Characterization of ammonia oxidizing bacteria associated with weeds in a Japanese paddy field using amoA gene fragments

Abstract

Ammonia oxidizing bacteria (AOB) are important microorganisms in rice paddy field ecosystems because they play a key role in the nitrogen (N) cycle by converting ammonia (NH₃) to nitrite (NO^? ₂). In this study, we investigated AOB associated with three types of weeds in a Japanese paddy field (semi-aquatic Echinochloa oryzicola Vasing, floating Lemna paucicostata Hegelm and submerged Najas graminea Delile) using molecular techniques polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and DNA sequencing targeting ammonia monooxygenase (amoA) gene. This work confirmed that rice paddy weeds harbor AOB and that the community composition is different for each type of weed. However, all AOB sequences associated with the tested weeds were closely related to known species of Nitrosospira-like AOB isolated from soil, suggesting that AOB associated with weeds were not specific to weeds and can also be found in the soil. Nitosomanas-like AOB were not detected on any of the weeds tested. In addition, the most dominant AOB strains present in the tested weeds were closely related to Nitrosospira sp. Ka3 and Nitrosospira sp. CT2F. The phylogenetic tree revealed that most of the AOB detected in the present study belonged to amoA cluster 1.     

Succession and phylogenetic profile of eukaryotic communities in rice straw incorporated into a rice field: Estimation by PCR-DGGE and sequence analyses

Abstract

Succession and the phylogenetic profile of eukaryotic communities associated with rice straw decomposition in a rice field were studied using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by 18S rDNA sequencing. Nylon mesh bags containing leaf sheaths or blades were buried in the plow layer of a rice field under flooded conditions after transplanting (Experiment 1) and under drained conditions during the off-crop season (Experiment 2). In addition, rice straw samples in Experiment 2 were taken out before plowing in spring and re-placed in the rice field under flooded conditions at transplanting. Statistical analyses based on DGGE patterns showed that eukaryotic communities were divided into two groups, namely group A before the placement in soil, after the mid-season drainage in Experiment 1 and under the drained conditions in Experiment 2 and group B before the mid-season drainage in Experiment 1 and under the flooded conditions in Experiment 2. Based on the sequence analysis of DGGE bands, which characterized the eukaryotic communities, succession of the communities was revealed, that is, most of the bands in group A were closely related to fungi, whereas the bands in group B were closely related to protozoa. These results indicated that eukaryotic communities associated with rice straw decomposition in the rice field are mainly affected by soil conditions, such as oxic or reduced conditions, irrespective of rice straw parts (leaf sheaths and blades).     

Community structure of bacteria and fungi responsible for rice straw decomposition in a paddy field estimated by PCR-RFLP analysis

Rice straw including leaf sheaths and blades put in nylon mesh bags was placed in the plow layer of a Japanese paddy field after harvest under upland conditions and after transplanting of rice seedlings under flooded conditions. In addition, rice straw that was decomposed under the upland conditions during the off-crop season in winter was placed again in soil at the time of transplanting. The materials were collected periodically to analyze the community structure of the bacteria and fungi responsible for rice straw decomposition by PCR-RFLP analysis. The PCR products with 27f and 1492r primers designed for bacterial 16S rDNA and with EF3 and EF4 primers designed for fungal 18S rDNA were digested with four restriction endonucleases (Hinf I, Sau3A I, Hae III, EeoR I). Bacterial communities in the decomposing rice straw were different from each other between upland and flooded conditions, between leaf sheaths and blades, and between straw samples with and without decomposition under upland conditions during the off-crop season. Fungal communities in the decomposing rice straw were also different between the leaf sheaths and blades under upland soil conditions. Score plots of bacterial and fungal communities in the principal component analysis were separated from the plot of the straw materials along with the duration of the placement, indicating the succession of bacterial and fungal communities in decomposing rice straw with time.     

Isolation and some properties of methane-oxidizing bacteria from a subtropical paddy field

Methane is the second most important greenhouse gas which contributes to global warming. As an important source of methane, rice paddy fields contribute an estimated lO% to the global methane emissions (IPCe 1992). Land use and agricultural practices significantly affect atmospheric methane fluxes (Bouwman 1989; Hütsch et al. 1994). Microbial oxidation of atmospheric methane in terrestrial environments is the only known net biological methane sink and the process consumes the equivalent of 1–l0% of the total global emission (Adamsen and King 1993). Methane-oxidizing bacteria (MOB, methanotrophic bacteria) are considered to be obligately or facultatively aerobic respiratory bacteria that can utilize methane as the sole source of carbon and energy for growth (Hanson et al. 1992; Roslev and King 1994). As a result, they are important regulators of atmospheric methane fluxes in nature (Mancinelli 1995). MOB have been isolated from a variety of environments including freshwater lakes, wetlands, and the open ocean (Whittenbury et al. 197Gb; Saralov et al. 1984; Holzapfel-Pschorn et al. 1985; Hanson et al. 1992; Omelchenko et al. 1993; Bowman et al. 1993a; Mancinelli 1995). However, reports on the isolation of MOB from rice paddy fields are limited. More information is needed on the ecology and taxonomy of MOB in paddy fields.     

稻田水分管理和秸秆还田对甲烷排放的微生物影响

为了探讨不同水分管理和秸秆还田对稻田甲烷排放的影响机理,依托中国科学院亚热带农业生态研究所长沙农业环境监测研究站长期定位试验,选择长期淹水(CF)、秸秆还田+长期淹水(HS+CF)、常规灌溉(IF)、秸秆还田+常规灌溉(HS+IF)4个处理,应用限制性片段长度多态技术(T-RFLP)和实时荧光定量PCR技术在DNA和c DNA水平对比研究不同水分管理和秸秆还田影响稻田土壤甲烷排放的微生物分子机理。结果表明:长期淹水处理的甲烷排放量显著高于常规灌溉处理,秸秆还田处理对甲烷排放的影响不显著。产甲烷菌和甲烷氧化菌群落结构只对土壤水分管理产生响应,对秸秆还田没有响应。水分管理和秸秆还田显著影响产甲烷菌的数量,却对甲烷氧化菌数量没有影响。产甲烷菌和甲烷氧化菌群落组成和表达群落组成存在明显的分异,c DNA水平上产甲烷菌和甲烷氧化菌表达群落组成对水分管理的响应比DNA水平群落组成更敏感。稻田甲烷排放通量与mcr A和pmo A基因丰度和表达丰度均没有显著相关性,只与DNA水平基因丰度比值(mcr A/pmo A)呈显著正相关关系。可以看出,长期淹水处理可以通过改变土壤中产甲烷菌和甲烷氧化菌的群落结构和数量及两者数量的比例来调控甲烷的排放量。     

Soil carbon budget in a single-cropping paddy field with rice straw application and water management based on soil redox potential

Abstract

An ideal state for agroecosystems to mitigate global warming should include both decreasing CO₂ and CH₄ emissions and increasing soil carbon storage. Two-year field experiments were carried out to examine the effects of water management (continuous flooding [CF] and Eh control [EH]) and rice straw management (application [+S] and removal [–S]) on the soil carbon budget in a single-cropping paddy field in Japan. The EH water management based on soil redox potential that the authors have proposed decreased the total CH₄ emission during the rice growing period compared with CF. The +S increased CO₂ emission as soil respiration during the non-flooded fallow period compared with –S, but also increased straw residues in the soil. However, there was little evidence for sequential carbon accumulation in the soil over the year by +S. The resultant annual budget of soil carbon was a loss of 32–103 g C m^?2 in the EH+S treatment compared with a loss of 166–188 g C m^?2 in the CF–S treatment. Taking into account the global warming potentials, the EH+S treatment also decreased the total CO₂-equivalent emission compared with the CF–S treatment. Consequently, a combination of appropriate water management and straw application will be an effective option in decreasing both CO₂-equivalent emission and sustaining soil carbon storage.     

Eukaryotic communities associated with the decomposition of rice straw compost in a Japanese rice paddy field estimated by DGGE analysis

To estimate the succession and phylogenetic composition of the eukaryotic communities responsible for the decomposition of rice straw compost under flooded conditions during the cultivation period of paddy rice, denaturing gradient gel electrophoresis (DGGE) analysis targeting 18S rDNA followed by sequencing was conducted in a Japanese paddy field. The eukaryotic communities in rice straw compost incorporated into the flooded paddy field were influenced by the mid-season drainage and mainly composed of fungi (Ascomycota, Zygomycota, and Chytridiomycota) and protozoa (Ciliophora, Euglyphida, and Dactylopodida), most of which existed continuously during the cultivation period of paddy rice. The results indicated that these eukaryotic members were associated with the decomposition of rice straw compost in paddy field soil directly or indirectly.     

稻草还田配施菌剂对晚稻生长和土壤酶活性的影响

探索秸秆腐解菌剂配施尿素和石灰在稻草全量还田中对晚稻生长、土壤酶活性和产量的影响。将人工收割后的早稻草按全量还田鲜重25 008 kg·hm-2,配施30 kg·hm-2秸秆腐解菌剂、150 kg·hm-2尿素和225 kg·hm-2石灰,施于稻田中,栽插晚稻秧苗,观察记录晚稻农艺性状,测定植株氮磷钾含量、土壤酶活性和有机质含量、生物量和产量。结果表明,稻草全量还田配施秸秆腐解菌剂、尿素和石灰,能够显著提高晚稻分蘖、土壤酶活性、产量构成农艺性状、植株氮磷钾含量、生物量和产量,相比于未施秸秆腐解菌剂处理,稻谷产量提高了13.12%,生物量提高了13.37%。稻草全量还田配施秸秆腐解菌剂对晚稻产量具有显著地增产效应。     

Microbial communities responsible for the decomposition of rice straw compost in a Japanese rice paddy field determined by phospholipid fatty acid (PLFA) analysis

To identify the microbial communities responsible for the decomposition of rice straw compost in soil during the rice cultivation period, phospholipid fatty acid (PLFA) composition of rice straw compost was determined by periodically sampling the compost from a Japanese rice field under flooded conditions. About 21% of the compost was decomposed within a period of 3 months. The total amount of PLFAs, as an indicator of microbial biomass, was significantly lower under drained conditions than under flooded conditions and was relatively constant during the flooding period. This indicates that the microbial biomass in the compost samples did not increase during the gradual decomposition of rice straw compost under flooded conditions. The proportion of branched-chain PLFAs (biomarker of Grampositive and anaerobic Gram-negative bacteria) slightly decreased during the early period after placement, and increased gradually afterwards. Among the branched-chain PLFAs, i15:0, ail5:0, i16:0 and i17:0 PLFAs predominated and their proportions increased gradually except for i16:0. The proportion of straight mono-unsaturated PLFAs (biomarker of Gramnegative bacteria) was almost constant throughout the period, and 18:1ω9 and 18:1ω7 PLFAs predominated. The proportion of straight poly-unsaturated PLFAs as a biomarker of eukaryotes including fungi was also constant throughout the period, except for a decrease under drained conditions. Straight poly-unsaturated PLFAs consisted mainly of 18:2ω6c PLFA. Therefore, these results suggest that the proportions of Gram-positive and anaerobic Gram-negative bacteria increased during the decomposition of rice straw compost in flooded paddy field. Statistical analyses enabled to divide PLFA patterns of microbiota in the rice straw compost into two groups, one group consisting of rice straw compost samples collected before mid-season drainage and the other of samples collected after mid-season drainage. Small squared distances among samples in cluster analysis indicated that the community structure of microbiota was similar to each other as a whole. These results suggest that the microbial communities changed gradually during the period of placement, and that mid-season drainage may have affected the community structure of microbiota. Principal component analysis of the PLFA composition suggested that the succession of microbiota along with the decomposition in flooded soil was similar between rice straw compost and rice straw and that the changes in the community structure during the decomposition in flooded soil were more conspicuous for rice straw than for rice straw compost.     

Effect of application of rice straw and cellulose on methane emission and biological nitrogen fixation in a subtropical paddy field

Methane-oxidizing bacteria (MOB, methanotrophs) limit the flux of methane to the atmosphere from sediments and soils, and consume atmospheric methane (King 1992; Oremland and Culbertson 1992). IPCC (1995) reported that an aerobic soil is equivalent to a sink of 10–20% of methane emissions. Hence MOB play an important role in regulating the atmospheric methane content (Mancinelli 1995). Over the last 20y, although a large amount of information has been supplied on the biochemistry of MOB, few ecological investigations have been devoted to them so far (Holzapfel-Pschorn et al. 1985; Oremland and Culbertson 1992).     

Effect of the long-term application of organic matter on soil carbon accumulation and GHG emissions from a rice paddy field in a cool-temperate region,Japan. -I. Comparison of rice straw and rice straw compost -

ABSTRACT

The influence of the long-term combination of rice straw removal and rice straw compost application on methane (CH₄) and nitrous oxide (N₂O) emissions and soil carbon accumulation in rice paddy fields was clarified. In each of the initial and continuous application fields (3 and 39?51 years, respectively), three plots with different applications of organic matter were established, namely, rice straw application (RS), rice straw compost application (SC) and no application (NA) plots, and soil carbon storage (0?15 cm), rice grain yield and CH₄ and N₂O fluxes were measured for three years. The soil carbon sequestration rate by the organic matter application was higher in the SC plot than in the RS plot for both the initial and continuous application fields, and it was lower in the continuous application field than in the initial application field. The rice grain yield in the SC plot was significantly higher than those in the other plots in both the initial and continuous application fields. Cumulative CH₄ emissions followed the order of the NA plot < the SC plot < the RS plot for both the initial and continuous application fields. The effect of the organic matter application on the N₂O emissions was not clear. In both the initial and continuous application fields, the increase in CH₄ emission by the rice straw application exceeded the soil carbon sequestration rate, and the change in the net greenhouse gas (GHG) balance calculated by the difference between them was a positive, indicating a net increase in the GHG emissions. However, the change in the GHG balance by the rice straw compost application showed negative (mitigating GHG emissions) for the initial application field, whereas it showed positive for the continuous application field. Although the mitigation effect on the GHG emissions by the combination of the rice straw removal and rice straw compost application was reduced by 21% after 39 years long-term application, it is suggested that the combination treatment is a sustainable management that can mitigate GHG emissions and improve crop productivity.     

稻草还田与施钾效果研究

采用田间小区试验研究了稻草还田对水稻生长发育、产量及土壤培肥的影响,结果表明:在洞庭湖贫钾地区,稻草还田能显著促进晚稻生长发育,提高结实率,增加千粒重。同时还可以提高土壤有机质含量,有利于培肥地力。在稻草还田的条件下,施用适量钾肥,对晚稻生长的促进作用和培土作用更加显著。