Ectomycorrhizal fungal community in alkaline-saline soil in northeastern China

Alkaline-saline soil is widespread in arid and semiarid regions of the world and causes severe environmental and agricultural problems. To advance our understanding of the adaptation of ectomycorrhizal fungi (EMF) to alkaline-saline soil, we investigated EMF communities on Mongolian willow (Salix linearistipularis) growing in alkaline-saline soil (up to pH 9.2) in northeastern China. In total, 75 root samples were collected from 25 willow individuals over 4.7 ha. To identify fungal species in ectomycorrhizal root tips, we used terminal restriction fragment length polymorphism and sequencing analyses of the internal transcribed spacer region of ribosomal DNA. We detected 11 EMF species, including species of Inocybe, Hebeloma, and Tomentella of the Basidiomycota and three Ascomycota species. The EMF richness of the study site was estimated to be 15–17 using major estimators. The most abundant species was Geopora sp. 1, while no Geopora-dominated EMF communities have been reported so far. Phylogenetic analysis showed that the phylogroup including Geopora sp. 1 has been found mostly in alkaline soil habitats, indicating its adaptation to high soil pH. Because EMF are indispensable for host plant growth, the EMF species detected in this study may be useful for restoration of alkaline-saline areas.     

Effects of N addition rates on the productivity ofPicea Sitchensis,Thuja plicata,andTsuga heterophylla seedlings

Seedlings ofPicea sitchensis, Thuja plicata andTsuga heterophylla were supplied N hydroponically at one of four exponentially increasing rates of addition (0.09, 0.07, 0.05, or 0.025 gN^-1 day^-1) for up to 3 months in a naturally illuminated glasshouse. Relative growth rates (RGR) were analyzed as a function of N uptake, the allocation of assimilated N to foliage (LNFR), foliar N concentrations (N_la) and met assimilation rates (NAR), which were combined to estimate N productivity (RGR per unit whole-plant N concentration). Nitrogen accumulation, biomass and N partitioning and RGR and its components varied with species in response to the different N regimes.T. heterophylla had the lowest maximum wholeplant N concentrations (wpN) and specific absorption rates for N and exhibited the least plasticity in root: shoot ratios as wpN increased from 11–21 mg g^-1. In all species, RGR increased linearly with wpN, while LNFR increased curvilinearly. Foliar N (N_la) increased linearly with wpN and NAR increased linearly with N_la. The RGRs ofT. heterophylla were highest at wpNs up to 18 mg g^-1, a result of higher foliar N use efficiencies (NAR/N_la). However, RGR increased more with wpN inT. plicata andP. sitchensis. Although LNFR increased with wpN in all species, foliar N use efficiency declined, possibly due to an increased partitioning of foliar soluble N to non-photosynthetic compounds. Thus, in each species, N productivity did not increase above intermediate levels of wpN: 14 mg g^-1 inT. heterophylla, 16 mg g^-1 inP. sitchensis and 17 mg g^-1 inT. plicata.     

Colonization of Sitka spruce stumps by decay-causing hymenomycetes in paired inoculations

Sitka spruce stumps were inoculated with decay fungi using colonized sawdust or dowel inoculum to investigate colonization in paired combinations. Estimates of domain sizes were made in the top 15 cm of stump after 13–14 or 21–23 months with sawdust or dowel inoculations, respectively. None of the co-inoculated species prevented colonization by Heterobasidion annosum; sapwood colonization by Resinicium bicolor may limit growth of H. annosum colonies out of heartwood, reducing the incidence of disease transfer at root contacts. H. annosum colonized stumps despite the presence of competing inoculum. Reduced colonization occurred in paired inoculations with R. bicolor, but not with other fungi. Co-inoculations with Stereum sanguinolentum increased colonization by H. annosum. R. bicolor largely remained in the upper 3–4 cm of stumps and reduced colonization by Melanotus proteus; growth of S. sanguinolentum was completely prevented. The results are discussed in relation to the colonization strategies of the decay fungi, their ability to colonize stumps in the presence of competitors and factors influencing development of communities of decay fungi in stumps.     

Ectomycorrhizal fungal species and strains differ in their ability to produce free and conjugated polyamines

Production of free and conjugated polyamines by one strain of Laccaria proxima (Boud.) Maire, three strains (H, O, K) of Paxillus involutus (Batsch) Fr., and one strain of Pisolithus tinctorius was studied in vitro. Spermidine (Spd) was the main polyamine in the 4-week-old mycelium of all the fungi. It was mainly present in the free form, but it also occurred in conjugated forms. Paxillus involutus strain H released large amounts of free putrescine (Put), and the Pisolithus tinctorius released a compound probably related to cadaverine (Cad). On the other hand, these two fungi contained less conjugated polyamines than the other fungi. In addition to the amounts, the forms (perchloric acid soluble and insoluble) of conjugated polyamines in the mycelium varied between species and strains. L. proxima contained nearly as much insoluble conjugated Spd as free Spd, whereas Paxillus involutus strains O and K contained relatively large amounts of soluble conjugated Spd. The results suggest that ectomycorrhizal fungal species and strains differ in their ability and need to produce conjugated polyamines. The small amounts of soluble conjugated polyamines found in the culture filtrates indicate that some specific conjugated polyamines may be involved in polyamine translocation across the plasma membrane.     

Effects of Soil Burn Severity on Post-Fire Tree Recruitment in Boreal Forest

Fire, which is the dominant disturbance in the boreal forest, creates substantial heterogeneity in soil burn severity at patch and landscape scales. We present results from five field experiments in Yukon Territory, Canada, and Alaska, USA that document the effects of soil burn severity on the germination and establishment of four common boreal trees: Picea glauca, Picea mariana, Pinus contorta subsp. latifolia, and Populus tremuloides. Burn severity had strong positive effects on seed germination and net seedling establishment after 3 years. Growth of transplanted seedlings was also significantly higher on severely burned soils. Our data and a synthesis of the literature indicated a consistent, steep decline in conifer establishment on organic soils at depths greater than 2.5 cm. A meta-analysis of seedling responses found no difference in the magnitude of severity effects on germination versus net establishment. There were, however, significant differences in establishment but not germination responses among deciduous trees, spruce, and pine, suggesting that small-seeded species experience greater mortality on lightly burned, organic soils than large-seeded species. Together, our analyses indicate that variations in burn severity can influence multiple aspects of forest stand structure, by affecting the density and composition of tree seedlings that establish after fire. These effects are predicted to be most important in moderately-drained forest stands, where a high potential variability in soil burn severity is coupled with strong severity effects on tree recruitment.     

Impact of biological (Rotstop) and chemical (urea) treatments on fungal community structure in freshly cut Picea abies stumps

To control the infections by root rot fungi Heterobasidion spp., surfaces of freshly cut Norway spruce stumps are covered either by a biological (Rotstop; spore suspension of competitive saprotrophic fungus Phlebiopsis gigantea), or by a chemical (35% aqueous solution of urea) compound. In Fennoscandia, Rotstop and urea are applied, respectively, on 47,000 ha and on 2000 ha of forestland each year. The aim of this work was to assess the impact of biological and chemical control on biodiversity in communities of non-target fungi in freshly cut (7-week-old) stumps. Isolation of fungi to pure culture was accomplished from 402 wood samples taken from 63 stumps, 21 treated with each of the compounds and 21 untreated. The isolations yielded 368 distinct fungal strains representing 47 species. Stump treatment led to decrease of species richness both in Rotstop-treated (by 15%) and in urea-treated (by 19%) stumps. Nevertheless, the stumps subjected to the biological compound were colonized mainly by the same fungi that occurred naturally in untreated stumps (Sorensen similarity indices; S_S=0.69; S_N=0.68). By contrast, chemical treatment strongly promoted stump colonization by Ascomycetes and Deuteromycetes, led to significant decrease of Zygomycetes, and almost completely eliminated Basidiomycetes (including Heterobasidion spp.). Thus, resemblance to a natural community was low (S_S=0.45; S_N=0.34). Rotstop treatment decreased significantly the extent of stump colonization by Heterobasidion spp., and increased that of P. gigantea. All strains of the latter were genetically identical among themselves and to the Rotstop strain. The mechanisms of biological and chemical control, and biodiversity aspects are discussed.     

Multilocus structure in Pinus contorta Dougl.

We studied isozyme variation at 21 loci in 66 populations from three subspecies of Pinus contorta Dougl.; 35 in spp. latifolia, 20 in spp contorta and 11 in spp. murrayana. The objectives were to assess gametic disequilibria and multilocus structure. There was considerable differentiation of allele frequencies at 19 polymorphic loci across the 66 populations and within the subspecies. Allele frequencies at many loci correlated with geographic variables. Genetic variability varied considerably among populations within subspecies but the subspecies means were similar. The mean number of polymorphic loci and the mean heterozygosity over 19 polymorphic loci were, respectively, 13 and 0.194 in latifolia, 12 and 0.196 in murrayana, and 12 and 0.180 in contorta. The mean heterozygosity correlated with longitude and altitude across the 66 populations and with latitude in latifolia. Gametic disequilibria were evident in 40 populations; 29 in latifolia, eight in murrayana and three in contorta. Gametic disequilibria correlated with latitude across the 66 populations and with longitude in latifolia. The single-locus F _ST averaged 0.0339 in latifolia, 0.0567 in murrayana, and 0.0764 in contorta. The multilocus F _STM was 0.1227 in latifolia, 0.2926 in murrayana, and 0.3328 in contorta. Multilocus Wahlund and founder effects, migration patterns, and natural selection, probably played significant roles in generating and maintaining the multilocus genetic structure in P. contorta in general and the subspecies latifolia in particular.     

Recovery from winter depression of photosynthesis in pine and spruce

Summary Recovery from winter depression of photosynthesis was studied in Pinus sylvestris, Pinus conforta and Picea abies by means of chlorophyll fluorescence and gas exchange measurements. During the winter 1986–1987 the fluorescence yield was low and no variable fluorescence was detectable before the end of March. In the field recovery of variable fluorescence/maximum fluorescence (F_v/F_m) during spring was slow for all three species studied. The temperature dependence of recovery was confirmed from measurements of the potential rate of recovery of F_v/F_m at different temperatures in the laboratory. At 20° C, F_v/F_m increased from 0.1 to 0.8 within 3 days. Recovery of F_v/F_m was paralleled by an increase in apparent photon yield. No significant differences could be demonstrated between the studied tree species in potential rate of recovery in the laboratory or in actual recovery in the field.     

Ectomycorrhizal syntheses with Picea abies and three fungal species: a case study on the use of an in vitro technique to identify naturally occurring ectomycorrhizae

Ectomycorrhizal syntheses between Picea abies and the fungal associates Scleroderma citrinum, Boletus luridus, and Tricholoma vaccinum were carried out using Melin's Erlenmeyer flask technique. The symbioses of S. citrinum were characterized by a mantle composed of an outer prosenchymatous and an inner synenchymatous layer. The mantles of B. luridus and T. vaccinum were solely prosenchymatous. Rhizomorphs were produced in all treatments, but only in association with S. citrinum were they differentiated with additional, enlarged hyphae. All synthesized ectomycorrhizae were white or whitish to light orange and greyishorange. On large-scale root sampling in two differing Picea abies forests in Switzerland, nine out of a total of 22 morphological types of ectomycorrhizae were white or yellow in colour and were, therefore, comparable with the synthesized ectomycorrhizae. These nine natural types generally had distinct mantle features (irregular synenchyma, gelatinous matrix, cystides, thick-walled hyphae), but mostly lacked clamp connections. Synthesized ectomycorrhizae, on the other hand, lacked distinct mantle characteristics and always had clamp connections. Natural and synthesized white or yellow ectomycorrhizae did not coincide morphologically and thus identification of the fungal partners of natural symbioses by means of in vitro-synthesis with potential ectomycorrhizal fungi was not possible in the present study.     

Lifespans of fungal rhizomorphs under nitrogen fertilization in a pinyon-juniper woodland

The lifespan of individual microbes in the soil influences nutrient cycling rates as well as population dynamics, but their responses to global change factors such as anthropogenic nitrogen deposition have been challenging to quantify in situ. We used minirhizotron images to track the abundance and turnover rate of individual fungal rhizomorphs under nitrogen fertilization in a pinyon-juniper woodland in New Mexico. We hypothesized that increases in nitrogen availability would alter rhizomorph lifespan and abundance. Sequential images were collected over eight sampling dates from November 1997 to August 1999, and a total of 278 rhizomorphs were examined. We found that neither standing stocks nor lifespans of rhizomorphs differed significantly between treatments. Lifespans of rhizomorphs lasted eleven months on average, indicating that nutrient immobilization in these structures could last for longer than a growing season in these sites.     

A comparison of rooting environments in containers of different sizes

Experiments on plants are often carried out in growth chambers or greenhouses which necessitate the use of an artificial rooting environment, though this is seldom characterized in detail. Measurements were made to compare the rooting environment in large boxes (0.25 m³) with that in small pots (0.19, 0.55 and 1.90 dm³) in naturally lit chambers. Diurnal temperature fluctuations of 14.6, 11.6 and 7.7°C occurred in the post compared with only 1.9°C in the boxes. Soil drying to a matric potential of-50 kPa was approximately 25 times faster in the pots. The mean heights of 2 year old Sitka spruce (Picea sitchensis (Bong.) Carr.) seedlings grown throughout their second growing season in the three sizes of pots were 38, 62 and 92% of the mean height of those grown in the boxes. Soil solution nutrient concentrations in the boxes were considerably increased by soil drying, an aspect which seems to have received little attention in experiments involving artificially imposed drought. An alternative system of constraining the roots of individual plants within nylon fabric bags, embedded in larger volumes of soil, to facilitate harvesting of complete root systems is described. The importance of the rooting environment in determining the outcome of physiological experiments is also briefly discussed.     

Fire Interval Effects on Successional Trajectory in Boreal Forests of Northwest Canada

Although succession may follow multiple pathways in a given environment, the causes of such variation are often elusive. This paper describes how changes in fire interval mediate successional trajectory in conifer-dominated boreal forests of northwestern Canada. Tree densities were measured 5 and 19 years after fire in permanent plots and related to pre-fire vegetation, site and fire characteristics. In stands that were greater than 75 years of age when they burned, recruitment density of conifers was significantly correlated with pre-fire species basal area, supporting the expectation of stand self-replacement as the most common successional pathway in these forests. In contrast, stands that were under 25 years of age at the time of burning had significantly reduced conifer recruitment, but showed no change in recruitment of trembling aspen (Populus tremuloides). As a result, young-burned stands had a much higher probability of regenerating to deciduous dominance than mature-burned stands, despite the dominance of both groups by spruce (Picea mariana and Picea glauca) and pine (Pinus contorta) before the fire. Once initiated, deciduous-dominated stands may be maintained across subsequent fire cycles through mechanisms such as low on-site availability of conifer seed, competition with the aspen canopy, and rapid asexual regeneration of aspen after fire. We suggest that climate-related increases in fire frequency could trigger more frequent shifts from conifer to deciduous-dominated successional trajectories in the future, with consequent effects on multiple ecosystem processes.     

Changes in ectomycorrhizal community structure on two containerized oak hosts across an experimental hydrologic gradient

Shifts in ectomycorrhizal (ECM) community structure were examined across an experimental hydrologic gradient on containerized seedlings of two oak species, Quercus montana and Quercus palustris, inoculated from a homogenate of roots from mature oak trees. At the end of one growing season, seedlings were harvested, roots were sorted by morphotype, and proportional colonization of each type was determined. DNA was subsequently extracted from individual root tips for polymerase chain reaction, restriction fragment length polymorphism, and rDNA sequencing of the ITS1/5.8S/ITS2 region to determine identities of fungal morphotypes. Twelve distinct molecular types were identified. Analysis of similarity showed that ECM fungal assemblages shifted significantly in composition across the soil moisture gradient. Taxa within the genus Tuber and the family Thelephoraceae were largely responsible for the changes in fungal assemblages. There were also significant differences in ECM community assemblages between the two oak host species. These results demonstrate that the structure of ECM fungal communities depends on both the abiotic and biotic environments and can shift with changes in soil moisture as well as host plant, even within the same genus.     

First fungal community analyses of endophytic ascomycetes associated with Viscum album ssp. austriacum and its host Pinus sylvestris

The endophytic fungal communities in the hemi-parasitic epiphyte Viscum album and in its phorophyte Pinus sylvestris were compared to reveal the fungal distribution patterns in their hosts. The ITS nrDNA of 208 multiple-isolated fungal strains was sequenced and a newly designed process was applied for assigning taxon names to the obtained sequences. Furthermore, the isolates were grouped as clusters, by subjecting a sequence similarity matrix to various cluster analyses, the results of which were compared and verified by data from phylogenetic reconstructions. In contrast to a previously reported dominance of Leotiomycetes among Pinus inhabiting fungi, the endophytic communities of the two host plant species studied here were dominated by Xylariaceae (Sordariomycetes). This is in accordance with the finding that host selectivity was only a minor factor in explaining the distribution patterns of the endophytic fungi in Viscum and Pinus. Organ and, probably, tissue selectivity had a more pronounced effect. The composition and condition of the woods in the surrounding, however, are concluded to be the major determinants, due to the following circumstantial evidence: The highest similarities in fungal community compositions were found for the leaves of the two host plant species, especially when considering only the older leaves. The finding that the inhabitants of matured or senescent organs are less host-selective is in accordance with decreasing defence capabilities of ageing host plant tissue and an increased nutrient supply for saprobic taxa. Therefore, the composition of the fungal communities in ageing leaves seems to be predominantly ascribed to contagious spread and to depend on the spectrum of nearby sporulating fungal taxa. We suggest that because a broad range of suitable substrates for Xylariaceae was present in immediate vicinity of the study sites, these fungi also dominated among the recorded endophytic taxa.     

The occurrence of flowering and fruiting on individual trees over 3 years and their effects on subsequent crown condition

Summary The level of fruiting in four forest trees species (Picea sitchensis, P. abies, Pinus sylvestris and Fagus sylvatica) was monitored in Great Britain over the period 1989–1991. In addition, assessments of crown transparency were available for many of the trees for 1987 and 1988. The monitoring period encompassed severe summer droughts in 1989 and 1990, with wetter conditions in 1991. Variations in the level of fruiting in spruce and beech (Fagus sylvatica L.) were seen, with a marked peak in 1990. No pattern was apparent in Scots pine (Pinus sylvestris L.). Coning, which was greater in trees with the least transparent crowns, had no discernible effect on the crown transparency of the conifers. Cupule production in beech was greatest in trees with the most transparent crowns, and trees with high numbers of cupules in 1990 tended to have greater crown dieback recorded in 1991.     

Influence of cold soil and snowcover on photosynthesis and leaf conductance in two Rocky Mountain conifers

Summary The influence of cold soil and snowcover on photosynthesis and conductance of Picea engelmannii and Pinus contorta was investigated early in the growing season in the Medicine Bow Mountains, Wyoming, USA. Trees of both species growing in cold soil (<1°C) associated with snowpack had 25–40% lower leaf photosynthesis than trees in warm soils (>10°C). In cold soils leaf conductance of both species was lower, but more so in Pinus, leading to lower intercellular CO₂ concentrations and greater stomatal limitation of photosynthesis. Soil temperature had no effect on predawn and midday shoot water potentials of Pinus and Picea and lower photosynthesis and conductance did not appear to be a result of lower bulk shoot water potential. Predawn, as well as midday, water potentials of Pinus were consistently higher than Picea suggesting that Pinus may have deeper roots, although trenching experiments indicated young Picea trees have more extensive lateral root systems than similar sized Pinus trees. Young Picea trees (<2 m in height) in snowbanks were capable of utilizing warmer soil 4 m from their base. Under similar conditions Pinus in snowbanks had lower photosynthesis and conductance than controls and Pinus did not appear capable of utilizing warmer soils nearby. Under full sunlight, PPFD reflected from the snow surface was 400–1400 mol m^-2 s^-1 higher than from snow-free surfaces. This reflected light resulted in a 10%–20% increase in photosynthesis of Picea. The beneficial effect of reflected light was apparent whether or not photosynthesis was reduced by low soil temperatures.     

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.     

Microsatellites as DNA markers in Sitka spruce

Nine microsatellite loci were found by screening a genomic DNA library of Sitka spruce (Picea sitchensis) with the four oligonucleotide probes (TG), (CAC), (GATA) and (AT). Pairs of flanking primers were generated for seven microsatellites. Five primer pairs were used to screen up to 58 Sitka spruce clones. The five loci SStg3a, SStg4, SStg4a, SStg4c and SSgataS were found to have 15, 13, 4, 3 and 6 different length alleles respectively, and in using a combination of them almost all 58 Sitka spruce genotypes could be identified. The five primer pairs were successful in amplifying DNA from two other spruce species (Picea albutilia and Picea smithiana), while only one primer pair could amplify DNA from the pine species, Pinus sylvestris and Pinus latifolia. The inheritance of microsatellites in Sitka spruce was co-dominant Mendelian.     

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