Application of ISSR-PCR as a rapid method for clustering and typing of yeasts isolated from table olives

Abstract

Table olives harbour a variable microbial population, dominated by lactic acid bacteria and yeasts. Clarifying the effect of the yeast population during table olive fermentation requires methods for yeast species identification. Until now, the most used molecular methods (culture-dependent and -independent) are limited to species level identification, whereas RAPD-PCR is primarily chosen when strain typing is required. In this work, species identification by sequencing of the ITS1–5.8S rDNA–ITS4 region, was followed by inter-simple-sequence-repeat-anchored PCR (ISSR-PCR) using the (CAG)4 primer, to evaluate its suitability for species clustering and strain typing of the yeast population from table olives. Yeast were isolated from ten table olive samples: seven samples were purchased from local markets (sliced, naturally fermented, crushed and Spanish-style fermented), and three samples were from pilot fermentations (Spanish-style fermented). Ten yeast species were identified by sequencing that were correctly clustered by ISSR-PCR, but without a clear association with the type of olive processing. ISSR-PCR revealed 57 different profiles into 254 total isolates. Citeromyces nyonsensis (39.4%) and Yarrowia deformans (22.2%), which have not previously been associated to table olives, showed high intraspecific variability. ISSR-PCR using (CAG)4 primer is a valuable method for identification and strain typing of yeast from table olives.