Gymnosperms of Idaho: Chemical Compositions and Enantiomeric Distributions of Essential Oils of Abies lasiocarpa, Picea engelmannii, Pinus contorta, Pseudotsuga menziesii, and Thuja plicata.

Conifers are of great economic value in terms of lumber production, important for construction and other uses such as pulp and paper. They are also important sources of essential oils. Conifer species have been vital to the ethnobotany and traditional herbal medicine of many different Native American groups. The objective of this work was to obtain and analyze the essential oils of several conifer species (Abies lasiocarpa, Picea engelmannii, Pinus contorta, Pseudotsuga menziesii, and Thuja plicata) growing in Idaho. The foliar essential oils were obtained by hydrodistillation and then analyzed by gas chromatographic methods, including GC-MS, GC-FID, and chiral GC-MS. The essential oils were obtained in varying yields from 0.66% up to 4.70%. The essential oil compositions were largely dominated by monoterpene hydrocarbons and oxygenated monoterpenoids. The chiral monoterpenoids were generally rich in the (-)-enantiomers for members of the Pinaceae, but the (+)-enantiomers predominated in the Cupressaceae. The essential oil compositions obtained in this work are qualitatively similar, but quantitatively different, to previously reported compositions and confirm and complement the previous reports. However, this is the first comprehensive analysis of the chiral terpenoid components in these conifer species. Additional research on essential oils of the Pinaceae and Cupressaceae is needed to describe the chemical profiles, chemical compositions, and enantiomeric distributions more reliably in the various species and infraspecific taxa of these two families.

Pseudotsuga menziesii (Pinaceae): Volatile Profiles, Antimicrobial Activity and Toxicological Evaluation of Its Essential Oil.

The present article investigates the chemical composition of volatiles of essential oil (EO) and headspace (HS) fraction, as well as biological activities of EO obtained from needles with twigs of Pseudotsuga menziesii var. menziesii cultivated in Serbia. The major class of compounds was monoterpene hydrocarbons with α-terpinolene, sabinene and ß-pinene (EO), and sabinene, α-terpinolene and ß-pinene (HS) as the dominant volatiles. Tested EO exhibited mostly low antimicrobial potential against investigated strains (ATCC and respiratory isolates), where MICs ranged 1.25-20.00 mg/mL. Nevertheless, based on presented results, where antimicrobial testing was done for the first time on human respiratory system isolates, there is a potential of this EO to be used as an adjuvant in the treatment of human respiratory infections, especially those caused by Pseudomonas aeruginosa or Candida albicans strains. Regarding toxicological evaluation, EO showed moderate toxicity in Artemia salina toxicity bioassay (LC50 =347.41, after 24 h) as well as week toxicity against Drosophila melanogaster with the ability only to moderately delay larval and pupal development.

Saprotrophic and ectomycorrhizal fungal sporocarp stoichiometry (C†:†N†:†P) across temperate rainforests as evidence of shared nutrient constraints among symbionts.

Quantifying nutritional dynamics of free-living saprotrophs and symbiotic ectomycorrhizal fungi in the field is challenging, but the stoichiometry of fruiting bodies (sporocarps) may be an effective methodology for this purpose. Carbon (C), nitrogen (N) and phosphorus (P) concentrations of soils, foliage and 146 sporocarp collections were analyzed from 14 Pseudotsuga menziesii var. menziesii stands across a podzolization gradient on Vancouver Island (Canada). N and P concentrations were considerably higher in saprotrophic fungi. Fungal N% increased with soil N content at a greater rate for saprotrophs than ectomycorrhizal fungi, while fungal P% of saprotrophs was more constrained. Fungal N : P was more responsive to soil N : P for ectomycorrhizal fungi (homeostatic regulation coefficient 'H' = 2.9) than saprotrophs (H = 5.9), while N : P of ectomycorrhizal fungi and host tree foliage scaled almost identically. Results underscore the role of ectomycorrhizal fungi as nutrient conduits, supporting host trees, whereas saprotrophs maintain a greater degree of nutritional homeostasis. Site nutrient constraints were shared in equal measure between ectomycorrhizal fungi and host trees, particularly for P, suggesting neither partner benefits from enhanced nutrition at the expense of the other. Sporocarp stoichiometry provides new insights into mycorrhizal relationships and illustrates pervasive P deficiencies across temperate rainforests of the Pacific Northwest.

Spatiotemporal activities of Douglas-fir BiP Pro1 promoter in transgenic potato.

MAIN CONCLUSION: The PmBiPPro1 promoter of the luminal binding protein (BiP) from Douglas-fir is fully functional in transgenic potato, responsive to wounding, and has high transcriptional activity in tubers. A predefined pattern and level of transgene expression targeted to specific tissues or organs and at a particular developmental stage is a pre-requisite for the successful development of plants with desired traits. Here, we evaluated the transcriptional activity of the PmBiPPro1 promoter of the luminal binding protein (BiP) from Douglas-fir, by expressing reporter ß-D-glucuronidase (GUS) gene constructs containing three different PmBiPPro1 promoter versions (2258 bp, 1259 bp, and 278 bp) in transgenic potato. In conifers, this promoter regulates the endoplasmic reticulum (ER) molecular chaperon of the HSP70 stress-related protein family and is essential for proper functioning of the ER. Stable expression analysis demonstrated that two of three PmBiPPro1 promoter versions (PmBiPPro1-1 and PmBiPPro1-3) were fully functional in the heterologous host, exhibited high transcriptional activities in the leaves of unstressed potatoes, and were responsive to wounding. Deletion analysis showed that the positive cis-active regulatory elements necessary for higher level expression resided within the - 1243 to - 261 region, whereas negative cis-active elements encompassed nucleotides - 2242 to - 1243. Histochemical staining revealed high level of GUS activities in tissues associated with a high rate of cell division and secretory activities. Most importantly, the PmBiPPro1 promoters, especially the full-length version, had activity in microtubers at a level that was much higher than in any other potato organ or tissue. The - 2242 to - 1243 bp region likely contains important cis element(s) that interact with tuber-specific transcription factors required for promoter activation in the storage organs. The organ-specific activity of the PmBiPPro1 promoters may be useful for targeted expression of heterologous molecules in potato tubers.

Temporal quantification of mating system parameters in a coastal Douglas-fir seed orchard under manipulated pollination environment.

Seed orchards main function is delivering breeding programs' gains in the form of genetically improved seedlings. They are unique experimental populations, perfectly suited for studying various pollination environments (natural or otherwise), affecting their mating system parameters. Here, under different pollination environment (natural and intrusive (pollen augmentation and/or bloom-delay)), the mating system of a second generation, wind-pollinated, coastal Douglas-fir (Pseudotsuga menziesii var. menziesii (Mirb.) Franco) seed orchard was evaluated over four years. Using DNA microsatellite markers and bulk seed samples, we conducted pedigree reconstruction to assign each seed's male and female parents, followed by determining the extent of pollen contamination (external gene flow), selfing rate, and, parental gametic contribution for each year. Overall, external pollen contamination rates ranged between 10 and 28%, selfing rate varied between 12 and 17%, and 80% of the seed crops were produced by 37-64% of the orchard's parents. Pollination environment and seed crop size substantially influenced the observed results, particularly for small crops as pollen contamination was high in natural (28%) vs. intrusive pollination (10%). Generally, irrespective of the crop size, seed produced under natural pollination had higher pollen contamination, confirming the role of pollination environment manipulation in improving seed crops' genetic quality.

Water availability as dominant control of heat stress responses in two contrasting tree species.

Heat waves that trigger severe droughts are predicted to increase globally; however, we lack an understanding of how trees respond to the combined change of extreme temperatures and water availability. Here, we studied the impacts of two consecutive heat waves as well as post-stress recovery in young Pseudotsuga menziesii (Mirb.) Franco (Douglas-fir) and Robinia pseudoacacia L. (black locust) growing under controlled conditions. Responses were compared under water supply close to the long-term average and under reduced irrigation to represent drought. Exposure to high temperatures (+10 °C above ambient) and vapour pressure deficit strongly affected the trees in terms of water relations, photosynthesis and growth. Douglas-fir used water resources conservatively, and transpiration decreased in response to mild soil water limitation. In black locust, heat stress led to pronounced tree water deficits (stem diameter shrinkage), accompanied by leaf shedding to alleviate stress on the hydraulic system. The importance of water availability during the heat waves became further apparent by a concurrent decline in photosynthesis and stomatal conductance with increasing leaf temperatures in both species, reaching the lowest rates in the heat-drought treatments. Stress severity determined both the speed and the amount of recovery. Upon release of stress, photosynthesis recovered rapidly in drought-treated black locust, while it remained below control rates in heat (t = -2.4, P < 0.05) and heat-drought stressed trees (t = 2.96, P < 0.05). In Douglas-fir, photosynthesis recovered quickly, while water-use efficiency increased in heat-drought trees because stomatal conductance remained reduced (t = -2.92, P < 0.05). Moreover, Douglas-fir was able to compensate for stem-growth reductions following heat (-40%) and heat-drought stress (-68%), but most likely at the expense of storage and other growth processes. Our results highlight the importance of studying heat waves alongside changes in water availability. They further suggest that we should look beyond the actual stress event to identify lagged effects and acclimation processes that may determine tree resilience in the long term.

Conifer somatic embryogenesis: improvements by supplementation of medium with oxidation-reduction agents.

A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue (ET) and continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment has been shown to decrease this recalcitrance. Glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid and dehydroascorbate analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiological concentrations. Major differences in stage-specific oxidation-reduction (redox) agents were observed. A simple bioassay was used to evaluate potential growth-promotion of natural and inorganic redox agents added to early-stage somatic embryo growth medium. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase initiation of loblolly pine. Low-cost reducing agents sodium dithionite and sodium thiosulfate increased ET initiation for loblolly pine and Douglas fir (Mirb) Franco. Germination medium supplementation with GSSG increased somatic embryo germination. Early-stage somatic embryos grown on medium with or without sodium thiosulfate did not differ in GSH or GSSG content, suggesting that sodium thiosulfate-mediated growth stimulation does not involve GSH or GSSG. We have developed information demonstrating that alteration of the redox environment in vitro can improve ET initiation, early-stage embryo development and somatic embryo germination in loblolly pine.

Phytoremediation of cadmium-contaminated soils by young Douglas fir trees: effects of cadmium exposure on cell wall composition.

Douglas fir trees grown on an artificially Cd-contaminated soil, can tolerate this trace element (up to 68 mg/kg in soil) during several months. Most of the absorbed Cd is retained in roots (25 mg/kg DM), but transfer to aerial part is also effective. Showing the highest content, up to 6 mg/kg DM, among all the aboveground parts, barks seem to be a preferred storage compartment. However, the transfer factor is quite low, about 0.3. Another objective of this study was to compare the cell wall components of trees exposed to increasing Cd amounts in soil. A decrease in lignin and an increase in pectin contents were observed in response to increasing soil cadmium concentration. A concurrent reduction in methyl-esterification of pectin suggests than the structure of this major binding site could therefore be modified as a reaction to cadmium contamination. Future prospects will focus on the modulation of pectin composition in response to Cd exposure.

Optimization of the arsenazo-III method for the determination of uranium in water and plant samples.

This work reports a reliable, fast and optimized photometric technique based on the specific chemical complexation of uranyl ion with arsenazo-III. In the case of solid samples (plant samples), for which mineralization under acidic and oxidative conditions was used, addition of ascorbic acid led to stabilization of the arsenazo-uranyl complex over time. The results, in total agreement with data obtained from α and γ spectrometries, demonstrate that the present technique is able to precisely quantify uranium in water as well as in plant samples, within the µg/L and mg/g ranges respectively.

Forest floor leachate fluxes under six different tree species on a metal contaminated site.

Trees play an important role in the biogeochemical cycling of metals, although the influence of different tree species on the mobilization of metals is not yet clear. This study examined effects of six tree species on fluxes of Cd, Zn, DOC, H(+) and base cations in forest floor leachates on a metal polluted site in Belgium. Forest floor leachates were sampled with zero-tension lysimeters in a 12-year-old post-agricultural forest on a sandy soil. The tree species included were silver birch (Betula pendula), oak (Quercus robur and Q. petraea), black locust (Robinia pseudoacacia), aspen (Populus tremula), Scots pine (Pinus sylvestris) and Douglas fir (Pseudotsuga menziesii). We show that total Cd fluxes in forest floor leachate under aspen were slightly higher than those in the other species' leachates, yet the relative differences between the species were considerably smaller when looking at dissolved Cd fluxes. The latter was probably caused by extremely low H(+) amounts leaching from aspen's forest floor. No tree species effect was found for Zn leachate fluxes. We expected higher metal leachate fluxes under aspen as its leaf litter was significantly contaminated with Cd and Zn. We propose that the low amounts of Cd and Zn leaching under aspen's forest floor were possibly caused by high activity of soil biota, for example burrowing earthworms. Furthermore, our results reveal that Scots pine and oak were characterized by high H(+) and DOC fluxes as well as low base cation fluxes in their forest floor leachates, implying that those species might enhance metal mobilization in the soil profile and thus bear a potential risk for belowground metal dispersion.

Post-pollination prefertilization drops affect germination rates of heterospecific pollen in larch and Douglas-fir.

Pollen of larch (Larix × marschlinsii) and Douglas-fir (Pseudotsuga menziesii) was used in homospecific and heterospecific crosses. Germination of heterospecific pollen in ovulo was reduced in post-pollination prefertilization drops. This provides evidence of selection against foreign pollen by open-pollinated exposed ovules in these two sister taxa, which share the same type of pollination mechanism. Of the other prezygotic stages in pollen-ovule interactions, uptake of pollen by stigmatic hairs did not show any selection. Pollen tube penetration of the nucellus was similar for hetero- and homospecific pollen tubes, but heterospecific tubes only delivered gametes in one cross. To test for differences in the post-pollination prefertilization drops of each species, drops were gathered and analysed. Glucose and fructose were present in similar amounts in Douglas-fir and larch, while sucrose was found in larch only. Other carbohydrates such as xylose and melezitose were species-specific. In P. menziesii, sucrose is absent due to its conversion to glucose and fructose by apoplastic invertases. In contrast, Larix × marschlinsii drops have sucrose because they lack apoplastic invertases. The presence of invertase activity shows that the composition of gymnosperm post-pollination prefertilization drops is not static but dynamic. Drops of these two species also differed in their calcium concentrations.

Sea sand disruption method (SSDM) as a valuable tool for isolating essential oil components from conifers.

Essential oils are one of nature's most precious gifts with surprisingly potent and outstanding properties. Coniferous oils, for instance, are nowadays being used extensively to treat or prevent many types of infections, modify immune responses, soothe inflammations, stabilize moods, and to help ease all forms of non-acute pain. Given the broad spectrum of usage of coniferous essential oils, a fast, safe, simple, and efficient sample-preparation method is needed in the estimation procedure of essential oil components in fresh plant material. Generally, the time- and energy-consuming steam distillation (SD) is applied for this purpose. This paper will compare SD, pressurized liquid extraction (PLE), matrix solid-phase dispersion (MSPD), and the sea sand disruption method (SSDM) as isolation techniques to obtain aroma components from Scots pine (Pinus sylvestris), spruce (Picea abies), and Douglas fir (Pseudotsuga menziesii). According to the obtained data, SSDM is the most efficient sample preparation method in determining the essential oil composition of conifers. Moreover, SSDM requires small organic solvent amounts and a short extraction time, which makes it an advantageous alternative procedure for the routine analysis of coniferous oils. The superiority of SSDM over MSPD efficiency is ascertained, as there are no chemical interactions between the plant cell components and the sand. This fact confirms the reliability and efficacy of SSDM for the analysis of volatile oil components.

Ectomycorrhizal hyphae structure components of the soil bacterial community for decreased phosphatase production.

Ectomycorrhizal fungi (EMF) provide nutrients to their hosts by means of hyphae that extend beyond nutrient-depleted rhizosphere soil. Soil bacteria may compete with EMF for nutrients or may act synergistically to enhance nutrient supply to hosts. To assess the interactions between hyphae and bacteria, two types of small, sand-filled mesh bags were incubated in a Pseudotsuga menziesii/Betula papyrifera forest. The bags allowed ingrowth by EMF (35-µm mesh) or excluded hyphae (0.5-µm mesh), while allowing migration of soil bacteria. After incubation, bacteria were isolated from bags using a method to enrich for Gram-positive bacteria. Isolates were assayed for phosphatase and N-acetyl glucosaminidase (NAGase) activities to assess the potential to access organic phosphorus and nitrogen. The average phosphatase activities were higher in exclusion than ingrowth bags, while NAGase activities did not differ. Streptomyces isolates, which are expected to be strong competitors and antagonists of EMF, were more prevalent in ingrowth bags and yet had lower phosphatase activities. Furthermore, there were no indications of antagonism between fungi and Streptomyces, as there were no increases in NAGase activities in ingrowth bags. We conclude that fungal hyphae can structure components of the soil bacterial community for decreased extracellular enzyme production.

Enzymatic saccharification of woody biomass micro/nanofibrillated by continuous extrusion process II: effect of hot-compressed water treatment.

An extrusion process involving a twin-screw extruder was used for the micro/nanofibrillation of Douglas fir and Eucalyptus treated with hot-compressed water (HCW). Partial removal of hemicellulose and lignin by HCW treatment effectively improved the fibrillation by extrusion. Only HCW treatment produced glucose less than 5 weight percent (wt.%) in Douglas fir in a temperature range of 140-180 degrees C by enzymatic hydrolysis. Glucose production yields of 18 and 26 wt.% were obtained by HCW treatment at 170 and 180 degrees C, respectively, in Eucalyptus. Use of extrusion after HCW treatment drastically improved monosaccharide production yield in both woods. In the case of Douglas fir, the obtained values were 5 times higher than those obtained by HCW treatment alone. Total monosaccharide production yields were higher in Eucalyptus than in Douglas fir. The extruded production had a fine fibrous morphology on a sub-micro/nanoscopic scale. This result shows the great potential of the extrusion process after HCW treatment as a cost-effective pretreatment for enzymatic saccharification of woody biomass.

Phylogeography of Douglas-fir based on mitochondrial and chloroplast DNA sequences: testing hypotheses from the fossil record.

The integration of fossil and molecular data can provide a synthetic understanding of the ecological and evolutionary history of an organism. We analysed range-wide maternally inherited mitochondrial DNA and paternally inherited chloroplast DNA sequence data with coalescent simulations and traditional population genetic methods to test hypotheses of population divergence generated from the fossil record of Douglas-fir (Pseudotsuga menziesii), an ecologically and economically important western North American conifer. Specifically, we tested (i) the hypothesis that the Pliocene orogeny of the Cascades and Sierra Nevada caused the divergence of coastal and Rocky Mountain Douglas-fir varieties; and (ii) the hypothesis that multiple glacial refugia existed on the coast and in the Rocky Mountains. We found that Douglas-fir varieties diverged about 2.11 Ma (4.37 Ma-755 ka), which could be consistent with a Pliocene divergence. Rocky Mountain Douglas-fir probably resided in three or more glacial refugia. More variable molecular markers would be required to detect the two coastal refugia suggested in the fossil record. Comparison of mitochondrial DNA and chloroplast DNA variation revealed that gene flow via pollen linked populations isolated from seed exchange. Postglacial colonization of Canada from coastal and Rocky Mountain refugia near the ice margin at the Last Glacial Maximum produced a wide hybrid zone among varieties that formed almost exclusively by pollen exchange and chloroplast DNA introgression, not seed exchange. Postglacial migration rates were 50-165 m/year, insufficient to track projected 21st century warming in some regions. Although fossil and genetic data largely agree, each provides unique insights.

Effects of growth medium, nutrients, water, and aeration on mycorrhization and biomass allocation of greenhouse-grown interior Douglas-fir seedlings.

Commercial nursery practices usually fail to promote mycorrhization of interior Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] seedlings in British Columbia, which may account for their poor performance following planting in the field. We tested the effects of four nursery cultivation factors (nitrogen fertilization, phosphorus fertilization, watering, and soil aeration) and field soil addition on mycorrhization, survival, growth, and biomass allocation of interior Douglas-fir seedlings in a series of greenhouse experiments. Where field soil was added to the growing medium, mycorrhization and root/shoot ratios were maximized at lower levels of mineral nutrient application and aeration. Where field soil was not added, mycorrhization was negligible across all fertilization and aeration treatments, but root/shoot ratio was maximized at lower levels of mineral nutrients and the highest level of aeration. Regardless of whether field soil was added, intermediate levels of soil water resulted in the best mycorrhizal colonization and root/shoot ratios. However, field soil addition reduced seedling mortality at the two lowest water levels. A cluster analysis placed ectomycorrhizal morphotypes into three groups (Mycelium radicis-atrovirens Melin, Wilcoxina, and mixed) based on their treatment response, with all but two morphotypes in the mixed group whose abundance was maximized under conditions common to advanced seedling establishment. For maximal mycorrhization and root development of interior Douglas-fir seedlings, nurseries should minimize addition of nitrogen and phosphorus nutrients, maximize aeration, provide water at moderate rates, and, where possible, add small amounts of field soil to the growing medium.

Conifer embryogenic tissue initiation: improvements by supplementation of medium with D-xylose and D-chiro-inositol.

A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (LP, Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue and to continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment may decrease this recalcitrance. Sugar and sugar alcohol analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiologic concentrations (Pullman, G.S. and M. Buchanan. 2008. Identification and quantitative analysis of stage-specific carbohydrates in LP (Pinus taeda) zygotic embryo and female gametophyte tissues. Tree Physiol. 28:985-996). Major differences in stage-specific sugars were observed. A simple bioassay was used to evaluate the potential growth promotion of individual carbohydrates added to initiation or multiplication media at physiologic concentrations. Seventeen sugars were screened. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase the initiation of LP. d-xylose and d-chiro-inositol produced statistically significant increases in early-stage embryo growth. When tested for improved initiation in P. taeda, Pseudotsuga menziesii (mirb) Franco and Picea abies L., Karst., d-xylose increased the averages of initiation by 6.5%, 7.3% and 16.7%, respectively. d-chiro-inositol increased the initiation in P. taeda by 7.3% in one test but not in the other, whereas in P. menziesii the initiation increases averaged 8.4% in two tests. Analyses of sugars and sugar alcohols in the seed environment coupled with a bioassay to screen potential media supplements for protocol improvement resulted in statistically significant increases in embryogenic tissue initiation for several coniferous species.

The influence of the ectomycorrhizal fungus Rhizopogon subareolatus on growth and nutrient element localisation in two varieties of Douglas fir (Pseudotsuga menziesii var. menziesii and var. glauca) in response to manganese stress.

Acidification of forest ecosystems leads to increased plant availability of the micronutrient manganese (Mn), which is toxic when taken up in excess. To investigate whether ectomycorrhizas protect against excessive Mn by improving plant growth and nutrition or by retention of excess Mn in the hyphal mantle, seedlings of two populations of Douglas fir (Pseudotsuga menziesii), two varieties, one being menziesii (DFM) and the other being glauca (DFG), were inoculated with the ectomycorrhizal fungus Rhizopogon subareolatus in sand cultures. Five months after inoculation, half of the inoculated and non-inoculated seedlings were exposed to excess Mn in the nutrient solution for further 5 months. At the end of this period, plant productivity, nutrient concentrations, Mn uptake and subcellular compartmentalisation were evaluated. Non-inoculated, non-stressed DFM plants produced about 2.5 times more biomass than similarly treated DFG. Excess Mn in the nutrient solution led to high accumulation of Mn in needles and roots but only to marginal loss in biomass. Colonisation with R. subareolatus slightly suppressed DFM growth but strongly reduced that of DFG (-50%) despite positive effects of mycorrhizas on plant phosphorus nutrition. Growth reductions of inoculated Douglas fir seedlings were unexpected since the degree of mycorrhization was not high, i.e. ca. 30% in DFM and 8% in DFG. Accumulation of high Mn was not prevented in inoculated seedlings. The hyphal mantle of mycorrhizal root tips accumulated divalent cations such as Ca, but not Mn, thus not providing a barrier against excessive Mn uptake into the plants associated with R. subareolatus.

Water transfer via ectomycorrhizal fungal hyphae to conifer seedlings.

Little is known about water transfer via mycorrhizal hyphae to plants, despite its potential importance in seedling establishment and plant community development, especially in arid environments. Therefore, this process was investigated in the study reported in this paper in laboratory-based tripartite mesocosms containing the shrub Arctostaphylos viscida (manzanita) and young seedlings of sugar pine (Pinus lambertiana) and Douglas-fir (Pseudotsuga menziesii). The objectives were to determine whether water could be transported through mycorrhizal symbionts shared by establishing conifers and A. viscida and to compare the results obtained using two tracers: the stable isotope deuterium and the dye lucifer yellow carbohydrazide. Water containing the tracers was added to the central compartment containing single manzanita shrubs. The fungal hyphae were then collected as well as plant roots from coniferous seedlings in the other two compartments to determine whether water was transferred via fungal hyphae. In addition, the length of the hyphae and degree of mycorrhizal colonisation were determined. Internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis was used to identify the fungal species involved in dye (water) transfer. Results of the stable isotope analysis showed that water is transferred via mycorrhizal hyphae, but isotopically labelled water was only detected in Douglas-fir roots, not in sugar pine roots. In contrast, the fluorescent dye was transported via mycorrhizal hyphae to both Douglas-fir and sugar pine seedlings. Only 1 of 15 fungal morphotypes (identified as Atheliaceae) growing in the mesocosms transferred the dye. Differences were detected in the water transfer patterns indicated by the deuterium and fluorescent dye tracers, suggesting that the two labels are transported by different mechanisms in the same hyphae and/or that different fungal taxa transfer them via different routes to host plants. We conclude that both tracers can provide information on resource transfer between fungi and plants, but we cannot be sure that the dye transfer data provide accurate indications of water transfer rates and patterns. The isotopic tracer provides more direct indications of water movement and is therefore more suitable than the dye for studying water relations of plants and their associated mycorrhizal fungi.

Fire resistance of Douglas fir (Pseudotsuga menzieesi) treated with borates and natural extractives.

The objective of this study was to determine fire resistance of Douglas fir (Pseudotsuga menzieesi (Mirb.) Franco) specimens treated with borate supplemented aqueous solutions of brutia pine bark powder, acorn powder, sumach leaf powder, and gall-nut powder. Boric acid (BA) and borax (BX) were used as borates which are the most commonly used fire retardants in wood preservation industry. Natural extractives (brutia pine bark powder, sumach leaf powder, acorn powder, and gall-nut powder) were also used which have toxic efficiency against insects and fungi due to their tannin contents. A commercial treatment compound Tanalith-CBC (copper-borate-chromate), which is an impregnation chemical, is used for comparison. The fire test method was performed in three stages: flame stage, without flame stage, and glowing stage. Results indicated that the lowest temperature for flame stage, without flame stage, and glowing stage were obtained for specimens treated with BA and BX mixture (7:3; weight:weight). The lowest mass loss was found for the specimens treated with a mixture of BA and BX. Natural extractives did not improve fire resistance of the samples. However, boric acid and borax had excellent fire retardant effectiveness over untreated and treated samples with natural extractives.