Methods for identifying and measuring the diversity of ectomycorrhizal fungi

Abstract

Mycorrhiza is a mutualistic symbiosis between some plant species and a diverse group of soil fungi. Mycorrhizal symbiosis is categorized into discrete types based on the structural differences and the taxonomic position of involved plants and fungi. The main types of mycorrhiza include arbuscular, ericoid, orchid, monotropoid and ectomycorrhiza, the latter being the main focus of this review. Identifying ectomycorrhizal fungi (EMF) is crucial for understanding the ecology and preservation of rare and endangered plants, fungi and habitats. Thus, since the discovery of ectomycorrhiza, new identification methods have been constantly developed. This review tracks the history of EMF identification methods, presents their limitations and aims to discern the frontier of the field. The early methods were based on the morphology and anatomy of EMF, which allowed ectomycorrhizae formed by different EMF to be distinguished. Recognizing the EMF species that formed them, however, was difficult. The introduction of molecular methods that analysed taxon-specific DNA barcodes was a breakthrough in EMF identification, and the internal transcribed spacer gene was adopted as a routine fungal barcoding sequence. Identifying EMF from randomly sampled roots by morphology or polymerase chain reaction (PCR)-based methods results in rare EMF taxa being missed and underreported. This is addressed by bulk sampling combined with environmental DNA metabarcoding methods, which, in turn, yield a lower resolution of taxonomic identification. Most EMF identification methods incorporate PCR reactions, which are biased against some groups of EMF; developing PCR-free molecular methods would constitute a significant advancement. As the traditional concept of species is challenging for fungi, studying EMF functional rather than taxonomic diversity appears to be promising.

Landscape-scale mapping of soil fungal distribution: proposing a new NGS-based approach

“…Compared to conventional sampling designs, the number of samples required for landscape-scale mapping of the soil fungi distribution is notably higher. While the conventional approach will remain a cheaper alternative, the per-sample costs in molecular studies are continually decreasing 30 . As a result, the proposed mapping method will become increasingly available to diverse research teams around the world.…”

“…The DNeasy PowerSoil (Qiagen) kit was used to extract and purify total soil DNA from each sample. The primers gITS7 (5′-GTG ART CAT CGA RTC TTT G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) were used for the PCR reactions to amplify the standard fungal barcoding gene ITS2 29 , 30 . Three independent PCR amplifications were conducted for each sample.…”

Ectomycorrhizal fungi are influenced by ecoregion boundaries across Europe

“…(R Core Team, 2023). We estimated the completeness of the sampling for each ecoregion based on a species accumulation curve using the 'iNEXT' package (Hsieh et al, 2022).…”

“…In addition, species abundances and local diversity generally cannot be reliably inferred from databases of species distribution data (e.g., Global Biodiversity Information Facility-GBIF. org) or eDNA (Janowski & Leski, 2023). This leads to ignoring a crucial component of diversity by underestimating the importance of abundant species while overestimating the contribution of rare species (Jost et al, 2011).…”

Ectomycorrhizal fungi are influenced by ecoregion boundaries across Europe

“…In addition, species abundances and local diversity generally cannot be reliably inferred from databases of species distribution data (e.g. Global Biodiversity Information Facility - GBIF.org) or eDNA (Janowski and Leski, 2023). This leads to ignoring a crucial component of diversity by underestimating the importance of abundant species while overestimating the contribution of rare species (Jost et al, 2011).…”