These yeast species with enhanced biological control efficacy have emerged as a potential alternative to the NVP-AUY922 datasheet conventional fungicide treatment. Considering the various importance and applications of the two species, there is a need for the development of accurate and reliable method to identify and distinctly discriminate the closely related species. Current methods of yeast identification, mostly in Napabucasin in vivo clinical practice, are mainly based on the conventional and rapidly evolving commercial phenotypic and biochemical methods. However, such methods are often unreliable for
accurate identification of closely related yeast species [13, 27]. According to recent studies, M. guilliermondii and M. caribbica are extremely difficult to differentiate by the phenotypic methods [28–31]. We also faced similar problem during differentiation of yeast isolates from soibum, an indigenous selleck chemical fermented bamboo shoot product of North East India (Additional file 1: Table S1). The widely used API 20 C AUX yeast identification system and sequencing of large subunit (LSU) rRNA gene D1/D2
domain failed to give proper species-level taxonomic assignment to these isolates (Additional file 1: Tables S2 and S3). Moreover, the phylogenetic tree reconstructed from the publicly available D1/D2 sequences of different strains of M. guilliermondii and M. caribbica failed to discriminate the two species (Additional file 2: Figure S1). Several attempts have been made using molecular approaches such as DNA base composition, electrophoretic karyotyping [6, 32], multi locus sequence typing (MLST) , multi Methocarbamol locus enzyme electrophoresis (MLEE), randomly amplified polymorphic DNA (RAPD) , sequencing of internal transcribed spacer (ITS) [28, 30], intergenic spacer restriction fragment length polymorphism (IGS-RFLP)  and RFLP of housekeeping genes such as riboflavin synthetase gene RIBO in order to resolve
the misidentification. Some recent studies have claimed that the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) is advantageous over previous approaches for reliable identification of clinically important NAC and non-Candida yeast species [28, 31, 33, 34]. Unfortunately, MALDI-TOF-MS requires reference spectra of accurately identified closely related strains otherwise the results may be erroneous. On the other hand, the sequence-based studies have considered the ITS1-5.8S-ITS2 region as universal DNA barcode for yeast identification  and the RFLP of ITS1-5.8S-ITS2 region has successfully separated the closely related species in the genera Candida and Pichia[36, 37]. Therefore, in this study, we targeted the ITS1-5.8S-ITS2 region to develop a simple RFLP method for accurate taxonomic assignment of M. guilliermondii and M. caribbica. With this background, the aim of the present study was (i) to perform in silico prediction of restriction enzymes to discriminate M.