Abstract
BackgroundMeyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Conventional phenotypic methods frequently misidentify the species within this complex and also with other species of the Saccharomycotina CTG clade. Even the long-established sequencing of large subunit (LSU) rRNA gene remains ambiguous. We also faced similar problem during identification of yeast isolates of M. guilliermondii complex from indigenous bamboo shoot fermentation in North East India. There is a need for development of reliable and accurate identification methods for these closely related species because of their increasing importance as emerging infectious yeasts and associated biotechnological attributes.ResultsWe targeted the highly variable internal transcribed spacer (ITS) region (ITS1-5.8S-ITS2) and identified seven restriction enzymes through in silico analysis for differentiating M. guilliermondii from M. caribbica. Fifty five isolates of M. guilliermondii complex which could not be delineated into species-specific taxonomic ranks by API 20 C AUX and LSU rRNA gene D1/D2 sequencing were subjected to ITS-restriction fragment length polymorphism (ITS-RFLP) analysis. TaqI ITS-RFLP distinctly differentiated the isolates into M. guilliermondii (47 isolates) and M. caribbica (08 isolates) with reproducible species-specific patterns similar to the in silico prediction. The reliability of this method was validated by ITS1-5.8S-ITS2 sequencing, mitochondrial DNA RFLP and electrophoretic karyotyping.ConclusionsWe herein described a reliable ITS-RFLP method for distinct differentiation of frequently misidentified M. guilliermondii from M. caribbica. Even though in silico analysis differentiated other closely related species of M. guilliermondii complex from the above two species, it is yet to be confirmed by in vitro analysis using reference strains. This method can be used as a reliable tool for rapid and accurate identification of closely related species of M. guilliermondii complex and for differentiating emerging infectious yeasts of the Saccharomycotina CTG clade.
Highlights
IntroductionMeyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex
Meyerozyma guilliermondii and Meyerozyma caribbica are closely related species of the genetically heterogenous M. guilliermondii complex
In silico analysis and restriction enzyme selection The full length ITS1-5.8S-ITS2 sequences of M. guilliermondii and M. caribbica were retrieved from NCBI and Centraalbureau voor Schimmelcultures (CBS-KNAW) yeast nucleotide databases
Summary
Meyerozyma guilliermondii (anamorph Candida guilliermondii) and Meyerozyma caribbica (anamorph Candida fermentati) are closely related species of the genetically heterogenous M. guilliermondii complex. Meyerozyma guilliermondii is a genetically heterogenous complex belonging to the Saccharomycotina CTG clade [1] This complex consists of phenotypically indistinguishable and closely related species namely Meyerozyma guilliermondii (anamorph Candida guilliermondii), Meyerozyma caribbica (anamorph Candida fermentati), Candida carpophila, Candida smithsonii, Candida athensensis, Candida elateridarum and Candida glucosophila [2,3,4,5,6]. Isolates of M. guilliermondii and M. caribbica have exhibited great potential in the biological control of fungi responsible for postharvest spoilage of fruits and vegetables [24,25,26] These yeast species with enhanced biological control efficacy have emerged as a potential alternative to the 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
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