Belowground ectomycorrhizal communities in three Norway spruce stands with different degrees of decline in the Czech Republic

We investigated the ectomycorrhizal communities on the roots of adult trees and seedlings associated with three Norway spruce stands in the Czech Republic using morphological and molecular tools. The stands had different degrees of forest decline due to air pollution. The aims of the study were to obtain information about the belowground ectomycorrhizal community in a heavily damaged spruce forest and to assess whether missing ectomycorrhizal fungal partners could be one of the reasons for the observed lack of regeneration. The ectomycorrhizal species richness on the roots of adult trees was significantly lower in the heavily damaged site Mumlavska hora than in the other two, but less drastically so than that found in a fruitbody survey. The roots of adult trees and seedlings were fully mycorrhizal at this site although they were less species-rich. The most abundant ectomycorrhizal species on the root system of adult trees in all three forest stands was Tylospora fibrillosa, a member of the athelioid clade. It made up over 60% of root tips in Mumlavska hora and its proportion was at least twice that in the other two sites. This species was also an efficient colonizer of roots from seedlings, in particular, in the most damaged site. The different soil properties in this site may have caused the observed differences in the ectomycorrhizal species richness and composition. For example, cation exchange capacity and soil base saturation were lower and the soil more often saturated. However, the number of living trees and their defoliation status may well directly impact the ectomycorrhizal species composition by presumably affecting the amount of carbon delivered to the symbiotic fungal partners. Athelioids and thelephoroids are an important component of the belowground ectomycorrhizal community in most temperate and boreal forests, but the role they play might even be more crucial in stressed forest ecosystems. Based on our results, we suggest that factors other than missing ectomycorrhizal inoculum constrain natural regeneration in the heavily damaged site Mumlavska hora.     

Ectomycorrhizal root growth in scots pine stands in response to manipulation of litter and humus layers

 The effect on ectomycorrhizal root growth in a nitrogen-enriched planted stand of Scots pine (Pinus sylvestris L.) on podzolic sandy soil to manipulation of litter and humus layers (removal, doubling and control treatments) was examined, and compared to ectomycorrhizal root growth in an untreated naturally established Scots pine stand on nutrient-poor non-podzolic sandy soil. Half a year after manipulation of litter and humus layers in the planted stand, ingrowth-cores to a depth of 60 cm were installed in both stands. Scots pine roots were sampled four times during two growing seasons. Ectomycorrhizal roots were found at all sampled soil depths to 60 cm in all plots. Root growth and ectomycorrhizal development were greater in the naturally established stand than in all plots in the planted stand. Numbers of ectomycorrhizal root tips in the litter and humus removal plots were generally higher than in the control plots in the planted stand until May 1992. Doubling litter and humus did not significantly affect root length or the numbers of ectomycorrhizal root tips. The N _dissolved , NH₄ ⁺ and NO₃ ^– concentrations and the organic matter content in the upper 5 cm of the mineral soil in the planted stand on podzolic sandy soil were generally higher and the pH significantly lower than in the naturally established stand on non-podzolic sandy soil. Root growth and ectomycorrhizal development in the secondary stand may have been negatively affected by the chemical composition of the podzolic sandy soil. Accepted: 19 March 1997     

Seasonal ectomycorrhizal fungal biomass development on loblolly pine (Pinus taeda L.) seedlings

Ergosterol, a membrane sterol found in fungi but not in plants, was used to estimate live mycelial biomass in ectomycorrhizae. Loblolly pine (Pinus taeda L.) seeds were sown in April 1993 and grown with standard nursery culture practices. Correlations between total seedling ergosterol and visual assessment of mycorrhizal colonization were high during July and August but low as ectomycorrhizal development continued into the growing season. Percentages of mycelial dry weight over lateral roots decreased from 9% in July to 2.5% in November because seedling lateral root dry weight accumulated faster than mycelial dry weight. Total ergosterol per seedling increased from July through February. As lateral root dry weight ceased to increase during winter months, ectomycorrhizal mycelia became the major carbohydrate sink of pine seedlings. No distinctive seasonal pattern of soil ergosterol content was observed. The impact of ectomycorrhizal fungi on plant carbohydrate source-sink dynamics can be quantitatively estimated with ergosterol analysis but not with conventional visual determination.     

Synthesis of Japanese Boletus edulis ectomycorrhizae with Japanese red pine

Boletus edulis is a well-known ectomycorrhizal mushroom. Although cultivation has been widely attempted, no artificial fruiting has been achieved owing to difficulties associated with mycorrhizal synthesis and acclimatization in fields. We collected fifteen B. edulis basidiomata samples from locations in Japan and identified them microscopically and by phylogenetic analysis of their nuclear ribosomal internal transcribed spacer (ITS) regions. Pure culture isolates of B. edulis were established efficiently on malt extract agar medium, and one isolate, EN-63, was inoculated to axenic Pinus densiflora seedlings in vitro. Brownish ectomycorrhizal tips were observed on the pine lateral roots within four months of inoculation. Ten pine seedlings that formed ectomycorrhizae were acclimatized under laboratory and greenhouse conditions. At four months after transplant, mycorrhizal colonization by B. edulis was observed on newly grown root tips under laboratory conditions, but no B. edulis ectomycorrhiza survived under greenhouse conditions. These results suggest that B. edulis ectomycorrhizae synthesized in vitro with P. densiflora requires additional steps for acclimatization to greenhouse conditions.     

Host plants and edaphic factors influence the distribution and diversity of ectomycorrhizal fungal fruiting bodies within rainforests from Tshopo,Democratic Republic of the Congo

Ectomycorrhizal fungi constitute an important component of forest ecosystems that enhances plant nutrition and resistance against stresses. Diversity of ectomycorrhizal (EcM) fungi is, however, affected by host plant diversity and soil heterogeneity. This study provides information about the influence of host plants and soil resources on the diversity of ectomycorrhizal fungal fruiting bodies from rainforests of the Democratic Republic of the Congo. Based on the presence of fungal fruiting bodies, significant differences in the number of ectomycorrhizal fungi species existed between forest stand types (p < 0.001). The most ectomycorrhizal species‐rich forest was the Gilbertiodendron dewevrei‐dominated forest (61 species). Of all 93 species of ectomycorrhizal fungi, 19 demonstrated a significant indicator value for particular forest stand types. Of all analysed edaphic factors, the percentage of silt particles was the most important parameter influencing EcM fungi host plant tree distribution. Both host trees and edaphic factors strongly affected the distribution and diversity of EcM fungi. EcM fungi may have developed differently their ability to successfully colonise root systems in relation to the availability of nutrients.     

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.     

Relationship between soil properties and incidence of pine wilt disease at stand level

This study was conducted to evaluate and compare soil properties between pine stands either damaged or undamaged by pine wilt disease in Jinju and Sacheon Cities, which are areas in Korea severely affected by the disease. Soil physical and chemical properties were generally similar between damaged and undamaged pine stands. There was no significant difference in soil factors related to soil nutrient fertility between damaged and undamaged pine stands, although the incidence of pine wilt disease was affected by soil clay content, which was significantly (P < 0.05) higher in the damaged (24.8%) than in the undamaged (20.1%) pine stands. However, this relationship should be treated with caution because small scale topographical variation may simply reflect the incidence of pine wilt disease. The results suggest that incidence of pine wilt disease was not influenced by soil nutrient properties at a small stand scale.     

Effects of manipulation of litter and humus layers on ectomycorrhizal colonization potential in Scots pine stands of different age

Effects of manipulation of litter and humus layers (removal, doubling and control treatments) on the colonization potential of ectomycorrhizal fungi were studied in two secondary stands of Pinus sylvestris (5 and 18 years old) in The Netherlands. Five-mont-hold, sterile-grown Scots pine seedlings, inoculated with Laccaria bicolor, Paxillus involutus or Rhizopogon luteolus and noninoculated seedlings were used as baits. The seedlings were harvested after one growing season. For comparison, sporocarps of ectomycorrhizal fungi were also investigated. Genus composition on the seedlings was independent of initial inoculum, but determined by both treatment and age of the stands. In both stands, removal of litter and humus layers increased, and addition of organic material decreased the number of ectomycorrhizal types on the seedlings. Not all indigenous genera were observed by either outplanting seedlings or sporocarp surveys.     

Ectomycorrhizal fungal diversity in orchards of cultivated pecan (Carya illinoinensis; Juglandaceae)

Carya illinoinensis (pecan) belongs to the Juglandaceae (walnut family) and is a major economic nut crop in the southern USA. Although evidence suggests that some species in the Juglandaceae are ectomycorrhizal, investigations on their ectomycorrhizal fungal symbionts are quite limited. Here we assessed the ectomycorrhizal fungal diversity in cultivated orchards of C. illinoinensis. Five pecan orchards in southern Georgia, USA, were studied, three of which were known to fruit the native edible truffle species Tuber lyonii. We sequenced rDNA from single ectomycorrhizal root tips sampled from a total of 50 individual trees. Mycorrhizae were identified by ITS and LSU rDNA sequence-based methods. Forty-four distinct ectomycorrhizal taxa were detected. Sequestrate taxa including Tuber and Scleroderma were particularly abundant. The two most abundant sequence types belonged to T. lyonii (17%) and an undescribed Tuber species (~20%). Because of our interest in the ecology of T. lyonii, we also conducted greenhouse studies to determine whether this species would colonize and form ectomycorrhizae on roots of pecan, oak, or pine species endemic to the region. T. lyonii ectomycorrhizae were formed on pecan and oak seedlings, but not pine, when these were inoculated with spores. That oak and pecan seedling roots were receptive to truffle spores indicates that spore slurry inoculation could be a suitable method for commercial use and that, ecologically, T. lyonii may function as a pioneer ectomycorrhizal species for these hosts.     

Partial restoration of fungal and plant species diversity by removal of litter and humus layers in stands of Scots pine in the Netherlands

A field experiment was set up in five mature Scots pine (Pinus sylvestris) stands in the Netherlands in order to study the effects of removal of litter and humus on ectomycorrhizal fungi and vegetation of higher plants, bryophytes, and lichens. Lime was also applied as a countermeasure to the removal of surplus cations. Application of lime did not have significant effects on mycorrhizal fungi. Removal of ectorganic material increased species richness of ectomycorrhizal fungi. The number of fruit bodies was also higher in the sod-cut plots, but the difference compared with the controls was not significant in all years. A major effect of removal of the ectorganic layer was a reversion of ectomycorrhizal succession to earlier stages. Liming increased the number and cover of ruderal, nitrophilous plant species. Sod-cutting caused a reversion of the green vegetation to earlier successional stages.     

Ectomycorrhizal and wood-decay macromycete communities along development stages of managed Pinus patula stands in Southwest Mexico

This study used fruit body surveys to evaluate the diversity and distribution of ectomycorrhizal and wood-decay macromycetes along developing stages of stands artificially reforested with Pinus patula in Southern Mexico. Three stand ages were selected: 1, 11, and 60 y old. Macromycetes were collected from June to October 2016 in 0.1 ha plots per stand age. A total of 106 taxa were recorded, 63 ectomycorrhizal and 43 wood-decay fungi. The highest diversity of ectomycorrhizal fungi was in 11-y stands, and 11- and 60-y stands had similar species composition. Wood-decay fungi showed a trend towards higher diversity in the 1- and 11-y stands, and species composition in these sites corresponded. Patterns of diversity and distribution were mainly determined by factors related to substratum availability and vegetation structure. Knowledge about the influence of artificially reforested stands on macrofungal communities is relevant to improve management plans and achieve a more sustainable use of forest timber and non-timber products.     

Ectomycorrhizal development on Scots pine (Pinus sylvestris L.) seedlings in different soils

In the present study ectomycorrhizal development of Laccaria bicolor, Rhizopogon luteolus and Suillus bovinus associated with Scots pine (Pinus sylvestris) seedings was studied as affected by primary stand humus, secondary stand humus, podsolic sandy soil or peat in perspex growth chambers. After 9 weeks, ectomycorrhizal development with S. bovinus was significantly greater in peat and primary stand humus than in secondary stand humus or podsolic sandy soil. Ectomycorrhizal development with R. luteolus in secondary stand humus was higher than in primary stand humus. Degree of ectomycorrhizal development of L. bicolor, R. lutuelus and S. bovinus on Scots pine was related to potassium concentration, organic matter content and pH of the soils suggesting that chemical composition of the soils affects ectomycorrhizal development.     

Difference of pine ectomycorrhizal biomass in relation to forest conditions

In this study, two plots in a secondary and another two in planted Pinus densiflora stands were exposed to different forest treatments, and the ectomycorrhizal (EM) biomass and its ergosterol content was measured for a year. The unmanaged plot in the secondary stand had greater EM biomass than those in any other plots. Whereas understory cutting had less effect on EM biomass, litter and humus removal decreased pine EM biomass and its ergosterol content, suggesting that such forest treatment alters EM biomass and its structure.     

An ectomycorrhizal fungus,Cenococcum geophilum,in a coastal pine forest has a high tolerance for an insecticide used to control pine wilt disease

Cenococcum geophilum Fr., one of several ectomycorrhizal species associated with black pine (Pinus thunbergii Parl.), is dominant in the coastal forests of Japan, even under adverse abiotic environmental conditions. In these forests, many tonnes of Sumipine® (fenitrothion) are applied every year to protect P. thunbergii from pine wilt disease, which is transmitted by a beetle. Here, we examined the effect of this insecticide on the species of fungi found as ectomycorrhizae on naturally regenerated P. thunbergii seedlings collected from coastal forest sites that had or had not been sprayed with fenitrothion. The proportion of C. geophilum ectomycorrhizae on black pine root tips was significantly higher in areas where fenitrothion had been applied than in areas where it had not. We measured the in vitro mycelial growth of C. geophilum as well as other ectomycorrhizal fungi of coastal black pine, Rhizopogon roseolus (Corda) Th. Fr. and Pisolithus arhizus (Scop.) Rauschert, at three levels of fenitrothion (density: 1.32 g/cm³), i.e., 0, 0.1 and 0.2 mL L^?1. The growth of all three species decreased significantly as the fenitrothion dosage increased. However, the reduction of mycelial growth in response to fenitrothion was lower in C. geophilum than in the other two species. These results suggest that C. geophilum has a high tolerance for fenitrothion, which may explain its dominance over other ectomycorrhizal species in coastal forests in Japan where fenitrothion is routinely sprayed.

     

Soil inoculation of lodgepole pine seedlings alters root-associated fungal communities but does not improve seedling performance in beetle-killed pine stands

Extensive tree mortality in forests can change the community composition of soil fungi altering seedling establishment, a process critical to forest restoration. Disturbances that result in the loss of ectomycorrhizal fungi, in particular, may impede the establishment of tree species reliant on these symbionts for their survival. Inoculation of seedlings with soil from intact forests may improve the establishment of seedlings in such disturbances but the method has rarely been tested in the field. Here, we assess whether soil inoculation improves lodgepole pine (Pinus contorta var. latifolia) seedling performance in conspecific stands with high levels of tree mortality caused by a mountain pine beetle (Dendroctonus ponderosae) outbreak and whether underlying soil type modifies inoculation effects. We first inoculated seedlings in a growth chamber with small amounts of soils (5% volume) originating from either intact (<10%) or “beetle-killed” (>70% pine basal area killed) conspecific stands or added no soil inoculum and, after 4 months, transplanted them into 15 beetle-killed stands. After two growing seasons, root-associated fungal communities of seedlings receiving inoculum from intact stands differed in composition from those receiving inoculum from beetle-killed stands or no inoculum. However, inoculation had no effect on seedling survival, height, or biomass. Site properties, including soil texture and the resident fungal community composition, overwhelmed the effect of soil inoculation on seedling performance. Seedling survival and shoot mass was higher in sandy than loamy soils. Restoration to improve seedling performance in beetle-killed stands should consider stand-level treatments as soil inoculation at the level evaluated was ineffective.     

Protozoan communities around conifer roots colonized by ectomycorrhizal fungi

Protozoan communities around roots with different types of ectomycorrhizae were distinct. These protozoan communities differed both qualitatively and quantitatively with the host (Pinus ponderosa, Pseudotsuga menziesii, Picea sitchensis, Tsuga heterophylla and Abies grandis) and the ectomycorrhizal fungal species. Based on the species identified and the numbers of individuals of each species, six communities of protozoa were found associated with specific ectomycorrhizae. Previous researchers have shown that mycorrhizal colonization of roots alters the amounts and types of exudates produced by roots, which in turn alters the bacterial community present. Most likely, mycorrhizal colonization of roots influences the protozoan community around roots by controlling the bacterial community. However, the protozoan community may in turn influence the successional dynamics of ectomycorrhizal fungi on different host root systems by a variety of mechanisms. These mechanisms could include: (1) preying upon individuals and perhaps removing particular species of bacteria from the mycorrhizosphere; and (2) controlling nitrogen mineralization in the rhizosphere. Further work needs to be performed to determine the interaction between these quadrate (plant-bacteria-fungi-protozoa) associations.     

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.     

In vitro synthesis of Pisolithus-Eucalyptus ectomycorrhizae: synchronization of lateral tip emergence and ectomycorrhizal development

 A simple and reproducible in vitro system is described for the synthesis of Pisolithus-Eucalyptus grandis ectomycorrhizae. Hyphal discs from actively growing colonies were placed in large petri dishes containing minimum nutrient agar overlaid with cellophane and allowed to grow for 7 days. Seeds were then surface sterilized and placed above the expanding fungal colonies and the plates slanted. Seedlings that germinated and grew in the presence of fungal hyphae had twice as many lateral root tips as seedlings that germinated before they were transferred onto hyphal mats. In addition, the lateral root tips of inoculated seedlings had a faster maturation rate and emerged closer to the primary root apex than non-inoculated seedlings. All lateral tips emerged in contact with fungal hyphae and the differentiation of ectomycorrhizae was followed by examining lateral tips basipetally along a single primary root. Typical ectomycorrhizae had formed on 4-day-old lateral tips, i.e. a mantle, radially elongated epidermal cells and a Hartig net commencing about 0.3 mm behind the lateral root apex. Thereafter, the mantle continued to thicken and the apical meristem diminished. The Hartig net often surrounded the apex of 11- to 12-day-old lateral root tips. This model system will facilitate detailed studies on synchronized ectomycorrhizal development and associated molecular and biochemical changes. Accepted: 12 January 1996     

Common environmental factors explain both ectomycorrhizal species diversity and pine regeneration variability in a post-fire Mediterranean forest

Natural seedling regeneration and establishment after stand replacing wildfires is influenced by a series of environmental and biological constraints. In this study, we characterized the diversity and structure of the ectomycorrhizal (ECM) fungal community associated with post-fire naturally regenerated maritime pine saplings, and individuate the environmental factors responsible for fungal species distribution. We also identify the main environmental factors responsible for maritime pine regeneration variability and assessed the relation between saplings performance and ECM fungal diversity indices. Fungal species were identified by direct sequencing of internal transcribed spacer regions. Five years after the disturbance event, a total of 30 taxa colonized the pine saplings. The ECM fungal community was dominated by ruderal species of the genus Rhizopogon (present in almost half of the samples). Almost one third of the identified ECM fungal species belonged to the family Thelephoraceae. Typical k-selected species like Amanita pantherina, Boletus aestivalis, Lactarius chrysorrheus, and Russula densifolia were found on pine saplings collected in proximity of unburnt pine trees, in correspondence with low erosion extents. Pine regeneration varied throughout the study areas and was enhanced at higher elevations, in correspondence with moderate slopes, shallower soils, and a reduced cover of ericaceous shrubs and bare ground. These conditions were found in close proximity to patches of pine trees that survived the disturbance event and were previously characterized by a higher pre-fire pine biomass. Even though no correlations were found between saplings performance and ECM fungal diversity indices, common environmental factors (i.e., ericaceous shrub cover, extent of erosion, slope, and soil depth) were responsible for shaping the ECM fungal distribution and for describing most of the explained regeneration variability.     

Site preparation alters soil distribution of roots and ectomycorrhizae on outplanted western white pine and Douglas-fir

This report documents root and ectomycorrhizal development on container-produced (1-0), outplanted, western white pine and Douglas-fir seedlings growing in site-prepared forest soils typical of the Inland Northwestern US. The following site preparations were used: 1) mounding organic and surface mineral horizons; 2) mounding with subsequent physical removal or chemical control of competing vegetation; 3) scalping to reduce competing vegetation; and, 4) a control or no post-harvest disturbance. Treatments were applied on relatively harsh and moderate sites in northern Idaho. Most ectomycorrhizae on the seedling population were found in the mineral substrates that dominated planting sites. However, compared to mineral substrates, highest seedling ectomycorrhizal tip counts were recorded in organic matter, particularly decayed wood or mixtures containing decayed wood. Strong ectomycorrhizal development was characteristic of western white pine. It supported highest ectomycorrhizal activity in organic substrates on the harshest treatments (scalps). Douglas-fir showed even stronger relative increases of ectomycorrhizae in organic substrates on harsh treatments. Three of the four common ectomycorrhizal morphological types were concentrated in mineral substrates with all treatments. A treatment-induced change of behavior was shown by the principal pine type. It occurred at highest numbers in organic substrates of the mound with competing vegetation treatment and in mineral substrates with the control. If relative availability to seedling roots was considered, organics (especially decomposed wood) were generally equal or superior to mineral substrates for supporting ectomycorrhizal activity on planted seedlings.