Microbial degradation as a powerful weapon in the removal of sulfonylurea herbicides.

Sulfonylurea herbicides have been widely used worldwide and play a significant role in modern agricultural production. However, these herbicides have adverse biological effects that can damage the ecosystems and harm human health. As such, rapid and effective techniques that remove sulfonylurea residues from the environment are urgently required. Attempts have been made to remove sulfonylurea residues from environment using various techniques such as incineration, adsorption, photolysis, ozonation, and microbial degradation. Among them, biodegradation is regarded as a practical and environmentally responsible way to eliminate pesticide residues. Microbial strains such as Talaromyces flavus LZM1, Methylopila sp. SD-1, Ochrobactrum sp. ZWS16, Staphylococcus cohnii ZWS13, Enterobacter ludwigii sp. CE-1, Phlebia sp. 606, and Bacillus subtilis LXL-7 can almost completely degrade sulfonylureas. The degradation mechanism of the strains is such that sulfonylureas can be catalyzed by bridge hydrolysis to produce sulfonamides and heterocyclic compounds, which deactivate sulfonylureas. The molecular mechanisms associated with microbial degradation of sulfonylureas are relatively poorly studied, with hydrolase, oxidase, dehydrogenase and esterase currently known to play a pivotal role in the catabolic pathways of sulfonylureas. Till date, there are no reports specifically on the microbial degrading species and biochemical mechanisms of sulfonylureas. Hence, in this article, the degradation strains, metabolic pathways, and biochemical mechanisms of sulfonylurea biodegradation, along with its toxic effects on aquatic and terrestrial animals, are discussed in depth in order to provide new ideas for remediation of soil and sediments polluted by sulfonylurea herbicides.

Development of immunoassay based on a specific antibody for sensitive detection of nicosulfuron in environment.

Nicosulfuron, one of the most widely used selective herbicides in corn field, can effectively control annual and perennial grass weeds, sedges, and some broadleaf weeds. The residual phytotoxicity of nicosulfuron in soil and water has become increasingly prominent. Therefore, an efficient method for detection of nicosulfuron was critical to ensure the sustainable and healthy development of agriculture and the ecological environment. In this paper, five nicosulfuron haptens which contained carboxyl group or aldehyde groups were designed and synthesized, and an indirect competitive immunoassay was developed for the first time. The assay showed an IC50 of 8.42 ng/mL and had negligible cross reactivities toward other sulfonylurea herbicides. In the spike and recovery studies, the recovery rate from soil samples was 95 %-104 %, and that of wheat roots was 92 %-98 %, which showed a good correlation with LC-MS analysis for nicosulfuron. The immunoassay was then used to quantify nicosulfuron concentration which could cause the obvious phytotoxic symptoms to wheat. Obvious symptoms of nicosulfuron phytotoxicity in wheat root was observed at the concentration of 0.068 ± 0.006 mg/kg (ELISA result) which was consistent with 0.072 ± 0.007 mg/kg obtained by LC-MS. The developed immunoassay method is an effective tool for environment contamination monitoring.

Citric acid modified ultrasmall copper peroxide nanozyme for in situ remediation of environmental sulfonylurea herbicide contamination.

Herbicide residues in the environment threaten high-quality agriculture and human health. Consequently, in situ remediation of herbicide contamination is vital. We synthesized a novel self-catalyzed nanozyme, ultrasmall (2-3 nm) copper peroxide nanodots modified by citric acid (CP@CA) for this purpose, which can break down into H2O2 and Cu2+ in water or soil. Ubiquitous glutathione reduces Cu2+ into Cu+, which promotes the decomposition of H2O2 into •OH through a Fenton-like reaction under mild acid conditions created by the presence of citric acid. The generated •OH efficiently degrade nicosulfuron in water and soil, and the maximum degradation efficiency could be achieved at 97.58% in water at 56 min. The possible degradation mechanisms of nicosulfuron were proposed through the 25 intermediates detected. The overall ecotoxicity of the nicosulfuron system was significantly reduced after CP@CA treatment. Furthermore, CP@CA had little impact on active components of soil bacterial community. Moreover, CP@CA nanozyme could effectively remove seven other sulfonylurea herbicides from the water. In this paper, a high-efficiency method for herbicide degradation was proposed, which provides a new reference for the in situ remediation of herbicide pollution.

Effects of long-term exposure to the herbicide nicosulfuron on the bacterial community structure in a factory field.

This study aims to investigate the effects of long-term nicosulfuron residue on an herbicide factory ecosystem. High-throughput sequencing was used to investigate the environmental microbial community structure and interactions. The results showed that the main contributor to the differences in the microbial community structure was the sample type, followed by oxygen content, pH and nicosulfuron residue concentration. Regardless of the presence or absence of nicosulfuron, soil, sludge, and sewage were dominated by groups of Bacteroidetes, Actinobacteria, and Proteobacteria. Long-term exposure to nicosulfuron increased alpha diversity of bacteria and archaea but significantly decreased the abundance of Bacteroidetes and Acidobateria compared to soils without nicosulfuron residue. A total of 81 possible nicosulfuron-degrading bacterial genera, e.g., Rhodococcus, Chryseobacterium, Thermomonas, Stenotrophomonas, and Bacillus, were isolated from the nicosulfuron factory environmental samples through culturomics. The co-occurrence network analysis indicated that the keystone taxa were Rhodococcus, Stenotrophomonas, Nitrospira, Terrimonas, and Nitrosomonadaceae_MND1. The strong ecological relationship between microorganisms with the same network module was related to anaerobic respiration, the carbon and nitrogen cycle, and the degradation of environmental contaminants. Synthetic community (SynCom), which provides an effective top-down approach for the critical degradation strains obtained, enhanced the degradation efficiency of nicosulfuron. The results indicated that Rhodococcus sp. was the key genus in the environment of long-term nicosulfuron exposure.

Nicosulfuron, a sulfonylurea herbicide, alters embryonic development and oxidative status of hatchlings at environmental concentrations in an amphibian species.

The widespread use of agrochemicals for controlling pests and diseases of crops is recognized as a main threat to biodiversity. Sulfonylurea herbicides are being increasingly used and display low levels of degradation in water which suggest that they might affect non-target organisms. In a common garden experiment, eggs of a widespread amphibian (Bufo spinosus) were exposed to sublethal environmentally relevant concentrations of a widely used sulfonylurea herbicide, nicosulfuron, during the whole embryonic development. We assessed development-related traits (i.e., development duration, hatching success, hatchling size and occurrence of malformation) as well as antioxidant markers in response to contamination (i.e., SOD, GPx, catalase, thiols and relevant ratios thereof). We found that sublethal concentrations of nicosulfuron increased embryonic development duration, increased hatchling size and tended to increase malformations. Embryos exposed to nicosulfuron displayed decreased thiols and increased catalase activity suggesting alteration of oxidative status. We did not find any effect of nicosulfuron on SOD and GPx levels. Interestingly, higher catalase activity was linked to higher proportion of malformed individuals, suggesting that exposure to nicosulfuron induced teratogenic effects. Our results suggest that alteration of antioxidant levels might be one physiological mechanism through which nicosulfuron might cause detrimental effects on amphibian embryos. Sublethal effects of pesticides at environmentally relevant concentrations have been overlooked and require further investigations, especially in non-target taxa occurring in agricultural landscapes.

Sub-structure-based category formation for the prioritisation of genotoxicity hazard assessment for pesticide residues: Sulphonyl ureas.

In dietary risk assessment, residues of pesticidal ingredients or their metabolites need to be evaluated for their genotoxic potential. The European Food Safety Authority recommend a tiered approach focussing assessment and testing on classes of similar chemicals. To characterise similarity and to identify structural alerts associated with genotoxic concern, a set of chemical sub-structures was derived for an example dataset of 74 sulphonyl urea agrochemicals for which either Ames, chromosomal aberration or micronucleus test results are publicly available. This analysis resulted in a set of seven structural alerts that define the chemical space, in terms of the common parent and metabolic scaffolds, associated with the sulphonyl urea chemical class. An analysis of the available profiling schemes for DNA and protein reactivity shows the importance of investigating the predictivity of such schemes within a well-defined area of structural space. Structural space alerts, covalent chemistry profiling and physico-chemistry properties were combined to develop chemical categories suitable for chemical prioritisation. The method is a robust and reproducible approach to such read-across predictions, with the potential to reduce unnecessary testing. The key challenge in the approach was identified as being the need for pesticide-class specific metabolism data as the basis for structural space alert development.

Cardiovascular outcomes of type 2 diabetic patients treated with DPP­4 inhibitors versus sulphonylureas as add-on to metformin in clinical practice.

DPP-4 inhibitors (DPP-4i) and sulphonylureas remain the most widely prescribed add-on treatments after metformin. However, there is limited evidence from clinical practice comparing major adverse cardiovascular events (MACE) in patients prescribed these treatments, particularly among those without prior history of MACE and from vulnerable population groups. Using electronic health records from UK primary care, we undertook a retrospective cohort study with people diagnosed type-2 diabetes mellitus, comparing incidence of MACE (myocardial infarction, stroke, major cardiovascular surgery, unstable angina) and all-cause mortality among those prescribed DPP-4i versus sulphonylureas as add-on to metformin. We stratified analysis by history of MACE, age, social deprivation and comorbidities and adjusted for HbA1c, weight, smoking-status, comorbidities and medications. We identified 17,570 patients prescribed sulphonylureas and 6,267 prescribed DPP-4i between 2008-2017. Of these, 16.3% had pre-existing MACE. Primary incidence of MACE was similar in patients prescribed DPP-4i and sulphonylureas (10.3 vs 8.5 events per 1000 person-years; adjusted Hazard Ratio (adjHR): 0.94; 95%CI 0.80-1.14). For those with pre-existing MACE, rates for recurrence were higher overall, but similar between the two groups (21.8 vs 17.2 events per 1000 person-years; adjHR: 0.93; 95%CI 0.69-1.24). For those aged over 75 and with BMI less than 25 kg/m2 there was a protective effect for DPP-I, warranting further investigation. Patients initiating a DPP-4i had similar risk of cardiovascular outcomes to those initiating a sulphonylurea. This indicates the choice should be based on safety and cost, not cardiovascular prognosis, when deciding between a DPP-4i or sulphonylurea as add-on to metformin.

Effects of sulfometuron-methyl on zebrafish at early developmental stages.

Sulfometuron methyl (SM) is a widely used herbicide and thus leading to accumulation in the environment. The toxicity assessments of SM in model organisms are currently rare. In the present study, zebrafish were utilized for evaluating the detrimental effects of SM in aquatic vertebrates. Zebrafish embryos were exposed to 0, 10, 20, and 40 mg/L SM from 5.5 to 72 h post-fertilization (hpf), respectively. Consequently, SM exposure resulted in increasing the mortality rate and reducing hatching rate in larval zebrafish at 10, 20, and 40 mg/L SM-treated groups. The reduced numbers of immune cells (neutrophils and macrophages) were observed after SM exposure by a dose-dependent manner. The inflammatory responses (TLR4, MYD88, IL-1ß, IL-6, IL-8, IFN-γ, IL-10, and TGF-ß) were measured to estimate immune responses. Anti-inflammatory factors (IL-10 and TGF-ß) were down-regulated in all the treated groups and significantly altered at 40 mg/L exposure group. Additionally, behavioral tests suggested that SM treatment significantly increased the total distance, average speed, and maximum acceleration of larval zebrafish during light-dark transition and subsequently enzymology test displayed the same trend to locomotor behaviors. The content significantly increased in oxidative stress, as reflected in ROS level in all the treated groups. The numbers of cell apoptosis were significantly increased at 20, and 40 mg/L and the highest concentration group induced the substantial increment (P < 0.001) of apoptosis-related genes including p53, Bax/Bcl-2, caspase-9, and caspase-3. In summary, our results demonstrated that exposure to SM caused toxicity of development, immune system, locomotor behavior, oxidative stress, and cell apoptosis at the early developmental stages of zebrafish.

Adaptation responses in C4 photosynthesis of sweet maize (Zea mays L.) exposed to nicosulfuron.

Nicosulfuron is an ingredient in photosynthesis-inhibiting herbicides and has been widely used in corn post-emergence weed control. In the current study, a pair of sister lines, HK301 (nicosulfuron-tolerence, NT) and HK320 (nicosulfuron-sensitive, NS), was used to study the effect of nicosulfuron in sweet maize seedlings on C4 photosynthetic enzymes and non-enzymatic substances, expression levels of key enzymes, and chloroplast structure. Nicosulfuron was sprayed at the four-leaf stage, and water was sprayed as a control. After nicosulfuron treatment, phosphoenolpyruvate carboxylase (PEPC), NADP-malic dehydrogenase (NADP-MDH), NADP-malic enzyme (NADP-ME), pyruvate orthophosphate dikinase (PPDK), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities of NT were significantly higher than those of NS. Compared to NT, malate, oxaloacetic acid, and pyruvic acid significantly decreased as exposure time increased in NS. Compared to NS, nicosulfuron treatment significantly increased the expression levels of PEPC, NADP-MDH, NADP-ME, PPDK, and Rubisco genes in NT. Under nicosulfuron treatment, chloroplast ultrastructure of NS, compared to that of NT, nicosulfuron induced swelling of the chloroplast volume and reduced starch granules in NS. In general, our results indicate that in different resistant sweet maize, C4 photosynthetic enzymes activity and key genes expression play a critical role in enhancing the adaptability of plants to nicosulfuron stress at a photosynthetic physiological level.

A potential role of calpains in sulfonylureas (SUs) -mediated death of human pancreatic cancer cells (1.2B4).

Sulfonylureas (SUs) are suggested to accelerate the pancreatic ß-cells mass loss via apoptosis. However, little is known whether calpains mediate this process. The aim of the present study is to evaluate the involvement of calpains in SUs-induced death of human pancreatic cancer (PC) cell line 1.2B4. The cells were exposed to: glibenclamide, glimepiride and gliclazide for 72 h. The expression analysis of caspase-3 (CASP-3), TP53, calpain 1 (CAPN-1), calpain 2 (CAPN-2) and calpain 10 (CAPN-10) was detected using RT-PCR method. Intracellular Ca2+ concentrations, CASP-3 activity and total calpain activity were also evaluated. Our results have shown that glibenclamide and glimepiride decrease 1.2B4 cells viability with accompanied increase in intracellular Ca2+ concentration and increased expression of apoptosis-related CASP-3 and TP53. Gliclazide did not affect 1.2B4 cell viability and Ca2+ concentration, however, it downregulated CASP-3 and upregulated TP53. Interestingly, 50 µM glimepiride increased expression of CAPN-1, CAPN-2 and CAPN-10 whereas 50 µM glibenclamide solely upregulated CAPN-2 expression. We have shown that 10 µM and 50 µM glibenclamide and glimepiride increased the activity of CASP-3, but decreased total calpain activity. Our results suggest that calpains may be involved in glibenclamide- and glimepiride-induced death of PC cells. However, further investigation is required to confirm the engagement of calpains in SUs-mediated death of PC cells, especially studies on protein level of particular isoforms of calpains should be conducted.

Protective efficacy of phenoxyacetyl oxazolidine derivatives as safeners against nicosulfuron toxicity in maize.

BACKGROUND: Herbicide safeners mitigate crop damage without reducing herbicide efficacy. Here, the protective effects of phenoxyacetyl oxazolidine derivatives as potential safeners were evaluated with a view toward reducing injury caused by sulfonylurea herbicide nicosulfuron to sensitive maize varieties. RESULTS: Growth indices demonstrated that the bioactivity of compound 9 (N-phenoxyacety-2-methyl-2,4-diethyl-1,3-oxazolidine) was superior to that of R-28725 and all other compounds tested. Compound 9 induced endogenous glutathione and upregulated glutathione-S-transferase (GST) in maize. Thus, it could enhance maize tolerance to nicosulfuron. Compared with the untreated water control group, the maximum reaction rate of GST was increased by 37.62%, while the maximum velocity of GST was decreased by 61.93% after treatment with compound 9. Acetolactate synthase relative activity was significantly enhanced in the case of treatment with compound 9, indicating the excellent protective effects of compound 9 against nicosulfuron in maize. CONCLUSIONS: The present work demonstrates that phenoxyacetyl oxazolidine derivatives are potentially efficacious as herbicide safeners and merit further investigation.

Design, Synthesis, and SAR of Novel 1,3-Disubstituted Imidazolidine or Hexahydropyrimidine Derivatives as Herbicide Safeners.

Herbicide safeners enhance herbicide detoxification in crops without reducing their herbicidal efficacy against target weeds. To alleviate maize injury caused by the sulfonylurea herbicide nicosulfuron, a series of 1,3-disubstituted imidazolidine or hexahydropyrimidine derivatives were rationally designed via bioisosterism and active subunit combinations. Thirty novel compounds were synthesized using an efficient one-pot method and low-cost raw materials and characterized by IR, 1H NMR, 13C NMR, and high-resolution mass spectrometer (HRMS). Bioactivity and structure-activity relationship (SAR) were evaluated for herbicide safeners tested against nicosulfuron injury. Most of the compounds effectively protected sensitive maize against nicosulfuron damage. The parent skeletons and substituents of the target compounds both substantially influenced their safener activity. Compound I-3 exhibited superior bioactivity compared to the safener isoxadifen-ethyl. Molecular docking simulations disclosed that compound I-3 competed with nicosulfuron for the acetolactate synthase active site and demonstrated that this is the protective mechanism of safeners. The target compound I-3 presented with strong herbicide safener activity in maize and is, therefore, a potential candidate for the development of a novel herbicide safener.

Integrated proteomics, metabolomics and physiological analyses for dissecting the toxic effects of halosulfuron-methyl on soybean seedlings (Glycine max merr.).

Halosulfuron methyl (HSM) is a herbicide widely used to control sedge and broad-leaved weeds during crop production, but its environmental residue may damage non-target crops. Here, proteomics and metabolomics methods were used to explore the phytotoxicity mechanisms of HSM against soybean (Glycine max Merr.). Soybean seedlings were exposed to 0.01, 0.05 and 0.5 mg/L HSM for 8 d. The HSM applications significantly reduced chlorophyll and carotenoid contents in HSM-treated seedlings. Additionally, chlorophyll a fluorescence was seriously affected. The glutathione, hydrogen peroxide and malondialdehyde contents, as well as antioxidant enzyme activities, significantly increased in seedlings exposed to HSM. Furthermore, five enzymes involved in the tricarboxylic acid (TCA) cycle, α-ketoglutarate dehydrogenase, isocitrate dehydrogenase, aconitase, malic dehydrogenase and succinate dehydrogenase, were inhibited to varying degrees in HSM-treated seedlings compared with controls. Proteomics results showed multiple differentially abundant proteins involved in chlorophyll synthesis, photosystem processes and chloroplast ATP synthetase were down-regulated. Metabolomics analyses revealed that metabolites involved in the TCA cycle decreased significantly. Moreover, metabolites and proteins related to reactive oxygen species detoxification accumulated. In conclusion, the phytotoxicity mechanisms of HSM against soybean mainly act by damaging the photosynthetic machinery, inhibiting chlorophyll synthesis, interrupting the TCA cycle and causing oxidative stress. These results provide new insights into the toxicity mechanisms of sulfonylurea herbicides against non-target crops.

Dipteryx alata, a tree native to the Brazilian Cerrado, is sensitive to the herbicide nicosulfuron.

The expansion of land use for agricultural interests and the excessive use of herbicides are among the causes of biodiversity losses in the Brazilian Cerrado biome. Therefore, we aimed to test the hypothesis that Dipteryx alata Vogel, a common species in this biome, is sensitive to nicosulfuron because of its high phytotoxicity. We evaluated physiological, biochemical and morphological responses in D. alata plants exposed to increasing doses of the herbicide. Young plants were transplanted to 10 L pots containing substrate composed of soil and sand (2:1) after fertilization. After an acclimation period, the following doses of nicosulfuron were applied: 0 (control), 6, 12, 24, 48, and 60 g a.e. ha-1. The experiment was conducted in a randomized block design factorial scheme with six doses of nicosulfuron, three evaluation times, and five replicates per treatment. The effects of the herbicide were assessed by measuring gas exchange, chlorophyll a fluorescence, photosynthetic pigments, membrane permeability, antioxidant enzymes and acetolactate synthase. Nicosulfuron altered the photosynthetic machinery and enzymatic metabolism of D. alata. Reductions in physiological traits, increased catalase and ascorbate peroxidase activities, enhanced malondialdehyde concentrations rate of electrolyte leakage and decreased acetolactate synthase activity in response to nicosulfuron all suggest that D. alata is sensitive to this herbicide.

Safeners Improve Maize Tolerance under Herbicide Toxicity Stress by Increasing the Activity of Enzymes in Vivo.

Tribenuron-methyl (TM), as one of the sulfonylurea (SU) herbicides, has been widely and effectively applied for many kinds of plants. SUs inhibit plant growth by restraining the biosynthetic pathway of branched-chain amino acids (BCAAs) catalyzed by acetolactate synthase (ALS). Safeners are agrochemicals that protect crops from herbicide injuries. To improve the crop tolerance under TM toxicity stress, this paper evaluated the protective effect of N-tosyloxazolidine-3-carboxamide. It turned out that most of the tested compounds showed significant protection against TM via enhancing the glutathione (GSH) content and glutathione S-transferase (GST) activity. Among all of the tested compounds, compound 16 exhibited more excellent protection than the contrast safener R-28725 and other target compounds. A positive correlation between the growth level, endogenous GSH content, and GST activity was observed in this research. The GST kinetic parameter Vmax of the maize was increased by 29.6% after treatment with compound 16, while Km was decreased by 51.9% compared to the untreated control. The molecular docking model indicated that compound 16 could compete with TM in the active site of ALS, which could interpret the protective effects of safeners. The present work demonstrated that N-tosyloxazolidine-3-carboxamide derivatives could be considered as potential candidates for developing new safeners in the future.

Respiration induced by substrate and bacteria diversity after application of diuron, hexazinone, and sulfometuron-methyl alone and in mixture.

After application, herbicides often reach the soil and affect non-target soil microorganisms, decreasing their population, diversity or affecting metabolic activity. Therefore, laboratory studies were performed to evaluate the effects of diuron, hexazinone and sulfometuron-methyl alone and mixed upon carbon transformation by soil microorganisms in clayey and sandy soils and the effect on bacterial diversity and structure. Control treatment without herbicide application was also performed. Sub-samples from the control and herbicide treatments (10 g - in triplicate) were collected before herbicide application and 7, 14, 28 and 42 days after treatment (DAT), then 1 mL of 14C-glucose solution was applied. The released 14CO2 was trapped in 2 M NaOH solution and the radioactivity was analyzed by liquid scintillation counting (LSC), 12 h after glucose application. The effect of herbicides on bacterial diversity was evaluated by T-RFLP. The experiment was conducted in a complete randomized design. Hexazinone did not affect 14CO2 evolution. Diuron showed a greater 14CO2 evolution in sandy and clayey soil, while sulfometuron-methyl led to an increase in sandy soil, at 42 DAT. A greater evolution of carbon was observed in the treatment with herbicide mixture in sandy soil, compared with the same treatment in clayey soil or control. However, the herbicide mixture application did not affect the soil biological activity measured by the respiration rate induced by substrate. On the other hand, the herbicide mixtures affected the bacterial diversity in both soils, being the strongest effect to diuron and sulfometuron-methyl in clayey soil and hexazinone in sandy soil.

Effects of bensulfuron-methyl residue on photosynthesis and chlorophyll fluorescence in leaves of cucumber seedlings.

A potted soil experiment was conducted to investigate the effects of bensulfuron-methyl (BSM) residue on the growth and photosynthesis of seedlings of a local cucumber variety (Xia Feng No.1). When the residue of bensulfuron-methyl in soil exceeded 50µg kg-1, it significantly inhibited the growth of cucumber, chlorophyll content and photosynthetic capacity of cucumber. BSM treatment caused significant decreases in the biomass, chlorophyll content, net photosynthesis rate, stomatal conductance, and transpiration rate, photosystem II (PSII) maximum quantum yield, actual quantum yield, photochemical quenching coefficient, and electron transport rate in cucumber seedlings, but increased the minimal fluorescence yield and dark respiration rate. Moreover, comparisons of the patterns of absorbed light energy partitioning revealed that the fractions of excess and thermally dissipated energy increased with rising concentrations of the BSM residue, but the fraction of PSII photochemistry declined. The BSM residues caused reversible destruction in the PSII reaction centers and decreased the proportion of available excitation energy used in PSII photochemistry. The results suggested that rice or wheat fields sprayed with BSM will not be suitable for planting cucumbers in succession or rotation.

Unravelling mesosulfuron-methyl phytotoxicity and metabolism-based herbicide resistance in Alopecurus aequalis: Insight into regulatory mechanisms using proteomics.

Non-target-site based resistance (NTSR), a poorly understood multigenic trait, has evolved as the greatest threat to crop production worldwide, by endowing weed plants an unpredictable pattern of resistance to herbicides. Our recent work with multiple-herbicide-resistant shortawn foxtail (Alopecurus aequalis Sobol.) biotype has preliminary indicated that cytochrome P450s-involved enhanced rate of mesosulfuron-methyl metabolism may involve in the NTSR. Here by further determining the differences in glutathione S-transferase (GST) activity and uptake and metabolic rates of mesosulfuron between resistant (R) and susceptible (S) A. aequalis plants, and associating them with endogenous differently regulated proteins (DEPs) identified from combinational proteomics analyses, we provided direct evidences on the enhanced herbicide degradation in resistant plants. Subsequently, the physiological phenotypes of photosynthesis, chlorophyll fluorescence, and antioxidation were compared between R and S plants and linked with correlative DEPs, indicating a series of key pathways including solar energy capture, photosynthetic electron transport, redox homeostasis, carbon fixation, photorespiration, and reactive oxygen species scavenging in susceptible plants were broken or severely damaged by mesosulfuron stress. In comparison, resistant plants have evolved enhanced herbicide degradation to minimize the accumulation of mesosulfuron and protect the photosynthesis and ascorbate-glutathione cycle against the adverse effects of chemical injury, giving A. aequalis plants a NTSR phenotype. Additionally, three key proteins respectively annotated as esterase, GST, and glucosyltransferase were identified and enabled as potential transcriptional markers for quick diagnosing the metabolic mesosulfuron resistance in A. aequalis species.

Rapid identification of a candidate nicosulfuron sensitivity gene (Nss) in maize (Zea mays L.) via combining bulked segregant analysis and RNA-seq.

KEY MESSAGE: A candidate nicosulfuron sensitivity gene Nss was identified by combining bulked segregant analysis and RNA-seq. Multiple mutations of this gene were discovered in nicosulfuron-sensitive maize compared with the tolerant. It has been demonstrated that variabilities exist in maize response to nicosulfuron. Two nicosulfuron-sensitive inbred lines (HB39, HB41) and two tolerant inbred lines (HB05, HB09) were identified via greenhouse and field trials. Genetic analysis indicated that the sensitivity to nicosulfuron in maize was controlled by a single, recessive gene. To precisely and rapidly map the nicosulfuron sensitivity gene (Nss), two independent F2 segregating populations, Population A (HB41 × HB09) and Population B (HB39 × HB05), were constructed. By applying bulked segregant RNA-Seq (BSR-Seq), the Nss gene was, respectively, mapped on the short arm of chromosome 5 (chr5: 1.1-15.3 Mb) and (chr5: 0.5-18.2 Mb) using two populations, with 14.2 Mb region in common. Further analysis revealed that there were 43 and 119 differentially expressed genes in the mapping intervals, with 18 genes in common. Gene annotation results showed that a cytochrome P450 gene (CYP81A9) appeared to be the candidate gene of Nss associated with nicosulfuron sensitivity in maize. Sequence analysis demonstrated that two common deletion mutations existed in the sensitive maize, which might lead to the nicosulfuron sensitivity in maize. The results will make valuable contributions to the understanding of molecular mechanism of herbicide sensitivity in maize.

Acute and subchronic effects of three herbicides on biomarkers and reproduction in earthworm Dendrobaena veneta.

Earthworms are exposed to herbicides both through their skin and digestive system. Herbicides can influence earthworms' survival, physiology and reproduction. However, there is a lack of data on herbicide effects on earthworms as they are often regarded as low or non-toxic. The aim of our study was to investigate whether widely used commercial formulations of glyphosate (GLF), tembotrione (TBT) and nicosulfuron (NCS) each applied at three environmentally relevant concentrations have adverse effects on various biomarkers and reproduction in epigeic earthworm Dendrobaena veneta. The activities of measured biomarkers varied depending on the herbicide used and the exposure duration and suggest that oxidative stress plays an important role in the toxicity of tested herbicides. Namely, GLF caused an acetylcholinesterase (AChE) activity induction after seven days, and NCS after 28 days, while TBT caused an inhibition up to 47% (6.6 µg kgdw soil-1) after seven days. Only TBT caused a significant change (H2 = 13.96, p = 0.002) to catalase (CAT) after seven days of exposure. Malondialdehyde concentrations (MDA) were increased all the time after NCS exposure, but only after seven days in GLF and 28 days in TBT treatments, respectively. The tested herbicides did not have a significant effect on reproduction success, expect of NCS which increased the number of juveniles (p < 0.05).