The environmental and intrinsic yeast diversity of Cuban cocoa bean heap fermentations.

The environmental yeast diversity of spontaneous cocoa bean fermentations in east Cuba was investigated. Seven fermentations, 25 equipment- and handling-related samples, and 115 environmental samples, such as flowers, leaf and cocoa pod surfaces, as well as drosophilid insects, were analysed. The basic fermentation parameters temperature and pH were recorded during five fermentations for at least six days. A total of 435 yeast isolates were identified by a combination of PCR-fingerprinting of genomic DNA with the M13 primer and sequence analysis of DNA from representative isolates, using the internal transcribed spacer region, the D1/D2 region of the large subunit rRNA gene, and an actin gene-encoding fragment, as required. Among 65 yeast species detected, Pichia manshurica and Hanseniaspora opuntiae were the most frequently isolated species, obtained from five and four fermentations, followed in frequency by Pichia kudriavzevii from two fermentations. Saccharomyces cerevisiae was isolated only occasionally. Cocoa fermentation yeast species were also present on processing equipment. The repeated isolation of a preliminarily as Yamadazyma sp. classified species, a group of strains similar to Saccharomycopsis crataegensis from fermentations and equipment, and the isolation of fifteen other potentially novel yeast species in low numbers provides material for further studies. Environmental samples showed higher yeast diversity compared to the fermentations, included the most frequent fermentation species, whereas the most frequently isolated environmental species were Candida carpophila, Candida conglobata, and Candida quercitrusa. Potential selective advantages of the most frequently isolated species were only partly explained by the physiological traits tested. For instance, tolerance to higher ethanol concentrations was more frequent in strains of Pichia spp. and S. cerevisiae compared to Hanseniaspora spp.; the ability to also assimilate ethanol might have conferred a selective advantage to Pichia spp. In contrast, high glucose tolerance was common among strains of Hanseniaspora spp., Torulaspora delbrueckii, and Candida tropicalis, among which only Hanseniaspora spp. were frequently isolated.

Starmerella neotropicalis f. a., sp. nov., a yeast species found in bees and pollen.

A novel yeast species was found repeatedly and in high cell densities in underground-nesting stingless bees of the species Melipona quinquefasciata and their provisions in northern Minas Gerais (Brazil). One additional strain was isolated from bee-collected pollen in Cuba. Phylogenetic analyses based on rRNA gene sequences (D1/D2 large subunit gene and internal transcribed spacer) indicated that the novel species belongs to the Starmerella clade and is most closely related to Candida (iter. nom. Starmerella) apicola. Growth reactions on carbon and nitrogen sources were typical of those observed in related species of the Starmerella clade. PCR-fingerprinting with mini- and microsatellite specific primers allowed the distinction of the novel species from Candida apicola, Candida bombi and a yet undescribed species represented by strain CBS 4353. On the basis of phylogenetic relationships, the novel species is assigned to the genus Starmerella despite the failure to observe sexual reproduction after extensive mating tests. We propose the name Starmerella neotropicalis f. a., sp. nov. (Mycobank MB 804285) and designate UFMG PST 09(T) ( = MUCL 53320(T) = CBS 12811(T)) as the type strain.

Metschnikowia cubensis sp. nov., a yeast species isolated from flowers in Cuba.

A novel yeast species is described from 19 strains isolated from flowers and insects in three provinces of Cuba. The species is so far known only from Cuba. Characteristic asci and ascospores as well as phylogenetic analysis of the rDNA sequence place the novel species in the genus Metschnikowia. The novel species belongs to the New World subclade of large-spored species of Metschnikowia. Mating tests with other members of the subclade resulted in the formation of sterile asci without ascospores, showing that the Cuban strains represent a distinct biological species. Intraspecies matings lead to the production of fertile asci containing large needle-shaped ascospores. The novel species was further distinguished from its close relatives by rDNA sequences and PCR fingerprinting using primers derived from mini- and microsatellites. We propose the name Metschnikowia cubensis sp. nov. and designate MUCL 45753(T) (=CRGF 279(T) =CBS 10832(T), h(+)) as the type strain and MUCL 45751 (=CRGF 278 =CBS 10833, h(-)) as the allotype.