Development and application of quantitative detection of cyanophages phylogenetically related to cyanophage Ma-LMM01 infecting Microcystis aeruginosa in fresh water

To develop a real-time PCR method for quantification of the abundance of cyanophages infecting Microcystis aeruginosa in aquatic environments, we characterized three cyanophage clones infecting M aeruginosa, and compared them to the cyanophage Ma-LMM01 which was isolated previously. The clones were similar to Ma-LMM01 I in morphological features and genome size. Further, the nucleotide sequences of the putative genes coding for the alpha- and beta-subunits of ribonucleotide reductase and the sheath protein from the three isolates were identical to those of Ma-LMM01 The isolates were closely related to Ma-LMM01 and designated Ma-MM01-type phages. We designed a real-time PCR primer set to amplify a conserved region of the gene encoding the sheath protein, and quantified Ma-LMM01-type phages in environmental samples. The phages were detected when Microcystis blooms occurred, however, the amino acid sequence deduced from the nucleotide sequence of the PCR products was relatively diverse. This will be a useful tool for studies of the ecological impact of cyanophages on the Microcystis bloom. However, throughout these experiments, we did not detect any phages lytic to M aeruginosa strain NIES298. This suggests three hypotheses: 1) diversity of host specificity in phages, 2) dominance of defective cyanophages in nature, and 3) lysogeny in the examined host strain NIES298.