Polish Journal of Microbiology ORIGINAL PAPER 2020, Vol. 69, Article in Press https://doi.org/10.33073/pjm-2020-027 Yeasts Associated with Various Amazonian Native Fruits CARLOS VEGAS1, AMPARO I. ZAVALETA1, PAMELA E. CANALES1* and BRAULIO ESTEVE-ZARZOSO2 1 Laboratory of Molecular Biology, Faculty of Pharmacy and Biochemistry, Universidad Nacional Mayor de San Marcos, Lima, Peru 2 Departament de Bioquímica i Biotecnologia, Facultat d’ Enologia, Universitat Rovira i Virgili, Tarragona, Spain Submitted 14 March 2020, revised 27 May 2020, accepted 28 May 2020 Abstract Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive com- pounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits. K e y w o r d s : yeast diversity, fruit, Amazonia, PCR-RFLP, 5.8S-ITS Introduction and yeast-like microbes associated with fruits and blos- soms of apple, plum, and pear orchards in Slovakia. Fruits constitute excellent habitats for yeasts, mainly Trindade et al. (2002) investigated yeasts inhabiting due to their low pH, availability of nutrients, and active the fresh and frozen pulps of Brazilian tropical fruits. fruit-associated vectors. These traits are variable across Notably, some fruits have been described as sources of the type and maturity of the fruit. Changes in the com- new yeasts (Bhadra et al. 2008; Sipiczki 2011). munity in response to varying availability of nutrients, Yeast diversity on the wide variety of Amazonian production of mycotoxins, and the arrival of new yeast native fruits (ANF) has not been widely investigated, species are evident (Tournas and Katsoudas 2005; with reports focusing mainly on other tropical fruits Starmer and Lachance 2011). like passion fruit (Passiflora edulis), mangaba (Hancor­ The majority of research has focused on the diver- nia speciosa), umbu (Spondias tuberosa), and acerola sity of yeasts on grapes and wine-related samples (Malpighia glabra) (Trindade et al. 2002; Da Silva et al. due to their application in the winemaking process 2005; Grondin et al. 2015). The Amazonian rainforest’s (Guillamón et al. 1998; Filho et al. 2017), although environmental characteristics suggest the possibility of some expansions have been made beyond this zone finding diverse yeast communities, including new spe- of interest. Koricha et al. (2019) identified yeasts from cies or strains with new characteristics of biotechno- lemon, mango, and guava fruits, with Candida albicans, logical interest (Morais et al. 1995; Da Silva et al. 2005). Debaryomyces hansenii, Kodamaea ohmeri, Rhodoto­ Yeasts represent a promising source for obtaining rula mucilaginosa, among others, found to be present. microbial enzymes (Trindade et al. 2002; Da Silva et al. Vadkertiová et al. (2012) studied the diversity of yeasts 2005; Raveendran et al. 2018), flavorings (Grondin et al. * Corresponding author: P. E. Canales, Laboratory of Molecular Biology, Faculty of Pharmacy and Biochemistry, Universidad Nacional Mayor de San Marcos, Lima, Peru; e-mail: [email protected] © 2020 Carlos Vegas et al. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License (https://creativecommons. org/licenses/by-nc-nd/4.0/). 2 Vegas C. et al. 3 2015), and can be used as biocontrol agents for posthar- using the 100 bp Plus DNA (Thermo Scientific, USA) vest fruit diseases (Janisiewicz et al. 2010; Ruiz-Moyano and Lambda DNA/EcoR I + Hind III Marker (Thermo et al. 2016). Yeasts or their metabolites isolated from Scientific, USA) ladders. One representative of each the Amazonian rainforest may display unique char- strain pattern obtained was chosen for the RFLP-PCR acteristics due to the particularities of their habitat. analysis of the 5.8S-ITS region. Accordingly, this study aimed to identify and char- PCRs were carried out using primers ITS1 (5’-TCCG- acterize yeasts isolated from Amazonian native fruits TAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTC- using molecular and phenotypic methods to glimpse CGCTTATTGATATGC-3’) (White et al. 1990) in also their potential biotechnological features. order to amplify the 5.8S rRNA gene and 2 internal transcribed spacers (ITS1 and ITS2), according to the methodology described by Esteve-Zarzoso et al. (1999). Experimental PCR products were digested by the restriction enzymes Hinf I, Cfo I, and Hae III (Thermo Scientific, USA) Materials and Methods following the manufacturer’s instructions. PCR prod- ucts and their restriction fragments were separated by Fruit samples. One hundred specimens from ten electrophoresis on 1 and 2% agarose gels, respectively. different species (ten of each species) of Amazonian Gels were stained with ethidium bromide, and DNA native fruits (ANF) were obtained from a small rus- fragments were visualized under UV. Sizes were esti- tic market in the city of Iquitos (Amazonian region mated by comparison against a DNA ladder (100 bp of Peru), which is supplied with fruits from different Plus; Thermo Fisher Scientific, USA). Preliminary localities of the region, in July 2015. At that time, tem- iden tification of restriction profiles was determined by perature was on average 25°C with the least amount comparison with those previously reported (Guillamón of rains of the year. Fruits were all ripe with no apparent et al. 1998; Esteve-Zarzoso et al. 1999). spoilage. Fruits were transported in refrigerated and Sequencing and phylogenetic analysis. The strains sterile bags to Lima for laboratory analysis. The fol- were subjected to sequencing. D1/D2 domains of the lowing ANF were employed in this study: aguaje (Mau­ 26S rRNA gene were amplified using primers NL-1 ritia flexuosa), camu camu (Myrciaria dubia), chari- (5’-GCATATCAATAAGCGGAGGAAAAG-3’) and chuelo (Garciniama crophylla), cidra (Citrus medica), NL-4 (5’-GGTCCGTGTTTCAAGACGG-3’) (Kurtz- cocona (Solanum sessiliflorum), pomarrosa (Syzygium man and Robnett 1998). PCR products were sent to jambos), taperiba (Spondias dulcis), ubos (Spondias Macrogen (Rockville, USA) for sequencing. Electro- mombin), umarí (Poraqueiba sericea), and ungurahui pherograms for both primers were evaluated using (Oenocarpus bataua). Sequencher version 4.1.4 (Gene Codes, USA) and Yeast isolation. For surface sampling, the same contigs for each sample were assembled. Sequences species of ANF were pooled and washed under aseptic are deposited in the GenBank database (https://www. conditions with sterile water which was used for further ncbi.nlm.nih.gov/genbank/) under the accession codes preparation of serial decimal dilutions in liquid YPD MF979591-MF979618 and MF979620. medium (1% yeast extract, 2% peptone, 2% glucose To construct our dataset, similar sequences were w/v), supplemented with chloramphenicol (100 mg/l; searched using the BlastN algorithm against the Gen- AppliChem GmbH, Germany). Aliquots of several Bank database. Identities were matched at 99–100% dilutions were spread onto YPD plates and incubated similarity. In addition, we searched for similar at 30°C for 24 h. Ten colonies from each fruit were sequences against the Mycobank database (http://www. selected based on different colony morphologies (form, mycobank.org) using its pairwise sequence alignment size, color, margin, and elevation) for further purifica- tool (MolecularID) for confirming results. Both of these tion. Yeast colonies were identified and characterized results provided preliminary information on the iden- genotypically and phenotypically. tification of each sample. Following this, preliminary DNA extraction, rep-PCR, and RFLP-PCR of the information was used to search for the correspond- 5.8S-ITS region. DNA extraction was performed as ing type strain sequences described by Kurtzman et al. per Querol et al. (1992) with a slight modification in (2011a). If accession codes for any type strain were the use of lyticase (3.3 U · µ/l; Sigma, USA) instead of not present, they were searched in GenBank (Fig. 1). zymolase. For discrimination at the strain level, PCR of For phylogenetic tree construction, we aligned multi- the repetitive extragenic palindromic sequences (rep- ple sequences using ClustalX 2.1 (Larkin et al. 2007). PCR) (Versalovic et al. 1991) was