Srdjan Bojović

Chemical and principal-component analyses of the essential oils of Apioideae taxa (Apiaceae) from central Balkan.

The volatile constituents of the essential oils of 23 taxa belonging to the Apioideae subfamily were studied in detail. The investigated taxa were Pimpinella serbica (Vis.) Bentham & Hooker, Libanotis montana Cr., Cnidium silaifolium (Jacq.) Simk. ssp. orientale (Boiss.) Tutin, Bupleurum praealtum L., B. sibthorpianum S. S. var. diversifolium (Roch.) Hay, Aegopodium podagraria L., Torilis anthriscus (L.) Gmel., Orlaya grandiflora (L.) Hoffm., Laserpitium siler L., Laser trilobum (L.) Brokh., Chaerophyllum aureum L., C. hirsutum L., C. temulum L., Pastinaca sativa L., P. hirsuta Pancic., Tordylium maximum L., Physospermum cornubiense (L.) DC., Peucedanum alsaticum L., P. oreoselinum (L.) Moench, P. cervaria (L.) Cuss., P. austriacum (Jacq.) Koch, P. longifolium W. et K., and P. officinale L. All of these species grow wild in the central part of the Balkan Peninsula. The essential oils were found to be complex mixtures of various compounds, more than 100 constituents being in each taxon, with contributions of main products never exceeding 25% of the total content. Sesquiterpene hydrocarbons were found to be the main group of constituents of all taxa, except for Peucedanum species, where monoterpene hydrocarbons were identified as the main components. The chemotaxonomic value of the essential‐oil composition is discussed according to results of principal‐component analysis (PCA). The essential‐oil composition mainly reflects current taxonomic relationships between the investigated taxa.

Origin identification of Pinus nigra populations in southwestern Europe using terpene composition variations

The geographical variation of terpenes of Pinus nigra populations from southwestern Europe was studied. Terpenes from the foliage of 16 populations from Corsica, Herault (France) and the East Pyrenees (France and Spain) were analyzed. A total of 42 terpenes were detected, with α-pinene the dominant monoterpene and germacrene-d and caryophyllene the dominant sesquiterpenes. The differences in quantitative content of selected compounds clearly divide populations into two basic geographical groups: on one side the populations from Herault and the East Pyrenees and on the other the populations from Corsica. β-Phellandrene and δ-cadinene have the greatest influence on this global discrimination. Some trees and populations show a similarity although they belong to different geographic locations. The similarity of some trees from Herault and the East Pyrenees and trees from Corsica points to their common origin (Corsica). Our results confirm the hypothesis that the afforestation of Herault and the East Pyrenees was also performed with black pine from Corsica.

Composition of n-alkanes in natural populations of Pinus nigra from Serbia - chemotaxonomic implications.

This is the first report on the composition and variability of the needle‐wax n‐alkanes in natural populations of Pinus nigra in Serbia. Samples of 195 trees from seven populations belonging to several infraspecific taxa (ssp. nigra, var. gocensis, ssp. pallasiana, and var. banatica) were analyzed. In general, the size of the n‐alkanes ranged from C16 to C33, with the exception of ssp. nigra, for which it ranged from C18 to C33. The most abundant were C23‐, C25‐, C27‐, and C29‐alkanes. The needle waxes of Populations I–III and V were characterized by a higher content of C23‐, C25‐, and C27‐alkanes and a lower content of C24‐, C26‐, C28‐, and C30‐alkanes, compared to the other populations, and the trees of these populations could be assigned to ssp. nigra. The samples of Population VI were characterized by higher amounts of C22‐, C24‐, C30‐, and C32‐alkanes and lower amounts of C25‐ and C27‐alkanes, and the trees could be considered as ssp. pallasiana. The samples of Population VII, consisting of trees belonging to var. banatica, were richer in C29‐, C31‐, and C33‐alkanes. The wax compositions of Populations IV and V, both composed of trees previously determined as P. nigra var. gocensis, showed a tendency of splitting. Indeed, the alkane composition of Population IV was closer to that of ssp. pallasiana pines, while that of Population V was more similar to that of ssp. nigra pines. From the results presented here, it is obvious that in the central part of the Balkan Peninsula, significant diversification and differentiation of the populations of black pine exists, and these populations could be defined as different intraspecific taxa. Our results also indicate the validity of n‐alkanes as chemotaxonomic characters within this aggregate.

Chemotaxonomic Significance of the Terpene Composition in Natural Populations of Pinus nigra J.F.Arnold from Serbia

The essential‐oil variability in seven native populations belonging to different infraspecific taxa of Pinus nigra (ssp. nigra, var. gocensis, ssp. pallasiana, and var. banatica) growing wild in Serbia was analyzed. In the needles of 195 trees from seven populations, 58 essential‐oil components were identified. The major components were α‐pinene (43.6%) and germacrene D (29.8%), comprising together 73.4% of the total oil composition. Based on the average chemical profile of the main terpene components (with contents >5%), the studied populations were found to be the most similar to populations from central Italy and Greece (ssp. nigra). Cluster analysis showed the division of the populations into three principal groups: the first group consisted of Populations I, II, III, IV, and V (considered as ssp. nigra group), the second of Population VI (ssp. pallasiana group), and the third of Population VII, which had the most distinct oil composition (ssp. banatica group). The taxonomic implications of the essential‐oil profiles of the investigated taxa of this very complex species are discussed.

Terpene Chemodiversity of Relict Conifers Picea omorika, Pinus heldreichii, and Pinus peuce, Endemic to Balkan

Terpenes are often used as ecological and chemotaxonomic markers of plant species, as well as for estimation of geographic variability. Essential oils of relic and Balkan endemic/subendemic conifers, Picea omorika, Pinus heldreichii, and P. peuce, in central part of Balkan Peninsula (Serbia and Montenegro), on the level of terpene classes and common terpene compounds were investigated. In finding terpene combinations, which could show the best diversity between species and their natural populations, several statistical methods were applied. Apart from the content of different terpene classes (P. omorika has the most abundant O‐containing monoterpenes and sesquiterpenes; P. heldreichii and P. peuce have the largest abundance of sesquiterpene and monoterpene hydrocarbons, resp.), the species are clearly separated according to terpene profile with 22 common compounds. But, divergences in their populations were established only in combination of several compounds (specific for each species), and they were found to be the results of geomorphologic, climatic, and genetic factors. We found similarities between investigated species and some taxa from literature with respect to terpene composition, possibly due to hybridization and phylogenetic relations. Obtained results are also important regarding to chemotaxonomy, biogeography, phylogeny, and evolution of these taxa.

Chemodiversity of Nonacosan-10-ol and n-Alkanes in the Needle Wax of Pinus heldreichii

This is the first report of individual variability and population diversity of the contents of nonacosan‐10‐ol and n‐alkanes in the needle cuticular waxes of Bosnian pines originated from Montenegro, regarded as Pinus heldreichii var. leucodermis, and from Serbia, regarded as P. heldreichii var. pančići. The amount of nonacosan‐10‐ol varied individually from 27.4 to 73.2% (55.5% in average), but differences between the four investigated populations were not statistically confirmed. The size of the n‐alkanes ranged from C18 to C33. The most abundant n‐alkanes were C23, C27, and C25 (12.2, 11.2, and 10.8% in average, resp.). The carbon preference index (CPI) of the n‐alkanes ranged from 0.8 to 3.1 (1.6 in average), while the average chain length (ACL) ranged from 20.9 to 26.5 (24.4 in average). Long‐chain and mid‐chain n‐alkanes prevailed (49.6 and 37.9% in average, resp.). It was also found that the populations of P. heldreichii var. leucodermis had predominantly a narrower range of n‐alkanes (C18C31) than the trees of the variety pančići (C18C33). Differences between the varieties were also significant for most of the other characteristics of the n‐alkane pattern (e.g., most abundant n‐alkanes, CPI, ACL, and relative proportion of short‐, mid‐, and long‐chain n‐alkanes). The principle component and cluster analyses of eleven n‐alkanes confirmed the significant diversity of these two varieties.

Biological activity of Pinus nigra terpenes-Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity

In the current work, in vitro antioxidant, antibacterial, and antifungal activites of the needle terpenes of three taxa of Pinus nigra from Serbia (ssp. nigra, ssp. pallasiana, and var. banatica) were analyzed. The black pine essential oils showed generally weak antioxidative properties tested by two methods (DPPH and ABTS scavenging assays), where the highest activity was identified in P. nigra var. banatica (IC50=25.08 mg/mL and VitC=0.67 mg (vitamin C)/g when tested with the DPPH and ABTS reagents, respectively). In the antimicrobial assays, one fungal (Aspergilus niger) and two bacterial strains (Staphylococcus aureus and Bacillus cereus) showed sensitivity against essential oils of all three P. nigra taxa. The tested oils have been shown to possess inhibitory action in the range from 20.00 to 0.62 mg/mL, where var. banatica exhibited the highest and ssp. nigra the lowest antimicrobial action. In order to determine potential compounds that are responsible for alternative mode of action, molecular docking simulations inside FtsZ (a prokaryotic homolog of tubulin) were performed. Tested compounds were the most abundant terpenoid (germacrene D-4-ol) and its structurally similar terpene (germacrene D), both present in all three essential oils. It was determined that the oxygenated form of the molecule creates stable bonds with investigated enzyme FtsZ, and that this compound, through this mechanism of action participates in the antimicrobial activity.

Chemotaxonomic implications of the n-alkane composition and the nonacosan-10-ol content in Picea omorika, Pinus heldreichii, and Pinus peuce.

The n‐alkane composition and the nonacosan‐10‐ol content in the needle cuticular waxes of Serbian spruce (Picea omorika), Bosnian pine (Pinus heldreichii), and Macedonian pine (Pinus peuce) were compared. The amount of nonacosan‐10‐ol in the needle waxes of P. omorika was higher than those in P. heldreichii and P. peuce. The range of n‐alkanes was also wider in P. omorika (C18–C35) than in P. heldreichii and P. peuce (C18–C33). The dominant n‐alkanes were C29 in the needle waxes of P. omorika, C23, C27, and C25 in those of P. heldreichii, and C29, C25, C27, and C23 in those of P. peuce. The waxes of P. omorika contained higher amounts of n‐alkanes C29, C31, and C33, while those of P. heldreichii and P. peuce had higher contents of n‐alkanes C21, C22, C23, C24, and C26. The principal component analysis of the contents of nine n‐alkanes showed a clear separation of the Serbian spruce populations from those of the two investigated pine species, which partially overlapped. The separation of the species was due to high contents of the n‐alkanes C29 and C31 (P. omorika), C19, C20, C21, C22, C23, and C24 (P. heldreichii), and C28 (P. peuce). Cluster analysis also showed a clear separation between the P. omorika populations on one side and the P. heldreichii and P. peuce populations on the other side. The n‐alkane and terpene compositions are discussed in the light of their usefulness in chemotaxonomy as well as with regard to the biogeography and phylogeny of these rare and endemic conifers.

The attraction of cerambycids and other xylophagous beetles, potential vectors of Bursaphelenchus xylophilus, to semio-chemicals in Slovenia

The attractiveness of different semio-chemicals to potential vectors of the phytoparasitic nematode Bursaphelenchus xylophilus was investigated in conifer forests in Slovenia. From 2007 to 2009, the presence of xylophagous beetles in Pinus nigra, P. sylvestris, P. halepensis, Picea abies and Abies alba stands was assessed at eight locations. Insects were collected at 1-month intervals during the growing season using four cross vane traps per location with a collecting container with propylene glycol and attractants (ethanol+α-pinene, Pheroprax® and Gallowit®). The trapped insects represented 24 families of the order Coleoptera, and we identified 94 species. The most numerous group was the weevil subfamily Scolytinae (76.55% of all insects collected), followed by the family Cerambycidae (8.12%), and the weevil subfamily Curculioninae (1.67%). With regard to species number, the most frequent wood-borers were Cerambycidae (24 taxa), Scolytinae (12 species) and Buprestidae (8 species). The most abundant species was Spondylis buprestoides, followed by Arhopalus rusticus, Monochamus galloprovincialis and Arhopalus ferus. At all locations, the largest catch of Cerambycidae occurred in July. The most effective attractant was ethanol+α-pinene, followed by Gallowit®; the least effective attractant was Pheroprax®. Among Monochamus species, M. galloprovincialis represented 17.54%, M. sutor 0.09% and M. sartor 0.04% of the long-horned beetles collected. Monochamus individuals were most numerous in the P. nigra stand and were attracted in the greatest numbers by Gallowit®, followed by ethanol+α-pinene. The cerambycid catch was highly correlated with the catch of non-target bark beetle predators (Cleridae, Staphylinidae, Histeridae, Trogositidae, Nitidulidae, Rhizophagidae) in the traps.

Population Variability of Nonacosan-10-ol and n-Alkanes in Needle Cuticular Waxes of Macedonian Pine (Pinus peuceGriseb.)

This is the first report on population variability of nonacosan‐10‐ol and n‐alkanes in needle epicuticular waxes of Macedonian pine (Pinus peuce Griseb.) Hexane extracts of needle samples, originating from two natural populations in Montenegro (Zeletin and Sjekirica) and from one population in Serbia (Mokra Gora) were analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The amount of nonacosan‐10‐ol varied individually from 41.3 to 72.31% (average 55.9%), with the Sjekirica population being statistically divergent (64.4% on average). The results showed n‐alkanes in epicuticular waxes ranging from C18 to C33. The most abundant alkanes were C29, C25, C27, and C23 (15.5, 11.1, 10.6, and 10.5% on average, resp.). The carbon preference index of Pinus peuce ranged from 1.0 to 4.3 (1.9 on average). Average chain length ranged from 18.4 to 27.7 (average 25.7). A high level of inidividual quantitative variation in all of these hydrocarbon parameters was also detected. These results were compared with published data on other species from the Pinus genus.