Karyotype Variability in Wild Narcissus poeticus L. Populations from Different Environmental Conditions in the Dinaric Alps.

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.

Allopolyploidy: An Underestimated Driver in Juniperus Evolution.

Allopolyploidy is considered as a principal driver that shaped angiosperms' evolution in terms of diversification and speciation. Despite the unexpected high frequency of polyploidy that was recently discovered in the coniferous genus Juniperus, little is known about the origin of these polyploid taxa. Here, we conducted the first study devoted to deciphering the origin of the only hexaploid taxon in Juniperus along with four of its closely related tetraploid taxa using AFLP markers with four primers combinations. Phylogenetic analysis revealed that the 10 studied species belong to 2 major clusters. J. foetidissima appeared to be more related to J. thurifera, J. sabina, and J. chinensis. The Bayesian clustering analysis showing a slight variation in genetic admixture between the studied populations of J. foetidissima, suggesting an allopolyploid origin of this species involving J. thurifera and J. sabina lineages, although a purely autopolyploidy origin of both J. thurifera and J. foetidissima cannot be ruled out. The admixed genetic pattern revealed for J. seravschanica showed that the tetraploid cytotypes of this species originated from allopolyploidy, whereas no clear evidence of hybridization in the origin of the tetraploid J. thurifera and J. chinensis was detected. This study provides first insights into the polyploidy origin of the Sabina section and highlights the potential implication of allopolyploidy in the evolution of the genus Juniperus. Further analyses are needed for a more in-depth understanding of the evolutionary scenarios that produced the observed genetic patterns.

Apomictic Mountain Whitebeam (Sorbus austriaca, Rosaceae) Comprises Several Genetically and Morphologically Divergent Lineages.

The interplay of polyploidisation, hybridization, and apomixis contributed to the exceptional diversity of Sorbus (Rosaceae), giving rise to a mosaic of genetic and morphological entities. The Sorbus austriaca species complex from the mountains of Central and South-eastern Europe represents an allopolyploid apomictic system of populations that originated following hybridisation between S. aria and S. aucuparia. However, the mode and frequency of such allopolyploidisations and the relationships among different, morphologically more or less similar populations that have often been described as different taxa remain largely unexplored. We used amplified fragment length polymorphism (AFLP) fingerprinting, plastid DNA sequencing, and analyses of nuclear microsatellites, along with multivariate morphometrics and ploidy data, to disentangle the relationships among populations within this intricate complex. Our results revealed a mosaic of genetic lineages-many of which have not been taxonomically recognised-that originated via multiple allopolyploidisations. The clonal structure within and among populations was then maintained via apomixis. Our results thus support previous findings that hybridisation, polyploidization, and apomixis are the main drivers of Sorbus diversification in Europe.

Phytochemical and Cytogenetic Study of Two Centaurea Species from Croatia: the Particular Case of Diploid and Tetraploid C. salonitana.

Natural wild populations of C. rupestris and C. salonitana were studied to determine possible relationships between the volatile oil (VO) composition and ploidy level. The chemical composition of the volatile oil was investigated using the GC/MS technique. The predominant components of the VO of diploid and tetraploid C. salonitana were hexadecanoic acid and α-linoleic acids, while in C. rupestris they were germacrene D and ß-caryophyllene in one population and heptacosane and germacrene D, in another. The nuclear DNA amounts (2 C DNA), determined by flow cytometry, were 3.54 pg for C. rupestris, 3.39 pg for the diploid and 6.79 pg for the tetraploid population of C. salonitana. Evidence that the degree of ploidy solely influences the chemical composition of the essential oil of C. salonitana was not found. The results presented are the first data to be reported on the DNA content of the studied Centaurea populations from Croatia, as well as on the chemical composition of C. salonitana volatile oil.

Sex Determination in Dioscorea dumetorum: Evidence of Heteromorphic Sex Chromosomes and Sex-Linked NORs.

Yams (Dioscorea spp.) are a pantropical genus located worldwide that constitute an important source of nutrients and pharmaceutical substances. Some Dioscorea crop species are widely grown in West Africa. One species that is mainly cultivated in Cameroon is Dioscorea dumetorum. This is a dioecious root crop whose sex-determining system was unknown until now. To address the possible presence of sex chromosomes in D. dumetorum, we performed a karyotype characterization of male and female individuals using classical and molecular cytogenetic approaches. It was determined that 2n = 40 was the most common number of chromosomes in all of the investigated samples. One chromosome pair was longer than the others in the chromosome set and was a heteromorph in male and homomorph in female individuals. This pair corresponded to sex chromosomes, and we also confirmed this with molecular cytogenetic experiments. The results of chromomycin banding revealed the presence of strong positive signals on this chromosome pair. The signals, corresponding to GC-rich DNA regions, were similar in size on the chromosomes of the female individuals, whereas they were different in size in the male individuals. This size difference in the GC-rich heterochromatin regions was also apparent in the interphase nuclei as one small and one large fluorescent spot. The results of the in situ hybridization experiment showed that these chromomycin positive signals on the sex chromosomes also corresponded to the 35S rDNA cluster. The mean 2C DNA value (genome size) obtained for D. dumentorum was 0.71 pg (±0.012), which represents a small genome size. We found no difference in the genome size between the male and female individuals. The results of this study contribute to increasing our knowledge of sex determination in D. dumetorum (standard sex-determining XX/XY system) and may have some agronomic applications.

Volatile Oil Chemical Composition of Wild, Edible Centaurea scabiosa L. and Its Cytotoxic Activity.

Centaurea species are well known as a source of phytopharmaceuticals having both beneficial and harmful influences on human health. Centaurea scabiosa L. is a wild edible plant used in Mediterranean cuisine in the Dalmatian region of Croatia. We have assessed the volatile oil's chemical composition using GC/MS chromatography and its cytotoxic activity on human fibroblasts using the MTT test. Data on chromosome number, obtained by classical karyological methods, and genome size, assessed by flow cytometry, of the same plant material of C. scabiosa, were also given. The major chemical compounds found in C. scabiosa volatile oil were heptacosane, caryophyllene oxide, alloaromadendrene epoxide, α-cyperone, and α-bisabolol. This volatile oil showed no cytotoxicity on human fibroblasts in a dose range of 0.01-1 g/L. The chromosome number of a C. scabiosa sample from Croatia showed 2n = 20 + 2B chromosomes. The total genome DNA amount of 2C = 3.3 ± 0.01 pg or 1 Cx = 1628 Mbp presents the first report on the genome size of this species from Croatia. The presented results support the idea of using this plant in the human diet. To our knowledge, this is the first report on edible C. scabiosa species in general and in particular from Croatia.

Genetic Variability in Balkan Paleoendemic Resurrection Plants Ramonda serbica and R. nathaliae Across Their Range and in the Zone of Sympatry.

The genus Ramonda includes three Paleoendemic and Tertiary relict species that survived in refugial habitats of the Balkan Peninsula (R. nathaliae and R. serbica) and the Iberian Peninsula (R. myconi). They are all "resurrection plants," a rare phenomenon among flowering plants in Europe. Ramonda myconi and R. nathaliae are diploids (2n = 2x = 48), while R. serbica is a hexaploid (2n = 6x = 144). The two Balkan species occur in sympatry in only two localities in eastern Serbia, where tetraploid potential hybrids (2n = 4x = 96) were found. This observation raised questions about the existence of gene flow between the two species and, more generally, about the evolutionary processes shaping their genetic diversity. To address this question, genetic markers (AFLP) and an estimate of genome size variation were used in a much larger sample and at a larger geographic scale than previously. The combination of AFLP markers and genome size results suggested ongoing processes of interspecific and interploidy hybridization in the two sites of sympatry. The data also showed that interspecific gene flow was strictly confined to sympatry. Elsewhere, both Ramonda species were characterized by low genetic diversity within populations and high population differentiation. This is consistent with the fact that the two species are highly fragmented into small and isolated populations, likely a consequence of their postglacial history. Within sympatry, enormous variability in cytotypes was observed, exceeding most reported cases of mixed ploidy in complex plant species (from 2x to >8x). The AFLP profiles of non-canonical ploidy levels indicated a diversity of origin pathways and that backcrosses probably occur between tetraploid interspecific hybrids and parental species. The question arises whether this diversity of cytotypes corresponds to a transient situation. If not, the question arises as to the genetic and ecological mechanisms that allow this diversity to be maintained over time.

A chromosome-level, haplotype-phased Vanilla planifolia genome highlights the challenge of partial endoreplication for accurate whole-genome assembly.

Vanilla planifolia, the species cultivated to produce one of the world's most popular flavors, is highly prone to partial genome endoreplication, which leads to highly unbalanced DNA content in cells. We report here the first molecular evidence of partial endoreplication at the chromosome scale by the assembly and annotation of an accurate haplotype-phased genome of V. planifolia. Cytogenetic data demonstrated that the diploid genome size is 4.09 Gb, with 16 chromosome pairs, although aneuploid cells are frequently observed. Using PacBio HiFi and optical mapping, we assembled and phased a diploid genome of 3.4 Gb with a scaffold N50 of 1.2 Mb and 59 128 predicted protein-coding genes. The atypical k-mer frequencies and the uneven sequencing depth observed agreed with our expectation of unbalanced genome representation. Sixty-seven percent of the genes were scattered over only 30% of the genome, putatively linking gene-rich regions and the endoreplication phenomenon. By contrast, low-coverage regions (non-endoreplicated) were rich in repeated elements but also contained 33% of the annotated genes. Furthermore, this assembly showed distinct haplotype-specific sequencing depth variation patterns, suggesting complex molecular regulation of endoreplication along the chromosomes. This high-quality, anchored assembly represents 83% of the estimated V. planifolia genome. It provides a significant step toward the elucidation of this complex genome. To support post-genomics efforts, we developed the Vanilla Genome Hub, a user-friendly integrated web portal that enables centralized access to high-throughput genomic and other omics data and interoperable use of bioinformatics tools.

Genome Size, Cytotype Diversity and Reproductive Mode Variation of Cotoneaster integerrimus (Rosaceae) from the Balkans.

Cotoneaster integerrimus represents a multiploid and facultative apomictic system of widely distributed mountain populations. We used flow cytometry to determine genome size, ploidy level, and reproduction mode variation of the Balkan populations, supplemented by analysis of nuclear microsatellites in order to address: (i) geographic distribution and variation of cytotypes among the populations; (ii) variation of reproduction mode and the frequency of sexuality; (iii) pathways of endosperm formation among the sampled polyploids and their endosperm balance requirements; (iv) genotypic diversity and geographic distribution of clonal lineages of polyploids. The prevalence of apomictic tetraploid cytotype followed by sexual diploids and extremely rare triploids was demonstrated. This prevalence of tetraploids affected the populations' structure composed from clonal genotypes with varying proportions. The co-occurrence of diploids and tetraploids generated higher cytotype, reproductive mode, and genotypic diversity, but mixed-ploidy sites were extremely rare. The endosperm imbalance facilitates the development and the occurrence of intermediate triploids in mixed-ploidy populations, but also different tetraploid lineages elsewhere with unbalanced endosperm. All these results showed that the South European populations of C. integerrimus have higher levels of cytotype and reproductive diversity compared to the Central European ones. Therefore, the South European populations can be considered as a potential reservoir of regional and global diversity for this species.

Molecular Cytogenetics (Fluorescence In Situ Hybridization - FISH and Fluorochrome Banding): Resolving Species Relationships and Genome Organization.

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Genome Size Evolution and Dynamics in Iris, with Special Focus on the Section Oncocyclus.

Insights into genome size dynamics and its evolutionary impact remain limited by the lack of data for many plant groups. One of these is the genus Iris, of which only 53 out of c. 260 species have available genome sizes. In this study, we estimated the C-values for 41 species and subspecies of Iris mainly from the Eastern Mediterranean region. We constructed a phylogenetic framework to shed light on the distribution of genome sizes across subgenera and sections of Iris. Finally, we tested evolutionary models to explore the mode and tempo of genome size evolution during the radiation of section Oncocyclus. Iris as a whole displayed a great variety of C-values; however, they were unequally distributed across the subgenera and sections, suggesting that lineage-specific patterns of genome size diversification have taken place within the genus. The evolutionary model that best fitted our data was the speciational model, as changes in genome size appeared to be mainly associated with speciation events. These results suggest that genome size dynamics may have contributed to the radiation of Oncocyclus irises. In addition, our phylogenetic analysis provided evidence that supports the segregation of the Lebanese population currently attributed to Iris persica as a distinct species.

Gene flow between diploid and tetraploid junipers - two contrasting evolutionary pathways in two Juniperus populations.

BACKGROUND: Gene flow and polyploidy have been found to be important in Juniperus evolution. However, little evidence has been published elucidating the association of both phenomena in juniper taxa in the wild. Two main areas were studied in Spain (Eastern Iberian Range and Sierra de Baza) with both diploid and tetraploid taxa present in sympatry. Gene flow and ploidy level were assessed for these taxa and the resulted offspring. RESULTS: Twenty-two allo-triploid hybrids between J. sabina var. sabina and J. thurifera were found in the Eastern Iberian Range population. However, in the Sierra de Baza population no triploids were found. Instead, 18 allo-tetraploid hybrids between two tetraploid taxa: J. sabina var. balkanensis and J. thurifera were discovered. High genetic diversity was exhibited among the tetraploid hybrids at Sierra de Baza, in contrast to the genetically identical triploid hybrids at the Eastern Iberian Range; this suggests meiotic difficulties within the triploid hybrids. In addition, unidirectional gene flow was observed in both studied areas. CONCLUSION: Polyploidy and hybridization can be complementary partners in the evolution of Juniperus taxa in sympatric occurrences. Juniperus was shown to be an ideal coniferous model to study these two phenomena, independently or in concert.

Polyploidy in the Conifer Genus Juniperus: An Unexpectedly High Rate.

Recent research suggests that the frequency of polyploidy may have been underestimated in gymnosperms. One notable example is in the conifer genus Juniperus, where there are already a few reports of polyploids although data are still missing for most species. In this study, we evaluated the extent of polyploidy in Juniperus by conducting the first comprehensive screen across nearly all of the genus. Genome size data from fresh material, together with chromosome counts, were used to demonstrate that genome sizes estimated from dried material could be used as reliable proxies to uncover the extent of ploidy diversity across the genus. Our analysis revealed that 16 Juniperus taxa were polyploid, with tetraploids and one hexaploid being reported. Furthermore, by analyzing the genome size and chromosome data within a phylogenetic framework we provide the first evidence of possible lineage-specific polyploidizations within the genus. Genome downsizing following polyploidization is moderate, suggesting limited genome restructuring. This study highlights the importance of polyploidy in Juniperus, making it the first conifer genus and only the second genus in gymnosperms where polyploidy is frequent. In this sense, Juniperus represents an interesting model for investigating the genomic and ecological consequences of polyploidy in conifers.

Cytological and genome size data analyzed in a phylogenetic frame: Evolutionary implications concerning Sisyrinchium taxa (Iridaceae: Iridoideae).

Sisyrinchium is the largest genus of Iridaceae in the Americas and has the greatest amount of cytological data available. This study aimed at investigating how genomes evolved in this genus. Chromosome number, genome size and altitude from species of sect. Viperella were analyzed in a phylogenetic context. Meiotic and pollen analyses were performed to assess reproductive success of natural populations, especially from those polyploid taxa. Character optimizations revealed that the common ancestor of sect. Viperella was probably diploid (2n = 2x =18) with two subsequent polyplodization events. Total DNA content (2C) varied considerably across the phylogeny with larger genomes detected mainly in polyploid species. Altitude also varied across the phylogeny, however no significant relationship was found between DNA content changes and altitude in our data set. All taxa presented regular meiosis and pollen viability (> 87%), except for S. sp. nov. aff. alatum (22.70%), suggesting a recent hybrid origin. Chromosome number is mostly constant within this section and polyploidy is the only source of modification. Although 2C varied considerably among the 20 taxa investigated, the diversity observed cannot be attributed only to polyploidy events because large variations of DNA content were also observed among diploids.

Epigenetic Differentiation of Natural Populations of Lilium bosniacum Associated with Contrasting Habitat Conditions.

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.

Cytological and genome size data analyzed in a phylogenetic frame: Evolutionary implications concerning Sisyrinchium taxa (Iridaceae: Iridoideae)

Abstract Sisyrinchium is the largest genus of Iridaceae in the Americas and has the greatest amount of cytological data available. This study aimed at investigating how genomes evolved in this genus. Chromosome number, genome size and altitude from species of sect. Viperella were analyzed in a phylogenetic context. Meiotic and pollen analyses were performed to assess reproductive success of natural populations, especially from those polyploid taxa. Character optimizations revealed that the common ancestor of sect. Viperella was probably diploid (2n = 2x =18) with two subsequent polyplodization events. Total DNA content (2C) varied considerably across the phylogeny with larger genomes detected mainly in polyploid species. Altitude also varied across the phylogeny, however no significant relationship was found between DNA content changes and altitude in our data set. All taxa presented regular meiosis and pollen viability (> 87%), except for S. sp. nov. aff. alatum (22.70%), suggesting a recent hybrid origin. Chromosome number is mostly constant within this section and polyploidy is the only source of modification. Although 2C varied considerably among the 20 taxa investigated, the diversity observed cannot be attributed only to polyploidy events because large variations of DNA content were also observed among diploids.

Evolutionary implications of heterochromatin and rDNA in chromosome number and genome size changes during dysploidy: A case study in Reichardia genus.

In this study we showed that constitutive heterochromatin, GC-rich DNA and rDNA are implicated in chromosomal rearrangements during the basic chromosome number changing (dysploidy) in Reichardia genus. This small Mediterranean genus comprises 8-10 species and presents three basic chromosome numbers (x = 9, 8 and 7). To assess genome evolution and differentiation processes, studies were conducted in a dysploid series of six species: R. dichotoma, R. macrophylla and R. albanica (2n = 18), R. tingitana and R. gaditana (2n = 16), and R. picroides (2n = 14). The molecular phylogeny reconstruction comprised three additional species (R. crystallina and R. ligulata, 2n = 16 and R. intermedia, 2n = 14). Our results indicate that the way of dysploidy is descending. During this process, a positive correlation was observed between chromosome number and genome size, rDNA loci number and pollen size, although only the correlation between chromosome number and genome size is still recovered significant once considering the phylogenetic effect. Fluorescent in situ hybridisation also evidenced changes in number, position and organisation of two rDNA families (35S and 5S), including the reduction of loci number and, consequently, reduction in the number of secondary constrictions and nuclear organising regions from three to one per diploid genome. The potential mechanisms of chromosomal and genome evolution, strongly implicating heterochromatin, are proposed and discussed, with particular consideration for Reichardia genus.

DNA Remodeling by Strict Partial Endoreplication in Orchids, an Original Process in the Plant Kingdom.

DNA remodeling during endoreplication appears to be a strong developmental characteristic in orchids. In this study, we analyzed DNA content and nuclei in 41 species of orchids to further map the genome evolution in this plant family. We demonstrate that the DNA remodeling observed in 36 out of 41 orchids studied corresponds to strict partial endoreplication. Such process is developmentally regulated in each wild species studied. Cytometry data analyses allowed us to propose a model where nuclear states 2C, 4E, 8E, etc. form a series comprising a fixed proportion, the euploid genome 2C, plus 2-32 additional copies of a complementary part of the genome. The fixed proportion ranged from 89% of the genome in Vanilla mexicana down to 19% in V. pompona, the lowest value for all 148 orchids reported. Insterspecific hybridization did not suppress this phenomenon. Interestingly, this process was not observed in mass-produced epiphytes. Nucleolar volumes grow with the number of endocopies present, coherent with high transcription activity in endoreplicated nuclei. Our analyses suggest species-specific chromatin rearrangement. Towards understanding endoreplication, V. planifolia constitutes a tractable system for isolating the genomic sequences that confer an advantage via endoreplication from those that apparently suffice at diploid level.

Essential Oil Profile, Phenolic Content and Antioxidant Activity of Geranium kikianum.

This work presents the first phytochemical investigation of Geranium kikianum Kit Tan & G. Vold (Geraniaceae), a species endemic to the southern Peloponnese, Greece. The essential oil from aerial parts of the plant was isolated by hydrodistillation and its chemical composition characterized by GC-MS. A total of 26 compounds were identified, constituting 98.3% of the oil composition. Oxygenated sesquiterpenes were the main group of constituents (75.4%). The major component of the oil was the sesquiterpene ketone germacrone (45.6%). Eleven alcohols represented the most diverse chemical class in the volatile oil (36.3%), and phytol was the most abundant acyclic diterpene alcohol (11.4%). The volatile compounds and residual water remaining after hydrodistillation were screened for their radical-scavenging activity by the DPPH method. IC50 values ranged from 0.2±0.03 mg/mL for the undiluted decoction to 69.7±0.5 mg/mL for the essential oil. The significant antioxidant activity of the residual water, comparable with that of the widespread synthetic antioxidant BHT. (0.2±0.01 mg/mL), and almost tenfold higher than thymol (1.90 t 0.04 mg/mL), correlates with a high content of total phenolic compounds (100.2±1.7 mg GAE/g), and flavonoids (11.9±1.2 mg GAE/g).

Phytochemical and Cytogenetic Characterization of Centaurea solstitialis L. (Asteraceae) from Croatia.

The cytogenetic characterization of Centaurea solstitialis L. (Asteraceae) showed a chromosome number of 2n = 16. Karyotype is composed by four pairs of metacentric, two pairs of submetacentric and two pairs of subtelocentric chromosomes. Physical mapping of two rDNA probes revealed two loci of 35S and one locus of 5S rRNA genes. Chromomycin fluorochrome banding revealed that all rDNA loci were GC rich. The genome size (2C-value) of 1.95 pg classes this species in the group of very small genomes. Chemical composition of C. solstitialis volatile oil (VO) from Croatia, studied with gas chromatography-mass spectrometry showed dominant components as it follows: hexadecanoic acid, α-linolenic acid, germacrene D and heptacosane. Antioxidant capacity, measured by ferric reducing power assay and 2,2-diphenyl-1-picrylhydrazyl methods, as well as inhibition of acetyl- and butyrylcholinesterase of VO was lower comparing to a standard solutions. Volatile oil tested with disc diffusion method showed good inhibitory potential against Pseudomonas aeruginosa, Escherichia coli and all tested fungi: Candida albicans, Penicillium funiculosum and Aspergillus fumigatus. The microdilution method showed best activity against Chronobacter sakazakii and A. fumigatus.