Growth of hydrogenotrophic and acetoclastic methanogens on substrate from rice plant callus cells in anaerobic soil: an estimation to the role of slough-off root cap cells to their growth

Flooded paddy fields are the major anthropogenic sources of methane (CH₄) emission, and organic materials of rice plant origin were estimated to be important as its source. This study used rice (Oryza sativa L. cv, Yukihikari) callus cells as a model material for slough-off root cap cells, and carbon-13 (¹³C)-labelled callus cells were subjected to decomposition in aerobic and anaerobic soil microcosms for 56 days. DNA was extracted from a soil incubated with carbon-12 (¹²C)- and ¹³C-callus cells and subjected to buoyant density gradient centrifugation to identify methanogenic archaeal species that assimilated carbon from the callus cells. ¹³C-labelled 16S rRNA gene (16S rDNA) fragments from methanogenic archaea were not polymerase chain reaction (PCR)-amplified in heavy fractions under aerobic soil conditions, while they were successfully done from day 3 onwards under anaerobic soil conditions. Eighty-four denaturing gradient gel electrophoresis (DGGE) bands in heavy fractions were sequenced, revealing that they were members of Methanosarcina spp. (20 clones), Methanosaeta spp. (18 clones), Methanocella spp. (25 clones), Methanomicrobiales (10 clones), Methanobacterium spp. (7 clones) and Cluster ZC-I (2 clones). They included hydrogenotrophic and acetoclastic methanogens and were phylogenetically different from those residing in rice roots and, presumably, from those assimilating root exudate and mucilage-derived carbon. This study indicates that carbon of slough-off root cap cells propagates specific methanogenic species in rice rhizosphere under anaerobic soil conditions and thus augments the diversity of the total rhizospheric methanogenic community.     

Bacterial populations assimilating carbon from C-labeled plant residue in soil: Analysis by a DNA-SIP approach

In this study, ¹³C-labeled rice callus was prepared as a model material for rice straw and was subjected to a DNA-SIP (stable isotope probing) experiment in which the bacterial population was monitored in a soil sample containing decomposing dried callus. Rice callus (¹³C = 78%) contained the more water-soluble organic carbon and less cellulose and lignin carbon than rice straw. The callus in the soil was 37% decomposed after 56 d of incubation in upland moisture conditions. PCR-DGGE analysis demonstrated that the bacterial community in the soil with the callus changed over time, showing a distinct difference between the first (up to 7 d) and second (14 d and later) stages. After isopycnic centrifugation, DNA in the fractions with a buoyant density between 1.759 and 1.734 g ml⁻¹ was subjected to population analysis (¹³C-assimilating populations). Diverse groups of bacterial sequences were retrieved from the ¹³C-labeled DNA fractions: Actinobacteria, Bacilli, γ-Proteobacteria, Chloroflexi, Sphingobacteria, Flavobacteria, Clostridia, Acidobacteria, Cyanobacteria and Candidate Division. Bacilli were detected mainly in the first stage, and Actinobacteria were detected throughout the incubation period. Several DGGE bands in the light fractions became more prominent in the soil with callus, which suggested that the addition of callus promoted the growth of bacteria that fed on soil organic matter, including α-Proteobacteria, γ-Proteobacteria, Bacilli, Actinobacteria, Nitrospira and Gemmatimonadetes.     

Abundance and composition of ammonia oxidizers in response to degradation of root cap cells of rice in soil microcosms

Purpose

Nitrification is a key process in the global nitrogen cycle, of which the first and rate-limiting step is catalyzed by ammonia monooxygenase. Root cap cells are one of substrates for microorganisms that thrive in the rhizosphere. The degradation of root cap cells brings about nitrification following ammonification of organic nitrogen derived from the root cap cells. This study was designed to gain insights into the response of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to mineralized N from root cap cells and the composition of active bacterial and archaeal ammonia oxidizers in rice soil.

Materials and methods

Rice callus cells were used as a model for root cap cells, and unlabelled (¹²C) and ¹³C-labelled callus cells were allowed to decompose in aerobic soil microcosms. Real-time quantitative polymerase chain reaction (PCR), DNA-based stable isotope probing (SIP), and denaturing gradient gel electrophoresis (DGGE) were applied to determine the copy number of bacterial and archaeal amoA genes and the composition of active AOB and AOA.

Results and discussion

The growth of AOB was significantly stimulated by the addition of callus cells compared with the growth of AOA with a much lesser extent. AOB communities assimilated ¹³C derived from the callus cells, whereas no AOA communities grew on ¹³C-callus. Sequencing of the DGGE bands in the SIP experiments revealed that the AOB communities belonging to Nitrosospira spp. dominated microbial ammonia oxidation with rice callus amendment in soil.

Conclusions

The present study suggests that root cap cells of rice significantly stimulated the growth of AOB, and the active members dominating microbial ammonia oxidation belonged to Nitrosospira spp. in rice rhizosphere.     

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.

     

Bacterial communities associated with nodal roots of rice plants along with the growth stages: Estimation by PCR-DGGE and sequence analyses

Bacterial communities in rice roots that developed from different nodes and at different growth stages were compared by using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA. Rice root samples were collected at three stages, namely tillering (July 2), maximum tillering (July 21), and ripening (September 12). The bacterial diversity in rice roots was found to increase along with the growth stages of the rice plants as well as the root age from the numbers of DGGE bands. The community structure of the bacteria was also found to change with the growth stages and root age from cluster analysis. Sequence analysis of the DGGE bands indicated that the dominant bacteria associated with rice roots were Gram-negative bacteria, especially β-Proteobacteria irrespective of the growth stages and root age. DGGE bands related to Janthinobacterium agaricidamnosum W1r3T and Clostridium sp. FCB90-3 were ubiquitous in many roots irrespective to the sampling date. Principal component analysis enabled to characterize the DGGE bands related to nitrogen-fixing Azoarcus spp., and Azovibrio sp. BS20-3 in the samples collected on July 2 and on July 21, and the myxobacteria collected on September 12, respectively, as representative bacteria in the bacterial communities. The habitat around older rice roots at every sampling date was more reductive than that around younger rice roots, and the DGGE bands related to Spirochaeta spp. were specific in older roots at every sampling date. Some specific bacteria that were most closely related to the DGGE bands were found from principal component analysis to characterize young and old. roots at each growth stage as follows: aerobes Flavobacterium sp. 90 clone 2 and Janthinobacterium agaricidamnosus W1r3T in young roots and facultative anaerobes Dechloromonas sp. MissR and Anaeromyxobacter dehalogenans 2CP-3 in old nodal roots on July 2, strict anaerobe Geobacter pelophilus Dfr2 and aerobes Nitrosospira sp. Nsp17 and uncultured Nitrospira sp. clone 4-1 in old roots on July 21, and different Clostridium spp. in both young and old roots and Desulfovibrio magneticus RS-1 in old roots on September 12, respectively. A larger number of the closest relatives of anaerobic bacteria grew at the late stage than at the early stages, and in old roots than in younger roots. Thus, the environment of paddy roots was remarkably heterogeneous as a bacterial habitat, where not only the whole root system but also a root may create oxic and anoxic environments.     

Identification of active bacteria involved in decomposition of complex maize and soybean residues in a tropical Vertisol using N-DNA stable isotope probing

As limited information is available about the relationship between microbial processes and community structure in tropical soils, we used ¹⁵N-DNA stable isotope probing (¹⁵N-DNA-SIP) to identify bacteria actively involved in decomposition of plant residues of different biochemical quality. ¹⁵N-labeled (90 atom%) and unlabeled (control) maize (C-to-N ratio: 32; cellulose content: 24.9%) and soybean (15; 15.5%) leaf residues were incubated in a tropical Vertisol for 15 days. Soil DNA was isolated, subjected to ¹⁵N-DNA-SIP and buoyant density-resolved DNA fractions were analyzed by 16S rRNA gene-based denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of selected DGGE bands. Residue addition induced new bands and changed relative intensity of already existing bands in ¹⁵N-enriched SIP fractions. Phylogenetic analysis of selected, cloned DGGE bands from ‘heaviest’ ¹⁵N-enriched fractions (57.8 atom% (maize), 87.1 atom% (soybean)) revealed that soils treated with maize residues were dominated by Pseudonocardia sp., while Arthrobacter sp. and Streptomyces sp. were found in the soybean residue treated soils. Sequences related to Bacillus sp. and Saccharopolyspora sp. were found in both organic residue treatments. Our study gave clear evidence that ¹⁵N-DNA-SIP combined with 16S rRNA gene-based community fingerprinting of density-resolved fractions and an unlabeled control was suited for detecting active bacteria involved in decomposition of complex maize and soybean residues. In conclusion, we could show that residue quality, inducing contrasting N assimilation by decomposing bacteria, was a substantial determinant of certain decomposing community members assayed in this study.     

Bacterial community in plant residues in a Japanese paddy field estimated by RFLP and DGGE analyses

Plant residues (PRs) are “hot spots” of microbial activities in soil. PRs with the size more than 0.5 mm were collected from a Japanese paddy field during rice cultivation period (from May to September) and fractionated into four categories by size (>4, 2-4, 1-2, and 0.5-1 mm) using sieves. Restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) patterns were compared among the fractions after DNA extraction from the PRs and PCR amplification. The total amount of PRs with the size over 0.5 mm decreased in the field with the first-order kinetics (r²=0.810, p<0.01) with time from rice transplanting to harvest. RFLP analysis showed that the bacterial community structure in PRs with the 0.5-2 mm fraction was different from that in PRs with the >2 mm fraction and the latter community structure changed after the midseason drainage. In contrast, the DGGE patterns of the bacterial community in the PRs indicated the succession from June to September during rice cultivation forming three major groups irrespective of the fraction size. Sequence analysis of DGGE bands showed that Firmicutes (clostridia), α-, γ-, δ-Proteobacteria (myxobacteria), Nitrospira, Acidobacteria, Bacteroidetes, Verrucomicrobia and Spirochaetes were predominant members in the PRs irrespective of fraction size.     

Denaturing gradient gel electrophoretic analysis of dominant 2,4-diacetylphloroglucinol biosynthetic phlD alleles in fluorescent Pseudomonas from soils suppressive or conducive to black root rot of tobacco

In Switzerland, similar types of rhizosphere pseudomonads producing the biocontrol compound 2,4-diacetylphloroglucinol (Phl) have been found in soils suppressive to Thielaviopsis basicola-mediated black root rot of tobacco as well as in conducive soils. However, most findings were based on the analysis of a limited number of Pseudomonas isolates, obtained from a single experiment and only from T. basicola-inoculated plants. Here, an approach based on denaturing gradient gel electrophoresis (DGGE) of dominant phlD alleles from tobacco rhizosphere provided different phlD migration patterns. Sequencing of phlD-DGGE bands revealed a novel phylogenetic cluster of phlD sequences found in both suppressive and conducive soils in addition to previously-documented phlD alleles. phlD-DGGE bands and alleles differed little from one plant to the next but more extensively from one sampling to the next during the three-year study. Three of the 13 bands and 12 of the 31 alleles were only found in suppressive soil, whereas five bands and 13 alleles were found exclusively in conducive soil. The population structure of phlD⁺ pseudomonads depended more on the individual soil considered and its suppressiveness status than on inoculation of tobacco with T. basicola. In conclusion, phlD-DGGE revealed additional phlD diversity compared with earlier analyses of individual Pseudomonas isolates, and showed differences in phlD⁺Pseudomonas population structure in relation to disease suppressiveness.     

Phylogenetic study on a bacterial community in the floodwater of a Japanese paddy field estimated by sequencing 16S rDNA fragments after denaturing gradient gel electrophoresis

Phylogenetic positions of characteristic bands of 16S rDNA that were obtained from the floodwater of a Japanese paddy field by denaturing gradient gel electrophoresis (DGGE) analysis in a previous work (Biol Fertil Soils 36:306–312, 2002) were determined to identify dominant bacterial members in the floodwater. Sequences of DGGE bands were affiliated with the Cytophaga–Flavobacterium–Bacteroides group, β-Proteobacteria, and Actinobacteria and showed phylogenetically close relationships with species inhabiting other aquatic environments, although most of their closest relatives were uncultured bacterial clones.     

Influence of the sunflower rhizosphere on the biodegradation of PAHs in soil

Reduced bioavailability to soil microorganisms is probably the most limiting factor in the bioremediation of polycyclic aromatic hydrocarbons PAH-polluted soils. We used sunflowers planted in pots containing soil to determine the influence of the rhizosphere on the ability of soil microbiota to reduce PAH levels. The concentration of total PAHs decreased by 93% in 90 days when the contaminated soil was cultivated with sunflowers, representing an improvement of 16% compared to contaminated soil without plants. This greater extent of PAH degradation was consistent with the positive effect of the rhizosphere in selectively stimulating the growth of PAH-degrading populations. Molecular analysis revealed that the increase in the number of degraders was accompanied by a dramatic shift in the structure of the bacterial soil community favoring groups with a well-known PAH-degrading capacity, such as Sphingomonas (α-Proteobacteria), Commamonas and Oxalobacteria (β-Proteobacteria), and Xhanthomonas (γ-Proteobacteria). Other groups that were promoted for which degrading activity has not been reported included Methylophyllus (β-Proteobacteria) and the recently described phyla Acidobacteria and Gemmatimonadetes. We also conducted mineralization experiments on creosote-polluted soil in the presence and absence of sunflower root exudates to advance our understanding of the ability of these exudates to serve as bio-stimulants in the degradation of PAHs. By conducting greenhouse and mineralization experiments, we separated the chemical impact of the root exudates from any root surface phenomena, as sorption of contaminants to the roots, indicating that sunflower root exudates have the potential to increase the degradation of xenobiotics due to its influence on the soil microorganisms, where sunflower root exudates act improving the availability of the contaminant to be degraded. We characterized the sunflower exudates in vitro to determine the total organic carbon (TOC) and its chemical composition. Our results indicate that the rhizosphere promotes the degradation of PAHs by increasing the biodegradation of the pollutants and the number and diversity of PAH degraders. We propose that the biostimulation exerted by the plants is based on the chemical composition of the exudates.     

DGGE fingerprinting of culturable soil bacterial communities complements culture-independent analyses

Culture-dependent DGGE (CD DGGE) fingerprinting of the 16S rRNA gene was used to characterize mixed bacterial communities recovered on agar plates. Using R2A Agar as a growth medium, CD DGGE analysis resulted in clear banding patterns of sufficient complexity (16-32 major bands) and reproducibility to investigate differences in bacterial communities in a silt loam soil. Replicate CD DGGE profiles from plates inoculated with less-dilute samples (10⁻³) had a higher band count and were more similar (72-77%) than profiles from more-dilute samples (51-61%). Different culture media and incubation conditions resulted in distinct community fingerprints and increased the cumulative number of unique bands detected. When CD DGGE fingerprints were compared to profiles constructed from 16S rRNA genes obtained from culture-independent clone libraries (CB DGGE profiles) 34% of the bands were unique to the culture-dependent profiles, 32% were unique to the culture-independent profiles and 34% were found in both communities. These data demonstrate that culture-independent DGGE profiles are supplemented by the distinct bands detected in culture-dependent profiles. CD DGGE can be a useful technique to follow the dynamics of distinct culturable fractions of the soil bacterial community.     

Seasonal dynamics of leaf- and root-derived C in arctic tundra mesocosms

We investigated contributions of leaf litter, root litter and root-derived organic material to tundra soil carbon (C) storage and transformations. ¹⁴C-labeled materials were incubated for 32 weeks in moist tussock tundra soil cores under controlled climate conditions in growth chambers, which simulated arctic fall, winter, spring and summer temperatures and photoperiods. In addition, we tested whether the presence of living plants altered litter and soil organic matter (SOM) decomposition by planting shoots of the sedge Eriophorum vaginatum in half of the cores. Our results suggest that root litter accounted for the greatest C input and storage in these tundra soils, while leaf litter was rapidly decomposed and much of the C lost to respiration. We observed transformations of ¹⁴C between fractions even when total C appeared unchanged, allowing us to elucidate sources and sinks of C used by soil microorganisms. Initial sources of C included both water soluble (WS) and acid-soluble (AS) fractions, primarily comprised of carbohydrates and cellulose, respectively. The acid-insoluble (AIS) fraction appeared to be a sink for C when conditions were favorable for plant growth. However, decreases in ¹⁴C activity from the AIS fraction between the fall and spring harvests in all treatments indicated that microorganisms consumed recalcitrant C compounds when soil temperatures were below 0 °C. In planted leaf litter cores and in both planted and unplanted SOM cores, the greatest amounts of ¹⁴C at the end of the experiment were found in the AIS fraction, suggesting a high rate of humification or accumulation of decay-resistant plant tissues. In unplanted leaf litter cores and planted and unplanted root litter cores most of the ¹⁴C remaining at the end of the experiment was in the AS fraction suggesting less extensive humification of leaf and root detritus. Overall, the presence of living plants stimulated decomposition of leaf litter by creating favorable conditions for microbial activity at the soil surface. In contrast, plants appeared to inhibit decomposition of root litter and SOM, perhaps because of microbial preferences for newer, more labile inputs from live roots.     

广谱抗真菌蛋白转基因水稻秸秆模拟还田对土壤真菌群落结构的影响

为探讨广谱抗真菌蛋白转基因水稻秸秆降解对土壤真菌群落结构的影响,本文在室温条件下进行田间秸秆还田模拟试验,设不添加秸秆(S)、添加转基因水稻‘转品1’秸秆(S-Z1)、添加转基因水稻‘转品8’秸秆(S-Z8)、添加非转基因水稻‘七丝软粘’秸秆(S-CK)4个土壤处理,采用传统的平板计数法和变性梯度凝胶电泳(denatured gradient gel electrophoresis,DGGE)技术,分析广谱抗真菌蛋白转基因水稻秸秆模拟还田过程中土壤可培养真菌数和土壤真菌群落的变化情况。平板计数结果表明,在秸秆降解的第40 d,转基因水稻秸秆处理(S-Z1、S-Z8)与非转基因水稻秸秆处理(S-CK)土壤之间的可培养真菌数差异显著,但秸秆降解中后期(50~90 d),S-Z1、S-Z8和S-CK之间土壤可培养真菌数的差异均不显著。真菌18S r RNA的PCR-DGGE图谱显示,S-Z1、S-Z8和S-CK在秸秆降解过程中没有显著不同的条带出现,仅有个别条带在亮度上存在差异。DGGE图谱条带多样性分析结果表明,在秸秆降解的个别时间段,S-Z1、S-Z8和S-CK之间在丰富度和Shannon-Wiener多样性指数上存在显著差异,而在秸秆降解的整个过程均匀度指数差异均不显著。对DGGE主要条带和差异性条带进行克隆测序后发现,子囊菌占最大比重,其次为担子菌、壶菌,而在转基因和非转基因土壤处理间亮度上存在差异的条带属于子囊菌。以上研究结果表明,广谱抗真菌蛋白转基因水稻秸秆降解对土壤真菌群落结构的影响是短暂的、不持续的。     

Soil microbial community responses to Bt transgenic rice residue decomposition in a paddy field

Purpose

Genetic modifications (GM) of commercial crops offer many benefits. However, microbial-mediated decomposition might be affected by GM crop residues in agricultural ecosystems. The objective of this study was to assess the possible impacts of cry1Ab gene transformation of rice on soil microbial community composition associated with residue decomposition in the paddy field under intensive rice cultivation.

Materials and methods

A 276-day field trial was set up as a completely randomized design for two types of rice residues, KMD (Bt) and Xiushui 11 (non-Bt parental variety) in triplicate by conventional intensive rice cropping system. The litterbag method was used in the rice residue decomposition and a total of 120 straw and root litterbags were either placed on the soil surface or buried at 10 cm depth in the field on Dec. 24, 2005. The litterbags were sampled periodically and their soil bacterial and fungal communities were determined by terminal restriction fragment length polymorphism (T-RFLP). The additive main effects with multiplicative interaction (AMMI) model were performed for the analysis of T-RFLP on binary variables of peak presence (presence/absence). The analysis of variance and linear regressions were performed for analysis of AMMI data.

Results and discussion

Total AMMI model analysis revealed that microbial community composition in the litterbags was affected by temporal and spatial factors. Compared with the non-Bt rice residue treatment, Bt rice straw had no significant effects on the soil bacterial and fungal community composition during the study period, regardless of the litterbags being placed on the surface or buried in the soil. There were no significant differences in the bacterial community composition profiles in root decomposition between Bt transgenic and non-Bt varieties. However, significant differences in soil fungal community composition between the buried Bt and non-Bt rice roots were observed in soils sampled on days 31, 68, and 137, indicating that Bt roots incorporated into paddy soil may affect soil fungal community during the initial stage of their decomposition.

Conclusions

There were some significant differences in fungal community composition between Bt rice root and non-Bt root treatments at the early stage of root decomposition in the paddy field. It is important that, before Bt rice is released for commercial production, more research should be conducted to evaluate the ecological effects of the Bt rice residues returned to paddy field upon grain harvesting.     

Effect of land use types on decomposition of 14C-labelled maize residue (Zea mays L.)

The effect of three land use types on decomposition of ¹⁴C-labelled maize (Zea mays L.) residues and soil organic matter were investigated under laboratory conditions. Samples of three Dystric Cambisols under plow tillage (PT), reduced tillage (RT) and grassland (GL) collected from the upper 5 cm of the soil profile were incubated for 159 days at 20 °C with or without ¹⁴C-labelled maize residue. After 7 days cumulative CO₂ production was highest in GL and lowest in PT, reflecting differences in soil organic C (SOC) concentration among the three land use types and indicating that mineralized C is a sensitive indicator of the effects of land use regime on SOC. ¹⁴CO₂ efflux from maize residue decomposition was higher in GL than in PT, possibly due to higher SOC and microbial biomass C (MBC) in GL than in PT. ¹⁴CO₂ efflux dynamics from RT soil were different from those of PT and GL. RT had the lowest ¹⁴CO₂ efflux from days 2 to 14 and the highest from days 28 to 159. The lowest MBC in RT explained the delayed decomposition of residues at the beginning. A double exponential model gave a good fit to the mineralization of SOC and residue-¹⁴C (R² > 0.99) and allowed estimation of decomposition rates as dependent on land use. Land use affected the decomposition of labile fractions of SOC and of maize residue, but had no effect on the decomposition of recalcitrant fractions. We conclude that land use affected the decomposition dynamics within the first 1.5 months mainly because of differences in soil microbial biomass but had low effect on cumulative decomposition of maize residues within 5 months.     

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.     

Succession and Phylogenetic Profile of Eubacterial Communities in Rice Straw Incorporated into a Rice Field: Estimation by PCR-DGGE Analysis

PCR-DGGE analysis followed by sequencing was conducted to estimate the succession and the phylogenetic profile of the eubacterial communities responsible for the decomposition of rice straw (RS) that was incorporated into a rice field. Leaf sheath and leaf blade parts were separately put in nylon mesh bags, and were placed in the rice field soil under drained conditions during the off-cropping season and under flooded conditions after transplantation of rice. In addition, RS samples that had been placed under drained conditions in the off-cropping season were placed again in flooded rice field soil after transplantation of rice. DGGE patterns of the bacterial communities in the RS samples were classified into two groups, namely leaf sheaths and leaf blades. Principal component analysis of the DGGE patterns revealed the succession along with the duration of placement. These results indicated that the RS part (sheath or blade) mainly determined the structure of the bacterial communities responsible for the RS decomposition, followed by the duration of placement. Sequence analysis of the characteristic DGGE bands indicated that most of the closest relatives associated with the bands belonged to α-, β-, γ-, and δ-Proteobacteria, CFB group, and Spirochaetes. Some bands were closely related to Acidobacteria and Verrucomicrobia. CFB members and α-Proteobacteria predominated commonly in both RS parts, while γ- and δ-Proteobacteria, and Spirochaetes and β-Proteobacteria specifically colonized sheath and blade parts, respectively. In addition, Proteobacteria and CFB members characterized the differences in the bacterial communities under flooded or drained conditions. These results suggest that Proteobacteria, CFB group, and Spirochaetes were responsible for RS decomposition in rice field soil under both flooded and drained conditions.     

Biochar soil amendment increased bacterial but decreased fungal gene abundance with shifts in community structure in a slightly acid rice paddy from Southwest China

Biochar’s role on greenhouse gas emission and plant growth has been well addressed. However, there have been few studies on changes in soil microbial community and activities with biochar soil amendment (BSA) in croplands. In a field experiment, biochar was amended at rates of 0, 20 and 40 t ha⁻¹ (C0, C1 and C2, respectively) in May 2010 before rice transplantation in a rice paddy from Sichuan, China. Topsoil (0–15 cm) was collected from the rice paddy while rice harvest in late October 2011. Soil physico-chemical properties and microbial biomass carbon (MBC) and nitrogen (MBN) as well as selected soil enzyme activities were determined. Based on 16S rRNA and 18S rRNA gene, bacterial and fungal community structure and abundance were characterized using terminal-restriction fragment length polymorphism (T-RFLP) combined with clone library analysis, denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR assay (qPCR). Contents of SOC and total N and soil pH were increased but bulk density decreased significantly. While no changes in MBC and MBN, gene copy numbers of bacterial 16S rRNA was shown significantly increased by 28% and 64% and that of fungal 18S rRNA significantly decreased by 35% and 46% under BSA at 20 and 40 t ha⁻¹ respectively over control. Moreover, there was a significant decrease by 70% in abundance of Methylophilaceae and of Hydrogenophilaceae with an increase by 45% in Anaerolineae abundance under BSA at 40 t ha⁻¹ over control. Whereas, using sequencing DGGE bands of fungal 18S rRNA gene, some bands affiliated with Ascomycota and Glomeromycota were shown inhibited by BSA at rate of 40 t ha⁻¹. Significant increases in activities of dehydrogenase, alkaline phosphatases while decreased β-glucosidase were also observed under BSA. The results here indicated a shift toward a bacterial dominated microbial community in the rice paddy with BSA.     

Interactive effects of functionally different earthworm species on aggregation and incorporation and decomposition of newly added residue carbon

The interactive effects of two functionally different earthworm species (Aporrectodea caliginosa (endogeic species) and Lumbricus rubellus (epigeic species)) on the incorporation of fresh residue into large macroaggregates and formation of microaggregates within these large macroaggregates were investigated during a short-term laboratory experiment using ¹³C-labelled sorghum (Sorghum bicolor (L.) Moench) residues. Soil was collected from a long-term no-tillage agricultural field, crushed through a 250-μm sieve and incubated under laboratory conditions. The following earthworm treatments were applied: (i) soil+¹³C-labelled residue+A. caliginosa; (ii) soil+¹³C-labelled residue+L. rubellus; (iii) soil+¹³C-labelled residue+A. caliginosa+L. rubellus and; (iv) soil+¹³C-labelled residue. Two residue placement treatments (i.e. surface and incorporated) were superimposed on the earthworm treatments. Earthworms were added after 8 days of incubation. Aggregate size distribution and total C and ¹³C were measured after 22 days. Microaggregates, fine inter-microaggregate particulate organic matter (inter-POM) and intra-microaggregate POM (intra-POM) were isolated from macroaggregates. Earthworms had a greater stimulating effect on the formation of large macroaggregates (>2000 μm) and microaggregates within large macroaggregates when residue was incorporated in the soil, especially in the presence of A. caliginosa. When residue was placed on the surface, residue-derived intra-POM C was highest when L. rubellus was present and significantly lower in the presence of A. caliginosa. Residue-derived inter-POM C was highest when a mix of both species was present. These results indicate that earthworm species differentially affect incorporation of fresh organic matter into stable microaggregates within macroaggregates, and that interactive effects of earthworm species might have important consequences for the incorporation and protection of C inside of microaggregates within macroaggregates especially when residues are placed on the soil surface.