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.     

Earthworms of different functional groups affect the fate of the Bt-toxin Cry1Ab from transgenic maize in soil

The fate of the insecticidal Cry1Ab protein from crop residues (leaves and roots) of the transgenic maize variety MON810 was studied in the presence and absence of two earthworm species (Lumbricus terrestris, Aporrectodea caliginosa; separate incubations) in soil microcosms. The recombinant Cry1Ab protein was quantified using a highly sensitive ELISA. Control microcosms received corresponding non-transgenic plant material. All earthworms survived in the microcosms over a period of 5 weeks, irrespective of whether they received MON810 or non-transgenic plant material. Weight loss was observed for both earthworm species, independent of the plant material or transgenic modification. A strong decline of immunoreactive Cry1Ab in plant residues (mean initial concentration approx. 5000 ng g⁻¹) of MON810 was observed in all treatments, but in microcosms with earthworms this decline was significantly higher with less than 10% of the initial Cry1Ab concentration remaining after 5 weeks. Cry1Ab concentrations in casts were only 0.1% of those found in remaining plant material of the respective microcosms. No immunoreactive Cry1Ab proteins were found in earthworm tissues (threshold of detection: 0.58 ng g⁻¹ fresh weight). No further decline was found for Cry1Ab concentrations in casts of A. caliginosa during a subsequent period of 3 months of incubation in bulk soil (<0.1 ng g⁻¹) after removal of the earthworms from the microcosms, while in casts of L. terrestris the concentration decreased from 0.4 to below 0.1 ng g⁻¹. In conclusion, this study demonstrates that earthworms enhance the decline of immunoreactive Cry1Ab proteins from maize residues.     

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.     

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.     

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.     

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.     

Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings

Azospirillum lipoferum CRT1 is a promising phytostimulatory PGPR for maize, whose effect on the plant is cell density-dependent. A nested PCR method is available for detection of the strain but does not allow quantification. The objective was to develop a real-time PCR method for quantification of A. lipoferum CRT1 in the rhizosphere of maize seedlings. Primers were designed based on a strain-specific RFLP marker, and their specificity was verified under qualitative and quantitative PCR conditions based on successful CRT1 amplification and absence of cross-reaction with genomic DNA from various rhizosphere strains. Real-time PCR conditions were then optimized using DNA from inoculated or non-inoculated maize rhizosphere samples. The detection limit was 60 fg DNA (corresponding to 19 cells) with pure cultures and 4 × 10⁴ CFU equivalents g⁻¹ lyophilized sample consisting of mixture of rhizosphere soil and roots. Inoculant quantification was effective down to 10⁴ CFU equivalents g⁻¹. Assessment of CRT1 rhizosphere levels in a field trial was in accordance with estimates from semi-quantitative PCR targeting another locus. This real-time PCR method, which is now available for direct rhizosphere monitoring of A. lipoferum CRT1 in greenhouse and field experiments, could be used as a reference for developing quantification tools for other Azospirillum inoculants.     

Earthworm populations in a northern U.S. Cornbelt soil are not affected by long-term cultivation of Bt maize expressing Cry1Ab and Cry3Bb1 proteins

Earthworms, which play a key role in biogeochemical processes in soil ecosystems, could be negatively affected by the cultivation of transgenic Bt crops. Studies to date have found few effects of Bt maize on earthworm species. If adverse effects occur, they are likely to be chronic or sub-lethal and expressed over large spatial and temporal scales. Our objective in the present study was to investigate potential effects on earthworm populations in soil cultivated with Bt maize in a large multiple-year field study. We surveyed the earthworm populations in 0.16-ha experimental field plots of two varieties of Cry1Ab Bt maize, one variety of Cry3Bb1 Bt maize, and three non-transgenic control varieties cultivated for four years. Four earthworm species were found in our sample: Aporrectodea caliginosa, Aporrectodea trapezoides, Aporrectodea tuberculata (collectively, the A. caliginosa species complex), and Lumbricus terrestris. We found no significant differences in the biomass of juveniles and adults for all four species between Bt and non-Bt maize varieties. From this and previous studies, we conclude that the effects of Cry1Ab and Cry3Bb1 Bt maize on the A. caliginosa species complex and L. terrestris are small. Nonetheless, general conclusions about the effects of Bt maize on earthworm populations are not warranted due to the small number of species tested. In future laboratory studies, earthworm species should be selected according to their association with a Bt crop and the impact of that species to valued soil ecosystem processes.     

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.     

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).     

Respiration pattern and microbial use of field-grown transgenic Bt-maize residues

A 49-day incubation experiment was carried out with the addition of field-grown maize stem and leaf residues to soil at three different temperatures (5, 15, and 25 °C). The aim was to study the effects of two transgenic Bt-maize varieties in comparison to their two parental non-Bt varieties on the mineralization of the residues, on their incorporation into the microbial biomass and on changes in the microbial community structure. The stem and leaf residues of Novelis-Bt contained 3.9 μg g⁻¹ dry weight of the Bt toxin Cry1Ab and those of Valmont-Bt only 0.8 μg g⁻¹. The residues of the two parental non-Bt varieties Nobilis and Prelude contained higher concentrations of ergosterol (+220%) and glucosamine (+190%) and had a larger fungal C-to-bacterial C ratio (+240%) than the two Bt varieties. After adding the Bt residues, an initial peak in respiration of an extra 700 μg CO₂-C g⁻¹ soil or 4% of the added amount was observed in comparison to the two non-Bt varieties at all three temperatures. On average of the four varieties, 19-38% of the maize C added was mineralized during the 49-day incubation at the three different temperatures. The overall mean increase in total maize-derived CO₂ evolution corresponded to a Q₁₀ value of 1.4 for both temperature steps, i.e. from 5 to 15 °C and from 15 to 25 °C. The addition of maize residues led to a strong increase in all microbial properties analyzed. The highest contents were always measured at 5 °C and the lowest at 25 °C. The variety-specific contents of microbial biomass C, biomass N, ATP and adenylates increased in the order Novelis-Bt ? Prelude<Valmont-Bt ? Nobilis. The mineralization of Novelis-Bt residues with the highest Bt concentration and lowest N concentration and their incorporation into the microbial biomass was significantly reduced compared to the parental non-Bt variety Nobilis. These negative effects increased considerably from 5 to 25 °C. The transgenic Bt variety Valmont did not show further significant effects except for the initial peak in respiration at any temperature.     

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.     

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 inoculation with ectomycorrhizal fungi on microbial biomass and bacterial functional diversity in the rhizosphere of Pinus tabulaeformis seedlings

This study investigated the effects of inoculation with three individual ectomycorrhizal (ECM) fungal species on soil microbial biomass carbon and indigenous bacterial community functional diversity in the rhizosphere of Chinese pine (Pinus tabulaeformis Carr.) seedlings under field experimental conditions. The results showed that ECM fungal inoculation significantly increased the ectomycorrhizal colonization compared with non-inoculated seedlings. ECM fungal inoculations have higher soil microbial biomass carbon than that of control, ranging from 49.6 μg C g^?1 dry soil in control to 134.02 μg C g^?1 dry soil in treatment inoculated with Boletus luridus Schaeff ex Fr. Multivariate analyses (PCA) of BIOLOG data revealed that the application of ECM fungi significantly influenced bacterial functional diversity in the rhizosphere of P. tabulaeformis seedlings. The highest average well-color development (AWCD) and functional diversity indices were also observed in treatment inoculated with B. luridus. A wider range of sole carbon sources were utilized by the bacterial community in the rhizosphere of inoculated seedlings. The data gathered from this study provides important information for utilization of ECM fungi in forest restoration project in the Northwestern China. The present study will also significantly broaden our understanding of practical importance in the application of ECM fungal inoculum to promote soil microbial community diversity of soil.     

Evaluation of effects of transgenic Bt maize on microarthropods in a European multi-site experiment

The effects of maize expressing the Bacillus thuringiensis Cry1Ab protein (Bt maize) on soil microarthropods were assessed in the field at four European locations (two in Denmark and two in France) that differ in their climatic conditions or soil properties. Each site was considered as a separate experiment, with separate statistical comparison. Effects of farming practices using Bt maize were compared with conventional farming practices using near-isogenic non-Bt maize and also (at some of the sites) other conventional varieties. Furthermore, at one field site (Foulum, Denmark), the effects of Bt crops were studied in both conventional tillage and reduced tillage contexts. At another field site (Askov, Denmark), Bt maize effects were also compared to the effects of the chemical insecticide dimethoate. Moreover, at three of the field sites (all except Narbons, France), the possibility of a localised Bt effect around the rhizosphere compared to the bulk soil was assessed by sampling within and between maize rows. There were some significant negative effects of Bt maize on microarthropods in soils with a high clay content. Significant differences of the same magnitude also occurred between different conventional varieties of maize, but the effect of dimethoate appeared clearly greater than Bt effects. It is thus debatable if the Bt maize effect is an effect of the Bt toxin or just an effect of the maize variety. Based on the results, it can be concluded that the effect of Bt maize on soil microarthropods was small and within the normal variation expected in conventional agricultural systems.     

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.     

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.     

Persistence of a biocontrol Pseudomonas inoculant as high populations of culturable and non-culturable cells in 200-cm-deep soil profiles

Little is known about the ecology of soil inoculants used for pathogen biocontrol, biofertilization and bioremediation under field conditions. We investigated the persistence and the physiological states of soil-inoculated Pseudomonas protegens (previously Pseudomonas fluorescens) CHA0 (10⁸ CFU g^?1 surface soil) in different soil microbial habitats in a planted ley (Medicago sativa L.) and an uncovered field plot. At 72 days, colony counts of the inoculant were low in surface soil (uncovered plot) and earthworm guts (ley plot), whereas soil above the plow pan (uncovered plot), and the rhizosphere and worm burrows present until 1.2 m depth (ley plot) were survival hot spots (10⁵–10⁶ CFU g^?1 soil). Interestingly, strain CHA0 was also detected in the subsoil of both plots, at 10²–10⁵ CFU g^?1 soil between 1.8 and 2 m depth. However, non-cultured CHA0 cells were also evidenced based on immunofluorescence microscopy. Kogure's direct viable counts of nutrient-responsive cells showed that many more CHA0 cells were in a viable but non-culturable (VBNC) or a non-responsive (dormant) state than in a culturable state, and the proportion of cells in those non-cultured states depended on soil microbial habitat. At the most, cells in a VBNC state amounted to 34% (above the plow pan) and those in a dormant state to 89% (in bulk soil between 0.6 and 2 m) of all CHA0 cells. The results indicate that field-released Pseudomonas inoculants may persist at high cell numbers, even in deeper soil layers, and display a combination of different physiological states whose prevalence fluctuates according to soil microbial habitats.     

The fate and toxicity of the flavonoids naringenin and formononetin in soil

The flavonoid class of plant secondary metabolites play a multifunctional role in below-ground plant–microbe interactions with their best known function as signals in the nitrogen fixing legume–rhizobia symbiosis. Flavonoids enter rhizosphere soil as a result of root exudation and senescence but little is known about their subsequent fate or impacts on microbial activity. Therefore, the present study examined the sorptive behaviour, biodegradation and impact on dehydrogenase activity (as determined by iodonitrotetrazolium chloride reduction) of the flavonoids naringenin and formononetin in soil. Organic carbon normalised partition coefficients, log K_oc, of 3.12 (formononetin) and 3.19 (naringenin) were estimated from sorption isotherms and, after comparison with literature log K_oc values for compounds whose soil behaviour is better characterised, the test flavonoids were deemed to be moderately sorbed. Naringenin (spiked at 50 μg g^?1) was biodegraded without a detectable lag phase with concentrations reduced to 0.13±0.01 μg g^?1 at the end of the 96 h time course. Biodegradation of formononetin proceeded after a lag phase of ～24 h with concentrations reduced to 4.5±1% of the sterile control after 72 h. Most probable number (MPN) analysis revealed that prior to the addition of flavonoids, the soil contained 5.4×10⁶ MPN g^?1 (naringenin) and 7.9×10⁵ MPN g^?1 (formononetin) catabolic microbes. Formononetin concentration had no significant (p>0.05) effect on soil dehydrogenase activity, whereas naringenin concentration had an overall but non-systematic impact (p=0.045). These results are discussed with reference to likely total and bioavailable concentrations of flavonoids experienced by microbes in the rhizosphere.     

Decomposition processes under Bt (Bacillus thuringiensis) maize: Results of a multi-site experiment

The effects of maize expressing the Bacillus thuringiensis Cry1Ab protein (Bt maize) on decomposition processes under three different European climatic conditions were assessed in the field. Farming practices using Bt maize were compared with conventional farming practices using near-isogenic non-Bt maize lines under realistic agricultural practices. The litter-bag method was used to study litter decomposition and nitrogen mineralization dynamics of wheat straw. After 4 months incubation in the field, decomposition and mineralization were mainly influenced by climatic conditions with no negative effect of the Bt toxin on decomposition processes.