A Three-Component Enzyme System Catalyzes the O Demethylation of the Herbicide Dicamba in Pseudomonas maltophilia DI-6

An enzyme activity which converts dicamba (2-methoxy-3,6-dichlorobenzoic acid) to 3,6-dichlorosalicylic acid in vitro has been detected in cell lysates of Pseudomonas maltophilia DI-6. Phenyl-Sepharose column chromatography of a partially purified lysate resulted in the separation of this enzyme into three separate protein components tentatively identified as an oxygenase, a ferredoxin, and a reductase. The activity of dicamba O-demethylase was dependent on oxygen and required NADH and Mg(sup2+).     

Specificity of aFlavobacterium in the metabolism of substituted chlorobenzoates

Summary A strain ofFlavobacterium breve capable of utilizing 3,5-dichlorosalicylate as a sole source of carbon and energy was identified. Degradation of 3,5-dichlorosolicylate, was specific as this strain did not metabolize dicamba (3,6-dichloro-2-methoxybenzoic acid), 3,5-dicamba (3,5-dichloros2-methoxybenzoic acid), or 3,6-dichlorosalicylate. The organism was able to remove completely 3,5-dichlorosalicylate in the presence of three times as much 3,6-dichlorosalicylate being degraded. The organism was able to utilize 3,5-dichlorosalicylate at concentrations up to 1000g/ml. A mixture of 3,5 and 3,6-dichlorosalicylate isomers purified by biological destruction of the unwanted isomer (3,5-dichlorosalicylate) would be useful for producing isomerically pure dicamba, an important herbicide.     

Recruitment of naphthalene dissimilatory enzymes for the oxidation of 1,4-dichloronaphthalene to 3,6-dichlorosalicylate, a precursor for the herbicide dicamba.

Pseudomonas putida expresses plasmid-encoded enzymes and regulatory proteins for the dissimilation of naphthalene through salicylate and the alpha-keto acid pathway. A strain of P. putida (NAH:Tn5/G67) defective in salicylate hydroxylase (nahG) was assessed for its ability to oxidize 1,4-dichloronaphthalene. Washed cell suspensions were shown to accumulate 3,6-dichlorosalicylate, which, after further chemical treatment, yields the herbicide dicamba (3,6-dichloro-2-methoxybenzoate). However, the rate of dichlorosalicylate formation from dichloronaphthalene was less than 1% of the rate of salicylate formation from unsubstituted naphthalene.     

A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: gene isolation, characterization, and heterologous expression

Dicamba O-demethylase is a multicomponent enzyme from Pseudomonas maltophilia, strain DI-6, that catalyzes the conversion of the widely used herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) to DCSA (3,6-dichlorosalicylic acid). We recently described the biochemical characteristics of the three components of this enzyme (i.e. reductase(DIC), ferredoxin(DIC), and oxygenase(DIC)) and classified the oxygenase component of dicamba O-demethylase as a member of the Rieske non-heme iron family of oxygenases. In the current study, we used N-terminal and internal amino acid sequence information from the purified proteins to clone the genes that encode dicamba O-demethylase. Two reductase genes (ddmA1 and ddmA2) with predicted amino acid sequences of 408 and 409 residues were identified. The open reading frames encode 43.7- and 43.9-kDa proteins that are 99.3% identical to each other and homologous to members of the FAD-dependent pyridine nucleotide reductase family. The ferredoxin coding sequence (ddmB) specifies an 11.4-kDa protein composed of 105 residues with similarity to the adrenodoxin family of [2Fe-2S] bacterial ferredoxins. The oxygenase gene (ddmC) encodes a 37.3-kDa protein composed of 339 amino acids that is homologous to members of the Phthalate family of Rieske non-heme iron oxygenases that function as monooxygenases. Southern analysis localized the oxygenase gene to a megaplasmid in cells of P. maltophilia. Mixtures of the three highly purified recombinant dicamba O-demethylase components overexpressed in Escherichia coli converted dicamba to DCSA with an efficiency similar to that of the native enzyme, suggesting that all of the components required for optimal enzymatic activity have been identified. Computer modeling suggests that oxygenase(DIC) has strong similarities with the core alphasubunits of naphthalene 1,2-dioxygenase. Nonetheless, the present studies point to dicamba O-demethylase as an enzyme system with its own unique combination of characteristics.     

Comparative effects of the herbicides dicamba, 2,4-D and paraquat on non-green potato tuber calli

The effects of the herbicides 1,1'-dimethyl-4,4'-bipyridylium dichloride (paraquat), 3,6-dichloro-2-metoxybenzoic acid (dicamba) and 2,4-dichlorophenoxyacetic acid (2,4-D) on cell growth of non-green potato tuber calli are described. We attempted to relate the effects with toxicity, in particular the enzymes committed to the cellular antioxidant system. Cell cultures were exposed to the herbicides for a period of 4 weeks. Cellular integrity on the basis of fluorescein release was strongly affected by 2,4-D, followed by dicamba, and was not affected by paraquat. However, the three herbicides decreased the energy charge, with paraquat and 2,4-D being very efficient. Paraquat induced catalase (CAT) activity at low concentrations (1muM), whereas at higher concentrations, inhibition was observed. Dicamba and 2,4-D stimulated CAT as a function of concentration. Superoxide dismutase (SOD) activity was strongly stimulated by paraquat, whereas dicamba and 2,4-D were efficient only at higher concentrations. Glutathione reductase (GR) activity was induced by all the herbicides, suggesting that glutathione and glutathione-dependent enzymes are putatively involved in the detoxification of these herbicides. Paraquat slightly inhibited glutathione S-transferase (GST), whereas 2,4-D and dicamba promoted significant activation. These results indicate that the detoxifying mechanisms for 2,4-D and dicamba may be different from the mechanisms of paraquat detoxification. However, the main cause of cell death induced by paraquat and 2,4-D is putatively related with the cell energy charge decrease.     

细胞色素P450与除草剂抗性转基因植物

介绍了除草剂代谢有关的细胞色素P450基因及其应用,已从动植物体中分离具有除草剂代谢活性的细胞色素P450基因,通过转基因方法,成功培育出抗除草剂的转基因植物。     

Comparative effects of four herbicides on physiological aspects in Triticum sativum L.

Four herbicide (topik, puma super, granstar and derby) treatments at the recommended field dose (RFD) were applied for 5 weeks on wheat (Triticum sativum L. cv. Giza 168) crop. These selected herbicides are recommended to control two types (narrow and broad) of weeds in this crop. The growth parameter, oxidative stress defence system, nitrate reduction, proline level and protein contents were used to evaluate the toxic effect of these herbicides on wheat crop. The toxicity of herbicides used on this crop was as follows: puma super > topik > derby > granstar. The correlation between the herbicide concentrations and the activities of the antioxidant enzymes was monitored.     

Heteroxanthinedemethylase,a new enzyme in the degradation of caffeine by Pseudomonas putida

Summary In Pseudomonas putida a new enzyme has been detected, which converts heteroxanthine into xanthine by oxidative demethylation. Evidence is presented that this heteroxanthinedemethylase is not responsible for the demethylation of caffeine, supporting the hypothesis that specific enzymes catalyze caffeine demethylation in Pseudomonas putida.     

Plasmid-mediated catabolism of dicamba by Pseudomonas species strain PXM

Pseudomonas species strain PXM, which is able to use dicamba (3,6-dichloro-2-methoxybenzoic acid; CAS 1918-00-9, Banvel) as its sole carbon source for growth, has been isolated. The catabolism of dicamba and some of its putative metabolic descendants correlates with the presence of a large and unstable plasmid.     

嗜麦芽窄食单胞菌OUC_Est10全基因组测序及脂类水解酶多样性分析

[目的]本研究的目的是研究嗜麦芽窄食单胞菌OUC_Est10中脂类水解酶的多样性。[方法]使用离子交换层析、全基因组测序和异源表达三种方法研究嗜麦芽窄食单胞菌OUC_Est10中脂类水解酶的多样性。[结果]离子交换层析结果显示嗜麦芽窄食单胞菌OUC_Est10可以分泌多种脂类水解酶。通过全基因组测序,我们给出了该菌的全基因组序列,该基因组大小为4668743 bp,GC含量为66.25%。通过详细的基因组序列分析,我们从该基因组中找到33个可能具有脂类水解酶活性的假定基因。通过异源表达OUC_Est10中的5个假定脂类水解酶基因,来研究其催化特性的多样性,结果显示这些脂类水解酶具有不同的催化特性。[结论]我们证明了嗜麦芽窄食单胞菌OUC_Est10拥有多样的脂类水解酶,这暗示了它在不同领域中的应用潜力。     

麦草畏室内生测方法建立及大豆对麦草畏耐受性分析

室内生物测定是植物对除草剂等化学物质耐受性鉴定的一种常用筛选方法,已广泛应用于大豆、稗草、棉花等植物对草甘膦、氯嘧磺隆等除草剂的耐受性研究,但麦草畏的室内生物测定方法和大豆对麦草畏耐受性相关研究尚未见报道。本研究以麦草畏对催芽大豆下胚轴伸长抑制率为评价指标,结合回归方程曲线分析和抑制中浓度分析,建立了大豆对麦草畏耐受性室内生物测定方法,确定以300μg/L麦草畏筛选浓度作为大豆室内生物测定临界筛选浓度。利用该方法对35份源自微核心种质的大豆品种进行鉴定,结果表明,随麦草畏浓度增加,不同品种对麦草畏的耐受性存在显著差异,大豆品种对麦草畏的耐受性降低,从中筛选出对麦草畏耐受性较高的大黄豆-1和什邡螺丝豆。本研究结果为培育抗麦草畏品种的亲本选配以及后代选择提供了理论依据、材料和技术支撑。     

Evaluation of Pre-Emergence and Post-Emergence Herbicides for Weed Management in <i>Miscanthus sacchariflorus</i> and <i>Miscanthus sinensis</i>

Miscanthus, is a promising bioenergy crop, considered superior to other bioenergy crops because of its higher water and nutrient use efficiency, cold tolerance, and higher production of biomass. Broadleaf weeds and grass weeds, cause major problems in the Miscanthus field. A field experiment was conducted in 2018 and 2019, to assess the effects of pre-emergence (alachlor and napropamide) and post-emergence herbicides (nicosulfuron, dicamba, bentazon, and glufosinate ammonium) on broadleaf and grass weeds in M. sinensis and M. sacchariflorus fields. The weed control efficiency and phytotoxicity of pre- and post-emergence herbicides were evaluated at 30 days after treatment (DAT) and compared to those of the control plots. The results showed wide variations in the susceptibility of the weed species to the treated herbicides. Treatment with nicosulfuron 40 g.a.i.ha⁻¹ provided the most effective overall weed control (with 10% visual injury), without affecting the height and biomass of neither Miscanthus species in the field. Post-emergence herbicides such as glufosinate ammonium 400 g.a.i.ha⁻¹ and dicamba 482 g.a.i.ha⁻¹ were effective and inhibited the growth and density of the majority of weeds to a 100%; however, they showed significant phytotoxicity (toxicity scale of 1–10) to both species of Miscanthus. The application of glufosinate ammonium caused severe injuries to the foliar region (90% visual injury) of both Miscanthus sps. Comparatively, M. sinensis showed a slightly higher tolerance to the herbicides nicosulfuron, bentazon and napropamide with 10% visual injury at the recommended dose than M. sacchariflorus. The present study clearly showed that infestation of broadleaf and grass weeds in Miscanthus fields can cause significant damage to the growth and biomass of Miscanthus and applying pre-emergence and post-emergence herbicides effectively controls the high infestation of these weeds.     

Microbiological degradation of the herbicide dicamba

Summary Pseudomonas paucimobilis was isolated from a consortium which was capable of degrading dicamba (3,6-dichloro-2-methoxybenzoic acid) as the sole source of carbon. The degradation of dicamba byP. paucimobilis and the consortium was examined over a range of substrate concentration, temperature, and pH. In the concentration range of 100–2000 mg dicamba L^–1 (0.5–9.0 mM), the degradation was accompanied by a stoichiometric release of 2 mol of Cl^– per mol of dicamba degraded. The cultures had an optimum pH 6.5–7.0 for dicamba degradation. Growth studies at 10°C, 20°C, and 30°C yielded activation energy values in the range of 19–36 kcal mol^–1 and an average Q₁₀ value of 4.0. Compared with the pure cultureP. paucimobilis, the consortium was more active at the lower temperature.     

农作物抗除草剂遗传工程研究进展

控制杂草提高农作物产量是农业生产中共同面临的问题,发展抗除草剂农作物将是最经济最方便控制杂草的技术。由于对除草剂的作用模式和除草剂代谢途径的了解,弄清了除草剂的关键靶酶及其基因,因此分离除草剂靶酶基因,克隆能解毒除草剂的酶基因,通过转化技术可获得抗除草剂农作物,大量的抗除草剂转基因农作物大田试验表明,将最有希望在２０００年进入市场。     

Accumulation of gold by microorganisms

Thirty species of microorganisms (8 bacteria, 9 actinomycetes, 8 fungi and 5 yeasts) were screened for maximal gold accumulation. Extremely high abilities to accumulate gold from a solution containing hydrogen tetrachloroaurate(III) were found in bacterial strains, such as Escherichia coli and Pseudomonas maltophilia. Most of the actinomycetes, fungi and yeasts had lower ability to accumulate gold than bacteria. Some microorganisms could accumulate similar amounts of gold from a solution containing sodium gold(I) thiomalate as those from gold(III) solution. However, most microorganisms tested accumulated far lesser amounts of gold from a solution containing sodium dicyanoaurate(I) than from the other two gold solutions. The accumulation of gold from the solution containing hydrogen tetrachloroaurate(III) by Pseudomonas maltophilia was very rapid, was affected by the pH of the solution, and obeyed the Langmuir adsorption isotherm. Pseudomonas maltophilia cells immobilized in polyacrylamide gel adsorbed gold effectively from the solution containing hydrogen tetrachloroaurate(III). The gold adsorbed on the cells was easily desorbed with 0.1 M thiourea solution. The immobilized Pseudomonas cells could be used repeatedly in the adsorption–desorption cycle using 0.1 M thiourea solution as desorbent.     

Weed Management in Rice: Wheat Rotation by Allelopathy

Rice is major crop in India and its cultivation in northwest India started 25 to 30 years ago in assured irrigation areas during the summer rainy season. In this region, rice-wheat rotation became most popular owing to its high yields; however, these crops are highly infested by the weeds, thus farmers use herbicides for their control. Hence, this rotation consumes a maximum quantity of herbicides in this region, which has resulted in several problems (environmental pollution, human health hazards, development of herbicide resistance in weeds). Thus, serious ecological questions about the reliance on herbicides for weed control in this rotation have been raised. One of the alternatives to overcome these problems is with the use of allelopathic strategies, including the use of weed-smothering crops for weed management and for the sustainability of agriculture. The field, pot culture, and laboratory studies have shown that inclusion of weed-smothering crops in rotation considerably reduced the weed population in the current and succeeding crops. Early summer (April-June) fodder crops of sorghum, pearlmillet and maize drastically smothered the weed population and biomass. The residual suppression effect of peralmillet also persisted in the next crop up to 45 days. Thus, it is conceptualized that the inclusion of such summer fodder crops before the rice crop in the rice-wheat rotation may provide satisfactory weed control in the succeeding rice crop and may minimize the use of herbicides. Likewise, the replacement of wheat by winter fodder crops of oat and berseem (Trifolium alexandrinum) may also help in the control of winter weeds. Hence, further studies in this direction may provide satisfactory weed management in rice-wheat rotation and may minimize the use of herbicides and thereby help indeveloping sustainable agricultural practices.     

Vegetative growth and sporulation ofBipolaris sorokiniana on sequentially older infected leaves ofPoa pratensis exposed to postemergence herbicides

Poa pratensis was exposed to the postemergence herbicides 2,4-D, MCPP, and dicamba at concentrations of 10^–6 M applied to the soil surface. Sequentially older leaf blades were inoculated withBipolaris sorokiniana and detached to determine the effect of the herbicides on saprophytic hyphal growth and sporulation on the tissue of each older leaf. 2,4-D and dicamba stimulated hyphal growth in vitro from leaf tissue of all ages; MCPP had no effect on hyphal growth. All herbicides increased sporulation byB. sorokiniana on leaf tissue of all ages. Both hyphal growth and sporulation increased progressively on tissue from sequentially older leaves ofP. pratensis exposed to dicamba.Journal Paper No. J-14985 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, USA, Project No. 2616.     

Evolution of atrazine-degrading capabilities in the environment

Since their first introduction in the mid 1950s, man-made s-triazine herbicides such as atrazine have extensively been used in agriculture to control broadleaf weed growth in different crops, and thus contributed to improving crop yield and quality. Atrazine is the most widely used s-triazine herbicide for the control of weeds in crops such as corn and sorghum. Although atrazine was initially found to be slowly and partially biodegradable, predominantly by nonspecific P450 monoxygenases which do not sustain microbial growth, microorganisms gradually evolved as a result of repeated exposure, started using it as a growth substrate and eventually succeeded in mineralizing it. Within three decades, an entirely new hydrolase-dependent pathway for atrazine mineralization emerged and rapidly spread worldwide among genetically different bacteria. This review focuses on the enzymes involved in atrazine mineralization and their evolutionary histories, the genetic composition of microbial populations involved in atrazine degradation and the biotechnologies that have been developed, based on these systems, for the bioremediation of atrazine contamination in the environment.     

Expression of Arabidopsis Bax Inhibitor‐1 in transgenic sugarcane confers drought tolerance

The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor‐1 from Arabidopsis thaliana (AtBI‐1), can confer increased tolerance of sugarcane plants to long‐term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C₄ grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long‐term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.     

The discovery of Arylex™ active and Rinskor™ active: Two novel auxin herbicides

Multiple classes of commercially important auxin herbicides have been discovered since the 1940s including the aryloxyacetates (2,4-D, MCPA, dichlorprop, mecoprop, triclopyr, and fluroxypyr), the benzoates (dicamba), the quinoline-2-carboxylates (quinclorac and quinmerac), the pyrimidine-4-carboxylates (aminocyclopyrachlor), and the pyridine-2-carboxylates (picloram, clopyralid, and aminopyralid). In the last 10 years, two novel pyridine-2-carboxylate (or picolinate) herbicides were discovered at Dow AgroSciences. This paper will describe the structure activity relationship study that led to the discovery of the 6-aryl-picolinate herbicides Arylex™ active (2005) and Rinskor™ active (2010). While Arylex was developed primarily for use in cereal crops and Rinskor is still in development primarily for use in rice crops, both herbicides will also be utilized in additional crops.