Derek T. Mitchell

The role of local environment and geographical distance in determining community composition of arbuscular mycorrhizal fungi at the landscape scale

Arbuscular fungi have a major role in directing the functioning of terrestrial ecosystems yet little is known about their biogeographical distribution. The Baas-Becking hypothesis (‘everything is everywhere, but, the environment selects’) was tested by investigating the distribution of arbuscular mycorrhizal fungi (AMF) at the landscape scale and the influence of environmental factors and geographical distance in determining community composition. AMF communities in Trifolium repens and Lolium perenne roots were assessed in 40 geographically dispersed sites in Ireland representing different land uses and soil types. Field sampling and laboratory bioassays were used, with AMF communities characterised using 18S rRNA terminal-restriction fragment length polymorphism. Landscape-scale distribution of AMF was driven by the local environment. AMF community composition was influenced by abiotic variables (pH, rainfall and soil type), but not land use or geographical distance. Trifolium repens and L. perenne supported contrasting communities of AMF, and the communities colonising each plant species were consistent across pasture habitats and over distance. Furthermore, L. perenne AMF communities grouped by soil type within pasture habitats. This is the largest and most comprehensive study that has investigated the landscape-scale distribution of AMF. Our findings support the Baas-Becking hypothesis at the landscape scale and demonstrate the strong influence the local environment has on determining AMF community composition.

Phosphatase activity of four ectomycorrhizal fungi found in a Sitka spruce-Japanese larch plantation in Ireland

Ectomycorrhizal fungi (Paxillus involutus, Suillus grevillei and two unidentified basidiomycetes from excised Sitka spruce mycorrhizas) were isolated from stands of Sitka spruce either in monoculture or in mixture with Japanese larch in an Irish conifer plantation. They were grown for 35 d on modified Melin-Norkrans liquid medium containing ferric phytate as the phosphorus source. The cultures were then separated into wall- and membrane-bound, cytoplasmic and extracellular fractions and assayed for phosphatase. Wall- and membrane-bound fractions contained the most active acid phosphatase. The unidentified basidiomycetes showed a lower substrate affinity and higher velocity of reaction than P. involutus and S. grevillei. Wall- and membrane-bound and cytoplasmic phosphatase activities were optimum over a broad pH range (4·0–6·0). Various methods were used to release the wall-bound phosphatase and a high proportion (49–88%) appeared to be tightly held within the wall. Phosphatase released from the wall by sonication had a similar Km to wall-bound phosphatase but Vmax was lower. The use of a number of substrates demonstrated a high affinity for inorganic pyrophosphate and sodium β-glycerophosphate but a low phytase activity.

Rapid predictions of cold tolerance in Douglas-fir seedlings using chlorophyll fluorescence after freezing

Chlorophyll fluorescence measurements were performed on the foliage of 3-year-old (11/2+11/2) nursery-grown Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] seedlings after exposure to controlled freezing temperatures, in the laboratory, to assess low temperature tolerance. The seedlings were propagated in an Irish nursery and lifted at monthly intervals overwinter 1999 and 1999–2000. Excised shoots from first-order laterals were frozen, in the dark. After freezing, needles were immediately assessed using chlorophyll fluorescence. The excised shoots were then maintained under controlled conditions for 14 days and visually assessed for needle damage. The chlorophyll fluorescence parameter, Fv/Fm, accurately predicted cold hardiness and was linearly related to visual needle damage and short-term survival. An equation was constructed using Fv/Fm data for determining the LT50, that is, the freeze temperature causing 50% seedling damage. The predictions of F.LT50 (fluorescence-based empirical determination of LT50) have been tested over two seasons (i.e., against a second independent data set) with variability between 0 and 1.8 °C of visual estimates, though predictions were often ≤ 1.1 °C of the visual assessment. This approach provided a simple, rapid and accurate prediction of cold tolerance, under climatic conditions where in situ measurements are unreliable. The method can be used to predict if Douglas-fir seedlings have developed sufficient tolerance for lifting to the cold-store, or for planting.

Colonization of root systems of Carex flacca and C. pilulifera by Cortinarius ( Dermocybe ) cinnamomeus

The root systems of Carex flacca and C. pilulifera, growing in the Burren (western Ireland), were shown to be colonized by Cortinarius (Dermocybe) cinnamomeus. This basidiomycete formed ectomycorrhiza-like structures, which possessed a distinct fungal mantle (85–100 μm thick), hyphal infection in epidermal cells, rhizomorphs and extramatrical hyphae, but lacked a Hartig net. These ectomycorrhiza-like structures were formed on first-order lateral roots but were distinct, morphologically and anatomically, from dauciform roots. Structures typical of arbuscular mycorrhizas were never observed. The colonization of root systems of C. flacca by C. cinnamomeus was confirmed by PCR/RFLPs and DNA sequencing of the ITS region of rDNA.

Diversity of fungi associated with hair roots of ericaceous plants is affected by land use

Culture-independent molecular studies have provided new insights into the diversity of fungi associating with ericaceous plant roots. However, there is little understanding of the distribution of these fungi across landscapes, or the effects of environmental heterogeneity on ericoid mycorrhizal (ERM) fungal diversity and distribution. Terminal-restriction fragment length polymorphism and selective sequence analyses of the internal transcribed spacer regions of rDNA were used to infer fungal diversity of bait Vaccinium macrocarpon grown in soils from nine peatland sites in Ireland, representing three different land uses (bog, rough grazing and forest plantation) and the fungal communities of field-collected Calluna vulgaris for five of these nine sites. A diverse range of potential ERM fungi were found, and the sampling approach significantly affected the diversity of the fungal community. Despite significant site groupings of the fungal communities associated with V. macrocarpon and C. vulgaris, fungal communities were significantly dissimilar between sites with different land uses. Soil nitrogen content significantly explained 52% of the variation in the V. macrocarpon fungal communities. Evidence suggests that environmental heterogeneity has a role in shaping ERM fungal community composition at the landscape scale.

Utilization of sucrose by Hymenoscyphus ericae (an ericoid endomycorrhizal fungus) and ectomycorrhizal fungi

Hymenoscyphus ericae utilized sucrose, whereas the ectomycorrhizal fungi either showed poor growth or responded to the presence of sucrose after a lag phase. On sucrose-containing media, fructose and glucose accumulated in the medium at the end of the linear phase of growth of H. ericae and then disappeared during the stationary phase. Dry mass of H. ericae increased with increasing concentration of both sucrose and glucose up to 2·0 g C l −1 , whereas on fructose, it increased up to 4 g C l −1 . Growth of H. ericae was doubled on media containing both glucose and fructose at 1:1 ratio compared with growth on either hexose alone at the same carbon concentration. Sucrose, fructose and glucose were detected in the mycelium of H. ericae , although the main soluble carbohydrates were mannitol and trehalose. Growth of ectomycorrhizal fungi (excluding Cenococcum geophilum ) on sucrose depended solely on the presence of hexoses in the medium and utilization of sucrose was poor. The use of ‘starter glucose’ had an effect on sucrose utilization by Laccaria laccata, Lactarius rufus, Paxillus involutus and Thelephora terrestris but growth was still less than that on equivalent amounts of glucose. These results confirm previous studies that the majority of ectomycorrhizal associations do not utilize sucrose directly and may have to rely on apoplastic host invertase. In contrast, host invertases may not be required in the utilization of sucrose by ericoid mycorrhizal associations.

Phosphatases of ericoid mycorrhizal fungi: kinetic properties and the effect of copper on activity.

Ericoid endomycorrhizal fungi (two isolates of Hymenoscyphus ericae obtained from unpolluted heathlands and two H. ericae-type endophytes isolated from Calluna vulgaris growing on Cu-contaminated mine spoil) were grown for 14 d on 10% Rorisons solution containing sodium phytate as the sole P source and either trace (0.16 microM) or elevated (0.25 mM) concentrations of Cu. The elevated levels of Cu in the medium had no effect on the growth of the two H. ericae-type endophytes from mine spoil sites but caused a significant reduction in growth of the two H. ericae isolates from unpolluted sites. Wall, cytoplasmic and extracellular fractions were assayed for phosphomonoesterase (PMEase) and phosphodiesterase (PDEase) activity. K(m) and V(max) values varied between the different endophytes and both were highest in the wall fractions. Wall-bound phosphatase activity, excluding PDEase of one H. ericae-type endophyte, was generally unaffected after the isolates had been grown on medium containing 0.25 mM Cu. Extracellular PDEase of the two H. ericae-type endophytes from mine spoil sites was stimulated by 0.25 mM Cu in the growth medium. Cu concentrations up to 5.0 mM in the assay medium did not inhibit wall-bound phosphatase activity whereas three of the isolates showed a stimulation of extracellular activity with increasing Cu. The results are discussed in relation to phosphatase activity of ericoid endophytes on Cu-contaminated substrates.

Nutritional influences on the solubilization of metal phosphate by ericoid mycorrhizal fungi

Four ericoid mycobionts (two isolates of Hymenoscyphus ericae, and two dark, sterile ericoid mycobionts isolated from metal-contaminated mine sites) were grown on solid agar plates supplemented with zinc phosphate (0.25 %) containing different forms of nitrogen (nitrate, ammonium or alanine) and different concentrations of carbon (glucose) and phosphorus (K2HPO4). The influence of nutrient variation on solubilizing ability of the fungi was assessed by measuring the zones of solubilization appearing beneath the growing colonies. All four mycobionts were capable of zinc phosphate solubilization in the presence of all three nitrogen sources and in media containing no nitrogen. No solubilization was observed at 0 mM glucose-C but was observed with increasing glucose concentration from 300 to 600 mM C. Increasing phosphorus concentration (0-5 mM P) had no effect on the solubilizing ability of the isolates. All but one of the mycobionts were capable of solubilizing calcium phosphate (CaHPO4), while no solubilization was observed in media containing aluminium phosphate (AlPO4), iron phosphate (FePO4 x 4H2O) or copper phosphate (Cu3O8P2 x 2H2O) under conditions which were found to be optimal for zinc phosphate solubilization. Under conditions of glucose at 300 mM C and alanine as the N source in the zinc phosphate-amended agar medium, one of the mycobionts produced new crystals, which were morphologically distinct from the original zinc phosphate crystals. It is concluded that medium composition influences the metal-phosphate solubilizing ability of ericoid mycobionts. The results are discussed in relation to the possible mechanisms involved in solubilization and the potential benefits of metal-phosphate solubilization to ericoid mycobionts and their host plants.

Properties of invertases in mycelium of Hymenoscyphus ericae and in endomycorrhizal association with cranberry seedlings

The properties of extracellular, soluble and insoluble invertases in mycelium of Hymenoscyphus ericae an ericoid mycorrhizal fungus, were investigated. Soluble and insoluble acid invertases were more active in sucrose- compared with glucose-grown mycelium, whereas extracellular invertase activities were similar in both glucose- and sucrose-grown cultures. Acid (pH 5·5) and alkaline (pH 9·0) invertases were detected in soluble and insoluble fractions but extracellular activity was predominantly acid (pH 5·5). Insoluble alkaline invertase was released completely by freeze-thawing. Freeze-thawing and Triton X-100 released similar proportions of acid invertase from the wall (42 and 31% respectively). The K m values varied from 9·1 m m (extracellular acid invertase) to 139·3 m m sucrose (soluble alkaline invertase). Fructose inhibited soluble alkaline invertase completely but it competitively inhibited soluble acid invertase. Increased soluble acid invertase activity was demonstrated in endomycorrhizas compared with uninoculated roots of cranberry.

Ectomycorrhizas associated with a relict population of Dryas octopetala in the Burren, western Ireland II. Composition, structure and temporal variation in the ectomycorrhizal community

The composition, structure and temporal variation of ectomycorrhizal (EM) communities associated with mountain avens (Dryas octopetala) in grass heaths of the Burren, western Ireland were assessed by using soil core sampling in two permanent plots and 30 other sites (196 cores in total). Of the 34 different EM types observed, 11 were common and constituted over 80% of the EM biomass. Four EM types, Craterellus lutescens, Tomentella sp., Dryadirhiza fulgens and Cenococcum geophilum were the most abundant as measured by EM length and frequency of occurrence in cores. The species profile and relative abundances were very similar in cores from the permanent plots and different sites in the Burren, indicating that they were all representative of the same EM community. The below-ground EM community in both plots was compared with production of basidiomes, and the latter was found to be an unreliable indicator of EM community structure. Temporal variation in the EM community was assessed by repeated core sampling of the two permanent plots over a 14-month period (between March 1998 and May 1999). No statistically significant shifts in EM abundance were found between sampling dates, probably as a consequence of the large variation in EM abundance between core samples over the sampling period. No significant relationship was found between rainfall, soil moisture or soil temperature and fluctuations in EM abundance. Patterns of total EM abundance and fluctuations in EM diversity were strongly correlated between the two permanent plots over the sampling period. Temporal fluctuations in the dominant EM type, Craterellus lutescens, were similar in both plots with respect to mycorrhizal length, biomass and relative abundance, and the patterns between both plots were positively correlated. EM diversity was negatively correlated with biomass of ectomycorrhizas of Craterellus lutescens in both plots, but it was significant only in plot 1.