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The aim of this study was to evaluate the phytotoxic, genotoxic, cytotoxic and antimicrobial effects of the Mentha arvensis L. essential oil (EO). The biological activity of M. arvensis EO depended on the analyzed variable and the tested oil concentration. Higher concentrations of EO (20 and 30 µg mL-1) showed a moderate inhibitory effect on the germination and growth of seedlings of tested weed species (Bellis perennis, Cyanus segetum, Daucus carota, Leucanthemum vulgare, Matricaria chamomilla, Nepeta cataria, Taraxacum officinale, Trifolium repens and Verbena × hybrida). The results obtained also indicate that the EO of M. arvensis has some genotoxic, cytotoxic and proliferative potential in both plant and human in vitro systems. Similar results were obtained for antimicrobial activity against eight bacteria, including multidrug-resistant (MDR) strains [Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Escherichia coli, extended-spectrum beta-lactamase-producing (ESBL) E. coli, Pseudomonas aeruginosa and Salmonella enterica subsp. enterica serovar Enteritidis], with the effect on multidrug-resistant bacterial strains. Research indicates that the EO of M. arvensis shows phytotoxic, genotoxic, cytotoxic and antimicrobial effects, as well as its potential application as a herbicide and against various human diseases.

F. Pustahija, N. Bašić, S. Siljak-Yakovlev

Narcissus poeticus L. (Amaryllidaceae), a facultative serpentinophyte, is a highly variable species and particularly important ancestor of cultivated daffodils, but is rarely studied in field populations. This study, based on natural populations in the Balkans, focused on karyotype variability, genome size, ploidy and the presence of B chromosomes. Thirteen native populations from different environmental and soil conditions were collected and analyzed using flow cytometry to estimate nuclear genome size, fluorescence in situ hybridization (FISH) for physical mapping of rDNA, fluorochrome labeling (chromomycin and Hoechst) for heterochromatin organization and silver nitrate staining of nucleoli for determining rRNA gene activity. The organization of rDNA and natural triploids is reported here for the first time. The presence of individuals with B chromosomes (in 9/13 populations) and chromosomal rearrangements was also detected. The observed B chromosome showed three different morphotypes. The most frequent submetacentric type showed four different patterns, mainly with active ribosomal genes. The results obtained show that N. poeticus has a dynamic genome with variable genome size due to the presence of polyploidy, B chromosomes and chromosomal rearrangements. It is hypothesized that the observed changes reflect the response of the genome to different environmental conditions, where individuals with B chromosomes appear to have certain adaptive advantages.

F. Pustahija, Sabina Rastoder, R. Duraković, M. Subašić, N. Bašić, A. Parić

Genus Silene (Caryophyllaceae) is very rich in secondary metabolites and has an antiviral, antimalarial, antitumor, antibacterial and antioxidant properties, but Balkan endemic Silene sendtneri (Sendtner's campion) is not analyzed from the aspect of phenolic composition and their biological activities. Evaluation of total phenolic contents (TPC) and total flavonoid contents (TFC), and antioxidant and antimicrobial activities of hydromethanolic extracts from inflorescences, stem, rhizome, and seeds of S. sendtneri, was done in this study for the first time. The TPC, TFC, and antioxidant activity (DPPH; 2,2-diphenyl-1-picrylhydrazyl) were determined by UV/VIS spectrophotometry. Antimicrobial activity was estimated against selected test microorganisms (Staphylococcus epidermididis, Staphylococcus aureus subsp. aureus, Salmonella abony, Escherichia coli, and Candida albicans) using a disc diffusion assay. The inflorescences had the highest (11.587 mg GAEg-1 DW) and rhizome the lowest TPC (2.017 mg GAEg-1 DW). The inflorescences extract exhibited the highest TFC (69.824 mg CEg-1 DW), while TFC was not detected in the rhizome extract. The stem’s extract had the highest antioxidant activity (IC50; 20.51%), while the rhizome had the lowest (61.89%). All extracts showed moderate antibacterial activity against Staphylococcus epidermididis and low activity against the three remaining tested organisms. Antifungal activity of inflorescence and rhizome extracts was moderate. Obtained results provide a basis for further investigations of various S. sendtneri extracts, which can be a potential natural antioxidant and antimicrobial agents

UDK: 582.661.51:631.53.027]:547.913 In order to investigate the effects of seed priming with silver fir and oregano essential oils on certain important seedling characteristic and seed vigour of interesting endemic and horticultural species Silene sendtneri, an experiment was conducted based on randomized completely design with three replications. Traits such as germination rate, water content, dry weight, vigour index and photosynthetic pigments were analysed. Results revealed that the seedlings obtained with priming seeds showed increased growth, water content, vigour and photosynthetic pigment contents but decreased germination rate and dry weight compared with that obtained with non-primed seeds. Analysis of seed priming effects had demonstrated even germination rate is smaller the seedling vigour is slightly higher especially with all oregano oil treatments. We suggest that oregano oil has a potential as a priming agent for improvement of seedling synchrony, although at lower rate of germination.

Hong‐Mei Liu, Libor Ekrt, P. Koutecký, J. Pellicer, O. Hidalgo, J. Marquardt, F. Pustahija, A. Ebihara et al.

Recent studies investigating the evolution of genome size diversity in ferns have shown that they have a distinctive genome profile compared with other land plants. Ferns are typically characterized by possessing medium‐sized genomes, although a few lineages have evolved very large genomes. Ferns are different from other vascular plant lineages as they are the only group to show evidence for a correlation between genome size and chromosome number. In this study, we aim to explore whether the evolution of fern genome sizes is not only shaped by chromosome number changes arising from polyploidy but also by constraints on the average amount of DNA per chromosome. We selected the genus Asplenium L. as a model genus to study the question because of the unique combination of a highly conserved base chromosome number and a high frequency of polyploidy. New genome size data for Asplenium taxa were combined with existing data and analyzed within a phylogenetic framework. Genome size varied substantially between diploid species, resulting in overlapping genome sizes among diploid and tetraploid spleenworts. The observed additive pattern indicates the absence of genome downsizing following polyploidy. The genome size of diploids varied non‐randomly and we found evidence for clade‐specific trends towards larger or smaller genomes. The 578‐fold range of fern genome sizes have arisen not only from repeated cycles of polyploidy but also through clade‐specific constraints governing accumulation and/or elimination of DNA.

F. Pustahija, N. Bašić, S. Siljak-Yakovlev

Pustahija, F., Bašić, N. & Siljak-Yakovlev, S.: Chromosome numbers and genome size data for some Balkan species. [In Kamari, G., Blanché, C. & Siljak-Yakovlev, S. (eds), Mediterranean plant karyological data 28]. – Fl. Medit. 28: 420-428. doi: 10.7320/FlMedit28.420 Chromosome numbers and metaphase plates are given for eight species, seven from Bosnia and Herzegovina (Alnus × pubescens, Erythronium dens-canis, Genista tinctoria, Leucanthemum vulgare, Melittis melissophyllum, Orchis mascula, Stachys recta), and one species from F.Y.R.O.M. (Scorzonera austriaca). Chromosome counts and genome sizes are discussed.

UDK 581.19:547.56]:582.736.3 The aim of this study was to determine the total concentrations of some phenolic compounds and antioxidant and antimicrobial activity of methanol extracts of different parts of Illyrian endemic Petteria ramentacea. Concentrations of phenolic compounds were determined with UV/VIS spectrophotometry. The antioxidant activity of plant extracts was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Antimicrobial activity of extracts was evaluated by measuring the inhibition's zone against six selected test bacteria and two fungi. The highest average total phenols concentrations were in seeds (10.78 mg GAE g-1 DW), root (10.51 mg GAE g-1 DW) and bark (10.40 mg GAE g-1 DW), and the lowest in inflorescences (2.99 mg GAE g-1 DW) and leaves (3.12 mg GAE g g-1 DW). The total flavonoids concentrations were determined only in leaves (8.25 mg CE g-1 DW) and in stem (5.66 mg CE g-1 DW). Both flavanols and proanthocyanidins (0.75 mg CE g-1 DW and 3.49 mg CE g-1 DW, respectively) were found only in leaves. Analysis of variance indicated presence of significant differences between total phenols and flavanols concentrations (p<0.05), and Duncan's test confirmed the presence of intraspecies variability according to their concentrations. The highest antioxidant activity (IC50) was observed for the seed's extract (6.86%) and the lowest for the bark’s (51.31%). All methanol extracts showed the most pronounced antibacterial activity against Staphylococcus epidermididis, and the lowest against S. aureus subsp. aureus. Antifungal activity against Candida albicans was moderate. Since Peteria ramentacea methanol extracts are potential natural antioxidant and antimicrobial preparations against selected microorganisms, it is necessary to continue with more detailed analysis.

581.19:547.56]:582.971.1 Methanol extracts obtained from the berries of three Symphoricarpos taxa (Caprifoliaceae) were used to determine their total phenols concentrations, and antioxidant and antimicrobial activities. According to our knowledge, total concentrations of phenolic compounds and antimicrobial activities for of S. × chenaultii and S. orbiculatus, as well as antioxidant activity of all three analyzed taxa are for the first time analyzed in this study. Based on obtained results, it was noticed that the highest average values of the total phenols, flavonoids and flavanols had methanol extracts of S. orbiculatus berries (3.525 mg GAE g-1 DW, and 11.037 and 0.881 mg CE g-1 DW, respectively), while methanol extracts of S. albus berries had the lowest (1.430 mg GAE g-1 DW, and 4.154 and 0.211 mg CE g-1 DW respectively). Symphoricarpos orbiculatus extract (IC50 = 5.299 mg mL-1) possessed the highest antioxidant activity while the lowest had S. albus (IC50 = 20.017 mg mL-1). The methanol extracts of analyzed Symphoricarpos taxa showed no antifungal activity against C. albicans. Moderate antibacterial activity of S. albus methanol extract is determined against five tested bacterial strains (Bacillus vulgatus, Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella abony, and Staphylococcus aureus), and S. × chenaultii extract showed a low antibacterial activity only against P. aeruginosa, B. vulgatus and S. aureus. Although S. orbiculatus methanolic extract had the highest antioxidant activity it did not show any antibacterial activity at all. Analyzed Symphoricarpos extracts are potentially effective and can be used as natural antioxidant and antimicrobial preparations against test microorganisms.

F. Nardi, F. Pustahija, A. Andreucci, D. Ciccarelli, S. Siljak-Yakovlev, L. Peruzzi

Solidago litoralis (Asteraceae) is a psammophile plant endemic to the northern coasts of Tuscany, Italy. During time, different authors have considered it either as a separate species, subspecies or variety of the European S. virgaurea , but few studies of experimental taxonomy have been investigating the relationship between these two taxa. Aim of this study is to compare S. litoralis and S. virgaurea from different points of view: cytogenetic (karyotype analysis, localization of rDNA loci and genome size estimation), molecular (using two plastidial molecular markers), and ecological (by comparing functional characters). A difference in ecological responses to habitats is confirmed, even though S. virgaurea is potentially capable of assuming ecological strategies similar to those of S. litoralis . Despite this, cytogenetic and molecular analyses failed to reveal any significant difference supporting a specific distinction of S. litoralis . The latter taxon is here hypothesized as being an ecotype at the initial steps of a speciation event, better recognized at subspecific level ( S. virgaurea subsp. litoralis ).

V. Zoldoš, I. Biruš, E. Muratović, Z. Šatović, Aleksandar Vojta, O. Robin, F. Pustahija, F. Bogunić et al.

Abstract Epigenetic variation in natural populations with contrasting habitats might be an important element, in addition to the genetic variation, in plant adaptation to environmental stress. Here, we assessed genetic, epigenetic, and cytogenetic structure of the three Lilium bosniacum populations growing on distinct habitats. One population was growing under habitual ecological conditions for this species and the other two were growing under stress associated with high altitude and serpentine soil. Amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses revealed that the three populations did not differentiate genetically, but were clearly separated in three distinct clusters according to DNA methylation profiles. Principal coordinate analysis showed that overall epigenetic variation was closely related to habitat conditions. A new methylation-sensitive amplification polymorphism scoring approach allowed identification of mainly unmethylated (φST = 0.190) and fully CpG methylated (φST = 0.118) subepiloci playing a role in overall population differentiation, in comparison with hemimethylated sites (φST = 0.073). In addition, unusual rDNA repatterning and the presence of B chromosomes bearing 5S rDNA loci were recorded in the population growing on serpentine soil, suggesting dynamic chromosome rearrangements probably linked to global genome demethylation, which might have reactivated some mobile elements. We discuss our results considering our earlier data on morphology and leaf anatomy of several L. bosniacum populations, and suggest a possible role of epigenetics as a key element in population differentiation associated with environmental stress in these particular lily populations.

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