Anne Winding

Soil networks become more connected and take up more carbon as nature restoration progresses

Soil organisms have an important role in aboveground community dynamics and ecosystem functioning in terrestrial ecosystems. However, most studies have considered soil biota as a black box or focussed on specific groups, whereas little is known about entire soil networks. Here we show that during the course of nature restoration on abandoned arable land a compositional shift in soil biota, preceded by tightening of the belowground networks, corresponds with enhanced efficiency of carbon uptake. In mid- and long-term abandoned field soil, carbon uptake by fungi increases without an increase in fungal biomass or shift in bacterial-to-fungal ratio. The implication of our findings is that during nature restoration the efficiency of nutrient cycling and carbon uptake can increase by a shift in fungal composition and/or fungal activity. Therefore, we propose that relationships between soil food web structure and carbon cycling in soils need to be reconsidered.

Effects of C60 fullerene nanoparticles on soil bacteria and protozoans

Nanotechnology should produce numerous new materials in the coming years. Because of the novel design of nanomaterials with new physicochemical characteristics, their potential adverse impact on the environment and human health must be addressed. In the present study, agglomerates of pristine C60 fullerenes (50 nm to microm-size) were applied to soil at 0, 5, 25, and 50 mg/kg dry soil to assess their effect on the soil microbiota by measuring total respiration; biomass, number, and diversity of bacteria; and total number and diversity of protozoans during 14 d. Respiration and microbial biomass were unaffected by the fullerenes at any time, whereas the number of fast-growing bacteria was decreased by three- to fourfold just after incorporation of the nanomaterial. Protozoans seemed not to be very sensitive to C60, because their number decreased only slightly in the beginning of the experiment. With polymerase chain reaction and denaturing gradient gel electrophoresis analysis of eubacteria and kinetoplastids from the soil, however, a difference between the fullerene treatments and nonamended controls was demonstrated. The fullerenes did not induce more than 20 to 30% of relative dissimilarity (with both bacteria and protozoans) between treatments, but this effect was persistent throughout the experiment. It therefore is recommended that fullerene nanomaterial not be spread deliberately in the environment and that their ecotoxicology be further clarified.

Linking of Microorganisms to Phenanthrene Metabolism in Soil by Analysis of 13C-Labeled Cell Lipids

ABSTRACT Phenanthrene-metabolizing soil microbial communities were characterized by examining mineralization of [14C]phenanthrene, by most-probable-number (MPN) counting, by 16S-23S spacer DNA analysis of the numerically dominant, culturable phenanthrene-degrading isolates, and by examining incorporation of [13C]phenanthrene-derived carbon into sterols and polar lipid fatty acids (PLFAs). An unpolluted agricultural soil, a roadside soil diffusely polluted with polycyclic aromatic hydrocarbons (PAHs), and two highly PAH-polluted soils from industrial sites were analyzed. Microbial phenanthrene degraders were not detected by MPN counting in the agricultural soil and the roadside soil. In the industrial soils, phenanthrene degraders constituted 0.04 and 3.6% of the total number of CFU. 16S-23S spacer DNA analysis followed by partial 16S DNA sequencing of representative isolates from one of the industrial soils showed that one-half of the isolates belonged to the genus Sphingomonas and the other half were closely related to an unclassified beta-proteobacterium. The 13C-PLFA profiles of the two industrial soils were relatively similar and resembled the profiles of phenanthrene-degrading Sphingomonas reference strains and unclassified beta-proteobacterium isolates but did not match the profiles of Pseudomonas, Mycobacterium, or Nocardia reference strains. The 13C-PLFA profiles of phenanthrene degraders in the agricultural soil and the roadside soil were different from each other and different from the profiles of the highly polluted industrial soils. Only in the roadside soil were 10me/12me18:0 PLFAs enriched in 13C, suggesting that actinomycetes metabolized phenanthrene in this soil. The 13C-PLFA profiles of the unpolluted agricultural soil did not resemble the profiles of any of the reference strains. In all of the soils investigated, no excess 13C was recovered in the 18:2ω6,9 PLFA, suggesting that fungi did not contribute significantly to assimilation of [13C]phenanthrene.

Non‐target effects of bacterial biological control agents suppressing root pathogenic fungi

Microorganisms have knowingly been used during the last century to control plant diseases. During the last decades, research and application of biological control agents (BCAs) as a pest control strategy have gained increasing attention. This review focuses specifically on non-target effects of bacterial BCAs that are used to suppress root pathogenic fungi. It attempts to critically evaluate the strengths and weaknesses of non-target effect studies published to date and relate them to the success of the BCA in fungal pathogen control. Significant non-target effects of BCAs have indeed been observed, but these are generally small in scale and limited to a growth season, and have not been proven to affect soil health. We discuss these studies and point out what we believe are notable deficiencies. Among the modes of disease suppression by BCAs, antibiotic production is believed to be of major importance. But assurances that in situ antibiotic production actually occurs in environmental samples are lacking in the non-target effect studies. Also the effectiveness of the BCA on the target pathogen, the absence of appropriate controls for inoculation effects, and the presence of pathogenic fungi are missing in most studies. In future non-target effect studies we recommend focusing on proven effective BCAs and clearly distinguishing effects of antimicrobial compounds from effects of general microbial activity.

Bacteria and protozoa in soil microhabitats as affected by earthworms

Abstract The effects of incorporation of elm leaves (Ulmus glabra) into an agricultural sandy loam soil by earthworms (Lumbricus festivus) on the bacterial and protozoan populations were investigated. Three model systems consisting of soil, soil with leaves, and soil with leaves and earthworms, respectively, were compared. The total, viable, and culturable number of bacteria, the metabolic potentials of bacterial populations, and the number of protozoa and nematodes were determined in soil size fractions. Significant differences between soil fractions were shown by all assays. The highest number of microorganisms was found in microaggregates of 2–53 μm and the lowest in the <0.2μm fraction. A major part of the bacteria in the latter fraction was viable, but non-culturable, while a relatively higher number of culturable bacteria was found in the macroaggregates. The number of colony-forming units and 5-cyano-2,3-ditolyl tetrazolim chloride (CTC)-reducing bacteria explained a major part of the variation in the number of protozoa. High protozoan activity and predation thus coincided with high bacterial activity. In soil with elm leaves, fungal growth is assumed to inhibit bacterial and protozoan activity. In soil with elm leaves and earthworms, earthworm activity led to increased culturability of bacteria, activity of protozoa, number of nematodes, changed metabolic potentials of the bacteria, and decreased differences in metabolic potentials between bacterial populations in the soil fractions. The effects of earthworms can be mediated by mechanical mixing of the soil constituents and incorporation of organic matter into the soil, but as the earthworms have only consumed a minor part of the soil, priming effects are believed partly to explain the increased microbial activity.

Succession of indigenous Pseudomonas spp. and actinomycetes on barley roots affected by the antagonistic strain Pseudomonas fluorescens DR54 and the fungicide imazalil.

ABSTRACT In recent years, the interest in the use of bacteria for biological control of plant-pathogenic fungi has increased. We studied the possible side effects of coating barley seeds with the antagonistic strain Pseudomonas fluorescens DR54 or a commercial fungicide, imazalil. This was done by monitoring the number of indigenous Pseudomonas organisms and actinomycetes on barley roots during growth in soil, harvest after 50 days, and subsequent decomposition. Bacteria were enumerated by traditional plate spreading on Goulds S1 agar (Pseudomonas) and as filamentous colonies on Winogradsky agar (actinomycetes) and by two quantitative competitive PCR assays. For this we developed an assay targeting Streptomyces and closely related genera. DR54 constituted more than 75% of thePseudomonas population at the root base during the first 21 days but decreased to less than 10% at day 50. DR54 was not successful in colonizing root tips. Initially, DR54 affected the number of indigenous Pseudomonas organisms negatively, whereas imazalil affected Pseudomonas numbers positively, but the effects were transient. Although plate counts were considerably lower than the number of DNA copies, the two methods correlated well for Pseudomonas during plant growth, but after plant harvest Pseudomonas-specific DNA copy numbers decreased while plate counts were in the same magnitude as before. Hence,Pseudomonas was 10-fold more culturable in a decomposition environment than in the rhizosphere. The abundance of actinomycetes was unaffected by DR54 or imazalil amendments, and CFU and quantitative PCR results correlated throughout the experiment. The abundance of actinomycetes increased gradually, mostly in numbers of DNA copies, confirming their role in colonizing old roots.

Indicators for Monitoring Soil Biodiversity

A Bispo,3 D Cluzeau,4 R Creamer,1 M Dombos,I U Graefe,# PH Krogh,33 JP Sousa,44 G Peres,4 M Rutgers,11 A Winding,33 and J Rombke*II 7French Agency for Environment and Energy Management, France 8University of Rennes, France 6Teagasc, Ireland IResearch Institute for Soil Science and Agricultural Chemistry, Hungary #Institut fur Angewandte Bodenbiologie, Germany 77Aarhus University, Denmark 88University of Coimbra, Portugal 66National Institute for Public Health and the Environment, Netherlands IIECT Oekotoxikologie GmbH, Germany * J-Roembke@ect.de

Biolog Substrate Utilisation Assay for Metabolic Fingerprints of Soil Bacteria: Incubation Effects

Bacterial communities can be described by their enzymatic potentials using the Biolog substrate utilisation assay. We have investigated tetrazolium reduction and cell growth during incubation of Pseudomonas fluorescens MM6 and soil bacteria in Biolog plates. Increasing the inoculum size shortened the lag phase before formazan formation. For the soil bacteria, increasing the inoculum also resulted in a higher rate constant of formazan formation, and the final number of wells with formazan formation increased. Both MM6 and soil bacteria proliferated in the wells both with and without specific carbon sources after inoculation. With soil bacteria, the presence of clay, humic substances, and dissolved organic matter increased the background coloration and may have resulted in cell growth. The growth led to increased culturability (CFU/AODC) and rate of colony-appearance and decreased the diversity of the bacterial communities within each well. Metabolic fingerprinting of bacterial communities using Biolog plates thus depends on aerobic growth of a fraction of the community.

Comparing the desorption and biodegradation of low concentrations of phenanthrene sorbed to activated carbon, biochar and compost

Carbonaceous soil amendments are applied to contaminated soils and sediments to strongly sorb hydrophobic organic contaminants (HOCs) and reduce their freely dissolved concentrations. This limits biouptake and toxicity, but also biodegradation. To investigate whether HOCs sorbed to such amendments can be degraded at all, the desorption and biodegradation of low concentrations of (14)C-labelled phenanthrene (≤5 μg L(-1)) freshly sorbed to suspensions of the pure soil amendments activated carbon (AC), biochar (charcoal) and compost were compared. Firstly, the maximum abiotic desorption of phenanthrene from soil amendment suspensions in water, minimal salts medium (MSM) or tryptic soy broth (TSB) into a dominating silicone sink were measured. Highest fractions remained sorbed to AC (84±2.3%, 87±4.1%, and 53±1.2% for water, MSM and TSB, respectively), followed by charcoal (35±2.2%, 32±1.7%, and 12±0.3%, respectively) and compost (1.3±0.21%, similar for all media). Secondly, the mineralization of phenanthrene sorbed to AC, charcoal and compost by Sphingomonas sp. 10-1 (DSM 12247) was determined. In contrast to the amounts desorbed, phenanthrene mineralization was similar for all the soil amendments at about 56±11% of the initially applied radioactivity. Furthermore, HPLC analyses showed only minor amounts (<5%) of residual phenanthrene remaining in the suspensions, indicating almost complete biodegradation. Fitting the data to a coupled desorption and biodegradation model revealed that desorption did not limit biodegradation for any of the amendments, and that degradation could proceed due to the high numbers of bacteria and/or the production of biosurfactants or biofilms. Therefore, reduced desorption of phenanthrene from AC or charcoal did not inhibit its biodegradation, which implies that under the experimental conditions these amendments can reduce freely dissolved concentration without hindering biodegradation. In contrast, phenanthrene sorbed to compost was fully desorbed and biodegraded.

Microbial succession in the rhizosphere of live and decomposing barley roots as affected by the antagonistic strain Pseudomonas fluorescens DR54-BN14 or the fungicide imazalil.

Abstract The protocol used in the present study was a long-term mesocosm experiment where the microbial succession around live barley roots and subsequent decomposing roots was assessed after seed coating with either the antagonistic strain Pseudomonas fluorescens DR54-BN14 or the fungicide imazalil. Four diversity measures were used: community level physiological profiles (CLPP), Bacteria-specific polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), actinomycete-specific PCR-DGGE and phospholipid fatty acid (PLFA), as well as total cell counts, colony-forming units (CFU) and culturable spore formers, and spore counts of the Bacillus cereus group. Analysis of non-treated plants provided a baseline description of the natural microbial succession from which effects of the treatments could be evaluated. A microbial succession occurred both in the rhizosphere and around decomposing roots, shown with all three diversity measures. A clear response to root death was found, and a clear distinction between root tip and root base samples. Using the recommended concentration of imazalil and a realistic number of DR54-BN14 for seed coating, transient, initial effects of both treatments on the microbial communities were observed at the root base with the PLFA analysis only. The lack of lasting significant side effects of DR54-BN14 is in agreement with an initial fast reduction in culturable DR54-BN14.