Until recently, direct TEM examination of aquatic viruses was the most common method of recording viral diversity in aquatic environments. Although morphological data give only a limited view of viral diversity, these data have been cited as evidence that bacteriophages comprise the majority of viruses within viral communities. Many observations of temporal and spatial changes in the frequency distribution of viral capsid sizes have been recorded for viral populations (Wommack and Colwell 2000). Now evaluation of viral diversity and exploration for novel genes in aquatic environments from genomic analyses open a new view of aquatic virus communities, the studies of which are the central topics of “the third age of virus ecology”.
Size distribution of viruses
Viruses in aquatic environments are generally dominated by the 30–60-nm capsid size class (Cochlan et al. 1993; Proctor 1997; Weinbauer 2004; Wommack and Colwell 2000), as shown in Table 4. The dominant viruses in the floodwater of paddy fields also fell into a similar size class (Nakayama et al. 2007b). Size distribution changes temporally and spatially from coastal to open ocean sites (Cochlan et al. 1993), with depth (Cochlan et al. 1993), and over a 1-month sampling period during spring diatom blooms (Bratbak et al. 1990; Maranger et al. 1994). A difference in viral size class distribution was also noted among water samples collected from 22 Canadian lakes (Maranger and Bird 1995).
Not every study has documented the change in the distribution of capsid size classes with a change in time or the location of the water samples collected. Mathias et al. (1995) noted no change in the frequency of four classes of capsid diameters among the virioplankton in river water samples collected over a 2-year period. Nakayama et al. (2007b) also did not find a change in capsid size distribution in the floodwater of a Japanese paddy field under different fertilizer treatments (no fertilization; chemical N, P and K fertilizers; chemical N, P, K and Ca fertilizers; and compost with chemical N, P, K and Ca fertilizers) during the entire period of field flooding from transplanting to harvesting.
Weinbauer and Peduzzi (1994) showed for Adriatic Sea water samples that the 30–60-nm capsid size class comprised 74 and 100% of intracellular viruses in rods and spirilla, respectively, whereas cocci more often contained larger viruses (60–110 nm) than the smaller 30–60 nm viruses (65%vs 35%) in the cells. This finding is indicative of the predominance of the 30–60-mn capsid size class of viruses in the marine environment because bacterial communities are predominated by rod-shaped members (Bratbak et al. 1990; Mathias et al. 1995; Weinbauer and Peduzzi 1994; Weinbauer et al. 1993).
The predominance of viruses within the 30–60-nm size class is attributed to the predominance of phages in viral communities and the selective predation of larger viruses by heterotrophic nanoflagellates. The average capsid size for viruses of eukaryotic algae is reported to be 152 nm, although 28% of them are less than 60 nm (Van Etten et al. 1991). In general, phages are smaller, ranging from 34 to 160 nm with a peak sharply at 60 nm based on 251 phages (Ackermann 1998). Ackermann and DuBow (1987b) also found that phages with isometric capsids have a clear preference for the 55–65-nm range from approximately 180 species descriptions of classified phages of various bacteria. Viruses with different capsid sizes were ingested at different rates, with the smallest virus being ingested at the slowest rate (González and Suttle 1993). And these researchers calculated that when there are 106 bacteria mL−1 and 107–108 viruses mL−1, viruses may represent 0.2–9% of the carbon, 0.3–4% of the nitrogen and 0.6–28% of the phosphorus that the flagellates obtain from the ingestion of bacteria (González and Suttle 1993).
The clonal diversity of viral communities is evaluated on genome size using pulsed-field gel electrophoresis (PFGE), although the resolution of PFGE is not very high and the number of bands on the agarose gel is a conservative estimation of the number of viral species (Weinbauer 2004). Wommack et al. (1999a) used PFGE to monitor the population dynamics of Chesapeake Bay virioplankton for an annual cycle. The PFGE analysis detected several distinct bands ranging from 50 to 300 kb, and statistical analyses elucidated changes in virioplankton community structure in relation to sampling time, geographical location and the extent of water column stratification. And the hybridization of PFGE-separated samples with DNA probes specific to single viral strains and a group of viruses with similar genome sizes demonstrated that the abundances of specific viruses changed in time and space in Chesapeake Bay (Wommack et al. 1999b).
Morphology of viruses
Although most studies on the morphology of free viruses in aquatic environments did not present quantitative data on the proportions of tailed versus non-tailed forms, most free viruses in aquatic environments appear to be predominantly tailed forms (Proctor 1997). The exception was the study by Tapper and Hicks (1998), in which they recorded the presence or absence of tails as well as capsid size distribution in water samples from Lake Superior, Canada (Table 4). On average, 70% of viruses were tailed and the proportion of tailed viruses increased with an increase in the capsid size from 65, 74 to 100% for those with < 30-nm, 30–60-nm and > 60-nm capsid diameters, respectively.
In six Delaware soils studied by Williamson et al. (2005), most soil viruses were phages belonging to Caudovirales. Among the tailed phages, podophages and siphophages were more dominant than myophages in these soils. In addition, a much higher frequency of filamentous and elongated capsids was found in these soils. In particular, approximately 10% of the viral community in a silt loam soil consisted of elongated capsid phages. The predominance of tailed viruses in aquatic and soil environments is natural because 96% of phages were tailed among over 5100 phages examined by TEM (Ackermann 1998, 2001, 2003). It is important to note that non-tailed forms may include free viruses that have lost their tails during the preparation for TEM observation.
Genomic diversity in aquatic viral communities
Although Siphoviridae or phages with long, non-contractile tails comprised 61% of tailed phages in the examinations using TEM since 1959 (Ackermann 1998, 2001, 2003), a predominance of Siphoviridae among tailed viruses is not necessarily the case in marine environments. Breitbart et al. (2002, 2004a) examined a viral-community DNA library created from the Mission Bay sediment, California, and from two marine waters at Mission Bay and Scripps Pier, California, using linker-amplified shotgun cloning. They found a predominance of T7-like podophages, λ-like siphophages and T4-like myophages in two marine waters and λ-like siphophages, T7-like podophages and T4-like myophages in the sediment, in this order, among clones significantly similar to previously reported phage sequences, although more than 65% of the sequences were not affiliated with any known sequences. In contrast, a majority of phage isolates from marine environments were generally myophages and siphophages (Demuth et al. 1993; Lu et al. 2001; Sullivan et al. 2003; Suttle and Chan 1993; Waterbury and Valois 1993; Wichels et al. 1998).
Until recently, it was commonly understood among viral taxonomists that there is no single gene that is common to all virus genomes and that a total diversity of uncultured virus communities cannot be evaluated using approaches analogous to 16S ribosomal DNA as for bacteria (Rohwer and Edwards 2002). However, the genome of a lytic phage P60 of marine Synechococcus WH7803 contained approximately 47,872 bp with 80 potential open reading frames that were mostly similar to the genes found in lytic podophages (T7, φYeO3-12 and SIO1). In addition, according to the 109 bacteriophage genomic databases available in 2001, lysogenic phage was the dominant form among the known myophage and siphophage genomes and integrase genes were found in all the lysogenic myophage and siphophage genomes, whereas eight of nine podophage genomes contained the DNA polymerase, primase and helicase genes (Chen and Lu 2002). It was also noted that three Prochlorococcus phages (a podophage and two myophages) were quite similar to T7-like (P-SSP7) and T4-like (P-SSM2 and P-SSM4) phages containing 15 of 26 core T7-like genes and 43 and 42 of 75 core T4-like genes, respectively (Sullivan et al. 2005). These findings indicate that comparable genomic information is preserved among viral subsets and that genomic information may be an effective tool for the phylogenetic classification of viruses and the phylogenetic evaluation of viral diversity in the environment (Weinbauer and Rassoulzadegan 2004). Metagenomic analyses are elucidating the remarkable diversity of environmental viral communities, and Breitbart and Rohwer (2005) estimated that there were possibly 5000 viral genotypes in 200 L of seawater and one million different viral genotypes in 1 kg of marine sediment.
Furthermore, some functional gene sequences in the host bacterial genomes were found to be highly conserved in viral genomes, examples of which are the DNA polymerase gene pol (Chen and Lu 2002; Culley et al. 2003), the photosynthesis genes psbA and hliP (Lindell et al. 2004; Sullivan et al. 2005), the aldolase family gene talC (Sullivan et al. 2005), and the phosphate-inducible genes phoH and pstS (Sullivan et al. 2005). These findings substantiate the possibility of phylogenetic classification of viral subsets in the environment by comparing these genomic sequences. Rohwer and Edwards (2002) presented the “Phage Proteomic Tree” based on the overall similarity of 105 completely sequenced phage genomes. The resulting taxonomy was compatible with the system of International Committee on the Taxonomy of Viruses (ICTV), indicating that phage taxonomy has entered the post-genomic era. The presence of common functional genes related to fundamental physiological processes, energy-acquiring processes and gene expression processes across virus, prokaryote and eukaryote kingdoms strongly indicates horizontal gene transfer among the kingdoms, which may contribute to an increase in the biodiversity of respective genes via transduction. In addition, phage-encoded virulence factors are found in a variety of phages, members of Myoviridae, Siphoviridae, Podoviridae and Inoviridae, with some phages having characteristics of more than one family. A phage–bacterium interaction is, therefore, not a simple parasite–host interaction, but an instance of coevolution of phages and prokaryotic cells (Boyd and Brüssow 2002).
The following sections provide brief summaries of the viral diversity in aquatic environments estimated from the structural genes (g20, g23) of T4-like phages, the polymerase gene (pol) of T7-like phages, and the photosynthesis gene (psbA) of marine cyanophages, as well as evidence of horizontal gene transfer among the living world.
Structure genes of T4-like phages
A marine cyanomyophage S-PM2 has a genome homology to coliphage T4 at a 10-kb region as a contiguous block from gene g18 to g23. In T4, g18 codes for the tail sheath, g19 for the tail tube, g20 for the head potal proteins, g21 for the prohead core protein, g22 for a scaffolding protein, and g23 for the major capsid protein (Hambly et al. 2001). The T4-like phage family was further classified into subgroups of the T-evens, PseudoT-evens, SchizoT-evens and ExoT-evens, with increasing divergence from T4 based on the sequence comparison of g18, g19 and g23 genes (Deplats and Krisch 2003; Tétart et al. 2001).
The polymerase chain reaction (PCR) primers CPS1 and CPS2, which specifically amplify a 165-bp region from the majority of cyanomyophages, were constructed first by Fuller et al. (1998), although priming efficiency of the primers exhibit phage-to-phage variability. The region has significant similarity to g20 of coliphage T4. Primer pairs of CPS1 and CPS8 constructed by Zhong et al. (2002) cover more nucleotide sequences of g20 and elucidated the presence of nine phylogenetic groups among 114 totally different g20 homologs in six natural virus concentrates from estuarine and oligotrophic offshore environments. Only three groups/clusters contained known cyanophage isolates, and the identities of the other six clusters remain unknown.
The analysis of cyanophage communities by the g20 gene is also useful in the natural freshwater environment. Dorigo et al. (2004) studied cyanophage communities over time in Lake Bourget, France, using CPS1–CPS8 primers and found 35 distinct cyanomyophage g20 genotypes among 47 sequences analyzed. Phylogenetic analyses showed that these sequences fell into seven genetically distinct operational taxonomic units (OTUs). Some of these freshwater cyanophage sequences were genetically more closely related to marine cyanophage sequences than to other freshwater sequences. Similar findings of a wide distribution of closely related hosts and/or horizontal gene exchange among phage communities from very different environments were also obtained by analyzing g20 genes in various water samples from the Gulf of Mexico, the Arctic, the Southern, Northeast and Southeast Pacific Oceans, an Arctic cyanobacterial mat, a catfish production pond, lakes in Canada and Germany, and a depth of approximately 3246 m in the Chuckchi Sea (Short and Suttle 2005). These researchers also found four novel phylogenetic groups of g20 genes, among which two were only found in freshwater.
In addition, Mühling et al. (2005) examined the control of Synechococcus genotypes by phages in the oligotrophic Gulf of Aquba, Red Sea, over an annual cycle from denaturing gradient gel electrophoresis (DGGE) patterns of a 118-bp g20 gene fragment of cyanophages and Restriction Fragment Length Polymorphism (RFLP) patterns of a 403-bp rpoC1 gene fragment of Synechococcus spp., in which they found that both the abundance and genetic diversity of cyanophage communities covaried with those of the Synechococcus communities. Wilhelm et al. (2006) observed the pervasive distribution of cyanophages in Lake Erie, USA, which can infect the marine Synechococcus sp. strain WH7803, and analyses of g20 indicate that these phages are related to marine cyanophages, but in some cases form a unique clade, leaving questions with regard to the natural hosts of these phages.
Filée et al. (2005) compared the phylogenies constructed from g23 gene segment sequences with those obtained from the T4-type phage genomes for 16 completely sequenced T4-type phages and found a very good similarity between the phylogenies. Therefore, they designed degenerate primers targeting the g23 gene of phage T4 (MZIA1 bis, MZIA6) and applied them to elucidate T4-like bacteriophage communities in diverse marine environments (fjords and bays of British Columbia, the eastern Gulf of Mexico, and the western Arctic Ocean). Although some of the sequences of the PCR products were closely related to well-studied subgroups of the T4-like phages, such as the T-evens, SchizoT-evens, PseudoT-evens and ExoT-evens, the majority belonged to five previously uncharacterized subgroups. Thus, the g23 gene has more appropriate regions than the g20 gene for the phylogenetic evaluation of T4-type phage communities in the environment.
However, g23 genes may not be specific for T4-type phages. Jenkins and Hayes (2006) recently compared amino-acid sequences of g23 fragments among 17 cyanophage isolates of the heterocystous, filamentous cyanobacterium Nodularia spumigena using-specific primer set (CAP 1 and CAP 2). Although they were diverse in terms of their morphology and host range and belonged to two families of Myoviridae and Siphoviridae in Caudovirales, the encoded protein was 99% identical to T4 g23 homologues across all cyanophages compared. This fact indicates that g23 genes may be shared among Myoviridae and Siphoviridae members and that amino-acid sequences of the major capsid protein (g23) are important in phage–host interactions, irrespective of the phylogenetic positions of the phage and the host.
Primer pairs (MZIA1 bis, MZIA6) were also used by Jia et al. (2007) to compare DNA extracts of surface soil and rice straw collected from a Japanese paddy field during the flooded rice cultivation period. The g23 genes in these samples were quite distinctive in sequence from those obtained from marine environments (Filée et al. 2005). Phylogenetic analysis showed that most of g23 sequences belonged to two novel subgroups of T4-type phages (Paddy Soil subgroup and Rice Straw subgroup), although some of them were distantly related to well-studied subgroups of T4-type phages, for example, exoT-evens, T-evens and Groups II, III and IV of marine clones (Jia et al. 2007). This finding strongly indicates that the virus communities in soil are different from those in marine environments and that soil environments store novel structural and functional genes of viral origins.
pol genes of T7-like phages
Eukaryotes, prokaryotes and some viruses possess B-family (α-like) DNA polymerases of exonuclease and polymerase domains with highly conserved amino acid sequence motifs (Braithwaite and Ito 1993; Grabherr et al. 1992; Ito and Braithwaite 1991). The conservation was at the amino-acid level, but not necessarily at the nucleotide level, and degenerate PCR primers (named AVS1 and AVS2) targeting algal-virus-specific pol genes were designed based on the successful amplification of viruses infecting Chlorella-like alga (Chlorophyceae), photosynthetic flagellate Micromonas pusilla (Prasinophyceae) and Chrysochromilina spp. (Prymnesiophyceae), although PCR products were not obtained from viruses infecting marine brown algae Ectocarpus siliculosis and Feldmannia sp. (Phaeophyceae) with the primers (Chen and Suttle 1995). The region of PCR amplification is at the catalytic site in the polymerase domain containing the most highly conserved amino acid sequence, YGDTDS. Phylogenetic trees of 13 microalgal viruses based on DNA pol sequences between AVS1 and AVS2 and on hybridization of total genomic DNA showed similar branching patterns, indicating that DNA pol sequences can be used to determine genetic relatedness and to infer phylogenetic relationships among these viruses. In addition, the phylogenetic tree constructed from the deduced amino-acid sequences of DNA pol genes of 24 dsDNA viruses, including phycodnaviruses, herpesviruses, poxviruses, baculoviruses and African swine fever virus, corresponded well with groupings based on the ICTV system (Chen and Suttle 1996).
Chen et al. (1996) applied these primers to the virus-sized fraction obtained from inshore and offshore water samples of the Gulf of Mexico and obtained five different genotypes or OTUs that were identified on the basis of RFLP-banding patterns, all of which belonged to the family Phycodnaviridae. The primers were applicable to DGGE analysis to rapidly analyze the PCR products of natural marine viral communities with resultant elucidation of spatial and temporal differences in algal-virus community structure (Short and Suttle 1999). Primers of AVS1 and AVS2 were also used for DGGE analysis of geographically isolated natural algal-virus communities from coastal sites in the Pacific Ocean in British Columbia, Canada, and the Southern Ocean near the Antarctic Peninsula. Of the 33 sequences of different DGGE bands, 25 successfully encoded pol gene fragments. Similar virus sequences (> 98% sequence identity) were recovered from British Columbia and Antarctica, demonstrating that closely related algal viruses occur in distant geographical locations (Short and Suttle 2002). Thus, sequence analyses of structural genes and the pol gene in various viral communities suggest that viral diversity could be high on a local scale, but relatively limited globally (Breitbart and Rohwer 2005).
Recently, primers (T7DPol230F, T7DPol510R) targeting pol genes in T7-like podophages were constructed to compare T7-like podophage communities among different environments (Breitbart et al. 2004b). DNA pol sequences of T7-like podophages occurred in all biomes investigated, including marine, estuarine, freshwater, sediment, terrestrial, hypersaline and metazoan-associated ones. The majority of these sequences belonged to a unique Polymerases from Uncultured Podophages (PUP) clade, distantly relating to cultured isolates. Some pol genes from this clade were > 99% conserved at the nucleotide level in multiple different environments, suggesting movement of these phages between biomes in recent evolutionary time (Breitbart et al. 2004b). These findings indicate that pol genes are diverse with at least some pol genes included in the PUP clade and the others remote from the clade, although pol genes specific to respective environments remain unknown.
In addition, picorna-like viruses belonging to positive-sense ssRNA viruses are pathogens of penaeid shrimp, seals and whales. Culley et al. (2003) designed primers (RdRp1, RdRp2) specific to RNA-dependent RNA polymerase (RdRp) sequence in picorna-like viruses, and applied the primers to assays for the presence of those viruses in the coastal waters of British Columbia, Canada. A diverse array of picorna-like viruses was found in the ocean, and all of the sequences were divergent from known picorna-like viruses falling within four monophyletic groups. Thus, polymerase sequences are well conserved among phylogenetic groups and this gene is an excellent molecular marker for examining the diversity of viruses in nature.
Analyses of phage communities in aquatic environments using primers specific to g20, g23 and pol genes elucidated the presence of abundant viruses belonging to clades distantly related to cultured isolates. This is because only a dozen phages are well characterized (Frost et al. 2005), and they are limited to the members infecting specific bacterial hosts. Therefore, more effort should be paid to the isolation of phages infecting common, indigenous bacteria in the environment concurrently with the elucidation of phage genomics with these primers.
psbA genes of cyanophages
Many cyanobacteria are obligate photolithoautotrophs and have two different classes of reaction centers for photosynthesis: Type I and Type II centers (PS I and PS II). The PS II centers consist of two key proteins (D1 and D2 proteins) and are crucial sites of damage in photoinhibition. As a phage known as S-PM2 also encodes the D1 and D2 proteins by psbA and psbD genes, respectively, their expression in infected cells would allow a repair cycle to operate in PS II after the host's protein synthesis had been shut down by photoinhibition (Bailey et al. 2004; Mann et al. 2003). The transcription and translation of photosynthesis genes of phage origin in infected cells were elucidated by Lindell et al. (2005) and Clokie et al. (2006b). The transcripts of the psbA gene of phage S-PM2 appeared soon after infection of Synechococcus sp. WH7803 and remained at high levels until lysis, whereas a considerable transient increase in the abundance of the host psbA transcripts occurred shortly after infection with a subsequent decline to a lower level than the level without infection. The photosynthetic capacity of the cells remained constant throughout the course of infection (Clokie et al. 2006). In the Prochlorococcus MED-4 and podophage P-SSP7 system, phage psbA and high-light-inducible protein (hli) genes were expressed during infection and were cotranscribed with essential phage capsid genes. The phage D1 protein increased steadily over the infective period, whereas the expression of the host photosynthesis genes declined over the course of infection. In addition, replication of the phage genome was a function of photosynthesis (Lindell et al. 2005).
As primary production in the sea is dominated by cyanobacteria, the abundance and community structure of cyanophages may be critical factors in primary production in respective marine ecosystems (Liu et al. 1997; Wilhelm and Suttle 1999). Primers (58-VDIDGIREP-66, 331-MHERNAHNFP-340) targeting conserved psbA genes were first constructed to elucidate the diversity of naturally occurring marine oxygenic picoplankton (Zeidner et al. 2003). The primers successfully amplified psbA gene fragments of both cyanobacterial groups and eukaryotic algae in the Red Sea and the Mediterranean Sea and also gave good separation of PCR products by DGGE (Zeidner and Béjà 2004; Zeidner et al. 2003).
Cyanophages belong to three families of tailed phages: Myoviridae, Siphoviridae and Podoviridae (Mann 2003; Suttle 2000). Three phages from two families (Myoviridae and Podoviridae) that infect a marine cyanobacterium Prochlorococcus contained genes that encode D1 protein (PsbA) and high-light-inducible protein (Hli). A myophage encoded the second photosystem II core reaction center protein D2 (PsbD) as well, whereas the other myophage encoded the photosynthetic electron transport proteins, plastocyanin (PetE) and ferredoxin (PetF), which suggests that they encode functional proteins that may help maintain photosynthetic activity during infection (Lindell et al. 2004). Furthermore, the distributions of phage D1, D2 and Hli proteins to the same clusters with those from Prochlorococcus spp. indicate that those phage proteins are of cyanobacterial origin (Lindell et al. 2004). Millard et al. (2004) also found that the phage psbA genes fall into a clade that includes the psbA genes from their potential Synechococcus and Prochlorococcus hosts, suggesting the idea of the acquisition of these genes through horizontal gene transfer from their hosts. In addition, the high degree of sequence identity of the psbAD cassettes of S-PM2 (obtained from the English Channel) and S-RSM88 (from the Gulf of Aqaba) suggests a fairly recent lateral transfer (Millard et al. 2004). Although another conclusion seemed to be drawn from a subsequent study that phage psbA sequences form a separate clade from the clade of their host Synechococcus (Zeidner et al. 2005), the separation was indistinguishable when the psbA sequences of Prochlorococcus hosts and their phages were included (Hambly and Suttle 2005). Furthermore, possible exchange and reshuffling of psbA genes between Synechococcus and Prochlorococcus via phage intermediates were indicated (Zeidner et al. 2005).
Recently, Sullivan et al. (2006) isolated 33 Prochlorococcus and Synechococcus phages (six podophages, 25 myophages and two siphophages) and examined the presence of psbA and psbD genes in their genomes, including nine other published cyanophages. Eighty-eight percent of the phage genomes contained psbA, and 50% contained both psbA and psbD. The psbA gene was found in all myophages (n = 32) and Prochlorococcus podophages (n = 5), but not detected in Prochlorococcus siphophages (n = 2) and Synechococcus podophages (n = 3). psbD was found only in phages that contained psbA and only in myophages, but not in all psbA-containing myophages. In general, phages of a broad host range encoded both psbA and psbD. Furthermore, phylogenetic clustering patterns of psbA and psbD genes indicate that transfers of these genes were predominantly from Prochlorococcus to their phages and from Synechococcus to their phages (Sullivan et al. 2006).
Sullivan et al. (2006) also examined the genetic diversity of psbA and psbD genes retrieved offshore from Hawaii where Prochlorococcus cells commonly outnumber Synechococcus cells by orders of magnitude, by constructing the phylogenetic trees with the Prochlorococcus and Synechococcus isolates mentioned above. More than half of the psbA sequences (42 of 81) formed a large cluster with cultured Prochlorococcus podophages, and other psbA sequences formed subclusters that also contained cultured Prochlorococcus myophages. Thus, cyanophage culture collections represented much of the naturally occurring Prochlorococcus cyanophage psbA gene sequence diversity of that area. In contrast, all psbD sequences from the Hawaiian seawater samples formed a single well-supported cluster that included only a cultured Prochlorococcus cyanophage P-SSM4, and this cluster consisted of several subclusters without ever-known, cultured phages.
Thus, an analysis of phage psbAD sequences enables not only the elucidation of the phylogenetic relationships among cyanophages, but also suggests frequent horizontal gene exchange between cyanophages and their hosts. This progress in phylogenetical phage classification is within the phage members of Synechococcus and Prochlorococcus, mainly because these cyanobacteria are the main primary producers in marine and freshwater environments. Many species of cyanobacteria, different from the species in these aquatic environments, proliferate in paddy fields in the world. For example, Nostoc, Anabaena, Calothrix and Aulosira species are predominant among N2-fixing cyanobacteria in South and Southeast Asia (Kimura 2005). Heckman (1979) identified 11 cyanophyta in rice fields in Udon Thani Province, Thailand, and Jutono (1973) identified seven genera of Nostocaceae, three genera of Rivulariaceae, three genera of Scytonemataceae, six genera of Chroococcaceae, and eight genera of Oscillatoriaceae in rice fields in the Jogjakarta district, Indonesia. Algal and cyanobacterial successions are a common occurrence in rice fields, for example, Spirogyra sp., Anabaena bharadwajae→A. bharadwajae→A. bharadwajae, Scytonema hofmanni and Nostoc passerianum in well-drained rice fields and A. bharadwajae→Scytonema coactile, Nostoc sp. and Aulosira fritschii in ill-drained lowland rice fields in India (Gupta 1966). Viruses infecting these cyanobacteria and algae are different from those infecting aquatic ones and may have very unique, varietal psbA genes. Rice field viruses provide a potentially rich source for the study of phylogenetic diversities of psbAD and other photosynthesis-related genes.