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1.
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.  相似文献   

2.
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.  相似文献   

3.
We surveyed the major capsid genes (g23) of T4-type bacteriophages using the primers MZIA1bis and MZIA6 and DNA extracted from seven upland black soils in Northeast China. In total, 99 different g23 clones were obtained. Approximately half of the clones fell into paddy groups, whereas the rest belonged to one of several groups containing only clones from upland black soils or remained ungrouped, suggesting that the T4-type phage communities in the upland black soil were relatively similar to those in paddy field soils but that specific communities exclusively inhabit the upland black soil. UniFrac analysis of all of the g23 clones obtained from various environments indicated that the T4-type phage communities varied among marine, lake, paddy field soil and upland soil environments and that the T4-type phage communities in upland black soils varied by sampling location.  相似文献   

4.
Although microbial communities in soil are well known to change with soil depth, the changes in viral communities with soil depth have not been documented. This study examined the soil depth profiles of T4-type phage communities in two Japanese rice fields from g23 clones in soil DNA extracts to a depth of 1 m. T4-type phage communities changed with soil depth, and the communities were grouped into two groups: the communities of the surface soil layers, where rice roots developed densely, and those of the subsoil layers. Although coarse- and fine-textured soils were stratified in the subsoil layers in both profiles, denaturing gradient gel electrophoresis band patterns and phylogenetic affiliation of g23 were highly similar to each other among the subsoil layers in both fields, indicating that soil texture did not affect T4-type phage communities in these fields. In addition, some clones had g23 sequences identical to those retrieved from rice fields in Northeast China, indicating that closely related viruses and their hosts distribute across the sea between rice fields in Japan and Northeast China.  相似文献   

5.
The present study compared the capsid gene ( g23 ) of T4-type bacteriophages (phages) in Mn nodules with those in the plow layer soil and subsoils of two Japanese paddy fields by applying the primers MZIA1bis and MZIA6 to DNA extracts from the nodules and soils. The deduced amino acid sequences of the g23 genes in the Mn nodules were similar to those in the plow layer soil and in the subsoils. This result indicated that similar T4-type phage communities developed at these sites and that the diversity of T4-type phage communities was wide enough to cover those in the plow layer soil and in the subsoils. The majority of g23 clones formed several clusters with the clones and phages obtained from far-apart paddy fields, and the sequences of two clones were completely identical to a phage and a clone from other paddy fields at the nucleotide or amino acid level, indicating horizontal transfer of g23 genes between those paddy fields. A clone with a long nucleotide residue (686 bp) and a distribution remote from the other clones in the phylogenetic tree indicated that there were many uncharacterized, novel g23 genes in the paddy fields.  相似文献   

6.
Abstract

The present study examined T4-type phage communities in rice straw (RS) under the composting process by analyzing the composition of the major capsid gene (g23) of T4-type bacteriophages. The g23 clones were obtained from RS throughout the composting process from RS materials to composting RS in the curing stage (for 124?days). Most of the g23 clones were phylogenetically closely related to those in rice field soils and rice field floodwaters, and Paddy Groups II and III appeared to characterize the g23 genes in the composting RS. The diversity of g23 genes in the composting RS was highest in the RS material (day 0 after the onset of composting) and in the early thermophilic stage (day 7), and decreased markedly in the middle and curing stages. This change was in contrast to that of the bacterial community, which showed higher diversity in the middle and curing stages. There was no specific clone that characterized any stage during the composting process. These findings indicate that the phage community is not the major controlling agent in determining eubacterial succession and that the thermophilic stage in the composting process efficiently annihilated T4-type phages in the composting pile.  相似文献   

7.
Bacteriophages (phages) are the most abundant biological entities on the planet and are important as the greatest genomic reservoirs in both marine and terrestrial environments. In this study, we analysed T4-type phage communities in an upland black soil by monitoring g23 clones in DNA extracted from seasonal soil samples with no fertilizer, chemical fertilizers, chemical fertilizers plus manure, and natural restoration treatments. PCR products with degenerate primers MZIA1bis and MZIA6 were subjected to denaturing gradient gel electrophoresis. In total, 46 clones with different g23 sequences were obtained. Phylogenetic analyses indicated that T4-type phage communities in the upland black soil were distinctly different from those in marine environments and in an Antarctic lake, which strongly suggested that T4-type phage communities in soil differed from those in aquatic environments. Among 46 clones, 18 clones formed clusters with the clones from rice field soils, 14 clones formed three new clusters, and 13 clones were left as ungrouped, which indicated that T4-type phage communities in the upland black soil were relatively similar to those in rice field soils but that specific communities also inhabit in the upland black soil exclusively.  相似文献   

8.
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.  相似文献   

9.
The present study examined T4-type phage communities in rice straw (RS) under the composting process by analyzing the composition of the major capsid gene ( g23 ) of T4-type bacteriophages. The g23 clones were obtained from RS throughout the composting process from RS materials to composting RS in the curing stage (for 124 days). Most of the g23 clones were phylogenetically closely related to those in rice field soils and rice field floodwaters, and Paddy Groups II and III appeared to characterize the g23 genes in the composting RS. The diversity of g23 genes in the composting RS was highest in the RS material (day 0 after the onset of composting) and in the early thermophilic stage (day 7), and decreased markedly in the middle and curing stages. This change was in contrast to that of the bacterial community, which showed higher diversity in the middle and curing stages. There was no specific clone that characterized any stage during the composting process. These findings indicate that the phage community is not the major controlling agent in determining eubacterial succession and that the thermophilic stage in the composting process efficiently annihilated T4-type phages in the composting pile.  相似文献   

10.
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.  相似文献   

11.
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.  相似文献   

12.
Abstract

Methane-oxidizing bacteria (MOB) are crucial to the reduction of CH4 emitted to the atmosphere. However, it is unclear how MOB in rice straw are affected by straw decomposition processes. In a Japanese rice field, a year-round experiment was set up to study the effects of agricultural practice (rice cultivation/winter fallow), straw parts (leaf sheath/blade) and the site of straw placement (plow layer/soil surface) on MOB communities in rice straw using denaturing gradient gel electrophoresis (DGGE) and DNA sequencing analyses of key MOB functional genes (pmoA and amoA). Thirty-eight different DGGE bands were observed over the entire investigation period. Principal component analysis of DGGE pattern suggested that agricultural practice is the key factor regulating the MOB communities. Sequencing of dominant DGGE bands showed that: (1) during the rice cultivation period, methanotrophs (particularly type I methanotrophs) dominated the MOB community, (2) during the winter fallow season both type I and type II methanotrophs were dominant in sheath segments placed both on the soil surface and in the plow layer, whereas ammonia oxidizers seemed to dominate blade segments placed in the plow layer. Alignment of diagnostic amino acid sequences of MOB suggested the presence of novel ammonia oxidizers in rice straw in rice fields.  相似文献   

13.
Denitrification is an important part of the nitrogen cycle in the environment, and diverse bacteria, archaea, and fungi are known to have denitrifying ability. Rice paddy field soils have been known to have strong denitrifying activity, but the microbes responsible for denitrification in rice paddy field soils are not well known. Present study analyzed the diversity and quantity of the nitrite reductase genes (nirS and nirK) in a rice paddy field soil, sampled four times in one rice-growing season. Clone library analyses suggested that the denitrifier community composition varied over sampling time. Although many clones were distantly related to the known NirS or NirK, some clones were related to the NirS from Burkholderiales and Rhodocyclales bacteria, and some were related to the NirK from Rhizobiales bacteria. These denitrifiers may play an important role in denitrification in the rice paddy field soil. The quantitative PCR results showed that nirK was more abundant than nirS in all soil samples, but the nirK/nirS ratio decreased after water logging. These results suggest that both diversity and quantity changed over time in the rice paddy field soil, in response to the soil condition.  相似文献   

14.
In a Japanese forest, CH4 uptake rate and methanotrophic community structure in the soil were investigated at four sites of different vegetation. At two of these sites, undergrowth was dominated by Sasa senanensis, and that of another was dominated by Sasa kurilensis. At the rest site, undergrowth had been removed artificially. The tree-layer composition differed between the two sites with S. senanensis, but tree layer of the other two sites were dominated by the same species. At the site lacking undergrowth, observed CH4 uptake rate was twice as high as that at the other sites. Under laboratory conditions, soil sample from the site lacking undergrowth exhibited CH4 consumption rate higher than that of the adjacent site with the same dominating tree species. The community structures of methanotrophs were investigated with denaturing gradient gel electrophoresis (DGGE) of the gene encoding particulate methane monooxygenase (pmoA). The banding patterns observed were different depending on the type of undergrowth vegetation. The sequences of the DGGE bands were closely related to each other and belonged to the “upland soil cluster alpha” (USCα). These results imply possible close relationship between the undergrowth vegetation and methanotrophic communities in forest soils.  相似文献   

15.
In-season diagnosis of crop nitrogen(N) status is crucial for precision N management. Critical N(N_c) dilution curve and N nutrition index(NNI) have been proposed as effective methods to diagnose N status of different crops. The N_c dilution curves have been developed for indica rice in the tropical and temperate zones and japonica rice in the subtropical-temperate zone, but they have not been evaluated for short-season japonica rice in Northeast China. The objectives of this study were to evaluate the previously developed N_c dilution curves for rice in Northeast China and to develop a more suitable N_c dilution curve in this region. A total of17 N rate experiments were conducted in Sanjiang Plain, Heilongjiang Province in Northeast China from 2008 to 2013. The results indicated that none of the two previously developed N_c dilution curves was suitable to diagnose N status of the short-season japonica rice in Northeast China. A new N_c dilution curve was developed and can be described by the equation N_c = 27.7 W~(-0.34) if W ≥ 1 Mg dry matter(DM) ha~(-1) or N_c = 27.7 g kg~(-1) DM if W 1 Mg DM ha~(-1), where W is the aboveground biomass. This new curve was lower than the previous curves. It was validated using a separate dataset, and it could discriminate non-N-limiting and N-limiting nutritional conditions. Additional studies are needed to further evaluate it for diagnosing N status of different rice cultivars in Northeast China and develop efficient non-destructive methods to estimate NNI for practical applications.  相似文献   

16.
Although root cap cells are an important substrate for microorganisms in the rhizosphere, little attention has been paid to the decomposition of sloughed root cap cells by microorganisms. This study used rice plant callus cells grown on medium containing 13C-labelled glucose as a model material for rice plant root cap cells. Harvested 13C-labelled callus cells (78 atom % 13C) were subjected to decomposition in an aerobic soil microcosm for 56 days. The low cellulose and lignin levels and the disaggregated nature of the callus cells indicated that these cells were an appropriate model material for root cap cells. DNA was extracted from a soil incubated with 12C- and 13C-callus cells and subjected to buoyant density gradient centrifugation to identify bacterial species that assimilated carbon from the callus cells. The stability of the total bacterial communities during the incubation was estimated. Many DGGE bands in light fractions of soil incubated with 13C-callus cells were weaker in intensity than those from soil incubated with 12C-callus cells, and those bands were shifted to heavier fractions after 13C-callus treatment. 13C-labelled DNA was detected from Day 3 onwards, and the DGGE bands in the heavy fractions were most numerous on Day 21. DGGE bands from heavy and light fractions were sequenced, revealing more than 70% of callus- C incorporating bacteria were Gram-negative, predominantly α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Sphingobacteria and Actinobacteria. These species were phylogenetically distinct from the bacteria reported to be present during plant residue decomposition and resident in rice roots. This study indicates that root cap cells are decomposed by specific bacterial species in the rhizosphere, and that these species augment the diversity of rhizospheric bacterial communities.  相似文献   

17.
Abstract

The phylogenetic positions of bacterial communities in manganese (Mn) nodules from subsoils of two Japanese rice fields were estimated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by sequencing of 16S rDNA. The DGGE band patterns and sequencing analysis of characteristic DGGE bands revealed that the bacterial communities in Mn nodules were markedly different from those in the plow layer and subsoils. Three out of four common bands found in Mn nodules from two sites corresponded to Deltaproteobacteria and were characterized as sulfate-reducing and iron-reducing bacteria. The other DGGE bands of Mn nodules corresponded to sulfate and iron reducers (Deltaproteobacteria), methane-oxidizing bacteria (Gamma and Alphaproteobacteria), nitrite-oxidizing bacteria (Nitrospirae) and Actinobacteria. In addition, some DGGE bands of Mn nodules showed no clear affiliation to any known bacteria. The present study indicates that members involved in the reduction of Mn nodules dominate the bacterial communities in Mn nodules in rice field subsoils.  相似文献   

18.
Pseudomonas spp. are one of the most important bacteria inhabiting the rhizosphere of diverse crop plants and have been frequently reported as biological control agents (BCAs). In this work, the diversity and antagonistic potential of Pseudomonas spp. in the rhizosphere of maize cultivars Nitroflint and Nitrodent grown at an organic farm in Brazil was studied by means of culture-dependent and -independent methods, respectively. Sampling of rhizosphere soil took place at three different stages of plant development: 20, 40 and 106 days after sowing. A PCR-DGGE strategy was used to generate specific Pseudomonas spp. fingerprints of 16S rRNA genes amplified from total community rhizosphere DNA. Shifts in the relative abundance of dominant populations (i.e. PCR-DGGE ribotypes) along plant development were detected. A few PCR-DGGE ribotypes were shown to display cultivar-dependent relative abundance. No significant differences in diversity measures of DGGE fingerprints were observed for different maize cultivars and sampling times. The characterisation and assessment of the antagonistic potential of a group of 142 fluorescent Pseudomonas isolated from the rhizosphere of both maize cultivars were carried out. Isolates were phenotypically and genotypically characterised and screened for in vitro antagonism towards three phytopathogenic fungi and the phytopathogenic bacterium Ralstonia solanacearum. Anti-fungal activity was displayed by 13 fluorescent isolates while 40 isolates were antagonistic towards R. solanacearum. High genotypic and phenotypic diversity was estimated for antagonistic fluorescent Pseudomonas spp. PCR-DGGE ribotypes displayed by antagonists matched dominant ribotypes of Pseudomonas DGGE fingerprints, suggesting that antagonists may belong to major Pseudomonas populations in the maize rhizosphere. Antagonists differing in their genotypic and phenotypic characteristics shared the same DGGE electrophoretic mobility, indicating that an enormous genotypic and functional diversity might be hidden behind one single DGGE band. Cloning and sequencing was performed for a DGGE double-band which had no corresponding PCR-DGGE ribotypes among the antagonists. Sequences derived from this band were affiliated to Pseudomonas stutzeri and P. alcaligenes 16S rRNA gene sequences. As used in this study, the combination of culture-dependent and -independent methods has proven to be a powerful tool to relate functional and structural diversity of Pseudomonas spp. in the rhizosphere.  相似文献   

19.
Diversity of methanogenic archaeal communities in Japanese paddy field ecosystem was evaluated by the denaturing gradient gel electrophoresis (DGGE) after PCR amplification of the 16S rRNA genes (16S rDNAs), sequencing analysis and data evaluation by principal component analysis. Data were obtained from samples collected from the plowed soil layer, rice roots, rice straws incorporated in soil, plant residues (mixture of weeds, rice litters, rice roots, and rice stubbles) in soil, and composing rice straw. The number of bands of DGGE profiles ranged from 12 to 26 with the highest numbers in rice roots and rice straws incorporated in soil. However, the diversity indices based on both the numbers and intensity of bands indicated that the community of the plowed soil layer was the most diverse, even, and stable. Sequencing of the main DGGE bands showed the presence of Methanomicrobiales, Methanosarcinales, Methanobacteriaceae, and Methanocellales. The plowed soil layer included all phylogenetic groups of the methanogenic archaea of the other studied habitats, with prevalence of the members of Methanomicrobiales and Methanocellales. The phylogenetic diversity was compared with that of paddy soils collected in Italy, China, and the Philippines and that of 12 anaerobic environments (fen, waste, coast, permafrost, natural gas field, bovine rumen, riparian soil, termite, ciliate endosymboints, lake sediment, landfill, and seep rumen). The phylogenetic diversity was more similar among paddy soils than with the other anaerobic environments. Probably, the methanogenic archaeal communities of the paddy field soils were characterized by indigenous members and some of the members of the community of the plowed soil layer colonized rice roots, rice straws, and plant residues.  相似文献   

20.
Community structure of methanogenic archaea in paddy field soil under double cropping (rice [Oryza sativa L.] and wheat [Triticum aestivum L.]) was studied by the denaturing gradient gel electrophoresis (DGGE) method. Soil samples under flooded and upland conditions were collected 7 and 6 times, respectively, from two paddy fields throughout a year, and two primer sets, 0357F-GC/0691R and newly designed 1106F-GC/1378R, were used for DGGE analysis. The 25 and 29 different bands were observed on the DGGE gels with the primers 0357F-GC/0691R and 1106F-GC/1378R, respectively. DGGE band patterns of the methanogenic archaeal community were stable throughout a year including the cultivation periods of rice under flooded conditions and of wheat under upland conditions. Cluster analysis and principal component analysis suggested that the difference in the soil type (sampling region) largely influenced the community structures of methanogenic archaea in paddy field soil, while the effects of sampling period and different fertilizer treatments on them were small. Most of the sequences obtained from the DGGE bands were closely related to Methanomicrobiales, Methanosarcinaceae, Methanosaetaceae and Rice cluster-I.  相似文献   

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