Changes in physiology, antioxidant system, and gene expression in Microcystis aeruginosa under fenoxaprop-p-ethyl stress.

Fenoxaprop-p-ethyl (FE) is one of the typical aryloxyphenoxypropionate herbicides. FE has been widely applied in agriculture in recent years. Human health and aquatic ecosystems are threatened by the cyanobacteria blooms caused by Microcystis aeruginosa, which is one of the most common cyanobacteria responsible for freshwater blooming. Few studies have been reported on the physiological effects of FE on M. aeruginosa. This study analyzed the growth curves, the contents of chlorophyll a and protein, the oxidative stress, and the microcystin-LR (MC-LR) levels of M. aeruginosa exposed to various FE concentrations (i.e., 0, 0.5, 1, 2, and 5 mg/L). FE was observed to stimulate the cell density, chlorophyll a content, and protein content of M. aeruginosa at 0.5- and 1-mg/L FE concentrations but inhibit them at 2 and 5 mg/L FE concentrations. The superoxide dismutase and catalase activities were enhanced and the malondialdehyde concentration was increased by FE. The intracellular (intra-) and extracellular (extra-) MC-LR contents were also affected by FE. The expression levels of photosynthesis-related genes psbD1, psaB, and rbcL varied in response to FE exposure. Moreover, the expressions of microcystin synthase-related genes mcyA and mcyD and microcystin transportation-related gene mcyH were significantly inhibited by the treatment with 2 and 5 mg/L FE concentrations. These results might be helpful in evaluating the ecotoxicity of FE and guiding the rational application of herbicides in modern agriculture.

Developmental toxicity and metabolomics analyses of zebrafish (Danio rerio) embryos exposed to Fenoxaprop-p-ethyl.

Fenoxaprop-p-ethyl (FEN) is an aryloxy phenoxy propionate herbicide that has been widely used in paddy fields. Previous studies have indicated that FEN is highly toxic to aquatic organisms, but little is known about the developmental effects of FEN. This study investigated acute and developmental toxicity, malondialdehyde (MDA) levels, superoxide dismutase (SOD) and catalase (CAT) activities, and metabolomic analyses in zebrafish embryos after 96 h of exposure. FEN exhibited high acute toxicity to zebrafish embryos and larvae. Exposure to FEN could reduce heartbeat and hatching rates and increase malformation rates in embryos. Oxidative damage was also caused in embryos. The results of metabolomics analysis showed that 102 differentially abundant metabolites were found in zebrafish embryos in the 0.05 mg/L FEN treatment group, and 60 differentially abundant metabolites were found in the 0.20 mg/L FEN treatment group. These differentially abundant metabolites mainly belonged to 9 metabolic pathways, of which folate pathways and ABC transport protein pathways had the greatest impact. These results suggested that FEN induced high acute and developmental toxicity in zebrafish embryos.

Fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon resistance status in Beckmannia syzigachne from wheat fields across Anhui Province, China.

Severe infestations of American sloughgrass (Beckmannia syzigachne (Steud.) Fernald) in wheat fields throughout Anhui Province, China, pose a significant threat to local agricultural production. This study aims to evaluate the susceptibility of 37 B. syzigachne populations collected from diverse wheat fields in Anhui Province to three commonly used herbicides: fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon. Single-dose testing revealed that out of the 37 populations, 31, 26, and 11 populations had either evolved or were evolving resistance to fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon, respectively. Among them, 25 populations displayed concurrent resistance to both fenoxaprop-P-ethyl and mesosulfuron-ethyl, while eight exhibited resistance to all three tested herbicides. Whole-plant bioassays confirmed that approximately 84% of the fenoxaprop-P-ethyl-resistant populations manifested high-level resistance (resistance index (RI) ≥10); 62% of the mesosulfuron-ethyl-resistant populations and 82% of the isoproturon-resistant populations exhibited low- to moderate-level resistance (2 ≤ RI <10). Three distinct target-site mutations were identified in 27% of fenoxaprop-P-ethyl-resistant populations, with no known resistance mutations detected in the remaining herbicide-resistant populations. The inhibition of cytochrome P450s (P450s) and/or glutathione S-transferases (GSTs) substantially increased susceptibility in the majority of resistant populations lacking mutations at the herbicide target site. In conclusion, resistance to fenoxaprop-P-ethyl and mesosulfuron-ethyl was widespread in B. syzigachne within Anhui Province's wheat fields, while resistance to isoproturon was rapidly evolving due to its escalating usage. Target-site mutations were present in approximately one-third of fenoxaprop-P-ethyl-resistant populations, and alternative mechanisms involving P450s and/or GSTs could explain the resistance observed in most of the remaining populations.

Protective mechanisms of neral as a plant-derived safener against fenoxaprop-p-ethyl injury in rice.

BACKGROUND: The use of herbicide safeners effectively minimises crop damage while maintaining the full efficacy of herbicides. The present study aimed to assess the potential protective effects of neral (NR) as a safener, in order to mitigate injury caused by fenoxaprop-p-ethyl (FE) on rice. RESULTS: The alleviating effect of NR was similar to that of the safener isoxadifen-ethyl (IE). The root elongation of rice was significantly promoted under the FE + NR and FE + IE treatments, as compared to the FE treatment. The transcriptome analysis further suggested that the effects of NR treatment on plant metabolic pathways differed from those of IE treatment. In total, 895 and 47 up-differentially expressed genes induced by NR (NR-inducible genes) and IE (IE-inducible genes) were identified. NR-inducible genes were mainly enriched in phytohormone synthesis and signalling response, including 'response to brassinosteroid', 'response to jasmonic acid', 'response to ethylene', 'brassinosteroid metabolic process', 'brassinosteroid biosynthesis' and 'plant hormone signal transduction'. In contrast, IE-inducible genes were predominantly enriched in glutathione metabolism. The activity of glutathione S-transferase was found to be increased after IE treatment, whereas no significant increase was observed following NR treatment. Moreover, several transcription factor genes, such as those encoding AP2/ERF-ERF and (basic helix-loop-helix) bHLH were found to be significantly induced by NR treatment. CONCLUSION: This is the first report of the utilisation of NR as an herbicide safener. The results of this study suggest the toxicity of FE to rice is mitigated by NR through a distinct mechanism compared to IE. © 2023 Society of Chemical Industry.

Long-term profound investigation of Fenoxaprop-p-ethyl and indirect pesticide pollution in jute-cropped environmental ecosystem for dietary and ecological risk assessment.

Jute is a healthy vegetable due to its high content of carotenoids, vitamins, and minerals, in addition to its industrial utility. The purpose of this study was to look into the dissipation of fenoxaprop-p-ethyl and to track indirect sources of pesticide accumulation in jute ecosystems for three years in a row during tropical monsoon seasons. To avoid the intake of residues over approved limits, a rapid extraction technique was developed to evaluate the residues of this herbicide (Whip-Super 9% EC) in jute leaves, fiber, cropped soil, and water. A modified QuEChERS approach based on liquid chromatography and ultraviolet-visible (UV-VIS) detection was developed and successfully validated in accordance with SANCO requirements. Gas chromatography mass spectrometer (GC-MS) was used to examine fish and water samples from jute fields and retting pond for twenty widely-used multi-class pesticide contaminations from indirect sources. Relative standard deviations (RSD) (≤ 20) and recoveries (100-115%) are observed to be within acceptable ranges. In edible jute leaves, independent of season, first-order kinetics of dissipation was recorded, with half-lives ranging from 0.61 to 0.68 days. Based on the findings of the risk assessment, it is feasible to conclude that the consumer health risks of ingesting jute leaves are insignificant, even on day zero, when used at the recommended amount, and that using jute bags for food packaging and storage is safe. But jute field water was found to be less polluted with indirect pesticides than water from a nearby jute retted pond. Fish samples from this jute retted pond were also analyzed and found to be 26% contaminated. The presence of quinalphos and chlorpyriphos in jute retting water and fish might pose a concern to consumer health and environmental ecosystems.

Expression Pattern of Entire Cytochrome P450 Genes and Response of Defenses in a Metabolic-Herbicide-Resistant Biotype of Polypogon fugax.

Enhanced herbicide metabolism mediated by cytochrome P450s has been proposed as one of the major mechanisms of resistance to fenoxaprop-P-ethyl in a metabolic-herbicide-resistant biotype of Asia minor bluegrass (Polypogon fugax Nees ex Steud.). Upon pre-treatment with the P450 inhibitor piperonyl butoxide, a remarkable reduction in metabolic rates of the phytotoxic fenoxaprop-P has been observed in the resistant plants, implying that constitutive and/or fenoxaprop-P-ethyl-induced up-regulation of specific P450 isoforms are involved in the fenoxaprop-P-ethyl resistance. However, which P450 gene(s) were responsible for the metabolic resistance is still unknown. In this present study, based on the abundant gene resources of P. fugax established previously, a total of 48 putative P450 genes were isolated from the metabolic-herbicide-resistant plants and used for gene expression analysis. The most suitable reference genes for accurate normalization of real-time quantitative PCR data were first identified in P. fugax and recognized as actin (ACT), 18S rRNA (18S), and ribulose-1,5-bisphosphate carboxylase oxygenase (RUBP) under fenoxaprop-P-ethyl stress, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elongation factor 1α (EF1α) under mesosulfuron-methyl stress, and ACT, EF1α, eukaryotic initiation factor 4a (EIF4A), and 25S rRNA (25S) at different growth stages. Expression analysis of the putative P450 genes revealed that six genes, respectively, annotated as CYP709B1, CYP71A1-4, CYP711A1, CYP78A9, P450-11, and P450-39 were up-regulated more than 10-fold in the resistant plants by fenoxaprop-P-ethyl treatment, and all of them exhibited constitutively and/or herbicide-induced higher transcript levels in the fenoxaprop-P-ethyl-resistant than in the susceptible plants. Three genes, respectively, annotated as CYPRO4, CYP313A4, and CYP51H11 constantly up-regulated in the resistant than in the susceptible plants after fenoxaprop-P-ethyl treatment. Up-regulated expressions of these specific P450 genes were consistent with the higher P450 contents determined in the resistant plants. These results will help to elucidate the mechanisms for P450-mediated metabolic-herbicide resistance in P. fugax as well as other grass weed species.

Transcriptome analysis and the identification of genes involved in the metabolic pathways of fenoxaprop-P-ethyl in rice treated with isoxadifen-ethyl hydrolysate.

Fenoxaprop-P-ethyl (FE) is a highly effective weed control agent for rice fields, but it causes phytotoxicity in crops. A whole-plant bioassay has revealed that isoxadifen-ethyl hydrolysate (IH) can significantly improve the tolerance of rice to FE, but the molecular mechanisms underlying this phenomenon are still unclear. In this study, we performed RNA-Seq analysis using rice seedlings treated with FE and IH to determine the IH-regulated candidate genes involved in metabolic resistance to FE. We also analyzed spatiotemporal expression using quantitative reverse transcription polymerase chain reaction to reveal the expression patterns of these genes under different treatments. The results showed that genes encoding metabolic enzymes, such as cytochrome P450 monooxygenases, glutathione-s-transferases, UDP-glycosyltransferase, carboxylesterase, and ATP-binding cassette transporter, were influenced by the application of IH. Most of these genes were upregulated, and their products were involved in various stages of FE metabolism. Tolerance to FE was primarily mediated by CarE15, CYP86A1, GSTU6, GST4, UGT13248, UGT79, and ABCC4, all of which played a vital role in regulating the detoxification process of FE. Our findings elucidated the protective mechanisms of IH, which can help alleviate the phytotoxic effects of FE and expand its potential for application in agriculture.

Identification and expression of main genes involved in non-target site resistance mechanisms to fenoxaprop-p-ethyl in Beckmannia syzigachne.

BACKGROUND: Non-target-site resistance (NTSR) to herbicides is a serious threat to global agriculture. Although metabolic resistance is the dominant mechanism of NTSR, the molecular mechanisms are not yet well-characterized. This study aimed to uncover the likely metabolism-related genes in Beckmannia syzigachne (American sloughgrass) resistant to fenoxaprop-p-ethyl. RESULTS: Ultra-performance liquid chromatography - tandem mass spectrometry experiments showed that the resistant American sloughgrass biotype (R, SD-04-SS) showed enhanced degradation of this herbicide compared to the susceptible biotype (S, SD-12). R and S biotype were harvested at 24 h after fenoxaprop-p-ethyl treatment to conduct RNA sequencing (RNA-Seq) analysis to investigate the likely fenoxaprop-p-ethyl metabolic genes. The RNA-Seq libraries yield 417 969 980 clean reads. The de novo assembly generated 115 112 unigenes, of which 57 906 unigenes were annotated. Finally, we identified 273 cytochrome P450s, 178 oxidases, 47 glutathione S-transferases (GSTs), 166 glucosyltransferases (GTs) and 180 ABC transporter genes to determine the likely fenoxaprop-p-ethyl metabolism-related genes in R biotype. Twelve overlapping up-regulated genes in the R biotype (fenoxaprop-p-ethyl-treated R/non-treated R, fenoxaprop-p-ethyl-treated R/fenoxaprop-p-ethyl-treated S) were identified by RNA-Seq and the results were validated using qRT-PCR. Ten were identified as fenoxaprop-p-ethyl metabolism-related genes, including three P450s (homologous to CYP71D7, CYP99A2 and CYP71D10), one GST (homologous to GSTF1), two GTs (homologous to UGT90A1 and UGT83A1) and four oxidase genes. CONCLUSION: This work demonstrates that the NTSR mechanism by means of enhanced detoxification of fenoxaprop-p-ethyl in American sloughgrass is very likely driven by herbicide metabolism related genes. The RNA-Seq data presented here provide a valuable resource for understanding the molecular mechanism of NTSR in American sloughgrass. © 2020 Society of Chemical Industry.

Constructing sustained-release herbicide formulations based on poly-3-hydroxybutyrate and natural materials as a degradable matrix.

BACKGROUND: The purpose of the present study was to develop ecofriendly herbicide formulations. Its main aim was to develop and investigate slow-release formulations of herbicides (metribuzin, tribenuron-methyl, and fenoxaprop-P-ethyl) of different structure, solubility, and specificity, which were loaded into a degradable matrix of poly-3-hydroxybutyrate (P(3HB)) blended with available natural materials (peat, clay, and wood flour). RESULTS: Differences in the structure and physicochemical properties of the formulations were studied depending on the type of the matrix. Herbicide release and accumulation in soil were associated with the solubility of the herbicide. Fourier-transform infrared spectroscopy showed that no chemical bonds were formed between the components in the experimental formulations. Degradation of the formulations in agro-transformed soil in laboratory conditions was chiefly influenced by the shape of the specimens (granules or pellets) while the effect of the type of filler (peat, clay, or wood flour) was insignificant. The use of granules enabled more rapid accumulation of the herbicides in soil: their peak concentrations were reached after 3 weeks of incubation while the concentrations of the herbicides released from the pellets were the highest after 5-7 weeks. Loading of the herbicides into the polymer matrix composed of the slowly degraded P(3HB) and natural materials enabled both sustained function of the formulations in soil (lasting between 1.5 and ≥3 months) and stable activity of the otherwise rapidly inactivated herbicides such as tribenuron-methyl and fenoxaprop-P-ethyl. CONCLUSION: The experimental herbicide formulations enabled slow release of the active ingredients to soil. © 2019 Society of Chemical Industry.

Degradation of Fenoxaprop-p-Ethyl and Its Metabolite in Soil and Wheat Crops.

The dissipation kinetics of fenoxaprop-p-ethyl and its metabolite (fenoxaprop acid) at two application rates under wheat field conditions for two seasons was investigated. Herbicides were extracted by solid liquid extraction, cleaned up, and analyzed by a liquid chromatography-UV detector. Dissipation followed first-order kinetics; in soil, fenoxaprop-p-ethyl dissipated rapidly with an average half-life of 1.45 to 2.30 days, while fenoxaprop acid persisted for more than 30 days. The method was validated in terms of accuracy, linearity, specificity, and precision. Linearity was in the range of 5 to 5,000 ng, with a limit of detection (LOD) of 2 and 1 ng for fenoxaprop-p-ethyl and fenoxaprop acid, respectively. The quantitation limits in soil, grain, and straw were 5, 8, and 10 ng g-1 for fenoxaprop-p-ethyl and 5, 10, and 10 ng g-1 for fenoxaprop acid, respectively. Recovery in soil, grains, and straw ranged from 85.1 to 91.25%, 72.5 to 84.66%, and 77.64 to 82.24% for fenoxaprop-p-ethyl and 80.56 to 86.5%, 78 to 81.88%, and 75.2 to 79.68% for fenoxaprop acid, respectively. At harvest, no detectable residues of fenoxaprop-p-ethyl or acid were observed in soil, wheat grain, and straw samples. Owing to the short persistence under field conditions, fenoxaprop-p-ethyl is safe for use because parent and metabolite residues were below the European Union maximum residue limit and would not pose an adverse effect on the environment and human or animal foods.

Fenoxaprop-P-ethyl resistance conferred by cytochrome P450s and target site mutation in Alopecurus japonicus.

BACKGROUND: Alopecurus japonicus is a serious grass weed species in wheat fields in eastern Asia, and has evolved strong resistance to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. Although target-site resistance (TSR) to ACCase inhibitors in A. japonicus has been reported, non-target site resistance (NTSR) has not. This study investigated both TSR and NTSR in a fenoxaprop-P-ethyl-resistant A. japonicus population (AHFD-3), which was collected in Feidong County, Anhui Province, China. RESULTS: We found that AHFD-3 exhibited high resistance to fenoxaprop-P-ethyl and low resistance to flucarbazone-sodium. The sensitivity of AHFD-3 to fenoxaprop-P-ethyl increased significantly after treatment with cytochrome P450 (P450) inhibitors; however, such synergies between P450 inhibitors and fenoxaprop-P-ethyl were not found in two control populations. Sequences of the entire carboxyltransferase domain of A. japonicus ACCase were obtained, and AHFD-3 plants showed an Asp-2078-Gly substitution in the ACCase. With the derived cleaved amplified polymorphic sequence (dCAPS) method, we found that 85.4% of the plants of AHFD-3 carried this mutation. The P450 content in AHFD-3 plants was significantly higher than those of the two control populations after treatment with fenoxaprop-P-ethyl. Ten partial sequences of P450 genes in A. japonicus were cloned. Three P450 genes were up-regulated 12 h after fenoxaprop-P-ethyl treatment, which were all from the P450 subfamily CYP72A. Moreover, a P450 gene from the P450 family CYP81 was up-regulated after fenoxaprop-P-ethyl treatment in all populations studied. CONCLUSION: Fenoxaprop-P-ethyl resistance in AHFD-3 plants was conferred by up-regulation of cytochrome P450s in the CYP72A subfamily and target site mutation of the ACCase gene. © 2018 Society of Chemical Industry.

Acetyl-CoA carboxylase overexpression in herbicide-resistant large crabgrass (Digitaria sanguinalis).

BACKGROUND: The occurrence of herbicide-resistant weed biotypes is increasing and this report of an acetyl-CoA carboxylase (ACCase) inhibitor-resistant Digitaria sanguinalis L. Scop. from southwestern Ontario is another example. The identified weed escaped control in an onion and carrot rotation in which graminicides were used for several consecutive years. Our goal was to characterize the level and mechanism of resistance of the biotype. RESULTS: The biotype was resistant to all five ACCase inhibitor herbicides tested. Gene-expression profiling was performed because none of the mutations known to confer resistance in the ACCase gene were detected. RNASeq and quantitative reverse-transcriptase PCR (qRT-PCR) results indicated that transcription of ACCase was 3.4-9.3 times higher in the resistant biotype than the susceptible biotype. ACCase gene copy number was determined by qPCR to be five to seven times higher in the resistant compared with the susceptible biotype. ACCase gene overexpression was directly related to the increase of the ACCase gene copy number. CONCLUSION: Our results are consistent with the hypothesis that overexpression of the herbicide target gene ACCase confers resistance to the herbicide. This is the first reported case of target gene duplication conferring resistance to a herbicide other than glyphosate. © 2017 Society of Chemical Industry See related Article.

miR397/Laccase Gene Mediated Network Improves Tolerance to Fenoxaprop-P-ethyl in Beckmannia syzigachne and Oryza sativa.

Herbicide resistance can be either target-site or non-target-site based. The molecular mechanisms underlying non-target-site resistance (NTSR) are poorly understood, especially at the level of gene expression regulation. MicroRNAs (miRNAs) represent key post-transcriptional regulators of eukaryotic gene expression and play important roles in stress responses. In this study, the miR397 gene from Beckmannia syzigachne (referred to as bsy-miR397) was functionally characterized to determine its role in regulating fenoxaprop-P-ethyl resistance. We showed that (1) bsy-miR397 transcript level is constitutively higher in resistant than in sensitive B. syzigachne plants, whereas bsy-Laccase expression and activity show the opposite trend, and (2) bsy-miR397 suppresses the expression of bsy-Laccase in tobacco, indicating that it negatively regulates bsy-Laccase at the transcriptional level. We found evidences that miR397/laccase regulation might be involved in fenoxaprop-P-ethyl NTSR. First, the rice transgenic line overexpressing OXmiR397 showed improved fenoxaprop-P-ethyl tolerance. Second, following activation of bsy-Laccase gene expression by CuSO4 treatment, fenoxaprop resistance in B. syzigachne tended to decrease. Therefore, we suggest that bsy-miR397 might play a role in fenoxaprop-P-ethyl NTSR in B. syzigachne by down-regulating laccase expression, potentially leading to the enhanced expression of three oxidases/peroxidases genes to introduce an active moiety into herbicide molecules in Phase-2 metabolism. Bsy-miR397, bsy-Laccase, and other regulatory components might form a regulatory network to detoxify fenoxaprop-P-ethyl in B. syzigachne, supported by the differential expression of transcription factors and oxidases/peroxidases in the rice transgenic line overexpressing OXmiR397. This implies how down-regulation of a gene (laccase) can enhance NTSR. Our findings shed light on the daunting task of understanding and managing complex NTSR in weedy plant species.

Physiological effects and toxin release in Microcystis aeruginosa and Microcystis viridis exposed to herbicide fenoxaprop-p-ethyl.

Fenoxaprop-p-ethyl (FPE) was studied for possible ecotoxicity on two representative toxigenic cyanobacteria including Microcystis aeruginosa and Microcystis viridis. Growth curves, chlorophyll a content, protein content, microcystin levels, oxidative stress, and apoptosis rates were measured for the two cyanobacteria after exposure to different concentrations of FPE. Results showed that the changes in chlorophyll a content and protein content were consistent with cell density, and M. viridis was more sensitive than M. aeruginosa to FPE. The results of oxidative stress indicated that FPE induced the generation of malondialdehyde (MDA) and enhanced the activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in these two cyanobacteria. To further explore the toxicity of FPE, apoptosis rates and toxin levels were measured for the two cyanobacteria. Different degrees of apoptosis rates were observed in the two cyanobacteria, and the apoptosis rates increased with the increase concentration of FPE. The intracellular and extracellular MC-LR were both affect by FPE. The presence of FPE in aquatic ecosystem may stimulate the synthesis and release of MC-LR, which may cause serious water pollution and pose threats to human health. These results may be useful for the ecotoxicity assessment of FPE and guiding the rational use of pesticides in agriculture.

Establishing a herbicide-metabolizing enzyme library in Beckmannia syzigachne to identify genes associated with metabolic resistance.

Non-target site resistance (NTSR) to herbicides is an increasing concern for weed control. Metabolic herbicide resistance is an important mechanism for NTSR. However, little is known about metabolic resistance at the genetic level. In this study, we have identified three fenoxaprop-P-ethyl-resistant American sloughgrass (Beckmannia syzigachne Steud.) populations, in which the molecular basis for NTSR remains unclear. To reveal the mechanisms of metabolic resistance, the genes likely to be involved in herbicide metabolism (e.g. for cytochrome P450s, esterases, hydrolases, oxidases, peroxidases, glutathione S-transferases, glycosyltransferases, and transporter proteins) were isolated using transcriptome sequencing, in combination with RT-PCR (reverse transcription-PCR) and RACE (rapid amplification of cDNA ends). Consequently, we established a herbicide-metabolizing enzyme library containing at least 332 genes, and each of these genes was cloned and the sequence and the expression level compared between the fenoxaprop-P-ethyl-resistant and susceptible populations. Fifteen metabolic enzyme genes were found to be possibly involved in fenoxaprop-P-ethyl resistance. In addition, we found five metabolizing enzyme genes that have a different gene sequence in plants of susceptible versus resistant B. syzigachne populations. These genes may be major candidates for herbicide metabolic resistance. This established metabolic enzyme library represents an important step forward towards a better understanding of herbicide metabolism and metabolic resistance in this and possibly other closely related weed species. This new information may help to understand weed metabolic resistance and to develop novel strategies of weed management.

Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh).

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L(-1) FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.

Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh)

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.

Biodegradation of fenoxaprop-P-ethyl (FE) by Acinetobacter sp. strain DL-2 and cloning of FE hydrolase gene afeH.

Fenoxaprop-P-ethyl (FE) is widely used as a post-emergence aryloxyphenoxy propionate (AOPP) herbicide in agriculture. An efficient FE-degrading strain DL-2 was isolated from the enrichment culture and identified as Acinetobacter sp. and the metabolite fenoxaprop acid (FA) was identified by HPLC/MS analysis. The strain DL-2 could also degrade a wide range of other AOPP herbicides. A novel FE hydrolase esterase gene afeH was cloned from strain DL-2 and functionally expressed in Escherichia coli BL21(DE3). The specific activities of recombinant AfeH was 216.39 U mg(-1) for FE with Km and Vmax values of 0.82 µM and 7.94 µmol min(-1) mg(-1). AfeH could also hydrolyze various AOPP herbicides, p-nitrophenyl esters and triglycerides. The optimal pH and temperature for recombinant AfeH were 9.0 and 50°C, respectively; the enzyme was activated by Co(2+) and inhibited by Ca(2+), Zn(2+), Ba(2+). AfeH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by dimethyl sulfoxide.

An effective method, composed of LAMP and dCAPS, to detect different mutations in fenoxaprop-P-ethyl-resistant American sloughgrass (Beckmannia syzigachne Steud.) populations.

The decreased susceptibility of Beckmannia syzigachne to fenoxaprop-P-ethyl is due to the reliance on it to control grass weeds since the 1990s. Loop-mediated isothermal amplification (LAMP), which is a proven simple, rapid, specific, sensitive and inexpensive assay method, has been used to detect the I1781L mutation in B. syzigachne. In the present study, four sets of primers detected four mutations in B. syzigachne, W2027C, I2041A, D2078G and G2096A, using the LAMP method. Additionally, five newly derived cleaved amplified polymorphic sequence (dCAPS) markers were developed to detect five different mutations. With a method composed of LAMP and dCAPS, 19 fenoxaprop-P-ethyl-resistant B. syzigachne populations collected in 2013 were studied. An effective method, composed of LAMP and dCAPS, to detect five mutations, I1781L, W2027C, I2041A, D2078G and G2096A, in B. syzigachne populations was developed. With this method, a B. syzigachne population resistant to fenoxaprop-P-ethyl can be studied to confirm its constitution. And we determined that the resistance level might be relevant to the mutation type and mutation frequency. The type of mutation and its frequency in fenoxaprop-P-ethyl-resistant B. syzigachne populations can be confirmed to provide appropriate herbicide management.

Detection of the I1781L mutation in fenoxaprop-p-ethyl-resistant American sloughgrass (Beckmannia syzigachne Steud.), based on the loop-mediated isothermal amplification method.

BACKGROUND: The increasing use of fenoxaprop-p-ethyl has resulted in evolved resistance in American sloughgrass (Beckmannia syzigachne Steud.). Target-site-based resistance to acetyl-CoA carboxylase (ACCase) inhibitors in B. syzigachne occurs owing to an isoleucine-to-leucine substitution at residue 1781 (I1781L) of the ACCase enzyme. A rapid detection method is needed to identify the resistance-conferring substitution. RESULTS: Four populations of B. syzigachne that were resistant to fenoxaprop-p-ethyl and contained the I1781L substitution were identified. Conventional PCR and derived cleaved amplified polymorphic sequence (dCAPS) methods were used to detect the mutation. Additionally, a rapid nucleic acid detection method, loop-mediated isothermal amplification (LAMP), was successfully developed and used to detect the genetic mutation underlying the I1781L substitution in the B. syzigachne ACCase enzyme. CONCLUSION: This report is the first to describe the application of a LAMP assay for mutation detection in herbicide-resistant weeds. The assay does not require specialised equipment: only a standard laboratory bath is needed. This technique could be employed for detecting the I1781L substitution in B. syzigachne plants and seeds.