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

Anna Baturo-Cieśniewska,Wojciech Pusz,Katarzyna Patejuk

doi:10.5586/am.5512

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 Arthrinium arundinis, Beauveria bassiana, Boeremia exigua, Cladosporium cladosporioides, Epicoccum nigrum, Nigrospora oryzae, Sclerotinia sclerotiorum, or Sordaria fimicola and members of the genera Alternaria and Trichoderma. 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.

Highlights

For many years, molecular analysis based on polymerase chain reaction (PCR) in combination with morphological and phylogenetic analyses have been used in taxonomic studies (Hyde et al, 2013; Jayasiri et al, 2015; Liew et al, 2000; Lücking et al, 2014; White et al, 1990)
Due to the possibility of ambiguous results, identification based on rDNA internal transcribed spacer (ITS) is usually completed by morphological feature observations and sequencing of other loci such as actin (ACTB), plasma membrane ATPase (ATP1A1), calmodulin (CAM), translation elongation factor 1-alpha (EEF1A1), the second largest subunit of RNA polymerase II (RPB2), chitin synthase (CHS), and β-tubulin (TUBB) (Aveskamp, Verkley, et al, 2009; Bensch et al, 2018; Houbraken, Visagie, et al, 2014; Lawrence et al, 2013; Samson et al, 2014; Stielow et al, 2015; Visagie, Hirooka, et al, 2014, Vitale et al, 2011; Vu et al, 2019)
We were able to determine the species of nearly 80% of our isolates. Among them were those for which BLASTn analysis was potentially sufficient to identify a sequence with confidence; for most sequences, the analysis of additional data was helpful and complementary

Summary

Introduction

Molecular analysis based on polymerase chain reaction (PCR) in combination with morphological and phylogenetic analyses have been used in taxonomic studies (Hyde et al, 2013; Jayasiri et al, 2015; Liew et al, 2000; Lücking et al, 2014; White et al, 1990). Species are identified by comparing the sequences of the analyzed isolates with sequences deposited in international databases, such as the International Nucleotide Sequence Database (INSD) (http://www.insdc.org/) and User-Friendly Nordic ITS Ectomycorrhiza (UNITE) database, considered to be the largest database (Nillson et al, 2019; Yahr et al, 2016). They provide access to the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/), European Nucleotide Archive (ENA) (https://www.ebi.ac.uk/ena/), or DNA Database of Japan (DDBJ) (https://www.ddbj.nig.ac.jp/index-e.html), where over 100,000 ITS sequences are deposited. Despite criticism (Kiss, 2012), ITS sequences have been shown to be useful in delineating many fungal species and are commonly used in identification and analysis of the molecular diversity of fungi found on various plants, inhabiting soil, air or human tissues (Baturo-Ciesniewska et al, 2017; Chehri et al, 2011; Ingle, 2017; Irinyi et al, 2015; Meyer et al, 2019; Nilsson et al, 2014; Pusz et al, 2018; Visagie, Hirooka, et al, 2014), and for development of speciesspecific PCR primers, e.g., for Fusarium spp. (Mishra et al, 2003)

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