Alteration of Soil Rhizosphere Communities following Genetic Transformation of White Spruce

The application of plant genetic manipulations to agriculture and forestry with the aim of alleviating insect damage through Bacillus thuringiensis transformation could lead to a significant reduction in the release of pesticides into the environment. However, many groups have come forward with very valid and important questions related to potentially adverse effects, and it is crucial to assess and better understand the impact that this technology might have on ecosystems. In this study, we analyzed rhizosphere soil samples collected from the first B. thuringiensis-transformed trees [with insertion of the CryIA(b) toxin-encoding gene] grown in Canada (Val-Cartier, QC, Canada) as part of an ecological impact assessment project. Using a robust amplified rRNA gene restriction analysis approach coupled with 16S rRNA gene sequencing, the rhizosphere-inhabiting microbial communities of white spruce (Picea glauca) genetically modified by biolistic insertion of the cryIA(b), uidA (beta-glucuronidase), and nptII genes were compared with the microbial communities associated with non-genetically modified counterparts and with trees in which only the genetic marker genes uidA and nptII have been inserted. Analysis of 1,728 rhizosphere bacterial clones (576 clones per treatment) using a Cramér-von Mises statistic analysis combined with a Monte Carlo comparison clearly indicated that there was a statistically significant difference (P < 0.05) between the microbial communities inhabiting the rhizospheres of trees carrying the cryIA(b), uidA, and nptII transgenes, trees carrying only the uidA and nptII transgenes, and control trees. Clear rhizosphere microbial community alterations due to B. thuringiensis tree genetic modification have to our knowledge never been described previously and open the door to interesting questions related to B. thuringiensis genetic transformation and also to the impact of commonly used uidA and nptII genetic marker genes.     

Fluorescein Diacetate Hydrolysis as a Measure of Fungal Biomass in Soil

The fatty acid methyl esters of lipids extracted from an agricultural soil in the preharvest period of soybean or middle growth cycle from wheat were characterized and quantified by gas-liquid chromatography. The fatty acids 18:2ω6 and 16:1ω5 were used as markers of saprotrophic and arbuscular mycorrhizal fungi. In parallel, biomass estimation through plate counts in selective media for cellulolytic and saprotrophic fungi was also performed all throughout a soybean crop or middle growth cycle of wheat. As an enzymatic method, the fluorescein diacetate (FDA) hydrolytic activity of the samples was determined. Owing to the high relationship exhibited by FDA hydrolysis with organic carbon and total nitrogen content of soil, the enzymatic activity was correlated with the microbial biomass estimated through marker lipids or plate counts. The results obtained point out that FDA hydrolysis may be used as a rapid, cheap, and reliable estimator of fungal biomass. Received: 3 August 2000 / Accepted: 13 October 2000     

Fungal Growth and Biomass Development is Boosted by Plants in Snow-Covered Soil

Soil microbial communities follow distinct seasonal cycles which result in drastic changes in processes involving soil nutrient availability. The biomass of fungi has been reported to be highest during winter, but is fungal growth really occurring in frozen soil? And what is the effect of plant cover on biomass formation and on the composition of fungal communities? To answer these questions, we monitored microbial biomass N, ergosterol, and the amount of fungal hyphae during summer and winter in vegetated and unvegetated soils of an alpine primary successional habitat. The winter fungal communities were identified by rDNA ITS clone libraries. Winter soil temperatures ranged between -0.6°C and -0.1°C in snow-covered soil. We found distinct seasonal patterns for all biomass parameters, with highest biomass concentrations during winter in snow-covered soil. The presence of plant cover had a significant positive effect on the amount of biomass in the soil, but the type of plant cover (plant species) was not a significant factor. A mean hyphal ingrowth of 5.6 m g(-1) soil was detected in snow-covered soil during winter, thus clearly proving fungal growth during winter in snow-covered soil. Winter fungal communities had a typical species composition: saprobial fungi were dominating, among them many basidiomycete yeasts. Plant cover had no influence on the composition of winter fungal communities.     

Impact of Bacterial Biomass on Contaminant Sorption and Transport in a Subsurface Soil

The objective of this study was to investigate the impact of bacterial biomass on the sorption and transport of three solutes (quinoline, naphthalene, and ⁴⁵Ca) in a subsurface soil. Miscible displacement techniques were employed to measure sorption and transport of the above compounds during steady, saturated water flow in sterile and/or bacterium-inoculated soil columns. The soil was inoculated with either a quinoline-degrading bacterium, Pseudomonas sp. 3N3A isolate, or its mutant isolate, B53, which does not degrade quinoline. In soil columns inoculated with the B53 and 3N3A isolates, quinoline sorption was reduced by about 60 and 20%, respectively. In contrast, ⁴⁵Ca sorption was minimally reduced, which indicated that biomass did not significantly alter the cation-exchange capacity of the soil. Biomass impacts on sorption were solute specific, even when the sorption mechanism for both quinoline and ⁴⁵Ca was similar. Thus, the differential response is attributed to biomass-induced changes in quinoline speciation; an increase in pH at the sorbent-water interface would result in a larger proportion of the neutral species and a decrease in sorption. Sorption of naphthalene was reduced by about 30%, which was attributed to accessibility of hydrophobic regions. Minimal biosorption of all solutes indicated negligible biofacilitated transport. Alteration of the soil surfaces upon addition of bacterial biomass reduced sorption of quinoline and naphthalene, thereby enhancing transport.     

Impact of Bt Corn on Rhizospheric and Soil Eubacterial Communities and on Beneficial Mycorrhizal Symbiosis in Experimental Microcosms

A polyphasic approach has been developed to gain knowledge of suitable key indicators for the evaluation of environmental impact of genetically modified Bt 11 and Bt 176 corn lines on soil ecosystems. We assessed the effects of Bt corn (which constitutively expresses the insecticidal toxin from Bacillus thuringiensis, encoded by the truncated Cry1Ab gene) and non-Bt corn plants and their residues on rhizospheric and bulk soil eubacterial communities by means of denaturing gradient gel electrophoresis analyses of 16S rRNA genes, on the nontarget mycorrhizal symbiont Glomus mosseae, and on soil respiration. Microcosm experiments showed differences in rhizospheric eubacterial communities associated with the three corn lines and a significantly lower level of mycorrhizal colonization in Bt 176 corn roots. In greenhouse experiments, differences between Bt and non-Bt corn plants were detected in rhizospheric eubacterial communities (both total and active), in culturable rhizospheric heterotrophic bacteria, and in mycorrhizal colonization. Plant residues of transgenic plants, plowed under at harvest and kept mixed with soil for up to 4 months, affected soil respiration, bacterial communities, and mycorrhizal establishment by indigenous endophytes. The multimodal approach utilized in our work may be applied in long-term field studies aimed at monitoring the real hazard of genetically modified crops and their residues on nontarget soil microbial communities.     

土壤真菌研究进展

综述了土壤真菌的研究简史、研究方法的发展、多样性和现代真菌分类技术在土壤真菌研究中的应用和发展等。     

Influence of Culture Medium on Fungal Biomass Composition and Biosorption Effectiveness

Zygomycetes such as Cunninghamella elegans seem to be promising biosorbents for pollutants removal from wastewaters because of their particular cell wall characteristics. In this article the effect of ten culture media on C. elegans biomass composition was investigated by means of Fourier transform infra red spectroscopy (FTIR). Biomasses grown on starches from potatoes and cereals were characterised by high amount of chitin and polysaccharides, the glucose gave rise to a biomass rich in acidic polysaccharides and lipids. By contrast, biomasses grown on corn steep liquor were poor in acidic polysaccharides and, when N sources and micronutrients were added, rich in proteins. The lipid content of the biomass generally increased by halving nutrients. Biosorption yields of these biomasses towards four wastewater models were assessed in terms of colour, salts and toxicity reduction. The biomasses rich in proteins and acid polysaccharides were less effective in removing reactive and direct dyes, whereas those rich in cationic polysaccharides showed a higher affinity for these dyes. Both chromatography and FTIR analyses showed that biomasses cultured in halved C and N had the highest affinity for salts. The wastewaters detoxification was quite always achieved, with values often lower that the Italian legal threshold limit.     

Effects of Initial Age Structure of Managed Norway Spruce Forest Area on Net Climate Impact of Using Forest Biomass for Energy

We investigated how the initial age structure of a managed, middle boreal (62°N), Norway spruce-dominated (Picea abies L. Karst.) forest area affects the net climate impact of using forest biomass for energy. The model-based analysis used a gap-type forest ecosystem model linked to a life cycle assessment (LCA) tool. The net climate impact of energy biomass refers to the difference in annual net CO₂ exchange between the biosystem using forest biomass (logging residues from final felling) and the fossil (reference) system using coal. In the simulations over the 80-year period, the alternative initial age structures of the forest areas were (i) skewed to the right (dominated by young stands), (ii) normally distributed (dominated by middle-aged stands), (iii) skewed to the left (dominated by mature stands), and (iv) evenly distributed (same share of different age classes). The effects of management on net climate impacts were studied using current recommendations as a baseline with a fixed rotation period of 80 years. In alternative management scenarios, the volume of the growing stock was maintained 20% higher over the rotation compared to the baseline, and/or nitrogen fertilization was used to enhance carbon sequestration. According to the results, the initial age structure of the forest area affected largely the net climate impact of using energy biomass over time. An initially right-skewed age structure produced the highest climate benefits over the 80-year simulation period, in contrast to the left-skewed age structure. Furthermore, management that enhanced carbon sequestration increased the potential of energy biomass to replace coal, reducing CO₂ emissions and enhancing climate change mitigation.     

Evolutionary Impact of In tracellular Symbiosis

Intracellular symbiosis involving two or more species can influence the rapid development and evolution of both the participating organisms, and the environments where they are found. At the cellular level, such associations direct the evolution of metabolic pathways and organelle systems, providing degrees of flexibility not found in single organisms. At the environmental level, they are a significant biogeochemical force that shapes habitats and ecosystems. As such, they are major elements of production and stability. Specific examples of both the cellular and the environmental impact of intracellular symbiosis are presented. Their significance in the overall evolution of the organic world is discussed.     

Nutrient and Carbon Additions to the Microbial Soil Community and its Impact on Tree Seedlings in a Boreal Spruce Forest

We have added glucose and nutrients to manipulate soil microbial activity and nutrient availability in a boreal spruce forest to study the performance of birch and spruce seedlings in relation to the soil microbial community. The proportion of aboveground biomass in the seedlings was largest in plots amended with extra nutrients, while ectomycorrhizal (ECM) colonisation was low in these plots. ECM appeared beneficial for growth of both species, but only at low levels of colonisation (<25% ECM colonised root-tips). The soil microbial biomass, as determined by total PLFA, was largest in plots treated with glucose and there was a significant negative relationship between birch seedling size and levels of total PLFA in soil. This could be taken to suggest that poor seedling growth was due to nutrient limitation caused by microbial assimilation. However, the treatment response of the birch seedlings was generally weak, and spruce often showed no response at all to the addition of nutrients and glucose. The most consistent parameter for the variation in plant performance, as well as for the microbial soil community, was the block-effect. This suggests a strong spatial structure in the soil microbial community, and that this structure was robust with respect to our treatments even though they continued over a 3-year period.     

Translocation and Utilization of Fungal Storage Lipid in the Arbuscular Mycorrhizal Symbiosis

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Carbon is transferred from the plant to the fungus as hexose, but the main form of carbon stored by the mycobiont at all stages of its life cycle is triacylglycerol. Previous isotopic labeling experiments showed that the fungus exports this storage lipid from the intraradical mycelium (IRM) to the extraradical mycelium (ERM). Here, in vivo multiphoton microscopy was used to observe the movement of lipid bodies through the fungal colony and to determine their sizes, distribution, and velocities. The distribution of lipid bodies along fungal hyphae suggests that they are progressively consumed as they move toward growing tips. We report the isolation and measurements of expression of an AM fungal expressed sequence tag that encodes a putative acyl-coenzyme A dehydrogenase; its deduced amino acid sequence suggests that it may function in the anabolic flux of carbon from lipid to carbohydrate. Time-lapse image sequences show lipid bodies moving in both directions along hyphae and nuclear magnetic resonance analysis of labeling patterns after supplying 13C-labeled glycerol to either extraradical hyphae or colonized roots shows that there is indeed significant bidirectional translocation between IRM and ERM. We conclude that large amounts of lipid are translocated within the AM fungal colony and that, whereas net movement is from the IRM to the ERM, there is also substantial recirculation throughout the fungus.     

The Fungal and Bacterial Flora of Stored White Cabbage

1. The predominant organisms isolated from the outer wrapper leaves of freshly harvested white cabbages were: bacteria, yeasts, Alternaria spp., Aureobasidium pullulans, Botrytis cinerea, Cladosporium spp. and Penicillium spp.
2. Few qualitative or quantitative changes were seen in the leaf surface flora during storage at 2°C for up to 33 weeks.
3. Numbers of bacteria, particularly fluorescent and pectolytic pseudomonads, were considerably higher on cabbages drenched with fungicide or water than on corresponding undrenched cabbages.     

Fungal Growth Stimulation by CO2 and Root Exudates in Vesicular-Arbuscular Mycorrhizal Symbiosis

Transformed roots of carrot were used to determine the effects of root metabolites on hyphal development from spores of the vesicular-arbuscular mycorrhizal fungus Gigaspora margarita. Hyphal growth of this obligately biotrophic symbiont was greatly stimulated by a synergistic interaction between volatile and exudated factors produced by roots. Root volatiles alone provided little stimulation, and root exudates alone had no effect. For the first time, carbon dioxide was demonstrated to be a critical root volatile involved in the enhancement of hyphal growth. ¹⁴C-labeled root volatiles were fixed by the fungus and thus strongly suggested that CO₂ served as an essential carbon source.     

Microbial Biomass and Activity in Lead-Contaminated Soil

Microbial community diversity, potential microbial activity, and metal resistance were determined in three soils whose lead contents ranged from 0.00039 to 48 mmol of Pb kg of soil⁻¹. Biomass levels were directly related to lead content. A molecular analysis of 16S rRNAs suggested that each soil contained a complex, diverse microbial community. A statistical analysis of the phospholipid fatty acids indicated that the community in the soil having the highest lead content was not related to the communities in the other soils. All of the soils contained active microbial populations that mineralized [¹⁴C]glucose. In all samples, 10 to 15% of the total culturable bacteria were Pb resistant and had MIC of Pb for growth of 100 to 150 μM.     

Fungal Toxicity of Mobilized Soil Aluminum and Manganese

Inhibition of Aspergillus flavus growth from spores was used as a simple bioassay for toxicity of Al and Mn mobilized by simulated acid precipitation. Al was identified as being toxic in soil leachates resulting from acid inputs of pH less than 2.7. Inhibition by Mn was not detectable. The addition of fluoride significantly reduced Al toxicity, suggesting that biotoxicity of Al is partially dependent on the anionic composition of the soil solution.     

No Evidence of an Impact on the Rhizosphere Diazotroph Community by the Expression of Bacillus thuringiensis Cry1Ab Toxin by Bt White Spruce

Nitrogen fixation is one of the most important roles played by soil bacterial communities, as fixation supplies nitrogen to many ecosystems which are often N limited. As impacts on this functional group of bacteria might harm the ecosystem's health and reduce productivity, monitoring that particular group is important. Recently, a field trial with Bt white spruce, which constitutively expresses the Cry1Ab insecticidal toxin of Bacillus thuringiensis, was established. The Bt white spruce was shown to be resistant to spruce budworm. We investigated the possible impact of these genetically modified trees on soil nitrogen-fixing bacterial communities. The trial consisted of untransformed controls, GUS white spruce (transformed with the β-glucuronidase gene), and Bt/GUS white spruce (which constitutively expresses both the Cry1Ab toxin and β-glucuronidase) in a random design. Four years after planting, soil samples from the control and the two treatments from plantation as well as from two natural stands of white spruce were collected. Diazotroph diversity was assessed by extracting soil genomic DNA and amplifying a region of the nitrogenase reductase (nifH) gene, followed by cloning and sequencing. Analysis revealed that nitrogen-fixing communities did not differ significantly among the untransformed control, GUS white spruce, and Bt/GUS white spruce. Nevertheless, differences in diazotroph diversity were observed between white spruce trees from the plantation site and those from two natural stands, one of which grew only a few meters away from the plantation. We therefore conclude, in the absence of evidence that the presence of the B. thuringiensis cry1Ab gene had an effect on diazotroph communities, that either site and/or field preparation prior to planting seems to be more important in determining diazotroph community structure than the presence of Bt white spruce.     

Biodegradation of Trichloroacetic Acid in Norway Spruce/Soil System

Trichloroacetic acid (TCA) belongs to secondary atmospheric pollutants affecting the forest health. Distribution of [1,2-¹⁴C]TCA-residues and TCA biodegradation were investigated in 4-year-old nursery-grown trees of Norway spruce [Picea abies (L.) Karst.] in the whole plant/soil system. Radioactivity was monitored in needles, wood, roots and soil as well as in the air. During two weeks of exposure TCA was continuously degraded, especially in the soil. Estimates of radioactivity balance showed loss of radioactivity into the atmosphere in the form of ¹⁴CO₂; unincorporated [1,2-¹⁴C]TCA, chloroform, carbon monoxide and methane were not detected at all. TCA degradation to CO₂ was indicated also in the spruce needles. Moreover, it was found that soil litter contained [1,2-¹⁴C]TCA unavailable to microorganisms.     

土壤微生物量测定方法概述

土壤微生物量是土壤生态系统研究的重要参数之一。常用的土壤微生物量的测定方法主要包括:直接镜检法、熏蒸系列方法、底物诱导系列方法、成分分析法和比色法。对这些具体测定方法、原理及其优缺点进行了简要的评述,并指出了应用这些方法须注意的问题和今后的研究方向。