普通小球藻对嗪草酮、骠马和甲草胺的敏感性研究

通过96h的毒性实验,研究了普通小球藻对3种不同作用机制农田常用除草剂嗪草酮、骠马和甲草胺的敏感性．结果表明。在实验条件下,嗪草酮、甲草胺对普通小球藻的毒性随时间的推移有加重趋势。并呈现很好的剂量效应关系;最高抑制生长浓度(嗪草酮0．24mg·L^-1,甲草胺12．8mg·L^-1)处理组的最大比增长率分别为对照组的12．38％和31．58％;骠马低浓度对普通小球藻的抑制作用不明显,并呈一定的生长促进效应,0．08mg·L^-1浓度组普通小球藻最大比增长率为对照组的111．44％,而高浓度则具有明显的生长抑制作用,并随时间推移,毒性逐渐减弱．嗪草酮、骠马和甲草胺的96hEC50分别为0．021、0．937和5．54mg·L^-1．普通小球藻对嗪草酮最敏感。其次为骠马和甲草胺．3种除草剂在实验条件下对普通小球藻叶绿素a含量的影响和对普通小球藻生长的影响相似。表现出较好的剂量．效应关系．     

Sporophytic response to pollen selection for Alachlor tolerance in maize

In order to assess the efficiency of male gametophytic selection (MGS) for crop improvement, pollen selection for tolerance to herbicide was applied in maize. The experiment was designed to test the parallel reactivity to Alachlor of pollen and plants grown in controlled conditions or in the field, the response to pollen selection in the sporophytic progeny, the response to a second cycle of MGS, and the transmission of the selected trait to the following generations. The results demonstrated that pollen assay can be used to predict Alachlor tolerance under field conditions and to monitor the response to selection. A positive response to selection applied to pollen in the sporophytic progeny was obtained in diverse genetic backgrounds, indicating that the technique can be generally included in standard breeding programs; the analysis of the data produced in a second selection cycle indicated that the selected trait is maintained in the next generation.     

Isolation and characterization of alachlor-degrading actinomycetes from soil

Alachlor (2-cloro-N-(methoxymethyl)-N-(2,6-diethylphenyl)-acetamide) is an extremely toxic and highly mobile herbicide that is widely used for pre-emergence control of grasses and weeds in many commercial crops in Brazil. In order to select soil actinomycetes able to degrade this herbicide, fifty-three actinomycete strains were isolated from soil treated with alachlor using selective conditions and subjected to in vitro degradation assays. Sixteen isolates were shown to be tolerant to high concentrations of the herbicide (up to 720 mg L^-1), and six of these were able to grow and degrade 50 alachlor (72 mg L^-1) in mineral salts medium. Morphological and phylogenetic analysis enabled the assignment of the alachlor-degrading strains to the genus Streptomyces. Strain LS151 was related to the type strains of Streptomyces capoamus/Streptomyces galbus, whereas strains LS143 and LS153 were related to Streptomyces bikiniensis. The remaining strains, LS166, LS177 and LS182, were similar in morphological features and recovered in a single cluster based on 16S rDNA sequence analysis, but shown to be distinct on the basis of genomic fingerprint data (rep-PCR). Though a definitive taxonomic assignment of alachlor-degrading strains was not possible, these data indicate that ability to degrade this pesticide was detected in different Streptomyces taxa.     

Characterization of the safener-induced glutathione S-transferase isoform II from maize

The safener-induced maize (Zea mays L.) glutathione S-transferase, GST II (EC 2.5.1.18) and another predominant isoform, GST I, were purified from extracts of maize roots treated with the safeners R-25788 (N,N-diallyl-2-dichloroacetamide) or R-29148 (3-dichloroace-tyl-2,2,5-trimethyl-1,3-oxazolidone). The isoforms GST I and GST II are respectively a homodimer of 29-kDa (GST-29) subunits and a heterodimer of 29 and 27-kDa (GST-27) subunits, while GST I is twice as active with 1-chloro-2,4-dinitrobenzene as GST II, GST II is about seven times more active against the herbicide, alachlor. Western blotting using antisera raised against GST-29 and GST-27 showed that GST-29 is present throughout the maize plant prior to safener treatment. In contrast, GST-27 is only present in roots of untreated plants but is induced in all the major aerial organs of maize after root-drenching with safener. The amino-acid sequences of proteolytic fragments of GST-27 show that it is related to GST-29 and identical to the 27-kDa subunit of GST IV.Abbreviations CDNB 1-chloro-2,4-dinitrobenzene - DEAE di-ethylaminoethyl - FPLC fast protein liquid chromatography - GSH reduced glutathione - GST glutathione S-transferase - GST-26 26-kDa subunit of maize GST - GST-27 27-kDa subunit of maize GST - GST-29 29-kDa subunit of maize GST - R-25788 safener N,N-diallyl-2-dichloroacetamide - R-29148 safener 3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazolidone - RPLC reverse phase liquid chromatography We are grateful to M-M. Lay, ZENECA AG Products (formerly ICI Americas), Richmond, Calif., USA for providing [¹⁴C] R-25788. ZENECA Seeds in the UK is part of ZENECA Limited.     

Determination of alachlor and its metabolites in rat plasma and urine by liquid chromatography-electrospray ionization mass spectrometry

A method based on liquid chromatography (LC) in combination with mass spectrometry (MS) for the analysis of alachlor (ALA) and its metabolites, 2-chloro-N-[2,6-diethylphenyl]acetamide (CDEPA) and 2,6-diethylaniline (DEA), in rat plasma and urine has been developed. 13C-labeled ALA was used as the internal standard for quantitation. The analyte in plasma or urine was isolated using a Waters Oasis HLB extraction plate. The mass spectrometer was operated in the ESI MS-SIM mode with a programming procedure. The retention times for ALA, CDEPA and DEA were 1.84, 3.11 and 4.12 min, respectively. The limits of quantification (LOQ) for ALA, CDEPA and DEA were 2.3, 0.8 and 0.8 ng per injection, respectively. The linear fit of analyte to mass response had an R2 of 0.99. Reproducibility of the sample handling and LC-MS analysis had a RSD of < or = 10%. The average recoveries for these analytes in rat plasma were better than 90%. Similar results were obtained with rat urine.     

Cu/Zn-superoxide dismutase and glutathione are involved in response to oxidative stress induced by protein denaturing effect of alachlor in Saccharomyces cerevisiae

Alachlor is a widely used pre-emergent chloroacetanilide herbicide which has been shown to have many harmful ecological and environmental effects. However, the mechanism of alachlor-induced oxidative stress is poorly understood. We found that, in Saccharomyces cerevisiae, the intracellular levels of reactive oxygen species (ROS) including superoxide anions were increased only after long-term exposure to alachlor, suggesting that alachlor is not a pro-oxidant. It is likely that alachlor-induced oxidative stress may result from protein denaturation because alachlor rapidly induced an increased protein aggregation, leading to upregulation of SSA4 and HSP82 genes encoding heat shock proteins (Hsp) of Hsp70 and Hsp90 family, respectively. Although only SOD1 encoding Cu/Zn-superoxide dismutase (SOD), but not SOD2 encoding Mn-SOD, is essential for alachlor tolerance, both SODs play a crucial role in reducing alachlor-induced ROS. We found that, after alachlor exposure, glutathione production was inhibited while its utilization was increased, suggesting the role of glutathione in protecting cells against alachlor, which becomes more important when lacking Cu/Zn-SOD. Based on our results, it seems that alachlor primarily causes damages to cellular macromolecules such as proteins, leading to an induction of endogenous oxidative stress, of which intracellular antioxidant defense systems are required for elimination.     

Genetic dissection of herbicide tolerance in maize by molecular markers

In order to detect the genetic architecture of maize tolerance to Alachlor, a widely used chloroacetanilide, linkage analysis between the expression of the trait and allelic composition of molecular markers was performed. The experiment was carried out on a population of 142 recombinant inbred lines, developed starting from the F₁ between two lines with different reactivity to the herbicide, and self-fertilized for 10 generations; the lines were typed by 48 RFLP markers and 66 microsatellites (SSR). Besides seedling tolerance, evaluated as proportion of normal (non-injured) plants after herbicide treatment, other minor components of tolerance were studied: seed germination ability, pollen germination and tube growth in the presence of the herbicide. The analysis, performed by three statistical methods, revealed the presence of factors controlling seedling tolerance on seven chromosomal regions. Five QTLs appeared to be involved in seed germination ability in the presence of Alachlor, four QTLs in pollen tolerance in terms of germination and four in tube growth under stress were detected. Three loci, on chromosomes 1, 7 and 10, explained most of the variation of seedling tolerance, thus being interesting candidate for marker-assisted selection.     

Impact of two soil-applied herbicides on damping-off of cowpea caused byRhizoctonia solani

Summary Alachlor was more inhibitory toRhizoctonia solani growth than fluchloralin in potato dextrose broth (PDB). Infective capacity of the pathogen was not altered by growing it in a medium containing either of the herbicides. Cowpea seedlings grown in alachlor-treated soil were more susceptible toR. solani than those treated with fluchloralin and the untreated seedlings. Pre-sowing application of alachlor in soil (5 l a.i./kg) aggravated damping-off whereas fluchloralin decreased the disease incidence to nearly half of that in untreated soil in greenhouse pot tests (av. temperature 31±5°C). Both herbicides reduced damping-off in pots kept at constant temperature of 30°C and increased the disease incidence at 20°C. Fungus growth in culture was stimulated at 20° but was strongly inhibited at 30°C by both herbicides. Growth inhibition by herbicides was maximum in PDB of pH 8 and decreased steadily up to pH 5.Impact of fluchloralin and alachlor onR. solani damping-off of cowpea appears to be due to the predisposing effect by the herbicides on the susceptibility of the host and is influenced by atmospheric temperature.     

Kinetic analysis of maize glutathione <Emphasis Type="Italic">S</Emphasis>-transferase I catalysing the detoxification from chloroacetanilide herbicides

Glutathione S-transferases (GSTs, EC 2.5.1.18) are a family of multi-functional enzymes involved in biodegradation of several herbicide classes. The ability of the maize isoenzyme GST I to detoxify from the acetanilide herbicide alachlor was investigated by steady-state kinetics and site-directed mutagenesis studies. Steady-state kinetics fit well to a rapid equilibrium random sequential bi-bi mechanism with intrasubunit modulation between GSH binding site (G-site) and electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent and thermodynamic data suggest that product release is rate-limiting. Three residues of GST I (Trp12, Phe35 and Ile118), which build up the xenobiotic binding site, were mutated and their functional and structural roles during alachlor conjugation were investigated. These residues are not conserved, hence may affect substrate specificity and/or product dissociation. The work showed that the key amino acid residue Phe35 modulates xenobiotic substrate binding and specificity, and participates in k_cat regulation by affecting the rate-limiting step of the catalytic reaction. Trp12 and Ile118 do not seem to carry out such functions but instead, regulate the K_m for alachlor by contributing to its productive orientation in the H-site. The results of the present work have practical significance since this may provide the basis for the rational design of new engineered GSTs with altered substrate specificity towards herbicides and may facilitate the design of new, more selective herbicides.