The mycorrhizal community in a forest chronosequence of Sitka spruce [Picea sitchensis (Bong.) Carr.] in Northern England

Demography and fungal diversity of the belowground ectomycorrhizal community in a chronosequence of Sitka spruce [Picea sitchensis (Bong.) Carr.] in Northumberland, Northern England, were analysed; mycorrhizal root samples were taken from 6-, 12-, 30- and 40-year-old stands, and fungal fruiting bodies were collected in autumn to complement the survey. Naturally germinated seedlings less than 1 year of age (taken from the 30-year-old stand) were also examined. A total of 118,000 mycorrhizal root tips were extracted from 40 soil cores (ten per age class) and from the complete root systems of 25 seedlings and separated into active and senescent root tips according to their morphology and anatomy. Active tips were distinguished according to their mycobionts which were characterised and identified microscopically. Although almost 100% of all fine roots were mycorrhizal, EM fungal diversity throughout the chronosequence was low, consisting of a total of 16 species of which three were only found as fruiting bodies. Of the six mycobionts found most regularly below ground, Tylospora fibrillosa was the most common, colonising about 70% of all root tips and more than 90% of those of seedlings and young trees. Root density and mycorrhizal diversity increased, but percentage of vital root tips decreased with increasing tree age, levelling off in the 30- and 40-year-old stand. Among the five subdominant fungal species, Dermocybe crocea was found to have its peak of distribution in the 12-year-old stand and Russula emetica, Lactarius rufus, Hymenoscyphus ericae agg. and the unidentified Piceirhiza sulfo-incrustata in the 30- and 40-year-old stands. The possible correlations between the mycorrhizal community structure and biotic and abiotic factors are discussed.     

Effect of P supply upon seasonal growth and internal cycling of P in Sitka spruce (Picea sitchensis(Bong.)Carr.) seedlings

The availability of phosphorus in many UK forest soils limits growth of Sitka spruce (Picea sitchensis (Bong.) Carr.). Efficient cycling of P within such systems is therefore necessary for sustained tree growth. Internal cycling of P is an important component of the overall P cycle in forests and the current work aims to quantify the impact of P nutrition on internal cycling and seasonal growth of Sitka spruce.Two-year old seedlings of Sitka spruce were grown in sand culture in the glasshouse for one year. Two treatments were imposed in which trees received either a complete nutrient solution from which P was excluded (-P) or one in which P was applied as labelled ³²P (+P). Internal cycling of P was measured directly in plants which had received no P and by difference in those which received ³²P.The contrasting P treatments produced an eight-fold difference in P content and a three-fold difference in tree growth between May and October. Root:shoot ratios increased during the growing season from 0.29 to 0.38 and from 0.29 to 0.52 in +P and-P treatments, respectively. In both treatments P was translocated from old shoots to support new shoot growth. P supply did not affect the amount of P remobilised but there was evidence that the rate of remobilisation may have been affected. The partition of remobilised P was affected by current P supply and differed from the partition of current P uptake.Results are compared to those from studies of growth and internal cycling of nitrogen in Sitka spruce.     

Localised impact of Sitka spruce (Picea sitchensis (Bong.) Carr.) on soil permeability

A typical upland soil catena afforested with Sitka spruce (Picea sitchensis (Bong.) Carr.) was chosen to examine the localised effect of trees on soil permeability. A borehole permeameter was used to measure soil permeability at 0.2 m and approximately 2 m distance from the stem of 20 trees at a fixed measurement depth of 0.25 to 0.45 m. In the case of the near-tree measurements, this corresponded to soil beneath the main root plate of each conifer. Two principal elements of the soil catena: the ferric podzol of the mid-slope and histosol soil of the foot-slope were investigated.The preliminary data set shows that within the ferric podzol element, the permeability of the soil beneath individual conifers was a factor of 5.4 less than that of the adjacent soil. In contrast, within the histosol sub-tree permeabilities could not be distinguished from those of soil 2 m away from each tree. The decrease in sub-tree permeability within the podzol may be caused by sensitivity of the Bsl horizon to consolidation by tree weight or by enhanced illuviation resulting from changes in local soil chemistry. The histosol may be less sensitive to such processes. The results of a consolidation test applied to the Rawls and Brakensiek model of soil permeability supported the possible role of consolidation in the reduction of soil permeability beneath conifers in podzolic soil. Additional data on soil bulk density, porosity and texture are required to corroborate either the consolidation or illuviation hypotheses. As the Bsl horizon of ferric podzol soil is typically slowly permeable, a further decline may (i) restrict root development and thus, increase windthrow hazard, and (ii) increase the lateral flow of water within podzolic Eag horizons and thus affect stream acidification. Deep ploughing of a site prior to afforestation may mitigate such impacts.     

Somatic embryogenesis in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Embryonal-suspensor masses from immature embryos from cones of Sitka spruce (Picea sitchensis (Bong.) Carr.) proliferated on a modified Murashige & Skoog medium with N⁶-benzyl-aminopurine, kinetin, 2,4-dichlorophenoxyacetic acid and an organic nitrogen source. The slimy white embryonal-suspensor masses with proembryos were maintained on a solid proliferation medium with reduced amounts of growth regulators. Transfer of embryonal-suspensor masses to a non-woven polyester carrier with liquid maturation media containing ±2-cis-4-trans-abscisic acid and a reduced amount of inositol and organic nitrogen resulted in synchronized embryo formation. Further development was achieved on a medium without ±2-cis-4-trans-abscisic acid and organic nitrogen. Somatic embryos were successfully transferred ex vitrum.Abbreviations ABA ±2-cis-4-trans-abscisic acid - BAP N⁶-benzyl-aminopurine - ESM embryonal-suspensor masses - KIN kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid     

Effect of stand age on greenhouse gas fluxes from a Sitka spruce [Picea sitchensis (Bong.) Carr.] chronosequence on a peaty gley soil

The influence of forest stand age in a Picea sitchensis plantation on (1) soil fluxes of three greenhouse gases (GHGs – CO₂, CH₄ and N₂O) and (2) overall net ecosystem global warming potential (GWP), was investigated in a 2‐year study. The objective was to isolate the effect of forest stand age on soil edaphic characteristics (temperature, water table and volumetric moisture) and the consequent influence of these characteristics on the GHG fluxes. Fluxes were measured in a chronosequence in Harwood, England, with sites comprising 30‐ and 20‐year‐old second rotation forest and a site clearfelled (CF) some 18 months before measurement. Adjoining unforested grassland (UN) acted as a control. Comparisons were made between flux data, soil temperature and moisture data and, at the 30‐year‐old and CF sites, eddy covariance data for net ecosystem carbon (C) exchange (NEE). The main findings were: firstly, integrated CO₂ efflux was the dominant influence on the GHG budget, contributing 93–94% of the total GHG flux across the chronosequence compared with 6–7% from CH₄ and N₂O combined. Secondly, there were clear links between the trends in edaphic factors as the forest matured, or after clearfelling, and the emission of GHGs. In the chronosequence sites, annual fluxes of CO₂ were lower at the 20‐year‐old (20y) site than at the 30‐year‐old (30y) and CF sites, with soil temperature the dominant control. CH₄ efflux was highest at the CF site, with peak flux 491±54.5 μg m⁻² h⁻¹ and maximum annual flux 18.0±1.1 kg CH₄ ha⁻¹ yr⁻¹. No consistent uptake of CH₄ was noted at any site. A linear relationship was found between log CH₄ flux and the closeness of the water table to the soil surface across all sites. N₂O efflux was highest in the 30y site, reaching 108±38.3 μg N₂O‐N m⁻² h⁻¹ (171 μg N₂O m⁻² h⁻¹) in midsummer and a maximum annual flux of 4.7±1.2 kg N₂O ha⁻¹ yr⁻¹ in 2001. Automatic chamber data showed a positive exponential relationship between N₂O flux and soil temperature at this site. The relationship between N₂O emission and soil volumetric moisture indicated an optimum moisture content for N₂O flux of 40–50% by volume. The relationship between C : N ratio data and integrated N₂O flux was consistent with a pattern previously noted across temperate and boreal forest soils.     

The effect of long term CO2 enrichment on the growth,biomass partitioning and mineral nutrition of Sitka spruce (Picea sitchensis (Bong.) Carr.)

Sitka spruce [Picea sitchensis (Bong.) Carr.] seedlings were grown for 3 years in an outside control plot or in ambient (355 mol mol^-1) or elevated (ambient + 350 mol mol^-1) atmospheric CO₂ environments, within open top chambers (OTCs) at the Institute of Terrestrial Ecology, Edinburgh. Sequential harvests were carried out at the end of each growing season and throughout the 1991 growing season, five in all. Plants grown in elevated CO₂ had, (i) 35 and 10% larger root/shoot ratios at the end of the first and third season, respectively, (ii) significantly higher summer leader extension relative growth rates, which declined more rapidly in early autumn than ambient grown plants, (iii) after three growing seasons a significantly increased mean annual relative growth rate, (iv) consistently lower foliar nutrient concentrations, and (v) after two growing seasons smaller total projected needle areas. Plants grown inside OTCs were taller, heavier and had a smaller root/shoot ratio than those grown outside the chambers. There was no effect of CO₂ concentration on Sitka spruce leaf characteristics, although leaf area ratio, specific leaf area and leaf weight ratio all fell throughout the course of the 3 year experiment.     

The effect of cultivation technique on root and shoot biomass production by young Sitka spruce (Picea sitchensis (Bong.) Carr.) trees on surface water gley soils

In Ireland much of the land available for plantation establishment requires some degree of soil cultivation and drainage to improve its suitability for tree growth. The method of cultivation and drainage normally varies depending upon the soil type and its drainage characteristics. Little research has been carried out on the impact of practices such as mounding and mole drainage upon rooting and biomass production in young crops. The research reported in this paper was carried out on four sites where the general soil type was surface water gley. Three of the sites studied were afforestations on old farmland, while the fourth site was a reforestation of a windblown stand. The cultivation methods investigated included mounding, mole drainage with mounds, mole drainage only and ripping. Sitka spruce trees ranging in age from five to thirty years were excavated from these experimental sites to describe the impact of different soil cultivation techniques upon root architecture and above and below ground biomass production. The results showed that while the effect of mound drainage upon water table level varied from site to site, the establishment of trees on mounds led to an increase in above and below ground biomass production. Mole drainage of these sites without mounds, did not lead to any increase in root or shoot development when compared with trees growing on uncultivated/undrained ground. This revised version was published online in June 2006 with corrections to the Cover Date.     

A branch bag and CO2 control system for long-term CO2 enrichment of mature Sitka spruce [Picea sitchensis (Bong.) Carr.]

This paper describes the construction and performance of branch bags and a CO₂ control system used to fumigate branches of mature Sitka spruce trees with air enriched in CO₂ (700 μmolmol^-1). It contains some examples of results obtained using the system over the course of the first two growing seasons. The branch bags have run continuously for 2 years with very few problems. CO₂ concentrations were within 20 μmol mol^-1 of the target concentration for more than 90% of the time. Temperatures within the bags were slightly higher than ambient (1–2 °C) and this had some effect on phenology. Attenuation of quantum flux density (photosynthetically active radiation) was 10–15%. The branch bag system has enabled investigation into the effects of elevated CO₂ on mature tissue without the problems and expense of fumigating whole trees. Growth in elevated CO₂ resulted in an increase in starch and a decrease in soluble protein content of needles. Stomatal conductance was higher in elevated CO₂ grown needles, and there was some evidence of an increase in photosynthetic capacity.     

Effect of carbenicillin and cefotaxime on somatic embryogenesis of Sitka spruce (Picea sitchensis (Bong.) Carr.)

Carbenicillin prevented the development of mature somatic embryos,reduced early stage embryos by >90% and tissue growth by50%. Cefotaxime had no effect on overall tissue growth, butreduced the development of early and mature embryos by 20% and66–80%, respectively. Carbenicillin should not be usedfor transformation of Sitka spruce. Key words: Sitka spruce, somatic embryogenesis, antibiotic sensitivity     

Development of a set of highly polymorphic genomic microsatellites (gSSRs) in Sitka spruce (Picea sitchensis (Bong.) Carr.)

Robust, polymorphic microsatellite DNA markers (simple sequence repeats—SSRs) are valuable tools for a range of tree conservation and breeding applications. SSRs are routinely used in the study of population genetic structure and diversity, pedigree reconstruction and genetic linkage mapping. Their abundance in the genome, co-dominant inheritance and potential for cross-species amplification make microsatellites highly prized markers. This paper characterises 22 novel genomic polymorphic microsatellite loci for Sitka spruce (Picea sitchensis (Bong.) Carr.). Amplification of DNA from Sitka spruce material was carried out both with a set of unrelated trees to obtain diversity statistics for each locus, and with the progeny of a full-sib family to test simple Mendelian inheritance. Observed heterozygosity ranged from 0.38 to 0.91 and allele number per locus ranged from 6 to 21, with a mean of 12.2. In addition, the primer pairs were tested with DNA from Norway spruce (P. abies) and white spruce (P. glauca) to investigate their potential for cross-species amplification and ten loci amplified in all three species. The results from these genomic microsatellites are compared to data generated from microsatellites derived from Picea EST libraries. In summary, this novel, highly polymorphic markers represent a significant addition to the rapidly expanding Picea genomics tool-box. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The influence of culture vessel head-space volatiles on somatic embryo maturation in Sitka spruce [Picea sitchensis (Bong.) Carr.]

The maturation of somatic embryos of Sitka spruce [Picea sitchensis (Bong.) Carr.] was found to be highly dependent on the method used to seal plastic Petri dishes. Large numbers of well-formed mature embryos developed if dishes were sealed with PVC cling-film (CF) whilst sealing with Parafilm M (PF) greatly reduced the numbers of embryos forming. Inclusion of potassium permanganate oxidation traps, normally used to deplete the atmospheric ethylene, greatly stimulated somatic embryo maturation under PF sealing. Similarly, traps of adsorption agents (Tenax, activated charcoal or soft white paraffin), capable of removing volatiles from the culture vessel head-space, stimulated somatic embryo maturation under PF sealing although to a lesser extent than the oxidation traps. Incorporation of silver nitrate or 2-chloroethylphosphonic acid (ethephon) in the culture medium indicated that ethylene was not the agent supressing somatic embryo maturation under PF sealing.Abbreviations ABA abscisic acid - CF PVC cling-film - PF Parafilm M     

Nitrogen dynamics in humus and soil beneath Sitka spruce (Picea sitchensis (Bong.) Carr.) planted in pure stands and in mixture with Scots pine (Pinus sylvestris L.)

Sitka spruce planted on nutrient-poor soils in mixture with pine or larch, unlike pure spruce, does not become N deficient and does not require N fertilizer. To test the hypothesis that N availability in the soil is enhanced beneath mixed species, the seasonal changes in different N forms were compared in humus (L+F+H) and soil beneath 15-year-old Sitka spruce (SS) and mixed Sitka spruce-Scots pine (SS and SP) planted on a gleyed heathland soil. Amounts of mineral and organic N extracted from humus in spring were significantly (p < 0.05) higher in SS and SP than in SS. Larger amounts were measured in the underlying soil, which favoured the deeper-rooting spruce and pine in SS and SP plots. Annual net N mineralization, measured by in-situ incubation, was 32 and 47 kg N ha^-1 in the surface 10 cm beneath SS and (SS and SP), respectively. In spring, readily mineralized organic N (waterlogged incubation at 30°C) was higher in humus and soil from (SS and SP) than from SS by 15 kg N ha^-1. The larger N pools beneath (SS and SP) were consistent with the higher total N content of the humus beneath (SS and SP), 446 compared with 255 kg N ha^-1 beneath SS. This indicated that beneath (SS and SP) N had been transferred from the underlying soil.     

Cytokinin pulse-mediated shoot organogenesis from cotyledons of Sitka spruce [Picea sitchensis (Bong.) Carr.] and high frequency in vitro rooting of shoots

Shoot formation from cotyledons of Sitka spruce was induced by a 2 h incubation in an aqueous solution of 400 μM benzyladenine, followed by culture on medium lacking plant growth regulators. Several basal media were assessed. Murashige and Skoog (1962) based medium was found to be optimal on which, after 25 weeks, 62.5% of cotyledons produced shoots with a mean of 10.7 shoots per cotyledon. Shoots, detached from cotyledons and propagated individually by axillary bud proliferation, rooted with a frequency of 84% following culture in half-strength MS-based medium lacking growth regulators under low illumination (1.5 μmol m⁻²s⁻¹). Rooted plants were successfully transferred to ex vitro conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inhibition of somatic embryo maturation in Sitka spruce [Picea sitchensis (Bong.) Carr.] by butylated hydroxytoluene,a volatile antioxidant released by parafilm

Head-space volatiles above embryogenicPicea sitchensis (Bong.) Carr. (Sitka spruce) tissues cultured in glass Petri dishes sealed with Parafilm M or cling-film, were captured on Tenax adsorption traps and analysed by gas chromatography / mass spectrometry. Each sealing system released a single major compound into the head-space; butylated hydroxytoluene from Parafilm M and 2-ethyl-1-hexanol from cling-film. After two weeks sealed under Parafilm M butylated hydroxytoluene accumulated to 1.1 g g^–1 FW in tissues and subsequent somatic embryo maturation was prevented. When butylated hydroxytoluene was supplied via the head-space (100 g/250 ml flask) 0.5 g g^–1 FW accumulated in tissues after two weeks and no somatic embryo maturation occurred. Potentially phytotoxic metabolites of butylated hydroxytoluene included a substituted stilbenequinone, butylated hydroxytoluene quinone methide and butylated hydroxytoluene dimer.     

The Effect of Explant Type on the Establishment of Sitka Spruce [Picea sitchensis (Bong.) Carr.] in Culture

Five different types of shoot tip explant from three-year-oldand 13-year-old Sitka spruce trees were established on a rangeof media and shoot growth was recorded. While Schenk and Hildebrandtmedium gave the greatest shoot elongation, Webb and Street mediumproduced the healthiest-looking shoots. Poorest growth was recordedfor the explants cultured without the crown or any vasculartissues from the previous year's growth. [¹⁴C]sucrose tracerstudies indicated that poor uptake was not responsible for thereduced growth and it was suggested that damage due to inadequatewater uptake may be responsible. Results demonstrated that peelingthe protective scales from resting buds and trimming the shoottissues below the crown to a wedge shape and excluding the barkwas a reliable method of explant preparation for establishingmature and juvenile Sitka spruce in culture. Picea sitchensis, Sitka spruce, mature, juvenile, crown, [¹⁴C]sucrose, micropropagation     

The effects of culture medium rigidity on adventitious bud production and tissue vitrification in needle cultures of Sitka spruce [Picea sitchensis (Bong.) Carr.]

Adventitious bud formation on Sitka spruce [ Picca sitchensis (Bong.) Carr.] needle explants was strongly dependent upon the rigidity of the culture medium. In general, of organogenesis was greatest on weak gels and poorest on more rigid gels resulting from increased medium pH or agar strength. There was a significant interaction between agar strength and pH, with the optimum pH for organogenesis declining with increasing agar strength. Poor organogenesis at high agar concentrations was not due to toxic impurities since increased adventitious bud production could be stimulated by decreasing the medium pH whilst maintaining a high agar strength and an agar washing treatment had no significant effect. Although high levels of organogenesis could be sustained on weak gels the resultant adventitious shoots often showed severe vitrification. The frequency of shoots showing vitrification could be reduced by growing the tissues on harder media but this resulted in reduced shoot elongation. Vitrification of needle tissues did not stimulate the formation of adventitious buds in the absence of cytokinins.