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 mcyE), revealed that almost all microcystin-producers in the studied eutrophic waterbodies belong to the genus Microcystis. 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 Microcystis, spread in six clusters of the phylogenetic tree. By LM, seven Microcystis morphospecies were identified (M. aeruginosa, M. botrys, M. flos-aquae, M. natans, M. novacekii, M. smithii, and M. wesenbergii). 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 Microcystis is needed, proved the presence of toxigenic strains in M. aeruginosa and M. wesenbergii, and suppose their existence in M. natans. 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 Microcystis in the water.

The rising interest in harmful cyanoprokaryote blooms promotes an increase of phycological and ecological research on potentially toxic species and their hazardous substances. The present study aimed to identify the main microcystin (MC) producers and their contribution to the phytoplankton of shallow waterbodies in Bulgaria, applying different methods. The sampling was performed in August 2019 in nine lakes and reservoirs, two of which (reservoirs Kriva Reka and Izvornik 2) were studied for the first time. The high contribution of cyanoprokaryotes to the total species composition and phytoplankton abundance was proved by light microscopic (LM) observations and HPLC analysis of marker pigments. The LM identification of potential MC-producers was supported by PCR amplification of mcyE and mcyB genes. The MCs amounts, detected by HPLC-DAD, varied by sites with a range from undetectable concentrations to 0.46 µg L⁻¹ with only one recorded variant, namely MC-LR. It was found only in the reservoirs Mandra and Durankulak, while toxigenic MC-strains were obtained by PCR from five more waterbodies. Both LM and PCR demonstrated that the MC-producers were Microcystis aeruginosa and M. wesenbergii, despite their occurrence in low amounts (<0.5–5% of the total biomass) when filamentous cyanoprokaryotes dominated.

Fifty-one strains of Erwinia amylovora isolated from nine host plants in Bulgaria were characterized phenotypically and identified by the API 20E and BIOLOG system. The identification was confirmed by PCR amplification of a specific region of the plasmid pEA29 and the genome ams region. The phenotypic diversity of the strains was studied on the basis of their API 20E and BIOLOG metabolic profiles, as well as of their SDS-PAGE protein profile. Metabolic diversity among the strains was established, but no connection with the origin of the strains was revealed. The Bulgarian strains showed API 20E metabolic profiles not found in previous studies of E. amylovora. The strains formed a homogenous group on the basis of their protein profiles. All the strains were sensitive to the antibiotics streptomycin, tetracycline and oxytetracycline. This study was an initial step towards an investigation of the diversity and evolution in the Bulgarian population of E. amylovora, and it was the first characterization of E. amylovora strains isolated from different host plants in the period 1995-2005 in Bulgaria.

During the last few decades, the main focus of numerous studies has been on the human breast milk microbiota and its influence on the infant intestinal microbiota and overall health. The presence of lactic acid bacteria in breast milk affects both the quantitative and qualitative composition of the infant gut microbiota. The aim of this study was to assess the most frequently detected cultivable rod-shaped lactobacilli, specific for breast milk of healthy Bulgarian women and fecal samples of their infants over the first month of life, in 14 mother–infant tandem pairs. Additionally, we evaluated the strain diversity among the most common isolated species. A total of 68 Gram-positive and catalase-negative strains were subjected to identification using the MALDI-TOF technique. Predominant cultivable populations belonging to the rod-shaped lactic acid bacteria have been identified as Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Lacticaseibacillus paracasei, and Limosilactobacillus reuteri. Also, we confirmed the presence of Lactiplantibacillus plantarum and Lactobacillus gasseri. Up to 26 isolates were selected as representatives and analyzed by 16S rRNA sequencing for strain identity confirmation and a phylogenetic tree based on 16S rRNA gene sequence was constructed. Comparative analysis by four RAPD primers revealed genetic differences between newly isolated predominant L. rhamnosus strains. This pilot study provides data for the current first report concerning the investigation of the characteristic cultivable lactobacilli isolated from human breast milk and infant feces in Bulgaria.

The paper presents the first proof of planktonic cyanoprokaryote genus Cuspidothrix as an anatoxin A (ATX) producer in Bulgarian wetlands. The results from polymerase chain reaction (PCR) obtained from two summer sampling campaigns in 26 selected lakes and reservoirs demonstrated presence of the anaC gene, responsible for ATX production in 21 strains of the genus. They were found in three waterbodies sampled in 2018 (coastal lake Vaya, coastal reservoir Poroy, inland reservoir Sinyata Reka) and in four waterbodies sampled in 2019 (inland reservoirs Duvanli, Koprinka, Plachidol 2, Sinyata Reka). The detected genetic diversity generally corresponds to the observations conducted by conventional light microscopy, by which we distinguished three species of Cuspidothrix (Cuspidothrix issatschenkoi, Cuspidothrix elenkinii and Cuspidothrix tropicalis, the latter considered alien in the country). Eleven strains showed high similarity to two sequences of C. issatschenkoi available from the National Centre for Biotechnology Information (NCBI). Ten other strains assembled in a group, which—in lack of available from NCBI genetic sequences—were presumed related to C. tropicalis and C. elenkinii after comparison with the results from light microscopy. Cuspidothrix strains found in Bulgarian waterbodies showed high genetic similarity to those isolated and sequenced from Asia (Japan, China) and Northern Europe (Norway, Finland).

The general awareness of the threats on biodiversity and water quality raised the number of studies that use phytoplankton in assessment procedures. Since most metrics require obtaining mean values, this paper presents data that may help speed up field work and find indicators for a rapid water quality assessment based on single samplings, allowing simultaneous work on many sites. The phytoplankton from 43 Bulgarian waterbodies collected during three summer campaigns (2018, 2019, 2021) at sites selected after drone observations was studied by conventional light microscopy (LM) and an HPLC analysis of marker pigments. Our results allowed us to recommend drones and the HPLC application as reliable methods in rapid water quality assessments. In total, 787 algae from seven phyla (53 alien, new for Bulgaria) were identified. Chlorophyta was the taxonomically richest group, but Cyanoprokaryota dominated the biomass in most sites. New PCR data obtained on anatoxin and microcystin producers confirmed the genetic diversity of Cuspidothrix and Microcystis and provided three new species for the country’s toxic species, first identified by LM. A statistical analysis revealed significant correlations of certain algal phyla and classes with different environmental variables, and their species are considered promising for future search of bioindicators. This is especially valid for the class Eustigmatophyceae, which, as of yet, has been almost neglected in water assessment procedures and indices.