Identification of microbial communities that assimilate substrate from root cap cells in an aerobic soil using a DNA-SIP approach

Although root cap cells are an important substrate for microorganisms in the rhizosphere, little attention has been paid to the decomposition of sloughed root cap cells by microorganisms. This study used rice plant callus cells grown on medium containing ¹³C-labelled glucose as a model material for rice plant root cap cells. Harvested ¹³C-labelled callus cells (78 atom % ¹³C) were subjected to decomposition in an aerobic soil microcosm for 56 days. The low cellulose and lignin levels and the disaggregated nature of the callus cells indicated that these cells were an appropriate model material for root cap cells. DNA was extracted from a soil incubated with ¹²C- and ¹³C-callus cells and subjected to buoyant density gradient centrifugation to identify bacterial species that assimilated carbon from the callus cells. The stability of the total bacterial communities during the incubation was estimated. Many DGGE bands in light fractions of soil incubated with ¹³C-callus cells were weaker in intensity than those from soil incubated with ¹²C-callus cells, and those bands were shifted to heavier fractions after ¹³C-callus treatment. ¹³C-labelled DNA was detected from Day 3 onwards, and the DGGE bands in the heavy fractions were most numerous on Day 21. DGGE bands from heavy and light fractions were sequenced, revealing more than 70% of callus- C incorporating bacteria were Gram-negative, predominantly α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Sphingobacteria and Actinobacteria. These species were phylogenetically distinct from the bacteria reported to be present during plant residue decomposition and resident in rice roots. This study indicates that root cap cells are decomposed by specific bacterial species in the rhizosphere, and that these species augment the diversity of rhizospheric bacterial communities.     

Impact of incorporated fresh 13C potato tissues on the bacterial and fungal community composition of soil

Soil microbial communities play a major role in organic matter decomposition, however the importance of the individual species involved is still unclear. To identify the dynamics and identity of bacterial species involved in decomposition of potato tissue as well as the assimilation of carbon from fresh plant material, ¹³C-labeled green potato tissues (¹³C 99.2%) were incorporated in soil microcosm for 39 days at the level of 2.5% (w/dry weight soil). The DNA was extracted from the soil after 1, 6, 15, 25 and 39 days. The heavy (¹³C) and light (¹²C) fractions of DNA were separated by ultracentrifugation and the structures of the bacterial and fungal communities were characterized by DGGE. Primary and secondary ¹³C-sequestrators were identified by sequencing DGGE bands that had appeared only in the heavy DNA fractions. Over the course of the experiment, the most dominant ¹³C-labeled phylogenetic group (class or phylum) was γ-Proteobacteria (51.4%), followed by Actinobacteria (27%), β-Proteobacteria (8.1%) and α-Proteobacteria (5.4%). Two taxa, namely Firmicutes and Verrucomicrobia, were represented by just one sequence type. These bacterial taxa were differentiated into primary (Arthrobacter, Pseudomonas) and secondary sequestrators (Actinobacteria, Dyella, Mesorhizobium and Sphingomonas). The latter were possibly involved in either the redistribution of previously consumed carbon or in a possible degradation of the more complex plant compounds. On the basis of this analysis, only 5 to 8 bacterial taxa were involved in carbon sequestration at any one measured time point. Our results show the importance of specific microbial taxa in the decomposition and mineralization of plant residues in soil, which will allow us to better understand the role of such communities in carbon cycling.     

Bacterial community incorporating carbon derived from plant residue in an anoxic non-rhizosphere soil estimated by DNA-SIP analysis

Purpose

Plant residues are one of the main sources of soil organic matter in paddy fields, and elucidation of the bacterial communities decomposing plant residues was important to understand their function and roles, as the microbial decomposition of plant residues is linked to soil fertility. We conducted a DNA stable isotope probing (SIP) experiment to elucidate the bacterial community assimilating 13-carbon (¹³C) derived from plant residue under an anoxic soil condition. In addition, we compared the bacterial community with that under the oxic soil condition, which was elucidated in our previous study (Lee et al. in Soil Biol Biochem 43:814–822, 2011).

Materials and methods

We used the ¹³C-labeled dried rice callus cells as a model of rice plant residue. A paddy field soil was incubated with unlabeled and ¹³C-labeled callus cells. DNA extracted from the soils was subjected to buoyant density gradient centrifugation to fractionate ¹³C-enriched DNA. Then, polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rDNA band patterns and band sequencing method were used to evaluate bacterial community.

Results and discussion

DGGE analysis showed that the band patterns in the ¹³C-enriched fractions were distinctly changed over time, while the changes in the community structure before fractionation were minor. Sequencing of the ¹³C-labeled DGGE bands revealed that Clostridia were a major group in the bacterial communities incorporating the callus-derived carbon although Gram-negative bacteria, and Actinobacteria also participated in the carbon flow from the callus under the anoxic condition. The proportion of Gram-negative bacteria and Actinobacteria increased on 14 days after the onset of incubation, suggesting that the callus was decomposed by diverse bacterial members on this phase. When the bacterial groups incorporating the ¹³C were compared between under anoxic and oxic soil conditions, the composition was largely different under the two opposite conditions. However, some members of Gram-negative bacteria were commonly found under the anoxic and oxic soil conditions.

Conclusions

The majority of bacterial members assimilating the callus carbon was Clostridia in the soil under anoxic conditions. However, several Gram-negative bacterial members, such as Acidobacteria, Bacteroidetes, and Proteobacteria, also participated in the decomposition of callus under anoxic soil conditions. Our study showed that carbon flow into the diverse bacterial members during the callus decomposition and the distinctiveness of the bacterial communities was formed under the anoxic and oxic soil conditions.

     

In-situ enrichment and analysis of atrazine-degrading microbial communities using atrazine-containing porous beads

We examined the community composition of microbes that colonized atrazine-containing beads buried in agricultural soils that differed in atrazine treatment history. Bacterial abundance was 5-40-fold greater in atrazine-fortified beads. In beads containing 20 mg atrazine kg⁻¹ buried in soil with a history of atrazine application (conditioned soil), the abundance of Actinobacteria increased approximately 80-fold whereas in control soil, Actinobacteria were enriched only 10-fold and the gamma-Proteobacteria and Planctomycetes increased by 60- and 25-fold, respectively. The gamma-Proteobacteria were enriched by 120- and 230-fold in beads containing 200 mg atrazine kg⁻¹ in conditioned and control soil, respectively. The results demonstrate that BioSep^® beads are a suitable matrix for recruiting a diverse subset of the bacterial community involved in atrazine degradation.     

Impacts of different N management regimes on nitrifier and denitrifier communities and N cycling in soil microenvironments

Real-time quantitative PCR assays, targeting part of the ammonia monooxygenase (amoA), nitrous oxide reductase (nosZ), and 16S rRNA genes were coupled with ¹⁵N pool dilution techniques to investigate the effects of long-term agricultural management practices on potential gross N mineralization and nitrification rates, as well as ammonia-oxidizing bacteria (AOB), denitrifier, and total bacterial community sizes within different soil microenvironments. Three soil microenvironments [coarse particulate organic matter (cPOM; >250 μm), microaggregate (53-250 μm), and silt-and-clay fraction (<53 μm)] were physically isolated from soil samples collected across the cropping season from conventional, low-input, and organic maize-tomato systems (Zea mays L.-Lycopersicum esculentum L.). We hypothesized that (i) the higher N inputs and soil N content of the organic system foster larger AOB and denitrifier communities than in the conventional and low-input systems, (ii) differences in potential gross N mineralization and nitrification rates across the systems correspond with AOB and denitrifier abundances, and (iii) amoA, nosZ, and 16S rRNA gene abundances are higher in the microaggregates than in the cPOM and silt-and-clay microenvironments. Despite 13 years of different soil management and greater soil C and N content in the organic compared to the conventional and low-input systems, total bacterial communities within the whole soil were similar in size across the three systems (∼5.15 × 10⁸ copies g⁻¹ soil). However, amoA gene densities were ∼2 times higher in the organic (1.75 × 10⁸ copies g⁻¹ soil) than the other systems at the start of the season and nosZ gene abundances were ∼2 times greater in the conventional (7.65 × 10⁷ copies g⁻¹ soil) than in the other systems by the end of the season. Because organic management did not consistently lead to larger AOB and denitrifier communities than the other two systems, our first hypothesis was not corroborated. Our second hypothesis was also not corroborated because canonical correspondence analyses revealed that AOB and denitrifier abundances were decoupled from potential gross N mineralization and nitrification rates and from inorganic N concentrations. Our third hypothesis was supported by the overall larger nitrifier, denitrifier, and total bacterial communities measured in the soil microaggregates compared to the cPOM and silt-and-clay. These results suggest that the microaggregates are microenvironments that preferentially stabilize C, and concomitantly promote the growth of nitrifier and denitrifier communities, thereby serving as potential hotspots for N₂O losses.     

Pyrosequencing and mid-infrared spectroscopy reveal distinct aggregate stratification of soil bacterial communities and organic matter composition

This study integrated physical, chemical, and molecular techniques to assess relationships between soil bacterial community structures and the quantity and quality of soil organic carbon (SOC) at the soil microenvironment scale (e.g., within different aggregate size-fractions). To accomplish this goal, soil samples (0–5 cm) were collected from the Texas High Plains region under a variety of dryland and irrigated cropping systems. The soil was separated into macroaggregates, microaggregates, and silt + clay fractions that were analyzed for (1) bacterial diversity via pyrosequencing of the 16s rRNA gene and (2) SOC quantity and quality using a combustion method and mid-infrared diffuse reflectance spectroscopy (mid-IR), respectively. Results from pyrosequencing showed that each soil microenvironment supported a distinct bacterial community. Similarly, mid-IR data revealed distinct spectral features indicating that these fractions were also distinguished by organic and mineral composition. Macroaggregates showed relatively high abundance of Actinobacteria (excluding order Rubrobacteriales) and α-Proteobacteria and contained the most SOC. Microaggregates showed high relative abundance of Rubrobacteriales and the least amount of SOC. Predominance within the soil microenvironment and correlations along the mid-IR spectra were different between members of the order Rubrobacteriales compared with all other members of the Actinobacteria phyla, suggesting they have different ecological niches. Mid-IR results revealed microaggregates had greater absorbance in the 1370–1450 cm^?1 region for phenolic and alkyl groups (possibly recalcitrant C). Silt + clay fractions were distinguished by Gemmatimonadetes and OP10 phyla, which positively correlated with spectral absorption in the1250–1150 cm^?1 range (indicating both degradable and recalcitrant C forms). In contrast to general diversity index measurements, distributions of the more rare bacterial phyla (phyla representing <6% of the identified population) were more important for differentiating between communities in soil microenvironments. To our knowledge, this is the first study to investigate soil bacterial communities among soil aggregates using pyrosequenging and to associate these communities to specific soil C chemistries as indicated by mid-IR absorbance.     

Influence of earthworm activity and rice straw application on the soil microbial community structure analyzed by PLFA pattern

The earthworm Pheretima hilgendorfi, one of the most common anecic species in Japan, abounds in soils with applied rice-straw residues. The influences of the worm’s activity and/or those of rice-straw application on the soil microbial community structure were studied using a microcosm approach. Low Humic Andosol was incubated with or without earthworms in a jar for a month after the following treatments: 1) no treatment, 2) chopped rice straw top-dressed on the soil and 3) rice straw incorporated into the soil. The soil NO₃^–-N level increased with the earthworms’ presence even in the soil without rice straw. The soil NH₄⁺-N level was by far the highest in the soil treated with worms and no rice straw. Amounts of total and bacterial PLFAs increased due to the earthworms’ presence when rice straw was incorporated. The proportion of unsaturated fatty acids increased in the earthworm treatment while the saturated fatty acids decreased, suggesting an increase in the Gram-negative bacterial proportion. The results of principal component analysis indicated that the rice straw and the earthworms affected the soil microbial community structure independently. Any direct influence of the PLFAs contained in the worms’ bodies and in the rice straw on the soil’s PLFA profile was thought to be small.     

Structural differences in the rhizosphere communities of legumes are not equally reflected in community-level physiological profiles

The relationship of structural diversity and differences in the functional potentials of rhizosphere communities of alfalfa, common bean and clover was investigated in microcosms. PCR-SSCP (single strand conformation polymorphism) analysis of 16S rRNA genes revealed significant differences in the composition of the leguminous rhizosphere communities at the shoot stage of plants grown in the same soil. Sequencing of dominant SSCP-bands indicated the presence of plant specific organisms. The partial rRNA gene sequences were related to members of the α- and γ-Proteobacteria, Bacteroidetes and Actinobacteria. Besides the plant species, the soil also affected the structural diversity in rhizospheres. The dominant bacterial populations of alfalfa grown in soils with different agricultural histories were assigned to different taxonomic groups. Addressing the functional potentials, community-level physiological profiles (CLPP) were generated using BIOLOG GN^®. The three leguminous rhizosphere communities could be differentiated by principle component analysis, though the overall analysis indicated that the metabolic potential of all rhizosphere samples was similar. The functional variation examined in rhizospheres of alfalfa was minor in response to the soil origin and was found not to be significant different at different growth stages. The results indicate that similar functional potentials may be provided by structurally different bacterial communities.     

Re-establishment of suppressiveness to soil- and air-borne diseases by re-inoculation of soil microbial communities

The aim of this study was to investigate the potentials and limitations in restoring soil suppressiveness in disturbed soils. Soils from three sites in UK and Switzerland (STC, REC, THE) differing in their level of suppressiveness to soil-borne and air-borne diseases were γ-irradiated and this soil matrix was re-inoculated with 1% (w/w) of either parent native soil or native soil from the other sites (‘soil inoculum’). Suppressiveness to air-borne and soil-borne diseases was quantified by means of the host-pathogen systems Lepidium sativum (cress)-Pythium ultimum, an oomycete causing root rot and seedling damping-off, and Arabidopsis thaliana-Hyaloperonospora parasitica, an oomycete causing downy mildew. Soil microbial biomass, activity and community structure, as determined by phospholipid fatty acid (PLFA) profiles, were measured in native, γ-irradiated, and re-inoculated soils. Both, L. sativum and A. thaliana were highly susceptible to the pathogens if grown on γ-irradiated soils. Re-inoculation completely restored suppressiveness of soils to the foliar pathogen H. parasitica, independently of soil matrix or soil inoculum, whereas suppressiveness to P. ultimum depended on the soil matrix and, to a lesser extent, on the soil inoculum. However, the soil with the highest inherent suppressiveness did not reach the initial level of suppressiveness after re-inoculation. In addition, native microbial populations as defined by microbial biomass, activity and community structure, could not be fully restored in re-inoculated soils. As for suppressiveness to P. ultimum, the soil matrix, rather than the source of soil inoculum was identified as the key factor for re-establishing the microbial community structure. Our data show that soils do not or only slowly fully recover from sterilisation by γ-irradiation, indicating that agricultural soil management practices such as soil fumigation or heat treatments frequently used in vegetable cropping should be avoided.     

Fate of gram-negative bacterial biomass in soil—mineralization and contribution to SOM

Soil microorganisms contribute to the formation of non-living soil organic matter (SOM) by metabolic transformation of plant-derived material. After cell death, their biomass components with a specific molecular character become incorporated into SOM imprinting its chemical properties, although this process has not yet been quantified. In order to elucidate the contribution to SOM formation, we investigated the fate of gram-negative bacterial model biomass (Escherichia coli usually introduced into soil with manure or feces) during incubation of soil with isotopically (¹³C) and genetically (lux gene) labeled cells. The decline of living cells was monitored by the loss of bioluminescence. The carbon turnover and mineralization was balanced by bulk soil stable isotope analysis, and the persistence of nucleic acids was investigated by PCR amplification of the lux gene. During incubation, the number of viable E. coli cells decreased rapidly (99.9% within the first 42 d) serving as substrate for other microorganisms or for the formation of SOM, and bioluminescent cells could only be detected during the first 56 d. However, the lux gene was still detected after 224 d, which indicates stabilization of DNA in SOM. Although the survival of E. coli in soil is limited, only about 65% of the added labeled biomass carbon was mineralized to ¹³CO₂ and 51% remained in soil after 224 d with an average ¹³C recovery of 117%. The amount of ¹³C found in the PLFA representative of living cells had decreased to 25% of the initial value, suggesting a proportional decrease of the ¹³C in the soil microbial biomass. The extent of this decrease is higher than the mineralization of the bulk E. coli C and thus the difference of around 25% has to be stabilized as metabolites, or in non-living SOM. The data provide evidence that the genetic information and a considerable part of the carbon from dying bacterial biomass were retained in both the soil microbial food web and in non-living SOM.     

Rhizosphere priming of soil organic matter by bacterial groups in a grassland soil

Plants often impact the rate of native soil organic matter turnover through root interactions with soil organisms; however the role of root-microbial interactions in mediation of the “priming effect” is not well understood. We examined the effects of living plant roots and N fertilization on belowground C dynamics in a California annual grassland soil (Haploxeralf) during a two-year greenhouse study. The fate of ¹³C-labeled belowground C (roots and organic matter) was followed under planted (Avena barbata) and unplanted conditions, and with and without supplemental N (20 kg N ha⁻¹ season⁻¹) over two periods of plant growth, each followed by a dry, fallow period of 120 d. Turnover of belowground ¹³C SOM was followed using ¹³C-phospholipid fatty acid (PLFA) biomarkers. Living roots increased the turnover and loss of belowground ¹³C compared with unplanted soils. Planted soils had 20% less belowground ¹³C present than in unplanted soils after 2 cycles of planting and fallow. After 2 treatment cycles, unlabeled soil C was 4.8% higher in planted soils than unplanted. The addition of N to soils decreased the turnover of enriched belowground ¹³C during the first treatment season in both planted and unplanted soils, however no effect of N was observed thereafter. Our findings suggest that A. barbata may increase soil C levels over time because root and exudate C inputs are significant, but that increase will be moderated by an overall faster C mineralization rate of belowground C. N addition may slow soil C losses; however, the effect was minor and transient in this system. The labeled root-derived ¹³C was initially recovered in gram negative (highest enrichment), gram positive, and fungal biomarkers. With successive growing seasons, the labeled C in the gram negative and fungal markers declined, while gram positive markers continued to accumulate labeled belowground C. The rhizosphere of A. barbata shifted the microbial community composition, resulting in greater abundances of gram negative markers and lower abundances of gram positive, actinobacteria and cyclopropyl PLFA markers compared to unplanted soil. However, the longer-term utilization of labeled belowground C by gram positive bacteria was enhanced in the rhizosphere microbial community compared with unplanted soils. We suggest that the activities of gram positive bacteria may be major controllers of multi-year rhizosphere-related priming of SOM decomposition.     

Modeling aerobic decomposition of rice straw during the off-rice season in an Andisol paddy soil in a cold temperate region of Japan: Effects of soil temperature and moisture

Submerged rice paddies are a major source of methane (CH₄) which is the second most important greenhouse gas after carbon dioxide (CO₂). Accelerating rice straw decomposition during the off-rice season could help to reduce CH₄ emission from rice paddies during the single rice-growth season in cold temperate regions. For understanding how both temperature and moisture can affect the rate of rice straw decomposition during the off-rice season in the cold temperate region of Tohoku district, Japan, a modeling incubation experiment was carried out in the laboratory. Bulk soil and soil mixed with 2% of δ¹³C-labeled rice straw with a full factorial combination of four temperature levels (?5 to 5, 5, 15, 25°C) and two moisture levels (60% and 100% WFPS) were incubated for 24 weeks. The daily change from ?5 to 5°C was used to model the freezing–thawing cycles occurring during the winter season. The rates of rice straw decomposition were calculated by (i) CO₂ production; (ii) change in the soil organic carbon (SOC) content; and (iii) change in the δ¹³C value of SOC. The results indicated that both temperature and moisture affected the rate of rice straw decomposition during the 24-week aerobic incubation period. Rates of rice straw decomposition increased not only with high temperature, but also with high moisture conditions. The rates of rice straw decomposition were more accurately calculated by CO₂ production compared to those calculated by the change in the SOC content, or in its δ¹³C value. Under high moisture at 100% WFPS condition, the rates of rice straw decomposition were 14.0, 22.2, 33.5 and 46.2% at ?5 to 5, 5, 15 and 25°C temperature treatments, respectively. While under low moisture at 60% WFPS condition, these rates were 12.7, 18.3, 31.2 and 38.4%, respectively. The Q₁₀ of rice straw decomposition was higher between ?5 to 5 and 5°C than that between 5 and 15°C and that between 15 and 25°C. Daily freezing–thawing cycles (from ?5 to 5°C) did not stimulate rice straw decomposition compared with low temperature at 5°C. This study implies that to reduce CH₄ emission from rice paddies during the single rice-growth season in the cold temperate regions, enhancing rice straw decomposition during the high temperature period is very important.     

Incorporation of C-labeled rice-straw-derived carbon into microbial communities in submerged rice field soil and percolating water

Rice straw is a major organic material applied to rice fields. The microorganisms growing on rice-straw-derived carbon have not been well studied. Here, we applied ¹³C-labeled rice straw to submerged rice soil microcosms and analyzed phospholipid fatty acids (PLFAs) in the soil and percolating water to trace the assimilation of rice-straw-derived carbon into microorganisms. PLFAs in the soil and water were markedly enriched with ¹³C during the first 3 days of incubation, which indicated immediate incorporation of rice-straw-derived carbon into microbial biomass. The enrichment of PLFAs in the percolating water with ¹³C suggested that microorganisms other than the population colonizing rice straw also assimilated rice-straw-derived carbon or that some bacterial groups were selectively released from the straw. The microbial populations could be categorized into two communities based on the carbon isotope data of the PLFAs: those derived from rice straw and those derived from soil organic matter (SOM). The composition of the PLFAs from the two communities differed, which indicated the assimilation of rice-straw-derived carbon by a subset of microbial populations. The composition of rice-straw-derived PLFAs in the percolating water was also distinct from that in the soil.     

Endogeic earthworms alter carbon translocation by fungi at the soil-litter interface

The effect of endogeic earthworms (Octolasion tyrtaeum (Savigny)) on the translocation of litter-derived carbon into the upper layer of a mineral soil by fungi was investigated in a microcosm experiment. Arable soil with and without O. tyrtaeum was incubated with ¹³C/¹⁵N-labelled rye leaves placed on plastic rings with gaze (64 μm mesh size) to avoid incorporation of leaves by earthworms. The plastic rings were positioned either on or 3 cm above the soil surface, to distinguish between biotic and chemical/physical translocation of nutrients by fungi and leaching.Contact of leaves to the soil increased ¹³C translocation, whereas presence of O. tyrtaeum reduced the incorporation of ¹³C into the mineral soil in all treatments. Although biomass of O. tyrtaeum decreased during the experiment, more ¹³C and ¹⁵N was incorporated into earthworm tissue in treatments with contact of leaves to the soil. Contact of leaves to the soil and the presence of O. tyrtaeum increased cumulative ¹³CO₂-C production by 18.2% and 14.1%, respectively.The concentration of the fungal bio-indicator ergosterol in the soil tended to be increased and that of the fungal-specific phospholipid fatty acid 18:2ω6 was significantly increased in treatments with contact of leaves to the soil. Earthworms reduced the concentration of ergosterol and 18:2ω6 in the soil by 14.0% and 43.2%, respectively. Total bacterial PLFAs in soil were also reduced in presence of O. tyrtaeum, but did not respond to the addition of the rye leaves. In addition, the bacterial community in treatments with O. tyrtaeum differed from that without earthworms and shifted towards an increased dominance of Gram-negative bacteria.The results indicate that litter-decomposing fungi translocate litter-derived carbon via their mycelial network in to the upper mineral soil. Endogeic earthworms decrease fungal biomass by grazing and disruption of fungal hyphae thereby counteracting the fungal-mediated translocation of carbon in soils.     

Changes in soil organic carbon dynamics in an Eastern Chinese coastal wetland following invasion by a C4 plant Spartina alterniflora

The exotic C₄ grass Spartina alterniflora was intentionally introduced to tidal coastal wetlands in Jiangsu province of China in 1982. Since then it has rapidly replaced the native C₃ plant Suaeda salsa, becoming one of the dominant vegetation types in the coastal wetlands of China. Although plant invasion can change soil organic carbon (SOC) storage, little is known about how plant invasion influences C storage within soil fractions. We investigated how S. alterniflora invasion across an 8, 12 and 14-year chronosequence affected SOC and soil nitrogen (N), using soil fractionation and stable δ¹³C isotope analyses. SOC and N concentrations at 0-10 cm depth in S. alterniflora soil increased during the S. alterniflora invasion chronosequence, ranging from 3.67 to 4.90 g C kg⁻¹ soil, and from 0.307 to 0.391 g N kg⁻¹ soil. These were significantly higher than the values in the Suaeda salsa community, by 27.0-69.6% for SOC, and 21.8-55.2% for total N. The S. alterniflora-derived SOC varied from 0.40 to 0.92 g C kg⁻¹ according to mixing calculations, assuming the two possible SOC sources of S. alterniflora and S. salsa, and accounted for 10.8-18.7% of total SOC in the colonized soils. The estimated accumulative rate of SOC from C₄ (S. alterniflora) was 64.1 C kg⁻¹ soil year⁻¹ and from C₃ sources was 78.1 mg C kg⁻¹. The concentration of S. alterniflora-derived SOC significantly decreased from coarse fraction to fine fraction, and linearly increased as the period of S. alterniflora invasion increased. The highest accumulative rate of SOC from a C₄ source occurred in macroaggregates, while the highest rate from C₃ was in microaggregates. The storage of SOC derived from S. alterniflora in the macroaggregates was 0.27-0.44 g C kg⁻¹ soil, accounting for 43.1-49.1% of the total C₄derived SOC in the soil. Our results suggest that S. alterniflora invasion in coastal wetlands could facilitate SOC storage, because of the high potential for accumulation of the C which has been newly derived from S. alterniflora litter and roots.     

Quantification of Wautersia [Ralstonia] basilensis in the mycorrhizosphere of Pinus thunbergii Parl. and its effect on mycorrhizal formation

The bacterium Wautersia [Ralstonia] basilensis has been shown to enhance the mycorrhizal symbiosis between Suillus granulatus and Pinus thunbergii (Japanese black pine). However, no information is available about this bacterium under field conditions. The objectives of this study were to detect W. basilensis in bulk and mycorhizosphere soils in a Japanese pine plantation in the Tottori Sand Dunes, determine the density of W. basilensis in soil, and determine the optimal cell density of W. basilensis for mycorrhizal formation in pine seedlings. We designed and validated 16S rRNA gene-targeted specific primers for detection and quantification of W. basilensis. SYBR Green I real-time PCR assay was used. A standard curve relating cultured W. basilensis cell density (10³-10⁸ cells ml⁻¹) to amplification of DNA showed a strong linear relationship (R = 0.9968). The specificity of the reaction was confirmed by analyzing DNA melting curves and sequencing of the amplicon. The average cell density of W. basilensis was >4.8 × 10⁷ cells g⁻¹ of soil in the mycorrhizosphere and 7.0 × 10⁶ cells g⁻¹ in the bulk soil. We evaluated the W. basilensis cell density required for mycorrhizal formation using an in vitro microcosm with various inoculum densities ranging from 10² to 10⁷ cells g⁻¹ soil (10⁴-10⁹ cells ml⁻¹). Cell densities of W. basilensis of >10⁶ cells g⁻¹ of soil were required to stimulate mycorrhizal formation. In vivo and in vitro experiments showed that W. basilensis was sufficiently abundant to enhance mycorrhizal formation in the mycorrhizosphere of Japanese black pine sampled from the Tottori Sand Dunes.     

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.     

Rhizosphere activity, grass species and N availability effects on the soil C and N cycles

We examined whether grass species and soil nitrogen (N) availability could enhance Carbon (C) and N turnover during root litter decay in grassland. Three species with increasing competitiveness (Festuca ovina, Dactylis glomerata and Lolium perenne) were grown at two N fertiliser levels in an undisturbed grassland soil, in which soil organic fractions derived for the last 9 years from Lolium root litter which was ¹³C-depleted. During the subsequent experimental year, the C turnover was calculated using the respective δ¹³C values of the old and new C in the root phytomass, in two Particulate Organic Matter (POM) fractions above 200 μm and in the lightest part of the aggregated soil fraction between 50 and 200 μm. Soil N availability was monitored during the regrowth periods with ion exchange resins (IER). The C decay rates of each particle size fraction were calculated with a simple mechanistic model of C dynamics. The N mineralisation immobilisation turnover (MIT) was characterised by dilution of ¹⁵N-labelled fertiliser in the N harvestThe C:N ratio and the residence time of C in the fractions decreased with particle size. The presence of a grass rhizosphere increased the decay rate of old C. Accumulation of new C in particle size fractions increased with species competitiveness and with N supply. Species competitiveness increased C turnover in the aggregated fraction, as a result of greater accumulation of new C and faster decay of old C. Fertiliser N increased N turnover and C mineralisation in the SOM. Species competitiveness decreased soil -N exchanged with the IER and increased dissolved organic C (DOC) content. The nature of the current rhizosphere is thus an important factor driving C and N transformations of the old root litter, in relation with grass species strategy. Plant competitiveness may stimulate the C and N turnover in the more evolved SOM fractions in a similar way to the mineral N supply.     

Polyphasic characterization of the bacterial community in an urban soil profile with in situ and culture-dependent methods

Bacterial communities of urban soils have not been thoroughly investigated up to now. Therefore, soil samples from the urban park Tiergarten in the centre of Berlin were taken from a profile in 15, 30 and 90 cm depth. The total number of bacteria (4′,6-diamidino-2-phenylindole (DAPI) counts) as well as biomass declined one order of magnitude from topsoil to subsoil. Soil texture changed comparably and water content and amount of organic matter dropped 3–10-fold. The number of culturable bacteria (colony forming units = CFU) also decreased with increasing soil depth. Amplified ribosomal DNA restriction analysis (ARDRA) revealed similar bacterial communities in the two upper soil layers in contrast to the deepest layer. The number of bacterial cells which were detected with probe EUB338 in relation to total cell counts differed between 43 and 35% in the three soil layers. With the probe active count method (PAC) this number could be increased up to 72% of total cell counts in topsoil whereas activation of cells declined with increasing depth. In relation to total cell counts (DAPI) α-Proteobacteria and β-Proteobacteria are equally distributed in all three depths, whereas γ-Proteobacteria declined within the soil profile. With the BIOLOG system we observed the general trend that the capability of utilizing diverse substrates decreased with soil depth whereas a few substrates, such as Tween 40 and Tween 80 could be utilised by the bacteria of all soil depths.     

Variations in soil microbial biomass and crop roots due to differing resource quality inputs in a tropical dryland agroecosystem

The influence of exogenous organic inputs on soil microbial biomass dynamics and crop root biomass was studied through two annual cycles in rice-barley rotation in a tropical dryland agroecosystem. The treatments involved addition of equivalent amount of N (80 kg N ha⁻¹) through chemical fertilizer and three organic inputs at the beginning of each annual cycle: Sesbania shoot (high-quality resource, C:N 16, lignin:N 3.2, polyphenol+lignin:N 4.2), wheat straw (low-quality resource, C:N 82, lignin:N 34.8, polyphenol+lignin:N 36.8) and Sesbania+wheat straw (high-and low-quality resources combined), besides control. The decomposition rates of various inputs and crop roots were determined in field conditions by mass loss method. Sesbania (decay constant, k=0.028) decomposed much faster than wheat straw (k=0.0025); decomposition rate of Sesbania+wheat straw was twice as fast compared to wheat straw. On average, soil microbial biomass levels were: rice period, Sesbania?Sesbania+wheat straw>wheat straw?fertilizer; barley period, Sesbania+wheat straw>Sesbania?wheat straw?fertilizer; summer fallow, Sesbania+wheat straw>Sesbania>wheat straw?fertilizer. Soil microbial biomass increased through rice and barley crop periods to summer fallow; however, in Sesbania shoot application a strong peak was obtained during rice crop period. In both crops soil microbial biomass C and N decreased distinctly from seedling to grain-forming stages, and then increased to the maximum at crop maturity. Crop roots, however, showed reverse trend through the cropping period, suggesting strong competition between microbial biomass and crop roots for available nutrients. It is concluded that both resource quality and crop roots had distinct effect on soil microbial biomass and combined application of Sesbania shoot and wheat straw was most effective in sustained build up of microbial biomass through the annual cycle.