Problems, Limitations, and Challenges in Species Identification of Ascomycota Members on the Basis of ITS Regions

Abstract

The internal transcribed spacer (ITS) region is regarded as a formal fungal primary barcode with a high probability of the correct identification for a broad group of fungi. ITS sequences have been widely used to determine many fungal species and analysis of rDNA ITS is still one of the most popular tools used in mycology. However, this region is not equally variable in all groups of fungi; therefore, identification may be problematic and result in ambiguous data, especially in some species-rich genera of Ascomycota. For these reasons, identification based on rDNA ITS is usually complemented by morphological observations and analysis of additional genes. Reliable species identification of Ascomycota members is essential in diagnosing plant diseases, verifying air quality and the effectiveness of agronomic practices, or analyzing relationships between microorganisms. Therefore, the present study aimed to verify, using specific examples, the extent to which ITS sequence analysis is useful in species identification of pathogens and saprobionts from Ascomycota and demonstrate problems related to such identification in practice. We analyzed 105 ITS sequences of isolates originating from air and plant material. Basic local alignment search tool (BLASTn) significantly contributed to the reliable species identification of nearly 80% of isolates such as <em>Arthrinium arundinis</em>, <em>Beauveria bassiana</em>, <em>Boeremia exigua</em>, <em>Cladosporium cladosporioides</em>, <em>Epicoccum nigrum</em>, <em>Nigrospora oryzae</em>, <em>Sclerotinia sclerotiorum</em>, or <em>Sordaria fimicola </em>and members of the genera <em>Alternaria </em>and <em>Trichoderma</em>. However, for most isolates, additional morphological observations, information regarding the isolate origin and, where possible, a PCR with species-specific primers were helpful and complementary. Using our practical approach, we determined that ITS-based species identification and comparative analysis with GenBank sequences significantly helps identifying Ascomycota members. However, in many cases, this should be regarded as suggestive of a taxon because the data usually require the use of additional tools to verify the results of such analysis.

Predicting global numbers of teleomorphic ascomycetes

“…There is a 1% intra-species variation within Aspergillus niger (Henry et al 2000). The ITS sequence of Aspergillus niger (MK461010) is identical to ITS sequences of Alternaria alternata (Baturo-Cieśniewska et al 2020). Further, the ITS sequences of Aspergillus niger (MK461010) shows 10% inter-species variation of ITS sequences of other A. niger strains.…”

“…The ITS region is the formal primary fungal barcode with the highest probability of correct species identification of a broad group of fungi (Horton and Bruns 2001;Bridge et al 2005;Martin and Rygiewicz 2005;Seifert 2009;Bellemain et al 2010;Schoch et al 2012). More than 100,000 ITS sequences of teleomorphic ascomycetes are deposited in international nucleotide sequence databases (Baturo-Cieśniewska et al 2020). Even though, many sequences of correctly identified species are deposited in databases, there are some sequences with technical errors, incorrectly named species, atypical chimeric ITS sequences, and sequences verified only at the generic level or above which makes reliable problems in species identification (Nilsson et al 2009(Nilsson et al , 2014Hongsanan et al 2018).…”

“…Further, ITS based species identification may not be suitable when interspecies nucleotide variations are less (Baturo-Cieśniewska et al 2020). Sclerotinia sclerotiorum and S. trifoliorum are causative agents of clover rot disease and the symptoms are identical (Vleugels et al 2012).…”

“…Therefore, the taxonomic credibility in sequence databases is with errors (Binder et al 2005;Bridge et al 2005;Nilsson et al 2006). Blast similarities of sequences sometimes give uncultured or unverified sequences and names not identified to species level (Baturo-Cieśniewska et al 2020). The major reason for these unidentified or insufficiently identified sequences in public databases is that most international journals require all sequences used in the manuscript to be deposited (Ryberg et al 2008).…”

Using High-Throughput Amplicon Sequencing to Evaluate Intragenomic Variation and Accuracy in Species Identification of Cordyceps Species

“…The nuclear internal transcribed spacer (nrITS) regions of the ribosomal DNA (rDNA) is used as a universal barcoding region, because a broad range of fungi can be identified using this region [ 4 ]. nrITS is still the most widely used for species identification, barcoding and fungal phylogenetics [ 3 , 5 ], although many studies have shown that its utility for species identification was limited [ 6 , 7 , 8 ]. Another commonly used ribosomal marker is the Ribosomal Large Subunit (nrLSU).…”