High genetic diversity in a population of the ectomycorrhizal basidiomycete laccaria amethystina in a 150-year-old beech forest

The genetic structure of a population of the ectomycorrhizal basidiomycete Laccaria amethystina (Bolt. ex Hooker) Murr. was assessed in a closed 150-year-old beech (Fagus sylvatica L.) forest in the Vosges Mountains in northeastern France. During the autumn of 1994 and 1997, sporophores were collected from three 100-m2 sampling plots located along a 120-m transect crossing the beech stand. The genetic variation of 676 sporophores was initially estimated using heteroduplex analysis of the ribosomal DNA intergenic spacer (IGS1). Ten unique IGS1 heteroduplex/homoduplex patterns were identified, although three types represented most of the sporophores analysed. Each group of IGS1 type was then analysed using random amplified microsatellite analysis (RAMS). RAMS resolved 388 different genotypes amongst the 634 sporophores analysed from the three plots during the autumn of 1994 and 1997. Density as high as 130 genets per 100 m2 was observed during the autumn of 1994. The largest clone covered approximately 1 m2, but most genets covered a few cm2 and produced only one to three sporophores. Only eight genotypes identified in 1994 were found in 1997. Although L. amethystina has the capacity for vegetative persistence, the present study indicates that its populations maintain a genetic structure more consistent with a high frequency of sexual reproduction. This suggests that beech trees could be recolonized by new genotypes each year. Alternatively, this spatial distribution may also arise from erratic fruiting of underground persistent genets. These features (i.e. numerous genets of small size), typical of ruderal species, contrast with studies carried out on other ectomycorrhizal basidiomycetes occurring in mature closed forests.     

Development of microsatellite markers from an ectomycorrhizal fungus,Laccaria amethystina,by a dual‐suppression‐PCR technique

Laccaria amethystina is a major early successional ectomycorrhizal fungus. We isolated 10 polymorphic loci from L. amethystina using a dual‐suppression‐PCR (polymerase chain reaction) technique. These loci provided microsatellite markers with polymorphism of two to 10 alleles per locus. The observed and expected heterozygosities ranged from 0.136 to 0.545 and 0.206 to 0.877, respectively.     

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.     

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.     

Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete Laccaria bicolor in a French forest plantation

Selected strains of ectomycorrhizal fungi, such as the basidiomycete Laccaria bicolor , are currently being used as inoculants in nurseries to improve growth of forest trees after outplanting. Information is needed on the survival of these introduced strains in forests and their impact on indigenous biodiversity. Dissemination and persistence of an American strain, L. bicolor S238N, were studied 10 years after outplanting in a Douglas fir plantation located at Saint-Brisson (Morvan, France). About 430 Laccaria spp. sporophores were collected over 3 years. Inheritance of nuclear ribosomal DNA, as well as RAPD markers, was characterized in L. bicolor S238N, using a haploid progeny set of 91 monokaryons. More than 50 markers were identified (19 heterozygous and 33 homozygous or cytoplasmic markers), which unambiguously confirmed that the introduced strain was still present in the inoculated plots. Neither selfing ( P < 0.0008) nor introgression with indigenous strains was detected although in vitro interfertility between the American strain and indigenous L. bicolor was identified. No ingress of the introduced genet into adjacent uninoculated plots colonized by various local Laccaria genets was detected. It is proposed that the spatial distributions identified have developed through mycelial propagation of the introduced strain and intraspecific competition with native genets. Although longer-term data is still lacking, the stability of the inoculant strain and the limited disturbance to indigenous populations described support large-scale nursery production of this host-fungal combination.     

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.     

Dual colonization of Eucalyptus urophylla S.T. Blake by arbuscular and ectomycorrhizal fungi affects levels of insect herbivore attack

Abstract 1 Eucalypts are an important part of plantation forestry in Asia but, in south China, productivity is very low. This is due to infertile soils and lack of indigenous symbiotic mycorrhizal fungi. The genus Eucalyptus is unusual because it forms both arbuscular (AM) and ectomycorrhizal (ECM) associations. 2 Eucalyptus urophylla saplings were grown with and without AM (Glomus caledonium) and ECM (Laccaria laccata) fungi in a factorial design. Two experiments were performed: one to simulate nursery conditions and the other to simulate the early stages of plantation establishment. Plant growth was measured over 18 weeks and levels of insect attack were recorded. 3 The AM fungus reduced tree growth in the early stages, but the effect appeared to be transient. No effects of ECM were detected on tree growth, but the ectomycorrhiza reduced colonization by the arbuscular mycorrhiza. AM fungi appear to be rapid invaders of the root system, gradually being replaced by ECM. 4 Both fungal types affected levels of damage by insect herbivores. Most importantly, herbivory by the pest insects Anomala cupripes (Coleoptera) and Strepsicrates spp. (Lepidoptera) was decreased by ECM. 5 It is suggested that mycorrhizal effects on eucalypt insects may be determined by carbon allocation within the plant. Future studies of eucalypt mycorrhizas need to take into account the effects of the fungi on foliar‐feeding insects and also the effects of insect herbivory on mycorrhizal establishment.     

Survival of an introduced ectomycorrhizal Laccaria bicolor strain in a European forest plantation monitored by mitochondrial ribosomal DNA analysis

Mitochondrial and nuclear genes have different inheritance, thus studies of fungal populations should use both mitochondrial and nuclear markers. Using nuclear markers, the S238N strain of the ectomycorrhizal basidiomycete Laccaria bicolor ((Maire) Orton) has been previously shown to persist for at least 10 yr after outplanting in a plantation of Douglas fir ( Pseudotsuga menziesii (Mir.) Franco) inoculated with this strain. In the present study, we have sampled 539 sporophores of Laccaria spp. from this plantation, some of which had the S238N nuclear genotype, to study mitochondrial DNA polymorphism and persistence of the inoculated S238N mitochondrial genome. Length polymorphism in fragments of the large subunit of mitochondrial ribosomal DNA (LrDNA) allowed distinction of the haplotypes present in the plantation at the species level. In addition, heteroduplex analysis and sequencing revealed intraspecific polymorphism of LrDNA among the L. bicolor sporophores and enabled specific identification of S238N LrDNA. This haplotype was only retained in sporophores carrying the S238N nuclear genome, confirming the survival of this introduced strain in a natural population.     

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.     

Correspondence between genet diversity and spatial distribution of above- and below-ground populations of the ectomycorrhizal fungus Hebeloma cylindrosporum

Population studies of ectomycorrhizal fungal species have largely relied upon fruit body (the reproductive organ) sampling. Analysis of the fruit bodies alone supposes that they reflect the present and spatial organization of all below-ground genets (mycorrhizas and extramatrical mycelia). The relation between fruit bodies and ectomycorrhizas was investigated for the basidiomycete agaric Hebeloma cylindrosporum in four Pinus pinaster stands in south-west France. Genet identification was based on the comparison of polymorphisms within a hypervariable segment of the ribosomal intergenic spacer amplified by polymerase chain reaction (PCR) using a H. cylindrosporum species-specific primer. Mycorrhizas were sorted from soil samples collected underneath patches of fruit bodies or patches where fruit bodies had or had not been observed during the years prior to mycorrhiza collection. On average 65% of the 1026 mycorrhizas collected underneath fruit bodies were formed by H. cylindrosporum, whereas only 2% of the 954 collected in places from where fruit bodies were absent were formed by this species. All genotypes identified above ground were also identified below ground. In patches where one genotype formed all or more than 90% of the fruit bodies, the same genotype formed all or a large majority of the mycorrhizas. In patches occupied by several different fruiting genotypes, additional nonfruiting ones could be present on the root systems. In all cases, the mycorrhizas of one genotype were found no more than 10-20 cm away from its corresponding fruit bodies, and fruit body disappearance at a given place was associated with the disappearance of the corresponding mycorrhizas within 1 year. Although there was not a strict coincidence between the total numbers of genets present below ground and of those forming fruit bodies, fruit body analysis for H. cylindrosporum appears to reflect both the genetic diversity and the spatial structure of its below-ground populations. The results obtained also illustrate the rapid turnover of ectomycorrhizal fungal species on the root systems in the absence of any obvious major disturbance of the ecosystem.     

Solarization in a forest nursery: effect on ectomycorrhizal soil infectivity and soil receptiveness to inoculation with Laccaria bicolor

 Field experiments were carried out in a forest nursery during the summer of 1994 to examine the effect of soil solarization on ectomycorrhizal soil infectivity (ESI) and soil receptiveness to inoculation with Laccaria bicolor. Soil samples from solarized, steamed, fumigated and untreated plots were periodically collected and assayed for ESI. Untreated soil exhibited high ESI. Solarization was as effective as steaming or fumigation in reducing ESI in the uppermost layer. Solarization with a double layer of polyethylene film and fumigation were the only treatments which reduced ESI deeper in the soil. During July, the temperature of covered beds reached 50  °C at a soil depth of 5 cm. Ectomycorrhizal fungi were among the soil-borne fungi most sensitive to solar heating. Soil solarization provides an effective disinfection method for controlled mycorrhization in forest nurseries. Accepted: 10 April 1997     

Fine-scale structure of populations of the ectomycorrhizal fungus Hebeloma cylindrosporum in coastal sand dune forest ecosystems

The basidiomycete mushroom Hebeloma cylindrosporum is a frequently found pioneer ectomycorrhizal species naturally associated with Pinus pinaster trees growing in coastal sand dune ecosystems along the Atlantic south-west coast of France. The genotypic diversity and spatial structure of three populations of this fungal species have been studied. At each site the basidiocarps were mapped, sampled and propagated as pure mycelial cultures. For each of the isolates, we have studied polymorphisms in the mitochondrial genome, polymorphisms at two different nuclear loci and also fingerprints produced with a multicopy DNA probe. The comparison of the different polymorphisms obtained, with each of the four molecular methods used, allowed the identification of several of the different genets present in each site. In two of the studied sites most of the basidiocarps, which often occurred as dense patches of 10–30 in 1 m² or less, were of a unique genotype, suggesting the below-ground mycelia to be of a small size (from 50 cm² to approx. 7 m² for the larger mycelia) and that the root system of a single Pinus tree can host several genets of the same symbiotic fungus. In the two sites, which were studied again after a 3-year interval, none of the genotypes identified in the first year of sampling was re-identified 3 years later. These results contrast with those reported for other species of soilborne homobasidiomycete species, either ectomycorrhizal, parasitic or saprophytic, showing mostly large clones resulting from the vegetative growth and from persistence of below-ground mycelia. Sexual reproduction through meiospore dispersal seems to play a key role in the structuring of the populations of H. cylindrosporum. Mycelia associated with the root systems seem to be replaced after 1 or a few years, during which basidiocarp differentiation takes place. As opposed to the few other studied ectomycorrhizal species, H. cylindrosporum has the characteristics of ruderal species, with a short life-span adapted to pioneer situations, e.g. to nutrient-poor and unstable sandy soils of coastal sand dunes.     

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.     

Effects of repeated harvesting of forest residues on the ectomycorrhizal community in a Swedish spruce forest

The use of biofuels has been proposed as one possible substitute for fossil fuels, which contribute substantially to the increase in [CO₂] in the atmosphere. However, increased harvesting of forest residues for biofuel might affect the availability of base cations, P and N, as well as the development, community dynamics and function of ectomycorrhizas. This in turn might influence nutrient uptake and tree growth. In this study we investigated the effects of repeated forest residue harvesting on ectomycorrhizal species colonizing spruce roots in the humus layer of a 35-yr-old forest. Harvesting significantly decreased the thickness of the humus layer as well as decreasing the numbers of ectomycorrhizal root tips both per metre root length and per unit humus volume. Changes in mycorrhizal community structure were studied by ITS typing with the use of PCR–RFLP analysis. In total, 19 different ITS types were found on two different sampling occasions (autumn and spring); 11 of these were common to both samplings. Nine of the ITS types were identified to at least the genus level by comparison with RFLP patterns of identified fruiting bodies or axenic cultures. Five species, Cortinarius sp. 2, Thelephora terrestris (Ehrenb.) Fr., Lactarius theiogalus (Bull.:Fr.) S. F. Gray s.st. Neuhoff, Tylospora fibrillosa Donk and Tö-96-12, occurred on over 5% of the total sampled root tips. Together these five types colonized 63% of the mycorrhizas screened. A similarity index assessment showed no shift in mycorrhizal community structure as a result of harvesting. Our findings suggest that the repeated removal of forest residues might have a strong effect on the quantity and development of ectomycorrhizal roots in the organic horizon, but little effect on the species composition of the community.     

Spatio‐temporal colonization patterns of forest plant species in a mixed deciduous forest

Abstract. An integrated analysis of the colonization patterns of forest plant species was carried out in a 34‐ha, mixed deciduous forest in northern Belgium. First, we sought to describe the relationships between land use history and environmental conditions. Land use history and soil type were related and negative correlations between pH and secondary forest age were found. The density of the shrub layer increases with secondary forest age. Litter quantity and cover of Urtica dioica were mainly indirectly influenced by land use history. Litter starts accumulating at low pH values and high shrub density and Urtica dioica grows vigorously on nutrient enriched soils where much light can reach the ground. Next, the importance of these human‐altered environmental conditions for the colonization of forest plant species was assessed relative to the importance of dispersal limitation. Therefore, the distribution of 16 forest species was mapped and species‐specific spatio‐temporal isolation measures were calculated. The analysis revealed that the colonization patterns of the slowly colonizing species (i.e. ‘ancient forest plant species’) are best explained by a combination of spatio‐temporal isolation, soil type, pH and the (non‐)cover of Urtica dioica. By contrast, spatio‐temporal isolation was never a limiting factor for good colonizing forest species. Our results suggest that colonization of ‘ancient forest plant species’ is hampered by a combination of dispersal‐ and recruitment limitation and that the relative importance of both factors is species‐specific.