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.     

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.     

Specific respiration rates, adenylates, and energy budgets of soil microorganisms after addition of transgenic Bt-maize straw

An arable soil was incubated with straw (stem+leaves) of two transgenic Bt-maize varieties (Novelis: event MON810 and Valmont: event Bt176) and the two corresponding near-isogenic varieties (Nobilis and Prelude). The aim was to evaluate the use of these substrates for microbial growth and maintenance in soil during early decomposition. The addition of Bt-maize straw increased CO₂ production rates and the specific respiration rates CO₂-C/microbial biomass C and CO₂-C/ATP significantly compared with the addition of non-Bt maize straw. This extra energy in the Bt-maize straw could not be used for microbial biomass or ATP and ADP production, and was lost for maintenance. In addition, increased death rates of microbial biomass occurred in the soils treated with the Bt-maize straw from day 3 to 21. Generally, most of the energy was stored in microbial biomass, whereas only 10% of energy was stored in ATP, and only 1-2% in ADP. The AEC (adenylate energy charge: (ATP+0.5×ADP)/(AMP+ADP+ATP)) was not affected by any treatment. The reasons for the lower efficiency of microbial substrate use after adding Bt-maize straw cannot be fully explained by the present experiment. However, a risk assessment has to look at the impact of transgenic plant material on soil microorganisms at different maturity stages.     

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.     

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.     

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).     

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.     

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.     

Microbial response of soils with organic and conventional management history to the cultivation of Bacillus thuringiensis (Bt)-maize under climate chamber conditions

An experiment was carried out in a climate chamber to analyse if Bt-maize may cause particular changes in soils with different levels of microbial biomass and activity due to long-term management history. Among the soils selected, the ones managed organically for 30 years exhibited twice the microbial biomass and 2.6 times the dehydrogenase activity (DHA) of the soil from a field with long-term conventional maize monoculture. Soils were cultivated twice in a row with Bt-maize, its near-isogenic line and a conventional breeding line. We tested the hypotheses that (a) soil microbial biomass and activity are affected by the cultivation of Bt-maize and that (b) the influence of Bt-maize depends on the level of soil microbial biomass and activity. Shoot and root yield and shoot C-content of Bt-maize were higher than the ones of the near-isogenic line. DHA under Bt-maize was 6 % higher, and the metabolic quotient for CO₂ (qCO₂) was 9 % lower than under its near-isogenic line, giving some support to hypothesis (a). No significant interactions of the soils and the varieties used were found in this study, thus hypothesis (b) was not confirmed, and soils with different microbial biomass and activity appear to react in a similar way to the cultivation of Bt-maize.     

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.     

Partitioning root and microbial contributions to soil respiration in Leymus chinensis populations

Based on the enclosed chamber method, soil respiration measurements of Leymus chinensis populations with four planting densities (30, 60, 90 and 120 plants/0.25 m²) and blank control were made from July 31 to November 24, 2003. In terms of soil respiration rates of L. chinensis populations with four planting densities and their corresponding root biomass, linear regressive equations between soil respiration rates and dry root weights were obtained at different observation times. Thus, soil respiration rates attributed to soil microbial activity could be estimated by extrapolating the regressive equations to zero root biomass. The soil microbial respiration rates of L. chinensis populations during the growing season ranged from 52.08 to 256.35 mg CO₂ m⁻² h⁻¹. Soil microbial respiration rates in blank control plots were also observed directly, ranging from 65.00 to 267.40 mg CO₂ m⁻² h⁻¹. The difference of soil microbial respiration rates between the inferred and the observed methods ranged from −26.09 to 9.35 mg CO₂ m⁻² h⁻¹. Some assumptions associated with these two approaches were not completely valid, which might result in this discrepancy. However, these two methods' application could provide new insights into separating root respiration from soil microbial respiration. The root respiration rates of L. chinensis populations with four planting densities could be estimated based on measured soil respiration rates, soil microbial respiration rates and corresponding mean dry root weight, and the highest values appeared at the early stage, then dropped off rapidly and tended to be constant after September 10. The mean proportions of soil respiration rates of L. chinensis populations attributable to the inferred and the observed root respiration rates were 36.8% (ranging from 9.7 to 52.9%) and 30.0% (ranging from 5.8 to 41.2%), respectively. Although root respiration rates of L. chinensis populations declined rapidly, the proportion of root respiration to soil respiration still increased gradually with the increase of root biomass.     

Boron availability to plants from coal combustion by-products

Agronomic use of coal combustion by-products is often associated with boron (B) excess in amended soils and subsequently in plants. A greenhouse study with corn (Zea mays L.) as test plant was conducted to determine safe application rates of five fly ashes and one flue gas desulfurization gypsum (FDG). All by-products increased soil and corn tissue B concentration, in some cases above toxicity levels which are 5 mg hot water soluble B (hwsB) kg^?1 soil and 100 mg B kg^?1 in corn tissue. Acceptable application rates varied from 4 to 100 Mg ha^? for different by-products. Leaching and weathering of a high B fly ash under ponding conditions decreased its B content and that of corn grown in fly ash amended soil, while leaching of the same fly ash under laboratory conditions increased fly ash B availability to corn in comparison to the fresh fly ash. Hot water soluble B in fly ash or FDG amended soil correlated very well with corn tissue B. Hot water soluble B in fly ash amended soil could be predicted based on soil pH and B solubility in ash at different pH values but not so in the case of FDG. Another greenhouse study was conducted to compare the influence of FDG and Ca(OH)₂ on B concentration in spinach (Spinacia oleracea L.) leaves grown in soil amended with the high B fly ash. The Ca(OH)₂ significantly decreased tissue B content, while FDG did not affect B uptake from fly ash amended soil.     

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.     

Can a mixed stand of N2-fixing and non-fixing plants restrict N2O emissions with increasing CO2 concentration?

Initial effects of elevated atmospheric CO₂ concentration on N₂O fluxes and biomass production of timothy/red clover were studied in the laboratory. The experimental design consisted of two levels of atmospheric CO₂ (ca. 360 and 720 μmol CO₂ mol⁻¹) and two N fertilisation levels (5 and 10 g N m⁻²). There was a total of 36 mesocosms comprising sandy loam soil, which were equally distributed in four thermo-controlled greenhouses. In two of the greenhouses, the CO₂ concentration was kept at ambient concentration and in the other two at doubled concentration. Forage was harvested and the plants fertilised three times during the basic experiment, followed by harvest, a fertilisation with the double amount of nitrogen and rise of water level. Under elevated CO₂, harvestable and total aboveground dry biomass production of a mixed Trifolium/Phleum stand was increased at both N treatments compared to ambient CO₂. The N₂O flux rates under ambient CO₂ were significantly higher at both N treatments during the early growth of mixed Phleum/Trifolium mesocosms compared to the N₂O flux rate under elevated CO₂. However, when the conditions were favourable for denitrification at the end of the experiment, i.e. N availability and soil moisture were high enough, the elevated CO₂ concentration enhanced the N₂O efflux.     

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.     

Volatile organic compounds in the roots and rhizosphere of Pinus spp.

Plant roots normally release a complex mixture of chemicals which have important effects in the rhizosphere. Among these different root-emitted compounds, volatile isoprenoids have received very little attention, yet they may play important and diverse roles in the rhizosphere, contributing to the regulation of microbial activity and nutrient availability. It is therefore important to estimate their abundance in the rhizosphere, but so far, there is no reliable sampling method that can be used to measure realistic rates of root emissions from plants growing in field conditions, or even in pots. Here, we measured root content of volatile isoprenoids (specifically monoterpenes) for Pinus pinea, and explored the feasibility of using a dynamic bag enclosure method to measure emissions from roots of intact pot-grown plants with different degrees of root cleaning. We also investigated a passive diffusion method for exploring monoterpenes in soil at incremental distances from mature Pinus sylvestris trees growing in field conditions. Total monoterpene content of P. pinea roots was 415±50 μg g⁻¹ fresh wt in an initial screening study, and between 688±103 and 1144±208 μg g⁻¹ dry wt in subsequent investigations. Emissions from shaken-clean roots of intact plants and roots of intact plants washed to remove remaining soil after shaken-clean experiments were 119±14 and 26±5 μg g⁻¹ dry wt h⁻¹, respectively. Emissions from intact roots in soil-balls were an order of magnitude lower than from shaken-clean roots, and probably reflected the amount of emitted compounds taken up by physical, chemical or biological processes in the soil matrix surrounding the roots. Although monoterpene content was not significantly different in droughted roots, emission rates from droughted roots were generally significantly lower than from well-watered roots. Finally, passive sampling of monoterpenes in the soil at different distances from mature P. sylvestris trees in field conditions showed significantly decreasing sampling rates with increasing distance from the trunk. We conclude that it is feasible to measure volatile isoprenoid emissions from roots but the method of root preparation affects magnitude of measured emissions and therefore must be decided according to the application. We also conclude that the rhizosphere of Pinus species is a strong and previously un-characterized source of volatile isoprenoid emissions and these are likely to impact significantly on rhizosphere function.     

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.     

CO2 and inorganic N supply modify competition for N between co-occurring grass plants, tree seedlings and soil microorganisms

Plant-plant and plant-soil interactions play a key role in determining plant community structure and ecosystem function. However, the effects of global change on the interplay between co-occurring plants and soil microbes in successional communities are poorly understood. In this study, we investigated competition for nitrogen (N) between soil microorganisms, grass plants and establishing tree seedlings under factorial carbon dioxide (CO₂) and N treatments. Fraxinus excelsior seedlings were germinated in the presence or absence of grass competition (Dactylis glomerata) at low (380 μmol mol⁻¹) or high (645 μmol mol⁻¹) CO₂ and at two levels of N nutrition in a mesocosm experiment. Pulse ¹⁵N labelling was used to examine N partitioning among plant and soil compartments. Dactylis exerted a strong negative effect on Fraxinus biomass, N capture and ¹⁵N recovery irrespective of N and CO₂ treatment. In contrast, the presence of Dactylis had a positive effect on the microbial N pool. Plant and soil responses to N treatment were of a greater magnitude compared with responses to elevated CO₂, but the pattern of Fraxinus- and microbial-N pool response to N and CO₂ varied depending on grass competition treatment. Within the Dactylis competition treatment, decreases in Fraxinus biomass in response to N were not mirrored by decreases in tree seedling N content, suggesting a shift from below- to above-ground competition. In the Dactylis-sown pots, ¹⁵N recovery could be ranked Dactylis > microbial pool > Fraxinus in all N and CO₂ treatment combinations. Inequalities between Fraxinus and soil microorganisms in terms of ¹⁵N recovery were exacerbated by N addition. Contrary to expectations, elevated CO₂ did not increase plant-microbe competition. Nevertheless, microbial ¹⁵N recovery showed a small positive increase in the high CO₂ treatment. Overall, elevated CO₂ and N supply did not interact on plant/soil N partitioning. Our data suggest that the competitive balance between establishing tree seedlings and grass plants in an undisturbed sward is relatively insensitive to CO₂ or N-induced modifications in N competition between plant and soil compartments.     

Growth promoting effects of corn (Zea mays) bacterial isolates under greenhouse and field conditions

Fertilizer costs are a major component of corn production. The use of biofertilizers may be one way of reducing production costs. In this study we present isolation and identification of three plant growth promoting bacteria that were identified as Enterobacter cloacae (CR1), Pseudomonas putida (CR7) and Stenotrophomonas maltophilia (CR3). All bacterial strains produced IAA in the presence of 100 mg l⁻¹ of tryptophan and antifungal metabolites to several soilborne pathogens. S. maltophilia and E. cloacae had broad spectrum activity against most Fusarium species. The only strain that was positive for nitrogen fixation was E. cloacae and it, and P. putida, were also positive for phosphate solubilization. These bacteria and the corn isolate Sphingobacterium canadense CR11, and known plant growth promoting bacterium Burkholderia phytofirmans E24 were used to inoculate corn seed to examine growth promotion of two lines of corn, varieties 39D82 and 39M27 under greenhouse conditions. When grown in sterilized sand varieties 39M27 and 39D82 showed significant increases in total dry weights of root and shoot of 10-20% and 13-28% and 17-32% and 21-31% respectively. Plants of the two varieties grown in soil collected from a corn field had respective increases in dry weights of root and shoot of 10-30% and 12-35% and 11-19% and 10-18%. In sand, a bacterial mixture was highly effective whereas in soil individual bacteria namely P. putida CR7 and E. cloacae CR1 gave the best results with 39M27 and 39D82 respectively. These isolates and another corn isolate, Azospirillum zeae N7, were tested in a sandy soil with a 55 and 110 kg ha⁻¹ of nitrogen fertility at the Delhi research Station of Agriculture and Agri-Food Canada over two years. Although out of seven bacterial treatments, no treatment provided a statistically significant yield increase over control plots but S. canadense CR11 and A. zeae N7 provided statistically significant yield increase as compared to other bacteria. The 110 kg rate of nitrogen provided significant yield increase compared to the 55 kg rate in both years.