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PubMed2005

Development of a nested PCR detection procedure for Nectria fuckeliana direct from Norway spruce bark extracts.

A pair of primers specific for Nectria fuckeliana, a bark infecting pathogen predominantly of Norway spruce (Picea abies), were designed from comparisons of nucleotide sequences of the nuclear ribosomal internal transcribed spacer (ITS) regions of nine isolates from Norway, Lithuania, Switzerland, Austria, Slovakia, Scotland (Larix sp.) and New Zealand (Pinus radiata), and other closely related nectriaceous species, including Neo. Neomacrospora, and 'N'. mammoidea, to which it exhibits taxonomic similarities. Complete ITS sequence homology was observed between each of the nine N. fuckeliana isolates, regardless of geographic provenance, including a previously published Danish strain. Primers Cct1 and Cct2 consistently amplified a single product of 360 bp from DNA prepared from 20 isolates covering the principle range of the disease from Central and Northern Europe, but not from other Neonectria, 'Nectria' or a range of species commonly encountered in forest ecosystems, as well as P. abies or P. radiata DNA. A quick, simple and efficient mechanical lysis procedure for the extraction of high quality total DNA from bark, coupled with post-extraction polyvinylpolypyrrolidone (PVPP) chromatography purification, is described to facilitate successful PCR detection of N. fuckeliana direct from bark extracts. Detection of N. fuckeliana from bark preparations was only possible following nested PCR of PVPP purified extracts using universal primers ITS5 and 4 in first round amplification. The identity of products from bark tissues was confirmed by Southern hybridisation and sequencing. Using the above procedure, positive diagnosis of N. fuckeliana was achievable within 5 h and has the potential for full exploitation as both a forest management and ecological research tool. As the DNA extraction procedure described here has been successful in application against other tree species, it has potential for incorporation into other molecular diagnostic systems for other microorganisms responsible for other wood or tree bark diseases.

Blotting, Southern↗

PubMed2006

Quantitative detection of Lactarius deliciosus extraradical soil mycelium by real-time PCR and its application in the study of fungal persistence and interspecific competition.

Real-Time PCR has been applied to quantify extraradical soil mycelium of the edible ectomycorrhizal fungus Lactarius deliciosus in an interspecific competition experiment under greenhouse conditions. Couples of Pinus pinea seedlings inoculated with either L. deliciosus, Rhizopogon roseolus, or non-inoculated (control) were transplanted into pots filled with two types of soil in all the possible combinations. Total DNA was extracted from soil samples at 3 and 6 months after transplantation to perform real-time PCR analysis. DNA extractions from soil mixed with known amounts of mycelium of L. deliciosus were used as standards. Six months after transplantation, the percentage of mycorrhizas of L. deliciosus and seedling growth were significantly affected by the soil type. Extraradical soil mycelium of L. deliciosus was positively correlated with the final percentage of mycorrhizas and significantly affected by the sampling time and soil depth. The competition effect of R. roseolus was not significant for any of the measured parameters, probably due to the sharp decrease of the mycorrhizal colonization by this fungus. We conclude that real-time PCR is a powerful technique for extraradical mycelium quantification in studies aimed at evaluating the persistence of introduced strains of L. deliciosus in field plantations.

Agaricales↗

PubMed2006

Assessing the potential of native tree species for carbon sequestration forestry in Northeast China.

Although the native forests of China are exceptionally diverse, only a small number of tree species have been widely utilized in forest plantations and reforestation efforts. We used dendrochronological sampling methods to assess the potential growth and carbon sequestration of native tree species in Jilin Province, Northeast China. Trees were sampled in and near the Changbaishan Biosphere Reserve, with samples encompassing old-growth, disturbed forest, and plantations. To approximate conditions for planted trees, sampling focused on trees with exposed crowns (dominant and co-dominant individuals). A log-linear relationship was found between diameter increment and tree diameter, with a linear decrease in increment with increasing local basal area; no significant differences in these patterns between plantations and natural stands were detected for two commonly planted species (Pinus koraiensis and Larix olgensis). A growth model that incorporates observed feedbacks with individual tree size and local basal area (in conjunction with allometric models for tree biomass), was used to project stand-level biomass increment. Predicted growth trajectories were then linked to the carbon process model InTEC to provide estimates of carbon sequestration potential. Results indicate substantial differences among species, and suggest that certain native hardwoods (in particular Fraxinus mandshurica and Phellodendron amurense), have high potential for use in carbon forestry applications. Increased use of native hardwoods in carbon forestry in China is likely to have additional benefits in terms of economic diversification and enhanced provision of "ecosystem services", including biodiversity protection.

Biodiversity↗

PubMed2006

Analysis of condensed and hydrolysable tannins from commercial plant extracts.

High performance liquid chromatography (HPLC)/DAD and MS qualitative and quantitative analyses of polyphenols, hydrolysable and condensed tannins from Pinus maritima L. and tannic acid (TA) extracts were performed using normal and reverse phase. Normal-phase HPLC was more suitable for pine bark (PBE) and tannic acid extracts analysis. The chromatographic profile revealed that P. maritima L. extract was mainly composed by polymeric flavanols (containing from two to seven units) and tannic acid (characterized by a mixture of glucose gallates containing from three to seven units of gallic acid). Concerning their antimycotic properties, P. maritima L. extract exhibited a broad activity towards yeast strains of the genera Candida, Cryptococcus, Filobasidiella, Issatchenkia, Saccharomyces: MICs from 200 to 4000 microg/ml (corresponding to 140-2800 microg/ml of active polyphenols) were determined. Conversely, no activity of tannic acid was observed over the same target microorganisms. Taken into consideration the above results of HPLC analysis and on the basis of the current literature, we may conclude that only 70.2% of polyphenols (recognized as condensed tannins) occurring in P. maritima L. extract can be apparently considered responsible for its antimycotic activity.

Antifungal Agents↗

PubMed2006

Spermidine and the ectomycorrhizal fungus Pisolithus tinctorius synergistically induce maturation of Scots pine embryogenic cultures.

Exogenous spermidine (Spd) and the ectomycorrhizal (ECM) fungus Pisolithus tinctorius (Pers.) Coker and Couch had a synergistic effect on the maturation of Scots pine (Pinus sylvestris L.) somatic embryos. Induced maturation was expressed as a higher number of cell masses able to form embryos and a greater number of embryos formed per cell mass. In contrast, treatment with P. tinctorius alone on the hormone-free medium resulted in the lowest embryo-forming capacity. Retarded proliferation growth appeared to be required for maturation, but did not explain the synergistic effect of the fungus and exogenous Spd. Simultaneous treatment did not result in lower concentrations of putrescine (Put), Spd or spermine (Spm) in the embryogenic cell masses relative to the separate treatments. Our study is the first report on the use of a specific ECM fungus to induce maturation of somatic embryos, and it indicates that P. tinctorius was able to modify the maturation media in a way that, together with exogenous Spd, positively affected embryogenic cultures of Scots pine. Our study also shows that it is possible to enhance plant development other than root formation by using specific ECM fungi.

Basidiomycota↗

PubMed2006

Compound response of marine and terrestrial ecosystems to varying climate: pre-anthropogenic perspective from bivalve shell growth increments and tree-rings.

Annual growth increments were examined from shells of the ocean quahog (Arctica islandica L.) from northwest Norway and from tree-ring samples of the Scots pine (Pinus sylvestris L.) from nearby coastal areas. The reconstructed annual growth increments were used to compare growth variability in marine and terrestrial ecosystems. Spatiotemporal comparison of the growth records showed statistically significant correlation during the 19th century A.D., indicative of ecosystem-independent response to pre-anthropogenic climate variations. Geographical correlation between marine and terrestrial records was only observed at the local scale. Years with particularly low winter or high summer North Atlantic Oscillation (NAO) indices showed the best synchronization of marine and terrestrial growth. Despite strong correlation during historical time, our palaeoecological evidence suggests that marine and terrestrial ecosystems may show dissimilar growth reaction to recently observed positive winter-NAO phases.

Animals↗

PubMed2005

The effects of heat treatments on ectomycorrhizal resistant propagules and their ability to colonize bioassay seedlings.

The effect of disturbance on the resistant propagule community (RPC) of ectomycorrhizal fungi has been given relatively little attention. In this study we investigate the effects of heat, one important factor of fire disturbances, on the ability of ectomycorrhizal RPC fungi to colonize Pinus jeffreyi seedlings in greenhouse bioassays. Prior to planting the seed, soils were collected from an old growth mixed-conifer forest in the southern Sierra Nevada, California, USA and then subjected to four heat treatments of none, 45 degrees C, 60 degrees C, and 75 degrees C. After eight months, seedlings were harvested and the ectomycorrhizal fungi colonizing the roots were characterized by molecular methods (PCR-RFLP and DNA sequencing). Rhizopogon species increased in dominance on seedlings grown in soils receiving the 75 degrees C heat treatment. One species significantly increased in frequency, Rhizopogon olivaceotinctus, and two species (Cenococcum geophilum and Wilcoxina sp.) significantly decreased in frequency in the 75 degrees C treatment. The increase of R. olivaceotinctus, coupled with other features of its behavior, suggests that substantial heat disturbances may benefit this species in competing for roots.

Basidiomycota↗

PubMed2006

A new Sparassis species from Spain described using morphological and molecular data.

Sparassis miniensis, collected in Pinus pinaster forests in Galicia (northwest Iberian Peninsula) is described as a new species, based on morphological and molecular data. Sparassis miniensis is morphologically distinct from all other species in the genus Sparassis based on scattered flabellae, which are strongly laciniated, azonate, and arise from an orange to rose-purplish base. The sporadic presence of clamp connections is restricted to subhymenial hyphae. Molecular data from LSU-rDNA, ITS and partial gene coding RNA polymerase subunit II (rpb2) suggest a close relationship between the new species S. miniensis and S. brevipes, another European species producing large fruiting bodies but with entire flabellae and no clamp connections.

Basidiomycota↗

PubMed2005

Enhanced tolerance to salt stress in transgenic loblolly pine simultaneously expressing two genes encoding mannitol-1-phosphate dehydrogenase and glucitol-6-phosphate dehydrogenase.

A reproducible approach to improve salt tolerance of conifers has been established by using the technology of plant genetic transformation and using loblolly pine (Pinus taeda L.) as a model plant. Mature zygotic embryos of three genotypes of loblolly pine were infected with Agrobacterium tumefaciens strain LBA 4404 harboring the plasmid pBIGM which carrying two bacterial genes encoding the mannitol-1-phosphate dehydrogenase (Mt1D, EC 1.1.1.17) and glucitol-6-phosphate dehydrogenase (GutD) (EC 1.1.1.140), respectively. Transgenic plantlets were produced on selection medium containing 15 mg l(-1) kanamycin and confirmed by polymerase chain reaction (PCR) and Southern blot analysis of genomic DNA. The Mt1D and GutD genes were expressed and translated into functional enzymes that resulted in the synthesis and accumulation of mannitol and glucitol in transgenic plants. Salt tolerance assays demonstrated that transgenic plantlets producing mannitol and glucitol had an increased ability to tolerate high salinity. These results suggested that an efficient A. tumefaciens-mediated transformation protocol for stable integration of bacterial Mt1D and GutD genes into loblolly pine has been developed and this could be useful for the future studies on engineering breeding of conifers.

Agrobacterium tumefaciens↗

PubMed2004

Effects of nitrogen deposition on nitrogen cycling in an Aleppo pine stand in Athens, Greece.

High levels of nitrogen deposition measured in throughfall fluxes in 2002, reaching 38 kg ha(-1) year(-1), affected the biogeochemical cycle of N in an Aleppo pine (Pinus halepensis) stand situated in Athens, the capital of Greece. The N fluxes in bulk deposition were far lower than those in throughfall indicating high levels of dry deposition in the city of Athens. The amounts of N in litterfall and those stored in the forest floor and mineral soil were not high, while the mean residence time of N in the forest floor, derived from fluxes equations, decreased considerably due to the high levels of N fluxes in throughfall deposition. Nitrogen concentration in needle tissues was high and this resulted in a high ratio of N/Zn. Zinc availability was low due to the alkaline nature of soil in the study area. Fast N cycling may affect the living status of Aleppo pine, which is an oligotrophic plant species capable of occupying poor soil sites. Nitrogen eutrophication may adversely affect forests grown on calcareous soils by developing nutrition imbalances between N and micronutrients, the solubility of which depends on soil pH values.

Environmental Monitoring↗

PubMed2004

Elemental analyses of pine bark and wood in an environmental study.

Bark and wood samples were taken from the same individuals of Scots pine (Pinus sylvestris L.) from a polluted area close to a Cu-Ni smelter in Harjavalta and from some relatively unpolluted areas in western Finland. The samples were analysed by thick-target particle induced X-ray emission (PIXE) after preconcentration by dry ashing at 550 degrees C. The elemental contents of pine bark and wood were compared to study the impact of heavy metal pollution on pine trees. By comparison of the elemental contents in ashes of bark and wood, a normalisation was obtained. For the relatively clean areas, the ratios of the concentration in bark ash to the concentration in wood ash for different elements were close to 1. This means that the ashes of Scots Pine wood and bark have quite similar elemental composition. For the samples from the polluted area the mean concentration ratios for some heavy metals were elevated (13-28), reflecting the effect of direct atmospheric contamination. The metal contents in the ashes of pine bark and wood were also compared to recommendations for ashes to be recycled back to the forest environment. Bark from areas close to emission sources of heavy metal pollution should be considered with caution if aiming at recycling the ash. Burning of bark fuel of pine grown within 6 km of the Cu-Ni smelter is shown to generate ashes with high levels of Cu, Ni as well as Cd, As and Pb.

Environmental Monitoring↗

PubMed2006

From dust to dose: Effects of forest disturbance on increased inhalation exposure.

Ecosystem disturbances that remove vegetation and disturb surface soils are major causes of excessive soil erosion and can result in accelerated transport of soils contaminated with hazardous materials. Accelerated wind erosion in disturbed lands that are contaminated is of particular concern because of potential increased inhalation exposure, yet measurements regarding these relationships are lacking. The importance of this was highlighted when, in May of 2000, the Cerro Grande fire burned over roughly 30% of Los Alamos National Laboratory (LANL), mostly in ponderosa pine (Pinus ponderosa) forest, and through areas with soils containing contaminants, particularly excess depleted and natural uranium. Additionally, post-fire thinning was performed in burned and unburned forests on about 25% of LANL land. The first goal of this study was to assess the potential for increased inhalation dose from uranium contaminated soils via wind-driven resuspension of soil following the Cerro Grande Fire and subsequent forest thinning. This was done through analysis of post-disturbance measurements of uranium air concentrations and their relationships with wind velocity and seasonal vegetation cover. We found a 14% average increase in uranium air concentrations at LANL perimeter locations after the fire, and the greatest air concentrations occurred during the months of April-June when wind velocities are highest, no snow cover, and low vegetation cover. The second goal was to develop a methodology to assess the relative contribution of each disturbance type towards increasing public and worker exposure to these resuspended soils. Measurements of wind-driven dust flux in severely burned, moderately burned, thinned, and unburned/unthinned forest areas were used to assess horizontal dust flux (HDF) in these areas. Using empirically derived relationships between measurements of HDF and respirible dust, coupled with onsite uranium soil concentrations, we estimate relative increases in inhalation doses for workers ranging from 15% to 38%. Despite the potential for increased doses resulting from these forest disturbances, the estimated annual dose rate for the public was <1 microSv yr(-1), which is far below the dose limits for public exposures, and the upper-bound dose rate for a LANL worker was estimated to be 140 microSv yr(-1), far below the 5 x 10(4) microSv yr(-1) occupational dose limit. These results show the importance of ecosystem disturbance in increasing mobility of soil-bound contaminants, which can ultimately increase exposure. However, it is important to investigate the magnitude of the increases when deciding appropriate strategies for management and long-term stewardship of contaminated lands.

Air Pollutants, Radioactive↗

PubMed2005

Effects of composted sewage sludge on microbial biomass, activity and pine seedlings in nursery forest.

The investigation was carried out in a 2 year experiment to evaluate the benefits and hazards of the use of composted sewage sludge as a restoration agent for the soil of the nursery forest intended for growing Pinus sylvestris seedlings. The grey forest soil (Haplic Greyzem) was amended with compost at the 25, 50, 75, 100, 150 and 175 t ha(-1) application rates on a dry matter basis. The organic matter content increased with the increase in sludge amendment as well as the metal content. However, the concentrations of individual metals were below the current limits established for Russia and European countries. Sludge amendments enhanced the germination and decreased the mortality of the seedlings. The effects were more obvious for the soil with the highest sludge treatment. The beneficial effects on the biomass of seedlings and the height of the shoots as well as on the length of the roots of the pine seedlings were greater in plots with the highest rates of composted sludge. The application of composted sludge to soil was followed by an increase in microbial biomass and to a lesser extent in basal respiration. In the absence of any detrimental effect on microorganisms, this study lends support to using composted sewage sludge as the organo-mineral fertilizer for the soil of nursery forest.

Biomass↗

PubMed2005

Mitochondrial phylogeny of pine cone beetles (Scolytinae, Conophthorus) and their affiliation with geographic area and host.

Pine cone beetles (Conophthorus spp.) feed and kill immature cones of Pinus species, thereby reducing seed production and seriously impairing reforestation of forest ecosystems. Population variation of Conophthorus reproductive behavior has hampered the development of semiochemical control of these pests. This difficulty is compounded by a lack of taxonomic knowledge and species diagnostic characters. Researchers and managers rely, in part, on host associations and geographic locality for species identifications and these have arguable taxonomic utility. However, host use and/or geographic separation may influence Conophthorus lineage diversification. To improve Conophthorus taxonomy and understand the association of host and geography with lineage diversification, a phylogeny of 43 individuals, including all valid species and a robust sample of C. ponderosae from different hosts, is reconstructed using 785 nucleotides of the 3'-end of the mitochondrial cytochrome oxidase I gene. Thirty trees were recovered in a parsimony analysis and the strict consensus was well resolved and supported by branch support measures. Conophthorus was monophyletic but mitochondrial polyphyly was uncovered for several species. The data also suggested an underestimation of species diversity. Phylogenetically related Conophthorus lineages were significantly associated with geographic proximity but not with host, as indicated by comparisons of character optimized geographic distributions and host associations against randomized distributions of these attributes on the parsimony tree. These results suggest that geographic separation better explains the mode of Conophthorus lineage diversification than does host specialization. Based on these results, researchers and managers of Conophthorus should consider populations as potentially different evolutionary entities until species boundaries are delineated via a robust phylogenetic revision of Conophthorus.

Amino Acids↗

PubMed2002

Analyses of the wood preservative component N-cyclohexyl-diazeniumdioxide in impregnated pine sapwood by direct thermal desorption-gas chromatography-mass spectrometry.

Investigations concerning the qualitative and quantitative determination of the organic wood preservative component N-cyclohexyl-diazeniumdioxide (HDO) in treated timber were carried out by means of direct thermal desorption-gas chromatography-mass spectrometry (DTD-GC-MS). It could be shown that the identification of HDO in treated pine sapwood (Pinus sylyestris L.) is relatively simple using this analytical technique. Quantification of this active ingredient can be carried out using the peak area of the specific mass fragment m/z 114. A calibration curve with a high correlation coefficient was obtained in the range from 40 to 550 mg HDO per kg timber. Furthermore it can be deduced that the results obtained are characterised by an excellent reproducibility with standard deviations ranging from 5 to 10% in general. For the chosen experimental set up a detection limit of 4 mg HDO per kg treated pine sapwood was calculated, although merely 20% of the active ingredient was desorbed.

Cyclohexanes↗

PubMed2002

Trace element concentration in tree-rings biomonitoring centuries of environmental change.

Inductively coupled plasma mass spectrometry (ICP-MS) was used to examine trace element concentration in tree-rings over three and half centuries to assess macro-trends of environmental change. Tree-rings of a 350+ year old mammoth ponderosa pine (Pinus ponderosa) were analyzed for element concentration and evaluated versus local and global historical events. The ponderosa pine was located 100 miles south of the Canada/USA border and 180 miles east of the Pacific Ocean, and grew near apple orchards, a public road, and Swakane Creek in western Washington, USA. The elements tested did not all display the same time versus concentration patterns. Copper and chromium displayed cyclic concentration patterns over the last 350+ years, which appear to be associated with local events. Strontium, barium, zinc and cadmium were found to be relatively constant between the mid 1600s and the early 1800s. Strontium, barium, zinc, and cadmium then increased beginning in the early 1800s for approximately 50 years then decreased to present day 2000. Significantly, similar changes seen in Ca, Mg, and Zn in other studies have been attributed to acid rain, whereas, in our study area there is no history of anthropogenic acid rain. Most importantly, our data goes back to the mid-1600s several hundred years further back than most other studies of this nature. This additional time data provides for a better context of trend data not previously available.

Acid Rain↗

PubMed2003

Background ozone monitoring and phytodetection in the greater rural area of Corinth--Greece.

Natural background ozone levels were monitored in three places within the greater rural area of Corinth, namely Bogdani Hill, Astronomical Observatory of Krionerion, and Kiato, and compared with ambient ozone monitored in the metropolitan area of Athens. Measurements were made sequentially, for a few weeks at each place, during the summer of 2000. In addition, ozone phytodetection, using tobacco (Nicotiana tabacum L.) plants of the Bel-W3 and Zichnomirodata varieties, was conducted in 12 places (the above included). Moreover, stomatal conductance was measured in the Bel-W3 plants, as well as in leaves of cultivated grape-vines (Vitis vinifera L.) and in needles of Aleppo pine (Pinus halepensis Mill.) trees and compared with the diurnal pattern of ozone concentrations. The 24 and 12 (08:00-20:00) hourly averages of ozone concentrations were high in Athens (37; 51 ppb), at Bogdani Hill (53; 56 ppb) and at the Astronomical Observatory (56; 55 ppb), but relatively low in Kiato (30; 34 ppb). Furthermore, the average daily AOT40 (accumulated exposure over 40 ppb for the daylight hours) (ppbh) was 193 in Athens, 212 at Bogdani Hill, 192 at the Astronomical Observatory and 47 in Kiato. Ozone concentrations exhibited the usual diurnal pattern in Athens (altitude 50 m), where they were maximum during midday and early afternoon hours, as well as at Bogdani Hill (300 m) and in Kiato (5 m) where, however, they were maximal 1-3 h later. At the Astronomical Observatory (altitude 920 m) ozone remained constant during both daylight and night hours. The differences in diurnal patterns are consistent with those in places of different elevation, reported elsewhere. The Bel-W3 plants were injured at all 12 places; Zichnomirodata plants exhibited lower injury and only in some of the places; probable ozone symptoms were also observed on vine plants and pine trees. The greatest injury was observed at the high altitude places of Astronomical Observatory and Mougostos. Stomatal conductance, in all three species, peaked during morning and early midday hours when ozone levels were higher in the high altitude, and lower in the low altitude, places.

Environmental Monitoring↗

PubMed2003

Conifer needles as passive biomonitors of the spatial and temporal distribution of DDT from a point source.

Needles of two conifer species, Picea abies and Pinus nigra, were used as passive samplers for monitoring air contamination by sampling at increasing distances from a suspected point source of DDT. Needle concentrations declined with increasing distance downwind of the point source allowing to identify spatial and temporal trends of accumulation. This suggested that conifer needles are effective biomonitors of contamination levels in areas characterized by the presence of semi-volatile substances. Differences in uptake were apparent between the species. Needle morphology and structure were studied with scanning electron microscope (SEM) as were dimensional parameters (surface area, volume). The results suggest that the concentrations depend on a mechanism involving the inner structure of the needles, specifically the number and accessibility of resin channels rather than their surface area. Pine needles have more channels with greater accessibility than spruce. The results suggest that spruce is more suitable for short term measurement while pine for determining long term cumulative exposure.

Air Pollutants↗