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.     

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.     

Comparison of g23 gene sequence diversity between Novosphingobium and Sphingomonas phages and phage communities in the floodwater of a Japanese paddy field

Our previous study indicated that the diversity of the major capsid gene (g23) of T4-type bacteriophages (phages) of Novosphingobium and Sphingomonas strains isolated from the floodwater of a Japanese paddy field is comparable to those of the clones obtained from other Japanese paddy fields. For more strict comparison of the diversity, this study examined g23 sequences between Novosphingobium and Sphingomonas phages and phage communities in the identical floodwater of a Japanese paddy field. The clones were obtained by applying g23-specific primers to DNA extracted from the floodwaters. Many 23 clones in the floodwater were grouped into the same clusters of Paddy Groups I-VI with g23 genes of Novosphingobium/Sphingomonas phages with some clones belonging to an additional cluster. In addition, the remaining clones belonged to the clusters of marine clones and T4-type enterophages. These findings indicate that the g23 genes in the floodwater are more diversified than those of Novosphingobium/Sphingomonas phages including g23 genes closely related to the genes of enterophages and marine origins.     

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.     

Seasonal variations in the abundance of virus-like particles and bacteria in the floodwater of a Japanese paddy field

Abstract

Viruses are the most abundant biological entities in marine and freshwater environments. Many studies have shown the ecological importance of viruses in the primary production and microbial food web in aquatic environments. However, no studies have examined viral abundance in the floodwater of paddy fields. The present study surveyed the abundance of virus-like particles (VLPs) and bacteria in the floodwater of a Japanese paddy field under a long-term fertilizer trial since 1925 during the rice cultivation period. Virus-like particles and bacterial abundances in the floodwater ranged from 5.6 × 10⁶ to 1.2 × 10⁹ VLPs mL^?1 and from 9.2 × 10⁵ to 4.3 × 10⁸ cells mL^?1 with mean abundances of 1.5 × 10⁸ VLPs mL^?1 and 5.1 × 10⁷ cells mL^?1, respectively, and increased with an increase in the turbidity of the floodwater with suspended particles. The magnitude of seasonal variation was more than 50-fold for VLP abundance and 100-fold for bacterial abundance. The virus-to-bacterium ratios fluctuated over the rice cultivation period, ranging from 0.11 to 72 and their increase correlated with the decrease in bacterial abundance. Our results suggest that viral abundance in the floodwater of paddy fields is larger than in natural marine and freshwater environments.     

Survey of major capsid genes (g23) of T4-type bacteriophages in Japanese paddy field soils

Many studies have shown the ecological importance of viruses as the greatest genomic reservoirs on the planet. As bacteriophages (phages) comprise the majority of viruses in the environment, we surveyed the capsid genes (g23) of T4-type phages, Myoviridae, from DNA extracts of three paddy field soils located in northern, central and southern Japan using the degenerate primers MZIA1bis and MZIA6. Denaturing gradient gel electrophoresis (DGGE) was performed to separate PCR-amplified g23 products, and 56 DGGE bands were identified as g23 fragments. Only nine clones were grouped into T-evens, PseudoT-evens and ExoT-evens, and most of the other clones were classified into Paddy Groups I-VI. No significantly different distribution of g23 clones was observed among the paddy fields at the group level, indicating that phage communities estimated from the g23 composition were common on the nationwide level. Comparison of g23 sequences showed that g23 genes in paddy fields were different from those in marine environments, and more divergence of g23 genes was estimated in the paddy fields compared to the marine environment. Two novel g23 clones with very short amino acid residues were detected, suggesting the existence of uncharacterized, novel groups of g23 genes in paddy field soils.     

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.     

Unique viral capsid assembly protein gene (g20) of cyanophages in the floodwater of a Japanese paddy field

In order to evaluate the genetic diversity of cyanophage communities of rice fields, viral capsid assembly protein gene (g20) was amplified with primers CPS1 and CPS8. The DNA was extracted three times from viral concentrates obtained from floodwater samples collected in each of four different plots (no fertilizer; P and K chemical fertilizers; N, P, and K chemical fertilizers; and chemical fertilizers with compost). Denaturing gradient gel electrophoresis (DGGE) gave different g20 clones. The sequencing of DGGE bands revealed that the g20 genes of the floodwater were divergent and that the majority of clones formed several unique groups. However, they were more closely related to g20 sequences from freshwaters than to those from marine waters, suggesting that g20 genes in terrestrial aquatic environments are different from those in marine environments.     

Novel capsid genes (g23) of T4-type bacteriophages in a Japanese paddy field

Viruses exist everywhere on the planet. Recent development in viral genomics confirmed that genomic information is preserved among viral subsets and can be used for phylogenetic classification of viruses and for evaluation of viral diversity in the environment. The capsid gene of T4-type bacteriophages, g23, is the most widely applied gene for evaluating the diversity of the T4-type bacteriophage family. In this study, we applied denaturing gradient gel electrophoresis to PCR products of DNA with g23-specific primers that were extracted from a Japanese paddy field under long-term fertilizer trial and obtained 39 different g23 clones at the DNA level. They showed identities of 27–99% with the clones within the NCBI database at the amino acid level. They were quite distinctive from those obtained in marine environments and most of them formed six phylogenetically novel groups in the T4-type bacteriophage family with the clones obtained from another paddy field. The existence of six novel groups was confirmed from molecular analysis of all the amino acid sequences between the primers, of the amino acid sequences excluding hypervariable region, and of those of conserved regions. These findings indicate that T4-type bacteriophage communities in paddy fields consist of previously uncharacterized members phylogenetically distant from those in marine environments. The type of fertilizers and the stage during rice cultivation were not the major factors in determining T4-type bacteriophage communities in the paddy field.     

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.     

基于GIS的江西省农田划分等级研究

如何解决当前农业税征收中的不合理现象,探索一种科学、简明、易于实践操作的农用土地等级评价方法,是当前亟待解决的热点问题,通过引入先进的GIS技术手段,对江西省农田划分等级进行研究,将江西省303.25万hm^2农田分成6个等级,为农业税征收提供客观标准。     

Morphology, host range and phylogenetic diversity of Sphingomonas phages in the floodwater of a Japanese paddy field

Members of the Sphingomonas -related genera ( Sphingomonas , Sphingobium , Novosphingobium and Sphingopyxis ) are dominant in bacterial isolates from the floodwater of Japanese paddy fields. Fifty-eight Sphingomonas / Novosphingobium bacteriophages (phages) were isolated to elucidate their morphology, host range and phylogenetic diversity based on the capsid gene ( g23 ) sequence. All of the phages were siphoviruses with isometric or elongated, icosahedral capsids and a long, non-contractile tail. The genomes were double-stranded DNA measuring either 40, 60, 100 or 160 kb. The host range of the phages was examined by infecting 16 bacterial isolates from the floodwater, belonging to Sphingomonas , Novosphingobium , Sphingopyxis and Porphyrobacter . The host range was widely different and varied between infection of only the host used for isolation and infection of hosts belonging to the three genera of Sphingomonas , Novosphingobium and Porphyrobacter . All phages had g23 , indicating the ubiquity of the g23 gene among Myoviridae and Siphoviridae members. Every g23 sequence of the phages belonged to one of the six uncharacterized Paddy Groups proposed by Fujii et al . (2008 ). The g23 sequences were identical at the nucleotide level for several phages with isometric and elongated capsids with 60 and 160 kb genomes, and between some phages and the clones that were retrieved from distant paddy fields. This indicates the common occurrence of horizontal transfer of g23 in the paddy fields. The g23 sequence does not correlate with the host range of those phages. In addition, a larger degree of divergence of g23 from coliphage T4 in paddy fields compared to marine environments was estimated from the present study.     

Microbial community responsible for the decomposition of rice straw in a paddy field: estimation by phospholipid fatty acid analysis

To estimate the microbial communities responsible for rice straw decomposition in paddy field, phospholipid fatty acid (PLFA) composition of leaf sheaths and blades was analyzed during the decomposition of both residues under upland conditions after harvest and under flooded conditions at the time of transplanting of rice plants. In addition, rice straw that had been placed in the field under upland conditions (November to April) was taken out in spring, and placed again in the same field under flooded conditions at the time of transplanting. High proportions of the branched-chain PLFAs were observed under flooded conditions (June to September); the proportions of straight mono-unsaturated and straight poly-unsaturated PLFAs were high under upland conditions in the winter season for 4 months. The dominant PLFAs in straight mono-unsaturated, straight poly-unsaturated and branched-chain PLFA groups were 18:19, 18:17 and 16:17c, 18:26c and i15:0, i17:0 and ai15:0, respectively, under both upland and flooded conditions. These findings indicated the important roles of Gram-negative bacteria and fungi under upland conditions and of Gram-positive bacteria and anaerobic Gram-negative bacteria under flooded conditions. Cluster analysis of PLFA composition showed the difference of community structure of microbiota in rice straw between upland and flooded conditions. In addition principal component analysis revealed the difference between leaf sheaths and blades under upland conditions and indicated that the content of straight unsaturated PLFAs (sheaths > blades) characterized their community structures.     

稻田温度与甲烷排放通量关系的研究

稻田温度与CH4 排放通量有密切关系 ,通过灰关联分析发现稻田 5cm深处温度与CH4 排放通量关系最密切 ,水稻抽穗期CH4 排放通量达极大值 ,而完熟期CH4 排放通量达极小值。温度对CH4 排放通量的增效应明显 ,减效应较弱。稻田温度与CH4排放通量的关系呈S型曲线相关关系。     

DGGE法与常规培养法对稻田蓝细菌多态性分析结果比较

研究运用蓝细菌和硅藻16SrDNA特异引物,将晚季水稻生长后期稻田土壤中提取的总DNA进行PCR扩增后,以DGGE技术对PCR产物进行分析结果表明,14条DGGE带经克隆测序,经NCBI基因库比对得晚季水稻生长后期存在10种蓝细菌,包括4种Leptolyngbya、1种Chamaesiphon、1种Nostoc、1种Oscillatoria、2种Syne-chococcus和1种Chroococcidiopsis。同层不同位置土壤中蓝细菌种群亦有所不同,但每个取样点都有一些特有的蓝细菌种类。用常规方法对同一稻田土壤样品进行分离培养,根据蓝细菌鉴定图谱观察到类似Lyngbya、Oscillatori-a、Chroococcidiopsis及Nostoc的蓝细菌,但显微镜下无法准确分类。比较结果表明采用DGGE法比常规培养法能更准确进行蓝细菌多态性鉴定。     

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).