Ectomycorrhizal fungi - Molecular tools to study species and functional diversity

The extramatrical mycelium of ectomycorrhizal (EM) fungi represents a significant component of the ectomycorrhizal (EM) symbiosis, as the mycelium is primarily involved in the uptake of water and nutrients from the soil. The extramatrical mycelium may also actively mobilize nutrients from organic and mineral sources through excretion of enzymes and organic acids respectively. An observation of tunnel-like structures in weatherable mineral grains gave rise to the hypothesis that these were formed by the hyphae of EM fungal species. Tunneled minerals have been found in the weathered E horizon of podzols, a typical soil type for boreal forests. In these forests the EM fungal community is highly species-rich and this thesis describes the vertical distribution of fungal species throughout the podzol profile, with special emphasis on the weathered E horizon. Identification of EM fungal species from podzol horizons through morphological and molecular identification of EM root tips revealed a significant relationship between the EM fungal species composition and soil horizons. The vertical distribution of the EM extramatrical mycelium in podzol horizons was studied with molecular identification techniques based on total soil DNA extracts and a cloning method. The root tip study as well as the mycelial study showed that many EM fungal species exist in the mineral soil that do not occur in the organic layer of a podzol. A high correspondence was furthermore found between the basidiomycete diversity detected by the molecular analysis of root tips and of hyphae. This thesis further describes the use of molecular techniques for the quantification of EM fungal species in soil. By comparing molecular techniques with conventional quantification techniques, it was shown that molecular methods provide tools to determine the biomass of individual fungal species in soil. The molecular methods enabled identification and relative quantification of two EM fungal species separately in a more-species environment and showed consistent results. In conclusion, this thesis demonstrates that molecular techniques facilitate identification and relative quantification of individual soil fungi and in addition highlights the pitfalls of the molecular methodology. As differences in amount and organization of the extramatrical mycelium produced by different EM fungal species may reflect the different ecological roles that the fungi may have, the detection of hyphae initiates possibilities for in situ studies on substrate specificity, niche partitioning and succession of EM fungal species below ground.<strong/

Rock-eating mycorrhizas: their role in plant nutrition and biogeochemical cycles

A decade ago, tunnels inside mineral grains were found that were likely formed by hyphae of ectomycorrhizal (EcM) fungi. This observation implied that EcM fungi can dissolve mineral grains. The observation raised several questions on the ecology of these ¿rock-eating¿ fungi. This review addresses the roles of these rock-eating EcM associations in plant nutrition, biogeochemical cycles and pedogenesis. Research approaches ranged from molecular to ecosystem level scales. Nutrient deficiencies change EcM seedling exudation patterns of organic anions and thus their potential to mobilise base cations from minerals. This response was fungal species-specific. Some EcM fungi accelerated mineral weathering. While mineral weathering could also increase the concentrations of phytotoxic aluminium in the soil solution, some EcM fungi increase Al tolerance through an enhanced exudation of oxalate. Through their contribution to Al transport, EcM hyphae could be agents in pedogenesis, especially podzolisation. A modelling study indicated that mineral tunnelling is less important than surface weathering by EcM fungi. With both processes taken together, the contribution of EcM fungi to weathering may be significant. In the field vertical niche differentiation of EcM fungi was shown for EcM root tips and extraradical mycelium. In the field EcM fungi and tunnel densities were correlated. Our results support a role of rock-eating EcM fungi in plant nutrition and biogeochemical cycles. EcM fungal species-specific differences indicate the need for further research with regard to this variation in functional traits

Diversity of an ectomycorrhizal fungal community studied by a root tip and total soil DNA approach

Molecular methods based on soil DNA extracts are increasingly being used to study the fungal diversity of ectomycorrhizal (EM) fungal communities in soil. Contrary to EM root tip identification, the use of molecular methods enables identification of extramatrical mycelia in soil. To compare fungal diversity as determined by root tip identification and mycelial identification, six soil samples were analysed. Root tips were extracted from the six samples and after amplification, the basidiomycete diversity on the root tips was analysed by denaturing gradient gel electrophoresis (DGGE). The soil from the six samples was sieved, total soil DNA was extracted and after amplification, the basidiomycete diversity in the soil fractions was analysed by DGGE. Fourteen different bands were excised from the DGGE gel and sequenced; fungal taxon names could be assigned to eight bands. Out of a total of 14 fungal taxa detected in soil, 11 fungal taxa were found on root tips, of which seven were EM fungal taxa. To examine whether the sieving treatment would affect EM species diversity, two different sieve mesh sizes were used and in addition, the organic soil fraction was analysed separately. DGGE analysis showed no differences in banding pattern for the different soil fractions. The organic fraction gave the highest DGGE band intensities. This work demonstrates that there is a high correspondence between basidiomycete diversity detected by molecular analysis of root tips and soil samples, irrespective of the soil fraction being analyse

Molecular identification of ectomycorrhizal mycelium in soil horizons

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (greater than or equal to99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had greater than or equal to98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil

SSU Ribosomal DNA-Based Monitoring of Nematode Assemblages Reveals Distinct Seasonal Fluctuations within Evolutionary Heterogeneous Feeding Guilds

Soils are among the most complex, diverse and competitive habitats on Earth and soil biota are responsible for ecosystem services such as nutrient cycling, carbon sequestration and remediation of freshwater. The extreme biodiversity prohibits the making of a full inventory of soil life. Hence, an appropriate indicator group should be selected to determine the biological condition of soil systems. Due to their ubiquity and the diverse responses to abiotic and biotic changes, nematodes are suitable indicators for environmental monitoring. However, the time-consuming microscopic analysis of nematode communities has limited the scale at which this indicator group is used. In an attempt to circumvent this problem, a quantitative PCR-based tool for the detection of a consistent part of the soil nematofauna was developed based on a phylum-wide molecular framework consisting of 2,400 full-length SSU rDNA sequences. Taxon-specific primers were designed and tested for specificity. Furthermore, relationships were determined between the quantitative PCR output and numbers of target nematodes. As a first field test for this DNA sequence signature-based approach, seasonal fluctuations of nematode assemblages under open canopy (one field) and closed canopy (one forest) were monitored. Fifteen taxa from four feeding guilds (covering ~ 65% of the free-living nematode biodiversity at higher taxonomical level) were detected at two trophic levels. These four feeding guilds are composed of taxa that developed independently by parallel evolution and we detected ecologically interpretable patterns for free-living nematodes belonging to the lower trophic level of soil food webs. Our results show temporal fluctuations, which can be even opposite within taxa belonging to the same guild. This research on nematode assemblages revealed ecological information about the soil food web that had been partly overlooked

Ecto- and arbuscular mycorrhizal symbiosis can induce tolerance to toxic pulses of phosphorus in jarrah (Eucalyptus marginata) seedlings

In common with many plants native to low P soils, jarrah (Eucalyptus marginata) develops toxicity symptoms upon exposure to elevated phosphorus (P). Jarrah plants can establish arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) associations, along with a non-colonizing symbiosis described recently. AM colonization is known to influence the pattern of expression of genes required for P uptake of host plants and our aim was to investigate this phenomenon in relation to P sensitivity. Therefore, we examined the effect on hosts of the presence of AM and ECM fungi in combination with toxic pulses of P and assessed possible correlations between the induced tolerance and the shoot P concentration. The P transport dynamics of AM (Rhizophagus irregularis and Scutellospora calospora), ECM (Scleroderma sp.), non-colonizing symbiosis (Austroboletus occidentalis), dual mycorrhizal (R. irregularis and Scleroderma sp.), and non-mycorrhizal (NM) seedlings were monitored following two pulses of P. The ECM and A. occidentalis associations significantly enhanced the shoot P content of jarrah plants growing under P-deficient conditions. In addition, S. calospora, A. occidentalis, and Scleroderma sp. all stimulated plant growth significantly. All inoculated plants had significantly lower phytotoxicity symptoms compared to NM controls 7 days after addition of an elevated P dose (30 mg P kg−1 soil). Following exposure to toxicity-inducing levels of P, the shoot P concentration was significantly lower in R. irregularis-inoculated and dually inoculated plants compared to NM controls. Although all inoculated plants had reduced toxicity symptoms and there was a positive linear relationship between rank and shoot P concentration, the protective effect was not necessarily explained by the type of fungal association or the extent of mycorrhizal colonization