RESULTS:

Nostoc is a complex and tough genus to differentiate and its morphological plasticity makes it taxonomically complicated. Its cryptic diversity and almost no distinguishable morphological characteristics make this genus incredibly heterogeneous to evaluate on taxonomic scales. The strain NOS isolated from a eutrophic water body is being described as a new genus Aliinostoc with the strain showing motile hormogonia with gas vesicles as an atypical feature which is currently considered as the diacritical feature of the genus but should be subjected to critical evaluation in the near future. The phylogenetic placement of Aliinostoc along with some other related sequences of Nostoc clearly separated this clade from Nostoc sensu stricto with high bootstrap support and robust topology in all the methods tested thus providing strong proof of the taxa being representative of a new genus which morphologically appears to be Nostoc -like. Subsequent phylogenetic assessment using the rbcL psbA rpoC1 and tufA genes was done with the aim of facilitating future multi-locus studies on the proposed genus for better taxonomic clarity and resolution. Folding of the 16S–23S internal transcribed spacer region and subsequent comparisons with members of the genera Nostoc Anabaena Aulosira Cylindrospermum Sphaerospermopsis Raphidiopsis Desmonostoc and Mojavia gave entirely new secondary structures for the D1-D1′ and box-B helix. Clear and separate clustering from Nostoc sensu stricto supports the establishment of Aliinostoc gen. nov. with the type species being Aliinostoc morphoplasticum sp. nov. in accordance with the International Code of Nomenclature for algae fungi and plants.

PCR amplification of the internal transcribed spacer (ITS) between the 16S rRNA and 23S rRNA genes of the cyanobacterium Nostoc PCC 7120 gave three products. Two represented true ITS regions of different sizes while the third was a heteroduplex. The longer spacer (ITS-L) contained 512 nucleotides and carried tRNAIle and tRNAAla genes separated by a large stem–loop structure (V2) composed of short tandemly repeated repetitive sequences. Both tRNA genes and the 5′ half of the intervening stem were absent from the shorter spacer (ITS-S) of length 283 nucleotides which was otherwise almost completely identical to ITS-L. The two spacer regions of Nostoc PCC 7120 were aligned to published ITS sequences of cyanobacteria the cyanelle of Cyanophora paradoxa and Escherichia coli. Although the ITS regions of cyanobacteria vary in length from 283 to 545 nucleotides and contain either both tRNAIle and tRNAAla genes only the tRNAIle gene or neither there is no correlation between ITS size and coding capacity for tRNAs. Putative secondary structures were determined for the deduced transcripts of the rrn operons of several cyanobacteria and were compared to that of E. coli. Highly conserved motifs important for folding and for maturation of the rRNA transcripts were identified and regions homologous to bacterial antiterminators (box B–box A) were located. The conserved and variable regions of the cyanobacterial ITS are potential targets of PCR primers and oligonucleotide probes for detection and identification of cyanobacteria at different taxonomic levels.

Purpose. Bloodstream infections are major causes of morbidity and mortality that lead to prolonged hospital stays and higher medical costs. In this study we aimed to evaluate the MinION nanopore sequencer for the identification of the most dominant pathogens in positive blood culture bottles.

Methodology. 16S and ITS1-5.8S-ITS2 rRNA genes were amplified by PCR reactions with barcoded primers using nine clinical isolates obtained from positive blood bottles and 11 type strains including five types of Candida species. Barcoded amplicons were mixed and multiplex sequencing with the MinION sequencer was performed. In addition barcoded PCR amplicons were sequenced by Sanger sequencing to validate the performance of the MinION.

Results. The bacterial and Candida spp. identified by MinION sequencing based on the highest homology of reference sequences from the NCBI gene databases agreed with the matrix-assisted laser desorption ionization time of flight mass spectrometry results excepting the closely related species Streptococcusand Escherichia coli. The ‘pass’ reads obtained within about 10 min of sequencing were sufficient to identify the pathogens. The average values of sequence identities with 1D2 chemistry and the R9.5 flow cell were around 99 %; thus frequent sequence errors did not affect species identification based on amplicon sequencing.

Conclusion. We have established a rapid portable and economical technique for the identification of pathogens in positive blood culture bottles through a novel MinION nanopore sequencer amplicon sequencing scheme which replaces traditional Sanger sequencing.

Two Nostoc-like strains have been isolated purified cultured and identified on the basis of the polyphasic approach using morphological ecological molecular and phylogenetic methods. Both strains were found to have morphology similar to the genus Nostoc but clustered strongly in a group distant from the Nostoc sensu stricto clade. Further analysis using the folded structures of the 16S–23S ITS region revealed strong differences from closely related members of the genus Nostoc . Distinct phylogenetic clustering and strong tree topologies using Bayesian inference maximum-likelihood and maximum-parsimony methods indicated the need to revisit the taxonomy of the members of this particular clade with a clear need for giving a generic status distinct from the genus Nostoc . In accordance with the International Code of Nomenclature for Algae Fungi and Plants the name Desikacharya gen. nov. is proposed for the new genus along with the description of two new species Desikacharya nostocoides sp. nov. and Desikacharya soli sp. nov. and reclassification of Nostoc thermotolerans to Desikacharya thermotolerans comb. nov.

Cyanobacterial strain ARC8 was isolated from seepage coming into the river Dračice Františkov Czech Republic and was characterized using a polyphasic approach. Strain ARC8 showed a typical Nostoc -like morphology and in-depth morphological characterization indicated that it is a member of the genus Nostoc . Furthermore in the 16S rRNA gene phylogeny inferred using Bayesian inference maximum likelihood and neighbour joining methods strain ARC8 clustered within the Nostoc sensu stricto clade. The phylogenetic distance and the positioning of strain ARC8 also indicated that it is a member of the genus Nostoc . Furthermore the rbcL gene phylogeny along with the 16S–23S ITS secondary structure analysis also supported the findings from the 16S rRNA gene tree. In accordance with the International Code of Nomenclature for Algae Fungi and Plants we describe a novel species of Nostoc with the name Nostoc neudorfense sp. nov.

This study describes a novel fungal species belonging to the genus Gongronella. During a previous work focusing on metalaxyl degradation by Mucorales strains two isolates from vineyard soil samples collected in the Alentejo region south Portugal were identified as a putative novel species based on combined molecular and MALDI-TOF MS data. This new species is described here using a polyphasic approach that combines morphology internal transcribed spacer of ribosomal DNA (ITS) and 28S ribosomal DNA (LSU) sequence data analysis and proteomic profiling by MALDI-TOF MS. Phenotypic and molecular data enabled this novel species to be clearly distinguished from other Gongronella species with results of combined ITS+LSU analysis showing that the Gongronella species is related to Gongronella butleri and Gongronella brasiliensis. Therefore from the results of morphological and molecular analyses isolates MUM 10.262 and MUM 10.263 seem to represent a new Gongronella species and the name Gongronella eborensis sp. nov. is proposed with the ex-type strain MUM 10.262 (=CCMI 1100=CBS 128763).