Community structure of microaerophilic iron-oxidizing bacteria in Japanese paddy field soils

ABSTRACT

Redox cycle of iron (Fe) is the central process in the biogeochemistry of paddy field soil. Although Fe(II)-oxidizing process is mediated by both abiotic and biotic reactions, microorganisms involved in the process have not been well studied in paddy field soil. The present study investigated the community structure of microaerophilic Fe(II)-oxidizing bacteria (FeOB) in the family Gallionellaceae in the plow layer of paddy fields located in the central (Anjo) and northeastern (Omagari) Japan since the members in the family are the typical FeOB in circumneutral freshwater environments and possibly have the significant role for Fe(II) oxidation in paddy field soils. A primer set targeting 16S rRNA gene of Gallionella-related FeOB was newly designed for the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR (qPCR) analyses. DGGE analysis showed significant differences in the band patterns between the field sites. Besides, minor differences were observed in the patterns between the soil depths (0–1 cm and below 1 cm) in the Anjo field, while the patterns were relatively stable in the Omagari field during the annual rice cultivation practices. In total 54 bands were sequenced and clustered into 20 operational taxonomic units (OTUs) on the basis of the 97% similarity. Eighteen out of twenty OTUs (50 of 54 bands) were affiliated within the FeOB cluster of Gallionellaceae, some of which were clustered with known microaerophilic FeOB, Ferrigenium kumadai, Ferriphaselus amnicola, ‘Sideroxydans lithotrophicus’ and ‘S. paludicola’. The number of the 16S rRNA gene copies was 10⁵–10⁷ and 10⁶–10⁸ copies g^?1 dried soil in the two paddy fields and negatively correlated to the contents of acetate-extractable Fe(II) in the soils during the rice cultivation period. These results suggested inhabitance of considerable number of diverse Gallionella-related FeOB and their potential involvement in the Fe(II)-oxidizing process of soil, especially during the rice cultivation period in the paddy field soils.     

Methane oxidation in the soil surface layer of a flooded rice field and the effect of ammonium

Summary The CH₄ flux from intact soil cores of a flooded rice field in Italy was measured under aerobic and anaerobic incubation conditions. The difference between the anaerobic and aerobic CH₄ fluxes was apparently due to CH₄ oxidation in the oxic soil surface layer. This conclusion was supported by measurements of the vertical CH₄ profile in the upper 2-cm layer, and of the V _max of CH₄ oxidation in slurried samples of the soil surface layer. About 80% of the CH₄ was oxidized during its passage through the soil surface layer. CH₄ oxidation was apparently limited by the concentration of CH₄ and/or O₂ in the active surface layer. The addition of ammonium to the water layer on top of the soil core reversibly increased the aerobic CH₄ fluxes due to inhibition of CH₄ oxidation in the soil surface layer.     

Oligotrophic bacteria on organic debris and plant roots in a paddy field soil

The distribution of heterotrophic bacteria on organic debris and roots of rice plants in a paddy field were studied. The heterotrophic bacteria consisted of two main groups: those which grew on full-strength nutrient broth (NB) and those which did not grow on NB but on a 100-fold dilution of NB (DNB). The latter group was called ‘DNB organisms’ and were considered to be oligotrophic. In both manured and unmanured soils, DNB organisms were predominant in the bacterial communities on organic debris and the rice roots throughout most of the entire period of rice cultivation, although a transient decrease in the proportion of DNB organisms was observed immediately after an application of manure. Morphological and physiological characteristics of DNB isolates from organic debris and rice roots were studied: five types of cell shape were observed, (1) regular rods, (2) filament-forming rods, (3) irregular rods, (4) prosthecate organisms and (5) large oval cells. Regular rods (42% of the total DNB isolates) and irregular rods (46%) were abundant. The ecological roles of DNB organisms in paddy soil are discussed in relation to their physiological characteristics.     

Sulfate-reducing bacteria and nitrogen fixation in flooded rice soil

Acetyle reduction and ¹⁵N studies showed that the addition of sulfate to flooded soil with rice straw enhanced N₂ fixation. The extent of the enhancement was dependent on the sulfate concentration. Sulfate also increased the population of SO₄^2? reducing bacteria and was completely reduced to sulfide by those microorganisms. Purified cultures of soil isolates were capable of C₂H₂ reduction. Based on this evidence, SO₄^2? reducing bacteria were considered responsible for the increase in N₂ fixation but only 1–2 mg N₂ were fixed g^1? SO₄^2? reduced. We conclude that the contribution of SO₄^2? reducing bacteria to the total N₂ fixation in flooded soil is unimportant.     

Modeling within field variation of the compaction layer in a paddy rice field using a proximal soil sensing system

A key characteristic of flooded paddy fields is the plough pan. This is a sub‐soil layer of greater compaction and bulk density, which restricts water losses through percolation. However, the thickness of this compacted layer can be inconsistent, with consequences such as variable percolation and leaching losses of nutrients, which therefore requires precision management of soil water. Our objective was to evaluate a methodology to model the thickness of the compacted soil layer using a non‐invasive electromagnetic induction sensor (EM38‐MK2). A 2.7 ha alluvial non‐saline paddy rice field was measured with a proximal soil sensing system using the EM38‐MK2 and the apparent electrical conductivity (EC_a) of the wet paddy soil was recorded at a high‐resolution (1.0 × 0.5 m). Soil bulk density (n = 10) was measured using undisturbed soil cores, which covered locations with large and small EC_a values. At the same locations (within 1 m²) the depth of the different soil layers was determined by penetrometer. Then a fitting procedure was used to model the EC_a – depth response functions of the EM38‐MK2, which involved solving a system of non‐linear equations and a R² value of 0.89 was found. These predictions were evaluated using independent observations (n = 18) where a Pearson correlation coefficient of 0.87 with an RMSEE value of 0.03 m was found. The EC_a measurements allowed the detail estimation of the compacted layer thickness. The link between water percolation losses and thickness of the compacted layer was confirmed by independent observations with an inverse relationship having a Pearson correlation coefficient of 0.89. This rapid, non‐invasive and cost‐effective technique offers new opportunities to measure differences in the thickness of compacted layers in water‐saturated soils. This has potential for site‐specific soil management in paddy rice fields.     

Cyanobacterial communities of rice straw left on the soil surface of a paddy field

To estimate diversity, seasonal variation, and phylogeny of the cyanobacterial communities in rice straw placed in nylon mesh bags and left on the soil surface of a paddy field, total DNA was extracted from straw, amplified by polymerase chain reaction targeting 16S rRNA genes of cyanobacteria, and the amplicons were separated by denaturing gradient gel electrophoresis (DGGE). These DGGE bands were sequenced. The paddy field was under flooded condition after transplanting of rice (Experiment 1) and under drained conditions after harvest (Experiment 2). The residual samples on the soil surface under upland conditions were collected just before spring plowing and were placed again on the soil surface after transplanting under flooded conditions. DGGE band patterns of cyanobacterial communities of rice straw were different under drained conditions, under flooded conditions when fresh rice straw samples were placed (Experiment 1), and under flooded conditions when residual rice straw samples were replaced (Experiment 2), indicating that the communities were influenced by both water regime of the paddy field and the degree of the rice straw decomposition. Sequence analysis of DGGE bands indicated that most of the cyanobacteria in rice straw on the soil surface in the paddy field were filamentous members belonging to Subsections III and IV. Filamentous cyanobacterial cells were observed in rice straw under flooded conditions by epifluorescence microscopy.     

Populations of methanogenic bacteria in paddy field soil under double cropping conditions (rice-wheat)

The methanogenic populations able to use H₂–CO₂, methanol, and acetate were investigated in paddy field soil in situ under double cropping conditions [rice (Oryza sativa L.) as a summer crop under flooded conditions and wheat (Triticum aestivum L.) as an upland winter crop] over 2 years approximately bimonthly by the most probable number method. Three fields, one without fertilizer, one treated with inorganic fertilizer (mixed fertilizer including urea, ammonium phosphate, and potassium sulfate), and one treated with wheat straw plus inorganic fertilizer, were examined. The population of H₂–CO₂, methanol, and acetate utilizers in the paddy field soil at a depth of 1–6 cm was 10³–10⁴, 10⁴–10⁵, and 10⁴–10⁵ g^-1 dry soil, respectively. These values were almost constant during the 2 years irrespective of moisture regime (flooded or nonflooded), crop (rice or wheat), fertilizer treatment, and soil depth (0–1, 1–10, and 10–20 cm).     

稻田中藻类的生长状况及其对肥料氮的固持

为了确定藻类对稻田生态系统中氮素固持与迁移过程的影响程度,本研究采用田间小区和15N微区试验,在水稻生长的不同时期,观测了藻类在稻田中的生长状况,定量测定了藻类的生物量和氮素固持量.结果显示,稻田中的藻类具有明显的群落演替特征,水稻生育初期以球状藻为主,中后期则以丝状藻为主.藻类的生长可分为两个阶段,即生长期和衰亡期,其中,生长期从稻秧移栽初期至拔节期,衰亡期从拔节期至水稻收获.在衰亡期,藻类生物量随时间的变化符合生长曲线模式.藻类生物质干重在2007和2009年水稻生育期的变化范围分别为205～610 kg/hm2和12～353 kg/hm2,而藻类氮素固持量则分别为N 5.4～17.5 kg/hm2和N0.4～11.0 kg/hm2.15N微区试验结果显示,藻类对15N的固持量为N 0.3～6.0 kg/hm2,占氮素总固持量的54％～68％,平均为57％,表明藻类固持的氮素中大部分来源于当季施入的氮肥.     

Soil type determines the abundance and community structure of ammonia-oxidizing bacteria and archaea in flooded paddy soils

Purpose  

Ammonia oxidation plays an important role in global nitrogen cycle. However, little information is available on ammonia oxidizers in paddy soils. This study aimed to understand the controlling factors of ammonia oxidizers in the paddy soils.     

Application of membrane inlet mass spectrometry to directly quantify denitrification in flooded rice paddy soil

Denitrification has long been considered a major mechanism of N loss when N fertilizer is applied to flooded rice paddies. However, the direct determination of denitrification in soils is almost impossible because of the high atmospheric background of dinitrogen (N₂). Dissolved N₂ in a small water sample can be rapidly and precisely measured through membrane inlet mass spectrometry (MIMS). This study is the first to directly measure N₂ flux through MIMS in flooded rice paddy plots that received different amounts of urea. Ammonia (NH₃) volatilization was measured simultaneously to verify whether NH₃ volatilization and denitrification are complementary loss mechanisms. The average cumulative N₂–N loss measured by MIMS 21 days after fertilization was 4.7?±?1.7 % of the applied N, which was within the range of the reported values obtained by cumulative recovery of (N₂ + N₂O)–¹⁵N and ¹⁵N-balance technique. Underestimation or overestimation of denitrification can be prevented in MIMS given that N₂ can be measured directly without ¹⁵N-labeled fertilizer. A good positive correlation was found between the dissolved in situ N₂ concentrations of floodwater and the denitrification rates of intact soil cores. Urea incorporation reduced NH₃ volatilization unlike surface broadcasting. However, urea incorporation significantly increased cumulative N₂–N loss during the 21 days after fertilization. Correlation analysis showed that nitrate (NO₃ ^?–N) concentration in floodwater could be the primary restricting factor for soil denitrification in the experimental field. Results suggest that MIMS is a promising technique for the measurement of denitrification in a flooded rice paddy.     

Estimation of microbial biomass potassium in paddy field soil

Abstract

Potassium (K) in microbial cells, microbial biomass K, in soil has been recently recognized as a K pool for plant growth. We determined soil microbial biomass K in paddy fields to reveal its importance as a K pool in paddy field soil for the first time. Microbial biomass K ranged from 5 to 21 mg K kg^?1 in the soil samples periodically collected from a paddy field and the value corresponded to 41% of the exchangeable K on average. Both microbial biomass K and exchangeable K increased conspicuously due to the long-term application of livestock manure compost or rice straw compost. Biomass K was higher than exchangeable K under K-deficient conditions in the long-term experimental plots without K application. The present study revealed that the microbial biomass contained considerable amounts of K in paddy field soil, indicating the need for evaluation of the microbial biomass K as a source and a stock of K in soil that has been overlooked.     

Leak-preventing effect of synthetic soil conditioners in paddy field

Synthetic soil conditioners have been studied by many soil scientists, but their studies were mainly on the formation of the water-stable aggregate of soils. But it may be possible that, if synthetic soil conditioners, the water-soluble polymers, are spread on the plowsole of the leaky paddy field, the leakage of water is prevented by the thin layer of the polymer formed by the swelling with irrigation water.     

Bt-transgenic rice straw affects the culturable microbiota and dehydrogenase and phosphatase activities in a flooded paddy soil

There have been few investigations of the possible effects of genetically engineered plants on the microbiota and enzyme activities in flooded soil. We studied the influence of the transgenic rice KeMingDao (KMD) straw on the culturable microbiota and enzymatic activities in a flooded paddy soil under laboratory conditions. KMD contained a synthetic cry1Ab gene from Bacillus thuringiensis under the control of a maize ubiquitin promoter and linked in tandem with the gusA and hpt genes. The results showed that there were only some occasional significant differences (P<0.05) in the number of Colony forming units of aerobic bacteria, actinomycetes and fungi and in the number of anaerobic fermentative bacteria, denitrifying bacteria, hydrogen-producing acetogenic bacteria, and methanogenic bacteria between the paddy soil amended with Bt-transgenic rice straw and with the non-Bt parental rice straw during the early stages of incubation. From d14 to d84 there were significant increases (P<0.05) in soil dehydrogenase and soil neutral phosphatase activity in soils amended with rice straw compared to soil without added straw. The dehydrogenase activity was significantly greatly (almost 1.95-fold) in soil amended with Bt-transgenic straw from d7 to d14 but from d21 to d49 there was significantly greater activity (about 1.47-fold) in the soil amended with non-Bt-straw. There were no apparent differences between the activity of soil neutral phosphatase in the soils to which non-Bt-straw and Bt-straw had been added. However, both soils to which rice straws were added demonstrated significant differences in the number of microorganisms except for aerobic bacteria and enzymatic activities with respect to the control soil throughout the incubation. The above results indicated that the Bt-straw from KMD transgenic rice is not toxic to a variety of culturable microorganisms in the studied flooded paddy soil.     

Incorporation of winter grasses suppresses summer weed germination and affects inorganic nitrogen in flooded paddy soil

ABSTRACT

Laboratory experiments were conducted to investigate how incorporation of the winter grasses foxtail (Alopecurus aequalis) and milk vetch (Astragalus sinicus) into flooded paddy soil affects the germination of summer weeds and soil inorganic nitrogen (N) levels. Polyethylene terephthalate (PET) vessels (10.0 × 8.0 × 15.0 cm) were filled with 0.7 kg air-dried soil that had been collected from organic paddy fields located in Tochigi, Japan. Foxtail and milk vetch were then independently incorporated into the soil at rates of 0% (untreated control), 0.25%, 0.5%, 1%, and 1.5%, with four repetitions per treatment. The PET vessels were placed in a growth chamber (Biotron) set to day/night temperatures and humidities of 27/18°C and 70%/65%, respectively, and with an illuminance of 40,000 lx (12 h) for 28 days, and the total number of weeds and changes in the electrical conductivity (EC) of the surface water were investigated throughout this period, while changes in soil inorganic N were determined at the ends of the experiments. The incorporation of both foxtail and milk vetch significantly suppressed the germination of summer weeds by 56% and 50%, respectively, at incorporation rates of 1% and by 78% and 77%, respectively, at incorporation rates of 1.5% compared with their respective controls. There was a high and significant positive correlation between both the rate of winter grass incorporation and the degree of growth suppression and EC. Incorporation of foxtail at rates of 0.25% and 0.5% significantly increased the inorganic N contents of the sub-layer by an average of 68% compared with the control but had no significant effect in the top layer, whereas the incorporation of milk vetch at a rate of 1% significantly increased the inorganic N contents of both the top layer and sub-layer by 200% and 316%, respectively, compared with the control. Furthermore, the effect of milk vetch on inorganic N significantly increased with an increase in the rate of incorporation. These findings demonstrate that incorporation of the winter grasses foxtail and milk vetch into flooded organic paddy soil can suppress weed growth and act as a source of inorganic N.     

典型红壤水稻土剖面细菌、真菌分子生态网络特征研究

土壤表层及深层的微生物互作对土壤养分循环和物质转化等具有重要意义。为了揭示典型红壤水稻土剖面微生物分子生态网络特征,通过16S rRNA和18S rRNA高通量测序技术,运用CoNet分析方法,构建上层（0 ~ 20 cm）、中层（20 ~ 60 cm）和下层（60 ~ 100 cm）土壤细菌、真菌分子生态网络。结果表明,随土壤深度的增加,总碳和总氮含量、细菌和真菌的丰富度（P < 0.05）显著降低。微生物网络拓扑参数,如连通度、群聚系数和网络密度等均明显增加,表明微生物互作随深度增加而增强。与细菌和真菌界内网络互作特征相反,界间的网络互作随深度减弱。进一步利用随机森林模型和方差分解分析（VPA）,揭示了碳和氮是影响微生物分子生态网络的主要因素,且随深度增加,对微生物互作网络贡献最大的因素由氮转变为碳。     

落干对表层水稻土氮素转化及关键功能微生物丰度的影响

落干过程中稻田土壤氮素转化发生显著变化,但表层土壤不同层次的氮素转化特征及相关机制尚不清楚。通过室内土壤培养试验,探讨了落干过程中(9 d)上表层(0～5 cm)和下表层(5～10 cm)水稻土氧化还原电位(Eh)、土体氧化亚氮(N2O)浓度和排放通量等的动态变化特征,并利用实时荧光定量PCR测定氨氧化基因(古菌amoA和细菌amoA)和硝酸盐还原酶基因(narG和napA)的丰度变化。结果表明,经9 d落干上表层水稻土Eh由-200 mV上升至500 mV左右,而下表层在-200～0 mV之间波动。上表层水稻土NH4+-N含量下降速率和NO^3--N含量上升速率分别是下表层的2.8和1.8倍。落干过程中,上表层水稻土氨氧化作用可能是由氨氧化细菌(AOB)主导的,而驱动硝酸盐还原作用可能以含napA基因反硝化微生物为主。