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Soil aggregation is an important physical indicator of soil health. Aggregation result from biological and physicochemical processes in which primary soil particles and organic matter are bound. Biotic-mediated soil aggregation is mainly assisted by fungal hyphae facilitating macroaggregate formation through mechanical union and exudation of binding agents. In agricultural soils, many crops establish a symbiosis root- arbuscular mycorrhizal (AM) fungi where the extensive mycelium directly connects roots and soil aggregates. However, the relative importance and specific contributions of the belowground plant root associated microbiome to soil aggregation is still not known.We set up a pot experiment under controlled conditions to investigate the aggregate stability of different crop species in an organic agricultural sandy soil. We manipulated the soil microbiome by planting 10 common crop species with different mycorrhizal status, 5 species with AM fungal symbiosis (barley, clover, maize, oat and wheat) and 5 non-mycorrhizal species (buckwheat, lupine, quinoa, rapeseed and spinach). We filled each pot with sandy soil sieved to 2 mm and added two sealed mesh bags, with mesh size of 40 µm to avoid roots but promoting hyphal entrance. Mesh bags were filled with the same sandy soil further sieved to 1 mm and 0.25 mm, respectively. All pots were harvested in the 10th week and biomass, roots and soil samples were processed.Soil aggregate stability, measured by wet sieving method, showed that soils with AM fungal symbiosis became more aggregated as these soils presented a higher % in soil fractions > 500 µm and > 250 µm. Moreover, non-mycorrhizal crops soils showed a significantly higher free mineral fraction (< 63 µm), i.e. a loss of soil aggregation. Still, our results showed that no crop species, independently their mycorrhizal status, were able to form aggregates above the sieving size and not all crop species conferred the same soil aggregation. Among mycorrhizal crop species, barley and wheat showed a higher soil aggregation compared to oat and maize. Curiously, barley and wheat were the crop species with the highest AM root colonization, 74 and 69 %, respectively, as well as the soils with the highest microbial biomass C, N and their ratio. Our results showed that exists a positive correlation between microbial biomass, root colonization and soil aggregation. Further analysis will provide data on soil mycelial length as well as fungal and bacterial community profiles.In conclusion, the mycorrhizal status of different crop species revealed the key role of AM fungi in soil aggregation and its relationship with microbial biomass; however, not surprisingly, the effects are species dependent. Our forthcoming data of the soil microbial communities and their functionality will further reveal which groups have a direct effect on soil aggregation.

Cadmium’s presence in agricultural soils resulting from industrial activities, factories, fertilizers, and pesticides is an environmental challenge threatening public health. Therefore, this study was carried out to assess Trichoderma harzianum and indigenous rhizosphere soil microbes’ ability on cadmium absorption, translocation, and tolerance in barley. A pot experiment was conducted in a full factorial randomized design with three replicates. Treatments were T. harzianum (Tri) and rhizosphere soil (Rhi) and Cd at seven levels (0, 1.54, 3.84, 9.6, 24, 60, and 150 mg kg−1). We found that simultaneous application of T. harzianum and indigenous soil microorganisms had positive effects on barley tolerance index (TI), Cd uptake efficiency, shoot dry weight, and plant uptake. Co-inoculation of T. harzianum and soil microorganisms resulted in significantly higher chlorophyll content as compared to plants inoculated only with only T. harzianum or soil microorganisms. The maximum root uptake obtained at 150 mg kg−1 Cd added was in plants also co-inoculated with Tri and Rhi. Also, the Cd uptake efficiency was lower in plants inoculated with Tri and Rhi compared to non-inoculated plants. Meanwhile, Cd uptake efficiency increased with soil Cd up to 133 mg kg−1. The lowest Cd phytoextraction efficiency was observed with Tri and Rhi co-inoculated plants. The obtained results suggest that co-inoculation of T. harzianum and soil microbes ameliorates the negative effects of Cd contamination and could then be used as an option to remediate Cd-contaminated soils even in higher levels than this study.

Permafrost soils store a substantial part of the global soil carbon and nitrogen. However global warming causes abrupt erosion and gradual thaw, which make these stocks vulnerable to microbial decomposition into greenhouse gases. Here, we investigated the microbial response to abruptin situpermafrost thaw. We sequenced the total RNA of a 1 m deep soil core consisting of up to 26’500-year-old permafrost material from an active abrupt erosion site. We analysed the microbial community in the active layer soil, the recently thawed, and the intact permafrost and found maximum RNA:DNA ratios indicating a microbial bloom in recently thawed permafrost. Several fast-growing prokaryotic taxa dominated thawed permafrost, including Sphingobacteriales, Burkholderiales, and Nitrosomonadales. Overall, the thaw state and soil moisture consistently explained changes in community composition, with especially the permafrost community being significantly distinct from thawed soils. Predation correlated with changes in prokaryotic composition. Bacterial grazers were dominated by Myxococcales and abundant in the active layer. In contrast, protozoa, especially Cercozoa and Ciliophora, doubled in relative abundance in thawed layers. Our findings highlight the ecological importance of a rapid development of microbial blooms as well as the successive predation as biological control mechanism in abruptly thawing permafrost.One sentence summaryUsing total RNA from an up to 26’500-year-old abruptly eroding permafrost site in Greenland, we described a microbial bloom and its controls, including bacterial and microeukaryotic predators.Abstract Figure

Biochar is proposed as an option to sequester carbon (C) in soils and promote other soil-based ecosystem services. However, its impact on soil biota from micro to macroscale remains poorly understood. We investigated biochar effects on the soil biota across the soil food web, on plant community composition and on biomass production. We conducted a field experiment in a nature restoration grassland testing four treatments: two biochar types (herbaceous feedstock pyrolyzed at 400 °C or 600 °C – hereafter B400 and B600), and a positive (i.e. unpyrolysed biochar feedstock, hereafter Hay) and negative (no addition) control. Responses of plants and soil biota were evaluated one and three years after establishing the treatments. Soil pH and K concentrations increased significantly in the B600 treatment. Mite abundances were significantly higher in B400 whereas nematode abundances were highest in Hay (1st year) and lowest in B400 (3rd year). Other soil fauna groups (enchytraeids and earthworms) varied more between years than between treatments. Legume cover increased significantly in the biochar treatments but this effect was transient. Legumes, grasses and primary productivity also showed a statistically significant Treatment x Year interaction due to transitory effects that were no longer present by the 3rd year. Our results suggest that biochar produced from meadow cuttings and applied at the 10 t/ha rate cause transitory impacts on soil biota abundance and plant communities over the 3-year timeframe used for this experiment. Therefore, this type of biochar could potentially be used for soil carbon sequestration, with minimal impacts on soil biota abundance or diversity, within the groups studied here, or plant biodiversity and productivity. Further research is required to investigate the longer-term impacts of this potential soil C storage sink.

Biodiversity of soil microbiota is routinely assessed with environmental DNA-based methods, among which amplification and massive parallel sequencing of marker genes (eDNA metabarcoding) is the most common. Soil microbiota may for example be investigated in relation to biodiversity research or as a tool in forensic investigations.After sampling, the taxonomic composition of soil biotic communities may change. In order to minimize community changes after sampling, it is desirable to reduce biological activity, e.g. by freezing immediately after sampling. However, this may be impossible due to remoteness of study sites or, in forensic cases, where soil has been attached to a questioned item for protracted periods of time.Here we investigated the effect of storage duration and conditions on the assessment of the soil biota with eDNA metabarcoding. We extracted eDNA from freshly collected soil samples and again from the same samples after storage under contrasting temperature conditions.We used five different primer sets targeting bacteria, fungi, protists (cercozoans), general eukaryotes, and plants. For these groups, we quantified differences in richness, evenness and community composition. Subsequently, we tested whether we could correctly infer habitat type and original sample identity after storage using a large reference dataset.We found increased community composition differences with extended storage time and with higher storage temperature. However, for samples stored less than 28 days at a maximum of 20°C, changes were generally insignificant. Classification models could successfully assign most stored samples to their exact location of origin and correct habitat type even after weeks of storage. Even samples showing larger compositional changes generally retained the original sample as the best match (relative similarity).Our results show that for most biodiversity and forensic applications, storage of samples for days and even several weeks may not be a problem, if storage temperature does not exceed 20°C. Even after suboptimal storage conditions, significant patterns can be reproduced.

Wood ash recycling to forests is beneficial because it regains nutrients and prevents acidification, but wood ash application is restricted due to its cadmium (Cd) content. We question if Cd in wood ash represents a problem, since decreases in Cd bioavailability due to ash-induced pH changes may counteract increased total Cd concentration. We studied effects of wood ash (0, 3, 9 and 30 t ha −1 ) and lime (pH increase equivalent to the wood ash treatments) on growth and Cd uptake in Deschampsia flexuosa. After four months, we measured plant biomass and Cd accumulation, and extracted Cd from the soil using three different methods; HNO 3 (total), EDTA (chelator-based) and NH 4 NO 3 (salt-based). Wood ash and lime strongly stimulated plant growth. Cd concentration in the plant tissue decreased with wood ash and lime addition, and correlated positively with the NH 4 NO 3 extractable fraction of Cd in the soil. In contrast, HNO 3 and EDTA extracted more Cd with increased wood ash application. We conclude that wood ash amendment increases soil pH, total Cd concentration, nutrient levels and stimulates plant growth. However, it does not increase Cd accumulation in D. flexuosa, as pH-driven decreases in Cd bioavailability leads to reduced plant Cd uptake. Finally, soil bioavailable Cd is best determined using NH 4 NO 3 -extraction.

Cadmium (Cd) is one of the most toxic heavy metals found in soil. Arbuscular mycorrhiza (AM) is known to reduce Cd translocation in plants by immobilising Cd in the root system. The effect of mycorrhiza on plant Cd uptake is usually studied in simple systems with single strains of mycorrhizal fungi and few levels of Cd. Here we studied how a wide range of soil Cd concentrations affected plant AM colonisation, and how the species-specific differences in AM colonisation affected uptake, translocation, and toxicity of Cd in plants in a system with naturally occurring mycorrhizal fungi. Six plant species were grown in pots in a greenhouse across seven levels of Cd, which made it possible to model dose-response curves, and calculate EC50 for each plant species. We found a remarkable trend where Cd at moderate levels stimulated mycorrhizal colonisation until a certain threshold where the symbiosis breaks down. Our results support the existence of a protective effect of AM fungi against Cd, as the symbiosis reduces Cd translocation to shoots, especially in plants with very high AM colonisation. Thus, we conclude that it is the combination of plant species and AM colonisation that determines Cd uptake in plants. AM is therefore an essential trait to consider when growing plants in Cd-polluted soil.

Wood ash is a by-product from energy production that can be recycled to forests to regain nutrients and prevent acidification. However, low concentrations of nitrogen (N) in wood ash may reduce its potential positive effect on plant growth. In addition, wood ash can have a high content of toxic heavy metals such as Cd, thus there are concerns that it may increase Cd accumulation in plants. We grew Deschampsia flexuosa (Wavy hair-grass) in pots in acidic nutrient poor forest soil fertilized with different concentrations of wood ash (corresponding to field application of 0, 1.1, 3.3, 11 and 33 t ha−1). Additionally, to disentangle the pH and nutrient effects of wood ash application, we included treatments with either CaO, to simulate pH effects of wood ash, or potassium (K) + phosphorus (P) fertilizer to mimic the nutrient effects. After 4.5 months of growth, we measured soil pH, plant biomass, Cd accumulation in shoots and N concentration in the various compartments of the system. Wood ash addition stimulated plant growth, whereas CaO and K + P addition resulted in more moderate increases in biomass. Despite the low concentration of N in the wood ash, plant uptake of N increased in wood ash amendments, probably because wood ash stimulated mineralization of soil organic N. Plant Cd content significantly increased at the highest dose of wood ash. Our results suggest that addition of wood ash significantly stimulates plant growth due to the combined effect of increased pH, elevated nutrient levels and increased N mineralization. Furthermore, despite the rather high Cd content in used wood ash (16.3 mg kg−1), wood ash amendments up to 11 t ha−1 did not result in significantly increased plant uptake of Cd.

Accepted manuscript version, licensed CC BY-NC-ND 4.0. Aims - Microorganisms play a dichotomous role in the soil nitrogen cycle through mineralization and immobilization. We aimed to understand how nitrogen availability modifies the effect of microorganisms on plant growth. We hypothesized that soil microorganisms would increase plant biomass following amendment with a substrate rich in organic nitrogen (net mineralization), be neutral when adding inorganic nitrogen, and decrease biomass when adding organic nitrogen-limited substrate (nitrogen competition). Method - Barley (Hordeum vulgare L., cv. Evergreen) was exposed to either i) limited, organically bound nitrogen, ii) organically bound nitrogen or iii) inorganic nitrogen. In these amendments, we assessed the differences in plant biomass and physiology between plants with or without soil microbiome addition. Results - The soil microbiome reduced shoot biomass equally (12%) across all nitrogen amendments. However, nitrogen availability did modulate the effect of the soil microbiome on plant physiological parameters associated with nitrogen deficiency. Conclusions - The results indicate that the net negative effect of complex microbiomes on shoot biomass is independent of nitrogen availability. Thus, microbiome addition was deleterious to biomass even in a nutrient-stress-free environment. We suggest that strategies for improving plant growth through manipulation of microorganisms should not exclusively focus on beneficial and pathogenic microorganisms, but also include minimizing plant metabolic costs of microbiome interactions.

Global biodiversity is under pressure from human activities, and the effort for nature conservation and restoration and the allocation of economic resources for biodiversity policies remain insufficient. In such a context, volunteers can play an important role as a resource in nature conservation projects if their recreational activities interact with the objectives of nature management. In recent years, the number of volunteers in conservation work has increased in Denmark, with more people volunteering to contribute to nature conservation projects. Ensuring that volunteers remain motivated and engaged is crucial for the success of such conservation projects. In this study, we evaluated the motivation among members of grazing organizations, an activity that represents the most prominent voluntary nature conservation initiative in Denmark. We applied an exploratory factor analysis (EFA) and an ordinal regression to analyze survey data from 25 Danish grazing organizations. We found that five motivational factors determine the engagement of the volunteers, namely social, nature value, instrumental, identification, and personal benefit. Whereas the social, nature value and personal benefit are factors also identified in the existing literature, the instrumental and identification factors add new perspectives to the motivation of environmental volunteers. We found that place attachment is an important driver, and that the chairpersons/coordinators of the grazing organizations especially emphasized the sharing of values and knowledge with their members as a driver. Lastly, volunteers were reluctant to support the idea of forming a more formal setup in terms of a “grazing organization union”.

Small heterotrophic protists (flagellates and naked amoebae) are very abundant in soil and play a key role in maintaining soil services. Hence, knowledge on how xenobiotics affect these organisms is essential in ecosystem management. Cadmium (Cd) is an increasing environmental issue as both industrial deposition and recycling of heavy metal rich waste products have led to Cd enrichment of soils. Evaluation of toxicity of Cd to micro-organisms is often performed using a solution of pure Cd (e.g. CdCl) in liquid culture. This approach may be highly misleading as interactions between Cd and other substances, e.g. various ions or inherent soil components often strongly modify Cd toxicity. Hence, we compared the toxic effect of Cd to small heterotrophic protists in soil microcosms and liquid culture. We also evaluated how zinc (Zn) affects Cd toxicity, as Zn usually accompanies Cd in a ratio of c. 100:1, and is known to impede Cd toxicity. In the soil microcosms, we also monitored the primary food source of the protists, i.e. culturable bacteria, and used soil respiration as a proxy of soil functioning. Finally, we examined to what extent Cd actually sorbs to soil. We found 1) that c. 103 times more Cd was required to obtain the same effect in the soil microcosms compared to the liquid culture, 2) that soil sorption explains why Cd, even though highly toxic in aqueous solutions, has very limited effect when applied to soil, and 3) (very surprisingly) that in our experimental systems Zn was as toxic as Cd. Our study suggests that Cd toxicity to soil protists will be small because most Cd in soil will be sorbed to the soil matrix and because the Zn:Cd ratio of 100:1 in most substances, incl. pollutants, will mean that lethal Zn effects will occur before Cd reaches toxic levels.

To understand and manipulate the interactions between plants and microorganisms, sterile seeds are a necessity. The seed microbiome (inside and surface microorganisms) is unknown for most plant species and seed-borne microorganisms can persist and transfer to the seedling and rhizosphere, thereby obscuring the effects that purposely introduced microorganisms have on plants. This necessitates that these unidentified, seed-borne microorganisms are removed before seeds are used for studies on plant–microbiome interactions. Unfortunately, there is no single, standardized protocol for seed sterilization, hampering progress in experimental plant growth promotion and our study shows that commonly applied sterilization protocols for barley grains using H2O2, NaClO, and AgNO3 yielded insufficient sterilization. We therefore developed a sterilization protocol with AgNO3 by testing several concentrations of AgNO3 and added two additional steps: Soaking the grains in water before the sterilization and rinsing with salt water (1% (w/w) NaCl) after the sterilization. The most efficient sterilization protocol was to soak the grains, sterilize with 10% (w/w) AgNO3, and to rinse with salt water. By following those three steps, 97% of the grains had no culturable, viable microorganism after 21 days based on microscopic inspection. The protocol left small quantities of AgNO3 residue on the grain, maintained germination percentage similar to unsterilized grains, and plant biomass was unaltered. Hence, our protocol using AgNO3 can be used successfully for experiments on plant–microbiome interactions.

Due to their small size, microorganisms can be widely dispersed and they colonize sterilized soils fast. However, details regarding quickness and successional patterns are poorly understood. We investigated the ability of airborne microorganisms to colonize sterilized soil at different heights above ground, and at different distances to a tree. Soil below the tree was colonized by bacteria already after 1 day and the sites above and away from the tree after only 4 days. The site below the tree was the only one to be colonized by culturable protists during the 15 day experiment. Our results show a pattern of succession when sterile soils are colonized by bacteria and protists. The tree was found to have a substantial impact on the recruitment of microorganisms.

Wood ash, the by-product of biomass combustion to energy, can return important nutrients back to the soil and counteract acidification. However, the application of wood ash may affect the emission of greenhouse gases. Here, the effect of wood ash application on nitrous oxide (N

As groundwater-fed waterworks clean their raw inlet water with sand filters, a variety of pro- and eukaryotic microbial communities develop on these filters. While several studies have targeted the prokaryotic sand filter communities, little is known about the eukaryotic communities, despite the obvious need for knowledge of microorganisms that get in contact with human drinking water. With a new general eukaryotic primer set (18S, V1-V3 region), we performed FLX-454 sequencing of material from 21 waterworks' sand filters varying in age (3–40 years) and geographical location on a 250 km east–west axis in Denmark, and put the data in context of their previously published prokaryotic communities. We find that filters vary highly in trophic complexity depending on age, from simple systems with bacteria and protozoa (3–6 years) to complex, mature systems with nematodes, rotifers and turbellarians as apex predators (40 years). Unlike the bacterial communities, the eukaryotic communities display a clear distance–decay relationship that predominates over environmental variations, indicating that the underlying aquifers feeding the filters harbor distinct eukaryotic communities with limited dispersal in between. Our findings have implications for waterworks' filter management, and offer a window down to the largely unexplored eukaryotic microbiology of groundwater aquifers.

Recycling of wood ash from energy production may counteract soil acidification and return essential nutrients to soils. However, wood ash amendment affects soil physicochemical parameters that control composition and functional expression of the soil microbial community. Here, we applied total RNA sequencing to simultaneously assess the impact of wood ash amendment on the active soil microbial communities and the expression of functional genes from all microbial taxa. Wood ash significantly affected the taxonomic (rRNA) as well as functional (mRNA) profiles of both agricultural and forest soil. Increase in pH, electrical conductivity, dissolved organic carbon and phosphate were the most important physicochemical drivers for the observed changes. Wood ash amendment increased the relative abundance of the copiotrophic groups Chitinonophagaceae (Bacteroidetes) and Rhizobiales (Alphaproteobacteria) and resulted in higher expression of genes involved in metabolism and cell growth. Finally, total RNA sequencing allowed us to show that some groups of bacterial feeding protozoa increased concomitantly to the enhanced bacterial growth, which shows their pivotal role in the regulation of bacterial abundance in soil., We applied total RNA sequencing to simultaneously assess the impact of wood ash amendment on the active soil microbial communities and the expression of functional genes from all microbial taxa.

International audience; Promoting diverse and functioning biological communities is an important objective of agroecology, with increasing attention given to the important role of soil biodiversity. In an experimental study conducted under field conditions, we followed over four years the dynamic of soil organisms from various sizes and trophic niches in four variants of a cropping system which are differentiated by soil tillage, residue management and N fertilization rate. Differentiation in overall family diversity, as well as in the abundance and diversity of the multiple trophic groups was evaluated every two years. Our study demonstrated a delayed but effective differentiation in soil biota diversity following implementation of the agricultural practices. Soil biodiversity varied throughout time with some groups responding more readily than others, thereby highlighting differences related to trophic position and body size. The visualization of diversity profiles revealed an increasing impact of agricultural practices on group diversity towards higher trophic levels. While tillage appeared a main factor of influence, surprisingly little impact of residue management and nitrogen fertilization could be observed. Predicting the response of the soil biota to anthropogenic influence calls for an understanding of complex interactions between soil organisms in heterogeneous soil microhabitats. Through its multitaxonomic approach, the present study increases our understanding of the dynamic of soil communities in agricultural cropping systems and helps identify possible consequences for soil functioning.

Yellow River Delta undergoes intensive conversion from natural wetland to agricultural fields and artificial woodland. In this study, we analysed how the conversion affects bacterial community diversity and composition by Illumina Miseq sequencing combined with functional prediction. Compared to natural wetland, arable land and woodland were featured with higher soil organic matter, total nitrogen and bacterial diversity, but lower electrical conductivity. The bacteria Gemmatimonadetes related to soil organic matter and total nitrogen, was enriched in arable land, while salt-resistant bacteria (e.g. phylum Chloroflexi and its class Ardenticatenia) were abundant in natural wetland. Moreover, the relative abundances of the nitrifying bacteria Nitrospira and Nitrosospira were significantly higher in arable land and woodland than in natural wetland, suggesting that land use changes significantly affect the bacterial processes involved in nitrogen cycling. Redundancy analysis (RDA) showed that the differences in bacterial community were attributed to soil nutrient-related properties (i.e., total nitrogen and soil organic matter), soil salinity (i.e., electrical conductivity), and heavy metals (i.e. Cu and Cr). PICRUSt results revealed that land use conversion from natural wetland to arable land increased soil functions, e.g., biosynthesis process and oxidative phosphorylation. The data help us elucidate how land use changes affect terrestrial ecosystem function, and advise local farmers to apply the suitable land-use strategies and keep agricultural sustainable development.

International audience; The active layer of soil overlaying permafrost in the Arctic is subjected to annual changes in temperature and soil chemistry, which we hypothesize to affect the overall soil microbial community. We investigated changes in soil microorganisms at different temperatures during warming and freezing of the active layer soil from Svalbard, Norway. Soil community data were obtained by direct shotgun sequencing of total extracted RNA. No changes in soil microbial communities were detected when warming from -10 to -2 degrees C or when freezing from -2 to -10 degrees C. In contrast, within a few days we observed changes when warming from -2 to +2 degrees C with a decrease in fungal rRNA and an increase in several OTUs belonging to Gemmatimonadetes, Bacteroidetes and Betaproteobacteria. Even more substantial changes occurred when incubating at 2 degrees C for 16 days, with declines in total fungal potential activity and decreases in oligotrophic members from Actinobacteria and Acidobacteria. Additionally, we detected an increase in transcriptome sequences of bacterial phyla Bacteriodetes, Firmicutes, Betaproteobacteria and Gammaproteobacteria-collectively presumed to be copiotrophic. Furthermore, we detected an increase in putative bacterivorous heterotrophic flagellates, likely due to predation upon the bacterial community via grazing. Although this grazing activity may explain relatively large changes in the bacterial community composition, no changes in total 16S rRNA gene copy number were observed and the total RNA level remained stable during the incubation. Together, these results are showing the first comprehensive ecological evaluation across prokaryotic and eukaryotic microbial communities on thawing and freezing of soil by application of the Total RNA technique.

Wood ash, the by-product of biomass combustion to energy, can return important nutrients back to the soil and counteract acidification. However, the application of wood ash may affect the emission of greenhouse gases. Here, the effect of wood ash application on nitrous oxide (N2O) emissions from different soil environments were investigated in a 40 days incubation experiment comprising ten different soil types amended with five different wood ash concentrations (0, 3, 9, 20, and 54 t ash ha−1). The emitted N2O was measured continuously, and initial soil properties without ash application (carbon (C), nitrogen (N), ammonium (NH4+), nitrate (NO3−), and pH) and resulting soil properties (pH, NH4+, and NO3−) were measured prior and after the incubation period, respectively. The Random Forests (RF) model was used to identify which factors (initial and resulting soil properties, vegetation, management, wood ash doze, and respiration rate) were the most important to predict the development of emitted N2O after ash application. Wood ash either increased, decreased, or had no effect on the amount of emitted N2O depending on soil type and ash dose. The RF model identified the final resulting pH as the most important factor for the prediction of emitted N2O. The results suggest that wood ash can mitigate N2O emissions from soil, however, this effect depends on soil type where a mitigating effect of wood ash application was observed mainly in low pH soils with high soil organic matter whereas an increase in N2O emissions was observed in mineral soils that had previously received N fertilization. This study emphasises the importance of pH manipulation in regards to N2O emissions from soil.

Wood ash is a beneficial fertilizer and liming agent in nutrient depleted soils, but it also contains considerable amounts of cadmium (Cd), which can be toxic to organisms in the environment. Therefore, risk assessments regarding utilization of wood ash is required. Here, we studied how wood ash (applied in doses equivalent to 0, 3 and 6 t ha −1 ) and Cd (applied in doses of 0, 10, 150, 300, 600, 1200 and 2000 mg kg −1 ) affected growth of the soil nematode Caenorhabditis elegans. The treatments were combined in a full factorial design. Wood ash alone greatly stimulated both soil respiration and growth of C. elegans, whereas Cd alone had a toxic effect. However, unrealistically high Cd levels were needed to severely affect growth of C. elegans and soil respiration, especially soil respiration was very resilient to Cd amendment. Ash addition decreased Cd toxicity to C. elegans, with an EC 50 value of 390 mg Cd kg −1 in the 3 t ash ha −1 treatment, and an increase of EC 50 to 1894 mg Cd kg −1 in the 6 t ash ha −1 treatment. This is probably because ash increases the Cd sorption capacity of the soil, and thereby decreases the bio-availability of Cd. The results suggest that there is no acute toxic effect of Cd to nematodes associated with wood ash recycling; in fact, our results suggest that ash actually decrease Cd toxicity.

Ash from combustion of biofuels, for example wood chips, is often deposited as waste, but due to its high content of essential plant nutrients and alkalinity, it has been proposed to recycle ash as a fertilizer and liming agent in biofuel production forest. However, current legislation sets rather strict limitations for wood-ash application in biofuel production systems. The soil microfood web, that is microorganisms and their microfaunal grazers, protozoa and nematodes, is pivotal for essential ecosystem processes such as decomposition and plant nutrient release. Therefore, a thorough assessment of the impacts on microfood web structure and functioning must precede actions towards raising the currently allowed application rates. In a Danish Norway spruce plantation, we evaluate the impact of wood ash applied at dosages from 0 to the extreme case of 90 t ash ha −1 on the microfood web, the bacterial community structure, soil content of inorganic nitrogen, organic matter, dissolved organic carbon and nitrogen. Using structural equation modelling (SEM), we disentangled the direct effect of the disturbance imposed by ash per se, the associated pH increase and changes in prey abundance on individual organism groups in the microfood web. The SEM showed that the pH rise was the main driver of increasing abundances of culturable heterotrophic bacteria with increasing ash doses, and via trophical transfer, this also manifested as higher abundances of bacterial grazers. Fungal-feeding nematodes were unaffected by ash, whereas carnivorous/omnivorous nematodes decreased due to the direct effect of ash. Increasing ash doses enhanced the difference between bacterial communities of control plots and ash-amended plots. The ash-induced stimulation of culturable heterotrophic bacteria and bacterial grazers increased inorganic nitrogen availability at ash doses of 9 t ha −1 and above. Hence, raised limits for ash application may potentially benefit tree growth via enhanced N mineralization activity of the soil food web.

Source at https://doi.org/10.1038/s41598-018-24082-y. Here we show that a commercial blocking reagent (G2) based on modified eukaryotic DNA significantly improved DNA extraction efficiency. We subjected G2 to an inter-laboratory testing, where DNA was extracted from the same clay subsoil using the same batch of kits. The inter-laboratory extraction campaign revealed large variation among the participating laboratories, but the reagent increased the number of PCR-amplified16S rRNA genes recovered from biomass naturally present in the soils by one log unit. An extensive sequencing approach demonstrated that the blocking reagent was free of contaminating DNA, and may therefore also be used in metagenomics studies that require direct sequencing.

Protists include all eukaryotes except plants, fungi and animals. They are an essential, yet often forgotten, component of the soil microbiome. Method developments have now furthered our understanding of the real taxonomic and functional diversity of soil protists. They occupy key roles in microbial foodwebs as consumers of bacteria, fungi and other small eukaryotes. As parasites of plants, animals and even of larger protists, they regulate populations and shape communities. Pathogenic forms play a major role in public health issues as human parasites, or act as agricultural pests. Predatory soil protists release nutrients enhancing plant growth. Soil protists are of key importance for our understanding of eukaryotic evolution and microbial biogeography. Soil protists are also useful in applied research as bioindicators of soil quality, as models in ecotoxicology and as potential biofertilizers and biocontrol agents. In this review, we provide an overview of the enormous morphological, taxonomical and functional diversity of soil protists, and discuss current challenges and opportunities in soil protistology. Research in soil biology would clearly benefit from incorporating more protistology alongside the study of bacteria, fungi and animals.

Rapid sand filtration is essential at most waterworks that treat anaerobic groundwater. Often the filtration depends on microbiological processes, but the microbial communities of the filters are largely unknown. We determined the prokaryotic community structures of 11 waterworks receiving groundwater from different geological settings by 16S rRNA gene-based 454 pyrosequencing and explored their relationships to filtration technology and raw water chemistry. Most of the variation in microbial diversity observed between different waterworks sand filters could be explained by the geochemistry of the inlet water. In addition, our findings suggested four features of particular interest: (1) Nitrospira dominated over Nitrobacter at all waterworks, suggesting that Nitrospira is a key nitrifying bacterium in groundwater-treating sand filters. (2) Hyphomicrobiaceae species were abundant at all waterworks, where they may be involved in manganese oxidation. (3) Six of 11 waterworks had significant concentrations of methane in their raw water and very high abundance of the methanotrophic Methylococcaceae. (4) The iron-oxidizing bacteria Gallionella was present at all waterworks suggesting that biological iron oxidation is occurring in addition to abiotic iron oxidation. Elucidation of key members of the microbial community in groundwater-treating sand filters has practical potential, for example, when methods are needed to improve filter function.

We describe two novel flagellates isolated from soil, Regin rotiferus and Otto terricolus, genera et species novae, which we cultivated and characterized by light and transmission electron microscopy and by 18S rDNA sequence analysis. Both strains exhibit the key characteristic structural feature of Bicosoecida; i.e. the L-shaped cytostomal root system with an x-fiber, used for feeding. Otto terricolus displays unique novel morphological traits; thus, it has a basal swelling on each flagellum, a root 3/root 2 distribution of 10 + 1 microtubules, and an amoeboid stage in its life cycle. Regin rotiferus has flagella without swellings and a root 3/root 2 distribution of 7 + 3 microtubules, a pattern commonly observed in the Bicosoecida. We present an updated exhaustive maximum likelihood phylogeny of 48 cultured, complete or nearly complete (+1600 bp) 18S rDNA Bicosoecida sequences. Both new species fall into a well-supported freshwater Siluaniidae clade, without being particularly closely related. The morphology and phylogeny do not conclusively support Rictus as a member of Bicosoecida.

Isotope fractionation is a ubiquitous phenomenon in natural ecosystems. When chemical elements move through food chains, natural isotope ratios change because biological processes tend to discriminate against heavier isotopes. This effect can be used to trace flows of matter, estimate process-rates and determine the trophic level of organisms in biological systems. While it is widely accepted that 15N-accumulates in natural food-chains, it is disputed to which extent this is the case for 13C. We constructed sand-microcosms inoculated with a dilution series of soil organisms and amended with glucose as the source of organic carbon. We demonstrated that the proportion of 13C in respiratory CO2 correlated inversely with community complexity. Our results therefore suggest that increasing community complexity, with increasing synergy, competition and predation, facilitates increasing 12C–13C isotopic fractionation.

Phylogenetic analyses of Mycena sect. Calodontes using ITS previously suggested ten cryptic monophyletic ITS lineages within the Mycena pura morphospecies. Here, we compare ITS data (645 bp incl. gaps) from 46 different fruit bodies that represent the previously described ITS diversity with partial tEF-1-α (423 bp) and RNA polymerase II (RPB1) (492 bp) sequence data to test the genealogical concordance. While neither of the markers were in complete topological agreement, the branches differing between the tEF and RPB1 trees had a low bootstrap (

We grew plants in soil amended with either finely ground ( 2 ) of maize leaves. Finely ground material will be mineralized faster; hence nitrogen is released faster in the soil. We inoculated the soil with the plant growth inhibiting bacterium Arthrobacter BEM463, a relatively poor food source for bacterial feeders, and the plant growth promoter Sphingopyxis BEM760 which is a good food source. Protozoa and nematodes were introduced separately or in combination. In soil with larger maize leaf pieces (low turnover, slow nitrogen release), bacterial feeders generally increased plant nitrogen uptake and growth. In systems with finely ground material (high turnover, fast nitrogen release), plants hardly benefited from presence of grazers; bacterial mineralization activity alone probably provided enough N for the plants. The harmful Arthrobacter thrived better when grazed by protozoa than when grazed by nematodes, probably because the selective protozoa could avoid it, which resulted in reduced plant performance. We suggest that bacterial feeders generally increase plant performance when nutrients are limited. At high nutrient availability, however, bacterial feeders promote grazer resistant bacteria, and, if these bacteria are harmful to plants, the net effect of bacterial feeders on plant growth can be negative.

Lundholm N., Ribeiro S., Andersen T.J., Koch T., Godhe A., Ekelund F. and Ellegaard M. 2011. Buried alive – germination of up to a century-old marine protist resting stages. Phycologia 50: 629–640. DOI: 10.2216/11-16.1 We report on the survival and germination of up to a century-old marine protist resting stages naturally preserved in sediments from Koljo Fjord on the west coast of Sweden. This work has focused on germination of dinoflagellate cysts, but diatom resting stages were also observed. We record the longest known survival of dormant dinoflagellate cells. We individually isolated more than 1200 cysts of the three most abundant dinoflagellate taxa: Pentapharsodinium dalei, Lingulodinium polyedrum and Scrippsiella spp. Germination success decreased with core depth, and all successful germinations took place within the first 2 wk of incubation. Pentapharsodinium dalei had the highest germination success rate, with a maximum of up to 80% in 28-yr-old sediment, and could successfully germinate from co...

Different features can protect bacteria against protozoan grazing, for example large size, rapid movement, and production of secondary metabolites. Most papers dealing with these matters focus on bacteria. Here, we describe protozoan features that affect their ability to grow on secondary-metabolite-producing bacteria, and examine whether different bacterial secondary metabolites affect protozoa similarly. We investigated the growth of nine different soil protozoa on six different Pseudomonas strains, including the four secondary-metabolite-producing Pseudomonas fluorescens DR54 and CHA0, Pseudomonas chlororaphis MA342 and Pseudomonas sp. DSS73, as well as the two nonproducers P. fluorescens DSM50090T and P. chlororaphis ATCC43928. Secondary metabolite producers affected protozoan growth differently. In particular, bacteria with extracellular secondary metabolites seemed more inhibiting than bacteria with membrane-bound metabolites. Interestingly, protozoan response seemed to correlate with high-level protozoan taxonomy, and amoeboid taxa tolerated a broader range of Pseudomonas strains than did the non-amoeboid taxa. This stresses the importance of studying both protozoan and bacterial characteristics in order to understand bacterial defence mechanisms and potentially improve survival of bacteria introduced into the environment, for example for biocontrol purposes.

Kinetoplastids are a large group of free-living and parasitic eukaryotic flagellates, including the medically important trypanosomatids (e.g., Trypanosoma and Leishmania) and the widespread free-living and parasitic bodonids. Small subunit rRNA- and conserved protein-based phylogenies support the division of kinetoplastids into five orders (Prokinetoplastida, Neobodonida, Parabodonida, Eubodonida, and Trypanosomatida), but they produce incongruent results regarding their relative branching order, in particular for the position of the Trypanosomatida. In general, small subunit rRNA tends to support their early emergence, whereas protein phylogenies most often support a more recent origin from within bodonids. In order to resolve this question through a phylogenomic approach, we carried out massive parallel sequencing of cDNA from representatives of three bodonid orders (Bodo saltans -Eubodonida-, Procryptobia sorokini -Parabodonida-, and Rhynchomonas nasuta -Neobodonida-). We identified 64 well-conserved proteins shared by these species, four trypanosomatids, and two closely related outgroup species (Euglena gracilis and Diplonema papillatum). Phylogenetic analysis of a concatenated data set yielded a strongly supported tree showing the late emergence of trypanosomatids as a sister group of the Eubodonida. In addition, we identified homologues of proteins involved in trypanosomatid mitochondrial mRNA editing in the three bodonid species, suggesting that editing may be widespread in kinetoplastids. Comparison of expressed sequences from mitochondrial genes showed variability at U positions, in agreement with the existence of editing activity in the three bodonid orders most closely related to trypanosomatids (Neobodonida, Parabodonida, and Eubodonida). Mitochondrial mRNA editing appears to be an ancient phenomenon in kinetoplastids.

We know little about the ability of protozoa to colonize soils, including their successional patterns. To elucidate this issue, we investigated in which order different protozoan morpho-types colonize sterile soil. We used sterilized soils with different carbon content, and exposed them to the atmosphere for a period of 14 months. Bacteria and flagellates occurred in high numbers after 25 days. Ciliates reached high abundance levels after 137 days, followed by amoebae after 245 days. Numbers of distinguishable protozoan morpho-types increased until 245 days after exposure and declined thereafter. DGGE bands showed a remarkable shift in bacterial diversity with time and a significant increase of detectable gene-types until day 137.

Cercozoa are abundant free-living soil protozoa and quantitatively important in soil food webs; yet, targeted high-throughput sequencing (HTS) has not yet been applied to this group. Here we describe the development of a targeted assay to explore Cercozoa using HTS, and we apply this assay to measure Cercozoan community response to drought in a Danish climate manipulation experiment (two sites exposed to artificial drought, two unexposed). Based on a comparison of the hypervariable regions of the 18S ribosomal DNA of 193 named Cercozoa, we concluded that the V4 region is the most suitable for group-specific diversity analysis. We then designed a set of highly specific primers (encompassing ~270 bp) for 454 sequencing. The primers captured all major cercozoan groups; and >95% of the obtained sequences were from Cercozoa. From 443 350 high-quality short reads (>300 bp), we recovered 1585 operational taxonomic units defined by >95% V4 sequence similarity. Taxonomic annotation by phylogeny enabled us to assign >95% of our reads to order level and ~85% to genus level despite the presence of a large, hitherto unknown diversity. Over 40% of the annotated sequences were assigned to Glissomonad genera, whereas the most common individually named genus was the euglyphid Trinema. Cercozoan diversity was largely resilient to drought, although we observed a community composition shift towards fewer testate amoebae.

The existing data on the molecular phylogeny of filose testate amoebae from order Euglyphida has revealed contradictions between traditional morphological classification and SSU rRNA phylogeny and, moreover, the position of several important genera remained unknown. We therefore carried out a study aiming to fill several important gaps and better understand the relationships among the main euglyphid testate amoebae and the evolutionary steps that led to the present diversity at a higher level. We obtained new SSU rRNA sequences from five genera and seven species. This new phylogeny obtained shows that (1) the clade formed by species of genera Assulina and Placocista branches unambiguously at the base of the subclade of Euglyphida comprising all members of the family Trinematidae and genus Euglypha, (2) family Trinematidae (Trachelocorythion, Trinema, and Corythion) branches as a sister group to genus Euglypha, (3) three newly sequenced Euglypha species (E. cf. ciliata, E. penardi, and E. compressa) form a new clade within the genus. Since our results show that Assulina and Placocista do not belong to the Euglyphidae (unless the Trinematidae are also included in this family), we propose the creation of a new family named Assulinidae. Consequently, we give a family status to the genera Euglypha and (tentatively) Scutiglypha, which become the new family Euglyphidae. The evolutionary pattern suggested by SSU rRNA phylogeny shows a clear tendency towards increasing morphological complexity of the shell characterised by changes in the symmetry (migration of the aperture to a ventral position and/or compression of the shell) and the appearance of specialised scales at the aperture (in families Trinematidae and Euglyphidae).

We compared the abundance and diversity of cultivable protozoa (flagellates and amoebae) in a polycyclic aromatic hydrocarbon (PAH) polluted soil and an unpolluted control, by isolating and cultivating clonal strains. The number of cultivable protozoa was higher in the polluted soil; however, the polluted soil displayed an impoverished community, dominated by certain taxa, such as Acanthamoeba sp. We isolated a total of 31 protozoan strains to characterize them morphologically and by 18S rRNA gene sequence analysis. This approach, i.e. combining morphological and molecular information had the advantage of providing quantitative data, information on morphology but also an accurate positioning of the isolates in 18S rRNA trees.

Pseudomonas bacteria isolated during 52 days on Gould's S1 agar from soil spiked with 0, 3.5 and 15 mg Hg(II) kg soil−1 were characterised to reveal whether mercury affected them differently. Isolates from the treatments with 0 and 15 mg Hg kg−1 were characterised using FT-IR characterisation and subsequent 16S rDNA partial sequencing of representative isolates. To verify the selectivity of Gould's S1 agar and the FT-IR characterisation, all 450 isolates were subjected to the following tests: Gram-determination, catalase and oxidase activity, pigment production on PDA and growth at different temperatures. Furthermore, the isolates were tested for their ability to grow on agar amended with 10 mg Hg kg−1 as an indication of mercury resistance. We found that up to 80% of the isolates in soil amended with 15 mg Hg kg−1 were mercury-resistant, whereas only up to 20% were resistant in the treatments with 0 and 3.5 mg Hg kg−1. We found two groups of Pseudomonas, which probably represent non-described species since they did not group closely with any known species of Pseudomonas in the dendrogram. Hg-enhanced isolates were closely related to P. frederiksbergensis. Furthermore, Hg resistance was almost exclusively restricted to P. frederiksbergensis and P. migulae groups. We conclude that Hg caused a shift in the dominating species of culturable Pseudomonas.

The morpho species Bodo designis is widespread and abundant globally in highly contrasting terrestrial and aquatic ecosystems. Whether the forms of Bodo designis from contrasting environments are conspecific, i.e. largely genetically identical, or whether they merely share the external morphology is presently not known. We examined the ability of different strains of Bodo designis isolated from different environments at different geographical sites to survive and grow at a salinity range of 0.5-45%. The Bodo designis strains from marine, freshwater, and terrestrial environments showed a different ability to cope with altered physiological conditions. Most of the tested strains were only able to tolerate a small salinity range, whereas others were able to withstand all tested salinity levels. We further examined the phylogenetic relationship between the different strains by sequencing the small subunit (SSU) rRNA gene. The resulting phylogenetic analyses suggest a huge genetic variation within Bodo designis, and also imply that Dimastigella and Rhyncomonas are developed inside Bodo designis. If the biological species concept is used, the genetic differences as well as the physiological barriers between the different strains of Bodo designis, would suggest that they should be assigned to different species.