In Vivo Natural-Abundance C Nuclear Magnetic Resonance Studies of Living Ectomycorrhizal Fungi : Observation of Fatty Acids in Cenococcum graniforme and Hebeloma crustuliniforme

Natural-abundance ¹³C nuclear magnetic resonance spectroscopy has been used to study intact mycelia of the ectomycorrhizal fungi Cenococcum graniforme (Ascomycetes) and Hebeloma crustuliniforme (Basidiomycetes). A number of sharp resonances are observed in living fungi. These signals primarily arise from fatty acyl chains and carbohydrate nuclei. The spectra are interpreted in terms of relative concentrations of the major fatty acids present in the fungal triglycerides. The small line width of fatty acids (mainly oleic, linoleic, and palmitic acids) resonances and spin-lattice relaxation time are indicative of fast rotational reorientations and are consequently thought to arise from fatty acyl chains in fat droplets. We were able to locate the site of lipids accumulation within mycelia using light microscopy and histological staining. Many lipid droplets were observed in mycelia of both species.

These results suggest that fatty acids droplets could be involved in carbon storage and metabolism from ectomycorrhizal fungi.

     

Influence of ectomycorrhizal inoculation on Pinus wallichiana and Cedrus deodara seedlings under nursery conditions

A study was undertaken to examine the extent of root colonization by four locally isolated ectomycorrhizal （ECM） fungi （Hebeloma theobrominum, Boletus dryophilus, Scleroderma citrinum and Suillus luteus） and their effects on seedling growth in Pinus wallichiana and Cedrus deodara under nursery conditions. Seedlings of the two conifers were inoculated with mycelium of ECM fungi and were grown in pots containing sterilized forest soil for six months. The percentage of ECM colonization of roots was 38%-52% in Pinus wallichiana and 33%-48~ in Cedrus deodara. ECM colonization increased shoot height, needle number, shoot and root biomass and survival of inoculated seedlings. Among the four ECM fungi Hebeloma theobrominum was more effective with Pinus wallichiana and Scleroderma citrinum with Cedrus deodara in promoting seedling survival and overall growth. All the four ECM fungi used enhanced growth of inoculated seedlings and thus can be used in afforestation and regeneration programmes in degraded forests ecosystems.     

Effects of pH,water stress and temperature on in vitro cultures of ectomycorrhizal fungi from Mediterranean forests

A comparative study of the growth of eight species of ectomycorrhizal fungi [Hebeloma edurum Métrod ex Bon, Lactarius deliciosus (L.: Fr.) S.F. Gray., Rhizopogon roseolus (Corda) Th.M.Fr., Suillus collinitus (Fr.) Kuntze, Suillus granulatus (L.: Fr.) Roussel, Suillus luteus (L.: Fr.) Roussel, Tricholoma focale (Fr.) Ricken, Tricholoma striatum (Schaeff.) Sacc.] in pure culture at different pH values, water potentials and temperatures was carried out. The biomass production and mycelial growth of each strain were assessed and characterised morphologically. H. edurum and L. deliciosus showed a clear affinity for neutral or slightly basic media. Most species had the lowest colony diameters and biomass production at pH 2.5. Only H. edurum, R. roseolus and S. collinitus grew at the highest water stress levels (−13.85 and −15.45 bar). All species grew at the lowest values of temperature whereas only R. roseolus and S. collinitus did at 30 °C.     

The enigmatic Squamanita odorata (Agaricales, Basidiomycota) is parasitic on Hebeloma mesophaeum

Squamanita odorata is an agaric which is parasitic on unrecognizable, previously unidentifiable sporophores of a fungal host that is transformed into galls at the bases of the parasite's sporophore. Amplification and sequencing portions of the nuclear (ITS) and mt rDNA from three samplings originating from two sites (from France and from Switzerland) demonstrate that the galls produce sequences that are identical to that of co-occurring Hebeloma mesophaeum. This demonstrates that S. odorata is a biotrophic parasite on Hebeloma mesophaeum.     

What happens to the mycorrhizal communities of native and exotic seedlings when Pseudotsuga menziesii invades Nothofagaceae forests in Patagonia,Argentina?

Pseudotsuga menziesii is one of the most widely planted conifers in the Patagonian Andes of Argentina, having invading characteristics that are broadly reported. We studied the mycorrhizal status of seedlings along six Nothofagaceae + P. menziesii invasion matrices to investigate their role in the invasive process, according to these hypothesis: a) The abundance and richness of EM will be higher in seedlings grown in their own soil; b) In the presence of native EM inoculum, the invasive plant will be associated with generalist mycorrhizae (EM and/or AM), c) AM associations will be more abundant in P. menziesii seedlings grown in Interface or native forest soils, d) Mycorrhizal community differences between treatments will alter host fitness (growth and nutritional parameters). Seedlings from Nothofagus dombeyi, N. antarctica, Lophozonia alpina, L. obliqua and Pseudotsuga menziesii were set up in a soil-bioassay that included soils from non-invaded Nothofagaceae forests, pure P. menziesii plantations, and the interface between both. Pseudotsuga menziesii seedlings showed a decreasing, although never null, ectomycorrhizal (EM) colonization pattern from plantations to non-invaded forests, mainly with exotic EM species. Hebeloma mesophaeum and Wilcoxina sp. 1, two EM species with cosmopolitan distribution, were found to be shared by both tree species. Hebeloma hiemale and Wilcoxina sp. 1, common mycorrhizal partners of P. menziesii in Patagonia although not registered from Nothofagaceae forest, were found to be associated with N. antarctica, being the first report for both fungal species. Pseudotsuga menziesii seedlings showed the ability to form different arbuscular mycorrhiza (AM) colonization types (Paris-, Arum-, Both- and Intermediate-types) depending on the treatments, with significantly higher presence of Intermediate-type in the Interface treatment, where colonization was low. The shared EM species and the presence of different AM colonization types imply enhanced possibilities for invasive P. menziesii seedlings establishment and development. Seedling features and EM colonization rates evidenced that P. menziesii invasion could produce maladaptation (defined as a relative decline in host fitness due to altered mycorrhizal communities from native settings) of mycorrhizal communities, seriously injuring native ecosystem.     

Responses of ectomycorrhizal Populus tremuloides and Betula papyrifera seedlings to salinity

Roots of trembling aspen (Populus tremuloides Michx.) and paper birch (Betula papyrifera Marsh.) seedlings were inoculated with Hebeloma crustuliniforme or Laccaria bicolor and treated with 25 mM NaCl for 6 weeks. Both tree species appeared to be relatively tolerant of the applied NaCl treatment and did not develop visible leaf symptoms that are characteristic of salt injury. Salt treatment reduced total dry weights in aspen and birch, but did not significantly affect transpiration rates and root hydraulic conductance. Salt-treated ectomycorrhizal aspen maintained higher root hydraulic conductance compared with non-mycorrhizal plants. Na and Cl concentrations increased in shoots and roots of mycorrhizal and non-mycorrhizal aspen and birch in response to NaCl treatment. Roots of NaCl-treated aspen inoculated with H. crustuliniforme had over twofold higher concentrations of Na compared with non-mycorrhizal NaCl-treated plants. Similarly to aspen, Na and Cl concentrations increased in roots and shoots of NaCl-treated birch seedlings. However, in birch, there were no significant differences in Na and Cl concentrations between mycorrhizal and non-mycorrhizal plants. The results suggest that salt exclusion by the ectomycorrhizal associations is host-specific or/and that the processes leading to salt exclusion are activated in ectomycorrhizal plants by a threshold salt level which may vary between plant species.     

Microsatellite markers for Hebeloma species developed from expressed sequence tags in the ectomycorrhizal fungus Hebeloma cylindrosporum

The increasing number of expressed sequence tag (EST) projects dedicated to ectomycorrhizal fungi is translating into the release of large sets of ESTs. The aim of this study was to develop and test simple sequence repeat (SSR) markers from EST databases of the model ectomycorrhizal fungus Hebeloma cylindrosporum. Six SSR markers were found to be both unambiguously scorable and polymorphic among 12 H. cylindrosporum isolates. Two SSR markers were transferable to other Hebeloma species and one marker was interestingly found to be polymorphic among seven H. crustuliniforme isolates.     

Ammonia fungi of iriomote Island in the southern Ryukyus,Japan and a new ammonia fungus,Hebeloma luchuense

Species composition and fruiting season of ammonia fungi were investigated in Iriomote Island of the southern Ryukyus, Okinawa Prefecture, Japan.Castanopsis andPinus forests were surveyed and 10 species of ammonia fungi were collected, including one new agaric species,Hebeloma luchuense sp. nov. This new fungus is characterized by having a rooting, squamulose-scaly stipe and cortinate veil and forms ectomycorrhizae withCastanopsis cuspidata var.sieboldii. Although the general mushroom season in theCastanopsis forest in Iriomote island was very short and restricted to summer, ammonia fungi were observed to fruit throughout the year in urea-treated plots.     

Laboratory experiments imply the conditionality of mycorrhizal benefits for Salix repens: role of pH and nitrogen to phosphorus ratios

Responses of one arbuscular mycorrhizal fungus (Glomus mosseae) and two ectomycorrhizal fungi (Hebeloma leucosarx, Paxillus involutus) to a range of substrate conditions were investigated in the laboratory. Non-mycorrhizal controls were also included. Substrate conditions included three levels of nitrogen – phosphorus ratios, ranging from N limitation to P limitation (N/P ratio 5.4, 16.2, 48.6), and three pH's, ranging from acidic to alkaline (pH 4, 5.5, 7), in a full factorial experiment. Plant parameters (carbon gain, N and P-content, root length) were significantly affected by fungus, soil pH and soil N/P, and their interactions. Mycorrhizal benefits by ectomycorrhizal fungi (EcMF) were generally larger than by the arbuscular mycorrhizal fungi (AMF), when assessed by above-ground parameters. Glomus mosseae, despite low colonization, had a much larger positive effect on root length than those EcMF. Hebeloma leucosarx and P. involutus were equally effective, despite differences in proportional colonization. Hebeloma leucosarx was able to expand niche width of S. repens towards alkaline conditions. Results are discussed in the framework of a dune successional gradient from young, calcareous, humus-poor towards old, acidic, humus-rich soils.     

A revision of Hebeloma sect. Indusiata in the Nordic countries

A revision of Hebeloma sect. Indusiata in the Nordic countries has been undertaken. The type species of the genus, Agaricus fastibilis , is interpreted as being a non-veiled species, and therefore sect. Indusiata is the correct name for the section comprising the veiled, non-rooting species of Hebeloma. Subsect. Amygdalina subsect. nov. is proposed for species in sect. Indusiata possessing amygdaloid, dextrinoid spores. The following new taxa and combinations are proposed: Hebeloma mesophaeum var. crassipes var. non., H. mesophaeum var. lacteum nom. et stat. nov., H. polare sp. nov., H. sordescens sp. nov., H. atrobrunneum sp. nov., H. monticola sp. nov. and Agrocybe elatella comb. nov.     

New causal animal for the growth of <Emphasis Type="Italic">Hebeloma radicosum</Emphasis> (Agaricales): shrew, <Emphasis Type="Italic">Sorex</Emphasis> sp. (Mammalia,Insectivora)

Hebeloma radicosum fructification occurred in 2003 in Hokkaido where Talpidae moles, known causal animals for H. radicosum growth, are not distributed. An excavation carried out in 2005 revealed a mass of deserted latrines and a collapsed nest in the soil. The latrines, being full of deteriorating ectomycorrhizas, were considered to have borne that fungal growth. The responsible animal was identified as a shrew (Sorex) species by the characteristics of hairs remaining in the soil. Thus, in addition to the moles (Insectivora, Talpidae) and the wood mouse (Rodentia, Muridae; in Europe), a shrew species (Insectivora, Soricidae) is found to cause H. radicosum growth.     

Growth of mycorrhizal jack pine (Pinus banksiana) and white spruce (Picea glauca) seedlings planted in oil sands reclaimed areas

The effectiveness of ectomycorrhizal inoculation at the tree nursery seedling production stage on growth and survival was examined in jack pine (Pinus banksiana) and white spruce (Picea glauca) planted in oil sands reclamation sites. The seedlings were inoculated with Hebeloma crustuliniforme strain # UAMH 5247, Suillus tomentosus strain # UAMH 6252, and Laccaria bicolor strain # UAMH 8232, as individual pure cultures and in combinations. These treatments were demonstrated to improve salinity resistance and water uptake in conifer seedlings. The field responses of seedlings to ectomycorrhizal inoculation varied between plant species, inoculation treatments, and measured parameters. Seedling inoculation resulted in higher ectomycorrhizal colonization rates compared with non-inoculated control, which had also a relatively small proportion of roots colonized by the nursery contaminant fungi identified as Amphinema byssoides and Thelephora americana. Seedling inoculation had overall a greater effect on relative height growth rates, dry biomass, and stem volumes in jack pine compared with white spruce. However, when examined after two growing seasons, inoculated white spruce seedlings showed up to 75 % higher survival rates than non-inoculated controls. The persistence of inoculated fungi in roots of planted seedlings was examined at the end of the second growing season. Although the inoculation with H. crustuliniforme triggered growth responses, the fungus was not found in the roots of seedlings at the end of the second growing season suggesting a possibility that the observed growth-promoting effect of H. crustuliniforme may be transient. The results suggest that the inoculation of conifer seedlings with ectomycorrhizal fungi could potentially be carried out on a large scale in tree nurseries to benefit postplanting performance in oil sands reclamation sites. However, these practices should take into consideration the differences in responses between the different plant species and fungal strains.     

Dynamics of fruit-body production and mycorrhiza formation of ectomycorrhizal ammonia fungi in warm temperate forests in Japan

To investigate the quantitative correlation between dynamics of ectomycorrhiza (ECM) formation and fruit-body production of the ammonia fungi, we treated forest soils in two ECM forests, a Castanopsis cuspidata forest and a Quercus serrata forest, in warm temperate Japan with urea to induce fruiting of ammonia fungi. We identified the ectomycorrhizae of two known species of ammonia fungi, Alnicola lactariolens and Hebeloma vinosophyllum, using morphological typing and PCR-RFLP. ECM initiation, increase, and subsequent decrease preceded the start, increase, and decrease of fruit-body production for each species. We also found many kinds of ECM fungi that did not develop fruit bodies after urea treatment during the observation period, which suggests that it is necessary to expand the definition of ammonia fungi from one that refers solely to the species reproducing on the ground to one which includes those in somatic forms in the soil.     

Living on the edge: Ecology of an incipient <Emphasis Type="Italic">Betula-</Emphasis>fungal community growing on brick walls

Walls provide extreme habitats where plants are subjected to various environmental hazards, including water deficit, shortage or excess of irradiance, and nutrient unbalance, and soil communities are in initial stages of development. Here we analyzed the soil substrate, mycorrhizal status, leaf structure and chemistry, CO₂ exchange and water relations of birch trees growing on brick walls of a 19th century building. The substrate was characterized by high pH, Ca, S, Zn, Cl, and Na concentrations and low concentrations of P, K, Mg, and Fe. From 45 to 70% of live fine roots were colonized by ectomycorrhizal (ECM) species: Hebeloma mesophaeum, Hebeloma helodes, Lactarius pubescens, Tomentella lilacinogrisea. Birch on the wall had typical leaf N concentrations, but stomatal conductance was extremely low, and as a consequence, so was net CO₂ assimilation rate. This research showed how a rudimentary soil, plant, and microbial community could develop even in a vertical and chemically foreign environment. Birch plants were able to maintain balanced foliar stoichiometry in a highly unbalanced substrate and acquire enough nutrients to drive sufficient C gain to provide energy for themselves and fungal symbionts.     

Phosphorus acquisition from phytate depends on efficient bacterial grazing, irrespective of the mycorrhizal status of Pinus pinaster

Background and aims

Phosphorus from phytate, although constituting the main proportion of organic soil P, is unavailable to plants. Despite the well-known effects of rhizosphere trophic relationships on N mineralization, no work has been done yet on P mineralization. We hypothesized that the interactions between phytate-mineralizing bacteria, mycorrhizal fungi and bacterial grazer nematodes are able to improve plant P use from phytate.

Methods

We tested this hypothesis by growing Pinus pinaster seedlings in agar containing phytate as P source. The plants, whether or not ectomycorrhizal with the basidiomycete Hebeloma cylindrosporum, were grown alone or with a phytase-producing bacteria Bacillus subtilis and two bacterial-feeder nematodes, Rhabditis sp. and Acrobeloides sp. The bacteria and the nematodes were isolated from ectomycorrhizal roots and soil from P. pinaster plantations.

Results

Only the grazing of bacteria by nematodes enhanced plant P accumulation. Although plants increased the density of phytase-producing bacteria, these bacteria alone did not improve plant P nutrition. The seedlings, whether ectomycorrhizal or not, displayed a low capacity to use P from phytate.

Conclusions

In this experiment, the bacteria locked up the phosphorus, which was delivered to plant only by bacterial grazers like nematodes. Our results open an alternative route for better utilization of poorly available organic P by plants.     

Mycorrhizal effects on potassium fluxes by northwest coniferous seedlings

In ectomycorrhizae, the relative abilities of mycobiont and host plant to take up and store inorganic nutrients are not easily determined due to the intimate physical relationship of the two components forming the association. Since compartmental analysis of solute elution can estimate cellular compartment pool sizes and unidirectional fluxes across membranes, we have used this method to study ectomycorrhizal coniferous roots. Rubidium-86, used as a tracer for potassium, was loaded into and eluted from intact roots of nonmycorrhizal and mycorrhizal (with the fungus Hebeloma crustuliniformme [Bull.: St. Amans Quél] Douglas fir (Pseudotsuga menziesii [Mirb.] Franco), western hemlock (Tsuga heterophylla [Raf.] Sarg.) and Sitka spruce (Picea sitchensis [Bong.] Carr.) seedlings.

Mycorrhizas significantly increased ⁸⁶Rb uptake rates while decreasing the amount of ⁸⁶Rb released to the external solution. Using compartmental analysis, the flux data suggest that the primary mycorrhizal effects were to increase inward potassium fluxes across the fungal tonoplast and to decrease potassium efflux across the fungal tonoplast, as compared with nonmycorrhizal seedling roots. The result was greater potassium storage, presumably in the fungal vacuole. The three coniferous species responded differently to fungal infection with respect to potassium fluxes. Both cytoplasmic and vacuolar fluxes for mycorrhizal hemlock were 2-fold greater than for spruce and 3-fold greater than for Douglas fir. These results demonstrate the usefulness of compartmental analysis for study of ion fluxes in intact mycorrhizal root systems and suggest that the fungal tonoplast may be the site for regulation of potassium fluxes in these coniferous roots.

     

Genetic Diversity of Naturally Established Ectomycorrhizal Fungi on Norway Spruce Seedlings under Nursery Conditions

We have assessed ectomycorrhizal fungi colonizing Norway spruce (Picea abies L.) seedlings in nine forest nurseries using restriction fragment length polymorphism (RFLP) and sequencing analyses of the internal transcribed spacers (ITS1-5.8S-ITS2) amplicons. Restriction analysis of the amplified DNA fragments with HinfI, MboI, and TaqI enzymes allowed the definition of 17 RFLP genotypes; five of them could be unambiguously assigned to Thelephora terrestris, Hebeloma longicaudum, H. crustuliniforme, Tricharina ochroleuca, and Cenococcum geophilum species by comparison with the sporocarp RFLP-pattern database. The remaining genotypes have been sequenced and compared with sequences deposited in the GenBank database. The phylogenetic analysis of resulting sequences and their identified matches indicated that isolated genotypes have formed seven clades. The ascomycetes were predominant: we have determined eight species—Wilcoxina mikolae, Phialophora finlandia, Tuber sp., Cenococcum geophilum, Tricharina ochroleuca, Pulvinula constellatio, and two unidentified ascomycetes—whereas the basidiomycetes were less common (four species denoted: Amphinema byssoides, Hebeloma crustuliniforme, H. longicaudum, and Thelephora terrestris). Wilcoxina mikolae and Phialophora finlandia were the most frequent fungi. Analysis of variance revealed that ascomycetes abundance was higher in nurseries that used organic fertilizer.     

Nitrate reductase activity of nonmycorrhizal Douglas-fir rootlets and of some associated mycorrhizal fungi

Douglas-fir root tips reduced nitrate at much higher rates than seven mycorrhizal fungi, which also differed between species in rate of nitrate reduction. Results are related to nitrogen nutrition of Douglas-fir.Excised, nonmycorrhizal root tips of Douglas-fir reduced nitrate at much higher rates than seven mycorrhizal fungi commonly associated with Douglas-fir. The fungi differed significantly in degree of activity:Cenococcum geophilum, Piloderma bicolor, andRhizopogon vinicolor were highest;Amanita muscaria was intermediate; andHebeloma crustuliniforme, Thelephora terrestris, andLaccaria laccata were lowest.     

Ectomycorrhizal fungi of Pinus pinaster

A study was undertaken to determine the ability to form ectomycorrhizae with Pinus pinaster Ait. in pure culture syntheses of 98 isolates of putative mycorrhizal fungi, mainly collected in northern Spain. A total of 35 species in 16 genera — Amanita, Cenococcum, Collybia, Cortinarius, Hebeloma, Laccaria, Lactarius, Lyophyllum, Melanogaster, Paxillus, Pisolithus, Rhizopogon, Scleroderma, Suillus, Thelephora and Xerocomus — formed ectomycorrhizae. Many of these fungal species were not previously reported as symbiotic with Pinus pinaster. Results obtained increase the range of potential fungal candidates for inoculation of nursery seedlings.