Natsuko Nakayama

Ecology of viruses in soils: Past, present and future perspectives

Abstract Viruses play important roles in biogeochemical nutrient cycles and act as genomic reservoirs in marine and freshwater environments, the understanding of which brought about the so-called ‘third age’ of virus ecology in aquatic environments. Unfortunately, the third age is in oceanography and limnology and outside the soil world. The main reason why virus ecology in soils has shown less progress is that agronomical and epidemiological interests were the primary motivation of viral studies by soil microbiologists. In this review, past research on viruses in soils is summarized after the introduction of the ecological traits of viruses, which are the effects of viruses on beneficial bacteria and soil-borne plant pathogens, adsorption of viruses to soils, and soil factors affecting viral inactivation and survival in soils. Horizontal gene transfer (transduction) in soils is also reviewed. Second, the abundance of viruses and their roles in biogeochemical nutrient cycles are summarized in aquatic environments. Five to 25% of the carbon fixed by primary producers is estimated to enter into the microbial loop via virus-induced lysis at different trophic levels in aquatic environments. The diversity of virus communities in aquatic environments estimated from analyses of the frequency distribution of capsid sizes and the morphology of virus populations are reviewed, and recent findings on the genomic diversity of viruses and their roles as the greatest genomic reservoirs in aquatic environments follow in the subsequent section. Viral genomics is elucidating the viral diversity and phylogenetic relationships among viruses in different environments. As the soil environment is a more diverse habitat for viruses than aquatic environments, viruses in soils have great potential to play roles comparable in quantity, which are unique in quality, to those in aquatic environments. Therefore, the potentiality and characteristics of viruses in soils are discussed in the final section for future research on virus ecology in soils from the viewpoints of biogeochemistry and genomic diversity. Synecological approaches to viruses in soils may open up a new era of soil virus ecology.

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.

Phylogenetic distribution of bacteria isolated from the floodwater of a Japanese paddy field

Abstract Two hundred and seventy five bacterial strains were isolated from the floodwater of a Japanese paddy field during rice cultivation and the cells from 205 of these strains were negative for Gram stain. One hundred and nineteen isolates negative for Gram stain were randomly chosen and subjected to sequencing of partial 16S rDNA to compare with DNA clones obtained from floodwater using the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method. The isolates belonged to α-, β-, and γ-Proteobacteria and the Cytophaga–Flavobacterium–Bacteroides (CFB) group as well as high-GC and low-GC Gram-positive bacteria, with proportions of 47.9, 7.6, 11.8, 13.4, 4.2 and 15.1%, respectively. Although their DNA sequences did not match the sequences of the DNA clones obtained from the floodwater of a neighboring paddy field, some isolates showed identical DNA sequences to DNA clones from rice straw in soil and rice roots. Phylogenetic distribution of the isolates was wider than that of the DNA clones obtained from the floodwater using the DGGE method. As the CFB cluster consisted exclusively of the isolates and the DNA clones obtained from the floodwater and from microcrustaceans, and the other isolates shared the α-, β- and γ-Proteobacterial clusters with the DNA clones associated with rice straw in soil, microcrustaceans, percolating water and rice roots, bacterial communities in the floodwater were estimated to include floodwater-specific members as well as members associated with other habitats in the paddy fields.

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

Abstract 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.

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.

Abundance of bacteriophages of common heterotrophic bacteria in the floodwater of a Japanese paddy field

Abstract Bacteriophages of 18 heterotrophic bacterial strains (four Cytophaga–Flavobacterium–Bacteroides bacteria, six α-Proteobacteria, two β-Proteobacteria, two γ-Proteobacteria, one Firmicute and three Actinobacteria) that were isolated from floodwater were enumerated in the floodwater of four plots (no fertilization, N, P, and K fertilizers, N, P, K and Ca fertilizers, and N, P, K and Ca fertilizers with compost) in a Japanese paddy field under a long-term fertilizer trial throughout the rice cultivation period. All floodwater samples contained phages of some of the tested bacteria. In general, phage abundance was large at the first sampling, 6 days after transplanting rice, and decreased with time until the midseason drainage, except for two bacterial strains. Abundance tended to be more numerous after the midseason drainage than before drainage. There was no clear plot preference for phages of respective bacteria, and no relationship was recognized between the rice growth stage when the studied bacteria were isolated and the stage of abundant phages of respective bacteria. Phages of a Sphingomonas sp., an Enterobacter sp., a Cytophaga sp. and a Microbacterium sp. occurred commonly and abundantly during the rice cultivation period among the bacteria examined. And phage communities in the floodwater appeared to consist of phages of physiologically broad hosts without any appreciable deflection of specific phage groups at the specific rice cultivation stage. There was no difference in phage infectivity between bacterial strains with and without prophage in their chromosomes. Phages of Sphingomonas spp. in the floodwater were strain dependent, especially before the midseason drainage. No relationship was observed between viral abundance in the floodwater and the incidence of phages infectious to bacteria. In contrast, significant correlations were observed between phage abundances of 18 bacterial strains in 34 out of 153 combinations.

Ubiquitous Bacteriophage Hosts in Rice Paddy Soil

Viruses are the most abundant biological entities in rice fields, and bacteriophages comprised the majority among viral communities. Sphingomonas/Novosphingobium phages were exclusively siphoviruses with various host ranges. The high frequency of phage-infected bacterial cells indicated that the bacterial mortality from phage lysis could be significant enough to redirect the microbial food web and change the bacterial communities. There was no significant difference in the frequency of lysogeny between oligotrophs and copiotrophs in soil. Superinfection immunity was not important for bacteria in rice fields, although slower growth from the burden of prophage DNA synthesis was indicated among strains at the genus or species level. The majority of the g23 sequences of T4-type bacteriophages in rice fields were distantly related to those of marine origins. Horizontal gene transfer was suggested by the identical g23 sequences found in distant rice fields. The g23 genes in rice fields have apparently diverged more compared with marine g23 genes.