Determination of yeast community structure distribution by high-throughput methods
In order to characterize the structure of the yeast community in honey from three regions of Xinjiang, the OTU sequences were clustered and annotated with species. 40 genera belonging to 2 phyla, 10 orders, 13 families and 15 families were included in the OTU of honey from the three sampling sites. The top 20 genera in relative abundance and the lower genera in relative abundance were selected and grouped into others. Zygosaccharomyces, Aureobasidium, Clavispor, Pichia and Filobasidium were the main yeast genera in honey. The results for the main yeast genera levels in honey (Fig.1) showed that Zygosaccharomyces had the highest relative abundance of 85.86% and was the most dominant genus among the three honey, followed by Aureobasidium and Clavispora. And the top 20 genera were distributed in all three honey.The relative abundances of Oleaster honey and sunflower honey differed little at the yeast genus level, but there were large differences in the dominant yeast and their relative abundances with multifloral honey. The highest relative abundance of Zygosaccharomyces was found in sunflower honey at the Shihezi sampling site (96.64%), followed by Clavispora (1.24%) and Filobasidium (1.06%), while the relative abundance of Zygosaccharomyces reached (44.71%) in multifloral honey at the Ili sampling site, and followed by Aureobasidium (14.07%) and Pichia (9.80%).
Heat map analysis showed the relative abundance of the last 20 yeast genera in the three honey groups (Figure 2). The relative abundance of these yeast genera was generally low in all three samples. Among them, the genus Barnettozyma was present in FM1 and FM3 yeast, but no tested were available in FM2. Eremothecium was present in both FM2 and FM3, but not in FM1.
Morphological identification of yeast
Through isolation and purification of YM medium, 15, 13 and 7 yeast strains were isolated and purified from Shawan oleaster honey, Shihezi sunflower honey and Yili multifloral honey, and a total of 35 yeast strains were obtained (Table 1). According to the phenotypic characteristics of yeast on WL medium, it can be classified into 11 different phenotypes (Figure 3), and then, the cell morphology of each phenotypic representative strain was observed, a total of three microscopic forms were obtained. Single-ended sprouting of pike (phenotype FM1-1), round single end sprouting (phenotype FM1-2), Oval single-ended sprouts (phenotype FM1-3,FM1-4,FM1-8,FM1-10,FM1-12,FM2-10,FM3-1,FM3-3,FM3-7).
Physiological and biochemical experiments
Based on morphological characterization, further physiological and biochemical experiments were conducted on yeast, including sugar fermentation experiments, carbon source assimilation experiments, nitrogen source assimilation experiments, and maximum growth temperature experiments. In the sugar fermentation experiment, strains FM1-1, FM1-2, FM1-4 and FM1-8 were unable to ferment glucose, galactose, sucrose, maltose, lactose, raffinose and trehalose. Except glucose, FM1-3, FM3-3 and FM3-7 cannot ferment other sugars. FM1-10, FM1-12, FM2-12 and FM3-2 have different sugar fermentation conditions (Table 2). In addition to various organic acid salts as the sole carbon source, including lactate, succinate and citrate, they also assimilated a wide range of carbon sources (Table 3). They assimilated differently on various nitrogen sources, with FM1-1,FM1-2,FM1-3,FM1-4,FM3-2 being able to utilize sodium nitrate and sodium nitrite, while the rest of the strains were unable to assimilate (Table 4). In addition, in the maximum temperature growth experiment, it was found that all strains except FM1-4 and FM1-8 could grow at 35°C (Table 5). According to the morphological, physiological and biochemical characteristics of the above isolate combined with the "Characteristics and Identification Manual of Yeast"(Kurtzman and Cletus 2011; Kurtzman et al. 2011), it can be preliminarily judged that the FM1-1 strain is Aureobasidium, the FM1-2 strain is Naganishia, and the FM1-3 strain is Barnettozyma, Strains FM1-4 and FM1-8 belong to the genus Rhodotorula, strain FM1-10 belongs to the genus Zygosaccharomyces, strain FM1-12 belongs to the genus Saccharomyces, strain FM2-10 belongs to the genus Candia, strain FM3-1 belongs to the genus Wickerhamomyces, FM3-3 and The FM3-7 strain belongs to the genus Pichia.
Molecular biology identification and phylogenetic tree construction
Sequence analysis of 26S rDNA D1/D2 region was performed on yeast isolates in honey. Amplification of the NL1 and NL4 regions produced 400 to 600 bp fragments. According to the sequencing results, submit to the GenBank database of the National Center for Biotechnology Information (NCBI), and use the basic local alignment search tool (BLAST) for homology search, similarity> 99% was applied to the species-level identification of isolates (Fang et al. 2012). Identified strains represented 11 species belonging to 9 genera, namely Aureobasidium, Naganishia, Barnettozyma, Rhodotorula, Zygosaccharomyces, Saccharomyces, Wickerhamomyces, Candia and Pichia. (Table 1). The main species are designated as Zygosaccharomyces ssiamensis, Aureobasidium pullulans and Naganishia albida. Among the 35 identified yeast strains, 24 belonged to Basidiomycetes (approximately 68.6%), while the remaining 11 strains were designated as Ascomycetes (approximately 31.4%). The phylogenetic tree constructed based on the 26S rDNA D1/D2 region further confirmed its taxonomic status (Figure 4).
Yeast community structure composition
Yeast communities from different sources show different species diversity. The majority of strains isolated from oleaster honey are Z.siamensis and N. albida, a colony frequency of 33.33% and 26.67%; the strains isolated from sunflower honey are mainly Z.siamensis, with a colony frequency of 76.92%. The main strain isolated from honey of various flowers are also Z.siamensis, with a colony frequency of 42.86%. Although Z.siamensis is the dominant strain in the three honey, it is more common in sunflower honey. It is worth mentioning that there are rich types of yeasts in oleaster honey, including 7 species, including A.pullulans, C.albidus, B.californica, R.glutinis, Z.siamensis, R.kratochvilovae, R.dioboratum; There are only 3 species in Shihezi sunflower honey, while there are 5 species in Yili honey of various flowers (Table 6).