tissue of each plant using the DNAsecure Plant kit (Tiangen Biotech, Beijing, China) according to the manufacturer's protocol.DNA was electrophoresed on 1.0% agarose gels to check the quality of the extraction.
Three pairs of primers (ALS-F1/ALS-R1, ALS-F2/ALS-R2 and ALS-F3/ALS-R3) (Table 3) were designed based on the ALS genes of Bromus tectorum (GenBank MK492423.1)and Hordeum vulgare (GenBank LOC123401036) to amplify the complete ALS sequence of B. japonicus.Primers were designed using Primer Premier 5.0 software and were synthe-Plants 2024, 13, 1139 9 of 12 sized by BGI Tech (Beijing, China).Polymerase chain reactions (PCRs) were conducted in a final volume of 25 µL and contained 1.25 µL of genomic DNA, 12.5 µL of 2 × GC Buffer (Mg 2+ plus), 1 µL of each primer (10 µM), 4 µL of dNTPs (2.5 mM), 0.25 µL of LA-Taq DNA polymerase (5 U µL −1 ) and 5 µL of ddH 2 O (TaKaRa Biotechnology, Dalian, China).Amplification was performed using initial denaturing for 10 min at 95  Mesosulfuron-methyl (sulfonylurea [SU] herbicide), pyroxsulam (triazolopyrimidine [TP] herbicide) and flucarbazone-Na (sulfonylamino-carbonyl-triazolinone [SCT] herbicide), representing three different chemical families of ALS-inhibiting herbicides, were selected for the cross-resistance evaluation.Seeds of QM21-14, QM21-41, QM20-8, QM21-72, QM21-76 and QM21-79 were planted as described above.Seedlings were thinned to ten plants per pot with uniform growth and even distributions before herbicide treatment.Three replicate pots were made for each herbicide treatment (30 plants per dose).At the 3-leaf stage of seedlings, the herbicides were sprayed, and the doses are shown in (Table 4).The aboveground shoot tissue of the seedlings in each pot was harvested 21 days after treatment (DAT), placed in envelope paper bags and dried at 70 • C for 72 h, and the dry weight was measured.The GR 50 was calculated as above.The resistance index (RI) was calculated by dividing the GR 50 value of the resistant biotype by that of the susceptible biotype (QM21-14).

Conclusions
In China, 59 populations of B. japonicus with resistance to pyroxsulam were identified across six provinces, among which 17 populations with higher levels of resistance were mainly distributed in Hebei, Shandong and Shanxi Provinces.Within these 17 resistant populations, four different mutations of the ALS gene were detected.The patterns of cross-resistance to ALS inhibitors varied among populations with different mutations.

Figure 1 .
Figure 1.Levels and geographical distribution of pyroxsulam resistance in Bromus japonicus populations in China.

Figure 1 .
Figure 1.Levels and geographical distribution of pyroxsulam resistance in Bromus japonicus populations in China.

Table 1 .
Collection site RI values and the mutation types of ALS gene in Bromus japonicus populations.

Table 1 .
Collection site RI values and the mutation types of ALS gene in Bromus japonicus populations.
a , The RI value of the population was derived from the results of the resistance screening test.b , no mutation.

Table 2 .
GR 50 and RI values of different B. japonicus populations with respect to three ALS inhibitors.
a GR 50 , herbicide dose causing 50% growth reduction in the dry weight compared to untreated control.b SE, standard errors.c RI, GR 50 (R)/GR 50 (S).
• C, followed by 35 cycles consisting of 95 • C for 30 s, 53-58 • C for 30 s, 72 • C for 1 min and 72 • C for 10 min for a final extension.Amplified PCR products were detected by 1% agarose gel electrophoresis and sequenced by Sangon Biotech.The obtained sequencing chromatogram results were aligned and analyzed with DNAMAN version 5.2.2 software (Lynnon LLC, San Ramon, CA, USA).

Table 3 .
Primer pairs designed for the amplification of the complete ALS gene from Bromus japonicus.

Table 4 .
Details of the herbicides used for the cross-resistance whole-plant assays.