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.     

Comparison of phospholipid fatty acid composition in percolating water,floodwater, and the plow layer soil during the rice cultivation period in a Japanese paddy field

Azolla has been used as a green manure for rice in Vietnam and southern China (3). Recently it was adopted by farmers in the Koronadal area in South Cotabato Province, the Philippines (2). In that area Azolla pinnata grows reasonably well without phosphate fertilizer, which is generally used for stimulating Azolla growth (3, 5).     

Frequency of phage-infected bacterial cells in the floodwater of a Japanese paddy field

In aquatic environments, viruses play an important role in the microbial food web through microbial mortality from viral lysis. Bacteriophages (phages) compose the majority of viral communities in the floodwater of paddy fields. The present study evaluated bacterial mortality from phage lysis in the floodwater of a Japanese paddy field based on the frequency of phage-infected bacterial cells by transmission electron microscopy. Floodwater was sampled five times during the rice cultivation period from two plots of NPK plus lime (no-compost plot) and NPK plus lime and compost (compost plot) under a long-term and ongoing fertilizer trial that began in 1925. The frequency of visibly infected cells in the compost plot was larger, ranging from 2.4 to 3.6% (average 3.0%), than that in the no-compost plot, which ranged from 1.6 to 2.9% (average 2.0%). The fractions of bacterial mortality from phage lysis in the floodwater samples were estimated to range from 12.8 to 27.3% (average 17.2%) and from 21.7 to 35.0% (average 27.9%) for the samples collected from the no-compost plot and the compost plot, respectively. This is the first study to estimate bacterial mortality from phage lysis in the paddy field ecosystem, and the frequency of phage lysis in floodwater was found to be within the frequency ranges observed in other aquatic environments.     

Morphological analysis of viral communities in the floodwater of a Japanese paddy field

Capsid size distributions of viral communities in the floodwater of a Japanese paddy field under a long-term fertilizer trial were surveyed during the rice cultivation period by using transmission electron microscopy. The capsid size distributions were monophasic, and the median values fell within the range of 50-70 nm. The quartile intervals were narrow from either 40-50 nm or 50-60 nm to either 60-70 nm or 70-80 nm for most samples. There was no clear seasonal variation in the capsid size distribution of viral communities. The difference in size distributions among different fertilizer plots was also not found. Viral communities in the floodwater were predominated by isometric icosahedral forms. Viruses with octahedral capsids and elongated ones were rare and sporadic in the floodwater.     

Epibiotic bacteria associated with microcrustaceans in the overlying water of paddy fields

The surface of micro crustaceans collected from the overlying water of a submerged paddy soil was observed in detail by scanning electron microscopy. Epibiotic bacteria were less diverse compared to free-living bacteria in the overlying water. Specific bacteria appeared to preferentially colonize certain parts of microcrustaceans. The epibiotic bacteria, the sites they colonized and their density depended on the kinds of microcrustaceans (Moina macrocopa, Cyclopidae, Cypretta sp., and Tanycypris sp.). These results suggested the presence of significant associations in the overlying water between specific groups of bacteria and microcrustaceans.     

The distribution characteristics of the major capsid gene (g23) of T4-type phages in paddy floodwater in Northeast China

Our previous study revealed the high diversity of the major capsid gene (g23) of T4-type phages that existed in the paddy field soils in Northeast China. In this study, the phylogeny and genetic diversity of the g23 gene in the paddy floodwater samples collected from five sampling sites at three sampling times during the rice (Oryza sativa L.) growth season in Northeast China are reported. In total, 104 different g23 clones were isolated, among which 50% of the clones exhibited the highest identities with the clones retrieved in paddy soils and upland black soils. The remaining clones had the highest identities with lake origins. Phylogenetic analysis revealed that 43% of the g23 clones grouped into three novel subgroups which included the clones unique to paddy floodwater, and no g23 sequences obtained in paddy floodwater fell into the paddy soil groups II, III, IV, V, VI, VII and NPC-A. UniFrac analysis of g23 clone assemblages demonstrated that T4-type phage communities in paddy floodwater were changed spatially and temporally, and the communities were different from those in paddy soils. Further comparison of the g23 clone assemblages from different environments demonstrated that T4-type phages were biogeographically distributed, and the distribution was both affected by geographical separation and ecological processes across the biomes.     

稻田固氮解磷解钾菌筛选及其复合菌剂对土壤培肥作用

用固氮、解磷、解钾培养基,分别从稻田土壤中分离获得9个固氮菌株(A1-A9)、7个解磷菌株(B1-B7)和6个解钾菌株(C1-C6)。对各菌株固氮、解磷、解钾能力及水稻促生效果分析表明,最突出菌株分别为A5、B2、C5;各菌株等比例混合组成复合菌剂接入灭菌稻田土壤,30 d后可使土壤总氮、有效磷、速效钾分别增加21.32%、32.17%、45.57%;初步鉴定各菌株分别为固氮菌属(Azotobacter sp.)、假单胞菌属(Pseudomonas sp.)和芽孢杆菌属(Bacillus sp.)。复合菌剂处理稻田土壤,水稻苗高、鲜重、干重及植株全氮、全磷、全钾显著优于对照组,说明筛选菌株可在稻田土壤氮、磷、钾转换及培肥过程中起重要作用。     

长江中下游地区高产稻田施肥与产量的关系

根据对长江中下游的湖南、江西、浙江、安徽、湖北五省19个高产稻田产量、施肥、耕作制度及农田管理调查,研究高产稻田施肥与产量的关系。结果表明,长江中下游地区双季稻高产田N、P2O5和K2O最佳施用量分别约为461、130和430 kg/hm2,水稻年产量平衡在16 893 kg/hm2左右;水旱轮作高产田N、P2O5和K2O最佳施用量分别约为200、145和250 kg/hm2,作物年产量平衡在13 000 kg/hm2左右,其中水稻产量为9 500 kg/hm2左右。长江中下游地区双季稻高产田N∶P2O5∶K2O最佳施肥比例约为1∶0.28∶0.85,水旱轮作高产田N∶P2O5∶K2O最佳施肥比例约为1∶0.72∶1.23。     

磁场对稻谷干燥特性的影响

针对稻谷的传统干燥过程存在能量消耗大,发芽率低和爆腰率高等缺点,把磁场引入稻谷干燥过程中,能快速而经济地降低稻谷的水分并保持其良好品质。采用永磁场强化稻谷的干燥过程,比较干燥时间、发芽率和爆腰率的变化。结果表明：在相同功率下稻谷在磁场中干燥时间比无磁场时缩短5％,平均失水速率加大,稻谷的发芽率有所升高、爆腰率下降;随着干燥功率的增加,磁场对稻谷的发芽率和爆腰率的影响加大。在试验基础上探讨了磁场对稻谷干燥过程的影响机理。     

Molecular identification of methane oxidizing bacteria in a Japanese rice field soil

By using cultivation-independent techniques, community changes of methane-oxidizing bacteria (MOB) in rice bulk soils were investigated under field conditions in a Japanese rice field. The representative soil samples were collected during the typical rice growing season and nonrice growing period all year round. Statistical characterization of denaturing gradient gel electrophoresis (DGGE) community patterns of MOB pmoA/amoA functional gene fragments showed that MOB community structures in the rice bulk soils remained largely unchanged throughout the investigated period. The total intensity of six common DGGE bands that appeared consistently throughout the investigated period accounted for 64% of the total intensity of all 18 different DGGE bands detected. The low squared distance of the Ward cluster analysis of the DGGE pattern and the high Sorensen similarity coefficient (81%) also implied the high similarity of the MOB community structures. The stable MOB community structure did not couple well with the wide variation of soil water contents all year round. Sequencing analysis of the nine characteristic bands including six common bands revealed the presence of Type I, Type II methanotrophs, and β-proteobacterial ammonia oxidizers in rice bulk soils. In comparison with MOB type species, three DGGE bands showed a wide variation of the highly conserved amino acid residues, implying the presence of novel MOB bacteria inhabiting the rice bulk soil. The high diversity of MOB composition suggested that rice bulk soils might serve as an ideal reservoir for the dynamic changes of MOB in a rice field ecosystem in response to environment changes.     

Vertical Changes in Bacterial Communities in Percolating Water of a Japanese Paddy Field as Revealed by PCR-DGGE

Percolating water was sampled from the plow layer and subsoil layer in a Japanese paddy field, and the bacterial communities were compared together with floodwater by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) targeting a partial 16S rRNA gene and subsequent sequencing. The number of DGGE bands ranged from 16 to 28 with no significant differences among the sampling sites and times. Only 2 bands were common for the three sources of water samples. DGGE bands specific for the floodwater samples and percolating water samples from the plow layer were identified, while percolating water samples from the subsoil layer did not show specific bands but displayed common bands to those of the floodwater samples (7 bands) and percolating water samples from the plow layer (1 band). Cluster analysis of the DGGE banding patterns showed a distinct clustering in the samples of percolating water from the plow layer and a closer relationship between the others. These results suggest that the bacterial communities in percolating water changed during downward movement through the plow layer and subsoil layer. Sequences of the DGGE bands specific for the samples of percolating water from the plow layer showed a close relationship with anaerobic bacteria such as iron-reducers or uncultured bacterial DNA isolated from environments that are considered to be less oxic. On the other hand, the sequences of the bands specific for the samples of floodwater and percolating water from the subsoil layer showed a close relationship with uncultured bacterial DNA isolated from freshwater environments.     

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).     

Farm-scale variations in soil color as influenced by organic matter and iron oxides in Japanese paddy fields

To understand how soil color is influenced by soil components at the farm scale, we evaluated spatial variation in soil color and related soil properties in Japanese paddy fields. After harvest of rice, 246 surface soil samples were collected in 10-m grids from five contiguous irrigated paddy fields, each with an area of about 0.5 ha. The samples were analyzed to determine color parameters (L*, a*, and b*), and contents of total C, total N, Fe oxides, sand, and loss-on-ignition. The results obtained were modeled and mapped geostatistically. All color parameters indicated strong spatial dependence with long ranges (>85 m). In contrast, total C and N showed short ranges (about 40 m). The contents of Fe oxides, sand, and loss-on-ignition showed intermediate ranges (50–85 m). The ranges of these properties and their distribution patterns suggested that the contents of total C and N were influenced by long-term application of manure and that sand content was influenced by topography and past land consolidation. Further soil color analysis after removal of organic matter or silt plus clay particles revealed that soil organic matter, texture, and Fe oxides affected soil color parameters in a complex manner. Prediction of total C from soil darkness was hindered by the presence of silt plus clay particles containing Fe oxides. On the other hand, citrate-dithionite extractable Fe was estimated accurately from the b* value (yellowness), which can be useful for predicting the occurrence of akiochi (autumn decline) disease of rice at the farm scale.     

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.