Evaluating resistance to Bt toxin Cry1Ab by F2 screen in European populations of Ostrinia nubilalis (Lepidoptera: Crambidae).

The large-scale cultivation of transgenic crops producing Bacillus thuringiensis (Bt) toxins have already lead to the evolution of Bt resistance in some pest populations targeted by these crops. We used the F2 screening method for further estimating the frequency of resistance alleles of the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), to Bt maize, Zea mays L., producing the Cry1Ab toxin. In France, Germany, and Italy, 784, 455, and 80 lines of European corn borer were screened for resistance to Mon810 maize, respectively. In Slovakia, 26 lines were screened for resistance to the Cry1Ab toxin. The cost of F2 screen performed in the four countries varied from U.S. dollars 300 to dollars 1300 per line screened. The major difference in cost was mostly due to a severe loss of univoltine lines during the screen in Germany and Slovakia. In none of the screened lines did we detect alleles conferring resistance to Mon810 maize or to the Cry1Ab toxin. The frequency of resistance alleles were < 1.0 x 10(-3), < 1.6 x 10(-3), < 9.2 x 10(-3), and < 2.6 x 10(-2) in France, Germany, Italy, and Slovakia, with 95% probability, respectively. The average detection probability over all lines was approximately 90%. Making the assumption that European corn borer populations in these countries belong to the same genetic entity, the frequency of alleles conferring resistance to the Cry1Ab produced by the Mon810 maize in western and central Europe was 1.0 x 10(-4), with a 95% confidence interval of 0-3.0 x 10(-4).

Novel activation mechanism for the promutagenic herbicide diallate.

The potent bacterial mutagen 2-chloroacrolein is formed from the carcinogenic herbicide S-2,3-dichloroallyl diisopropylthiocarbamate (diallate) on incubation with hepatic microsomal monooxygenases or on reaction with m-chloroperbenzoic acid. A proposed activation mechanism for this promutagen involves sulfoxidation followed by [2,3] sigmatropic rearrangement and 1,2-elimination reactions. A portion of the highly reactive intermediate, diallate sulfoxide (proximate mutagens), is attacked by glutathione in a reaction which competes with its transformation to the ultimate mutagen, 2-chloroacrolein.

Monitoring the Cry1Ab susceptibility of European corn borer in Germany.

The European corn borer, Ostrinia nubilalis (Hübner), is one of the most important insect pests in corn, Zea mays L. Transgenic corn cultivars expressing Bacillus thuringiensis (Bt) toxin provide a promising crop protection strategy against European corn borer; however, management is needed to avoid resistance development of the target pest species. The aim of this work was to establish the baseline susceptibility of different European corn borer populations in Germany to be able to forecast a possible development of resistance at an early stage. To standardize test procedures for future resistance management, the efficiency of Cry1Ab toxins from different suppliers and different production was assessed. Furthermore, two different test methods, surface treatment and the incorporation method, were compared with regard to their practicability and efficiency. Neither method provided significant differences in the baseline susceptibility of populations from different German regions. Overall, the data suggested little differentiation among German populations in terms of their susceptibility to Bt toxin and their genetic background. Future monitoring could therefore use a single European corn borer population as a representative for southwestern Germany. However, toxins from different suppliers and different production batches produced a vast range of LC50 values. Changes because of different toxin batches may be mistaken as a change in baseline susceptibility or even as the start of a resistance development. Thus, it is important throughout insect resistance management that the same toxin batches will be available for baseline susceptibility bioassays and for future tests.

Impact of gene flow from cultivated beet on genetic diversity of wild sea beet populations

Gene flow and introgression from cultivated plants may have important consequences for the conservation of wild plant populations. Cultivated beets (sugar beet, red beet and Swiss chard: Beta vulgaris ssp. vulgaris) are of particular concern because they are cross-compatible with the wild taxon, sea beet (B.vs. ssp. maritima). Cultivated beet seed production areas are sometimes adjacent to sea beet populations; the numbers of flowering individuals in the former typically outnumber those in the populations of the latter. In such situations, gene flow from cultivated beets has the potential to alter the genetic composition of the nearby wild populations. In this study we measured isozyme allele frequencies of 11 polymorphic loci in 26 accessions of cultivated beet, in 20 sea beet accessions growing near a cultivated beet seed production region in northeastern Italy, and 19 wild beet accessions growing far from seed production areas. We found one allele that is specific to sugar beet, relative to other cultivated types, and a second that has a much higher frequency in Swiss chard and red beet than in sugar beet. Both alleles are typically rare in sea beet populations that are distant from seed production areas, but both are common in those that are near the Italian cultivated beet seed production region, supporting the contention that gene flow from the crop to the wild species can be substantial when both grow in proximity. Interestingly, the introgressed populations have higher genetic diversity than those that are isolated from the crop. The crop-to-wild gene flow rates are unknown, as are the fitness consequences of such alleles in the wild. Thus, we are unable to assess the long-term impact of such introgression. However, it is clear that gene flow from a crop to a wild taxon does not necessarily result in a decrease in the genetic diversity of the native plant.

High-performance liquid chromatographic determination of the rhamnolipids produced by Pseudomonas aeruginosa.

The bacterial biosurfactants 3-[3'-(L-rhamnopyranosyloxy)decanoyloxy]decanoic acid (RL-1) and 3-[3'-(2"-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyloxy) decanoyloxy]decanoic acid (RL-2) were isolated from Pseudomonas aeruginosa DSM 2659 cultures. An HPLC method was developed for the p-bromophenacyl esters of the rhamnolipids. Separation was obtained within 25 min on a RP C18 column using a linear gradient of water-acetonitrile (30:70 to 0:100) and UV detection (265 and 320 nm). Linearity of response existed for 1.2-25 microg of the RL-1 and 1.8-25 microg of the RL-2-p-bromophenacyl ester (0.9-19.2 microg RL-1 and 1.3-18.0 microg RL-2). The reproducibility of the entire analytical method (extraction, derivatisation) was tested.

Environmental fate and distribution of sodium [14C] pentachlorophenate in a section of urban wasteland ecosystem.

An urban terrestrial microecosystem has been used under outdoor conditions to study the transfer of chemical residues within the system components. The microecosystem consisted of soil monoliths obtained from a site with an established vegetation cover dominated by goldenrod, Solidago gigantea. The microecosystem contained integrated food chain elements composed of primary producers, herbivores and carnivores. The system was stocked with indicator insect species, snails (Cepaea nemoralis) and earthworms (Allolobophora caliginosa), captured from the original site. Evaluation of the system was made using radiolabelled sodium pentachlorophenate (PCP-Na) which was applied as a single application at an equivalent rate of 5 kg ha-1. The mass balance revealed that, after 131 days, in the autumn, and after 222 days, in the winter, that 43 and 39% radiocarbon, respectively, was recoverable from the microecosystem. The unaccounted radiocarbon was very probably removed through volatilization and photomineralization of the compound. PCP residues on foliage decreased rapidly, 50% of which were metabolised within 15 days. Most of the radiocarbon remaining in the system after 131 days was in the top soil and plant litter, transmitted mainly through washing off by rain and leaf litter fall. There was a variation in the uptake of PCP-Na residues in the food chain organisms, where the total radiocarbon concentrations during the first 19 days of exposure ranged, e.g. in snails, from 3 to 0.6 micrograms g-1, in springtails from 5 to 105; in beetles (Amara fusca) from 3 to 1, in spiders from 13 to 11, and in harvestman from 31 to 77 micrograms g-1. The ecological magnification indices (EM) of all the organisms with respect to their main food source, i.e. plant litter, demonstrated no bioconcentration effects. This is attributed to the metabolism of PCP-Na by the organisms and its rapid excretion. The urban wasteland ecosystem contained in outdoor lysimeters employed as a model gives valuable information and has considerable value in predicting the ecological fate of industrial chemicals.

Metabolism of the environmental estrogen bisphenol A by plant cell suspension cultures.

The metabolism of the environmental estrogen bisphenol A (BPA) was studied in heterotrophic plant cell suspension cultures of soybean (Glycine max), wheat (Triticum aestivum), foxglove (Digitalis purpurea), and thorn apple (Datura stramonium), which were regarded as metabolic model systems for intact plants. Three main metabolic routes of BPA were observed in the tissues. Most of the radioactivity found in the cell extracts consisted of carbohydrate conjugates of BPA amounting to about 85% (foxglove), 80% (wheat), 7% (soybean) and 15% (thorn apple) of applied 14C. The second main route was formation of non-extractable residues. Portions detected were low in foxglove (3.9% of applied 14C), moderate in wheat (13.5%), high in thorn apple (27.4%) and soybean (49.4%). With thorn apple, BPA derived bound residues were preponderantly resistant towards acid treatment; only traces of BPA were released. The third route was the formation of a highly polar, presumably polymeric material detected in media of soybean and thorn apple (29.3% and 36.0% of applied 14C, respectively). The mechanism of its formation remained unknown. In thorn apple, this highly polar material was formed extremely rapidly, and was considerably stable. Only traces of BPA were liberated by hydrolytic treatment with cellulase or acid. During hydrolysis experiments with glycoside fractions, non-extractable residues and highly polar materials, low amounts of presumably primary metabolites of BPA (up to 6% of applied 14C) were detected besides the parent compound; their chemical structures remained unclear.

Metabolism of the herbicide glufosinate-ammonium in plant cell cultures of transgenic (rhizomania-resistant) and non-transgenic sugarbeet (Beta vulgaris), carrot (Daucus carota), purple foxglove (Digitalis purpurea) and thorn apple (Datura stramonium).

The metabolism of the herbicide glufosinate-ammonium was investigated in heterotrophic cell suspension and callus cultures of transgenic (bar-gene) and non-transgenic sugarbeet (Beta vulgaris). Similar studies were performed with suspensions of carrot (Daucus carota), purple foxglove (Digitalis purpurea) and thorn apple (Datura stramonium). 14C-labelled chemicals were the (racemic) glufosinate, L-glufosinate, and D-glufosinate, as well as the metabolites N-acetyl L-glufosinate and 3-(hydroxymethylphosphinyl)propionic acid (MPP). Cellular absorption was generally low, but depended noticeably on plant species, substance and enantiomer. Portions of non-extractable residues ranged from 0.1% to 1.2% of applied 14C. Amounts of soluble metabolites resulting from glufosinate or L-glufosinate were between 0.0% and 26.7% of absorbed 14C in non-transgenic cultures and 28.2% and 59.9% in transgenic sugarbeet. D-Glufosinate, MPP and N-acetyl L-glufosinate proved to be stable. The main metabolite in transgenic sugarbeet was N-acetyl L-glufosinate, besides traces of MPP and 4-(hydroxymethylphosphinyl)butanoic acid (MPB). In non-transgenic sugarbeet, glufosinate was transformed to a limited extent to MPP and trace amounts of MPB. In carrot, D stramonium and D purpurea, MPP was also the main product; MPB was identified as a further trace metabolite in D stramonium and D purpurea.

Oxidative metabolic profiling of xenobiotics by human P450s expressed in tobacco cell suspension cultures.

Elucidation of metabolic pathways of xenobiotics (pesticides, pharmaceuticals and industrial pollutants) in human, animals and plants and chemical identification of corresponding metabolites are required for comprehensive (eco-) toxicological evaluation of the compounds prior to their usage. The most important metabolic products are oxidized metabolites, and most of these are formed by catalytic activity of P450s (cytochrome P450 mono-oxygenases). In human, 11 P450 isoenzymes exhibiting broad and overlapping substrate specificities are responsible for approx. 90% of drug metabolism. As support for inevitable metabolism studies with intact organisms under relevant conditions, tobacco cell cultures were transformed separately with cDNA sequences of human P450 isoenzymes CYP1A1, CYP1A2 and CYP3A4. The resulting P450-transgenic cell suspensions were used for metabolism studies with pesticides, industrial pollutants, a secondary plant metabolite and human sex hormones. A summary of basic results is provided; these are discussed regarding application of the method for screening of the oxidative metabolism of xenobiotics and the large-scale production of metabolites.