Pinaceae Trees Research Articles

Characterization of Larix decidua Mill. (Pinaceae) oleoresin's essential oils composition using GC-MS.

Larch oleoresin has been described regarding several biological activities and medicinal applications, such as wound healing and treatment of ulcers, but little is known about its chemical composition. Eight oleoresins from Larix decidua Mill. obtained from four companies and one adulterated control were therefore investigated to determine their content of essential oils and to verify possible differences in their composition in relation to the harvest and manufacturing processes. Essential oils (EOs) were isolated by distillation and the yield was analysed. The yield of EO varied among all samples. The yield of the pure larch samples covered a range of 7.8% to 15.5%. A higher yield (19.0%) was observed for adulterated control, which contained oleoresins from different Pinaceae trees. Age of samples had no impact on yield. However, there was a significant statistical variation (p<0.05) in the yields of the mid-summer oleoresins (>10%) compared to early or late summer (<10%), emphasising the importance of the time of collection. Samples were subsequently analysed by GC-MS. EO samples confirmed the presence of various chemical classes, such as monoterpenes, sesquiterpenes, and diterpenes. α-pinene was the compound with the highest concentrations (>50%), followed by β-pinene (>6%), D-limonene (>2.5%), α-terpineol (>0.9%), β-myrcene (>0.2%), and 3-carene (>0.05%). Samples were grouped using multivariate data analysis (MVDA) with respect to the chemical variation between the oleoresins' EOs. The resulting four clusters were named low (low yield obtained for the samples), mixed (mixed oleoresin from different Pinaceae species, adulteration control), old (old oleoresin kept in the institute), and normal (other oleoresins) samples, each presenting distinct chemical biomarkers. There were considerable differences between site and time of collection. Essential oil yield did not always meet requirements as defined by the German Homeopathic Pharmacopoeia. In addition, adulterated or aged samples could be identified as compared to pure and fresh larch oleoresins. We conclude that larch oleoresin used for pharmaceutical applications has to be carefully analysed and standardised to guarantee reproducible product quality.

Production and exchange of goods in Early Byzantine Hierapolis of Phrygia (Turkey): Organic residue analyses on local/regional ovoid amphorae

The paper presents the results of an interdisciplinary investigation aimed at recognizing the contents of the ovoid amphorae produced between the 5th and the 7th century CE in the territory of Hierapolis of Phrygia (Turkey). The specific content of this amphora class has never been investigated although the large amounts of local or regional amphorae recovered in several areas of the city are good indicators of the relationship between Hierapolis and its territory regarding the exchange of locally/regionally produced goods during the Early Byzantine period. Furthermore, the study of pottery assemblages shows a relatively insignificant amount of imported amphorae in the general framework of exchange of the regional cities of the Lykos Valley, such as Hierapolis. Organic residues analysis were carried out by Gas Chromatography-Mass Spectrometry (GC–MS) on twenty-one amphorae samples from the residential contexts excavated in three different areas of the city. The morphology of the selected vessels is well representative of the local/regional amphora type attested in other Early Byzantine contexts of the city. The results obtained from the analyzed samples indicate that most of the amphorae were coated with pitch from Pinaceae trees and that contained grape derivatives, while some of them contained plant oils. These results, combined with morphological and typological data, together with fabric analyses, provide interesting insights into the production and exchange of goods in Hierapolis as well as into the reconstruction of the local economies of inland regions of Anatolia in the Early Byzantine period.

Effects of Hericium erinaceus Hedgehog mushroom on the endophytic microbial community of the host plant.

Hedgehog mushroom is a Hericium erinaceus associated with fagaceae and pinaceae trees in the northern hemisphere. It is still unknown whether this symbiotic relationship will affect the endophytic microbial community of the host plants. In this study, the endophytic microbial communities of different Quercus aliena tissues (root, stem, and leaf) with or without H. erinaceus partner were analyzed by bar-coded pyrosequencing. About 29,000 clean reads were obtained per sample representing 28 phyla of bacteria and 6 phyla of fungi. A total of 26,838 operational taxonomic units (OTUs) of bacteria and 4323 OTUs of fungi were observed at a 97% similarity level. Three bacterial phyla, Proteobacteria, Cyanobacteria and Bacteroidetes, and fungal phylum Ascomycota were dominant in all tissues. The relative abundance of these dominant communities showed significantly differences between Q. aliena tissues with or without H. erinaceus. Bacterial genus Pseudomonas and fungal genus Cryptosporiopsis were species-rich in Q. aliena root infected by H. erinaceus hyphae. This study demonstrated that the endophytic microbial community structure and dominant species varied in Q. aliena mycorrhized with H. erinaceus.

Soil spore bank communities of ectomycorrhizal fungi in Pseudotsuga japonica forests and neighboring plantations.

Ectomycorrhizal (EcM) fungal spores play an important role in seedling establishment and forest regeneration, especially in areas where compatible host tree species are absent. However, compared to other Pinaceae trees with a wide distribution, limited information is available for the interaction between the endangered Pseudotsuga trees and EcM fungi, especially the spore bank. The aim of this study was to investigate EcM fungal spore bank communities in soil in remnant patches of Japanese Douglas-fir (Pseudotsuga japonica) forest. We conducted a bioassay of 178 soil samples collected from three P. japonica forests and their neighboring arbuscular mycorrhizal artificial plantations, using the more readily available North American Douglas-fir (Pseudotsuga menziesii) as bait seedlings. EcM fungal species were identified by a combination of morphotyping and DNA sequencing of the ITS region. We found that EcM fungal spore banks were present not only in P. japonica forests but also in neighboring plantations. Among the 13 EcM fungal species detected, Rhizopogon togasawarius had the second highest frequency and was found in all plots, regardless of forest type. Species richness estimators differed significantly among forest types. The community structure of EcM fungal spore banks differed significantly between study sites but not between forest types. These results indicate that EcM fungal spore banks are not restricted to EcM forests and extend to surrounding forest dominated by arbuscular mycorrhizal trees, likely owing to the durability of EcM fungal spores in soils.

Production and Transport of Goods in the Roman Period: Residue Analysis and Wine Derivatives in Late Republican Baetican Ovoid Amphorae

ABSTRACT Amphorae are key materials in the investigation of the production and transport of goods in ancient times. For the Roman period, many typologies of amphorae are standardised and there are hypotheses concerning their uses and contents mainly based on the shape, provenance, tituli picti and, when preserved, the solid contents. However, there are still many amphora types that have to be investigated in order to better understand the economy of the regions where they were produced and filled. This is the case of the amphorae object of this paper: the so-called ovoid amphorae of Hispania Ulterior/Baetica. This paper presents new results of an interdisciplinary investigation aimed to discover the commodities contained in ovoid amphorae. This amphora type and its specific use have never been investigated, except for a preliminary test. Here, organic residues analysis of twenty-four amphorae produced in two different locations in Hispania Ulterior/Baetica (Bay of Cadiz and the Guadalquivir Valley) and excavated at the site of El Olivillo in Cadiz (Spain), are presented. The findings suggest that the majority of the amphorae were coated with abundant pitch derived from Pinaceae trees and that most of them contained grape derivatives, although other products were also identified. Not only is this documentation of Hispania Ulterior/Baetican wine production in the late Republican period important, but the use of ovoid amphorae for carrying wine is somewhat unexpected because it is usually thought that amphorae of this type in southern Italy and in the Corinthia probably carried olive oil.

Sustaining the growth of Pinaceae trees under nutrient-limited edaphic conditions via plant-beneficial bacteria.

Lodgepole pine, a prominent Pinaceae tree species native to western North America, is well-known for its ability to thrive in highly disturbed and degraded areas. One such area is the Sub-Boreal Pine-Spruce xeric-cold (SBPSxc) region in British Columbia, Canada, which is characterized by weakly-developed, parched soils that lack an organic forest floor and essential plant-available nutrients. We hypothesized that plant growth-promoting bacteria could play a significant role in sustaining the growth of lodgepole pine trees in the SBPSxc region. Testing this hypothesis, we evaluated plant growth-promoting abilities of six endophytic bacterial strains previously isolated from lodgepole pine trees growing in this region. These bacterial strains significantly enhanced the length and biomass of their natural host (lodgepole pine) as well as a foreign host (hybrid white spruce) in a 540-day long greenhouse trial. This growth stimulation could be linked to the diverse plant growth-promoting (PGP) abilities detected in these strains using in vitro assays for inorganic/organic phosphate-solubilization, siderophore production IAA production, ACC deaminase activity, lytic enzymes (chitinase, β-1,3-glucanase, protease, and cellulase) activity, ammonia production and catalase activity. ACC deaminase activity was also detected in vivo for all strains using ethylene-sensitive plants–canola and tomato. Notably, strains belonging to the Burkholderiaceae family (HP-S1r, LP-R1r and LP-R2r) showed the greatest potential in all PGP assays and enhanced pine and spruce seedling length and biomass by up to 1.5-fold and 4-fold, respectively. Therefore, such bacterial strains with multifarious PGP abilities could be crucial for survival and growth of lodgepole pine trees in the SBPSxc region and could potentially be utilized as bioinoculant for Pinaceae trees in highly disturbed and nutrient-poor ecosystems.

Identifying new associations between invasive aphids and Pinaceae trees using plant sentinels in botanic gardens

Despite progress in prevention and management of biological invasions, challenges remain, including difficulties with assessing future invasion risks. Predicting the identity of potentially damaging invaders is complex because they are often unknown as pests in their natural range. We used a plant sentinel approach to assess host ranges of invasive aphids across 62 conifer species from around the world in Christchurch Botanic Gardens, as a model for risk assessment such as for the ‘International Plant Sentinel Network’. Using standardised sampling, we obtained ca. 600 observations of aphid abundances (4731 individuals), mainly of the pine aphids Eulachnus brevipilosus and Essigella californica and the spruce aphids Cinara pilicornis and Elatobium abietinum. These aphids were highly genus-specific, despite the spatially mixed distribution of genera. A phylogenetic analysis of pine aphid host preferences showed that abundances of species varied among pine subgenera and the geographic origin of trees, with significantly more aphids on pines in the subgenus Pinus than the subgenus Strobus. Essigella californica occurred abundantly on many pines across most subsections in the subgenus Pinus whereas E. brevipilosus was largely restricted to a few species in the subsection Pinus. Our study revealed several new host records (previously unknown aphid-host relationships) including the abundant occurrence of E. brevipilosus on Japanese red pine, P. densiflora, and of E. californica on P. yunnanensis, P. serotina, P. brutia, and P. nigra, among others. Our study identified numerous novel insect-plant interactions that are likely to materialise if these aphids colonise new host plants, confirming the utility of the plant sentinel approach.

Anti-tumorigenic and anti-angiogenic effects of natural conifer Abies sibirica terpenoids in vivo and in vitro.

The natural terpenoid compound was explored in vitro and in vivo to investigate the anti-HCC properties. For our study we used Abisilin®- a novel natural pharmacological terpenoid compound extracted and purified from coniferous Pinaceae trees. Anti-tumorigenic properties of different concentrations of Abisilin® were tested on murine hepatoma Hepa 1-6 cell lines. The analysis of proliferation and apoptosis was performed using immunofluorescence microscopy, FACS and qPCR. As an in vivo approach, we tested Abisilin® (400mg/kg/day, 14 days, orally) in xenograft mouse models of liver cancer and investigated tumor growth, proliferation, apoptosis and angiogenesis by means of Western blotting, immunofluorescence microscopy and qPCR. Application of Abisilin® for 24h at a dosage ranging from 0.03 to 0.045mg significantly reduced the number of viable Hepa 1-6 cells and induced apoptotic cell death with microscopic evidence of changes in cell morphology, and positive TUNEL, cleaved caspase 3 and Annexin V/Propidium Iodide (PI) stainings. Furthermore, treatment with Abisilin® strongly inhibited proliferation, impaired mitosis and prompted cell cycle arrest by down-regulation of the Cyclin D1, E1 and A2 expression levels. In Hepa 1-6 xenograft in vivo model, Abisilin® considerably decreased the xenograft tumor size and tumor volume. Consistently with in vitro Abisilin® administration elicited apoptosis and inhibit proliferation in the xenograft tumor. We also found that Abisilin® remarkably decrease microvessel density, diminished tumor angiogenesis and reduced expression of ICAM-1. Moreover, the expression of pAMPK, a cellular energy sensor, was up-regulated after Abisilin® application. Anti-proliferative, pro-apoptotic activity and anti-angiogenic potential of natural conifer terpenoids might turn these compounds into an attractive drug candidate for combination therapy against liver cancer.

Two new species of Rhizopogon associated with Pinus pumila from Japan

Rhizopogon (Boletales) is an ectomycorrhizal fungal genus that forms symbiotic associations with Pinaceae trees, especially Pinus and Pseudotsuga, and produce hypogeous sporocarps. The taxonomy of Rhizopogon has been well-studied in Europe and North America, but little work has been conducted in Asia. In Japan, previous studies described several Rhizopogon species, each of which was associated with two-leaved pines (subgenus Pinus) or Pseudotsuga japonica. In the present study, we describe R. alpinus sp. nov. and R. nitidus sp. nov. associated with Pinus pumila (subgenus Strobus) distributed in alpine regions of Japan. These two new species are phylogenetically and morphologically distinct from other Rhizopogon species, especially those associated with the subgenus Pinus.

Bursaphelenchus niphades n. sp. (Tylenchina: Aphelenchoididae) amensally associated with Niphades variegatus (Roelofs) (Coleoptera: Curculionidae)

ABursaphelenchusspecies was isolated from a Japanese native wood-boring weevil,Niphades variegatus, and dead Pinaceae trees. The nematode is associated with the weevils as dauer (dispersal third stage) juveniles and the dauers enter the weevil tracheal system forming an abnormal expansion on the weevil trachea (atrium). Thus, the nematode is hypothesised to be an amensal/phoretic associate of the weevil because the abnormal expansion appeared to inhibit weevil respiration. The propagative stages of the nematode are associated with dead trees (wood and bark materials) and are thought to feed there on naturally propagated fungi. Morphologically, the new species is considered an undescribed species close toB. antoniae,B. chengiandB. hylobianum. Within these four species, the new species, which is described herein asB. niphadesn. sp., is closest toB. chengi,i.e., the typological character of these two species are almost identical to each other and is distinguished by some minor characters (structure of the male P4 genital papillae and spicule length). The molecular phylogenetic analysis supported the morphological observations.Bursaphelenchus niphadesn. sp. formed a well supported subclade with the four species and is intermediate betweenB. hylobianumandB. chengi; however, it is distinguished by the molecular sequences of some ribosomal RNA genes. Because three of these four species are associated with weevil species, the subclade is considered a ‘weevil-associated’ species group.

Exotic mammals disperse exotic fungi that promote invasion by exotic trees.

Biological invasions are often complex phenomena because many factors influence their outcome. One key aspect is how non-natives interact with the local biota. Interaction with local species may be especially important for exotic species that require an obligatory mutualist, such as Pinaceae species that need ectomycorrhizal (EM) fungi. EM fungi and seeds of Pinaceae disperse independently, so they may use different vectors. We studied the role of exotic mammals as dispersal agents of EM fungi on Isla Victoria, Argentina, where many Pinaceae species have been introduced. Only a few of these tree species have become invasive, and they are found in high densities only near plantations, partly because these Pinaceae trees lack proper EM fungi when their seeds land far from plantations. Native mammals (a dwarf deer and rodents) are rare around plantations and do not appear to play a role in these invasions. With greenhouse experiments using animal feces as inoculum, plus observational and molecular studies, we found that wild boar and deer, both non-native, are dispersing EM fungi. Approximately 30% of the Pinaceae seedlings growing with feces of wild boar and 15% of the seedlings growing with deer feces were colonized by non-native EM fungi. Seedlings growing in control pots were not colonized by EM fungi. We found a low diversity of fungi colonizing the seedlings, with the hypogeous Rhizopogon as the most abundant genus. Wild boar, a recent introduction to the island, appear to be the main animal dispersing the fungi and may be playing a key role in facilitating the invasion of pine trees and even triggering their spread. These results show that interactions among non-natives help explain pine invasions in our study area.

An inoculation experiment of Japanese Bursaphelenchus nematodes on Japanese black and red pine, Pinus thunbergii and P. densiflora

The pinewood nematode (PWN) Bursaphelenchus xylophilus is an invasive pathogen that was introduced from North America to Asian countries and Portugal and is devastating native pine forests. Some native European and Asian Bursaphelenchus nematodes also have weak to moderate pathogenicity to native pine species. To evaluate the potential risk of native Bursaphelenchus species, we inoculated ten Japanese Bursaphelenchus species into native pine species (the dominant forest species) in Japan, and evaluated their pathogenicity using mortality and tracheal tissue damage as indices. Inoculation was conducted on August 3, 2007, and the symptoms were observed every 2 weeks until February 1, 2008. None of the inoculated trees, excluding the pathogenic PWN inoculated control, showed external disease symptoms; however, four species [a less pathogenic PWN isolate, B. luxuriosae, Bursaphelenchus sp. NK215 (undescribed), and NK224 (undescribed)] caused tracheal tissue damage in inoculated seedlings and showed weak pathogenicity. Therefore, we conclude that there are some potentially pathogenic native species of nematodes distributed in Japan. Interestingly, two of these weakly pathogenic species, B. luxuriosae and NK215, are not associated with Pinaceae trees, suggesting that nematode pathogenicity may be a pre-adaptive character. More experimental studies under different conditions are necessary to accurately evaluate the potential risk of these pathogens.

Terpenoids and steroids from several euphorbiaceae and pinaceae plants

During the course of a search for biologically active constituents from unexamined plant sources, several biogenetically interesting new di- and tri-terpenes and steroids were isolated from several weeds and shrubs of Euphorbiaceae and the bark, leaves and cones of several Pinaceae trees which had been treated as wastes in the forestry industry. Euphorbia supina contained 3,4-seco-5 alpha- and 5 beta-adian-4(23)-ene-3,5-diols and related oxides, oxygenated fern-8-en-3 beta-ols named supinenolones A-E and unusually migrated oxyfernanes having (9S)- and (9R)-7(8-->9)abeo-9-D:C-friedo-B':A'-neogammacerane skeletons named spirosupinane and neospirosupinane, while E. chamaesyce contained 3,4-seco-oleana-4(23), 18-dien-3-oic acid, 3,4-seco-8 beta H-ferna-4(23),9(11)-dien-3-oic acid and two oxygenated obtusifoliols. The bark of Phyllanthus flexuosus (Euphorbiaceae) contained 11 beta-hydroxy-D:A-friedo-olean-1-en-3-one, lup-20(29)-ene-3 beta, 15 alpha-diol, olean-12-ene-3 beta,15 alpha-diol and olean-12-ene-3 beta,15 alpha,24-triol together with trichadenic acid B for which we revised the structure to 3 beta-hydroxy-D:A-friedo-oleanan-27-oic acid. Two 26-nor-D:A-friedo-olean-14-enes were isolated from P. watsonii. Regarding Pinaceae trees, an unusually migrated abieslactone [(3R, 7S, 9R, 23R)-7-hydroxy-3-methoxy-8-oxo-7(8-->9)abeo-lanost-24-eno-26,23-lactone], named spiroveitchionolide, was isolated from the bark of Abies species, besides nine abieslactone analogues. Two pairs of unusually migrated serratanes, piceanonols A and B and jezananals A and B having novel skeletal systems of 14(13-->12) abeo- and 16(15-->14) abeo-serratanes named piceanane and jezanane, respectively, were also isolated from the stem bark of Picea species, besides three 14 beta,15 beta-epoxyserratanes and two 13 alpha,14 alpha-epoxyserratanes. The leaves of Larix kaempferi contained two deformed abietanes named karamatsuic acid (9,10-seco-9,10 alpha-epoxyabieta-8,11,13-trien-18-oic acid) and larikaempferic acid [9 alpha,13 alpha-epoxy-8-oxo-9(8-->7)abeo-7 beta-abietan-18-oic acid], as well as the cones to contain 8 alpha,12 alpha-epidioxy-15-hydroxyabiet-13-en-18-oic acid, three diepoxy-abietan-18-oic acids and two new dehydroabietic acid analogues. Several of the above compounds exhibited inhibitory effects against tumor-promoting and DNA topoisomerase II activities.