Morphological and Molecular Identification of Microcystin-Producing Cyanobacteria in Nine Shallow Bulgarian Water Bodies

Abstract

The paper presents results from the first application of polyphasic approach in studies of field samples from Bulgaria. This approach, which combined the conventional light microscopy (LM) and molecular-genetic methods (based on PCR amplified fragments of microcystin synthetase gene <i>mcyE</i>), revealed that almost all microcystin-producers in the studied eutrophic waterbodies belong to the genus <i>Microcystis</i>. During the molecular identification of toxin-producing strains by use of HEPF × HEPR pair of primers, we obtained 57 sequences, 56 of which formed 28 strains of <i>Microcystis,</i> spread in six clusters of the phylogenetic tree. By LM, seven <i>Microcystis</i> morphospecies were identified (<i>M. aeruginosa</i>, <i>M. botrys</i>, <i>M. flos</i>-<i>aquae, M. natans</i>, <i>M. novacekii, M. smithii</i>, and <i>M. wesenbergii</i>). They showed significant morphological variability and contributed from <1% to 98% to the total biomass. All data support the earlier opinions that taxonomic revision of <i>Microcystis</i> is needed<i>,</i> proved the presence of toxigenic strains in <i>M. aeruginosa</i> and <i>M. wesenbergii</i>, and suppose their existence in <i>M. natans</i>. Our results demonstrated also that genetic sequencing, and the use of HEPF × HEPR pair in particular, can efficiently serve in water quality monitoring for identifying the potential risk from microcystins, even in cases of low amounts of <i>Microcystis</i> in the water.