Fungal DNA barcoding
In this attempt, DNA barcoding relies on universal genes that are ideally present in all fungi with the same degree of sequence variation.
These morphs usually differ drastically in their phenotypic appearance, preventing a straightforward association of the asexual anamorph with the sexual teleomorph.
[3] Fungal DNA barcoding can help to identify and associate anamorphic and teleomorphic stages of fungi, and through that to reduce the confusing multitude of fungus names.
Without a database covering a broad taxonomic range of fungi, many identification queries will not result in a satisfyingly close match.
When the identity of a certain taxon (or a genetic sequence in the case of DNA barcoding) is in doubt, the original specimen can be re-examined to review and ideally solve the issue.
[13] In fungi, the Internal transcribed spacer (ITS) is a roughly 600 base pairs long region in the ribosomal tandem repeat gene cluster of the nuclear genome.
Optimised primers specifically for ITS sequencing in Dikarya (comprising Basidiomycota and Ascomycota) have been proposed by Toju et al.
A major advantage of using the ITS region as molecular marker and fungal DNA barcode is that the entire ribosomal gene cluster is arranged in tandem repeats, i.e., in multiple copies.
[15] This allows for its PCR amplification and Sanger sequencing even from small material samples (given the DNA is not fragmented due to age or other degenerative influences).
[19] The tandem repeats of the ribosomal gene cluster cause the problem of significant intragenomic sequence heterogeneity observed among ITS copies of several fungal groups.
Furthermore, because of the non-coding nature of the ITS region that can lead to a substantial amount of indels, it is impossible to consistently align ITS sequences from highly divergent species for further bigger-scale phylogenetic analyses.
This variability, however, can range from 0% for example in Serpula lacrymans (n=93 samples) over 0.19% in Tuber melanosporum (n=179) up to 15.72% in Rhizoctonia solani (n=608), or even 24.75% in Pisolithus tinctorius (n=113).
Solely relying on ITS barcode data for the identification of such species pairs or complexes may thus obscure the actual diversity and might lead to misidentification if not accompanied by the investigation of morphological and ecological features and/or comparison of additional diagnostic genetic markers.
[19][24][26][27] For some taxa, ITS (or its ITS2 part) is not variable enough as fungal DNA barcode, as for example has been shown in Aspergillus, Cladosporium, Fusarium and Penicillium.
[36] Stielow et al. (2015) investigated the TEF1α gene, among a number of others, as potential genetic marker for fungal DNA barcoding.
Despite this, the authors conclude that TEF1α is the most promising candidate for an additional DNA barcode marker in fungi as it also features sequence regions of higher mutation rates.
IGS has been successfully used for the differentiation of strains of Xanthophyllomyces dendrorhous[47] as well as for species distinction in the psychrophilic genus Mrakia (Cystofilobasidiales).
Furthermore, a part of the β-Tubulin A (BenA) gene exhibits a higher taxonomic resolution in distinguishing Penicillium species as compared to COI and ITS.
[52] COI also performs poorly in the identification of basidiomycote rusts of the order Pucciniales due to the presence of introns.
[52][54] Agaricus bisporus was found to contain up to 19 introns, making the COI gene of this species the longest recorded, with 29,902 nucleotides.
[58] Xiang et al. (2013) showed that using ITS sequences, the commercially highly valuable the caterpillar fungus Ophiocordyceps sinensis and its counterfeit versions (O. nutans, O. robertsii, Cordyceps cicadae, C. gunnii, C. militaris, and the plant Ligularia hodgsonii) can be reliably identified to the species level.
[59] A study by Vi Hoang et al. (2019) focused on the identification accuracy of pathogenic fungi using both the primary (ITS) and secondary (TEF1α) barcode markers.
[62] Fungal DNA barcoding has been successfully applied to the investigation of foxing phenomena, a major concern in the conservation of paper documents.
Aspergillus versicolor, Cladosporium cladosporioides, C. sphaerospermum, C. tenuissimum, Epicoccum nigrum, Parengyodontium album, Penicillium brevicompactum, P. crustosum, P. glabrum, Talaromyces amestolkiae and T. stollii were the most common species isolated from the samples.
[64] Another study concerning objects of cultural heritage investigated the fungal diversity on a canvas painting by Paula Rego using the ITS2 subregion of the ITS marker.
