Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is increasingly infesting imidazolinone-tolerant (IMI-T) rice (Oryza sativa L.) fields in China, and E. crus-galli imazamox resistance has become the major concern for weed management in IMI-T rice fields. In this study, the susceptible population JLGY-3 (S) and the suspected resistant population JHXY-2 (R) collected from IMI-T rice fields were used as research subjects. When treated with imazamox, the JHXY-2 (R) population showed a high level of herbicide resistance with a resistance index of 31.2. JHXY-2 (R) was cross-resistant to all five acetolactate synthase (ALS) inhibitors from different chemical families, but sensitive to herbicides inhibiting acetyl-CoA carboxylase. To understand the reason why JHXY-2 (R) was resistant to imazamox, we performed experiments to characterize potential target-site resistance (TSR) and non–target site resistance (NTSR) mechanisms. A Trp-574-Leu amino acid mutation in ALS and low imazamox ALS sensitivity were the main mechanisms underlying imazamox resistance in this JHXY-2 (R) population. There was no significant difference in ALS gene expression and ALS protein abundance between R and S populations. High-performance liquid chromatography–tandem mass spectrometry analysis showed enhanced metabolism of imazamox in JHXY-2 (R), which was in contrast to the results of pretreatment with a metabolic enzyme inhibitor. Treatments with cytochrome P450 monooxygenase/glutathione S-transferase (P450/GST) inhibitors did not alter the resistance level of JHXY-2 (R) against imazamox. Transcriptome sequencing showed that there was almost no significant difference in the expression of P450 and GST metabolic enzyme genes between R and S populations, and only GSTU1 showed a significant upregulation in the R population, further clarifying the NTSR mechanism of JHXY-2 (R). In conclusion, amino acid mutation and higher enzyme activity of ALS are the main causes of imazamox resistance in JHXY-2 (R). However, given the differences in imazamox residues in the leaves of E. crus-galli, there may still be undetectable NTSR mechanisms that are causing imazamox resistance in the R population.

Isoproturon phytotoxicity to wheat (Triticum aestivum L.) is a worry for many farmers in chemical control of weeds in wheat fields, especially in subzero weather conditions. Iron chlorin e6 (ICe6), a new plant growth regulator, has been reported to enhance crop stress resistance to alleviate damage caused by stress; however, it is not clear whether ICe6 has an alleviative effect on isoproturon phytotoxicity to wheat. We determined the alleviative effect of ICe6 on isoproturon phytotoxicity to wheat, and 0.018 g ai ha−1 was the optimal dose. Meanwhile, we also studied the photosynthetic pigment content, photosynthetic parameters, oxidative stress indicators, and antioxidant enzyme activity of wheat treated with the three different treatments. We found that the photosynthetic pigment content, antioxidant enzyme activity, and photosynthesis of wheat damaged by isoproturon were significantly lower than those of the control, and the hydrogen peroxide (H2O2) and malondialdehyde (MDA) content increased. These results indicate that isoproturon stress significantly weakened the photosynthetic and antioxidant capacity of wheat. The photosynthetic pigment content, photosynthetic parameters (excluding intercellular CO2 concentration), and antioxidant enzyme activity of isoproturon+ICe6– treated wheat were significantly higher than those of isoproturon-treated wheat. The H2O2 and MDA content was significantly lower than that of isoproturon-treated wheat. These results indicate that ICe6 treatment maintained the photosynthetic pigment content of wheat and relatively improved photosynthetic capacity, allowing photosynthesis to proceed normally. ICe6 treatment also limits the decrease in antioxidant enzyme activity, effectively clearing excess reactive oxygen species and ultimately alleviating membrane lipid peroxidation damage. In summary, ICe6 not only enhances stress resistance and increases yield in crops such as soybean [Glycine max (L.) Merr.] and canola (Brassica napus L.), but also has an alleviating effect on the isoproturon phytotoxicity to wheat, which is manifested by the improvement of photosynthetic and antioxidant abilities, ultimately leading to an increase in wheat shoot height and shoot fresh weight.

The identification of herbicide tolerance is essential for effective chemical weed control. According to whole-plant dose–response assays, none of 29 pond lovegrass [Eragrostis japonica (Thunb.) Trin.] populations were sensitive to penoxsulam. The effective dose values of penoxsulam causing 50% inhibition of fresh weight (GR50: 105.14 to 148.78 g ai ha−1) in E. japonica populations were much higher than the label rate of penoxsulam (15 to 30 g ai ha−1) in the field. This confirmed that E. japonica was tolerant to penoxsulam. Eragrostis japonica populations showed 52.83- to 74.76-fold higher tolerance to penoxsulam than susceptible barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.]. The mechanisms of tolerance to penoxsulam in E. japonica were also identified. In vitro activity assays revealed that the penoxsulam concentration required to inhibit 50% of the acetolactate synthase (ALS) activity (IC50) was 12.27-fold higher in E. japonica than in E. crus-galli. However, differences in the ALS gene, previously found to endow target-site resistance in weeds, were not detected in the sequences obtained. Additionally, the expression level of genes encoding ALS in E. japonica was approximately 2-fold higher than in E. crus-galli after penoxsulam treatment. Furthermore, penoxsulam tolerance can be significantly reversed by three cytochrome P450 monooxygenase (CytP450) inhibitors (1-aminobenzotriazole, piperonyl butoxide, and malathion), and the activity of NADPH-dependent cytochrome P450 reductase toward penoxsulam in E. japonica increased significantly (approximately 7-fold higher) compared with that of treated E. crus-galli. Taken together, these results indicate that lower ALS sensitivity, relatively higher ALS expression levels, and stronger metabolism of CytP450s combined to bring about penoxsulam tolerance in E. japonica.

Resistance to penoxsulam among barnyardgrass populations is prevalent in rice fields in China. Seeds of penoxsulam-resistant (AXXZ-2) and penoxsulam-susceptible (JLGY-3) barnyardgrass populations, as well as the seeds of two rice varieties, including Wuyungeng32 (WY) and Liangyou669 (LY), were planted in plastic pots and then treated with a rate titration of acetochlor, anilofos, butachlor, clomazone, oxadiazon, pendimethalin, pretilachlor, pyraclonil, or thiobencarb. The two barnyardgrass populations exhibited similar susceptibility to acetochlor, anilofos, butachlor, oxadiazon, pretilachlor, or pyraclonil. However, the susceptibility differed between the barnyardgrass populations in response to clomazone, pendimethalin, and thiobencarb. For AXXZ-2, herbicide rates that caused 50% reduction in shoot biomass from the nontreated control (GR50) were 179, >800, and 1,798 g ha−1 for clomazone, pendimethalin, and thiobencarb, respectively; whereas JLGY-3 GR50 values were 61, 166, and 552 g ha−1, respectively. Both rice varieties demonstrated excellent tolerance to acetochlor, butachlor, oxadiazon, pretilachlor, and thiobencarb. However, substantial rice damage was observed when anilofos and clomazone were used. Anilofos at 352 g ha−1 and clomazone at 448 g ha−1 reduced rice shoot biomass by 41% and 50% from the nontreated, respectively. Averaged across herbicide rates, clomazone use resulted in a reduction in rice shoot biomass from that of the nontreated control by 52% and 34% for WY and LY, respectively; and pendimethalin use resulted in a reduction in rice shoot biomass from the nontreated control by 25% and 9% for WY and LY, respectively.

Late watergrass [Echinochloa phyllopogon (Stapf) Koso-Pol.] is one of the most persistent weeds in rice fields and shows resistance to some acetolactate synthase (ALS)-inhibiting herbicides, such as penoxsulam. Previous studies of E. phyllopogon’s herbicide resistance have focused on non–target site resistance mechanisms. In this study, E. phyllopogon populations from Heilong Jiang Province, China, that were possibly resistant to penoxsulam were used to identify the target site–based mechanisms of resistance. Population HSRH-520 showed a 25.4-fold higher resistance to penoxsulam than the sensitive population, HSRH-538. HSRH-520 was resistant to other ALS inhibitors, with resistance indexes ranging from 17.1 to 166. Target-gene sequence analysis revealed two different ALS genes in E. phyllopogon; a Pro-197-Ser substitution occurred in the ALS-2 gene of HSRH-520. In vitro activity assays revealed that the penoxsulam concentrations required to inhibit 50% of the ALS activity were 13.7 times higher in HSRH-520 than in HSRH-538. Molecular-docking tests showed that the Pro-197-Ser mutation reduced the binding affinity between ALS and ALS inhibitors belonging to the triazolopyrimidine, sulfonylaminocarbonyltriazolinone, and sulfonylurea families, and there were almost no effects on binding affinity when the ALS inhibitors were of the pyrimidinylthiobenzoate and imidazolinone families. Overall, the results indicated and verified that the Pro-197-Ser mutation leads to increased ALS activity by reducing the binding affinity of the inhibitor and ALS. This is the first report on the Pro-197-Ser mutation in the complete ALS gene of E. phyllopogon and will aid future research of target site–based resistance mechanisms of E. phyllopogon to ALS inhibitors.

Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is acknowledged to be the most troublesome weed in rice fields in Anhui and Jiangsu provinces of China. It cannot be effectively controlled using certain acetolactate synthase (ALS)-inhibiting herbicides, including penoxsulam. Echinochloa crus-galli samples with suspected resistance to penoxsulam were collected to identify the target site–based mechanism underlying this resistance. Populations AXXZ-2 and JNRG-2 showed 33- and 7.3-fold resistance to penoxsulam, respectively, compared with the susceptible JLGY-3 population. Cross-resistance to other ALS inhibitors was reported in AXXZ-2 but not in JNRG-2, and occasionally showed higher sensitivity than JLGY-3. In vitro ALS activity assays revealed that penoxsulam concentrations required to inhibit 50% of ALS activity were 11 and 5.2 times greater in AXXZ-2 and JNRG-2, respectively, than in JLGY-3. DNA and predicted amino acid sequence analyses of ALS revealed Ala-205-Val and Ala-122-Gly substitutions in AXXZ-2 and JNRG-2, respectively. Our results indicate that these substitutions in ALS are at least partially responsible for resistance to penoxsulam.

Italian ryegrass has invaded wheat fields in China and is becoming a predominant, troublesome weed. Fenoxaprop-P-ethyl has been widely used for weed control on Chinese farms since the 1990s. However, overuse has led to fenoxaprop-P-ethyl resistance in Italian ryegrass in Chinese wheat fields. In this study, we identified a putative fenoxaprop-P-ethyl–resistant population of Italian ryegrass, HZYC-6, from Henan province, China. Mutations involving Asp-2078-Gly and Ile-1781-Leu substitutions were identified in the carboxyl-transferase domain of acetyl-coenzyme A carboxylase in this population, and these mutations are the likely cause of the target site–based resistance to fenoxaprop-P-ethyl. In addition, we identified cytochrome P450–mediated metabolism of herbicides (non–target site based resistance) in the HZYC-6 population, indicating that multiple mechanisms of resistance may be segregating in this population. Furthermore, HZYC-6 was also highly resistant to haloxyfop-R-methyl and quizalofop-P-ethyl, moderately resistant to clodinafop-propargyl and sethoxydim, and had low resistance to clethodim and pinoxaden.

Herbicide-resistant weeds pose a considerable threat to agriculture, but their resistance mechanisms are poorly understood. Differential gene expression analysis of a weed subjected to herbicide treatment is a key step toward more mechanistic studies. Such an analysis, often involving quantitative real-time PCR (qPCR), requires suitable reference genes as internal controls. In this study, we identified optimal reference genes in the noxious weed, Japanese foxtail. This weed has evolved resistance to acetyl-coenzyme A carboxylase (ACCase) inhibitors. We analyzed the stability of eight commonly used candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]; ubiquitin [UBQ]; capsine phosphatase [CAP]; beta-tubulin [TUB]; eukaryotic initiation factor 4a [EIF4A]; elongation factor-1 alpha [EF1]; 18S ribosomal RNA [18S]; 25S ribosomal RNA [25S]) from root, stem, and leaf tissue of plants that were either resistant or sensitive to ACCase inhibitors, with or without herbicide stress, using qPCR. The results were further ranked and analyzed using geNorm, NormFinder, and BestKeeper software. These analyses identified EF1 and UBQ in roots, EF1, TUB, CAP, and 18S in stems, and EF1, GAPDH, and 18S in leaves as suitable references for qPCR normalization. We have identified a set of reference genes that can be used to study herbicide resistance mechanisms in Japanese foxtail.

Japanese foxtail is a grass weed in eastern China. This weed is controlled by fenoxaprop-P-ethyl, one of the most common acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. Some Japanese foxtail populations have developed resistance to fenoxaprop-P-ethyl, owing to target-site mutations (amino acid substitutions) located within the carboxyl transferase domain of ACCase. In the present study, three mutations were detected in three fenoxaprop-P-ethyl–resistant Japanese foxtail populations: Ile-1781-Leu in JCJT-2, Ile-2041-Asn in JZJR-1, and Asp-2078-Gly in JCWJ-3. Two copies of ACCase (Acc1-1 and Acc1-2) were identified, but mutations were detected only in Acc1-1. The derived cleaved amplified polymorphic sequence (dCAPS) method detected these mutations successfully in Japanese foxtail. The mutation frequencies in JCJT-2, JZJR-1, and JCWJ-3 were approximately 98%, 92%, and 87%, respectively. Different cross-resistance patterns to ACCase inhibitors were found in the three resistant populations. JCJT-2 (Ile-1781-Leu) and JZJR-1 (Ile-2041-Asn) showed cross-resistance to haloxyfop-R-methyl, clodinafop-propargyl, and pinoxaden, but were susceptible to clethodim. JCWJ-3 (Asp-2078-Gly) showed cross-resistance to all tested ACCase-inhibiting herbicides.

Keng stiffgrass is a serious farmland grass weed distributed globally in winter wheat fields and rice–wheat double-cropping areas. The intensive use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has led to the evolution of resistance in a growing number of grass weeds. In this study, whole-plant pot bioassay experiments were conducted to establish that a Keng stiffgrass population from eastern China, JYJD-2, has evolved high-level resistance to fenoxaprop-P-ethyl and moderate resistance to quizalofop-P-ethyl and pinoxaden. Using the derived cleaved amplified polymorphic sequence method, a tryptophan-to-cysteine mutation at codon position 1999 (W1999C) was detected in the ACCase gene of the resistant population JYJD-2. Of the 100 JYJD-2 plants tested, we found 47 heterozygous resistant and 53 homozygous sensitive individuals. In vitro ACCase assays revealed that the IC50 value of the ACCase activity of the resistant population JYJD-2 was 6.48-fold higher than that of the susceptible population JYJD-1. To the best of our knowledge, this is the first report of the occurrence of W1999C mutation in the ACCase gene of fenoxaprop-P-ethyl–resistant Keng stiffgrass. This study confirmed the resistance of Keng stiffgrass to the ACCase inhibitor fenoxaprop-P-ethyl, cross-resistance to other ACCase inhibitors, and the resistance being conferred by specific ACCase point mutations at amino acid position 1999.

Little published information is available related to seed germination and seedling establishment of Asia Minor bluegrass, a problematic grass weed in some regions of China. The objective of this study was to examine the effects of different environmental factors on Asia Minor bluegrass seed germination. The optimum temperature for germination was around 10 to 20 C, and more than 90% of seeds germinated under 20/10 and 25/15 C temperature regimes. Also, light and pH did not appear to have any effect on seed germination. Asia Minor bluegrass was sensitive to osmotic stress, but tolerant of NaCl. No seedlings emerged when seeds were buried 4 cm deep. The result suggested that Asia Minor bluegrass has the potential to spread into a large area in China. To prevent its spreading, measures such as soil cultivation can be used to limit seed germination from increased burial depth and/or nonselective herbicides can be applied to kill early-germinating weed seedlings in late summer.

Japanese foxtail is a predominant tetraploid grass weed in wheat and oilseed rape fields in eastern China. In China, pyroxsulam is mainly used to manage annual grass weeds, especially those resistant to acetyl coenzyme A carboxylase (ACCase)-inhibiting herbicides. Using dose–response studies, a pyroxsulam-resistant population, ACTC-1, was identified with a resistance index value of 58. Additionally, ACTC-1 was cross-resistant to sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl-benzoates, and sulfonylaminocarbonyl-triazolinones and multiresistant to ACCase and photosystem II inhibitors. Sequence analysis revealed four gene fragments encoding acetolactate synthase (ALS) from ACTC-1, and three from JNXW-1, a pyroxsulam-sensitive population. An Asp-376-Glu substitution was found in ALS1;2 and an Ile-2041-Asn in Acc1;1, which may be responsible for its resistance to pyroxsulam and ACCase inhibitors, respectively. In vitro assays of ALS activity revealed that in ACTC-1, the sensitivity of ALS to pyroxsulam was lower, and the basal ALS activity was twofold higher than that of sensitive population JNXW-1. Additionally, the combined application of pyroxsulam with malathion or piperonyl butoxide increased the sensitivity of ACTC-1 to pyroxsulam, although it could not completely overcome the resistance. It was inferred that both target-site-based resistance and nontarget-site-based resistance may be involved in the resistance to pyroxsulam.

Herbicide-resistant (R biotype) and -sensitive (S biotype) individuals were identified from the same population, and seed was increased for each biotype for three generations. We conducted laboratory experiments to determine the effects of temperature, light, salt stress, osmotic stress, pH, and burial depth on the germination and emergence of resistant and sensitive biotypes of Japanese foxtail. The results revealed that there was no difference in the final germination rate between the two biotypes under different temperature conditions, but time to obtain 50% germination or emergence (tE50) and mean germination time of the R biotype were higher than that of the S biotype at 10 C and 15/10 C 12-h day/night regime. In dark conditions, the final germination rate of the S biotype was higher and lower than that of the R biotype at 10 and 25 C, respectively. The overall germination rate of the R biotype was lower than that in the S biotype, and extended germination time was required in extreme conditions, such as 250 mM NaCl and −0.4 MPa osmotic potential. The change in environmental pH had no effect on the germination of the two biotypes. Emergence of the R biotype was lower than the S biotype when seed was buried at least 8 cm deep in an organic matter substrate. This study demonstrated the pleiotropic effects of a resistance allele on seed germination and emergence under different environmental conditions. Deep tillage could be used to reduce the growth and spread of resistant Japanese foxtail individuals.

The influence of temperature, light, solution pH, water stress, salt stress, and burial depth on seed germination and seedling emergence of catchweed bedstraw and the sensitivity of that weed to commonly available herbicides in China were studied in laboratory and greenhouse. Germination occurred at day/night temperatures from 5/0 C to 30/25 C, with optimum germination at 15/10 C. Catchweed bedstraw germinated equally well under a 12-h photoperiod and continuous darkness; however, a 24-h photoperiod inhibited seed germination. Catchweed bedstraw seed is moderately sensitive to osmotic potential and salt stress, with 15 and 3% germination rates at an osmotic potential of −0.5 Mpa and salinity level of 120 mM, respectively. Maximum seed germination was observed in near neutral pH; germination was greater than 80% over a broad pH range from 5 to 8. Seedling emergence of the seeds buried at a depth of 1 cm was higher (74%) than those placed on the soil surface (20%), but declined with burial depth increasing. Few (10%) seedlings emerged when seeds were placed at a depth of 5 cm. Bensulfuron-methyl, and ethametsulfuron-methyl applied PRE and tribenuron-methyl, fluroxypyr, and florasulam applied POST can be used to provide greater than 80% control of catchweed bedstraw. The results of this study have contributed to more complete understanding of the germination and emergence of catchweed bedstraw.

The objective of this study was to investigate the resistance level of Japanese foxtail to haloxyfop, an acetyl coenzyme A carboxylase (ACCase; EC 6.4.1.2)–inhibiting herbicide. Eleven biotypes were collected from oilseed rape fields in different areas in Jiangsu and Anhui provinces where haloxyfop had been continuously applied for various periods. Biotypes were assessed by two different methods, a seed bioassay and whole-plant assay, to identify the most resistant and susceptible biotypes for further studies on the activity of the target enzyme ACCase. A good correlation was obtained between the two different bioassay methods. The Jurong and Chuzhou biotypes were the most resistant and susceptible biotypes, respectively, whereas the other nine biotypes showed variable and relatively low degrees of haloxyfop resistance. Furthermore, target-site enzyme sensitivity results confirmed that the Jurong biotype was resistant to haloxyfop with a concentration of herbicide causing 50% inhibition of ACCase activity (IC50) of 9.19 µM, whereas the IC50 of the susceptible biotype (Chuzhou) was 0.76 µM, giving a resistance index of 12.

The influence of environmental factors on seed germination and seedling emergence of American sloughgrass was studied in laboratory and greenhouse conditions. The optimum temperature for seed germination was 10 C and light was not necessary. Seed germination was sensitive to osmotic potential and completely inhibited at an osmotic potential of −0.6 MPa, but it was quite tolerant to salinity: germination occurred even at 160 mM NaCl (36%). More than 80% of seeds germinated at pH values ranging between 4 and 10. Seedling emergence was highest when seeds were placed on the soil surface (91%) but declined with burial depth. Few (3%) seedlings emerged when seeds were planted at a depth of 5 cm. Information gained in this study will lead to a better understanding of the requirements for American sloughgrass germination and emergence.