Presence Of Selenate Research Articles

Fabrication of sulfur-based functionalized activated carbon as solid phase extraction adsorbent for selective analysis of selenite in water

Based on the important feature of sulfur with excellent selectivity toward selenite in the presence of selenate, a simple and low-cost adsorbent of solid phase extraction known as sulfur loading activated carbon (SAC-6) was successfully prepared and applied for selenite (Se(IV)) analysis in water. Microstructure and morphological characteristics of SAC-6 had been identified by XRD, TEM, BET and FT-IR. In the static adsorption experiments, Se(IV) could be separated in a wide range of pH values (pH=3–11). The retention process of Se(IV) onto SAC-6 was characterized as spontaneous exothermic reaction. An obvious change of adsorption mechanism occurred in static and dynamic adsorption processes shown that the behaviors followed monolayer and hybrid adsorption. The theoretical maximum adsorption capacity of SAC-6 calculated by Langmuir-Freundlich was 13.48 mg/g. The microcolumn filled with SAC-6 was applied to extract Se(IV) in water solution. The detection limit of Se(IV) analytical procedure was confirmed as 0.27 μg/L within a linear range of 10–1000 μg/L. A good precision with relative standard deviation of 1.34 % (100 μg/L, n = 6) was achieved. The high adaptability and accuracy of SAC-6 microcolumn was validated by analyzing natural water samples and certified reference materials. Our work successfully excavated the application value of the sulfur selectivity, and also provided a new adsorbent for Se(IV) extraction and analysis.

Foraging for selenium: a comparison between hyperaccumulator and non-accumulator plant species

Selenium (Se) hyperaccumulators are a unique group of plants that can accumulate this element in their aerial parts at concentrations exceeding 100 mg kgDW−1. These plants actively search for Se in the soil, a phenomenon known as root foraging, reported to date only by few studies. In this study, the effect of localized Se enrichment, in the form of selenite and selenate, was investigated on the root architecture of two Se-hyperaccumulators (Stanleya pinnata and Astragalus bisulcatus) and two non-accumulators (Brassica juncea and Medicago sativa). Rhizoboxes were divided into two halves: one half was filled with control soil while the other with selenate or selenite (30 mg kgDW−1) spiked soil. Seedling were transferred into the interface of the two soils and allowed to grow for three weeks under controlled light and temperature conditions. Staneya pinnata exhibited equal root density in both halves of the rhizobox when grown in control/control and selenite/control soil treatments. However, in the presence of selenate, S. pinnata developed 76% of the roots towards the selenate-enriched half, indicating an active root foraging. In contrast, A. bisulcatus and the non-accumulators B. juncea and M. sativa did not show any preferential distribution of roots. This study revealed that only S. pinnata showed the ability to detect and forage for Se when provided as selenate. Non-accumulators did not show any morphological or Se-accumulation difference associated with the presence of Se in soil in either form.

Simultaneous removal of lead and selenium through biomineralization as lead selenide by anaerobic granular sludge

This study demonstrated the simultaneous removal of lead (Pb) and selenium (Se) as lead selenide biomineralization using anaerobic granular sludge. The microbial community of the granular sludge was first enriched for 140 days in the presence of Pb(II) only, selenate and selenite only, Pb(II)+selenate, and Pb(II)+selenite. In the absence of Se, removal of Pb(II) mainly occurred via biosorption and deposited on the biomass as lead oxide and lead carbonate. The Pb removal efficiency (94% of initial 50 mg L-1) was reduced to 90% and 86% in the presence of selenate and selenite, respectively, due to biosorption. Addition of Pb(II) didn’t exert any toxic effect on the Se-reducing microbial community, on the contrary: Pb(II) addition improved the Se removal efficiency for selenate from 85% to 90%, but did not affect selenite removal after 14 d of incubation. The bioreduction of the Se-oxyanions produced elemental Se (Se(0)) and selenide, which later interacted with Pb(II) to produce lead selenide (PbSe). Adsorption of Pb(II) onto the Se(0) nanoparticles and precipitation as the Se(0)-Pb complex might also have contributed to the simultaneous removal of Pb and Se. XPS and XRD analysis further confirmed the immobilization of Pb as PbSe, PbO and PbCO3 in the biomass.

The effects of selenate on goethite synthesis and selenate sorption kinetics onto a goethite surface - A three-step process with an unexpected desorption phase

Iron oxyhydroxides are widely spread in soils and sediments. They can considerably affect the migration of selenate in the environment, due to their high affinity towards oxyanions. To assess the sorptive properties of one of the most common oxyhydroxides, the selenate sorption onto goethite and its effects on goethite's structural properties during its crystallization were investigated. The sorption kinetics of selenate onto goethite at different pH revealed the presence of a desorption phase that had manifested extensively, especially at higher pH values. Also, an isotherm study indicated that the selenate sorption onto goethite is multi-layered at higher concentrations. Phosphate, which is one of the common naturally occurring oxyanions, affected selenate sorption onto goethite significantly, while chloride and sulfate influenced sorption only marginally. The Mössbauer spectrometry showed no structural changes after iron oxyhydroxides crystallization in the presence of selenate. However, changes outside the goethite lattice were manifested by the alterations in distributions of hyperfine magnetic fields obtained from Mössbauer spectra analyses, as well as by the changes in values of the selenate-treated samples' surface charge.

Effect of selenate and thiosulfate on anaerobic methanol degradation usingactivated sludge.

Anaerobic bioconversion of methanol was tested in the presence of selenate (SeO42-), thiosulfate (S2O32-), and sulfate (SO42-) as electron acceptors. Complete SeO42- reduction occurred at COD:SeO42- ratios of 12 and 30, whereas ~ 83% reduction occurred when the COD:SeO42- ratio was 6. Methane production did not occur at the three COD:SeO42- ratios investigated. Up to 10.1 and 30.9% of S2O32- disproportionated to SO42- at COD:S2O32- ratios of 1.2 and 2.25, respectively, and > 99% reduction was observed at both ratios. The presence of S2O32- lowered the methane production by 73.1% at a COD:S2O32- ratio of 1.2 compared to the control (no S2O32-). This study showed that biogas production was not preferable for SeO42- and S2O32--rich effluents and volatile fatty acid production could be a potential resource recovery option.

Graphene Oxide Decorated with Cerium(IV) Oxide in Determination of Ultratrace Metal Ions and Speciation of Selenium.

Graphene oxide decorated with cerium(IV) oxide (GO/CeO2) was synthesized and applied in adsorption of several metal ions such as As(III), As(V), Se(IV), Cu(II), and Pb(II) from aqueous samples. The important feature of GO/CeO2 nanocomposite is also its selectivity toward selenite in the presence of selenate. The structure of GO/CeO2 has been proven by microscopic and spectroscopic techniques. The maximum adsorption capacities of GO/CeO2 calculated by Langmuir model toward arsenic, selenium, copper, and lead ions are between 6 and 30 mg g-1. An interesting feature of this adsorbent is its excellent dispersibility in water. Thus, GO/CeO2 nanocomposite is ideal for fast and simple determination of heavy metal ions using dispersive microsolid phase extraction (DMSPE). Moreover, coupling DMSPE with energy-dispersive X-ray fluorescence spectrometry (EDXRF) is extremely beneficial because it allows direct analysis of adsorbent. Thus, the analyte elution step, as needed in many analytical techniques, was obviated. The influence of sample volume and the sorption time as well as the influence of foreign ions and humic acid on the recovery of determined elements are discussed in the paper. The results showed that developed methodology provided low limits of detection (0.07-0.17 μg/L) and good precision (RSD < 4%). The GO/CeO2 nanocomposite was applied to analysis of real water samples and certified reference materials (CRM) groundwater (BCR-610) and pig kidney (ERM-BB186).

Cadmium uptake dynamics and translocation in rice seedling: Influence of different forms of selenium

Selenium (Se) can alleviate the toxicity of cadmium (Cd), but little is known about its mechanism in Cd uptake and translocation in plants. We investigated the effects of exogenous selenite, selenate, and selenomethionine (SeMet) on Cd uptake and translocation within rice (Oryza sativa L., Zhunliangyou 608) seedlings, and the concentration-dependent uptake kinetics of Cd into rice roots (with or without Se) were determined. The effect of the endogenous Se pool on Cd uptake was also investigated. Results of uptake kinetics showed that selenite slightly promoted Cd influx during 1h of exposure, compared with no selenite addition; Vmax of Cd uptake increased by 13.8% in 10μM selenite treatment; while the presence of selenate had no effect on the influx of Cd. When exposed to Cd (5μM) over 20h (with selenite) or 30h (with selenate or SeMet), Se addition (5μM) decreased Cd uptake and root-to-shoot translocation; after 30h selenite, selenate, or SeMet addition decreased Cd uptake by roots by 28.6%, 17.7% or 12.1%, respectively. Besides, as the selenite levels in the treatment solutions (1μMCd) increased (0, 0.1, 1, and 5μM, Se), Cd uptake and translocation were both significantly reduced, while the inhibitive effect was more significant at lower levels of selenate. Pretreatment of selenite or selenate (5μM) also decreased Cd uptake by 24.9% or 15.7%, and reduced the root-to-shoot transfer factor by 41.4% or 36.2% after 144h of subjection to Cd (5μM), respectively. The presence of selenite decreased Cd content more effectively than did selenate. Our results demonstrated that Se can effectively reduce the Cd translocation from roots to shoots in rice seedlings.

The dual effects of two inorganic selenium forms on the growth, selected physiological parameters and macronutrients accumulation in cucumber plants

The boundary between beneficial and phytotoxic levels of selenium (Se) is narrow, and both induce alteration in plant growth and their physiology. In this study, the influence of two Se forms (selenite or selenate) with different concentrations (2–80 µM) on cucumber plants was investigated. The toxicity threshold for selenate and selenite was determined at the concentrations of 80 and 20 µM, respectively. In the Se-exposed plants, the growth-promoting effect was found at 6 µM of selenite and at 6–20 µM of selenate. The root activity considerably increased with increasing selenite concentrations suggesting the upregulation of mitochondrial dehydrogenases activity. Selenite treatment also impaired photosynthetic pigments accumulation and chlorophyll fluorescence parameters. Moreover, Se exerted a dual effect on lipid peroxidation in roots: at low concentrations it inhibited this process, whereas at high concentrations it enhanced the accumulation of harmful lipid peroxides. Under low Se concentrations (<10 µM), the accumulation of Se in shoots was similar in the presence of selenate and selenite. When Se concentration was >10 µM, the accumulation of Se in shoots was greater in selenate-exposed than selenite-exposed plants. However, in the roots the Se concentrations were always higher after selenite exposure comparing to selenate. The N level in plants was generally maintained constant, while the remaining macronutrients (especially K, P, and S) concentrations were significantly changed depending on the form and concentrations of Se. These results imply that an application of either selenate or selenite at concentrations <10 µM may be potentially used for biofortification of cucumber with Se and changes in plant macronutrient contents are not expected under these conditions.

Sequestration of Selenium on Calcite Surfaces Revealed by Nanoscale Imaging

Calcite, a widespread natural mineral at the Earth's surface, is well-known for its capacity to sequester various elements within its structure. Among these elements, selenium is important because of its high toxicity in natural systems and for human health. In the form of selenite (Se((IV))), selenium can be incorporated into calcite during growth. Our in situ atomic force microscopy observations of calcite surfaces during contact with selenium-bearing solutions demonstrate that another process of selenium trapping can occur under conditions in which calcite dissolves. Upon the injection of solutions containing selenium in two states of oxidation (either Se((IV)) or Se((VI))), precipitates were observed forming while calcite was still dissolving. In the presence of selenate (Se((VI))), the precipitates formed remained small during the observation period. When injecting selenite (Se((IV))), the precipitates grew significantly and were identified as CaSeO3·H2O, based on SEM observations, Raman spectroscopy, and thermodynamic calculations. An interpretation is proposed where the dissolution of calcite increases the calcium concentration in a thin boundary layer in contact with the surface, allowing the precipitation of a selenium phase. This process of dissolution-precipitation provides a new mechanism for selenium sequestration and extends the range of thermodynamic conditions under which such a process is efficient.

Evaluation of the antioxidant capacity of lupin sprouts germinated in the presence of selenium

Antioxidant capacity of 5 DAS (days after seeding) lupin sprouts germinated in the presence of selenate or selenite was determined by cyclic voltammetry (CV), Folin-Ciocalteu reagent (FCR) application and photochemiluminescence (PCL) methods. The hydrophilic (80% methanol) and lipophillic (hexane/methanol) extracts of 5 DAS lupin sprouts germinated in the absence of selenium (control sprouts) showed higher antioxidant capacity than ungerminated seeds. Five DAS sprouts produced in the presence of higher concentration of selenate or selenite (6–8 mg/L) showed increased total antioxidant capacity formed by both hydrophilic and lipohillic antioxidants when evaluated by CV and FCR methods. The hydrophilic extracts from sprouts produced in the presence of low concentration of selenate (2–6 mg/L) showed a significantly higher antioxidant capacity of water-soluble compounds (PCL ACW) while no changes were noted in those germinated in the presence of the highest concentration in relation to the control sprouts. In contrast, the antioxidant capacity of lipid-soluble compounds (PCL ACL) lowered significantly in relation to the control sprouts. The similar changes in antioxidant capacity of sprouts produced in the presence of selenite were found by both PCL assays. Comparison of PCL with CV and FCR reducing capacity assays has shown that these methods yielded considerably different chemical information. Moreover, the changes in total antioxidant and total reducing capacity of sprouts germinated in the presence of either selenate or selenite provided by CV and FCR assays were higher than those obtained by photochemiluminesce measurements. It can be suggested to use 6–8 mg/L of selenite rather than selenate in order to obtain a lupin sprouts with high antioxidant capacity.

Characterization of a selenate-resistant Arabidopsis mutant. Root growth as a potential target for selenate toxicity.

Screening an Arabidopsis (Arabidopsis thaliana) T-DNA mutant library for selenate resistance enabled us to isolate a selenate-resistant mutant line (sel1-11). Molecular and genetic characterization showed that the mutant contained a lesion in the SULTR1;2 gene that encodes a high affinity root sulfate transporter. We showed that SULTR1;2 is the only gene among 13 mutated genes of the Arabidopsis sulfate transporter family whose mutation conferred selenate resistance to Arabidopsis. The selenate resistance phenotype of the sel1-11 mutant was mirrored by an 8-fold increase of root growth in the presence of selenate as shown by the calculated lethal concentration values. The impairment of SULTR1;2 activity in sel1-11 resulted in a reduced (35)S-sulfate uptake capacity by both roots and calli and a reduced sulfate and selenate content in root, shoot, and calli. Comparing sulfate-to-selenate ratios instead of absolute sulfate and selenate contents in roots and shoots enabled us to gain better insight into the mechanism of selenate toxicity in Arabidopsis. Roots of the sel1-11 mutant line showed a higher sulfate to selenate ratio than that of wild-type roots, while there were no significant differences in sulfate to selenate ratios in shoots of wild-type and mutant lines. These results indicated that the mechanism that confers the selenate resistance phenotype to the sel1-11 line takes place rather in the roots. It might be in part the result of a lower selenate uptake and of a protective effect of sulfate against the toxic effects of selenate on root growth. These results revealed in plants a central and specific role of the transporter SULTR1;2 in selenate sensitivity; they further suggested that root growth and potentially the root tip activity might be a specific target of selenate toxicity in Arabidopsis.

Development of a viologen-based microtiter plate assay for the analysis of oxyanion reductase activity: Application to the membrane-bound selenate reductase from Enterobacter cloacae SLD1a-1

The membrane-bound selenate reductase of Enterobacter cloacae SLD1a-1 is purified in low yield and has relatively low activity in the pure form compared to that of other oxyanion reductases, such as the membrane-bound and periplasmic nitrate reductases. A microtiter plate assay based on the original quartz cuvette viologen assay of Jones and Garland (R.W. Jones, P.B. Garland, Biochem. J 164 (1977) 199–211) was developed specifically for analysis of such low-abundant, labile oxyanion reductases. The plate assay detects the enzyme-dependent reoxidation of reduced methyl viologen spectrophotometrically at 600 nm. The assay is quick, uses a minimal sample volume (<5 μl), can simultaneously test a range of alternative substrates, and permits activity measurements on multiple samples. We demonstrate the accuracy and versatility of the microtiter plate assay by application to the kinetic analysis, inhibition, and pH optimization of the membrane-bound selenate reductase from E. cloacae SLD1a-1. Results show that the membrane–bound selenate reductase has optimum activity at pH ∼ 8 and its active site is able to accommodate larger inhibitory complexes resulting in mixed-type inhibition, in the presence of selenate and potassium thiocyanate.

Identification of biogenic dimethyl selenodisulfide in the headspace gases above genetically modified Escherichia coli

Escherichia coli JM109 cells were modified to express the genes encoded in a 3.8-kb chromosomal DNA fragment from a metalloid-resistant thermophile, Geobacillus stearothermophilus V. Manual headspace extraction was used to collect the gases for gas chromatography with fluorine-induced sulfur chemiluminescence analysis while solid-phase microextraction was used for sample collection in gas chromatography/mass spectrometry (GC/MS) analysis. When grown in the presence of selenate or selenite, these bacteria produced both organo-sulfur and organo-selenium in the headspace gases above the cultures. Organo-sulfur compounds detected were methanethiol, dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide. Organo-selenium compounds detected were dimethyl selenide and dimethyl diselenide. Two mixed sulfur-selenium compounds, dimethyl selenenyl sulfide and a chromatographically late-eluting compound, were detected. Dimethyl selenodisulfide, CH 3SeSSCH 3, and dimethyl bis(thio)selenide, CH 3SSeSCH 3, were synthesized and analyzed by GC/MS and fluorine-induced chemiluminescence to determine which corresponded to the late-eluting compound that was bacterially produced. CH 3SeSSCH 3 was positively identified as the compound detected in bacterial headspace above Se-amended cultures. Using GC retention times, the boiling point of CH 3SeSSCH 3 was estimated to be approximately 192 °C. This is the first report of CH 3SeSSCH 3 produced by bacterial cultures.

Interactions between different selenium compounds and zinc, cadmium and mercury

In this paper, we present a polarographic study of systems containing different inorganic and organic selenium compounds (sodium selenite, sodium selenate, seleno-methionine and seleno-urea) and metal ions (Zn2+, Cd2+ Hg2+) of the 12th group of elements in the periodic table. While zinc is a trace element known to be essential for plants and animals, cadmium and mercury are exogenous elements and are harmful pollutants that accumulate during aging; selenium is also recognized as an important micronutrient and is sometimes added to the diet. Experiments investigating the interactions were carried out using polarographic techniques in unbuffered systems. The three metal cations originated complexes with different strength and solubility in the presence of selenite anions; in the presence of selenate, polarography was not able to detect formation of complexes with these metal ions, at least under the experimental conditions used: a decrease of Hg2+ ion concentration was observed. Seleno-methionine did not react with Cd2+; in the presence of Zn2+, a soluble complex with a co-ordination number 1 was formed, while, again, the concentration of Hg2+ decreased in the presence of increasing concentrations of the selenium derivative. Seleno-urea did not react with Zn2+, but formed a complex with Cd2+ with limited solubility. Finally, this ligand could not be studied with Hg2+ because of the overlapping of the reduction potentials of both the ligand and the metal cation. Overall equilibrium constants for complex formation (Kf) and the solubility product (Ksp) for poorly soluble species are also reported.

Chromatographic speciation of anionic and neutral selenium compounds in Se-accumulating Brassica juncea (Indian mustard) and in selenized yeast

Selenium-accumulating plants such as Brassica juncea (Indian mustard) concentrate the element in plant shoots and roots. Such behavior may provide a cost-effective technology to clean up contaminated soils and waters that pose major environmental and human health problems (phytoremediation). Such ability to transform selenium into bioactive compounds has important implications for human nutrition and health. Element selective characterization of B. juncea grown in the presence of inorganic selenium under hydroponic conditions provides valuable information to better understand selenium metabolism in plants. The present work determines both previously observed organoselenium species such as selenomethionine and Se-methylselenocysteine and for the first time detects the newly characterized S-(methylseleno)cysteine in plant shoots and roots when grown in the presence of selenate or selenite as the only selenium source. A key feature of this study is the complementary role of selenium and sulfur specific chromatographic detection by HPLC with interfaced inductively coupled plasma mass spectrometry (ICP-MS) detection and by derivatization GC with interfaced atomic spectral emission. HPLC–ICP-MS limits of detection for such species were in the range 5–50 ng Se mL−1 in the injected extracts. Speciation profiles are compared with those of selenium-enriched yeast by both HPLC–ICP-MS and GC–AED.

Chromatographic speciation of anionic and neutral selenium compounds in Se-accumulating Brassica juncea (Indian mustard) and in selenized yeast

Selenium-accumulating plants such as Brassica juncea (Indian mustard) concentrate the element in plant shoots and roots. Such behavior may provide a cost-effective technology to clean up contaminated soils and waters that pose major environmental and human health problems (phytoremediation). Such ability to transform selenium into bioactive compounds has important implications for human nutrition and health. Element selective characterization of B. juncea grown in the presence of inorganic selenium under hydroponic conditions provides valuable information to better understand selenium metabolism in plants. The present work determines both previously observed organoselenium species such as selenomethionine and Se-methylselenocysteine and for the first time detects the newly characterized S-(methylseleno)cysteine in plant shoots and roots when grown in the presence of selenate or selenite as the only selenium source. A key feature of this study is the complementary role of selenium and sulfur specific chromatographic detection by HPLC with interfaced inductively coupled plasma mass spectrometry (ICP-MS) detection and by derivatization GC with interfaced atomic spectral emission. HPLC–ICP-MS limits of detection for such species were in the range 5–50 ng Se mL −1 in the injected extracts. Speciation profiles are compared with those of selenium-enriched yeast by both HPLC–ICP-MS and GC–AED.

Uptake and transport of selenite and selenate by soybean seedlings of two genotypes

In an attempt to address the role of biological behavior on Se uptake by soybean crop and the genotype effects, experiments with time and concentration sequences of Se uptake by seedlings in Hoagland solution are conducted using selenite and selenate respectively. Two soybean cultivars Tong-ai 405 (TA) and Qidong Green-skin (QG) are used as different genotypes. In presence of selenite, Se uptake by both roots and shoots exhibited a linear increase with the growing time at 5 μM and with the solution Se concentrations. However, in presence of selenate, the linear response to growing time is only valid before 24 h of growing. While root Se uptake is much slower under selenate than under selenite in the time sequence experiment, shoot Se levels are similar between the two different Se form treatments. Nevertheless, in the experiment of concentration sequence, either root Se or shoot Se responses linearly to solution Se concentration regardless of the Se forms supplied. A big discrepancy of root Se level with a similarity of shoot Se between the two cultivars is observed in the concentration sequence experiment. This supports a much faster passive uptake of selenite but more or less an active uptake of selenate by soybean seedlings. Comparatively, cultivars TA have a consistently higher Se concentration than QG both in roots and shoots under selenate, while no difference of concentration ratio of shoot to root is recognized between them. The higher Se level in seed grains, therefore, may be accounted for not by Se transport form root to shoot but by greater ability of Se uptake and retention under selenate by the former cultivar. Therefore, not only forms of Se supply but also genotype difference affects the Se bioavailability by different soybean cultivars. This should be taken into account for screening the high Se-efficiency plants or cultivars to improve the Se supply of the food chain.

Study on a highly sensitive determination of toxic metals in waste water with high matrix contents using coprecipitation separation techniques

Selective coprecipitations of selenium species and arsenic species in complex high-salts wastewater, followed by graphite-furnace AAS, were developed. Selective separations of selenium species were based on the reductive coprecipitations on tellurium and palladium collector using hydrazinium sulfate, L-ascorbic acid and tin (II) chloride as reducing agents. In particular, the selective reductive coprecipitation of selenite in the presence of selenate was achieved on a 50∼500 μg amount of tellurium collector under 1 M HCl for 15 min boiling using L-ascorbic acid and tin (II) chloride, and was achieved on a palladium collector under 0.2 M HCl for 30 min boilng by using hydrazinium sulfate. On the other hand, the selective coprecipitation of inorganic arsenic (III,V) in the presence of organic arsenics was achieved by lanthanum hydroxide coprecipitation at pH 12 or more. Moreover, organic arsenics (dimethylarsinic acid, monometylarsonic acid and p-aminophenylarsonic acid) were quantitatively distilled as chloroarsine compounds under 0.2 M SnCl2, 2.75 M H2SO4 and 4 M HCl. The developed selective determinations of selenium species and arsenic species were satisfactorily applied to real waste water containing ppb levels of selenium and arsenic.

One-electron oxidation of plasmid DNA by selenium(V) species

Purpose : To employ the γ-radiation-generated selenium(V) one-electron-oxidizing agent SeO 3 •- for the preparation of guanyl radicals in plasmid DNA, and to compare the behaviour of this reagent with that of other similarly reactive oxidant species. Materials and methods : Plasmid DNA in aerobic aqueous solution was irradiated with 137 Cs γ-rays (662 keV). The solutions also contained up to 4 ×10 -2 mol dm -3 sodium selenate (Na 2 SeO 4) and/or up to 10 -1 mol dm -3 sodium biselenite (NaHSeO 3) , as well as auxiliary scavengers such as DMSO or glycerol. In some cases, reducing agents such as ferrocyanide were also present. After irradiation, the plasmid was incubated with the Escherichia coli base excision-repair endonuclease formamidopyrimidine-DNA N -glycosylase (FPG). These treatments produced strand breaks in the plasmid. The yields of these strand breaks were quantified by agarose gel electrophoresis. Results : In general, γ-irradiation produced single-strand breaks (SSB) in plasmid DNA. Subsequent incubation with the endonuclease FPG increased the SSB yield by a factor of 2-100-fold. The smallest effects of FPG were observed when only DMSO or glycerol were present during irradiation. FPG incubation produced significantly larger increases in the SSB yield after γ-irradiation in the additional presence of selenate and/or biselenite. The largest effect of FPG was observed after γ-irradiation in the presence of 10 -2 mol dm -3 sodium selenate and 10 -1 mol dm -3 glycerol. This was indicative of extensive oxidative damage to the plasmid under these conditions and provided evidence for guanine oxidation mediated by SeO 3 •-. The large effect of FPG was strongly attenuated by the addition of reducing agents such as ferrocyanide. The observations suggest that these reducing agents exert their effects through the reduction of an intermediate guanyl radical. Conclusion : By comparing the yields of breaks produced after γ-irradiation under a range of conditions, it is possible to formulate a reaction scheme that describes the chemical reactions responsible for the formation of strand breaks and FPG-sensitive sites. By applying this scheme to the data, we can quantify rate constants for the reduction of DNA guanyl radicals by reducing agents. This reaction is of particular interest to radiation biology because it is the equivalent of the repair of DNA damage by the direct effect of ionizing radiation.

New genetic markers for Botrytis cinerea ( Botryotinia fuckeliana)

Selenate-resistant (SelR) mutants of Botrytis cinerea were selected by plating conidia or mycelial plugs onto minimal medium amended with selenate and taurine. Ultraviolet irradiation of conidia to give 5% survival increased mutant yield twenty-fold. Mutants could be divided into three classes based on growth in the presence of selenate or chromate and on response to taurine in minimal media. Nitrate non-utilizing (Nit) mutants, generated as spontaneous sectors on minimal media amended with chlorate, behaved as nit1 mutants in growth tests (i.e. putatively defective in nitrate reductase apoenzyme). When nit1 mutants were paired on medium with nitrate as sole nitrogen source some pairings complemented, behaviour attributed to intragenic complementation. Selected crosses of SelR and nit1 mutants with wild-type strains gave 1[ratio ]1 segregation of both phenotypes and no evidence of linkage to either Mbc1 (benzimidazole resistance) or Daf1 (dicarboximide resistance) markers; loose linkage was confirmed between Mbc1 and Daf1. Strains showing the SelR phenotype may result from mutations in different genes; the genotypic symbol Sel 1 is allocated to one of these mutations. Four-point crosses involving the markers at Sel 1, nit1, Mbc1 and Daf1 generated progeny with all 16 genotypes. Both Sel1R and nit1 mutants were stable following subculture and retained pathogenicity in a bean seedling assay. Complementation was demonstrated between SelR and nit1 mutants selected from the same parent.