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Page 4 of 16 Renzi et al. Microbiome Res Rep 2024;3:2 https://dx.doi.org/10.20517/mrr.2023.27

Culture-dependent approaches
Traditionally, culture-dependent approaches been employed to investigate microorganisms’ diversity,
including fungi. However, these techniques have well-known limitations. For instance, many species remain
undetected because appropriate culture conditions are either unknown or challenging to reproduce .
[41]
Moreover, culture methods are time-consuming and hardly suitable for high-throughput analysis.
Culturomic approaches offer undeniable benefit as they provide access to the fungus itself, allowing for the
assessment of its viability, metabolites, phenotypical and functional characterization, and other host-
[42]
adaptation features . In recent years, the integration between culture- dependent and culture-independent
approaches has increased, thanks to molecular techniques. Sequencing of large portions or entire microbial
genomes has provided the necessary information for fine-tuning the growth conditions of even those
microorganisms considered “unculturable” until a few years ago [43,44] . As a result, culture-dependent
approaches remain useful and of great interest [45,46] . This is especially important given that some fungal
strains cannot be accurately identified by a culture-independent method. This underrepresentation of some
species might result from factors such as cell wall structure or the inadequacy of the chosen PCR primers
[47]
and/or barcode sequence . However, the identification process for isolated fungal strains is not yet
complete and requires further steps, often involving culture-independent approaches.

Culture-independent approaches
The use of DNA as an identifying marker in culture-independent approaches avoids some of the
aforementioned issues. However, this method strongly relies on the choice and efficiency of DNA recovery
methods, and it also introduces new limits and hurdles [Figure 1]. Fungi, unlike bacteria, have a strong and
complex cell wall rich in glucans and chitin [48-51] . Consequently, the efficient destruction of the fungal cell
wall is crucial for genomic DNA extraction. Several bead-beating stages followed by enzymatic cell lysis are
[47]
required for successful mycobiota analysis of any sample matrix . Following DNA extraction, different
approaches can be used to detect and identify fungi. This methods may include PCR , metabarcoding
[52]
sequencing analysis, or whole genome sequencing (WGS) metagenomics.

Amplicon-based sequencing: a matter of target
While amplicon sequencing techniques have successfully revealed the microbiome of a plethora of
organisms [53-56] , the choice of the marker to use is crucial as it drastically affects the type of organisms that
can be detected. In the micro-eukaryotic world, mainly composed of fungi, protists, algae, and other
microorganisms known to inhabit almost all ecological niches explored on Earth, the selection of
“universal” targets is limited [Table 1]. Only a few available pipelines are available to cope with markers
different from the well-known bacterial 16S rRNA gene [57-60] .

Similarly to bacterial metabarcoding, the usual fungal barcode is the rRNA gene locus, which includes the
genes for 18S rRNA, 5.8S rRNA, and 28S rRNA, separated by the internal transcribed spacers (ITS1 and
ITS2). This approach seems to discriminate better at higher taxonomic ranks than the 16S rRNA gene .
[61]
After exploring fungal rRNA genes, Schoch et al. in 2012 identified the ITS as the possible universal DNA
barcode identifier for fungi [24,62] , although currently, it is still not clear which of the two ITS components has
the better resolution in strain prediction. Recent findings has shown that both regions suffer from
amplification biases, resulting in an uneven representation of synthetic fungal communities [63-65] : ITS1-based
PCR appears to favor Basidiomycota, whereas Ascomycota seems to be favored by ITS2-based PCR [66-68] ,
although this consideration should not be generalized. In fact, there are known ascomycetes species (such as
the ones belonging to the genera Saccharomyces and Komagataella) that are discriminated with greater
resolution by employing the ITS1 marker . Hoggard et al. recommend the selection of the ITS2 region in
[69]
human mycobiota investigation after comparing four sets of primers targeting the small subunit (SSU)
rRNA (18S), ITS1, ITS2, and large subunit (LSU) rRNA (26S) genomic regions . In yeast, the D1/D2
[70]