Abstract
Three isolates belonging to the ascomycetous genus Zygotorulaspora were obtained from the fruits of Cornus officinalis and Smilax china, and flowers of Dendranthema zawadskii var. latilobum in Gongju-si, Korea. Phylogenetic Analyses of the LSU D1/D2 domain and ITS region sequences supported the recognition of two new species: Zygotorulaspora cornina sp. nov. (type strain NIBRFGC000500475 = KACC93346PPP) and Zygotorulaspora smilacis sp. nov. (type strain NIBRFGC000500476 = KACC93347PPP). The two novel species revealed no growth on D-Galactose, unlike the other six species in the genus Zygotorulaspora. They are distinguished from each other by their phylogenetic differences and phenotypic characteristics such as assimilation of xylitol, 5-keto-D-gluconate, and ethanol. All species in the genus Zygotorulaspora including the two novel species have phenotypic traits of genus Zygotorulaspora: asci are persistent, sucrose and raffinose are assimilated, and m-inositol is not required for growth, and they are mainly associated with plants.
Keywords: Zygotorulaspora cornina sp. nov., Zygotorulaspora smilacis sp. nov., ascomycete yeast, new yeast species
The genus Zygotorulaspora (Saccharomycetales, Saccharomycetaceae) was described by Kurtzman [1] to accommodate two species, Z. florentina and Z. mrakii, which had previously been assigned to the genus Zygosaccharomyces. As the clade containing the two species shows weak association with both Zygosaccharomyces and Torulaspora, Kurtzman suggested the Zygotorulaspora as a sister genus [2]. The genus Zygotorulaspora is characterized by vegetative reproduction with multilateral budding, persistent asci, assimilation of ribitol, mannitol, glucitol and succinate, and no use of m-inositol for growth [1]. At the time of writing, the genus Zygotorulaspora consists of six sexual species. Strains of Z. florentina have mainly been found in soft drinks, juices, and plants. The two strains of Z. mrakii, the type species of the genus, were isolated from silage. Carvalho et al. [3] reported two new species, Z. chibaensis and Z. danielsina, which were isolated using a Saccharomyces enrichment protocol and low temperatures from tree bark and soil under trees. Moreira et al. [4] described a new species, Z. cariocana, which was isolated from tree bark in Brazil. In a recent study [5], Z. dagestanica was proposed as new species of the genus Zygotorulaspora. The type strain of Z. dagestanica was isolated from soil underneath Georgian honeysuckle (Lonicera, Caprifoliaceae).
Through a survey of the indigenous yeast species of Korea, we discovered undescribed ascomycetous yeast species. As a result of phylogenetic analyses and physiological testing, we suggest these strains as two novel ascomycetous yeast species in the genus Zygotorulaspora. Zygotorulaspora cornina sp. nov. was found in the fruits of Cornus officinalis and Z. smilacis sp. nov. were found in fruits of Smilax china and flowers of Dendranthema zawadskii var. latilobum in Gongju-si, Korea.
Plant materials were collected from a mixed pine-oak forest (Pinus densiflora and Quercus spp.) in Gongju-si in the Midwest of South Korea (36°26’N, 127°07’E) in October 2017. To isolate yeast strains, wild fruits and flowers were collected aseptically, and approximately 3 g of each sample was added to conical tubes containing 10% malt extract media. The tubes were tightly capped and incubated at 30 °C without shaking. Turbidity and gas formation in the tubes were periodically surveyed for two weeks. Samples exhibiting yeast growth were spread onto YPD media plates were incubated at 30 °C for two weeks. Yeast colonies with different colors and morphologies were isolated. All yeast isolates were purified by repeatedly streaking the YPD agar plates three times, and the isolates were preserved as 10% glycerol stocks at −80 °C in the culture collection repository at the NIBR (National Institute of Biological Resources), Korea. The isotypes of two novel species were deposited KACC (patent-pending).
DNA was extracted from the yeast colonies using a Nucleospin plant kit (Macherey-Nagel, Düren, Germany). The extracted DNA was amplified using the primers ITS1f [6] and ITS4 [7] for the ITS region, and NL1 and NL4 [8] for the LSU D1/D2 domain with the following thermal cycling parameters: initial denaturation for 5 min at 94 °C, 30 cycles each 1 min at 94 °C, 30 sec at 55 °C, and 1 min at 73 °C, and final elongation for 10 min at 72 °C. The amplicons were sequenced by Macrogen (Seoul, Korea).
Combined alignment of LSU D1/D2 domain and ITS region was used for phylogenetic analysis. The phylogenetic tree was constructed using the program provided with the MEGA7 [9] software package. Phylogenetic analysis of the novel species was based on Tamura-Nei, using a discrete gamma distribution model and the maximum-likelihood method, as suggested by the implemented model test. Bootstrap analysis was carried out using 1000 replicates to estimate the confidence of the tree nodes, and the other parameters retained their default settings.
Carbon assimilation was assessed in glass vials containing yeast nitrogen base liquid media. Nitrogen assimilation was carried out on yeast carbon base agar. Other physiological and chemotaxonomical tests were performed following standard protocols [10]. For microscopy, isolates were grown at 25 °C on YM agar and evaluated using phase contrast optics. Strains were examined for the sexual state after growth and incubation at 25 °C on 5% malt extract agar, McClary acetate agar, corn meal agar (CMA), yeast morphology agar, potato dextrose agar, V8 juice agar, water agar, and YM agar. All experiments were independently carried out in three vials or on three plates. For the analysis of the composition of ubiquinone, type strain of Z. chibaensis (CBS 15364) and Z. danielsina (CBS 15365) was purchased from CBS.
Thirteen ascomycetous yeast strains were isolated using the enrichment method with 10% malt extract. All of the strains were identified by analyzing the sequences of LSU D1/D2 domain and ITS region. These isolates belonged to Hanseniaspora opuntiae (one strain), Hanseniaspora uvarum (three strains), Kazachstania sp. (one strain), Lachancea thermotolerans (three strains), Pichia kluyveri (one strain), Schizosaccharomyces japonicus (one strain), and two noble Zygotorulaspora species (three strains). The strains isolated are listed in Table S1.
Phylogenetic analysis of the novel species strains confirmed the placement of the three strains in the genus Zygotorulaspora (order Saccharomycetales, subphylum Saccharomycotina; one Z. cornina [NIBRFGC000500475T] and two Z. smilacis [NIBRFGC000500476T, NIBRFGC000500477] strains) (Figure 1). Two novel species are closely related with Z. chibaensis, Z. florentina and Z. danielsina group, but exhibit 130–172 substitutions in ITS and 24–28 substitutions in D1/D2 regions. The sequences of the two strains NIBRFGC000500476 and NIBRFGC000500477 of Z. smilacis were identical in the ITS and D1/D2 regions. In terms of pairwise sequence similarity, Z. cornina (NIBRFGC000500475T) and Z. smilacis (NIBRFGC000500476T) were the most closely related species, but had nine nucleotide substitutions in the LSU D1/D2 region and two-nucleotide substitution and one gap in the ITS region (98.5% and 99.5%, respectively). Kurtzman and Robnett [8] reported that strains having greater than 1% substitutions in the nucleotide D1/D2 domain (ca. 600) are likely to be different species, supporting the contention that Z. cornina and Z. smilacis are different species. The genetic distance of the two novel species is similar to the distance between Z. chibaensis and Z. florentina, with eight nucleotide substitutions in the LSU D1/D2 region, and between two and four nucleotide substitutions in the ITS region [3]. BLAST search revealed that the D1/D2 region sequence of VdF2-P092, IFO 11070 was identical to that of Z. cornina, and CE41 and IFO 11069 were identical to Z. smilacis, but only in D1/D2 in those cases (Figure S1). It implies additional strains of Z. cornina and Z. smilacis Carvalho et al. [3] identified, based on D1/D2 and ITS sequences, a possible new Zygotorulaspora species (strain MM1) which we reveal here to constitute an additional representative of Z. cornina because of its identical sequences in the LSU D1/D2 domain and ITS region.
Figure 1.
Phylogenetic tree drawn from maximum-likelihood analysis of combined sequences of the LSU D1/D2 domain and ITS regions, showing positions of the two novel species. The tree was rooted with Kluyveromyces marxianus and K. wickerhamii. Numbers at the nodes indicate bootstrap support percentages, derived from 1000 samples (values below 70% not shown). GenBank accession numbers are indicated after strain designations.
Since the two novel species share similar cell morphology with existing Zygotorulaspora species (globose to ellipsoid; white to tannish white, beige and cream-colored; multilateral budding), it is difficult to distinguish them by only morphological characteristics, so the phenotypic characteristics must be considered. Carvalho et al. [3] suggested physiological differences among species in the genus such as the assimilation of five sugars (D-xylose, L-arabinose, maltose, L-sorbose, D-mannitol) and growth at 35 °C for species recognition. We suggest the use of additional physiological traits to distinguish the species in this genus (Table 1). The two novel species differ in their fermentation of D-galactose and assimilation of D-galactose, DL-lactate, and ethylamine, compared to other Zygotorulaspora species. Z. cornina and Z. smilacis are distinguished from each other not only based on the sequences of LSU D1/D2 domain and ITS region but also based on phenotypic characteristics such as the assimilation of xylitol, 5-keto-D-gluconate, and ethanol. Kurtzman described the genus Zygotorulaspora to accommodate two species, Z. florentina and Z. mrakii, having Q-6 as major ubiquinone [11]. Since then, four additional species have been described in Zygotorulaspora. Their composition of ubiquinone has not been studied. As a result of analysis the ubiquinone of four type strains (Z. chibaensis, CBS 15364; Z. danielsina CBS 15365; Z. corninna NIBRFGC000500475; Z. smilacis NIBRFGC000500476), it was found that Q-7 is the major ubiquinone of four species. Genus Zygotorulaspora may have six or seven isoprene units in the side chain.
Table 1.
Phenotypic characteristics that differentiate the two new species from each other and from the other six species in Zygotorulaspora.
| Yeast species |
||||||||
|---|---|---|---|---|---|---|---|---|
| Characteristics | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Fermentation of: | ||||||||
| D-Galactose | − | − | + | + | + | + | n | + |
| Maltose | + | + | + | − | + | + | n | − |
| α,α-Trehalose | + | + | + | − | − | − | n | − |
| Assimilation of carbon compounds: | ||||||||
| D-Galactose | − | − | + | + | + | + | + | + |
| L-Sorbose | + | + | + | − | + | − | + | − |
| Melibiose | − | − | + | + | + | v | + | − |
| Xylitol | w | − | n | n | + | d | + | n |
| 5-Keto-D-gluconate | − | + | − | − | n | n | n | − |
| DL-Lactate | + | + | − | − | − | − | + | − |
| Ethanol | − | + | + | v | + | + | + | − |
| Assimilation of nitrogen compounds: | ||||||||
| Ethylamine | + | + | n | n | − | − | n | n |
| Major ubiquinone | 7 | 7 | 6 | 6 | 7 | 7 | n | n |
Species: 1, Zygotorulaspora cornina sp. nov.; 2, Z. smilacis sp. nov.; 3, Z. florentina; 4, Z. mrakii; 5, Z. chibaensis; 6, Z. danielsina; 7, Z. cariocana; 8, Z. dagestanica. Data for species 1 and 2 are from the present study, for species 3 and 4 are from Kurtzman [11], for species 5 and 6 are from Carvalho et al. [3], for 7 is from Moreira et al. [4], and for species 8 is from Kachalkin et al. [5]. Growth reactions: +, strong growth; d, delayed growth; w, weak growth; −, no growth; n, no data; v, variable results.
All species in the genus Zygotorulaspora, including the two novel species, are associated with plants. The species were isolated from plants (bark, exudate, flower, and fruit), plant products (silage, soft drinks, and juice), and soil under trees. Known cultures of this genus, except for Z. cariocana, are from the temperate region of the northern hemisphere in Europe (Italy, Austria, France, Netherlands and Russia), Asia (Korea and Japan), and the southern hemisphere (New Zealand). Z. cariocana strains were isolated from a rainforest in Brazil.
Taxonomy
Zygotorulaspora cornina sp. nov. C. Ahn and C. Kim
Zygotorulaspora cornina (cor.ni’na. N.L. fem. n. cornina of cornus refers to the host plant Cornus officinalis from which this yeast was isolated).
After 1 week on YM agar at 25 °C, colonies are shiny, smooth, with an entire margin and white to tannish-white in color. After 3 days on YM agar at 25 °C, cells are globose to sub-globose (2.9–6.0 × 2.8–5.4 µm) and proliferation is by multilateral budding on a narrow base (Figure 2a). On Dalmau plates after 2 weeks at 25 °C, rudimentary pseudohyphae are present but true hyphae are not formed. Sexual reproduction is observed on corn meal agar plate after 7 days at 25 °C. Asci are persistent and form after conjugation between either a cell and its bud or two independent cells. Asci produce one to two smooth, globose ascospores, measuring 2–3 µm in diameter (Figure 2b–d). The studied strains appear to be homothallic.
Figure 2.
Micrographs of Zygotorulaspora cornina sp. nov. NIBRFGC000500475T (a–d) and Zygotorulaspora smilacis sp. nov. NIBRFGC000500475T (e–h). (a) Budding cells on YM agar 3 days at 25 °C. (b–d) Asci and ascospores on corn meal agar. (e) Budding cells on YM agar after 3 days at 25 °C. (f–h) Asci and ascospores on corn meal agar. Bar, 10 µm.
Carbon compounds fermented: D-glucose, maltose, α-methyl-D-glucoside, sucrose, α,α-trehalose, melezitose, raffinose, and inulin. No fermentation of D-galactose, melibiose, lactose, cellobiose, soluble starch, or D-xylose.
Carbon compounds assimilated: D-glucose, L-sorbose, sucrose, maltose, α,α-trehalose, methyl α-D-glucoside, raffinose, melezitose, inulin, xylitol(weak), D-glucitol, D-mannitol, D-glucono-1,5-lactone, 2-keto-D-gluconate, D-gluconate, DL-lactate, succinate, palatinose, and L-malic acid (weak).
No growth on D-galactose, D-glucosamine, D-ribose, D-xylose, L-arabinose, D-arabinose, L-rhamnose, cellobiose, salicin, arbutin, melibiose, lactose, soluble starch, glycerol, erythritol, ribitol, L-arabinitol, galactitol, myo-Inositol, 5-Keto-D-gluconate, D-glucuronate, D-galacturonate, citrate, methanol, ethanol, propane 1,2 diol, butane 2,3 diol, quinic acid, D-glucarate, levulinate, L-tartaric acid, D-tartaric acid, meso-tartaric acid, galactaric acid, uric acid, gentobiose, ethylene glycol, Tween 40, Tween 60, or Tween 80.
Nitrogen compounds assimilated: ethylamine, L-lysine, cadaverine, D-tryptophan, D-proline, and putrescine. No growth on nitrate, nitrite, creatine, creatinine, glucosamine, or imidazole. Growth on 0.01 % and 0.1% cycloheximide containing medium was positive. Grows at 35 °C but negative at 37 °C. Hydrolysis of urea and DBB reaction are negative. Grows in the absence of vitamins. The major ubiquinone system is Q-7.
Mycobank: MB837980
Type: Korea, Gongju-si, 18 Oct 2017, isolated from fruit of Cornus officinalis (holotype: NIBRFGC000500475 preserved in a metabolically inactive state at the National Institute of Biological Resources [NIBR], Incheon, Korea; isotype culture: KACC93346PPP); ex-type: ITS (MT340888) and LSU D1/D2 (MT340891) sequences.
Zygotorulaspora smilacis sp. nov. C. Ahn and C. Kim
Zygotorulaspora smilacis (smi.la’cis. N.L. fem. n. smilacis of smilax, refers to the host plant Smilax china from which this yeast was isolated).
After 1 week on YM agar at 25 °C, colonies are shiny, smooth, with an entire margin and white to tannish-white in color. After 3 days on YM agar at 25 °C, cells are globose to sub-globose (3.1–6.4 × 2.7–6.1 µm) and proliferation is by multilateral budding on a narrow base (Figure 2e). On Dalmau plates after 2 weeks at 25 °C, rudimentary pseudohyphae are present but true hyphae are not formed. Sexual reproduction is observed on corn meal agar plate after 7 days at 25 °C. Asci are persistent and form after conjugation between either a cell and its bud or two independent cells. Asci produce one to two smooth, globose ascospores, measuring 2–3 µm in diameter (Figure 2f–h). The studied strains appear to be homothallic.
Carbon compounds fermented: D-glucose, maltose, α-methyl-D-glucoside, sucrose, α,α-trehalose, melezitose, raffinose, and inulin. No fermentation of D-galactose, melibiose, lactose, cellobiose, soluble starch, or D-xylose.
Carbon compounds assimilated: D-glucose, L-sorbose, sucrose, maltose, α,α-trehalose, methyl α-D-glucoside, raffinose, melezitose, inulin, D-glucitol, D-mannitol, D-glucono-1,5-lactone, 2-keto-D-gluconate, 5-keto-D-gluconate, D-gluconate, DL-lactate, succinate, ethanol, and palatinose.
No growth on D-galactose, D-glucosamine, D-ribose, D-xylose, L-arabinose, D-arabinose, L-rhamnose, cellobiose, salicin, arbutin, melibiose, lactose, soluble starch, glycerol, erythritol, ribitol, xylitol, L-arabinitol, galactitol, myo-Inositol, D-glucuronate, D-galacturonate, citrate, methanol, propane 1,2 diol, butane 2,3 diol, quinic acid, D-glucarate, levulinate, L-malic acid, L-tartaric acid, D-tartaric acid, meso-tartaric acid, galactaric acid, uric acid, gentobiose, ethylene glycol, Tween 40, Tween 60, or Tween 80. Nitrogen compounds assimilated: ethylamine, L-lysine, cadaverine, D-tryptophan, D-proline, and putrescine. No growth on nitrate, nitrite, creatine, creatinine, glucosamine, or imidazole.
Growth on 0.01 % and 0.1% cycloheximide containing medium was positive. Grows at 35 °C but negative at 37 °C. Hydrolysis of urea and DBB reaction are negative. Grows in the absence of vitamins. The major ubiquinone system is Q-7.
Mycobank: MB837981
Type: Korea, Gongju-si, 18 Oct 2017, isolated from the fruit of Smilax china (holotype: NIBRFGC000500476 preserved in a metabolically inactive state at National Institute of Biological Resources [NIBR], Incheon, Korea; isotype culture: KACC93347PPP; paratype: NIBRFGC000500477); ex-type: ITS (MT340889) and LSU D1/D2 (MT340892) sequences.
Supplementary Material
Funding Statement
This research was supported by the project on the survey and excavation of Korean indigenous species of the National Institute of Biological Resources (NIBR 202002116) under the Ministry of Environment, Republic of Korea.
Author contributors
Chorong Ahn: Investigation; Writing – Original Draft Preparation, Minkyeong Kim: Investigation; Review and Editing, Changmu Kim: Supervision; Writing – Review and Editing and Funding.
Repositories
The GenBank accession numbers of the LSU and ITS sequences of Zygotorulaspora cornina NIBRFGC000500475T and Zygotorulaspora smilacis NIBRFGC000500476T are MT340891, MT340888, MT340892 and MT340889, respectively. The MycoBank accession numbers are MB837980 and MB837981, respectively.
Disclosure statement
The type strains described in this paper are patent-pending (details of the patent application).
References
- 1.Kurtzman CP. Phylogenetic circumscription of Saccharomyces, Kluyveromyces and other members of the Saccharomycetaceae, and the proposal of the new genera Lachancea, Nakaseomyces, Naumovia, Vanderwaltozyma and Zygotorulaspora. FEMS Yeast Res. 2003;4(3):233–245. [DOI] [PubMed] [Google Scholar]
- 2.Kurtzman CP, Robnett CJ.. Phylogenetic relationships among yeasts of the ’Saccharomyces complex’ determined from multigene sequence analyses. FEMS Yeast Res. 2003;3(4):417–432. [DOI] [PubMed] [Google Scholar]
- 3.Carvalho C, Tomas A, Libkind D, et al. Zygotorulaspora chibaensis sp. nov. and Zygotorulaspora danielsina sp. nov., novel ascomycetous yeast species from tree bark and soil. Int J Syst Evol Microbiol. 2018;68(8):2633–2637. [DOI] [PubMed] [Google Scholar]
- 4.Moreira JD, Santos ARO, Oliveira FL, et al. Zygotorulaspora cariocana sp. nov., a yeast species isolated from tree bark in Brazil. Int J Syst Evol Microbiol. 2020;70(4):2677–2681. [DOI] [PubMed] [Google Scholar]
- 5.Kachalkin AV, Abdullabekova DA, Magomedova ES, Yurkov AM.. Zygotorulaspora dagestanica sp. nov., a novel ascomycetous yeast species associated with the Georgian honeysuckle (Lonicera iberica M. Bieb.). Int J Syst Evol Microbiol. 2021;71:004785. [DOI] [PubMed] [Google Scholar]
- 6.Gardes M, Bruns TD.. ITS primers with enhanced specificity for basidiomycetes–application to the identification of mycorrhizae and rusts. Mol Ecol. 1993;2(2):113–118. [DOI] [PubMed] [Google Scholar]
- 7.White TJ, Bruns T, Lee S, et al. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR protocols: a guide to methods and applications. New York: Academic Press; 1990. p. 315–322. [Google Scholar]
- 8.Kurtzman CP, Robnett CJ.. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leewenhoek. 1998;73(4):331–371. [DOI] [PubMed] [Google Scholar]
- 9.Kumar S, Stecher G, Tamura K.. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–1874. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Kurtzman CP, Fell JW, Boekhout T, et al. Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman CP, Fell JW, Boekhout T, editors. The yeasts, a taxonomic study. 5th ed. Amsterdam: Elsevier; 2011. p. 87–110. [Google Scholar]
- 11.Kurtzman CP. 85. Zygotorulaspora Kurtzman (2003). In: Kurtzman CP, Fell JW, Boekhout T, editors. The yeasts, a taxonomic study. 5th ed. Amsterdam: Elsevier; 2011. 949–950. [Google Scholar]
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