Evaluation of the yield, chemical composition and biological properties of essential oil from bioreactor-grown cultures of Salvia apiana microshoots.

Microshoot cultures of the North American endemic Salvia apiana were established for the first time and evaluated for essential oil production. Stationary cultures, grown on Schenk-Hildebrandt (SH) medium, supplemented with 0.22 mg/L thidiazuron (TDZ), 2.0 mg/L 6-benzylaminopurine and 3.0% (w/v) sucrose, accumulated 1.27% (v/m dry weight) essential oil, consisting mostly of 1,8-cineole, ß-pinene, α-pinene, ß-myrcene and camphor. The microshoots were adapted to agitated culture, showing biomass yields up to ca. 19 g/L. Scale-up studies demonstrated that S. spiana microshoots grow well in temporary immersion systems (TIS). In the RITA bioreactor, up to 19.27 g/L dry biomass was obtained, containing 1.1% oil with up to ca. 42% cineole content. The other systems employed, i.e. Plantform (TIS) and a custom made spray bioreactor (SGB), yielded ca. 18 and 19 g/L dry weight, respectively. The essential oil content of Plantform and SGB-grown microshoots was comparable to RITA bioreactor, however, the content of cineole was substantially higher (ca. 55%). Oil samples isolated from in vitro material proved to be active in acetylcholinesterase (up to 60.0% inhibition recorded for Plantform-grown microshoots), as well as hyaluronidase and tyrosinase-inhibitory assays (up to 45.8 and 64.5% inhibition observed in the case of the SGB culture).

Recombination Events in Putative Tail Fibre Gene in Litunavirus Phages Infecting Pseudomonas aeruginosa and Their Phylogenetic Consequences.

Recombination is the main driver of bacteriophage evolution. It may serve as a tool for extending the phage host spectrum, which is significant not only for phages' ecology but also for their utilisation as therapeutic agents of bacterial infections. The aim of this study was to detect the recombination events in the genomes of Litunavirus phages infecting Pseudomonas aeruginosa, and present their impact on phylogenetic relations within this phage group. The phylogenetic analyses involved: the whole-genome, core-genome (Schitoviridae conserved genes), variable genome region, and the whole-genome minus variable region. Interestingly, the recombination events taking place in the putative host recognition region (tail fibre protein gene and the adjacent downstream gene) significantly influenced tree topology, suggesting a strong phylogenetic signal. Our results indicate the recombination between phages from two genera Litunavirus and Luzeptimavirus and demonstrate its influence on phage phylogeny.

Species Phylogeny versus Gene Trees: A Case Study of an Incongruent Data Matrix Based on Paphiopedilum Pfitz. (Orchidaceae).

The phylogeny of the genus Paphiopedilum based on the plastome is consistent with morphological analysis. However, to date, none of the analyzed nuclear markers has confirmed this. Topology incongruence among the trees of different nuclear markers concerns entire sections of the subgenus Paphiopedilum. The low-copy nuclear protein-coding gene PHYC was obtained for 22 species representing all sections and subgenera of Paphiopedilum. The nuclear-based phylogeny is supported by morphological characteristics and plastid data analysis. We assumed that an incongruence in nuclear gene trees is caused by ancestral homoploid hybridization. We present a model for inferring the phylogeny of the species despite the incongruence of the different tree topologies. Our analysis, based on six low-copy nuclear genes, is congruent with plastome phylogeny and has been confirmed by phylogenetic network analysis.

Genetic History of the Remnant Population of the Rare Orchid Cypripedium calceolus Based on Plastid and Nuclear rDNA.

The lady's slipper orchid (Cypripedium calceolus), which inhabits shady deciduous and mixed forests and meadows, is now threatened with extinction in many European countries, and its natural populations have been dramatically declining in recent years. Knowledge of its evolutionary history, genetic variability, and processes in small populations are therefore crucial for the species' protection. Nowadays, in south-west Poland, it is only distributed in seven small remnant and isolated populations, which we examined. One nuclear (ITS rDNA) and two plastid (accD-psa1, trnL-F) markers were analyzed and compared globally in this study. Based on the nuclear marker, the most common ancestor of C. calceolus and Cypripedium shanxiense existed about 2 million years ago (95% HPD: 5.33-0.44) in Asia. The division of the C. calceolus population into the European and Asian lineages indicated by C/T polymorphism started about 0.5 million years ago (95% HPD: 1.8-0.01). The observed variation of plastid DNA, which arose during the Pleistocene glacial-interglacial cycles, is still diffuse in Poland. Its distribution is explained by the result of fragmentation or habitat loss due to human impact on the environment.

Identification of Three Type II Toxin-Antitoxin Systems in Model Bacterial Plant Pathogen Dickeya dadantii 3937.

Type II toxin-antitoxin (TA) systems are genetic elements usually encoding two proteins: a stable toxin and an antitoxin, which binds the toxin and neutralizes its toxic effect. The disturbance in the intracellular toxin and antitoxin ratio typically leads to inhibition of bacterial growth or bacterial cell death. Despite the fact that TA modules are widespread in bacteria and archaea, the biological role of these systems is ambiguous. Nevertheless, a number of studies suggests that the TA modules are engaged in such important processes as biofilm formation, stress response or virulence and maintenance of mobile genetic elements. The Dickeya dadantii 3937 strain serves as a model for pathogens causing the soft-rot disease in a wide range of angiosperm plants. Until now, several chromosome-encoded type II TA systems were identified in silico in the genome of this economically important bacterium, however so far only one of them was experimentally validated. In this study, we investigated three putative type II TA systems in D. dadantii 3937: ccdAB2Dda, phd-docDda and dhiTA, which represents a novel toxin/antitoxin superfamily. We provide an experimental proof for their functionality in vivo both in D. dadantii and Escherichia coli. Finally, we examined the prevalence of those systems across the Pectobacteriaceae family by a phylogenetic analysis.

The influence of Nordic walking on the general functioning and cognitive performance of patients with Alzheimer's disease.

Purpose: Most studies show that physical activity (PA) can inhibit the progression of Alzheimer's disease (AD). This research aimed to investigate the effect of 3-month moderate PA on the general functioning and cognitive abilities of AD patients. Methods: The study included 32 patients with mild or moderate AD who scored 11-23 points in Mini-Mental State Examination (MMSE). Half of the participants were qualified to perform the exercises; the other half constituted the control group. The recommended form of activity was Nordic walking, 30 minutes, 3 days a week, for 3 months, under the supervision of the patient's carer. General functioning and the severity of cognitive deficits were assessed using the Activities of Daily Living (ADL) scale and the Montreal Cognitive Assessment scale (MoCA) before and after the 3-month study. Results: No improvement was observed in the overall functioning of the patients after 3 months of regular, moderate PA. Neither were there any differences in this respect between the active and inactive groups, both in patients with mild (MMSE 19-23) and moderate (MMSE 11-18) disease severity. Men showed a deterioration in overall functioning regardless of PA. No differences in cognitive functioning were observed between the active and inactive groups, the two genders or between mild (MMSE 19-23) and moderate (MMSE 11-18) subjects. Conclusions: The 3-month, moderate, Nordic walking PA did not improve the overall general functioning or cognitive performance in patients with mild or moderate AD.

Bacteriophages vB_Sen-TO17 and vB_Sen-E22, Newly Isolated Viruses from Chicken Feces, Specific for Several Salmonella enterica Strains.

Two newly discovered bacteriophages, isolated from chicken feces and infecting Salmonella enterica strains, are described in this report. These phages have been named vB_Sen-TO17 and vB_Sen-E22, and we present their molecular and functional characterization. Both studied viruses are able to infect several S. enterica strains and develop lytically, but their specific host ranges differ significantly. Electron microscopic analyses of virions have been performed, and full genome sequences were determined and characterized, along with molecular phylogenetic studies. Genomes of vB_Sen-TO17 (ds DNA of 41,658 bp) and vB_Sen-E22 (dsDNA of 108,987 bp) are devoid of homologs of any known or putative gene coding for toxins or any other proteins potentially deleterious for eukaryotic cells. Both phages adsorbed efficiently (>95% adsorbed virions) within 10 min at 42 °C (resembling chicken body temperature) on cells of most tested host strains. Kinetics of lytic development of vB_Sen-TO17 and vB_Sen-E22, determined in one-step growth experiments, indicated that development is complete within 30-40 min at 42 °C, whereas burst sizes vary from 9 to 79 progeny phages per cell for vB_Sen-TO17 and from 18 to 64 for vB_Sen-E22, depending on the host strain. Virions of both phages were relatively stable (from several percent to almost 100% survivability) under various conditions, including acidic and alkaline pH values (from 3 to 12), temperatures from -80 °C to 60 °C, 70% ethanol, chloroform, and 10% DMSO. These characteristics of vB_Sen-TO17 and vB_Sen-E22 indicate that these phages might be considered in further studies on phage therapy, particularly in attempts to eliminate S. enterica from chicken intestine.

Characterization of the Bacteriophage vB_EfaS-271 Infecting Enterococcus faecalis.

A newly isolated bacteriophage infecting Enterococcus faecalis strains has been characterized, including determination of its molecular features. This phage, named vB_EfaS-271, has been classified as a Siphoviridae member, according to electron microscopy characterization of the virions, composed of a 50 nm-diameter head and a long, flexible, noncontractable tail (219 × 12.5 nm). Analysis of the whole dsDNA genome of this phage showed that it consists of 40,197 bp and functional modules containing genes coding for proteins that are involved in DNA replication (including DNA polymerase/primase), morphogenesis, packaging and cell lysis. Mass spectrometry analysis allowed us to identify several phage-encoded proteins. vB_EfaS-271 reveals a relatively narrow host range, as it is able to infect only a few E. faecalis strains. On the other hand, it is a virulent phage (unable to lysogenize host cells), effectively and quickly destroying cultures of sensitive host bacteria, with a latent period as short as 8 min and burst size of approximately 70 phages per cell at 37 °C. This phage was also able to destroy biofilms formed by E. faecalis. These results contribute to our understanding of the biodiversity of bacteriophages, confirming the high variability among these viruses and indicating specific genetic and functional features of vB_EfaS-271.

Characteristics of a Series of Three Bacteriophages Infecting Salmonella enterica Strains.

Molecular and functional characterization of a series of three bacteriophages, vB_SenM-1, vB_SenM-2, and vB_SenS-3, infecting various Salmonella enterica serovars and strains is presented. All these phages were able to develop lytically while not forming prophages. Moreover, they were able to survive at pH 3. The phages revealed different host ranges within serovars and strains of S. enterica, different adsorption rates on host cells, and different lytic growth kinetics at various temperatures (in the range of 25 to 42 °C). They efficiently reduced the number of cells in the bacterial biofilm and decreased the biofilm mass. Whole genome sequences of these phages have been determined and analyzed, including their phylogenetic relationships. In conclusion, we have demonstrated detailed characterization of a series of three bacteriophages, vB_SenM-1, vB_SenM-2, and vB_SenS-3, which reveal favorable features in light of their potential use in phage therapy of humans and animals, as well as for food protection purposes.

A Revision of the Taxonomy and Identification of Epipactis greuteri (Orchidaceae, Neottieae).

Epipactis greuteri is an obligate autogamous orchid species. Due to large differences in the interpretation of the diagnosis of this species, it is often mistakenly identified by botanists, which results in erroneous information provided in the literature about its distribution in Europe. In the present paper we review its description, including flower details, gynostemium features, and papillae morphology and compare it to E. helleborine, with which it is often confused. Based on thorough study of herbarium material (including holotype and isotype) and field research in Greece, Romania, and Poland, we confirm that gynostemium of E. greuteri has strongly reduced clinandrium and does not produce viscidium. We also discuss taxonomic treatment of E. preinensis and E. flaminia, two recently described taxa related to E. greuteri. The results of genetic analyses, as well as the range of phenotypic variability of E. greuteri individuals from various regions of Europe were presented and discussed. The analysis based on the ITS (internal transcribed spacer) nuclear marker showed no differences among E. helleborine, E. purpurata, E. albensis, and E. greuteri, which probably indicates their close relationship. Based on the analysis of plastid regions, six haplotypes were detected in all investigated samples. An exhaustive description of morphological features of E. greuteri is provided.

Revision of Gyrodactylus salaris phylogeny inspired by new evidence for Eemian crossing between lineages living on grayling in Baltic and White sea basins.

In this research, grayling-specific Gyrodactylus salaris Malmberg, 1957 isolates from Baltic Sea basin were collected in Sweden for the first time. Samples were obtained in three drainage systems: Kalixälven (River Kaitum), Ljungan (River Sölvbacka strömmar), and Umeälven (River Juktån). Three molecular markers were analysed: nuclear ITS rDNA (Internal Transcribed Spacer) and ADNAM1 (Anonymous DNA Marker 1), and mitochondrial cox1 gene. As a result, four new mitochondrial haplotypes were identified (III-C1tt, III-C1ttht, IX-A1tt and X-A1tt). The ADNAM1 analyses resulted in revealing two new alleles (WS4 and BS9) and two new genotypes (T6 and T7). T7 seems to be an indicator of ancient crossing between Baltic and White Sea lineages of the parasite which happened during a first 3000-year period of Eemian interglacial about 130,000 years ago in the connection between Baltic and White Sea. Molecular clock estimates were adjusted, revealing the mean substitution rate and the divergence rate among branches of 3.6% (95% HPD: 2.2%-5.2%) and 7.2% per million years, respectively. As a result, cox1 phylogeny rooted with the introgressed haplotypes has been revised and altered in accordance to new data, revealing fourteen equidistant lineages five of which have been excluded from the study. Based on the new phylogenetic approach, including the molecular clock, this work suggests an overall revision of G. salaris phylogeny and attempts at precisely drawing the division of lineages within this polytypic species as well as proposes unification in nomenclature for its strains.

Establishing the cell biology of apomictic reproduction in diploid Boechera stricta (Brassicaceae).

Background and aims: In the Brassicaceae family, apomictic development is characteristic of the genus Boechera. Hybridization, polyploidy and environmental adaptation that arose during the evolution of Boechera may serve as (epi)genetic regulators of apomictic initiation in this genus. Here we focus on Boechera stricta, a predominantly diploid species that reproduces sexually. However, apomictic development in this species has been reported in several studies, indicating non-obligate sexuality. Methods: A progressive investigation of flower development was conducted using three accessions to assess the reproductive system of B. stricta. We employed molecular and cyto-embryological identification using histochemistry, transmission electron microscopy and Nomarski and epifluorescence microscopy. Key Results: Data from internal transcribed spacer (ITS) and chloroplast haplotype sequencing, in addition to microsatellite variation, confirmed the B. stricta genotype for all lines. Embryological data indicated irregularities in sexual reproduction manifested by heterochronic ovule development, longevity of meiocyte and dyad stages, diverse callose accumulation during meiocyte-to-gametophyte development, and the formation of triads and tetrads in several patterns. The arabinogalactan-related sugar epitope recognized by JIM13 immunolocalized to one or more megaspores. Furthermore, pollen sterility and a high frequency of seed abortion appeared to accompany reproduction of the accession ES512, along with the initiation of parthenogenesis. Data from flow cytometric screening revealed both sexual and apomictic seed formation. Conclusion: These results imply that B. stricta is a species with an underlying ability to initiate apomixis, at least with respect to the lines examined here. The existence of apomixis in an otherwise diploid sexual B. stricta may provide the genomic building blocks for establishing highly penetrant apomictic diploids and hybrid relatives. Our findings demonstrate that apomixis per se is a variable trait upon which natural selection could act.

Taxonomy of Cyrtochilum-alliance (Orchidaceae) in the light of molecular and morphological data.

BACKGROUND: The generic separateness and specific composition of the orchid genus Cyrtochilum was discussed for almost two centuries. Over the years several smaller taxa were segregated from this taxon, but their separateness was recently questioned based on molecular studies outcomes. The aim of our study was to revise concepts of morphological-based generic delimitation in Cyrtochilum-alliance and to compare it with the results of genetic analysis. We used phylogenetic framework in combination with phenetical analysis to provide proposal of the generic delimitation within Cyrtochilum-alliance. Two molecular markers, ITS and matK were used to construct phylogenetic tree. A total of over 5000 herbarium specimens were included in the morphological examination and the phenetical analysis included 29 generative and vegetative characters. RESULTS: Comparative morphology of the previously recognized genera: Buesiella, Dasyglossum, Neodryas, Rusbyella, Siederella and Trigonochilum is presented. A new species within the the latter genus is described. Fourteen new combinations are proposed. The key to the identification of the genera of the Cyrtochilum-alliance and morphological characteristics of each genus are provided. CONCLUSIONS: A total of six separated genera are recognized within Cyrtochilum-alliance. The reasons of the incompatibility between morphological differences observed within studied taxa and phylogenetic tree are argued and the taxonomic implications of such inconsistency, resulting in fragmentation or lumping of taxonomic units, are discussed.

First Guatemalan record of natural hybridisation between Neotropical species of the Lady's Slipper orchid (Orchidaceae, Cypripedioideae).

The first natural hybrid in the section Irapeana of the orchid genus Cypripedium is described and illustrated based on Guatemalan material. A molecular evaluation of the discovery is provided. Specimens with intermediate flowers between C. irapeanum and C. dickinsonianum within ITS and Xdh sequences have the signal sequence of both these species. The analysis of plastid sequences indicated that the maternal line is C. irapeanum. Information about the ecology, embryology and conservation status of the novelty is given, together with a distribution map of its parental species, C. irapeanum and C. dickinsonianum. A discussion of the hybridization between Cypripedium species is presented. The potential hybrid zones between the representatives of Cypripedium section Irapeana which were estimated based on the results of ecological niche modeling analysis are located in the Maya Highlands (C. dickinsonianum and C. irapeanum) and the eastern part of Southern Sierra Madre (C. molle and C. irapeanum). Moreover, all three Cypripedium species could inhabit Cordillera Neovolcánica according to the obtained models; however, it should be noticed that this region is well-distanced from the edges of the known geographical range of C. molle.

Phylogenetic placement and taxonomy of the genus Hederorkis (Orchidaceae).

Three plastid regions, matK, rpl32-trnL and rpl16 intron and the ITS1-5.8S-ITS2 nuclear ribosomal DNA were used to demonstrate a phylogenetic placement of the genus Hederorkis (Orchidaceae) for the first time. The taxonomic position of this genus has been unclear thus far. The phylogenetic and morphological relations of Hederorkis to the most closely related genera Sirhookera, Adrorhizon, Bromheadia and Polystachya are also discussed. A hypothesis concerning an origin and evolution of Hederorkis is proposed. Hederorkis is an epiphytic two-leaved orchid genus with lateral inflorescence, non-resupinate flowers, elongate gynostemium and rudimentary column foot. It is native to the Indian Ocean Islands. Two species of Hederorkis are recognized worldwide, H. scandens endemic to Mauritius and Réunion and H. seychellensis endemic to Seychelles. For each of the species treated a full synonymy, detailed description and illustration are included. The distribution map and dichotomous keys to the species have also been provided.

Taxonomic position and phylogeny of the genus Vargasiella (Orchidaceae, Vandoideae) based on molecular and morphological evidence.

Since the description of the Neotropical genus Vargasiella in 1952, its taxonomic position has remained unclear, mainly due to a lack of sufficient data. In this study, the taxonomic position of Vargasiella was revised based on the outcomes of macro- and micromorphological studies, analyses of selected molecular markers and ecological methods of niche distribution modeling. The phylogenetic relationships were inferred using three DNA markers: matK, trnL-F and ITS sequences. The morphological studies included the analysis of macromorphological features of herbarium specimens as well as micromorphological examination of preserved flowers. The ecological niche modeling was applied to identify the distribution of the suitable niches of the studied taxa. The relationships between Vargasiella and most similar taxa remain unresolved based on the molecular analysis. The outcomes from the morphological studies indicated significant differences between Vargasiella, Warrea and Warreopsis. Moreover, a niche shift in response to changing climate after the last glacial maximum is observed in Vargasiella, while no substantial changes in the occupied habitats were identified in the other related taxa. The clocktree of the Zygopetaleae estimated from the matK gene indicated that the most recent common ancestors of Vargasiella, Warrea and Warreopsis originated in the Miocene, while the divergence time for Vargasiella and Warrea was assessed at approximately 5.4 Ma ago. Vargasiella seems to be an outshoot of the main branch of evolution of the Zygopetaleae. It is noteworthy that the Vargasiella-Warrea dichotomy could have taken place later than the divergence of Warreopsis from the mutual lineage. The molecular analysis and morphological data suggest that Vargasiella and Warrea could have evolved from a common ancestor. Accumulation of morphological differences and acceleration of the evolution of Vargasiella were more intensive than in other Warreinae and this could probably be synchronized with adaptation to different climatic conditions.

Taxonomic placement of Paphiopedilum canhii (Cypripedioideae; Orchidaceae) based on cytological, molecular and micromorphological evidence.

Paphiopedilum canhii was discovered in Northern Vietnam. Since its description in 2010, it has caused a stir among taxonomists due to its interesting mixture of morphological features, i.e. marbled, relatively thick leaves, flowers similar to species classified in the section Barbata, and unique, large staminodial shield. On the basis of these features, it is difficult to classify the species to existing infrageneric units. Using cytological data, phylogenetic analyses based on plastid and nuclear genes and the study of the adaxial epidermis of the leaves and gynostemium structure obtained from Scanning Electron Microscopy (SEM) and Light Microscopy (LM), the taxonomic position of P. canhii was determined. These results suggest that P. canhii forms an independent phylogenetic line within the genus Paphiopedilum deserving subgeneric status, already proposed by Braem and Gruss (2011) as Megastaminodium.

Phylogenetic relationships within Orchidaceae based on a low-copy nuclear coding gene, Xdh: Congruence with organellar and nuclear ribosomal DNA results.

Using parsimony and Bayesian analyses, we estimated higher-level relationships within Orchidaceae, focusing on subfamilies and tribes. DNA sequences of part of the low-copy nuclear protein gene Xdh were obtained for 154 taxa including 126 genera of Orchidaceae and outgroup families of Asparagales. The general topology of the Xdh trees is congruent with those published previously based on plastid protein-coding genes and non-coding nuclear ribosomal DNA. The five subfamilies previously recognized are monophyletic and well supported. The results indicate that monandrous condition evolved independently in Vanilloideae and Epidendroideae/Orchidoideae. The analysis clarifies relationships between tribes of Epidendroideae such as Vandeae sensu lato to Collabieae, Epidendreae to Calypsoeae and Malaxideae to Dendrobieae. Also relationships of Bromheadia, Imerinaea, Sirhookera, and achlorophyllous species of Corallorhiza, Gastrodia, Limodorum, Neottia, Wullschlaegelia are for the first time evaluated in a broad molecular phylogenetic framework.