The ectomycorrhizal flora of primary and secondary stands of Pinus sylvestris in relation to soil conditions and ectomycorrhizal succession

Abstract. Ectomycorrhizal species composition and sporocarp abundance in two 15–20-yr old primary stands of Pinus sylvestris in the central part of the Netherlands was compared with those in two 16- and 27-yr old secondary stands of P. sylvestris in the Northeast of the country. The trees of the primary stands were spontaneously seeded in a drifting sand area. Only thin litter and humus layers were present. The trees of the 16-yr old secondary stand were planted on podzolic sandy soil and those of the 27-yr old stand on non-podzolic sandy soil. In both secondary stands thick litter and humus layers had developed. The litter and humus partly originated from former stands at the same sites and partly from the present stands. In the secondary stands the thick litter and humus layers and herb vegetation were removed (‘sod-cutting’) in order to simulate the thin litter and humus layers in the primary stands. Control treatments were present. Surveys in 1991, 1992 and 1993 showed that sod-cutting enhanced both abundance and diversity of ectomycorrhizal fungi. However, species richness and diversity were higher in the primary stands than in the secondary ones, also even in the sod-cut plots. High species richness and diversity were associated with low concentrations of nitrogen and relatively high pH in the litter and humus layers, and in the mineral soil.     

Lactarius deliciosus and Pinus radiata in New Zealand: towards the development of innovative gourmet mushroom orchards

The cultivation of Lactarius deliciosus (saffron milk cap) in New Zealand began in 2002 when fruiting bodies were produced in an Otago plantation of Pinus radiata seedlings artificially mycorrhized by L. deliciosus. In 2007, 42 P. radiata seedlings mycorrhized by L. deliciosus under controlled conditions were planted in a grass field at Plant and Food Research (Lincoln, Canterbury). The effects of pine bark mulch application and initial degree of mycorrhization of seedlings were examined to determine their influence on tree growth, development of mycorrhizae (i.e. their multiplication on the root system and their degree of branching) and fruiting body production. Mulch application increased tree growth significantly over 4 years. High initial mycorrhization slightly stimulated tree growth over 2 years. The initial degree of mycorrhization was positively, but not strongly, related to the persistence and development of L. deliciosus mycorrhizae and rhizomorphs based on root sample analyses 2 years after planting. However, mulching strongly reduced the proportion of highly branched L. deliciosus mycorrhizae compared with poorly ramified ones. A positive correlation was observed between the fruiting of L. deliciosus and the development of mycorrhizae. Mulching delayed the onset of fruiting body production. In 2010, fruiting bodies were produced only from non-mulched trees with eight of these (38 %) producing a total of 12 fruiting bodies. In 2011, 19 non-mulched trees (90 %) and 9 mulched trees (45 %) produced 143 and 47 fruiting bodies, respectively, totalling 190 fruiting bodies. By 2012, 19 non-mulched trees (90 %) and 13 mulched trees (65 %) produced 333 and 236 fruiting bodies, respectively, totalling 569 fruiting bodies (c. 30 kg). This study presents new information on factors influencing the onset of fruiting and the development of yields in a plantation of P. radiata mycorrhized by L. deliciosus. Projected yields as high as c. 300 kg/ha from the third year of production reiterate the feasibility of farming saffron milk cap in P. radiata plantations in New Zealand. Continued monitoring of this site and development of similar trials will provide important knowledge for the optimisation of yields in commercial saffron milk cap orchards.     

Bacterial microbiomes of individual ectomycorrhizal Pinus sylvestris roots are shaped by soil horizon and differentially sensitive to nitrogen addition

Plant roots select non‐random communities of fungi and bacteria from the surrounding soil that have effects on their health and growth, but we know little about the factors influencing their composition. We profiled bacterial microbiomes associated with individual ectomycorrhizal Pinus sylvestris roots colonized by different fungi and analyzed differences in microbiome structure related to soils from distinct podzol horizons and effects of short‐term additions of N, a growth‐limiting nutrient commonly applied as a fertilizer, but known to influence patterns of carbon allocation to roots. Ectomycorrhizal roots growing in soil from different horizons harboured distinct bacterial communities. The fungi colonizing individual roots had a strong effect on the associated bacterial communities. Even closely related species within the same ectomycorrhizal genus had distinct bacterial microbiomes in unfertilized soil, but fertilization removed this specificity. Effects of N were rapid and context dependent, being influenced by both soil type and the particular ectomycorrhizal fungi involved. Fungal community composition changed in soil from all horizons, but bacteria only responded strongly to N in soil from the B horizon where community structure was different and bacterial diversity was significantly reduced, possibly reflecting changed carbon allocation patterns.     

Effects of experimental conditions on mycorrhizal relationships between Pinus sylvestris and Lactarius deliciosus and unprecedented fruit-body formation of the Saffron milk cap under controlled soilless conditions

The mycorrhizal relationships between pines and two edible species of Lactarius sect. Dapetes were investigated by optimizing the experimental conditions of mycelial growth and of mycorrhizal colonization of pine seedlings. In vitro mycelial growth of Lactarius deliciosus and L. sanguifluus was improved on a buffered medium containing glucose, amino acids, and vitamins. Two methods of mycorrhization of pines with Lactarius deliciosus were tested. The mycorrhizal colonization was rapid and intense under non-aseptic conditions with a low nutrient supply and without exogenous glucose. A positive influence of mycorrhizal colonization on Pinus sylvestris growth was subsequently observed. Under axenic conditions and with a high nutrient supply, mycorrhization was stimulated at 10 g/L of exogenous glucose, irrespective of the phosphorus concentration. At high phosphorus level (1 mM) and 0.1, 1.0, or 10.0 g/L glucose, growth of Pinus sylvestris was reduced by inoculation. Stability and development of Pinus spp./Lactarius deliciosus symbioses were assayed in a climatic chamber using containers filled with a synthetic substrate. Over a 2-year culture period, the root systems of the pine seedlings were heavily colonized by Lactarius deliciosus. One year following inoculation, Lactarius deliciosus fruit-body primordia appeared associated with Pinus sylvestris seedlings. Six months later, two mature basidiomata were obtained. This is the first report of soilless fruit-body formation of this edible mushroom.     

Different soil moisture conditions change the outcome of the ectomycorrhizal symbiosis between Rhizopogon species and Pinus muricata

The outcome of species interactions often depends on the environmental conditions under which they occur. In this study, we tested how different soil moisture conditions affected the outcome of the ectomycorrhizal symbiosis between three Rhizopogon species and Pinus muricata in a factorial growth chamber experiment. We found that when grown in 7% soil moisture conditions, ectomycorrhizal plants had similar biomass, photosynthesis, conductance, and total leaf nitrogen as non-mycorrhizal plants. However, when grown at 13% soil moisture, ectomycorrhizal plants had significantly greater shoot biomass, higher photosynthetic and conductance rates, and higher total leaf nitrogen than non-mycorrhizal plants. The differences in plant response by mycorrhizal status in the two soil moisture treatments corresponded with evidence of water limitation experienced by the fungi, which had much lower colonization at 7% compared to 13% soil moisture. Our results suggest that the outcome of the ectomycorrhizal symbiosis can be context-dependent and that fluctuating environmental conditions may strongly affect the way plants and fungi interact. Peter G. Kennedy and Kabir G. Peay contributed equally to this work and order was determined by a coin toss.     

The effect of ectomycorrhizal fungi on Zn uptake and distribution in seedlings of Pinus sylvestris L

Seedlings of pine, infected with two different ectomycorrhizal fungi, Suillus bovinus (Fr.) O. Kuntze and an unidentified isolate (BP), were exposed to various external Zn concentrations. An additional strain of S. bovinus, cultured on a Zn-enriched medium, was also used. The effect of mycorrhizal associations on Zn uptake and distribution within the plant was determined by AAS.The results show that nonmycorrhizal seedlings have the capability to control the uptake and translocation of Zn to the shoot until the external Zn concentration reaches a threshold level, where no limitation of uptake is possible. Excess Zn is accumulated in the root system to protect the shoot against toxic tissue concentrations. The effect of an ectomycorrhizal infection on Zn uptake and distribution depends on (1) the fungal species (2) the external concentration and (3) the Zn content of the fungal culture medium. Under conditions of low external Zn supply, especially a mycorrhizal infection with S. bovinus led to an increased Zn uptake in root and needles of Pinus sylvestris. Under high external conditions the mycobionts varied considerably in their capability to reduce the transport of Zn to the shoot. Only by an infection with S. bovinus the plant was able to maintain the shoot tissue concentration on a low level. This effect can be enhanced by pretreatment of S. bovinus with high Zn concentrations.     

The communities of ectomycorrhizal fungal species associated with Betula pendula Roth and Pinus sylvestris L. growing in heavy-metal contaminated soils

Plant and Soil - Pioneer tree species such as Betula pendula and Pinus sylvestris encroach soils contaminated with heavy metals (HMs). This is facilitated by ectomycorrhizal fungi colonizing tree...     

Effects of forest management on understorey vegetation in a Pinus sylvestris L. Plantation in NE Scotland

Summary

This paper reports on a six-year experiment which assessed the effects of crop thinning, fencing, and ground preparation (screefing) on the development of understorey vegetation in a first rotation Pinus sylvestris plantation. Thinning or fencing had no effect on species richness or diversity, but richness increased to a greater degree in screefed plots than in unscreefed controls. Fencing benefited the establishment of Goody era repens colonies. Vegetation was matched against the National Vegetation Classification W18a community (Pinus sylvestris-Hylocomium splendens woodland: Erica cinerea-Goodyera repens sub-community. Pinewoods have relatively species-poor floras in comparison to other native woodland types, and these results indicate the importance of striking a balance between management for ‘general’ biodiversity and the conservation of rare and specialized species assemblages.     

Nitrogen mineralization and H+ transfers in a Scots pine (Pinus sylvestris L.) forest soil as affected by liming

H⁺ production due to N uptake in a mature Scots pine stand subjected to high NH₄ ⁺ deposition was previously estimated to amount to approx. 2.2 kmol ha^-1 y^-1. The question whether H⁺ transfers related to N mineralization (ammonification and nitrification) offset or corroborate this proton production is investigated in the present research. To determine N mineralization, soil cores were used of which both ends were closed with layers of ion exchange resin (IER) to prevent influx and efflux of ions. The effect of liming on N mineralization and the resulting H⁺ production was investigated in 7 incubation periods of each ca. 8 wk. Because of its high mobility NO₃ accumulated in both IER layers at the expense of that in the incubated forest floor and mineral soil. Net N mineralization in the soil cores as a whole amounted to 40 and 77 kg N ha^-1 in 384 d in the control and limed plots, respectively. In both treatments ca. 65% of mineralized N was nitrified. H⁺ production due to N mineralization amounted to approx. 1.2 kmol ha^-1 y^-1 in the control and limed plots. Liming reduced the amount of C in the forest floor, but not forest floor mass, because of an increased mixing with mineral particles.     

Sensitivity to Cd or Zn of host and symbiont of ectomycorrhizal Pinus sylvestris L. (Scots pine) seedlings

Pinus sylvestris seedlings infected with either the ectomycorrhizal (ECM) fungus Paxillus involutus or Suillus variegatus were exposed to a range of Cd or Zn concentrations. This was done to investigate the relationship between the sensitivity of ECM fungi and their host plants over a wide range of concentrations. P. involutus ameliorated the toxicity of Cd and Zn to P. sylvestris with respect to root length, despite significant inhibition of ECM infection levels by Cd (Cd EC₅₀ [effective concentration which inhibits ECM infection by 50%] values were: P. involutus 3.7 μg g^-1 Cd; S. variegatus 2.3 μg g^-1 Cd). ECM infection by P. involutus also decreased Cd and Zn transport to the plant shoots at potentially toxic concentrations and also influenced the proportion of Zn transported to the roots and shoots, with a higher proportion retained in the roots of the seedlings. ECM infection did increase host biomass production, but this was not affected by the presence of Cd or Zn. Root and shoot biomass production by P. sylvestris, in both the presence and absence of ECM fungi, was unaffected by Cd and Zn at all concentrations tested. This revised version was published online in August 2006 with corrections to the Cover Date.     

Community structure of ectomycorrhizal fungi in a Pinus muricata forest: minimal overlap between the mature forest and resistant propagule communities

We have investigated colonization strategies by comparing the abundance and frequency of ectomycorrhizal fungal species on roots in a mature Pinus muricata forest with those present as resistant propagules colonizing potted seedlings grown in the same soil samples. Thirty-seven fungal species were distinguished by internal transcribed spacer (ITS) restriction fragment length polymorphisms (RFLPs); most were identified to species level by sporocarp RFLP matches or to genus/family level by using sequence databases for the mitochondrial and nuclear large-subunit rRNA genes. The below-ground fungal community found in the mature forest contrasted markedly with the resistant propagule community, as only four species were found in both communities. The dominant species in the mature forest were members of the Russulaceae, Thelephorales and Amanitaceae. In contrast, the resistant propagule community was dominated by Rhizopogon species and by species of the Ascomycota. Only one species, Tomentella sublilacina (Thelephorales), was common in both communities. The spatial distribution of mycorrhizae on mature roots and propagules in the soil differed among the dominant species. For example, T. sublilacina mycorrhizae exhibited a unique bias toward the organic horizons, Russula brevipes mycorrhizae were denser and more clumped than those of other species and Cenococcum propagules were localized, whereas R. subcaerulescens propagules were evenly distributed. We suggest that species differences in resource preferences and colonization strategies, such as those documented here, contribute to the maintenance of species richness in the ectomycorrhizal community.     

Ectomycorrhizal fungal biomass in roots and uptake of P from apatite by Pinus sylvestris seedlings growing in forest soil with and without wood ash amendment

Forest soil from an experimental Norway spruce forest with four levels of wood ash addition (0, 1, 3 and 6 tonnes ha^–1) was used to inoculate pine (Pinus sylvestris) seedlings with indigenous ectomycorrhizal (EM) fungi. Uptake of ³²P and ⁸⁶Rb in a root bioassay was used to estimate the demand for P and K by seedlings grown in the different soils. Utilisation of P from apatite was tested in a laboratory system where uptake by the ectomycorrhizal mycelium was separated from uptake by roots. The demand for P and K in the seedlings was similar regardless of the ash treatment. Variation in EM levels, estimated as fungal biomass (ergosterol) in roots, was large in the different soils, but not related to ash addition. Uptake of P from apatite was, on average, 23% of total seedling P and was not related to EM levels. It was concluded that the improved P uptake from apatite by EM fungi found in earlier studies is probably not a general phenomenon among EM fungi. The small effect of ash addition on EM levels and P uptake suggests that addition of granulated wood ash is a forest management treatment that will have only minor influence on ectomycorrhizal symbiosis.     

Evaluating interactions between soil drainage and seedling performance in a restoration of Pinus sylvestris woodland, Scotland

• 1 This paper evaluates the role of soil drainage in tree seedling performance at a site being restored from Calluna vulgaris moorland to Pinus sylvestris woodland, in Glen Affric, Scotland. The investigation focuses on the relationships between height of planted seedlings, type of ground vegetation and drainage conditions.
• 2 Slope, aspect, and soil depth were assessed as potential surrogates for direct measures of soil drainage, all of which were derived from digital terrain data.
• 3 Six variables related to drainage were recorded at 58 seedling locations and used in a factor analysis to understand links between soil moisture conditions, topographic variables and soil depth characteristics.
• 4 Factor analysis generated two factors that accounted for 70.5% of the variance in the correlation matrix of these variables: Factor 1 correlated strongly with variables that controlled peat accumulation and Factor 2 correlated strongly with topographic controls upon drainage patterns.
• 5 These two factors explained a significant amount of the variance in height of the Pinus seedlings planted at these locations. Significant differences were found between the factor scores associated with different types of ground vegetation, as well as between the seedling heights observed at locations with different vegetation types.
• 6 Multiple regressions were developed that indicated that slope, aspect, and soil depth were significant as independent variables in models where soil moisture content and aerobic soil depth were the dependent variables.

     

Uptake of 15N-labelled alanine,ammonium and nitrate in Pinus sylvestris L. ectomycorrhiza growing in forest soil treated with nitrogen,sulphur or lime

The uptake of ¹⁵N-labelled alanine, ammonium and nitrate was studied in ectomycorrhizal morphotypes of intact Pinus sylvestris seedlings. PCR-RFLP analysis of the ITS-region of fungal rDNA was used to identify the morphotypes. Seedlings were grown in forest soil collected at an experimental site in southern Sweden. The treatments compared were a control, N fertilisation (600 kg N ha^-1 as urea), sulfur application (1200 kg S ha^-1) and lime application (6000 kg CaCO₃ ha^-1). The forest, which had been dominated by Picea abies, was clear-cut two years before the forest soil was sampled. Soil was also collected from an adjacent standing forest. The aim of the present study was to detect changes in the ectomycorrhizal communities in forest soils and relate these changes to the functional parameter of uptake of nitrogen from organic (alanine and protein) and inorganic (ammonium and nitrate) sources.Liming resulted in the detection of a morphotype not found in other samples, and one morphotype was only found in samples from the standing forest (the fungi in these two morphotypes could not be identified). All mycorrhizal root tips showed a higher ¹⁵N concentration after exposure to different nitrogen forms than non-mycorrhizal long roots. Uptake of¹⁵ N from a labelled solution of alanine or ammonium was higher (about tenfold) than uptake from a ¹⁵N-labelled solution of nitrate. Uptake of ammonium and alanine varied between 0.2 and 0.5 mg N g^-1 h^-1 and between 0.1 and 0.33 mg N g^-1 h^-1, respectively, among the different morphotypes.In seedlings grown in the control soil and in soil from standing forest, alanine and ammonium were taken up to a similar degree from a supply solution by all morphotypes, whereas ammonium uptake was higher than alanine uptake in seedlings grown in lime-treated soil (about twofold) and, to a lesser extent, in the nitrogen- and sulfur-treated soils. The higher ammonium uptake by morphotypes from the limed soil was confirmed in pure culture studies. In cases where ammonium was used as the N source, an isolate of the S. variegatus morphotype collected in the limed soil produced more biomass compared with isolates of S. variegatus collected in nitrogen- or sulphur-treated soil. One isolate of a silvery white morphotype produced about equal amounts of biomass on alanine and ammonium, whereas all S. variegatus isolated performed better with ammonium as their N source. Based on the results it is hypothesised that liming can induce a shift in the ectomycorrhizal community, favouring individuals that mainly utilise inorganic nitrogen over those that primarily utilise organic nitrogen.