Feeding activity of the earthworm Eisenia andrei in artificial soil

Quantitative information on the feeding activity of earthworms is scarce but this information is valuable in many eco(toxico)logical studies. In this study, the feeding activity of the compost worm Eisenia andrei is examined in artificial soil (OECD medium), with and without a high-quality food source (cow manure), and at two temperatures (10 and 20 °C). Methods are provided to estimate the most important parameters: gut load, selection of organic matter (OM), digestion efficiency, compaction, gut retention time, and fraction of manure in the diet. Lanthanides (Lu and Tm) were successfully used as inert markers in soil and manure, and we applied Bayesian statistics to analyse the data and fully capture the compounded uncertainty in the parameter estimates. Results show that the compost worm does not feed on soil indiscriminately but is able to select an OM-enriched diet from apparently homogeneous OECD medium. When manure is present on the soil surface, approximately three-quarters of the diet still consists of soil particles. The gut load of the worms was approximately 10% (dwt gut/wwt empty worm), varying little with the treatments. Unfortunately, the digestion efficiency could only be reliably estimated at 20 °C, and was approximately 40%. Temperature clearly affected feeding as a 10° temperature decrease nearly doubled the gut retention time (from 2.9 to 5.5 h), which corresponds to a two-fold decrease in feeding rate. The present data may be used to interpret toxicity and accumulation studies with E. andrei in OECD medium. However, care must be taken, as it seems possible that feeding is influenced by the size of the worm and subtle differences in experimental set-up.     

Environmental fate and effects of Bacillus thuringiensis (Bt) proteins from transgenic crops: a review

This paper reviews the scientific literature addressing the environmental fate and nontarget effects of the Cry protein toxins from Bacillus thuringiensis (Bt), specifically resulting from their expression in transgenic crops. Published literature on analytical methodologies for the detection and quantification of the Cry proteins in environmental matrices is also reviewed, with discussion of the adequacy of the techniques for determining the persistence and mobility of the Bt proteins. In general, assessment of the nontarget effects of Bt protein toxins indicates that there is a low level of hazard to most groups of nontarget organisms, although some investigations are of limited ecological relevance. Some published reports on the persistence of the proteins in soil show short half-lives, whereas others show low-level residues lasting for many months. Improvements in analytical methods will allow a more complete understanding of the fate and significance of Bt proteins in the environment.     

Neither transgenic Bt maize (MON863) nor tefluthrin insecticide adversely affect soil microbial activity or biomass: A 3-year field analysis

Laboratory and greenhouse studies on transgenic Bacillus thuringiensis (Bt) maize have drawn attention to the persistence and activity of the Cry proteins in soil and their potential effects on soil microorganisms, but there have been few field assessments that evaluate the effects of Bt maize with those of insecticides on soil microbial populations. This study was conducted to determine the effects of Cry3Bb Bt maize with those of the insecticide tefluthrin on soil microbial biomass and activity in the field over a 3-year cropping cycle. The recently commercialized maize variety YieldGard^® Rootworm (MON863), which produces the Cry3Bb protein, was grown along with a non-Bt isoline with and without tefluthrin applied at planting. Microbial biomass, nitrogen (N) mineralization potential, short-term nitrification rate, and respiration rate were measured in rhizosphere and bulk soil samples collected from three replicate field plots just before planting, at anthesis, and at harvest in each year. There were clear seasonal effects on microbial biomass and activity in the field soils—as represented by the consistent changes in all measured variables across years and sampling times. Differences in the measured variables were also sometimes observed between bulk and rhizosphere soil. However, there were no adverse effects of either the Bt or non-Bt maize with insecticide applied compared to the non-Bt controls; on the contrary, microbial biomass and soil respiration data suggested a stimulatory effect of the Bt genotype, particularly in comparison to the non-Bt isoline. Although ‘higher’ does not necessarily mean ‘better’, the higher microbial biomass and respiration rates observed in the Bt and insecticide-applied soils compared to non-Bt soils does allay concerns that either the Bt protein or the tefluthrin typically used to control the corn rootworm reduce microbial biomass or its respiratory activity in field soils. Similarly, the higher N mineralization potential and nitrification rates observed in some soil samples from the Bt and tefluthrin-treated plots indicate higher activity of N-mineralizing microorganisms, a potentially positive consequence as both ammonium and nitrate are effective N sources for maize during grain filling. Our data suggest that cropping MON863 Bt maize is unlikely to adversely affect soil ecology in the short term. Longer-term monitoring of transgenic cropping systems should assure that the biotic functioning of the soil is maintained as a part of studies on overall ecosystem integrity.     

转Bt基因棉粉碎叶、蕾样Bt蛋白降解

以转Bt基因棉Bt冀668为材料,室内采用ELISA法,测定粉碎叶、蕾样Bt蛋白在土壤、水中不同时间的残留量以及不同环境处理下的残留量,以研究转Bt基因棉粉碎叶、蕾样Bt蛋白在不同基质中的降解动态以及与环境影响因子的关系.结果显示,粉碎叶、蕾样Bt蛋白分别在熟土、生土和水中前40d、48d取样天数间的残留量差异显著,中...     

Effects of physicochemical interactions and microbial activity on the persistence of Cry1Aa Bt (Bacillus thuringiensis) toxin in soil

Genetically modified crops, that produce Cry insecticidal crystal proteins (Cry) from Bacillus thuringiensis (Bt), release these toxins into soils through root exudates and upon decomposition of residues. The fate of these toxins in soil has not yet been clearly elucidated. Persistence can be influenced by biotic (degradation by microorganisms) and abiotic factors (physicochemical interactions with soil components, especially adsorption). The aim of this study was to follow the fate of Cry1Aa Bt toxin in contrasting soils subjected to different treatments to enhance or inhibit microbial activity, in order to establish the importance of biotic and abiotic processes for the fate of Bt toxin. The toxin was efficiently extracted from each soil using an alkaline buffer containing a protein, bovine serum albumin, and a nonionic surfactant, Tween 20. The marked decline of extractable toxin after incubation of weeks to months was soil-dependent. The decrease of extractable toxin with incubation time was not related to microbial degradation but mainly to physicochemical interactions with the surfaces that may decrease immunochemical detectability or enhance protein fixation. Hydrophobic interactions may play an important role in determining the interaction of the toxin with surfaces.     

Risk assessment of the cultivation of a stacked Bt-maize variety (MON89034 × MON88017) for nematode communities

Genetically modified Bt-maize MON89034 × MON88017 contains three different genes derived from Bacillus thuringiensis (Bt) which enable protection against insect pests, due to expression of three different insecticidal crystal proteins (Cry proteins), i.e., Cry1A.105 and Cry2Ab2 against the European corn borer and Cry3Bb1 against the Western corn root worm. Nematodes are important organisms in agricultural soil ecosystems, and on fields with Bt-maize cultivation they will be exposed to Cry proteins released into the soil from roots or plant residues. The objective of this study was to analyze in a field experiment the effect of Bt-maize MON89034 × MON88017 on nematodes as non-target organisms. Nematode communities from soil planted with the Bt-maize were compared to those from soil planted with the near-isogenic cultivar (with and without chemical insecticide treatment) and two conventional maize cultivars. The experimental field consisted of 40 plots in a completely randomized block design (eight plots for each treatment), which were monitored over two growing seasons (2008 and 2009) at six sampling dates for nematode diversity at the genus level in the rhizosphere soil. Physicochemical soil properties and Cry protein concentrations were also analyzed. Nematodes showed very high abundances, as well as a high diversity of taxa and functional guilds, indicating the relevance of maize fields as their habitat. Neither Bt-maize cultivation, nor insecticide treatment adversely affected abundance or community structure of nematode assemblages in field plots compared to several non-Bt cultivars including a near-isogenic cultivar. This confirmed the risk estimations based on the analyzed soil concentrations of extractable Cry protein, not exceeding 4.8 ng g⁻¹ soil dry weight and thus revealing a safe toxicity-exposure ratio of >20.     

Proteins Extracted by Water or Aqueous Ethanol During Refining of Developed Wheat Dough to Vital Wheat Gluten and Crude Starch as Determined by Capillary‐Zone Electrophoresis (CZE)

Fluids applied to large‐sale, technical separation of wheat starch and protein also extract soluble proteins. The degree and rate of extraction and the specific components extracted depend on the flour, the flour hydration and development, the starch‐displacing fluid composition, the temperature, and the mechanical processing method. This study sought to identify major extracted protein groups using high‐performance capillary zone electrophoresis (CZE) applied directly to fluids obtained during laboratory‐scale technical separations. A dough‐ball or compression separation method was applied using a Glutomatic system and a batter or dispersion method was applied using a a McDuffie mixer and Pharmasep vibratory separator. Process fluids were water at 22°C to model commercial practice and 70 vol% ethanol in water at ‐13°C to model the cold ethanol process being developed here. Data were referenced to use of 70 vol% ethanol in water at 22°C in the Glutomatic compression method. The dough processed by each method was developed by mixing to a separable state. When flooded with excess water, this dough immediately released starch and water‐soluble or albumin proteins. When flooded with excess cold aqueous ethanol, neither the albumin nor gliadin proteins appeared in significant amounts until the bulk of the starch had been displaced, regardless of the mechanical method. Even with extraction and manipulation well beyond that necessary for starch displacement, the net amount of gliadin proteins dissolved was only ≈10% of that available from wet developed dough using 70 vol% ethanol at 22°C. There was more gliadin protein in the fluids at earlier stages of processing when the batter dispersion method was applied using cold ethanol. The most common soluble proteins revealed in the electrophoresis patterns for the batter compression method using cold aqueous ethanol were initially albumins and later γ‐gliadins. Albumins not appearing as soluble in cold 70 vol% ethanol were found in the insoluble crude starch, suggesting their precipitation in the dough fluids during the change from free water to cold aqueous ethanol. These results establish that some protein is dissolved during starch displacement by cold aqueous ethanol, but that the amounts may be limited by control of the mechanical working of the dough in the presence of the displacing fluids.     

基于Illumina测序技术挖掘Bt杀虫基因资源

苏云金芽孢杆菌作为应用最为广泛的生物农药,已经成为化学合成农药必要的补充或替代品,其杀虫蛋白基因已成功的应用到转基因抗虫作物中。本文基于海南五指山和吊罗山热带原始雨林区土壤样品筛选分离的Bt菌株资源,利用第二代测序技术Illumina对分离的特异Bt菌株的杀虫基因进行全面分析,鉴定出新型Bt杀虫蛋白基因25个,其中包括5个新型的营养期杀虫蛋白基因（vip）和3个新型cyt基因。本研究表明第二代DNA测序技术可以高效用于挖掘Bt杀虫基因资源,而获得的新型杀虫蛋白基因将进一步丰富我国能够应用的杀虫基因种类。     

Assessing daily egestion rates in earthworms: using fungal spores as a natural soil marker to estimate gut transit time

Earthworms have an important role in ‘bioturbation’—the mixing of soil due to biological processes. Quantification of earthworm bioturbation relies on estimating earthworm egestion rates which in turn depend on two parameters: the gut content of the worms and the gut transit time (GTT). Gut content can be determined relatively easily, but determining GTT is problematic. The present study aimed at estimating daily soil egestion rates of Aporrectodea caliginosa and Lumbricus terrestris, refining the most common approach for estimating GTT by using fungal spores as natural markers in ingested soil. This approach avoids the use of artificial markers that may adversely affect the earthworms. Gut transit time was estimated by tracking the passage of marked soil through the gut by the appearance of the spores in the egested faeces. Gut transit time was estimated to be 9.6?±?0.3 h for A. caliginosa and 11.6?±?0.5 h for L. terrestris. Gut content averaged 465?±?40(± standard error (SE))?mg dw g^?1 dw worm for A. caliginosa and 265?±?80 mg dw g^?1 dw worm for L. terrestris. From these values, daily egestion rates of 1.16 and 0.66 g dw faeces g^?1 dw worm d^?1 were calculated for A. caliginosa and L. terrestris, respectively. Both values compare well to literature values for each species. The presented method for GTT estimation is inexpensive, rapid and easy to evaluate, with spores being a good alternative to existing markers.     

Soil persistence of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> (Bt) toxin from transgenic Bt cotton tissues and its effect on soil enzyme activities

A silty loam soil was incubated with the leaves and stems of two transgenic Bacillus thuringiensis (Bt) cotton varieties and nontransgenic Bt cotton to study the soil persistence of the Bt toxin from the decomposing transgenic Bt cotton tissues and its effect on soil enzyme activities. The results showed that after Bt cotton tissue amendment, Bt toxin was introduced into soil upon decomposition; about 50% of the introduced Bt toxin persisted in soil for at least 56 days. No Bt toxin was detected in the nontransgenic Bt cotton-amended soil; the amount of Bt toxin was the highest in the soil treated with the residue with the higher Bt toxin content. Activities of soil urease, acid phosphomonoesterase, invertase, and cellulase were stimulated by the addition of Bt cotton tissues, whereas activity of soil arylsulfatase was inhibited. Probably cotton tissue stimulated microbial activity in soil, and as a consequence, enzyme activities of soil were generally increased. This effect can mask any negative effect of the Bt toxin on microbial activity and thus on enzyme activities.     

Application of immunoaffinity column as cleanup tool for an enzyme linked immunosorbent assay of phosphinothricin-N-acetyltransferase detection in genetically modified maize and rape

We have developed a new immunoassay method to detect genetically modified (GM) maize and rape containing phosphinothricin-N-acetyltransferase (PAT). PAT encoded by Bialaphos resistance gene (bar) was highly expressed in soluble form in Escherichia coli BL21(DE3) and purified to homogeneity by Ni2+ affinity chromatography. A simple and efficient extraction and purification procedure of PAT from GM maize and rape was developed by means of the immunoaffinity column (IAC) as a cleanup tool. Purified polyclonal antibodies against PAT was produced and coupled covalently to CNBr-activated Sepharose 4B. Both the binding conditions and elution protocols were optimized. The IAC was successfully employed to isolate and purify the PAT from the various tissues of GM maize (Bt11 and Bt176) and rapes (MS1/RF1 and MS8/RF3). Enzyme linked immunosorbent assay (ELISA) procedures were established further on to measure the PAT protein. GM maize cannot be differentiated from non-GM maize by ELISA. But IAC-ELISA allowed 0.5% GMOs to be detected in MS1/RF1 and MS8/RF3 and 10% GMOs to be detected in Bt11 and Bt176, which makes this method an acceptable method to access PAT protein in GM rapes and maize.     

Soil solid phases effects on the proteomic analysis of Cupriavidus metallidurans CH34

Cupriavidus metallidurans CH34 is a completely sequenced soil-borne beta-proteobacterium with known genome and proteome. Comparative 2-D electrophoresis and protein mass spectrometry were used to compare the proteome of C. metallidurans CH34 from liquid culture and after incubation for 1, 3, and 12?days in microcosms containing quartz sand, kaolinite, montmorillonite, or an artificial soil. Results showed that proteome from liquid culture was similar to CH34 proteins extracted from sand and kaolinite, whereas the proteins extracted from artificial soil differed significantly and no proteins were detected from C. metallidurans CH34 incubated in the montmorillonite microcosms. Protein recovery decreased on prolonging incubation time in all microcosms. Mass spectrometry identification showed that the trend of lower recovery upon incubation time was independent on the putative function of protein. These results suggest that the soil solid phase influences the protein recovery and soil proteomic analysis and that distinction between protein recovery and protein expression in soil will be a challenging for soil proteomic researchers.     

转基因棉种植对土壤氧化还原酶活性的影响

转Bt基因棉和转Bt+CpTI基因棉及相应非转基因棉的盆栽试验研究表明,转基因作物生长30天时可向土壤中释放Bt杀虫晶体蛋白,而双抗棉种植时CpTI杀虫晶体蛋白的释放量与作物品种有关;转基因棉种植对土壤过氧化氢酶、脱氢酶和硝酸还原酶的活性影响研究表明,生长30天时,与等价基因系非转基因棉相比,转Bt基因棉和双抗棉A的种植并未使三种酶活性发生显著变化,而双抗棉B的种植使土壤脱氢酶活性显著下降。从杀虫晶体蛋白的释放和对酶活性的影响来看,双抗棉B的种植对土壤的生物活性扰动更大。     

黑龙江凉水自然保护区苏云金芽孢杆菌的收集与鉴定

黑龙江凉水自然保护区是我国现有保存下来的较大片原始红松林基地之一,总面积6394hm^2,森林覆盖率95％以上。本研究从小兴安岭凉水自然保护区采集土样782份,采用醋酸钠培养基结合高温方法筛选土壤中的芽孢杆菌,通过光学显微镜观察鉴定产生伴胞晶体的苏云金芽孢杆菌（Bacillus thuringiensis．Bt）。总计分离得到芽孢杆菌和苏云金芽孢杆菌分别为1735株和33株,Bt菌株的分离率和出菌率分别为1．90％和4．22％。利用SDS-PAGE和PCR—RFLP方法对筛选获得的成分离株进行了杀虫晶体蛋白和基因型分析,结果表明14株产菱形伴胞的＆菌株,SDS-PAGE电泳分析芽孢后期产生分子量大小130kD蛋白带,PCR-RFLP分析初步鉴定为crylAc基因,其它产圆形或其它不定形晶体蛋白的＆分离菌中,芽孢后期主要蛋白大小为20～150kD不等,PCR—RFLP方法鉴定结合PCR片段测序分析这些菌株含有新型cry4、cry39和cry40基因等。本研究是“中国助资源收鉴与利用”项目组成部分之一,凉水自然保护区苏云金芽孢杆菌的收集与鉴定目的是要对整个东北地区成资源分布和多样性作一个初步评估,实验结果表明东北森林地区具有丰富多样的威菌株和杀虫基因资源。     

Rapid degradation of the Cry1F insecticidal crystal protein in soil

The gene for the core Cry1F insecticidal crystal protein (ICP) from Bacillus thuringiensis Berliner (Bt) has been incorporated into the genome of maize plants, Zea mays L. Plants expressing this ICP are protected from attack by various Lepidopteran pests including the European corn borer, Ostrinia nubilalis (Hübner). The stability of the Cry1F ICP in soil was assessed in a laboratory study designed to determine the persistence of the active protein residue in soil over time, using insect bioassay as the analytical quantification method. The GI(50) (concentration estimated to inhibit growth by 50%) rose at each consecutive incubation interval, indicating a consistent decline in Cry1F activity over time. The residue data were poorly described by a first-order model when fit to either the full data or a truncated data set where the last interval (28 days) was excluded. Data were well described by a shift-log model, and this model predicted DT(50) (time until 50% decay) and DT(90) (time until 90% decay) values of 0.6 and 6.9 days, respectively. This rapid degradation rate was consistent with other Bt proteins evaluated in our laboratory.     

Mechanisms of stabilization of earthworm casts and artificial casts

Summary Fresh casts were collected from the earthworm species Aporrectodea caliginosa, and artificial casts were also made. The casts were subjected to ageing, drying-rewetting, and sterilization by hexanol vapour. Clay dispersion was determined, as a measure of the lack of stability of the casts. Two soils were used, the topsoil of a recently reclaimed polder soil in the Netherlands and the topsoil from a South Australian duplex soil. For both soils the fresh worm casts had higher dispersible clay than the artificial casts. During ageing, both types of casts became more stable. There are strong indications that this was mainly due to changes on the surface of the casts. Fungi developed on the surface of 6-day-old worm casts made of Australian soil. This gave a higher stability to the casts compared to artificial casts of the same age without fungal growth. With both types of casts, hexanol inhibited fungal growth on the surface of the casts, reducing the stabilizing effect of ageing. The fungus did not develop on Dutch soil casts until after 42 days, and the development of a higher stability with age was also less marked than in the Australian soil. When the casts were subjected to a drying and rewetting cycle before analysis, they became much more stable than the casts that were analyzed wet. The drying-rewetting cycle removed most of the differences between the worm casts and the artificial casts, and also removed any effects of ageing.Dedicated to the late Prof. Dr. W. Kühnelt     

转基因植物释放Bt毒素的土壤环境行为与生物学效应

Bt(Bacillus thuringiensis)杀虫晶体蛋白基因是植物抗虫基因工程中应用最广泛的基因,随着大批转Bt基因作物的商品化,Bt毒素对土壤生态系统的影响已引起了人们的高度关注。本文对转基因植物释放Bt毒素的土壤环境行为与生物效应进行了综述,包括Bt毒素蛋白进入土壤的途径、在土壤中的运动、与土壤颗粒的结合与存留及其对土壤生物和土壤酶的影响。     

Effect of Transgenic Bt Cotton on Bioactivities and Nutrients in Rhizosphere Soil

The influence of transgenic Bacillus thuringiensis (Bt) cotton (BtXincai1) and its corresponding nontransgenic isoline (Xincai1) on the microorganisms, enzyme activity, and nutrient content of rhizosphere soil was studied through experiments in potted plants. The calcareous drab soil samples were collected (0–15 cm deep) from an experimental field in Shanxi Agricultural University (China) in 2005. The pots were categorized in different groups with replicates for each variety (transgenic BtXincai1 and general Xincai1). The rhizosphere soil samples were collected at different growth periods (seedling, bud, flowering, peak boll, boll opening, and harvest). The Bt protein and other microbial properties in the soil samples were determined by using selected methods (material and methods session). The results demonstrated that the concentration of the Bt protein in the rhizosphere soil of BtXincai1 reached a peak at 56.14 ng g^?1 during the flowering period. However, the Bt protein would not continuously accumulate in the soil. The rhizosphere soil of BtXincai1 was more suitable for the growth and proliferation of bacteria and fungi but it had no significant impact on the number of actinomycetes. BtXincai1 had some inhibitory effects on alkaline phosphatase activity in the rhizosphere soil, and it might promote dehydrogenase activity during the blooming period. However, it had no significant influence on protease, urease, or sucrase activities. Further, it had no significant impact on the contents of organic matter, total nitrogen, available nitrogen, or potassium in rhizosphere soil. It could significantly decrease the content of available phosphorus during the flowering period. Based on this study, the sensitive reactions of microorganisms and the activities of alkaline phosphatase and dehydrogenase might be considered as three potential indexes for assessing the risk posed by transgenic Bt cotton to soil ecology.     

Bt水稻杀虫蛋白时空变化及秸秆还田后在土壤中的持留规律

以Bt抗虫水稻华池B6、TT51及其非转基因水稻亲本嘉早935、明恢63,以及与它们亲缘较远但农艺性状相近的水稻品种中九B、R9311为试验材料,研究了田间种植条件下Bt抗虫水稻杀虫蛋白的时空变化及其在根际土中的持留规律,同时,还研究了秸秆还田后Bt蛋白在土壤中的持留规律。结果表明：1）Bt抗虫水稻华池B6植株各个部位...     

Bt杀虫晶体蛋白的土壤残留及其对土壤磷酸酶活性的影响

研究表明以不同形式导入土壤中的杀虫晶体蛋白在土壤中的残留特性及其对土壤磷酸酶活性的影响有所不同。以Bt菌体向土壤导入杀虫晶体蛋白的试验表明 :随着培养时间的延长 ,土壤中杀虫晶体蛋白含量逐渐增加 ,到 1 5d时达到一个峰值 ,而后下降 ,在培养 30d时 ,杀虫晶体蛋白含量基本与初始含量相同。以不同Bt棉组织添加土壤的试验表明 :随着培养时间的延长 ,土壤中的杀虫晶体蛋白含量降低 ,在培养初期下降的速度较快 ,随后下降的速度较慢 ,在培养的中后期基本稳定 ,在培养 5 6d时 ,杀虫晶体蛋白含量为初始值的 4 4 7%(ZK)和 5 6 1 %(GK)。不同Bt棉的盆栽试验表明 :在整个生育期内 ,Bt棉花种植后根际土壤中杀虫晶体蛋白含量均明显比非Bt棉高。Bt菌体和Bt棉组织处理的土壤磷酸酶活性均呈现出比对照高的趋势 ,而在Bt棉种植过程中Bt棉根际土壤的磷酸酶活性则呈现出比非Bt棉低的趋势。无论以何种方式向土壤中导入杀虫晶体蛋白 ,土壤磷酸酶活性在不同杀虫晶体蛋白浓度处理间的差异显著。