Ericoid Mycorrhizal Fungus Hymenoscyphus Ericae Research Articles

Host Range of a Select Isolate of the Ericoid Mycorrhizal Fungus Hymenoscyphus ericae

Studies were conducted to examine the host range of a select isolate of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf and Kernan [American Type Culture Collection (ATCC) #32985]. Host status was tested for 15 ericaceous species, including: Calluna vulgaris (L.) Hull, Enkianthus campanulatus (Miq.) Nichols, Gaultheria procumbens L., Kalmia latifolia L., Leucothoe fontanesiana Sleum., Oxydendrum arboreum (L.) DC.,Pierisfloribunda (Pursh) Benth. & Hook.,Rhododendron calendulaceum (Michx.) Torr.,Rhododendron carolinianum Rehd., Rhododendron catawbiense Michx., Rhododendron maximum L., Rhododendron mucronulatumTurcz., Vaccinium corymbosum L., and Vaccinium macrocarpon Ait. Arbutus unedo L., an ericaceous species that forms arbutoid, not ericoid, mycorrhizae, was used as a negative control. All of the species were colonized by the ericoid isolate with the exception of Enkianthus campanulatus and the negative control. Inoculation with this isolate of H. ericae resulted in a significant increase in shoot growth. However, intensity of root colonization was not correlated to amount of shoot growth. In fact, an increase in growth was observed in the two species that lacked fungal colonization.

Arsenate resistance in the ericoid mycorrhizal fungus Hymenoscyphus ericae

• Differential resistance to arsenate (AsO4 3- ) is demonstrated here among populations of the ericoid mycorrhizal fungus Hymenoscyphus ericae isolated from Calluna vulgaris in natural heathland soils and soils contaminated with AsO4 3- . • Isolates (c.25) of the fungus from each of two As and Cu mine sites, and a natural heathland site, were screened for AsO4 3- and Cu2+ resistance by growing isolates in media containing a range of AsO4 3- and Cu2+ concentrations. • H. ericae populations from the mine sites demonstrated resistance to AsO4 3- compared with the heathland population; the mine-site populations producing significant growth at the highest AsO4 3- concentration (4.67mol m-3 ), whereas growth of the heathland population was almost completely inhibited. EC50 values for mine-site isolates were estimated to be 5-41-times higher than the heathland population. All isolates produced identical responses to increasing Cu2+ concentrations, with no differences observed between mine-site and heathland isolates. • Populations of H. ericae on the contaminated mine sites have developed adaptive resistance to AsO4 3- . By contrast, Cu2+ resistance appears to be constitutive.

Mechanism of arsenate resistance in the ericoid mycorrhizal fungus Hymenoscyphus ericae.

Arsenate resistance is exhibited by the ericoid mycorrhizal fungus Hymenoscyphus ericae collected from As-contaminated mine soils. To investigate the mechanism of arsenate resistance, uptake kinetics for arsenate (H(2)AsO(4)(-)), arsenite (H(3)AsO(3)), and phosphate (H(2)PO(4)(-)) were determined in both arsenate-resistant and -non-resistant H. ericae. The uptake kinetics of H(2)AsO(4)(-), H(3)AsO(3), and H(2)PO(4)(-) in both resistant and non-resistant isolates were similar. The presence of 5.0 microM H(2)PO(4)(-) repressed uptake of H(2)AsO(4)(-) and exposure to 0.75 mM H(2)AsO(4)(-) repressed H(2)PO(4)(-) uptake in both H. ericae. Mine site H. ericae demonstrated an enhanced As efflux mechanism in comparison with non-resistant H. ericae and lost approximately 90% of preloaded cellular As (1-h uptake of 0.22 micromol g(-1) dry weight h(-1) H(2)AsO(4)(-)) over a 5-h period in comparison with non-resistant H. ericae, which lost 40% of their total absorbed H(2)AsO(4)(-). As lost from the fungal tissue was in the form of H(3)AsO(3). The results of the present study demonstrate an enhanced H(3)AsO(3) efflux system operating in mine site H. ericae as a mechanism for H(2)AsO(4)(-) resistance. The ecological significance of this mechanism of arsenate resistance is discussed.

Piceirhiza bicolorata- the ectomycorrhizal expression of the Hymenoscyphus ericae aggregate?

The mycobionts of Piceirhiza bicolorata, a distinct ectomycorrhizal morphotype of conifers and hardwoods, have been identified by internal transcribed spacer 1 (ITS1) nuclear ribosomal DNA (rDNA) sequence comparison of the fungi involved. Samples of Piceirhiza bicolorata were obtained from seedlings of Picea abies, Pinus sylvestris, Betula pubescens, Populus tremula, Quercus robur and Salix phylicifolia. In an initial screening, the fungus amplified with universal ITS primers from ectomycorrhizal root samples of P. bicolorata shared approx. 95% ITS1 sequence identity with the ericoid mycorrhizal fungus Hymenoscyphus ericae. A total of 77 out of 88 (= 87.5%) DNA samples (i.e. 52/56 root samples and 25/32 axenic culture isolates) of P. bicolorata were successfully amplified with a taxon-selective primer designed for exclusive amplification of H. ericae-like strains. Forty-seven amplicons were sequenced, yielding 15 different ITS1 genotypes that differed by 1-14 nucleotide character state changes. An inferred ITS1 phylogeny (maximum parsimony) showed that a single major evolutionary lineage of P. bicolorata embraced the historically important H. ericae isolates in a 100% bootstrap-supported clade. The 15 P. bicolorata genotypes were positioned in four subclades, roughly corresponding to morphological groups of P. bicolorata isolates observed in axenic culture. Culture isolates of H. ericae and P. bicolorata share some common morphological features including slow, dense growth and formation of short aerial hyphal aggregates. Our results suggest that members of the H. ericae aggregate participate in the formation of the distinct ectomycorrhizal morphotype P. bicolorata. This opposes the widely accepted discrimination of ericoid and ectomycorrhizal mycobionts of the boreal forest ecosystem. The high prevalence of the P. bicolorata morphotype on pioneer seedlings of P. sylvestris, B. pubescens and S. phylicifolia at a copper mine spoil was remarkable. Hypotheses of possible nutrient mobilization and detoxification potentials of the fungal associates of P. bicolorata are discussed. We hypothesize that ericoid and ectomycorrhizal plants may share mycobionts of the H. ericae aggregate.

Carbohydrate oxidases in ericoid and ectomycorrhizal fungi: a possible source of Fenton radicals during the degradation of lignocellulose

Isolates of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf et Kernan, and the ectomycorrhizal fungi Suillus variegatus (Swartz ex Fr.) and Pisolithus tinctorius (Pers.) Coker & Couch, along with a Cortinarius sp. and the white rot Phanerochaete chrysosporium Burdsall were examined for the ability to oxidize carbohydrates to their corresponding lactones and to excrete the H2O2 produced thereby. All except Phanerochaete chrysosporium were found to express cellobiose oxidase (cellobiose dehydrogenase, EC 1.1.19.88) and glucose oxidase (β‐d‐glucose∶oxygen 1‐oxidoreductase, EC 1.1.3.4) when grown on cellobiose and glucose respectively. Production of extracellular H2O2 was visualized during growth on both substrates using ABTS as the chromogen. According to the Fenton reaction, H2O2 will react with hydrated or chelated Fe(II) in the environment to produce hydroxyl (Fenton) radicals, HO·. Mycelial extracts from each of the mycorrhizal fungi produced HO· in the presence of cellobiose and Fe(II), presumably mediated by H2O2 produced by cellobiose oxidase activity in the extracts. Conditions favourable to HO· production were shown to exist in Modified Melin–Norkrans medium, and the data discussed in relation to previously observed lignin degradation by mycorrhizal fungi.

The biology of mycorrhiza in the Ericaceae. XX. Plant and mycorrhizal necromass as nitrogenous substrates for the ericoid mycorrhizal fungus Hymenoscyphus ericae and its host

In the ‘F’ horizons of acid mor‐humus soils of heathland ecosystems, mycorrhizal roots of the dominant ericaceous species form a large fraction of the soil biomass. Rapid turnover of these roots provides the potential for recycling of substantial amounts of nitrogen contained in their fungal and plant components. Here, we first determine the amount of N in the biomass of ericoid roots growing in heathland and show it to constitute a large proportion of total soil N. In order to assess the accessibility of this N to ericaceous plants, experiments were then conducted using aseptically produced shoot and root necromass of Vaccinium macrocarpon Ait., the roots being grown with or without mycorrhizal colonization. These materials were provided as sole nitrogenous substrates in growth experiments using the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan in pure culture and V. macrocarpon in the mycorrhizal (M) or non‐mycorrhizal (NM) condition as test organisms. The experiments were designed to test the hypothesis that the N contained in these substrates can be mobilized by the mycorrhizal endophyte. The ability of the endophyte to utilize the substrates was determined by measuring fungal yields and by assessing the presence of its extra‐cellular protease and chitinase enzymes. Transfer of N to the host by the endophyte was determined through measurements of plant yield and tissue N contents. H. ericae produced a significantly greater yield on shoot and mycorrhizal root necromass than on non‐mycorrhizal root necromass. The extra‐cellular enzymes protease and chitinase were produced by the fungus when grown on the M root necromass. The fungus also transferred N to the host plant, up to 76% of N contained in the substrate being found in M plants whereas less than 5% was present in their NM counterparts.

Phosphodiesterase as mycorrhizal P sources: I. Phosphodiesterase production and the utilization of DNA as a phosphorus source by the ericoid mycorrhizal fungus Hymenoscyphus ericae.

These results provide the first account of the breakdown and utilization of DNA by an ericoid mycorrhizal fungus, and of its phosphodiesterase activity in vitro. Hymenoscyphus ericae (Read) Korf & Kernan grew well on DNA as a sole source of phosphorus (P), achieving greater mycelium dry weight than on an equivalent concentration of P supplied as orthophosphate. Some characteristics of the production and activity of extracellular (culture filtrate) and cell-wall bound phosphodiesterase in H. ericae are reported. At least part of the phosphodiesterase activity is attributed to the exonuclease F nucleotide diesterase on the basis of its high affinity for a specific substrate of this enzyme, The pH optima for extra-cellular and cell-wall-bound diesterase are acidic (pH 4.0-5.5), with considerable activity maintained in the pH range typical of organic soils under ericaceous plants (pH 3.0-4.5). The production of phosphodiesterase was not substrate-induced, since highest specific activity for wall-bound enzyme was found in culture grown without organic or inorganic P. The results are discussed in relation to the ecology and biology of acid organic soils on which ericaceous plants are dominant.

The biology of mycorrhiza in the Ericaceae

summaryIn acid mor‐humus soils of heathland ecosystems fungi are a significant part of the soil biomass. In these organic soils chitin and hyphal wall hexosamines are major potential sources of nitrogen (N). The ability of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan to degrade purified chitin and the hexosamines, N‐acetylglucosamine, glucosamine and galactosamine when supplied as sole sources of N, was investigated in aseptic liquid culture. The fungus grew rapidly on all the organic N sources, producing the largest dry weight increase when supplied with galactosamine and similar yields on the other nitrogenous sources. Mycelial N contents of fungus grown on the hexosamines reflected the dry weight yields with the fungus grown on galactosamine haying the highest N content. After 20 d only tract quantities of N‐acetylglucosamine and glucosamine were present in culture filtrate, and 15% of the galactosamine remained at the final harvest. Plants of Vaccinium macrocarpon Ait. and Calluna vulgaris (L.) Hull were grown aseptically in the mycorrhizal or non‐mycorrhizal condition on agar media containing chitin or a hexosamine and compared with yields and N contents in the absence of an N source. It was revealed that significant quantities of N were transferred to the host in the mycorrhizal condition that led to enhanced growth rates. The highest yields of V. macrocarpon were observed with N‐acetylglucosamine, with the other hexosamines giving yields slightly below that on chitin. The yields were reflected in the calculation of the percentage of available substrate present in the plant; up to 40% of the N‐acetylglucosamine was present in the plants after 40 d.

Analysis of acid invertase and comparison with acid phosphatase in the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf and Kernan

Fractions of acid invertase and acid phosphatase of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan were compared by column chromatography and polyacrylamide gel electrophoresis. Acid invertase levels were measured during the exponential phase after 14 days growth in pure culture. Most acid invertase was wall associated (50%) with 41% forming an extracellular fraction and 9% a soluble, cytoplasmic fraction. The wall-bound fraction was partially solubilized by 1 M NaCl, bulked with the extracellular fraction and separated by gel filtration into two acid invertase activity peaks. These peaks corresponded closely to two acid phosphatase activity peaks measured in the same eluates. Anion exchange chromatography under a continuous salt gradient separated the invertase and phosphatase isoforms from each other. Non-denaturing polyacrylamide gel electrophoresis demonstrated that the more active isoforms of each enzyme have different electrophoretic properties and are high mannose-type glycoproteins with a high affinity for the lectin, concanavalin A. The results are discussed in terms of the functional aspects of the two enzymes and their cytochemical localization.

The mycorrhizal status of Arbutus unedo in relation to compatible and incompatible fungi

Ectendomycorrhizae of Arbutus unedo L. were synthesized in the laboratory and were observed over the first 80 days of development. After 40 days some roots showed many hyphae running around enlarged tips, on the 60th day short roots had assumed a clavate form, and on the 80th day mycorrhizae could be observed with the naked eye. The laboratory-synthesized ectendomycorrhizae had a cruciform structure and were smooth, shiny, and pale orange-brown. They presented a loose sheath and a Hartig net. The first layer of cortical root cells was invaded by the fungus. Running hyphae and vesicles of Glomus microcarpum invaded A. unedo roots, but no root invasion was observed in the presence of five other vesicular–arbuscular mycorrhizal fungal species. The ericoid mycorrhizal fungus Hymenoscyphus ericae caused a darkening of cortical cells and root tips and the death of many seedlings. The correspondence between specific plant species and particular kinds of mycorrhizae and the possibility of intracellular colonization by nonspecific fungi are also discussed.