Investigations of ectomycorrhizal fungal communities in terrestrial and canopy environments

Abstract

Methodologies to detect ectomycorrhizal fungi from below-ground environments, including canopy soil environments, and canopy root formation were investigated in native and non-native vegetation of New Zealand. The methodologies tested include both molecular (DNA sequence barcoding and fingerprinting analyses) and non-molecular (hyphal ingrowth bags, ergosterol assays and artificial environment manipulations) approaches. Soil diversity of Cortinarius (an ectomycorrhizal fungal genus) communities associated with native (Nothofagus spp. and Kunzea ericoides) and exotic (Pinus radiata) ectomycorrhizal hosts were compared using genus-specific sequence barcoding. Analysis of the soil-clones detected from the three ectomycorrhizal hosts identified all clones were from the genus Cortinarius and covered a range of the genus’ large diversity. A statistical comparative measure of the 138 soil-clones showed the Kunzea and Pinus soil-clone communities were significantly different from the Nothofagus community but were not significantly different from each other. Thirty-one unique molecular operational taxonomic units (MOTUs) were described from the soil-clone sequences, 3 MOTUs were shared between all hosts; Pinus and Kunzea shared 9 MOTUs, while only sharing 1 MOTU each with Nothofagus. The soil-clone MOTU richness was compared with taxonomic richness of Cortinarius species for the three hosts in New Zealand. The 11 MOTUs found from N. menziesii and N. solandri var. cliffortioides is, as expected, much lower than the 176 Cortinarius species recorded in association with all five Nothofagus species throughout New Zealand. In contrast, the 21 MOTUs collected from K. ericoides are greater than the 14 Cortinarius species currently recorded in association with this host nationwide. Sixteen unique MOTUs were described from the P. radiata soil-clones, while there are no published records of Cortinarius in association with P. radiata in New Zealand. The DNA fingerprinting technique terminal restriction fragment length polymorphism (TRFLP) analysis was investigated to determine accuracy of detecting ectomycorrhizal fungal species richness from pools of mixed-species DNA. Mixed-species samples (pools) with low known species richness (total species ≤ 10) were estimated at ~ 2–12 times greater by TRFLP analysis, and pools with high species richness (total species ≥ 19) were estimated at 0.6–1 times lower by the analysis. The raw data show that the high variation observed from replicates of each enzyme is predominantly caused by disagreement between the TRFs detected by the 6FAM and PET dyes; the two dyes represent the terminal ends of sequence fragments amplified by the primers ITS1F (6FAM) and ITS4 (PET). Intragenomic variation within the ITS region of ectomycorrhizal sporocarps is the probable cause for the observed high variation in TRF profiles, especially in causing overestimation of low richness. Accumulation into hyphal ingrowth bags of hyphal diversity and mass of ectomycorrhizal fungi associated with Nothofagus spp. was investigated over a period of 10 months, with a combined approach of molecular (TRFLP analysis) and non-molecular (ergosterol assay) analysis. Fungal mass (measured as ergosterol per gram of sand) extracted from the incubated hyphal ingrowth bags was found to significantly increase over a period of 10 months (from 0.1–5.2 μg g-1). Ectomycorrhizal fungal diversity and richness also increased over time (from 106– 187 unique TRFs), although this relationship was not significant. Ordination and cluster analyses show that there are no time dependent groupings of the ectomycorrhizal fungal communities, showing that while diversity and richness increase over time, the communities of TRFs (i.e., effective species) were not greatly altered. Thus, species succession was not observed from the accumulation of ectomycorrhizal fungi in hyphal ingrowth bags. The accumulation of humus soil in a canopy environment (along the upper surface of branches) was investigated as the cue for canopy root growth in native New Zealand N. menziesii and N. solandri var. cliffortioides. After an application of “artificial canopy soil” over a period of 12 months, no canopy root growth was observed in either of the Nothofagus host trees. This shows that (i) accumulation of soil alone is not the cue for canopy root growth, (ii) the application period (max 12 months) was not long enough to allow for root growth, or (iii) multiple factors cue the growth of canopy roots in Nothofagus systems. Below-ground ectomycorrhizal fungal communities associated with canopy and terrestrial root systems of N. menziesii were investigated using TRFLP analysis and ergosterol assays on hyphae accumulated in hyphal ingrowth bags. The diversity and richness of ectomycorrhizal fungi was greater in the terrestrial root systems in comparison to the canopy environments. The TRF communities from the terrestrial environment were highly diverse, while the canopy communities were very similar to each other. However, the canopy and terrestrial TRF communities were shown to share some ectomycorrhizal fungi (27% of TRFs), showing that while the terrestrial communities are more species rich and diverse there are some ectomycorrhizal fungi that are present in both communities. This thesis concluded that the TRFLP analysis method proposed was more appropriate than the sequence barcoding protocol with genus-specific primers. While both methods had limitations, TRFLP analysis descriptions of below-ground ectomycorrhizal fungal communities were of greater benefit. Hyphal ingrowth bags were shown to accumulate ectomycorrhizal fungal communities without an effect of incubation time on the detected communities. The previously proposed cue initiating canopy roots growth was concluded to not be the only influencing factor in Nothofagus species. Finally, Nothofagus canopy ectomycorrhizal fungal communities were similar independent of the tree host and were less diverse than terrestrial communities, although all communities were found to share some fungi