Increased expression of Nav1.6 of reactive astrocytes in the globus pallidus is closely associated with motor deficits in a model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease in elderly people, which is characterized by motor disabilities in PD patients. Nav1.6 is the most abundant subtype of voltage-gated sodium channels (VGSCs) in the brain of adult mammals and rodents. Here we investigated the role of Nav1.6 in the external globus pallidus (GP) involved in the pathogenesis of motor deficits in unilateral 6-OHDA(6-hydroxydopamine)lesioned rats. The results show that Nav1.6 is dramatically increased in reactive astrocytes of the ipsilateral GP in the middle stage, but not different from the control rats in the later stage of the pathological process in 6-OHDA lesioned rats. Furthermore, the down-regulation of Nav1.6 expression in the ipsilateral GP can significantly improve motor deficits in 6-OHDA lesioned rats in the middle stage of the pathological process. The electrophysiological experiments show that the down-regulation of Nav1.6 expression in the ipsilateral GP significantly decreases the abnormal high synchronization between the ipsilateral M1 (the primary motor cortex) and GP in 6-OHDA lesioned rats. Ca2+ imaging reveals that the down-regulation of Nav1.6 expression reduces the intracellular concentration of Ca2+ ([Ca2+ ]i) in primary cultured astrocytes. These findings suggest that the increased Nav1.6 expression of reactive astrocytes in the GP play an important role in the pathogenesis of motor dysfunction in the middle stage in 6-OHDA lesioned rats, which may participate in astrocyte-neuron communication by regulating [Ca2+ ]i of astrocytes, thereby contributing to the formation of abnormal electrical signals of the basal ganglia (BG) in 6-OHDA lesioned rats.

Cytochrome P450 CYP709C56 metabolizing mesosulfuron-methyl confers herbicide resistance in Alopecurus aequalis.

Multiple herbicide resistance in diverse weed species endowed by enhanced herbicide detoxification or degradation is rapidly growing into a great threat to herbicide sustainability and global food safety. Although metabolic resistance is frequently documented in the economically damaging arable weed species shortawn foxtail (Alopecurus aequalis Sobol.), relevant molecular knowledge has been lacking. Previously, we identified a field population of A. aequalis (R) that had evolved metabolic resistance to the commonly used acetolactate synthase (ALS)-inhibiting herbicide mesosulfuron-methyl. RNA sequencing was used to discover potential herbicide metabolism-related genes, and four cytochrome P450s (CYP709C56, CYP71R18, CYP94C117, and CYP94E14) were identified with higher expressions in the R vs. susceptible (S) plants. Here the full-length P450 complementary DNA transcripts were each cloned with identical sequences between the S and R plants. Transgenic Arabidopsis overexpressing CYP709C56 became resistant to the sulfonylurea herbicide mesosulfuron-methyl and the triazolo-pyrimidine herbicide pyroxsulam. This resistance profile generally but does not completely in accordance with what is evident in the R A. aequalis. Transgenic lines exhibited enhanced capacity for detoxifying mesosulfuron-methyl into O-demethylated metabolite, which is in line with the detection of O-demethylated herbicide metabolite in vitro in transformed yeast. Structural modeling predicted that mesosulfuron-methyl binds to CYP709C56 involving amino acid residues Thr-328, Thr-500, Asn-129, Gln-392, Phe-238, and Phe-242 for achieving O-demethylation. Constitutive expression of CYP709C56 was highly correlated with the metabolic mesosulfuron-methyl resistance in A. aequalis. These results indicate that CYP709C56 degrades mesosulfuron-methyl and its up-regulated expression in A. aequalis confers resistance to mesosulfuron-methyl.

Method validation and dissipation kinetics of the novel HPPD-inhibiting herbicide cypyrafluone in winter wheat using QuEChERS method coupled with UPLC-MS/MS.

Cypyrafluone, a novel hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide, can successfully control a wide species of grass and broadleaf weed in wheat fields. However, the dissipation behaviors and terminal residues of cypyrafluone in wheat fields remain unclear. Here, a simple, accurate, and dependable approach for the analysis of cypyrafluone in soil, wheat plant, and grain was constructed utilizing an adapted QuEChERS extraction combined with UPLC-MS/MS. For accurate quantification, matrix-matched calibrations with high linearity (R2 >0.99) were employed to eliminate matrix interference. The method possessed high accuracy with recoveries in the range of 85.5%- 100.6% and precision with relative standard deviations < 14.3%, as well as high sensitivity with limits of quantifications of 0.001 mg kg-1 in the three matrixes. The dissipation kinetics and terminal residues of cypyrafluone were determined at two separate locations with different climates, soil types and cropping systems in 2018. The half-lives of cypyrafluone in soil and wheat plant were 1.47-1.55 d and 1.00-1.03 d, respectively. At harvest, the terminal residue values of cypyrafluone detected in wheat plants were 0-0.0025 mg kg-1 and 0.0044-0.0057 mg kg-1 at the recommended dose and 1.5 times of the recommended dose, respectively, and 0.0049 mg kg-1 of this herbicide was detected in grain at 1.5 times of the recommended dose, which was below the maximum residue limit (MRL). Finally, the risk quotient for cypyrafluone ranged from 0.33% to 0.81% (<1) for different age groups in China, indicating that the impact of residues from the cypyrafluone application on wheat was acceptable. These findings above will offer scientific guidelines for cypyrafluone application in the wheat field ecosystem.

Resistance to ALS inhibitors conferred by non-target-site resistance mechanisms in Myosoton aquaticum L.

Myosoton aquaticum L. is a competitive broadleaf weed commonly found in wheat fields in China and has become challenging due to its evolving herbicide resistance. In this study, one subpopulation, RF1 (derived from the tribenuron-methyl-resistant population HN10), with none of the known acetolactate synthase (ALS) resistance mutations was confirmed to exhibit resistance to tribenuron-methyl (SU), pyrithiobac­sodium (PTB), florasulam (TP), flucarbazone-Na (SCT), and diflufenican (PDS). In vitro ALS activity assays showed that the total ALS activity of RF1 was lower than that of the susceptible (S) population. However, there was no difference in ALS gene expression induced by tribenuron-methyl between the two populations. The combination of the cytochrome P450 monooxygenase (P450) inhibitor malathion and tribenuron-methyl resulted in the RF1 population behaving like the S population. The rapid P450-mediated tribenuron-methyl metabolism in RF1 plants was also confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. In addition, approximately equal glutathione S-transferase (GST) activity was observed in RF1 and S plants of untreated and tribenuron-methyl treated groups. This study reported one M. aquaticum L. population without ALS resistance mutations exhibiting resistance to ALS inhibitors and the PDS inhibitor diflufenican, and the non-target-site resistance mechanism played a vital role in herbicide resistance.

Effects of the novel HPPD-inhibitor herbicide QYM201 on enzyme activity and microorganisms, and its degradation in soil.

QYM201 is a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibiting herbicide recently registered in China for controlling grass and broadleaf weeds in wheat. It is a novel herbicide, and its potential harm to soil ecosystems has not yet been reported. This study investigates the influence of QYM201 on soil enzyme activity and microorganism quantities in two different soils at concentrations of 0.1, 1, and 5 mg kg-1 soil. Results indicate that QYM201 initially inhibited soil protease, urease, and sucrase activity and this effect increased with concentration. During the later stages of incubation, inhibitory effects gradually weakened and by the end of the experiment (45 days), enzyme activity was restored to control levels. Catalase activity was stimulated by QYM201, with significant differences observed between the QYM201-treated groups and the control at the onset of exposure. This stimulation effect decreased during the later stages of the experiment. However, catalase activity was still significantly higher at the end of the experiment compared to the control. The effects of QYM201 on soil microorganisms differed. Initially, bacteria and actinomycetes quantities were decreased by QYM201 (10 days). As the incubation progressed, microorganism quantities in the lower concentration groups (0.1 and 1 mg kg-1 soil) were restored to control levels, while those of the high concentration group (5 mg kg-1 soil) did not fully recover. QYM201 did not significantly impact the quantity of fungi. The half-life and degradation rate constant (k) of QYM201 for the two studied soil types were 23.1 days and 16.1 days, and 0.030 and 0.043 day-1, respectively.

Florasulam resistance status of flixweed (Descurainia sophia L.) and alternative herbicides for its chemical control in the North China plain.

Flixweed (Descurainia sophia L.) is widely distributed in winter wheat (Triticum aestivum L.) fields in the North China Plain and has evolved resistance to herbicides, including the acetolactate synthase (ALS) inhibitor florasulam. However, the florasulam resistance status of flixweed in the North China Plain is poorly understood, which hinders the integrated management of this weed in winter wheat production systems. Thus, 45 flixweed populations were collected in wheat fields in these areas, and their sensitivity to florasulam and ALS-inhibitor-resistant mutation diversity were assessed. Meanwhile, alternative herbicides/herbicide mixtures for the control of florasulam-resistant flixweed were screened and evaluated under greenhouse and field conditions. Of the populations, 30 showed florasulam resistance (RRR and RR), 9 had a high risk of evolving florasulam resistance (R?) and 6 were susceptible. These populations had 5.3 to 345.1-fold resistance to florasulam, and 4 ALS resistance mutations (P197H, P197S, P197T and W574L) were observed. The subsequent herbicide sensitivity assay showed that the SD-06 population (with ALS1 P197T and ALS2 W574L mutations) exhibited cross-resistance to all ALS inhibitors tested, but was sensitive to MCPA-Na, fluroxypyr, carfentrazone-ethyl and bipyrazone. Meanwhile, the other HN-07 population with non-target-site resistance (NTSR) also showed resistance to all tested ALS inhibitors, and it was "R?" to MCPA-Na while sensitive to fluroxypyr, carfentrazone-ethyl and bipyrazone. The field experiments were conducted at the research farm where the SD-06 population was collected, and the results suggested that florasulam at 3.75-4.5 g ai ha-1 had little efficacy (0.6-12.1%), whereas MCPA-Na + carfentrazone-ethyl (87.1-91.2%) and bipyrazone+fluroxypyr (90.1-97.8%) controlled the resistant flixweed.

Unravelling the effect of two herbicide resistance mutations on acetolactate synthase kinetics and growth traits.

Gene mutations conferring herbicide resistance are hypothesized to have negative pleiotropic effects on plant growth and fitness, which may in turn determine the evolutionary dynamics of herbicide resistance alleles. We used the widespread, annual, diploid grass weed Alopecurus aequalis as a model species to investigate the effect of two resistance mutations-the rare Pro-197-Tyr mutation and the most common mutation, Trp-574-Leu-on acetolactate synthase (ALS) functionality and plant growth. We characterized the enzyme kinetics of ALS from two purified A. aequalis populations, each homozygous for the resistance mutation 197-Tyr or 574-Leu, and assessed the pleiotropic effects of these mutations on plant growth. Both mutations reduced sensitivity of ALS to ALS-inhibiting herbicides without significant changes in extractable ALS activity. The 197-Tyr mutation slightly decreased the substrate affinity (corresponding to an increased Km for pyruvate) and maximum reaction velocity (Vmax) of ALS, whereas the 574-Leu mutation significantly increased these kinetics. Significant decrease or increase in plant growth associated, respectively, with the 197-Tyr and 574-Leu resistance mutations was highly correlated with their impact on ALS kinetics, suggesting more likely persistence of the 574-Leu mutation than the 197-Tyr mutation if herbicide application is discontinued.

Method validation and dissipation kinetics of the new HPPD inhibitor QYR301 in rice, paddy water and paddy soil using a QuEChERS-based method and LC-MS/MS.

A rapid and simple method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) of sample preparation using QuEChERS was developed for detecting residues of QYR301, a new HPPD-inhibiting herbicide, in rice plant (straw), water, soil, rice hull and brown rice (BR). To eliminate matrix interference, matrix-matched calibrations with satisfactory linearity (R2 > 0.99) were used for accurate quantification. The method showed recoveries of 90.3-108.1% and relative standard deviations (RSDs) < 11%. The limits of quantification (LOQ) for QYR301 were 0.005 mg kg-1 in all five matrixes. Furthermore, the dissipation kinetics and terminal residues of QYR301 were determined at two sites in 2018. The days for 50% dissipation (DT50) of QYR301 in rice plants, water and soil were 3.6-4.4, 0.7-3.0 and 4.3-8.0 d, respectively, which indicated that QYR301 is a short-persistence herbicide. Moreover, no QYR301 residues were detected in BR, rice hull and straw collected at harvest following its application at 1.0 or 1.5 × of the recommended high rate. These results will help organizations and governments establish related principles/laws regarding the use of QYR301 in terms of environmental protection, food safety and other potential aspects.

A Trp-574-Leu mutation in the acetolactate synthase (ALS) gene of Lithospermum arvense L. confers broad-spectrum resistance to ALS inhibitors.

Lithospermum arvense is a troublesome dicotyledonous winter annual weed of wheat in China. A L. arvense population (HN01) suspected of being resistant to acetolactate synthase (ALS) inhibitors was found in Henan Province, China. This study aimed to testify the sensitivity of this HN01 population to eight herbicides from 3 different modes of action, and to explore the potential target-site-resistance mechanism to tribenuron-methyl. The whole-plant bioassays indicated that the population was highly resistant to tribenuron-methyl (SU, 350-fold), pyrithiobac sodium (PTB, 151-fold), pyroxsulam (TP, 62.7-fold), florasulam (TP, 80.6-fold), and imazethapyr (IMI, 136-fold), but was sensitive to carfentrazone-ethyl and fluroxypyr-meptyl. ALS gene sequencing revealed that the Trp (TGG) was substituted by Leu (TTG) at codon 574 in resistant plants. In in vitro ALS assays, the concentration of tribenuron-methyl required to inhibit 50% ALS activity (I50) for HN01 was 117-fold greater than that required to inhibit a susceptible population (HN05), indicating that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron-methyl. To the best of our knowledge, this is the first report of ALS gene Trp-574-Leu amino acid mutation confer resistance to tribenuron-methyl in L. arvense.

Isolation and expression of acetolactate synthase genes that have a rare mutation in shepherd's purse (Capsella bursa-pastoris (L.) Medik.).

Acetolactate synthase (ALS) inhibitor-resistant biotypes are the fastest growing class of herbicide-resistant weeds. Shepherd's purse (Capsella bursa-pastoris (L.) Medik.), a tetraploid species and one of the most troublesome weeds in wheat production, has evolved ALS inhibitor resistance. To confirm and characterize the resistance of shepherd's purse populations to ALS-inhibiting herbicides, whole-plant bioassays were conducted. To investigate the molecular basis of resistance in shepherd's purse, the ALS gene was sequenced and compared between susceptible (S) and resistant (R) biotypes. Two partial intronless ALS genes (ALS-1 and ALS-2) were identified, and two heterozygous mutations (CCT to TCT in ALS-1 and CCT to CAT in ALS-2) at position 197 (Pro197Ser and Pro197His) providing resistance were simultaneously found in a single plant in a resistant population. Our results confirmed that the resistant shepherd's purse population showed high-level resistance to tribenuron-methyl (RI = 59.8), pyroxsulam (RI = 38.7) and flucarbazone-Na (RI = 88.0). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) results suggested that the difference in ALS gene expression was small between S and R populations, which may be insufficient to cause herbicide resistance, and according to the results of in vitro ALS activity, insensitivity of ALS may be the main mechanism of high resistance to tribenuron-methyl in resistant populations.

Target-site and non-target-site-based resistance to tribenuron-methyl in multiply-resistant Myosoton aquaticum L.

Myosoton aquaticum L., a widespread and competitive winter weed of wheat in China, has evolved resistance to many classes of herbicides. In one M. aquaticum population (AH03), collected from Anhui Province, where tribenuron-methyl and florasulam had been used to control this weed resistance to both herbicides had evolved. Compared with the sensitive population, HN03(S), the resistant (R) population, AH03, was highly resistant to tribenuron-methyl, flucarbazone-Na and pyroxsulam, moderately resistant to pyrithiobac­sodium, and florasulam, and had low resistance to diflufenican. AH03 was still controlled by imazethapyr, 2,4-D butylate, fluroxypyr-meptyl, and isoproturon. Pretreatment with the P450 inhibitor malathion reduced the GR50 value of tribenuron-methyl by 43% in the R population, and by 25% in the S population. This indicates that P450-mediated enhanced metabolism is one likely mechanism for tribenuron-methyl resistance in M. aquaticum. Glutathione-S-transferase (GST) activity could be induced by tribenuron-methyl in both the R and S populations. However, both the basal and induced GST activity of the R population was lower than that of the S population. The in vitro ALS assay confirmed that the ALS from the R plants showed a high resistance (52.93-fold) to tribenuron-methyl. ALS gene sequencing revealed a Pro197Ala substitution in the R plants. Based on the ALS gene sequence analysis, molecular markers were also developed to identify the specific Pro197Ala mutation. This population of M. aquaticum has multiple resistance and target-site (ALS Pro197Ala) and non-target-site resistance mechanisms contribute to tribenuron-methyl resistance.

Non-target site-based resistance to tribenuron-methyl and essential involved genes in Myosoton aquaticum (L.).

BACKGROUND: Water chickweed (Myosoton aquaticum (L.)) is a dicot broadleaf weed that is widespread in winter fields in China, and has evolved serious resistance to acetolactate synthase (ALS) inhibiting herbicides. RESULTS: We identified a M. aquaticum population exhibiting moderate (6.15-fold) resistance to tribenuron-methyl (TM). Target-site ALS gene sequencing revealed no known resistance mutations in these plants, and the in vitro ALS activity assays showed no differences in enzyme sensitivity between susceptible and resistant populations; however, resistance was reversed by pretreatment with the cytochrome P450 (CYP) monooxygenase inhibitor malathion. An RNA sequencing transcriptome analysis was performed to identify candidate genes involved in metabolic resistance, and the unigenes obtained by de novo transcriptome assembly were annotated across seven databases. In total, 34 differentially expressed genes selected by digital gene expression analysis were validated by quantitative real-time (qRT)-PCR. Ten consistently overexpressed contigs, including four for CYP, four for ATP-binding cassette (ABC) transporter, and two for peroxidase were further validated by qRT-PCR using additional plants from resistant and susceptible populations. Three CYP genes (with homology to CYP734A1, CYP76C1, and CYP86B1) and one ABC transporter gene (with homology to ABCC10) were highly expressed in all resistant plants. CONCLUSION: The mechanism of TM resistance in M. aquaticum is controlled by NTSR rather than TSR. Four genes, CYP734A1, CYP76C1, CYP86B1, and ABCC10 could play essential role in metabolic resistance to TM and justify further functional studies. To our knowledge, this is the first large-scale transcriptome analysis of genes associated with NTSR in M. aquaticum using the Illumina platform. Our data provide resource for M. aquaticum biology, and will facilitate the study of herbicide resistance mechanism at the molecular level in this species as well as in other weeds.

Fenoxaprop-P-ethyl and mesosulfuron-methyl resistance status of shortawn foxtail (Alopecurus aequalis Sobol.) in eastern China.

Resistance to the acetyl-coenzyme A carboxylase (ACCase)- and acetolactate synthase (ALS)- inhibiting herbicides in shortawn foxtail (Alopecurus aequalis) has been reported in wheat fields of eastern China. To better understand the distribution of the resistant populations and the occurrence of the target-site mutations, 74 populations collected from Anhui, Jiangsu and Shandong province were surveyed, and the ACCase and ALS gene fragments, encompassing all the documented mutant codon positions, were amplified and sequenced. Plants from 37 and 34 populations survived fenoxaprop-P-ethyl and mesosulfuron-methyl treatment at 62.1 g a.i. ha-1 and 9 g a.i. ha-1 respectively, with different survival rates. Twenty-seven populations exhibited multiple resistance to fenoxaprop-P-ethyl and mesosulfuron-methyl. Whole-plant dose-response experiments showed that the resistance index ranged from 6.2 to 167.8 for fenoxaprop-P-ethyl, and from 7.8 to 139.5 for mesosulfuron-methyl. Four ACCase (I1781L, I2041N, I2041T and D2078G) and four ALS (P197R, P197S, P197T and W574 L) resistance mutations were detected respectively. Individuals containing two amino acid substitutions were also found. D2078G and W574 L were predominant ACCase and ALS gene mutations respectively. This study has shown that fenoxaprop-P-ethyl and mesosulfuron-methyl resistance was prevalent in A. aequalis in eastern China, and target site mutations in the ACCase and ALS gene were one of the most common mechanisms.

Comparison of ALS functionality and plant growth in ALS-inhibitor susceptible and resistant Myosoton aquaticum L.

Herbicide target-site resistance mutations may cause pleiotropic effects on plant ecology and physiology. The effect of several known (Pro197Ser, Pro197Leu Pro197Ala, and Pro197Glu) target-site resistance mutations of the ALS gene on both ALS functionality and plant vegetative growth of weed Myosoton aquaticum L. (water chickweed) have been investigated here. The enzyme kinetics of ALS from four purified water chickweed populations that each homozygous for the specific target-site resistance-endowing mutations were characterized and the effect of these mutations on plant growth was assessed via relative growth rate (RGR) analysis. Plants homozygous for Pro197Ser and Pro197Leu exhibited higher extractable ALS activity than susceptible (S) plants, while all ALS mutations with no negative change in ALS kinetics. The Pro197Leu mutation increased ALS sensitivity to isoleucine and valine, and Pro197Glu mutation slightly increased ALS sensitivity to isoleucine. RGR results indicated that none of these ALS resistance mutations impose negative pleiotropic effects on relative growth rate. However, resistant (R) seeds had a lowed germination rate than S seeds. This study provides baseline information on ALS functionality and plant growth characteristics associated with ALS inhibitor resistance-endowing mutations in water chickweed.

Tribenuron-methyl resistance and mutation diversity of the AHAS gene in shepherd's purse (Capsella bursa-pastoris (L.) Medik.) in Henan Province, China.

Shepherd's purse is a troublesome dicot weed that occurs in the major wheat-producing areas in China. Twenty-eight shepherd's purse populations were collected from winter wheat-planting areas in Henan Province and used to evaluate tribenuron-methyl resistance and acetohydroxyacid synthase (AHAS) gene-mutation diversity. The results indicate that all 28 shepherd's purse populations were resistant to tribenuron-methyl at different levels compared with the susceptible population. Mutation of the 197 codon (CCT) changed proline (Pro) into tyrosine (Tyr), histidine (His), leucine (Leu), serine (Ser), arginine (Arg), alanine (Ala) and threonine (Thr), whereas mutation of the 574 codon (TGG) changed tryptophan (Trp) into leucine (Leu). Among these amino acid changes, a co-concurrence of Pro197Leu and Trp574Leu substitutions was identified for the first time in resistant weed species. Furthermore, Pro197Tyr, Pro197Arg and Pro197Ala substitutions have not been previously reported in shepherd's purse. The results of the in vitro AHAS assay suggest that an insensitive AHAS is likely involved in the resistance to tribenuron-methyl in the R populations with AHAS gene mutations, and the non-target-site based resistance might exist in some populations.

Cross-resistance patterns to ACCase-inhibitors in American sloughgrass (Beckmannia syzigachne Steud.) homozygous for specific ACCase mutations.

American sloughgrass is a troublesome annual grass weed in winter wheat field rotated with rice in China. The overreliance on acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides has resulted in resistance evolution in this weed. In this study, the cross-resistance patterns to fenoxaprop-p-ethyl, clodinafop-propargyl, fluazifop-p-butyl, haloxyfop-p-methyl, sethoxydim, clethodim and pinoxaden were established using purified plants individually homozygous for specific mutant ACCase alleles. Results indicated that 1781Leu allele endows high-level resistance to APPs, CHDs and pinoxaden while confers moderate resistance to haloxyfop-p-methyl. The 2027Cys and 2041Asn alleles endow high-level resistance to APPs and pinoxaden and lower level resistance to CHDs. The 2078Gly allele confers high-level resistance to all herbicides tested in this study, however, moderate resistance to sethoxydim. The 2096Ala very likely endows high-level resistance to fluazifop-p-butyl, haloxyfop-p-methyl and moderate resistance to sethoxydim. In addition, one undefined resistance mechanism was involved in population SD-04.

Molecular basis of multiple resistance to ACCase- and ALS-inhibiting herbicides in Alopecurus japonicus from China.

Fenoxaprop-P-ethyl-resistant Alopecurus japonicus has become a recurring problem in winter wheat fields in eastern China. Growers have resorted to using mesosulfuron-methyl, an acetolactate synthase (ALS)-inhibiting herbicide, to control this weed. A single A. japonicus population (AH-15) resistant to fenoxaprop-P-ethyl and mesosulfuron-methyl was found in Anhui Province, China. The results of whole-plant dose-response experiments showed that AH-15 has evolved high-level resistance to fenoxaprop-P-ethyl (95.96-fold) and mesosulfuron-methyl (39.87-fold). It was shown via molecular analysis that resistance to both fenoxaprop-P-ethyl and mesosulfuron-methyl was due to an amino acid substitution of Ile1781 to Leu in acetyl-CoA carboxylase (ACCase) and a substitution of Trp 574 to Leu in ALS, respectively. Whole-plant bioassays indicated that the AH-15 population was resistant to the ACCase herbicides clodinafop-propargyl, clethodim, sethoxydim and pinoxaden as well as the ALS herbicides pyroxsulam, flucarbazone-Na and imazethapyr, but susceptible to the ACCase herbicide haloxyfop-R-methyl. This work reports for the first time that A. japonicus has developed resistance to ACCase- and ALS-inhibiting herbicides due to target site mutations in the ACCase and ALS genes.

Molecular basis for resistance to ACCase-inhibiting herbicides in Pseudosclerochloa kengiana populations.

Pseudosclerochloa kengiana is a troublesome annual grass weed of wheat fields in the rice-wheat double cropping areas in China. Resistance has evolved in P. kengiana under continuously selective pressure of ACCase-inhibiting herbicides. Whole-plant experiments showed that two suspected resistant populations 12-SD-12 and 12-SD-13 were highly resistant to fenoxaprop-P-ethyl (69.9- and 57.2-fold); moderately resistant to clodinafop-propargyl (5.9- and 4.1-fold) and pinoxaden (4.4- and 3.5-fold); lowly resistant to fluazifop-P-butyl (2.2- and 2.0-fold) and sethoxydim (1.8- and 1.6-fold), but were sensitive to clethodim (1.0- and 0.9-fold) and mesosulfuron-methyl (1.1- and 0.9-fold). Molecular analyses confirmed that a Trp1999 to Ser mutation was present in the resistant populations. Two dCAPS markers were also developed to positively determine the wild type Trp and mutant Ser alleles at ACCase position 1999. All 350 individual plants of 12-SD-12 population analysed were heterozygous mutants. Meanwhile, 318 mutant heterozygotes and 32 wild types were detected from the 12-SD-13 population. In addition, the analysis of plant genotype and phenotype showed that all wild type plants were killed after treatment with any one of the ACCase-inhibiting herbicides used, while individual plants carrying the W1999S mutation survived except when treated with clethodim. To our knowledge, this is the first report of pinoxaden resistance and a Trp-1999-Ser mutation in the plastid ACCase for P. kengiana.

A novel Pro197Glu substitution in acetolactate synthase (ALS) confers broad-spectrum resistance across ALS inhibitors.

Water chickweed (Myosoton aquaticum L.), a competitive broadleaf weed, is widespread in wheat fields in China. Tribenuron and pyroxsulam failed to control water chickweed in the same field in Qiaotian Village in 2011 and 2012, respectively. An initial tribenuron resistance confirmation test identified a resistant population (AH02). ALS gene sequencing revealed a previously unreported substitution of Glu for Pro at amino acid position 197 in resistant individuals. A purified subpopulation (WRR04) that was individually homozygous for the Pro197Glu substitution was generated and characterized in terms of its response to different classes of ALS inhibitors. A whole-plant experiment showed that the WRR04 population exhibited broad-spectrum resistance to tribenuron (SU, 318-fold), pyrithiobac sodium (PTB, > 197-fold), pyroxsulam (TP, 81-fold), florasulam (TP, > 36-fold) and imazethapyr (IMI, 11-fold). An in vitro ALS assay confirmed that the ALS from WRR04 showed high resistance to all the tested ALS inhibitors. These results established that the Pro197Glu substitution endows broad-spectrum resistance across ALS inhibitors in water chickweed. In addition, molecular markers were developed to rapidly identify the Pro197Glu mutation.

Multiple resistance to ACCase and AHAS-inhibiting herbicides in shortawn foxtail (Alopecurus aequalis Sobol.) from China.

Shortawn foxtail (Alopecurus aequalis) is a troublesome grass weed infesting winter wheat and oilseed rape productions in China. Fenoxaprop-p-ethyl and mesosulfuron-methyl failed to control shortawn foxtail of AHSX-1 population collected from a wheat field in Shou County, Anhui province. Molecular analyses revealed that Asp2078Gly mutation of ACCase and Trp574Leu mutation of AHAS were present in plants of the AHSX-1 population. The homozygous plants were isolated and cultured until seed maturity. Whole-plant herbicide bioassays were conducted in the greenhouse using the purified seeds of F1 generation. Dose-response experiments showed that the AHSX-1 population has evolved a very high level resistance to fenoxaprop-p-ethyl (RI = 275) and mesosulfuron-methyl (RI = 788). To determine the sensitivity to other herbicides, assays were conducted at the single recommended rate of each herbicide. Based on the results, the AHSX-1 population was considered to be highly resistant to clodinafop-propargyl, pyroxsulam and flucarbazone-sodium, moderately or highly resistant to quizalofop-p-ethyl, clethodim, sethoxydim and pinoxaden, and susceptible to isoproturon and chlorotoluron. This is the first report of Asp2078Gly mutation in shortawn foxtail and the two robust dCAPS markers designed could quickly detect Asp2078 and Trp574 mutations in ACCase and AHAS gene of shortawn foxtail, respectively.