Community structure of arbuscular mycorrhizal fungi in a primary successional volcanic desert on the southeast slope of Mount Fuji

Community structure of arbuscular mycorrhizal fungi (AMF), evaluated as spore samples and mycorrhizal roots of four herbaceous plant species, was investigated at different altitudes in a primary successional volcanic desert on Mount Fuji using molecular methods (fragment and sequence analysis of the large ribosomal subunit RNA gene). In total, 17 different AMF clades were identified, and most were members of the Glomaceae, Acaulosporaceae, and Gigasporaceae. The AMF community structures detected by spore sampling were inconsistent with those from plant roots. Of all AMF clades, six (35.3%) were detected only on the basis of spores, six (35.3%) only in roots, and five corresponded to both spores and roots (29.4%). Although an Acaulospora species was the most dominant among spores (67.1%), it accounted for only 6.8% in root samples. A species analysis of AMF communities at different altitudes demonstrated that AMF species diversity increased as altitude decreased and that the species enrichment at lower altitudes resulted from the addition of new species rather than species replacement. The inconsistencies in the species composition of spore communities with those in roots and the change in species diversity with altitude are discussed.     

Bacterial community on ectomycorrhizal roots of Laccaria laccata in a chestnut plantation

This study investigated the bacterial communities present on the ectomycorrhizal roots of Laccaria laccata using culture- and cloning-based methods. One soil core was collected from five locations where sporocarps of L. laccata occurred in a chestnut plantation. A total of 10 and 50 ectomycorrhizal root tips of L. laccata were collected from each soil core and subjected to bacterial isolation and cloning. In total, 223 isolates were classified into 35 molecular operational taxonomic units (MOTUs). The majority of MOTUs were infrequent, whereas one MOTU each of Rhizobium and Bradyrhizobium were common across the sampling locations. In the cloning-based method, 77 MOTUs were detected. The majority of these MOTUs were infrequent, whereas one MOTU each of Bradyrhizobium and one taxon of Gammaproteobacteria was common across the sampling locations. These results indicate that Bradyrhizobium is a common member of bacteria present on the ectomycorrhizal roots of L. laccata in the chestnut plantation.     

Linear fusigen as the major hydroxamate siderophore of the ectomycorrhizal Basidiomycota Laccaria laccata and Laccaria bicolor

A screening for siderophores produced by the ectomycorrhizal fungi Laccaria laccata and Laccaria bicolor in synthetic low iron medium revealed the release of several different hydroxamate siderophores of which four major siderophores could be identified by high resolution mass spectrometry. While ferricrocin, coprogen and triacetylfusarinine C were assigned as well as other known fungal siderophores, a major peak of the siderophore mixture revealed an average molecular mass of 797 for the iron-loaded compound. High resolution mass spectrometry indicated an absolute mass of m/z = 798.30973 ([M + H]⁺). With a relative error of Δ = 0.56 ppm this corresponds to linear fusigen (C₃₃H₅₂N₆O₁₃Fe; MW = 797.3). The production of large amounts of linear fusigen by these basidiomycetous mycorrhizal fungi may possibly explain the observed suppression of plant pathogenic Fusarium species. For comparative purposes Fusarium roseum was included in this study as a well known producer of cyclic and linear fusigen.     

Isolation and characterization of five microsatellite loci in an ectomycorrhizal fungus Laccaria laccata

Laccaria laccata is an early successional ectomycorrhizal fungus. We isolated five polymorphic microsatellite loci from L. laccata using a dual‐suppression polymerase chain reaction technique. The number of alleles per locus ranged from three to six. The observed and expected heterozygosities ranged from 0.269 to 0.462, and 0.249 to 0.775, respectively. These microsatellite markers would be valuable molecular tools for population genetic studies of L. laccata.     

Ultrastructural and cytochemical aspects of the interaction between the ectomycorrhizal fungus Laccaria laccata and the saprotrophic fungi, Trichoderma harzianum and T. virens

Different interactions between soil fungi competing in the rhizosphere with each other are necessary to understand their influence on plant growth and health. The interactions between the ectomycorrhizal (ECM) fungus Laccaria laccata and soil saprotrophic fungi (T. harzianum, T. virens) were studied by transmission electron microscopy, and by gold cytochemistry to assess the potential role of cell wall lytic enzymes in mycoparasitism. Anti-β-1,3-glucan antibody, WGA/ovomucoid-gold complex and PATAg test were used to localize β-1,3-glucan, chitin and polysaccharides. Cytoplasm disorganisation of the saprotrophic fungi occurred concurrently with dissolution of β-1,3-glucan in walls of hyphae and conidia of the saprotrophic fungi. Then digestion of polysaccharides and chitin of colonised fungal structures occurred. The studies suggest sequential contribution of cell wall lytic enzymes and importance of disturbing the host's cell integrity during mycoparasitism. We conclude that the ECM fungus can parasitise on the saprotrophic fungi not only in dual culture on artificial medium but also in the rhizosphere of Scots pine.     

Extensive gene flow over Europe and possible speciation over Eurasia in the ectomycorrhizal basidiomycete Laccaria amethystina complex

Biogeographical patterns and large-scale genetic structure have been little studied in ectomycorrhizal (EM) fungi, despite the ecological and economic importance of EM symbioses. We coupled population genetics and phylogenetic approaches to understand spatial structure in fungal populations on a continental scale. Using nine microsatellite markers, we characterized gene flow among 16 populations of the widespread EM basidiomycete Laccaria amethystina over Europe (i.e. over 2900 km). We also widened our scope to two additional populations from Japan (10(4) km away) and compared them with European populations through microsatellite markers and multilocus phylogenies, using three nuclear genes (NAR, G6PD and ribosomal DNA) and two mitochondrial ribosomal genes. European L. amethystina populations displayed limited differentiation (average F(ST) = 0.041) and very weak isolation by distance (IBD). This panmictic European pattern may result from effective aerial dispersal of spores, high genetic diversity in populations and mutualistic interactions with multiple hosts that all facilitate migration. The multilocus phylogeny based on nuclear genes confirmed that Japanese and European specimens were closely related but clustered on a geographical basis. By using microsatellite markers, we found that Japanese populations were strongly differentiated from the European populations (F(ST) = 0.416), more than expected by extrapolating the European pattern of IBD. Population structure analyses clearly separated the populations into two clusters, i.e. European and Japanese clusters. We discuss the possibility of IBD in a continuous population (considering some evidence for a ring species over the Northern Hemisphere) vs. an allopatric speciation over Eurasia, making L. amethystina a promising model of intercontinental species for future studies.     

The mid-domain effect in ectomycorrhizal fungi: range overlap along an elevation gradient on Mount Fuji,Japan

Mid-domain effect (MDE) models predict that the random placement of species'' ranges within a bounded geographical area leads to increased range overlap and species richness in the center of the bounded area. These models are frequently applied to study species-richness patterns of macroorganisms, but the MDE in relation to microorganisms is poorly understood. In this study, we examined the characteristics of the MDE in richness patterns of ectomycorrhizal (EM) fungi, an ecologically important group of soil symbionts. We conducted intensive soil sampling to investigate overlap among species ranges and the applicability of the MDE to EM fungi in four temperate forest stands along an elevation gradient on Mount Fuji, Japan. Molecular analyses using direct sequencing revealed 302 EM fungal species. Of 73 EM fungal species found in multiple stands, 72 inhabited a continuous range along the elevation gradient. The maximum overlap in species range and the highest species richness occurred at elevations in the middle of the gradient. The observed richness pattern also fit within the 95% confidence interval of the mid-domain null model, supporting the role of the MDE in EM fungal richness. Deviation in observed richness from the mean of the mid-domain null estimation was negatively correlated with some environmental factors, including precipitation and soil C/N, indicating that unexplained richness patterns could be driven by these environmental factors. Our results clearly support the existence of microbial species'' ranges along environmental gradients and the potential applicability of the MDE to better understand microbial diversity patterns.     

Beech roots are simultaneously colonized by multiple genets of the ectomycorrhizal fungus Laccaria amethystina clustered in two genetic groups

In this study, we characterize and compare the genetic structure of aboveground and belowground populations of the ectomycorrhizal fungus Laccaria amethystina in an unmanaged mixed beech forest. Fruiting bodies and mycorrhizas of L. amethystina were mapped and collected in four plots in the ?wi?tokrzyskie Mountains (Poland). A total of 563 fruiting bodies and 394 mycorrhizas were successfully genotyped using the rDNA IGS1 (intergenic spacer) and seven simple sequence repeat markers. We identified two different genetic clusters of L. amethystina in all of the plots, suggesting that a process of sympatric isolation may be occurring at a local scale. The proportion of individuals belonging to each cluster was similar among plots aboveground while it significantly differed belowground. Predominance of a given cluster could be explained by distinct host preferences or by priority effects and competition among genets. Both aboveground and belowground populations consisted of many intermingling small genets. Consequently, host trees were simultaneously colonized by many L. amethystina genets that may show different ecophysiological abilities. Our data showed that several genets may last for at least 1 year belowground and sustain into the next season. Ectomycorrhizal species reproducing by means of spores can form highly diverse and persistent belowground genets that may provide the host tree with higher resilience in a changing environment and enhance ecosystem performance.     

Pioneer dwarf willow may facilitate tree succession by providing late colonizers with compatible ectomycorrhizal fungi in a primary successional volcanic desert

To advance understanding of the contribution of ectomycorrhizal (ECM) fungi to tree successional processes, natural establishment patterns of secondary colonizing hosts and their ECM fungal communities were investigated with special reference to pioneer hosts. In the volcanic desert on Mount Fuji, Japan, vegetation is sparsely distributed, resembling islands in a sea of scoria. Of 509 vegetation islands in the research area, 161 contained Salix reinii (Salix), the first colonizing ECM host species. The spatial coincidence between secondary colonizing timber species and Salix was analysed, and ECM fungal communities were studied using molecular identification methods. I found 39 and 26 individuals of Betula ermanii and Larix kaempferi, respectively. Without exception, these individuals were all accompanied by Salix. The ECM fungal communities of these timber species showed high similarity to that of Salix and were dominated by generalists that were compatible with two or more plant families. In this desert, available ECM propagules are limited. Pioneer Salix may contribute to tree succession by providing adjacent late colonizers with compatible ECM fungal symbionts.     

Mycorrhizal Cirsium purpuratum improves the growth of Clematis stans in volcanic scoria of Mount Fuji

We performed a planting experiment to investigate the effects of mycorrhizal Cirsium purpuratum on the growth of Clematis stans using sterilized scoria collected from Mount Fuji, Japan. Mycorrhizal or non-mycorrhizal C. purpuratum seedlings of different ages were planted on one side of a rhizobox, and C. stans were sown at 5, 10, and 15 cm from the C. purpuratum. After the growth period, C. stans seedlings closer to C. purpuratum exhibited higher dry weights. Our results suggest that earlier colonizers may promote vegetation succession by providing a common mycelial network of mycorrhizal fungi that facilitates the establishment of later colonizers.     

Survival of inoculated Laccaria bicolor in competition with native ectomycorrhizal fungi and effects on the growth of outplanted Douglasfir seedlings

The survival, development and mycorrhizal efficiency of a selected strain of Laccaria bicolor along with naturally occurring ectomycorrhizal fungi in a young plantation of Douglas fir was examined. Symbionts were identified and their respective colonization abilities were determined. Eight species of symbiotic fungi, which may have originated in adjacent coniferous forests, were observed on the root systems. Mycorrhizal diversity differed between inoculated (5 taxa) and control (8 taxa) seedlings. Ectomycorrhizal fungi which occurred naturally in the nursery on control seedlings (Thelephora terrestris and Suillus sp.) did not survive after outplanting. Both inoculated and naturally occurring Laccaria species, as well as Cenococcum geophilum, survived on the old roots and colonized the newly formed roots, limiting the colonization by other naturally occurring fungi. Other fungi, such as Paxillus involutus, Scleroderma citrinum and Hebeloma sp. preferentially colonized the old roots near the seedling's collar. Russulaceae were found mainly in the middle section of the root system. Mycorrhizal colonization by Laccaria species on inoculated seedlings (54%) was significantly greater than on controls (13%) which were consequently dominated by the native fungi. Significant differences (up to 239%) were found in the growth of inoculated seedlings, especially in root and shoot weight, which developed mainly during the second year after outplanting. Seedling growth varied with the species of mycorrhizae and with the degree of root colonization. Competitiveness and effectiveness of the introduced strain on improving growth performances of seedlings are discussed.     

Effect of dual inoculation of Douglas fir with the ectomycorrhizal fungus Laccaria laccata and mycorrhization helper bacteria (MHB) in two bare-root forest nurseries

Douglas fir (Pseudotsuga menziesii) seedlings in two bare-root forest nurseries were inoculated with the ectomycorrhizal fungus Laccaria laccata, together or not with one of five mycorrhization helper bacteria isolated from L. laccata sporocarps or mycorrhizas and previously selected by in vitro and glasshouse screenings. With the most efficient MHB isolates, when compared to the control with no bacteria, the percent of mycorrhizal short roots was increased from 60 to 90 or from 80 to 100, depending on the nursery, with inoculation doses as low as 10⁶ living cells per m². A dual inoculum made of calcium alginate beads containing the two microorganisms appears to be a valuable technique for increasing the efficiency of ectomycorrhizal inoculation of planting stocks in forest nurseries.     

Primary structure and specificity of a new member of galectin family from the Amethyst deceiver mushroom Laccaria amethystina

A new galectin was characterized in the Amethyst deceiver mushroom Laccaria amethystina. The complete amino acid (AA) sequence of the lectin, which exhibited β-galactoside specificity, was deduced from its peptide sequences. The AA sequence of L. amethystina galectin (LAG) showed high homology with those of the same genus, at 75.6% identity to Laccaria bicolor, and 35.5-65.0% to galectins of Agrocybe spp. and Coprinopsis cinerea. The AA sequence of LAG contained all but one conserved residue known to be involved in β-galactoside binding, with His at the position 57 residue replaced by Thr in LAG. Analysis of binding specificity by hemagglutination inhibition assay and enzyme-linked lectin-sorbent assay revealed high specificity of LAG towards O-glycoproteins.     

Arbuscular mycorrhizal colonization of the dominant plant species in primary successional volcanic deserts on the Southeast slope of Mount Fuji

Arbuscular mycorrhizal (AM) colonization was observed on four plant species in primary successional volcanic deserts on the Southeast slope of Mount Fuji. The AM colonization of the dominant species, Polygonum cuspidatum, contradicts the conclusion that Polygonaceae are often regarded as being non-mycorrhizal species. The secondary dominant species, Polygonum weyrichii var. alpinum, formed no mycorrhizas. The roots of Cirsium purpuratum, Clematis stans and Campanula punctata ssp. hondoensis, showed a higher percentage of AM colonization than P. cuspidatum. AM colonization and spore density in the rhizosphere soil of P. cuspidatum significantly decreased as elevation increased. AM colonization in roots of Cirsium purpuratum and Clematis stans also tended to decrease with increased altitudes. Cirsium purpuratum and Campanula punctata ssp. hondoensis formed single structural types of Arum- and Paris-type, respectively, whereas P. cuspidatum and Clematis stans formed both Arum- and Paris-type morphologies.     

Differences in nitrogen redistribution between early and late plant colonizers through ectomycorrhizal fungi on the volcano Mount Koma

Relationships involving the transfer of nitrogen (N) among Salix reinii (willow), Larix kaempferi (larch), and mycorrhizal fungi were investigated in a ridge and hillslope on the volcano Mount Koma in northern Japan using a two-pool fungal model. This model estimated N transfer among the examined taxa by measuring changes in the stable isotope ratio of N (δ¹⁵N). Although N content in tephra was low at both sites, it was higher on the ridge than on the hillslope, and higher in the willow patch than on bare ground or in the larch understory. The non-mycorrhizal sedge (Carex oxyandra) exhibited non-significant differences between the two sites regarding δ¹⁵N for N obtained from tephra. Larches developed a relationship with larch-specific Suillus mycorrhizal fungal species in the roots, and had a lower foliar δ¹⁵N on the hillslope than on the ridge. The larch δ¹⁵N increased during the growing season, while the willow δ¹⁵N remained stable. The dependence of larch on mycorrhizal fungi for N uptake was 3–5 % on the ridge and 56–76 % on the hillslope in autumn. Therefore, larches exhibited a flexible symbiotic relationship with mycorrhizal fungi for obtaining N. Over 45 % of the N taken up by willow plants was obtained from mycorrhizal fungi at both sites. In conclusion, willow plants promoted N deposition in tephra through the litter supply, and formed a stable relationship with mycorrhizal fungi. This enabled successful revegetation with larch plants, which exhibited flexibility in terms of N uptake (i.e., dependent on mycorrhizae or from tephra).     

Genetic structure of Cenococcum geophilum populations in primary successional volcanic deserts on Mount Fuji as revealed by microsatellite markers

Polymorphic simple sequence repeat (SSR) markers were used to investigate the genetic structure in a Cenococcum geophilum population associated with Salix reinii in an early successional volcanic desert at Gotenba, on the south-eastern slope of Mount Fuji in Japan, and in three other populations associated with the same host at more developed sites on the mountain, one at Fujinomiya and two at Subashiri. The genotype richness of C. geophilum tended to be higher in more developed vegetation patches as well as in more developed sites, suggesting that genotype richness increased with advanced succession because new genotypes might have been introduced into these sites over time. High genotypic similarity was observed between the Gotenba and Fujinomiya populations but not between the Gotenba and Subashiri populations, suggesting that C. geophilum genotypes in Gotenba were introduced from the direction of Fujinomiya. Genotypes in the Gotenba population were clearly distinguishable into two groups. The absence of any intermediate genotype suggests the absence of frequent recombination in this C. geophilum population associated with early successional vegetation.     

Interaction between Laccaria laccata and Trichoderma virens in co-culture and in the rhizosphere of Pinus sylvestris grown in vitro

Interactions between the ectomycorrhizal fungus Laccaria laccata and the soil fungus Trichoderma virens in co-culture and in the rhizosphere of Pinus sylvestris seedlings growing in vitro were investigated by light and scanning electron microscopy. The growth of T. virens was inhibited in co-culture. Shortened, more branched and sometimes deformed or injured hyphae of T. virens were observed in the zone of inhibition. Two-month-old mycorrhizae of P. sylvestris/ L. laccata were inoculated with a conidial suspension of T. virens and examined at intervals of 7-24 h and 2, 3, and 6 days post-inoculation (p.i.). On non-mycorrhizal roots, conidia germination was high and long hyphae formed 3 days p.i. On mycorrhizal roots, short germ tubes were observed only sporadically. At 3 days p.i., the mantle hyphae of L. laccata grew towards the conidia and coiled around them. Extremely dense coils of hyphae were found around clusters of conidia. Deformation of conidia, breaks in conidial walls and their partial degradation were observed 6 days p.i.     

Effects of arbuscular mycorrhizal fungi and soil developmental stages on herbaceous plants growing in the early stage of primary succession on Mount Fuji

A pot culture experiment was conducted to examine the effects of arbuscular mycorrhizal (AM) fungi and soil developmental stages on the growth and nutrient absorption of pioneer plants growing in the early stage of primary succession on Mt. Fuji. Four herbaceous plants, Polygonum cuspidatum (Polygonaceae), Miscanthus oligostachyus (Gramineae), Aster ageratoides var. ovatus (Compositae), and Hedysarum vicioides (Leguminosae), were grown from seed in soils collected from two different successional stages, bare ground and an herbaceous plant community. Spores of indigenous AM fungi collected from the herbaceous plant community were used as inoculum. The initial colonizer P. cuspidatum showed very low levels of AM colonization (<0.4%), whereas the average AM colonization levels of M. oligostachyus, A. ageratoides var. ovatus, and H. vicioides were within the range of 13–49%. AM fungi had positive effects on the growth and N acquisition of the leguminous species (H. vicioides) irrespective of soil developmental stages. In contrast, AM colonization did not increase the plant dry weight and N content of the non-leguminous species (P. cuspidatum, M. oligostachyus, and A. ageratoides var. ovatus) in both soil developmental stages. A positive effect of AM colonization on P content was observed in M. oligostachyus, A. ageratoides var. ovatus, and H. vicioides only in soil collected from the herbaceous plant community. P. cuspidatum showed no or a negative response to AM colonization in all cases. These results suggest that the effect of AM fungi on plant growth depends more on the plant species than soil developmental stages in the early stage of primary succession in this volcanic area.