Effect of Temperature on Structure and Function of the Methanogenic Archaeal Community in an Anoxic Rice Field Soil

Soil temperatures in Italian rice fields typically range between about 15 and 30°C. A change in the incubation temperature of anoxic methanogenic soil slurry from 30°C to 15°C typically resulted in a decrease in the CH₄ production rate, a decrease in the steady-state H₂ partial pressure, and a transient accumulation of acetate. Previous experiments have shown that these changes were due to an alteration of the carbon and electron flow in the methanogenic degradation pathway of organic matter caused by the temperature shift (K. J. Chin and R. Conrad, FEMS Microbiol. Ecol. 18:85–102, 1995). To investigate how temperature affects the structure of the methanogenic archaeal community, total DNA was extracted from soil slurries incubated at 30 and 15°C. The archaeal small-subunit (SSU) rRNA-encoding genes (rDNA) of these environmental DNA samples were amplified by PCR with an archaeal-specific primer system and used for the generation of clone libraries. Representative rDNA clones (n = 90) were characterized by terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis. T-RFLP analysis produced for the clones terminally labeled fragments with a characteristic length of mostly 185, 284, or 392 bp. Sequence analysis allowed determination of the phylogenetic affiliation of the individual clones with their characteristic T-RFLP fragment lengths and showed that the archaeal community of the anoxic rice soil slurry was dominated by members of the families Methanosarcinaceae (185 bp) and Methanosaetaceae (284 bp), the kingdom Crenarchaeota (185 or 284 bp), and a novel, deeply branching lineage of the (probably methanogenic) kingdom Euryarchaeota (392 bp) that has recently been detected on rice roots (R. Großkopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). The structure of the archaeal community changed when the temperature was shifted from 30°C to 15°C. Before the temperature shift, the clones (n = 30) retrieved from the community were dominated by Crenarchaeota (70%), “novel Euryarchaeota” (23%), and Methanosarcinacaeae (7%). Further incubation at 30°C (n = 30 clones) resulted in a relative increase in members of the Methanosarcinaceae (77%), whereas further incubation at 15°C (n = 30 clones) resulted in a much more diverse community consisting of 33% Methanosarcinaceae, 23% Crenarchaeota, 20% Methanosaetaceae, and 17% novel Euryarchaeota. The appearance of Methanosaetaceae at 15°C was conspicuous. These results demonstrate that the structure of the archaeal community in anoxic rice field soil changed with time and incubation temperature.     

Effects of Biosolid Soil Amendment on Heterodera glycines Populations

The high degree of parasitic variability in Heterodera glycines and its distribution in a wide range of soybean production systems present multiple challenges for management, which necessitate increased understanding of the biology of H. glycines. Soil amendments are being considered either as stand-alone and/or as part of integrated management approaches. A recycled municipal biosolid with nutrition supplement and liming qualities, N-Viro Soil (NVS) has potential as a multi-purpose soil amendment. In three greenhouse experiments, the effects of 0, 1.0 or 4.0 g NVS/100 cm³ of sandy loam soil on three H. glycines populations (GN1, GN2 and GN3) were investigated on soybean grown for 557 ± 68 degree-days (base 10°C). The response of the three H. glycines populations to NVS treatment varied by experiment. The overall numbers of preadult stages and cysts generally decreased with increasing levels of NVS in all experiments, and the high rate was more effective than the low rate in suppressing H. glycines numbers. This suggests that the high NVS treatment can affect the three populations in the experimental soil type under controlled conditions. Field studies to determine efficacy of the soil amendment in a wide range of environments should be initiated.     

Impact of Protists on the Activity and Structure of the Bacterial Community in a Rice Field Soil

Flooded rice fields have become a model system for the study of soil microbial ecology. In Italian rice fields, in particular, aspects from biogeochemistry to molecular ecology have been studied, but the impact of protistan grazing on the structure and function of the prokaryotic community has not been examined yet. We compared an untreated control soil with a γ-radiation-sterilized soil that had been reinoculated with a natural bacterial assemblage. In order to verify that the observed effects were due to protistan grazing and did not result from sterilization, we set up a third set of microcosms containing sterilized soil that had been reinoculated with natural assemblage bacteria plus protists. The spatial and temporal changes in the protistan and prokaryotic communities were examined by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis, respectively, both based on the small-subunit gene. Sequences retrieved from DGGE bands were preferentially affiliated with Cercozoa and other bacteriovorous flagellates. Without protists, the level of total DNA increased with incubation time, indicating that the level of the microbial biomass was elevated. Betaproteobacteria were preferentially preyed upon, while low-G+C-content gram-positive bacteria became more dominant under grazing pressure. The bacterial diversity detectable by T-RFLP analysis was greater in the presence of protists. The level of extractable NH₄⁺ was lower and the level of extractable SO₄²⁻ was higher without protists, indicating that nitrogen mineralization and SO₄²⁻ reduction were stimulated by protists. Most of these effects were more obvious in the partially oxic surface layer (0 to 3 mm), but they could also be detected in the anoxic subsurface layer (10 to 13 mm). Our observations fit well into the overall framework developed for protistan grazing, but with some modifications pertinent to the wetland situation: O₂ was a major control, and O₂ availability may have limited directly and indirectly the development of protists. Although detectable in the lower anoxic layer, grazing effects were much more obvious in the partially oxic surface layer.     

Effect of Temperature on Carbon and Electron Flow and on the Archaeal Community in Methanogenic Rice Field Soil

Temperature is an important factor controlling CH₄ production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH₄ production rates increased with temperature, with an apparent activation energy of 61 kJ mol⁻¹. Steady-state partial pressures of the methanogenic precursor H₂ also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H₂ plus CO₂-dependent methanogenesis was kept at −20 to −25 kJ mol of CH₄⁻¹ over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 μM. Simultaneously, the relative contribution of H₂ as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to ¹⁴CH₄, so that the carbon and electron flow to CH₄ was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.     

Succession of Bacterial Populations during Plant Residue Decomposition in Rice Field Soil

The incorporation of rice residues into paddy fields strongly enhances methane production and emissions. Although the decomposition processes of plant residues in rice field soil has been documented, the structure and dynamics of the microbial communities involved are poorly understood. The purpose of the present study was to determine the dynamics of short-chain fatty acids and the structure of bacterial communities during residue decomposition in a rice field soil. The soil was anaerobically incubated with the incorporation of rice root or straw residues for 90 days at three temperatures (15, 30, and 45°C). The dynamics of fatty acid intermediates showed an initial cumulative phase followed by a rapid consumption phase and a low-concentration quasi-steady state. Correspondingly, the bacterial populations displayed distinct successions during residue decomposition. Temperature showed a strong effect on the dynamics of bacterial populations. Members of Clostridium (clusters I and III) were most dominant in the incubations, particularly in the early successions. Bacteroidetes and Chlorobi were abundant in the later successions at 15 and 30°C, while Acidobacteria were selected at 45°C. We suggest that the early successional groups are responsible for the decomposition of the easily degradable fraction of residues, while the late successional groups become more important in decomposing the less-degradable or resistant fraction of plant residues. The bacterial succession probably is related to resource availability during residue decomposition. The fast-growing organisms are favored at the beginning, while the slow-growing bacteria are better adapted in the later stages, when substrate availability is limiting.Rice residues, including root and straw residues, serve as the major carbon source in paddy fields. It has been estimated that the amounts of organic matter supplied annually to paddy fields range from 1,700 to 3,470 kg ha⁻¹, and more than 65% of them were derived from plant residues (19, 23). The incorporation of rice residues into paddy fields helps sustain soil organic matter, improve physical and chemical properties, and increase nutrient availability (15, 39). However, it also strongly enhances methane production and emissions (6, 45, 46).Numerous studies have been carried out on residue decomposition and CH₄ production in rice field soils (11-13, 22). The rate of decomposition usually is separated into a fast phase and a slowdown phase (24). According to the dynamics of the intermediates H₂ and fatty acids and the activities of polysaccharolytic enzymes, Glissmann and Conrad (13) proposed five stages for residue decomposition, including the production and consumption of reducing sugars, the production and consumption of H₂ and fatty acids, and the production of CH₄. The rate of decomposition was affected by the composition of residues (11, 18). Root residues generally are decomposed slower than straw residues (24). The fermentation pathway also could differ depending on the residue material, resulting in different fatty acid intermediates (13).A complex microbial assemblage consisting of hydrolytic, fermenting, homoacetogenic, syntrophic, and methanogenic microorganisms are involved in the anaerobic decomposition of organic residues (5, 41, 48). Plant residues are composed of complex components. With the decomposition process, the proportion of labile component decreases while the resistant components relatively accumulate. Changes in the activity and structure of the microbial community thus are anticipated during the processes of residue decomposition. However, little has been known about the dynamics of microbial populations during residue decomposition in anoxic rice soil. Using culture-independent methods, Weber et al. (47) showed that the structure of the bacterial community shifted between early and late stages. Microscopic observation revealed the differential colonizations of bacterial populations on different parts of straw residue, indicating the effects of residue quality and niche condition (18). The microbial community appears to form a spatially well organized architecture, with the fermenting bacteria colonizing the residue particles, while the syntrophic bacteria and methanogens mainly inhabit the adjacent soil during the decomposition process (12).Air temperature exhibits a large seasonal variation in southeastern Asia, where rice is widely cultivated. The lowest and highest records in our research site, for instance, were −5 and 40°C, respectively, in 2006, when we collected the soil samples. It has been demonstrated that temperature has a strong effect on residue decomposition and CH₄ production (8, 22, 33). However, it is uncertain whether the effect also is reflected in the structure and function of degrading microbial communities in the soil. Therefore, the purpose of our project was to determine the effect of temperature on microbial communities during the processes of plant residue decomposition in a Chinese rice field soil. The dynamics of methanogenesis and methanogenic archaea have been reported previously (33). Here, we show the results on fatty acid intermediates and the responsible bacterial communities.     

Effect of Inoculation and Leaf Litter Amendment on Establishment of Nodule-Forming Frankia Populations in Soil

High-N₂-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [¹⁵N]NO₃ and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N₂ fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N₂ fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% ± 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% ± 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% ± 6%) than by group IV (81% ± 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N₂ fixation rates by ¹⁵N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N₂-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N₂-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

Soil pH Effects on Nematode Populations Associated with Soybeans

''Amsoy'' soybeans were grown for 2 months in nonsterilized Jackson silt loam amended to pH 4.0, 6.0, and 8.0. Nematodes were extracted biweekly from soil and roots. The greatest numbers of Pratylenchus alleni colonized soybean roots at pH 6.0. Hoplolaimus galeatus and members of the Tylenchinae-Psilenchinae survived best at pH 6.0, while numbers o f the Dorylaimoidea were greatest at both pH 6.0 and 8.0. The non-stylet nematodes were recovered in greater numbers from pH 8.0 soil. Potassium, manganese, and phenols were highest in soybean plants grown in pH 4.0 soil, the pH at which there were the fewest nematodes. A thicker suberized outer layer o f root tissue occurred in plants grown at pH 4.0.     

Effects of a Combined Amendment on Pb,Cd, and As Availability and Accumulation in Rice Planted in Contaminated Paddy Soil

The objectives of the present study were to investigate the mitigation of lead (Pb), cadmium (Cd), and arsenic (As) in a multi-metal contaminated soil and their accumulation in rice plants (Oryza sativa L., cv II You 93) using a combined amendment (CMF, calcium carbonate + metakaolin + fused calcium–magnesium phosphate fertilizer). The results showed that application of CMF was effective in reducing the acid-extractable concentrations of soil Pb and Cd. The exchangeable concentrations of soil As showed an initial decrease followed by a gradual increase. The application of 0.2% CMF notably reduced the concentrations of Pb, Cd, and As in brown rice by 46.5%, 43.6%, and 32.0%, respectively. The concentration of As in brown rice was 0.179 mg kg^?1 at 0.2% CMF, which met the maximum levels of contaminants in foods of China (MLs) (the ML of Pb, Cd, and As is 0.2 mg kg^?1 according to the China national standard GB 2762-2012). At 1.6% CMF, the concentrations of Pb and Cd in brown rice were 0.002 and 0.185 mg kg^?1, respectively, i.e., reductions of 99.6% and 74.1%, and these values also fell within the MLs.     

脱硫石膏改良强度苏打盐渍土效果的研究

石膏改良盐碱土在我国已是成功的经验，但是，由于受到资源条件的限制及其价格昂贵等原因，影响着大面积的推广应用。目前，利用煤炭作为能源的工厂，在其煤炭燃烧过程中产生着大量的污染物质硫氧化物，而日本国旨在消除此类污染物所研究的除硫装置新工艺中的副产物脱硫石...     

Screening for Drought Resistance of Rice Recombinant Inbred Populations in the Field

In a 2-year experiment, 187 genotypes were grown under well-watered and drought stress conditions, imposed at panicle initiation stage. The relationship of genotypic variation in yield under drought conditions to potential yield, heading date and flowering delay, reduction in plant height, and to a drought response index （DRI） was detected. Grain yield under drought stress conditions was associated with yield under well-watered conditions （r= 0.47^＊＊, and r= 0.61^＊＊ during 2 years of tests）. The delay of heading date ranged from -1 （no delay） to 24days, and was negatively associated with grain yield （r =-0.40^＊）, spikelet fertility percentage （r =-0.40^＊＊）, harvest index （r =-0.58^＊＊）, but positively associated with yield reduction percentage （r = 0.60^＊＊）. The reduction in plant height was negatively associated with grain yield （r =-0.24^＊＊, and r=-0.29^＊＊）, spikelet fertility percentage （r = -0.23^＊＊, and r= -0.21^＊）, harvest index （r = -0.37^＊＊, and r= -0.54^＊＊）, and positively associated with yield reduction percentage （r = 0.58^＊＊, and r= 0.58^＊＊） in 2003 and 2004, respectively. The DRI of genotypes was strongly associated with grain yield （r = 0.87^＊＊, and r= 0.77^＊＊）, fertility percentage （r = 0.66^＊＊ and r= 0.54^＊＊）, harvest index （r= 0.67^＊＊ and r= 0.61^＊＊）, and negatively associated with grain reduction percentage （r=-0.70^＊＊, and r=-0.73^＊＊） under drought stress. The results indicate that genotypes with drought resistance can be identified by measuring yield potential, delay in flowering, reduction in plant height, or DRI under test environments of well-watered and drought stress.     

Screening for Drought Resistance of Rice Recombinant Inbred Populations in the Field

In a 2-year experiment, 187 genotypes were grown under well-watered and drought stress conditions, imposed at panicle initiation stage. The relationship of genotypic variation in yield under drought conditions to potential yield, heading date and flowering delay, reduction in plant height, and to a drought response index (DRI) was detected. Grain yield under drought stress conditions was associated with yield under well-watered conditions (r= 0.47**, and r= 0.61** during 2 years of tests). The delay of heading date ranged from -1 (no delay) to 24days, and was negatively associated with grain yield(r=-0.40*), spikelet fertility percentage (r=-0.40**), harvest index (r=-0.58**), but positively associated with yield reduction percentage (r= 0.60**). The reduction in plant height was negatively associated with grain yield (r =-0.24**, and r =-0.29**), spikelet fertility percentage (r =-0.23**, and r =-0.21*), harvest index (r =-0.37**, and r = -0.54**), and positively associated with yield reduction percentage (r = 0.58**, and r = 0.58**) in 2003 and 2004, respectively. The DRI of genotypes was strongly associated with grain yield (r = 0.87**, and r= 0.77**), fertility percentage (r= 0.66** and r = 0.54**), harvest index (r=0.67** and r=0.61**), and negatively associated with grain reduction percentage (r=-0.70**, and r=-0.73**)under drought stress. The results indicate that genotypes with drought resistance can be identified by measuring yield potential, delay in flowering, reduction in plant height, or DRI under test environments of well-watered and drought stress.     

Diversity and Structure of the Methanogenic Community in Anoxic Rice Paddy Soil Microcosms as Examined by Cultivation and Direct 16S rRNA Gene Sequence Retrieval

A dual approach consisting of cultivation and molecular retrieval of partial archaeal 16S rRNA genes was carried out to characterize the diversity and structure of the methanogenic community inhabiting the anoxic bulk soil of flooded rice microcosms. The molecular approach identified four groups of known methanogens. Three environmental sequences clustered with Methanobacterium bryantii and Methanobacterium formicicum, six were closely related but not identical to those of strains of Methanosaeta concilii, two grouped with members of the genus Methanosarcina, and two were related to the methanogenic endosymbiont of Plagiopyla nasuta. The cultivation approach via most-probable-number counts with a subsample of the same soil as an inoculum yielded cell numbers of up to 10⁷ per g of dry soil for the H₂-CO₂-utilizing methanogens and of up to 10⁶ for the acetate-utilizing methanogens. Strain VeH52, isolated from the terminal positive dilution on H₂-CO₂, grouped within the phylogenetic radiation characterized by M. bryantii and M. formicicum and the environmental sequences of the Methanobacterium-like group. A consortium of two distinct methanogens grew in the terminal positive culture on acetate. These two organisms showed absolute 16S rRNA gene identities with environmental sequences of the novel Methanosaeta-like group and the Methanobacterium-like group. Methanosarcina spp. were identified only in the less-dilute levels of the same dilution series on acetate. These data correlate well with acetate concentrations of about 11 μM in the pore water of this rice paddy soil. These concentrations are too low for the growth of known Methanosarcina spp. but are at the acetate utilization threshold of Methanosaeta spp. Thus, our data indicated Methanosaeta spp. and Methanobacterium spp. to be the dominant methanogenic groups in the anoxic rice soil, whereas Methanosarcina spp. appeared to be less abundant.     

Bacterial Populations Colonizing and Degrading Rice Straw in Anoxic Paddy Soil

Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera Bacillus (3 clones), Nitrosospira (1 clone), Fluoribacter (1 clones), and Acidobacterium (2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes) Bacteria detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus Clostridium, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum, 4% belonged to the α, β, and γ Proteobacteria, and 1.3% belonged to the Bacillus subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa.