Two New Species of Cryptococcus sp. and Candida sp. from Wild Flowers in Korea.

Among 80 types of yeast isolated from wild flowers in Daejeon, Korea, two species that have not yet been identified by phylogenetic analysis of the internal transcribed spacer-2 (ITS2) genes and 26S rDNA sequences were identified as Candida sp. 44-C-1 and Cryptococcus sp. 9-D-1. Neither of the newly identified species formed ascospores, while Candida sp. 44-C-1 formed pseudomycelium and Cryptococcus sp. 9-D-1 did not.

Several species of yeast belonging to the GRAS strain have long been used in the preparation of alcoholic beverages and foods such as bread and soy sauce. Since yeast have been reported to produce bioactive compounds with effects such as anti-hypertension [1, 2], anti-dementia [3], and anti-angiogenesis [4-9], the use of yeast for the production bioactive agents in alternative drugs and functional foods has been investigated.

Despite many studies of various yeasts isolated from fermented foods including alcohol fermentation products [10-13], little is known about yeasts present on wild flowers and fruits in the natural environment in Korea.

In a previous study [14], we isolated and identified 80 types of yeast from wild flowers in the City of Daejeon, Korea and its vicinity. Among the isolated yeasts, two novel species that had not yet been identified by phylogenetic analyses were recovered. These novel yeasts are characterized in the present study.

Yeasts were isolated and identified as followed. Wild flowers from mountains or fields in and around Daejeon, Korea were collected and suspended in sterilized distilled water. The supernatants were then inoculated in yeast extract-peptone-dextrose agar medium (10 g yeast extract, 20 g peptone, 20 g dextrose, 15 g agar and 1 L of distilled water) containing antibacterial streptomycin (50 µg/mL) and ampicillin (50 µg/mL) and incubated for 2 days at 30℃.

Extraction of the genomic DNA from the isolated yeasts was accomplished using a Genomic DNA prep kit for yeast (Solgent, Daejeon, Korea). Primers ITS1 and ITS4 were used for amplification of the internal transcribed spacer (ITS) region [14, 15]. The D1/D2 region of 26S rRNA was amplified by PCR using NL-1 (5'-GCA TAT CAA TAA GCG GAG GAA AAG-3') and NL-4 (5'-GGT CCG TGT TTC AAG ACG G-3') as the forward and reverse primers, respectively. Amplification reactions were performed in a total volume of 25 µL containing 2.5 µL 10 × PCR buffer, 0.5 µL 10 mM dNTPs, 0.5 µL of each primer at 20 pmol/µL, 0.13 µL Taq DNA polymerase (5 U/µL). PCR was performed using a GeneAmp PCR system 9700 (Applied Biosystem, Foster City, CA, USA) with and initial denaturation stage of 5 min at 95℃, followed by 30 cycles of denaturation for 30 sec at 95℃, annealing for 1 min at 52℃, extension for 1 min at 72℃ and a final extension at 72℃ for 5 min. Amplification products were analyzed by gel electrophoresis on 1.5% agarose gel using a 1 kb DNA ladder as a marker. A gel extraction kit (Qiagen, Valencia, CA, USA) was used for purification of the amplified PCR products and DNA strands of the amplified DNA fragments were then sequenced with the same PCR primers. Sequences of the amplified gene for each isolated yeast were merged in a single nucleotide alignment. Multiple sequence alignment by Florence Corpet was used for alignment of the resulting sequence and relevant sequences available in the GenBank database maintained by the NCBI with manual adjustment when necessary.

Screening for new species among yeasts isolated from wild flowers.

Among 80 yeasts isolated from wild flowers in Daejeon, Korea, 78 were identified based on ITS gene sequence analysis [14, 15]. However, Candida sp. 44-C-1 (GenBank accession No. KC242238) and Cryptococcus sp. 9-D-1 (GenBank accession No. KC242237) were only identified as Candida sp. TrB1-1 (GenBank accession No. AY562397.0) and Cryptococcus sp. KCTC 17072 (GenBank accession No. AF459689.1) based on 26S rDNA sequence analysis (Table 1). Furthermore, these organisms were only identified Candida sp. TrB1-1 (GenBank accession No. AY559471.1) and Cryptococcus sp. SJ8L05 (GenBank accession No. FJ153175.1) based on ITS2 region sequence analysis (Table 1). Therefore, we concluded that these two organisms were novel yeasts. A phylogenetic tree of these new yeasts is shown in Figs. 1 and 2.

Table 1

ITS2 region and 26S rDNA sequence analysis of the new yeasts

ITS, internal transcribed spacer.

aGenBank accession No. KC242237.

bGenBank accession No. KC242238.

Fig. 1

Neighbor-joining phylogenetic tree based on the D1/D2 region of the 26S rDNA sequence of Cryptococcus sp. 9-D-1 isolated from Rhododendron mucronulatum in Daejeon, Korea.

Fig. 2

Neighbor-joining phylogenetic tree based on the D1/D2 region of the 26S rDNA sequence of Candida sp. 44-C-1 isolated from Albizia julibrissin Durazz in Daejeon, Korea.

Cryptococcus sp. 9-D-1 and Candida sp. 44-C-1 were isolated from Rhododendron mucronulatum and Albizia julibrissin Durazz, respectively, in Daejeon, Korea.

Morphological characteristics of new yeasts strains.

Taxonomic descriptions and microbiological structures of unrecorded Candida sp. 44-C-1 and Cryptococcus sp. 9-D-1 are shown in Fig. 3 and Table 2. Cryptococcus sp. 9-D-1 was oval and did not form ascospores or pseudomycelium. However, Candida sp. 44-C-1 was ellipsoidal, and formed pseudomycelium, but not ascospores.

Fig. 3

Microscopic features of newly identified yeasts. A, Cryptococcus sp. 9-D-1; B, Candida sp. 44-C-1 (scale bars: A, B = 5 µm).

Table 2

Morphological characteristics of yeast recovered from wild flowers of Daejeon, Korea

O, oval; E, ellipsoidal; MB, multilateral budding.