Transgenic Bt plants decompose less in soil than non-Bt plants

Bt plants are plants that have been genetically modified to express the insecticidal proteins (e.g. Cry1Ab, Cry1Ac, Cry3A) from subspecies of the bacterium, Bacillus thuringiensis (Bt), to kill lepidopteran pests that feed on corn, rice, tobacco, canola, and cotton and coleopteran pests that feed on potato. The biomass of these transgenic Bt plants (Bt+) was decomposed less in soil than the biomass of their near-isogenic non-Bt plant counterparts (Bt−). Soil was amended with 0.5, 1, or 2% (wt wt⁻¹) ground, dried (50 °C) leaves or stems of Bt corn plants; with 0.5% (wt wt⁻¹) ground, dried biomass of Bt rice, tobacco, canola, cotton, and potato plants; with biomass of the near-isogenic plants without the respective cry genes; or not amended. The gross metabolic activity of the soil was determined by CO₂ evolution. The amounts of C evolved as CO₂ were significantly lower from soil microcosms amended with biomass of Bt plants than of non-Bt plants. This difference occurred with stems and leaves from two hybrids of Bt corn, one of which had a higher C:N ratio than its near-isogenic non-Bt counterpart and the other which had essentially the same C:N ratio, even when glucose, nitrogen (NH₄NO₃), or glucose plus nitrogen were added with the biomass. The C:N ratios of the other Bt plants (including two other hybrids of Bt corn) and their near-isogenic non-Bt counterparts were also not related to their relative biodegradation. Bt corn had a significantly higher lignin content than near-isogenic non-Bt corn. However, the lignin content of the other Bt plants, which was significantly lower than that of both Bt and non-Bt corn, was generally not statistically significantly different, although 10-66% higher, from that of their respective non-Bt near-isolines. The numbers of culturable bacteria and fungi and the activity of representative enzymes involved in the degradation of plant biomass were not significantly different between soil amended with biomass of Bt or non-Bt corn. The degradation of the biomass of all Bt plants in the absence of soil but inoculated with a microbial suspension from the same soil was also significantly less than that of their respective inoculated non-Bt plants. The addition of streptomycin, cycloheximide, or both to the soil suspension did not alter the relative degradation of Bt+ and Bt− biomass, suggesting that differences in the soil microbiota were not responsible for the differential decomposition of Bt+ and Bt− biomass. All samples of soil amended with biomass of Bt plants were immunologically positive for the respective Cry proteins and toxic to the larvae of the tobacco hornworm (Manduca sexta), which was used as a representative lepidopteran in insect bioassays (no insecticidal assay was done for the Cry3A protein from potato). The ecological and environmental relevance of these findings is not clear.     

Release of the recombinant Cry3Bb1 protein of Bt maize MON88017 into field soil and detection of effects on the diversity of rhizosphere bacteria

A three-year experimental field study with a genetically engineered Bt maize (event MON88017) and three conventionally bred cultivars was conducted to quantify the recombinant Cry3Bb1 protein released into soil and detect effects on the diversity of soil bacteria. Protein extraction and an enzyme-linked immunosorbent assay (ELISA) allowed a threshold detection of 0.01 ng Cry3Bb1 g^?1 soil. The maximum amount found in field plots with Bt maize was 1.0 ng Cry3Bb1 g^?1 rhizosphere soil. Average concentrations during the growing seasons varied between years from 0.07 to 0.29 ng g^?1. No accumulation of Cry3Bb1 in soil occurred over the three growing seasons. Four weeks after harvest, the major Cry3Bb1 reservoirs on the field were the remaining root stubbles, but their Cry3Bb1 concentration declined by 98.30–99.99% in the following seven months. During the three consecutive years of study there were never significant differences between the rhizosphere bacterial community structure of the Bt maize and the other cultivars, as detected by cultivation independent profiling of PCR-amplified 16S rRNA genes. The low concentrations of soil extractable Cry3Bb1, its degradation in decaying roots, and the lack of effects on rhizosphere bacteria give no indications of adverse effects of MON88017 cultivation on soil ecology.     

Litterbag decomposition of residues from Bacillus thuringiensis (Bt) rice hybrids and the parental lines under multiple field conditions

Purpose

The cultivation of genetically modified (GM) crops has raised environmental concerns, since large amounts of plant materials remain in the field after harvesting. Specific proteins of GM crops might negatively impact soil ecosystem by changing residue decomposition dynamics. Particularly, the residue decomposition of crop-wild hybrids, which were formed through transgene escape to wild population, remains unexplored.

Materials and methods

We used litter bags to assess residue (leaves, stems and roots) decomposition dynamics of two stacked genes from Bacillus thuringiensis (Bt) Cry1Ac and the sck (a modified CpTI gene encoding a cowpea trypsin-inhibitor) (Bt/CpTI) rice lines (Kefeng-6 and Kefeng-8), a non-transgenic rice near isoline (Minghui86), wild rice (Oryza rufipogon) and Bt wild rice at three sites. The enzyme-linked immunosorbent assay (ELISA) was used to monitor the changes of the Cry1Ac protein in Bt rice residues.

Results and discussion

Mass remaining, total N and total C concentrations of rice residues declined over time and varied among plant tissues, with significant differences among cultivar, crop-wild hybrids and wild rice, but no differences between Bt and non-Bt rice cultivars. The initial concentration of Cry1Ac was higher in leaves and stems than in roots and was different between rice types. The degradation dynamics of Cry1Ac fitted best to a first-order kinetics model and correlated with the level of total nitrogen in residues but did not correlate with the mass decomposition rate. The predicted DT50 (50 % degradation time) of the protein ranged from 10.7 to 63.6 days, depending on plant types, parts and burial sites. By the end of the study (~170 days), the protein was present in low concentration in the remaining residues.

Conclusions

Our results suggest that the impacts of the stacked Bt/CpTI gene inserts on the decomposition dynamics of rice residues are insignificant.     

Decomposition of Bacillus thuringiensis (Bt) transgenic rice residues (straw and roots) in paddy fields

Background, aim, and scope  

Genetic modification of commercial crops may affect their decomposition and nutrient cycling processes in agricultural ecosystems. Intensive rice cultivation under partially submerged conditions (paddy rice) is an important and widespread cropping system, particularly in the tropics, yet there is little data on the decomposition of Bt rice residue under field conditions. We investigated straw and root decomposition of rice modified to express the Cry1Ab protein of Bacillus thuringiensis (Bt) to kill lepidopteran pests, compared with a parental non-Bt isoline. The objective of this study was to assess the possible impacts of cry gene transformation of rice on residue decomposition under intensive rice cultivation with long period of submergence.     

Fate and effects of insect-resistant Bt crops in soil ecosystems

Recent applications of biotechnology, especially genetic engineering, have revolutionized crop improvement and increased the availability of valuable new traits. A current example is the use of the insecticidal Cry proteins from the bacterium, Bacillus thuringiensis (Bt), to improve crops, known as Bt crops, by reducing injury from various crop pests. The adoption of genetically modified (GM) crops has increased dramatically in the last 11 years. However, the introduction of GM plants into agricultural ecosystems has raised a number of questions, including the ecological impact of these plants on soil ecosystems. Crop residues are the primary source of carbon in soil, and root exudates govern which organisms reside in the rhizosphere. Therefore, any change to the quality of crop residues and rhizosphere inputs could modify the dynamics of the composition and activity of organisms in soil. Insect-resistant Bt crops have the potential to change the microbial dynamics, biodiversity, and essential ecosystem functions in soil, because they usually produce insecticidal Cry proteins through all parts of the plant. It is crucial that risk assessment studies on the commercial use of Bt crops consider the impacts on organisms in soil. In general, few or no toxic effects of Cry proteins on woodlice, collembolans, mites, earthworms, nematodes, protozoa, and the activity of various enzymes in soil have been reported. Although some effects, ranging from no effect to minor and significant effects, of Bt plants on microbial communities in soil have been reported, using both culturing and molecular techniques, they were mostly the result of differences in geography, temperature, plant variety, and soil type and, in general, were transient and not related to the presence of the Cry proteins. The respiration (i.e., CO₂ evolution) of soils cultivated with Bt maize or amended with biomass of Bt maize and other Bt crops was generally lower than from soils cultivated with or amended with biomass of the respective non-Bt isolines, which may have been a result of differences in chemical composition (e.g., the content of starch, soluble N, proteins, carbohydrates, lignin) between Bt plants and their near-isogenic counterparts. Laboratory and field studies have shown differences in the persistence of the Cry proteins in soil, which appear to be the result primarily of differences in microbial activity, which, in turn, is dependent on soil type (e.g., pH, clay mineral composition, other physicochemical characteristics), season (e.g., temperature, water tension), crop species (e.g., chemical composition, C:N ratio, plant part), crop management practices (e.g., till vs. no-till), and other environmental factors that vary with location and climate zones. This review discusses the available data on the effects of Cry proteins on below-ground organisms, the fate of these proteins in soil, the techniques and indicators that are available to study these aspects, and future directions.     

Adsorption and Desorption of Cry1Ab Proteins on Differently Textured Paddy Soils

In recent years, selected cry genes from Bacillus thuringiensis (Bt) encoding the production of Cry proteins (Bt toxins) have been engineered into crop plants (Bt-crops). Through the cultivation of Bt crops and the application of Bt pesticides, Cry proteins could be introduced into arable soils. The interaction between the proteins and soils was analyzed in this study to investigate the affinity of Cry proteins in paddy soil ecosystems. Four Paddy soils were selected to represent different soil textures. Cry proteins were spiked in soils, and the amount of protein adsorbed was measured over 24 h. Desorption of Cry1Ab proteins from paddy soils was performed by washing with sterile Milli-Q water (H₂O_MQ), and subsequently extracted with an extraction buffer. The paddy soils had a strong affinity for Cry1Ab proteins. Most of the Cry1Ab proteins added (> 98%) were rapidly adsorbed on the paddy soils tested. More Cry1Ab proteins were adsorbed on non-sterile soils than on sterile soils. Less than 2% of the adsorbed Cry1Ab proteins were desorbed using H₂O_MQ, while a considerable proportion of the adsorbed proteins could be desorbed with the buffer, ranging from 20% to 40%. The amount of proteins desorbed increased with the increases in the initial amount of Cry1Ab proteins added to the paddy soils. The concentration of Cry1Ab proteins desorbed from the paddy soils was higher for sterile soils than non-sterile ones. Our results indicate that Bt toxins released via the cultivation of Bt crops, the application of Bt pesticides can be adsorbed on paddy soils, and soil texture could impose an impact on the adsorption capability.     

Exposure and effects assessments of Bt-maize on non-target organisms (gastropods, microarthropods, mycorrhizal fungi) in microcosms

Potential differences between Bt-maize (MEB307 expressing the insecticidal Cry1Ab protein) and a near-isogenic non-Bt variety (Monumental) in their influence on the garden snail (Helix aspersa), soil microarthropods (Collembola, Actinedida, Acaridida, Gamasida and Oribatida) and mycorrhizal fungi were studied. Growing snails were caged in microcosms allowing the development of Bt or non-Bt-maize (Zea mays L.) on a sandy loam soil. After 3 months exposure, survival and growth of snails were similar in both treatments. Cry1Ab protein was detected in the Bt-maize leaves (22–42.2 μg Bt protein g⁻¹ dry wt), in the snail tissues (0.04–0.11 μg Bt-protein g⁻¹ dry wt) and in their faeces (0.034–5 μg Bt-protein g⁻¹ dry wt). Total soil microarthropod abundance and diversity were similar between control (non-Bt-maize) and the genetically modified (GM) Bt-maize microcosms. The mycorrhizal colonization of roots did not differ between Bt and non-Bt-maize (frequency of mycorrhizal roots was 88.7% and 83.3% respectively). The mycorrhizal infectivity of soils, expressed as MI₅₀ (minimum soil dry weight required to colonize 50% of plants) was measured using red clover. MI₅₀ was similar for soils where Bt or non-Bt-maize was cultivated for 4 months. The detection of Cry1Ab protein in the viscera and faeces of H. aspersa exposed to Bt-maize indicates that snails contribute to the transfer of the Bt-protein from plant to soil or snail predators. This may constitute an alternative route of exposure for Bt-protein in soil, but this was without a negative influence on mycorrhizal fungi or microarthropods. Results showed that Bt-maize was not toxic for the selected non-target species exposed for 3 or 4 months. The microcosms and analyses used in this study represent new methods for assessing effects of chronic exposure to GM plants of several diverse, yet ecologically and temporally associated species. As the soil organisms we studied can also be used in standardized ecotoxicological tests (XP X31-205-2 for mycorrhizal fungi, ISO 11267 for Collembola and ISO 15952 for snails), microcosm exposures represent a way to link laboratory and field methods for the ecotoxicological evaluation of GM plants.     

Fruiting-branch removal enhances endotoxin expression and lint yield in Bt cotton

Abstract

A two-year field experiment was conducted to determine the effects of removal of early-fruiting branches (REFB) on yield, quality, and endotoxin expression in transgenic Bt (Bacillus thuringiensis) cotton (Gossypium hirsutum L.). Two early-fruiting branches of field-grown cotton plants were removed and retained at squaring to form the REFB and the control treatments, respectively. Lint yield, yield components, fibre quality, and Cry1Ac protein concentration in the first fully expanded young leaves on the main stem were measured. Results show that lint yields were increased by 5.1 and 5.5% with REFB compared with control in 2004 and 2005, respectively. There was no difference in fibre quality in the first two harvests between REFB and control, but fibre strength and micronarie in the third harvest were improved with REFB. Levels of total N, soluble protein, and Cry1Ac protein as well as glutamic-pyruvic transaminase (GPT) activity in leaves were higher in REFB than in the control. Laboratory bioassay showed significant enhancement of the control efficacy by REFB in terms of Helicoverpa armigera (Hübner) neonate mortality for both years. It is suggested that REFB might be a potential practice for enhancing transgenic Bt cotton production.     

Agricultural studies of GM maize and the field experimental infrastructure of ECOGEN

Within the ECOGEN project, long-term field experiments with genetically modified maize, Zea mays L. were conducted to study agro-ecological effects on the soil fauna and agro-economic implications of the technology. Here, we describe the study-sites, experimental layout and results of agro-economic relevance. Experiments were conducted during 2002–2005 in Denmark (Foulum), northern France (Varois) and the Midi-Pyrenees region of southern France (Narbons). MON810 Bacillus thuringiensis (Bt)-varieties expressing the Cry1Ab protein, and a T25 glufosinate-ammonium (Basta) tolerant variety expressing the pat-gene encoding phosphinotrinacetyl-transferase were compared with near-isogenic non-Bt varieties, and conventional maize varieties. At Foulum, the maize was harvested for silage. There were no significant differences in yield between Bt-maize and a near-isogenic non-Bt variety, while a small difference in N-concentration of dry matter was detected in 1 year in a range of a measured quality parameters. Similar yield and quality were found in ploughed and reduced tillage treatments in all varieties. At Varois, the maize was harvested at ripeness and no significant differences in grain yield between Bt-maize and near-isogenic non-Bt varieties were found. These results were expected, as only Narbons harbours significant corn-borer populations. At Narbons, the number of Sesamia and Ostrinia corn-borer larvae were significantly lower in the Bt-maize than in a near-isogenic non-Bt variety and for Sesamia even less than in conventional varieties sprayed with pesticides to control corn-borer infestation. Here, Bt-maize produced a higher grain yield and grain size than a near-isogenic non-Bt variety or allowed a significant reduction in pesticide use. Concentrations of Cry1Ab in the Bt-varieties were sufficient to effectively control corn-borer larvae. In soil, Cry1Ab was close to the limit of detection and the protein did not accumulate in the soil year on year.     

泥鳅生长及抗氧化 解毒酶系统对水体中转Cry1Ab/Ac基因水稻残遗物的响应

在泥鳅养殖水体中添加稻秆粉模拟水稻残遗物生境,研究了泥鳅生长和肝胰脏抗氧化酶(SOD、CAT)与解毒酶(GST)活性对转Cry1Ab/Ac基因水稻‘华恢1号’(HH1)的响应。设计以HH1稻秆粉10 mg·L?1、50 mg·L?1、100 mg·L?1和200 mg·L?1 4个梯度浓度处理泥鳅为试验组,以非转Bt基因水稻‘明恢63’(MH63)稻秆粉处理组为阴性对照,不加稻秆粉的基础饲养组为空白对照。结果显示:在4种稻秆粉浓度下,HH1组与MH63对照组泥鳅的特定生长率、肥满度、内脏系数及SOD、CAT和GST酶活性均无显著差异(P0.05);与空白对照比较,稻秆粉浓度升高对泥鳅生长的抑制逐渐增强,当浓度达到200 mg·L?1时,HH1组和MH63对照组泥鳅的特定生长率、内脏系数与CAT活性降低。研究结果表明,水体中低含量的转融合基因Cry1Ab/Ac水稻HH1稻秆粉对泥鳅的生长与生理酶活性没有明显影响,高浓度HH1和MH63稻秆粉均使泥鳅的生长和生理酶活性显著降低,这可能与养殖水体中浓度较高的悬浮稻秆粉妨碍了泥鳅的呼吸和滤食,及稻秆粉的分解降低了水体p H和溶氧量有关。     

The specific responses of Acari community to Bt cotton cultivation in agricultural soils in northern China

Transgenic cotton expressing the Bacillus thuringiensis (Bt) insecticidal crystal (Cry1A) protein effectively controls the cotton bollworm and thus has been planted extensively in China. However, the large-scale release of Bt cotton may have undesirable effects on soil fauna due to Bt protein accumulation and the pleiotropic effects of genetic manipulation. A survey of soil mites was carried out monthly for two consecutive years (2009–2010) in Bt and non-Bt cotton fields. The soil mites were extracted using modified Tullgren funnels and were identified to the genus level, when possible. The results suggested that the effects of Bt cotton on soil mite community size were time dependent and taxonomic group specific. The cumulated abundance over a year was always higher in non-Bt fields for Oribatida; this effect was statistically significant in 2010 for Prostigmata and Astigmata. The changes in the community variables tested were similar between Bt and non-Bt cotton fields in 2009, whereas the taxonomic group richness, Shannon–Weaver index and evenness index were significantly different between Bt and non-Bt cotton fields in 2010. Additionally, sharp inter-annual fluctuations in the community composition of the soil mites were found, accompanied with the replacement of some taxonomic groups. Finally, the dominances of some taxonomic groups were significantly different between Bt and non-Bt cotton fields. Bt cotton cultivation fostered Laelapidae populations while inhibited Tectocepheus abundance in 2009. However, Bt cotton cultivation negatively impacted the abundances of Scheloribates and Nothrus in 2010. In conclusion, Bt cotton cultivation exerted specific impacts on soil mites.     

Detection and decay of the Bt endotoxin in soil from a field trial with genetically modified maize

Genetically modified plants and their residues may have direct effects on ecosystem processes. We aimed to determine the amount in soil of the insecticidal δ‐endotoxin, originally from the bacterium Bacillus thuringiensis, introduced into soil by root exudates and residues from genetically modified maize, to compare the short‐term rates of decay of Bt‐maize and non‐Bt‐maize, and to determine the rate at which the toxin in Bt‐maize leaves decomposes in soil. Intact soil, size fractions of soil, soluble fractions from soil and fractions of organic residues from a field where Bt‐maize had been cultivated for 4 years were analysed for the Btδ‐endotoxin. Traces of the δ‐endotoxin were detected in the whole (unfractionated) soil, the water‐soluble fractions, and some of the particle‐size fractions, but it was sufficiently concentrated only in the > 2000‐µm size fraction to be quantified. The δ‐endotoxin concentrations in this fraction ranged between 0.4 and 4.4 ng toxin g^?1 fraction, which equated to 70, 6 and 50 mg toxin m^?2 in the 0–15, 15–30 and 30–60 cm depths, respectively (or 126 mg toxin m^?2 over the 0–60 cm depth) in the field in June (early summer). The > 2000‐µm size fraction was a mixture of light‐ and dark‐coloured organic material and mineral material comprising sand grains and stable aggregates. For samples collected early in the growing season, most of the detected δ‐endotoxin was present in the light‐coloured organic material, which was comprised of primarily live roots. However, recognizable maize residues, probably from previous years' crops, also contained δ‐endotoxin. In a laboratory incubation study, Bt‐ and non‐Bt‐maize residues were added to soil and incubated for 43 days. There was no detectable difference in the decomposition of plant material from the two lines of maize, as determined by CO₂ production. The quantity of δ‐endotoxin in the decomposing plant material and soil mixtures declined rapidly with time during the incubation, with none being detectable after 14 days. The rapid disappearance of the δ‐endotoxin occurred at a rate similar to that of the water‐soluble components of the maize residues. The results suggested that much of the δ‐endotoxin in crop residues is highly labile and quickly decomposes in soil, but that a small fraction may be protected from decay in relatively recalcitrant residues.     

Are survival and reproduction of Enchytraeus albidus (Annelida: Enchytraeidae) at risk by feeding on Bt-maize litter?

Enchytraeids are saprophagous soil organisms, appearing in high abundances and contributing to ecological processes within the soil. For decades they have been used as model species for biological research. In the framework of research on genetically modified plants, however, they have not been considered to date. Following the ISO/DIS guideline, survival and reproduction of Enchytraeus albidus, fed with diets containing Bt-maize (N4640Bt Cry1Ab, DKC5143Bt Cry3Bb1) leaf material were analysed. For comparison, diets with the corresponding untransformed near-isolines (N4640, DKC5143) were examined. Additionally a high quality control diet (oat flakes) was included. Survival and reproduction showed no significant differences between the Cry3Bb1 treatment and the treatment with the untransformed counterpart. For the Cry1Ab treatment survival was significantly higher than for the treatment with the corresponding near-isoline. In contrast, reproduction was significantly lower for the Cry1Ab treatment compared to that for the isoline. For the Cry3Bb1 treatment, no effect was shown on survival or reproduction. For the Cry1Ab variety and its untransformed counterpart, a contrasting result was detected, which is unlikely to be caused by the Bt-protein but rather by differences in other plant components. Overall survival and reproduction were highest for the control.     

Persistence of Cry1Ac Protein from Transgenic Bt Cotton Cultivation and Residue Returning in Fields and Its Effect on Functional Diversity of Soil Microbial Communities

The persistence of Cry1Ac protein in the soil and its effect on soil microbial communities are a core issue in assessing the ecological risk of transgenic Bacillus thuringiensis (Bt) cotton. In this study a field experiment was conducted on the cultivation of transgenic Bt cotton (Jin 26 and BtJi 668) with the immediate returning of residues to the fields, in order to quantify the Cry1Ac protein content in the fields and investigate its effects on the functional diversity of soil microbial communities. Cry1Ac protein in the residue-soil mixture was gradually degraded in the transgenic Bt cotton fields. After transgenic Bt cotton straw was returned to the fields for 30 d, 63.73% and 58.33% of the initial amounts of Cry1Ac protein were degraded in the Jin 26 and BtJi 668 fields, respectively. Before the crops were sown in the following year (180 d after returning the straw), no Cry1Ac protein was detected in the fields. After returning the cotton straw to the fields for 30 d, the Shannon-Wiener and McIntosh indices of soil microbial communities in the transgenic Bt cotton fields were significantly higher than those in the non-transgenic cotton fields. Meanwhile, the utilization of carbon sources including amino acids, amines, and carbohydrates by the soil microbial communities significantly increased. Both the McIntosh index and the utilization of carbohydrates increased until 180 d. Principal component analysis revealed that amino acids, amides, and carbohydrates were the main carbon sources distinguishing the two principal component factors. These findings indicated that Cry1Ac protein did not accumulate in the fields after transgenic Bt cotton was planted for one year and the residues were immediately returned to the fields; however, the original functional diversity of soil microbial communities was affected continuously.     

Impact of coleopteran targeting toxin (Cry3Bb1) of Bt corn on microbially mediated decomposition

Genetically engineered corn expressing crystalline proteins for insect control and encoded by genes derived from soil bacterium Bacillus thuringiensis (Bt) are widely adopted in the United States. Among the seven different events of Bt corn available commercially, YieldGard^® Rootworm (MON863) expresses a variant of the cry3Bb1 protein in the root tissue to control corn rootworm larvae. Although numerous laboratory and field studies show no unexpected ecological risks at the insect community-level above-ground, few studies have addressed the possible impact of cry proteins released from living or decaying roots of Bt corn on soil microbial communities. Here, we test the hypothesis that coleopteran-active Bt corn does not affect nontarget ecological processes, such as decomposition or the function of the associated saprophytic microbial community. Experimental treatments were: (1) a Bt hybrid; (2) a non-Bt, isogenic hybrid treated with a conventional soil insecticide; and (3) a non-Bt, isogenic hybrid without insecticide. Soil and root samples were collected at various times throughout 2 years from experimental plots to estimate microbial community function by quantifying activity of extracellular enzymes on 10 substrates. Decomposition was measured as mass loss by root decay in litter bags. Bt corn (MON863) exuding the cry3Bb1 toxin does not appear to have adverse effects on saprophytic microbial communities of soil and decaying roots or on decomposition. The addition of the soil insecticide had greater effects on microbial function in soil and decaying roots than Bt corn. Our results are similar to those found previously for the cry3Bb1 protein that showed no adverse effects on microbial community composition in controlled and natural environments. This field study is one of the first to report the use of extracellular enzyme assays to examine the effect of transgenic crops on the functional activity of microbes in soil and decaying roots.     

The role of laboratory, glasshouse and field scale experiments in understanding the interactions between genetically modified crops and soil ecosystems: A review of the ECOGEN project

The interactions of genetically modified (GM) crops with soil species and ecosystems is complex, requiring both specific and broad spectrum assessments. In the ECOGEN project we undertook experiments at three scales of increasing complexity, using Bt maize expressing the Cry1Ab protein from Bacillus thuringiensis as an example. Test species were selected for laboratory-scale experiments to represent taxonomic groups that we could also monitor at glasshouse and field scales (e.g., nematodes, protozoa, micro-arthropods, earthworms, and snails). In the laboratory, single species were exposed to purified Cry1Ab protein or to Bt maize leaf powder incorporated into simplified diets under controlled conditions. In the glasshouse, multiple test species and soil microbial communities taken from ECOGEN's field sites were exposed to Bt maize plants growing under glasshouse or mesocosm conditions. In the field, evaluations were conducted on our selected indicator groups over multiple sites and growing seasons. Field evaluation included assessment of effects due to the local environment, crop type, seasonal variation and conventional crop management practice (tillage and pesticide use), which cannot be assessed in the glasshouse. No direct effects of Cry1Ab protein or Bt leaf residues were detected on our laboratory test organisms, but some significant effects were detected in the glasshouse. Total nematode and protozoan numbers increased in field soil under Bt maize relative to conventional maize, whilst microbial community structure and activity were unaffected. Field results for the abundance of nematodes and protozoa showed some negative effects of Bt maize, thus contradicting the glasshouse results. However, these negative results were specific to particular field sites and sampling times and therefore were transient. Taking the overall variation found in maize ecosystems at different sites into account, any negative effects of Bt maize at field scale were judged to be indirect and no greater than the impacts of crop type, tillage and pesticide use. Although the ECOGEN results were not predictive between the three experimental scales, we propose that they have value when used with feedback loops between the scales. This holistic approach can used to address questions raised by results from any level of experimentation and also for putting GM crop risk:benefit into context with current agricultural practices in regionally differing agro-ecosystems.     

Effects of physical and chemical properties of soils on adsorption of the insecticidal protein (Cry1Ab) from Bacillus thuringiensis at Cry1Ab protein concentrations relevant for experimental field sites

The adsorption of the insecticidal Cry1Ab protein of Bacillus thuringiensis (Bt) on Na-montmorillonite (M-Na) and soil clay fractions was studied. The aim of this study was not to find the adsorption capacity of the soils from the experimental field site, where Bt corn (MON810) was cultivated, but rather to characterize the adsorption behavior of the Cry1Ab protein at concentrations typically found at experimental field sites. In kinetic experiments, the Cry1Ab protein adsorbed rapidly (<60 min) on M-Na. As the concentration of M-Na was varied and the added Cry1Ab protein concentration was kept constant (20 and 45 ng ml⁻¹), the adsorption per unit weight of Cry1Ab protein decreased with increasing concentrations of M-Na. Adsorption of Cry1Ab protein on M-Na decreased as the pH value of the suspension increased. All adsorption isotherms could be described mathematically by a linear regression with the parameter k, the distribution coefficient, being the slope of the regression line. Although their mineralogical composition was nearly identical, the soil clay fractions showed different k values. The different k values were correlated with the physical and chemical properties of the soil clay fractions, such as the organic carbon content, the specific external surface area, and the electrokinetic charge of the external surfaces of the clays, as well as with the external surface charge density. An increase in the amount of soil organic matter, as well as an increase in the electrokinetic external surface charge of the soil clays, decreased the distribution coefficient k. An increase of the specific external surface areas of the soil clays resulted in a higher distribution coefficient k.Less than 10% of adsorbed Cry1Ab protein was reversibly adsorbed on the soil clays and, thus, desorbed. The desorption efficiency of distilled water was higher than that of a solution of CaCl₂ (2.25 mmol) and of dissolved organic carbon (50 mg C l⁻¹).     

Does stable isotope analysis separate transgenic and traditional corn (Zea mays L.) detritus and their consumers?

Transgenic corn crops (including the Bt variety) are expanding rapidly worldwide, and the large amounts of cultural residues remaining after harvest pose questions about the fate of this novel source of plant detritus in soil. To verify whether transgenic and conventional corn litters were different in their isotopic signatures, the ¹³C and ¹⁵N stable isotopes of different portions of Bt and non-Bt Aristis corn plants after harvest were analysed. Laboratory feeding experiments were then conducted to assess the transfer of corn isotopic signals to detritivores using the isopod Trachelipus sp., reared on either Bt or non-Bt corn dead leaves as the only food source. δ¹⁵N differed significantly between Bt and non-Bt corn in kernels and stems, whereas both δ¹⁵N and δ¹³C were different in leaves before and after exposure to isopods. During feeding, the N and C isotopic signatures of isopods shifted towards the diet values. Significant differences existed both between sampling dates and corn treatments. The results suggest that detritus from transgenic and conventional corn crops may have different isotopic signatures and the isotopic differences can persist through the trophic levels, making corn detritus feeders suitable sentinel species for the Bt corn isotopic signal in agroecosystems.     

Effects of Bt-maize material on the life cycle of the land snail Cantareus aspersus

Insect resistant Bt-maize (MON 810) expresses active Cry1Ab endotoxin derived from Bacillus thuringiensis (Bt). Snails constitute non-target soil species potentially exposed to Bt-toxin through consumption of plant material and soil in fields where transgenic plants have been grown. We studied the effect of the Cry1Ab toxin on survival, growth and egg hatchability of the snail Cantareus aspersus. From the age of 4 to 88 weeks, snails were fed either powdered Bt-maize or non-Bt-maize and exposed to soil samples collected after harvesting either the Bt-maize or non-Bt-maize. We applied four treatments: non-Bt soil + non-Bt-maize (MM); Bt soil + Bt-maize (BB), non-Bt soil + Bt-maize (MB), Bt soil + non-Bt-maize (BM). Eggs laid by snails not exposed to Bt-toxin were also exposed to the two types of soils (Bt and non-Bt soil).At the end of growth (47 weeks of exposure), snails exposed to Bt-toxin in food and soil (BB) had a growth coefficient (GC) 25% lower than unexposed snails (MM). After the first period of reproduction (68 weeks) a significant difference remained for body mass GC between the BB and MM treatments. Differences in body mass were not significant at the end of exposure (88 weeks). For snails not previously exposed to Bt material, hatchability of eggs was similar in the soils tested. The outcome of the experiments indicates that, in growing snails, long-term exposure is needed to reveal an effect of Bt-maize. The hazard analysis of Bt-maize which we performed, based on a worst-case scenario, i.e. snails having no food choice, should now be complemented by other simple measurements, e.g. food intake, to understand the underlying mechanisms involved.     

Different responses of ash and beech on nitrate versus ammonium leaf labeling

The effects of tree species on the N cycle in forest systems are still under debate. However, contradicting results of different ¹⁵N labeling techniques of trees and N tracers in the individual studies hamper a generalized mechanistic view. Therefore, we compared Ca(¹⁵NO₃)₂ and ¹⁵NH₄Cl leaf‐labeling method to investigate: (1) N allocation patterns from aboveground to belowground, (2) the cycles of N in soil‐plant systems, and (3) to allow the production of highly ¹⁵N enriched litter for subsequent decomposition studies. 20 beeches (Fagus sylvatica ) and 20 ashes (Fraxinus excelsior ) were ¹⁵N pulse labeled from aboveground with Ca(¹⁵NO₃)₂ and 40 beeches and 40 ashes were ¹⁵N pulse labeled from aboveground with ¹⁵NH₄Cl. ¹⁵N was quantified in tree compartments (leaves, stem, roots) and in soil after 8 d. Beech and ash incorporated generally more ¹⁵N from the applied ¹⁵NH₄Cl compared to Ca(¹⁵NO₃)₂ in all measured compartments, except for ash leaves. Ash had highest ¹⁵N incorporation [45% of the applied with Ca(¹⁵NO₃)₂] in its leaves. Both tree species kept over 90% of all fixed ¹⁵N from Ca(¹⁵NO₃) in their leaves, whereas only 50% of the ¹⁵N from the ¹⁵NH₄Cl tracer remained in the leaves and 50% were allocated to stem, roots, and soil. There was no damage of the leaves by both salts, and thus both ¹⁵N tracers enable long‐term labeling in situ field studies on N rhizodeposition and allocation in soils. Nonetheless, the ¹⁵N incorporation by both salts was species specific: the leaf labeling with ¹⁵NH₄Cl results in a more homogenous distribution between the tree compartments in both tree species and, therefore, ¹⁵NH₄Cl is more appropriate for allocation studies. The leaf labeling with Ca(¹⁵NO₃)₂ is a suitable tool to produce highly enriched ¹⁵N leaf litter for further long term in situ decomposition and turnover studies.