Colacogloea armeniacae sp. nov., a novel pucciniomycetous yeast species isolated from apricots

Abstract

A survey of yeasts associated with apricots was carried out in May 2019. Two strains isolated from two fresh apricots were identified as a novel species of the genus Colacogloea based on a multi locus phylogenetic analysis, and physiological and biochemical tests. The two strains differed from any hitherto known Colacogloea species by at least 18 nucleotide substitutions (3%) in the D1/D2 domains of the LSU rRNA gene and by more than 10% mismatches in the ITS region. Also, the two strains differed phylogenetically from their closest relatives, C. falcata and C. diffluens, in its inability to assimilate galactose, D-xylose and citrate. The new species C. armeniacae sp. nov. is proposed here to accommodate those two strains.

The genus Colacogloea (Colacogloeaceae, Microbotryomycetes incertae, Microbotryomycetes, Pucciniomycotina) was described by Oberwinkler et al. (1990) to accommodate a single species, C. peniophorae (Bourdot & Galzin) Oberw., R. Bauer & Bandoni, (originally described as Platygloea peniophorae; Bourdot & Galzin, 1909). Later, another species of the genus Platygloea, P. bispora Hauerslev, was transferred to Colacogloea as C. bispora (Hauerslev) Oberw. & R. Bauer (Oberwinkler et al., 1999). Two new species of Colacogloea, namely C. papilionacea R. Kirschner & Oberw. and C. allantospora Ginns & Bandoni, were proposed by Kirschner & Oberwinkler (2000) and Bandoni et al. (2002), respectively. The genus comprised four mycoparasitic species, reproducing sexually only in a host, with transversely septate basidia, “simple” septal pores and colacosomes (Oberwinkler et al., 1990; Sampaio et al., 2011). Among them, C. peniophorae and C. papilionacea have a dimorphic life cycle with an asexual yeast state, which resembled yeasts from the formerly polyphyletic anamorphic genus Rhodotorula species, namely R. cycloclastica Thanh, M.S. Smit, Moleleki & Fell, R. terpenoidalis Thanh, M.S. Smit, Moleleki & Fell, R. philyla (Van der Walt, Klift & D.B. Scott) Rodr. Mir. & Weijman, and R. retinophila Thanh, M.S. Smit, Moleleki & Fell. These yeasts were isolated from a soil sample collected in an eucalyptus plantation, beetle-infested Harpephyllum caffrum Bernh. ex C.Krauss tree and plant residues under a pine tree (Sampaio et al., 2011). Phylogenetic analyses showed that in addition to the four Rhodotorula species, the Colacogloea clade (Sampaio et al., 2011) included R. foliorum (Ruinen) Rodr. Mir. & Weijman, R. diffluens (Ruinen) Arx & Weijman and Sporobolomyces falcatus Nakase, Itoh & M. Suzuki, all of which were isolated from leaves (Sampaio et al., 2011; Wang et al., 2015a). Considering the results of multi-locus phylogenetic analyses performed by Wang et al. (2015a, 2015b), the aforementioned asexual yeasts were transferred in the genus Colacogloea. The genus Colacogloea was emended to include anamorphic species. In total, twelve species, including four teleomorphic and eight anamorphic species were accepted (Wang et al., 2015b). Recently, four new species and one new combination of Colacogloea, C. demeterae Yurkov, A.M. Schäfer & Begerow from grassland soil in Germany, and C. aletridis Q.M. Wang, F.Y. Bai & A.H. Li, C. hydrangeae Q.M. Wang, F.Y. Bai & A.H. Li and C. rhododendri Q.M. Wang, F.Y. Bai & A.H. Li from leaves in China and C. subericola (Belloch, Villa-Carv., Álv.-Rodríg. & Coque) Q.M. Wang & F.Y. Bai (originally described as Rhodotorula subericola; Belloch et al., 2007) from bark of cork oak in Spain were described by Yurkov et al. (2016) and Li et al. (2020), respectively.

Huangcangyu National Forest Park situated in Xiaoxian, Anhui province, China, covers a total area of 26.5 square kilometers with the forest coverage rate of 97%. The area represents a secondary forest with more than 200 species of woody plants. The climate is humid subtropical and warm (average temperature 15.2 ºC, precipitation 728 mm). The majority of wild fruit trees growing in the forest park are apricots (Prunus armeniaca L, syn. Armeniaca vulgaris Lam.), which are distributed in blocks across the area. During a survey of yeasts diversity on wild fruits in the Huangcangyu National Forest Park, a total of 60 yeast strains were isolated from four fruit samples. Two of them, strains AnhuiB6 and AnhuiD28, were isolated from two apricot samples collected from two different locations (34°2′24″ N, 117°3′10″ E, 130 m asl. and 33°57′29″ N, 117°4′37″ E, 110 m asl, respectively) in May 2019. The two strains were identified as a potential novel species of the genus Colacogloea, based on phylogenetic analyses of nucleotide sequences of the internal transcribed spacer (ITS) region and D1/D2 domains of the large subunit (LSU) rRNA gene.

Fresh fruits were cut into small pieces of the size 0.5–1 cm, and four to six pieces of each fruits were placed in YM (yeast extract-malt extract-peptone-glucose) liquid medium (1.0% glucose, 0.5% peptone [Difco, Detroit, USA], 0.3% malt extract [Difco, Detroit, USA], 0.3% yeast extract [Oxoid, Basingstoke, England], pH 6.0, 100 µg/mL chloramphenicol) and incubated at 25 °C for 24–48 h in a shaking bed (150 rpm). Yeast strains were isolated from the enrichment culture following the method described by Sun et al. (2020).

Morphological, physiological and biochemical characteristics were examined according to standard methods (Kurtzman et al., 2011). Assimilation tests of carbon and nitrogen sources were carried out in liquid media. In order to induce sexual cycle, of the new species, strains were incubated (singly and mixed) on YM agar, PDA (Potato-dextrose agar, 0.4% potato starch (from infusion), 2% dextrose, 1.5% agar, Difco, Detroit, USA), V8 agar (2% V8 juice [Campbell Soup, Camden, USA], 2% agar) and CM agar (corn meal, 5% infusion corn meal, 1.5% agar, Difco, Detroit, USA) at 25 °C for two mo.

Genomic DNA was extracted from yeast cells according to Li et al. (2020). The ITS region and the D1/D2 domains of the LSU rRNA gene were amplified and sequenced as described by Wang and Bai (2004). The SSU rRNA gene and fragments of genes encoding the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2), translation elongation factor 1-α (TEF1) and the mitochondrial gene cytochrome b (CYTB) were amplified and sequenced according to the protocol described in Wang et al. (2015a). The SSU, ITS, D1/D2, RPB1, RPB2, TEF1 and CYTB sequences were aligned with Muscle software (Edgar, 2004), and concatenated in MEGA 7.0 software (Kumar et al., 2016). Two maximum-likelihood phylogenetic trees were calculated using the best-fit model GTR+I+G determined in MEGA 7.0 software (Kumar et al., 2016) for two datasets, (i) LSU rRNA and (ii) seven-loci combined. Statistical support for clades was calculated from bootstrap analyses with 1000 replicates (Felsenstein, 1985).

Strains AnhuiB6 and AnhuiD28 shared identical LSU sequences. ITS sequences of the two strains differed in 2 nucleotides (nt) that suggested that they belong to the same species. A BLAST search (blastn), ITS sequences of the two strains queried against the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/), showed the best match with C. falcata (KY102573) and C. diffluens (KY102571) with 89% and 90% similarity, respectively. Comparison of LSU sequences showed that the two strains differed in 3–5% (18–30 nt) to six described Colacogloea species, C. diffluens (AF075485), C. demeterae (FN428967), C. falcata (AF075490), C. foliorum (AF317804), C. hydrangeae (MK050451) and C. rhododendri (MK050452) (Fig. 1). LSU sequences of the two strains from bromeliads, ‘Rhodotorula’ sp. BI240 (EU678935) and ‘Rhodotorula’ sp. BI108 (EU678940), differed from AnhuiB6 and AnhuiD28 in 2 nucleotides substitutions, suggesting that they may be conspecific. The phylogenetic analysis based on concatenated sequences of seven locus analysis demonstrated that the two strains from apricots had a close relationship with C. falcata, C. diffluens, C. hydrangeae and C. rhododendri (Fig. 2). The results of phylogenetic analyses suggested that these strains represent a novel species in the genus Colacogloea, for which the name C. armeniacae sp. nov is proposed.

Fig. 1. Phylogenetic tree drawn from the maximum-Likelihood analysis of nucleotide sequences of the D1/D2 domains of the LSU rRNA gene depicting the placement of Colacogloea armeniacae sp. nov. in the genus Colacogloea. The numbers given on branches are frequencies (>50 %) with which a given branch appeared in 1000 replicates. The tree is rooted with Udeniozyma ferulica (J.P. Samp. & Uden) Q.M. Wang, F.Y. Bai, M. Groenew. & Boekhout JCM 8231. The scale indicates the number of expected substitutions accumulated per site.

Table 1. Physiological and biochemical characteristics of Colacogloea armeniacae and its closest relatives

Characteristic	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26
C. aletridisa	-	-	+	w	-	-	+	-	-	-	-	-	+	-	-	l	-	w	-	-	-	-	+	+	+	+
C. armeniacae sp. nov.	-	-	-	-	+	-	-	-	lw,-	-	lw,-	+	+	lw,-	-	-	-	-	lw,-	-	+	-	+	+	+	+
C. cycloclasticab	-	-	-	-	-	-	-	v	-	-	-	v	+	v	-	+	-	-	-	+	+	+	-	-	+	+
C. demeteraec	+	-	+	n	n	+	+	+	+	+	-,w	-	-	w	n	w	n	n	n	+	+	+	+	+	n	n
C. diffluensb	+	+	+	+	-	+	+	+	-	+	+	+	+	+	+	+	+	-	+	-	+	+	+	+	+	-
C. eucalypticab	-	-	+	+	-	-	+	+	-	-	-	+	+	-	-	-	-	w	-	-	+	+	-	-	+	n
C. falcatad	+	-	-	-	+	-	-	+	-	-	-	+	+	+	-	-	-	-	+	-	+	+	+	+	-	-
C. foliorumb	-	-	-	-	+	-	-	+	+	+	+	+	+	+	-	+	-	-	-	+	+	+	+	+	+	-
C. hydrangeaea	-	-	+	+	-	-	l	-	-	-	-	+	+	-	-	l	-	-	+	-	-	-	+	+	w	-
C. papilionaceab	+	-	-	+	-	-	-	-	-	-	-	-	+	+	-	-	-	-	-	-	+	-	-	-	-	+
C. peniophoraeb	-	-	-	-	-	-	-	-	-	v	-	+	v	-	-	-	-	-	-	v	+	v	-	-	+	+
C. philylab	-	-	-	-	-	-	-	+	-	+	-	+	+	v	-	+	-	-	-	v	+	+	-	-	+	+
C. retinophilab	-	-	-	-	-	-	-	+	-	+	-	+	+	v	-	+	-	-	-	+	+	+	-	-	+	+
C. rhododendria	v	-	+	+	v	-	+	-	-	-	-	w	+	l	-	v	-	-	-	-	-	-	w	v	+	-
C. subericolae	-	-	+	-	-	-	-	+	-	+	-	+	+	-	-	+	-	-	-	-	+	w	+	+	+	+
C. terpenoidalisb	-	-	+	+	-	-	+	+	-	+	-	+	+	+	-	+	-	+	-	+	+	+	-	-	+	+

Note: 1:Galactose; 2:L-Sorbose; 3:Sucrose; 4:Maltose; 5:Cellobiose; 6:Raffinose; 7:Melezitose; 8:D-Xylose; 9:L-Arabinose; 10:D-Arabinose; 11:D-Ribose; 12:D-Glucosamine; 13:Ethanol; 14:Glycerol; 15:Erythritol; 16:Ribitol; 17:Galactitol; 18:Methyl-α-D-glucoside; 19:Salicin; 20:DL-Lactate; 21:Succinate; 22:Citrate; 23:Potassium nitrate; 24:Sodium nitrite; 25:Growth in vitamin-free medium; 26:Growth at 30 °C.

Abbreviations: +, positive; -, negative; l, latent; w, weak; lw, latent and weak; v, variable; n, not available.

^a Result obtained from Li et al. (2020); ^b Result obtained from Sampaio (2011); ^c Result obtained from Yurkov et al. (2016); ^d Result obtained from Hamamoto et al. (2011); ^e Result obtained from Belloch et al. (2007).

Figure 2 Phylogenetic tree drawn from the maximum-Likelihood analysis of concatenated sequences of LSU, ITS, SSU, RPB1, RPB2, TEF and CYTB regions depicting the placement of Colacogloea armeniacae sp. nov. in the genus Colacogloea. The numbers given on branches are frequencies (>50 %) with which a given branch appeared in 1000 replicates. The tree is rooted with Udeniozyma ferulica (J.P. Samp. & Uden) Q.M. Wang, F.Y. Bai, M. Groenew. & Boekhout JCM 8231. The scale indicates the number of expected substitutions per nucleotide site.

Our phylogenetic analyses showed that the genus of Colacogloea has two subclades (Fig. 1, Fig. 2). The novel species C. armeniacae was placed in the subclade I, which is comprised by anamorphic species only. The subclade II includes teleomorphic species C. peniophorae (the type species of the genus Colacogloea), C. papilionacea and seven anamorphic species. In previous studies, the two sub-clades were supported in phylogenetic trees from the four protein-coding genes and the combined seven-loci analysis (Wang et al., 2015a; Li et al., 2020), whereas not supported in phylogenetic tree from the three rRNA locus analysis (Wang et al., 2015a). The two sub-clades of the genus Colacogloea seem to represent two genera, however, further analyses using more molecular data or genomic data are needed in the future.

Taxonomy

Colacogloea armeniacae Q.-M. Wang, G.-S. Wang, Wangmu, Y. Sun sp. nov. Fig. 3. MycoBank no.: MB 835274.

Fig. 3. Light microscopic images illustrating cell morphology of Colacogloea armeniacae sp. nov. after 7 d at 25 ºC in YM broth. Bar: 10 μm.

Etymology: the specific epithet armeniacae refers to the former generic name of the apricot tree, Armeniaca vulgaris, from which the type strain was isolated.

Typus: China, Xiaoxian county, Anhui province, obtained from a fruit of Prunus armeniaca (syn. Armeniaca vulgaris), collected in May 2019, Y. Sun. Holotype CGMCC 2.6134 (strain AnhuiB6) preserved in a metabolically inactive state in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China. Metabolically inactive isotypes are preserved in the Vietnam Collection of Industrial Microorganisms, Hanoi, Vietnam (VCIM 4237) and the Agricultural Culture Collection, Beijing, China (ACCC 21433). Paratype CGMCC 2.6419 (AnhuiD28) from the same source.

Molecular characteristics (type strain): Holotype GenBank accession numbers SSU+ITS+LSU: MT252007, RPB2: MT268686, TEF1: MT268691, CYTB: MT268700; paratype GenBank accession numbers SSU+ITS+LSU: MT252008, RPB2: MT268687, TEF1: MT268692, CYTB: MT268701.

Culture characteristics: In YM broth, after 7 d at 25 ºC, cells are ovoid, ellipsoidal to cylindrical, 2.0–3.5 ×3.8–10.6 µm, occur singly or in pairs, and proliferating by polar budding (Fig. 3). A sediment is present. After 1 mo at 25 ºC on YM agar, the streak culture is cream, butyrous, and smooth. The margin is entire. In Dalmau plate culture on corn meal agar, pseudohyphae and hyphae are not formed. Sexual structures are not observed on YM, PDA, V8 and CM agar after 2 mo of incubation at 25 ºC. Ballistoconidia are not observed.

Physiological and biochemical characteristics: fermentation of glucose is negative. Growth on glucose, cellobiose, trehalose, D-glucosamine (or delayed and weak), ethanol, ribitol (or delayed and weak), D-mannitol, D-glucitol, D-Glucuronic acid and succinate. No growth on galactose, L-sorbose, sucrose, maltose, lactose, melibiose, raffinose, melezitose, inulin, soluble starch, D-xylose, L-arabinose (or delayed and weak), D-arabinose, D-ribose (or delayed and weak), L-rhamnose, N-Acetyl-D-glucosamine, methanol, glycerol (or delayed and weak), erythritol, galactitol, Methyl-α-D-glucoside, salicin (or delayed and weak), DL-lactate, citrate, hexadecane and myo-inositol. Nitrogen sources ammonium sulfate, potassium nitrate, sodium nitrite and ethylamine hydrochloride are assimilated, but not on L-lysine and cadaverine dihydrochloride. Maximum growth temperature: 32 ºC. Minimal growth temperature: 4 ºC. Optimal growth temperature: 24 ºC. Growth in vitamin-free medium is positive. Starch-like substances are not produced. Urease activity is positive. Diazonium Blue B reaction is positive.

The novel species can be physiologically distinguished from other members of the genus Colacogloea (Table 1). It differs from the two closely related species (Fig. 2), C. falcata and C. diffluens, by its inability to assimilate galactose, D-xylose and citrate.

Disclosure

The authors declare no conflicts of interest.

Acknowledgements

We thank Dr. A.M. Yurkov for correcting the English language of this manuscript. This study was supported by grants No.31570016, 31770018 and No. 31961133020 from the National Natural Science Foundation of China (NSFC), and No. 521000981388 from Advanced Talents Incubation Program of the Hebei University.

References

• Bandoni, R.J. , Krug, J. , & Ginns J. (2002) On some Colacogloea species from Canada. Czech Mycology, 54, 31-43.
• Belloch, C. , Villa-Carvajal, M. , Alvarez-Rodríguez M. L. , & Coque J.J. (2007). Rhodotorula subericola sp. nov., an anamorphic basidiomycetous yeast species isolated from bark of Quercus suber (cork oak). International Journal of Systematic and Evolutionary Microbiology, 57, 1668-1671. https://doi.org/10.1099/ijs.0.64766-0
• Bourdot, H. , & Galzin, L. (1909). Hyménomycètes de France (I. Héterobasidiés).Bulletin trimestriel de la Société mycologique de France, 2515-36.
• Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32, 1792-1797. https://doi.org/10.1093/nar/gkh340.
• Felsenstein, J. ( 1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783-791. https://doi.org/10.1111/j.1558-5646.1985. tb00420.x.
• Hamamoto, M. , Boekhout, T. , & Nakase, T. (2011). Sporobolomyces Kluyver & van Niel (1924). In: C. P. Kurtzman, J. W. Fell, & T. Boekhout (Eds). The Yeasts, A Taxonomic Study (5th ed., pp. 1929–1990). Amsterdam, Netherland: Elsevier.
• Kirschner, R. , & Oberwinkler, F. (2000). A new species of Colacogloea with zygoconidia. Sydowia, 52, 195-203.
• Kumar, S. , Stecher, G. , & Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Molecular Biology and Evolution, 33, 71870-1874. https://doi.org/10.1093/molbev/msw054.
• Kurtzman, C.P. , Fell, J.W. , Boekhout, T. , & Robert, V. (2011). Methods for isolation, phenotypic characterization and maintenance of yeasts. In: C. P. Kurtzman, J. W. Fell, & T. Boekhout (Eds). The Yeasts, A Taxonomic Study (5th ed., pp. 87– 110). Amsterdam, Netherland: Elsevier.
• Li, A.H. , Yuan, F.X. , Groenewald, M. , Bensch, K. , Yurkov, A.M. , & Li, K. , et al. (2020). Diversity and phylogeny of basidiomycetous yeasts from plant leaves and soil: Proposal of two new orders, three new families, eight new genera and one hundred and seven new species. Studies in Mycology, 96, 17-140. https://doi:10.1016/j.simyco.2020.01.002
• Oberwinkler, F. , Bauer, R. , & Bandoni, R.J. (1990). Colacogloea: a new genus in the auricularioid Heterobasidiomycetes. Canadian Journal of Botany, 68, 2531-2536. https://doi.org/10.1139/b90-318.
• Oberwinkler, F. , Bauer, R. , & Tschen, J. (1999). The mycoparasitism of Platygloea bispora. Kew Bulletin, 54, 763-769 https://www.jstor.org/stable/4110873.
• Sampaio, J.P. , (2011). Rhodotorula Harrison (1928). In: C. P. Kurtzman, J. W. Fell, & T. Boekhout (Eds). The Yeasts, A Taxonomic Study (5th ed., pp. 1873–1927). Amsterdam, Netherland: Elsevier.
• Sampaio, J.P. , Kirschner, R. , & Oberwinkler, F. (2011). Colacogloea Oberwinkler (1990). In: C. P. Kurtzman, J. W. Fell, & T. Boekhout (Eds). The Yeasts, A Taxonomic Study (5th ed., pp. 1403–1408).Amsterdam, Netherland: Elsevier.
• Sun, Y. , Wang, G.S. , Li, A.H. , Wangmu, Chui, X.Q. , & Jiang, J.H. , et al. (2020). Phaeotremella camelliae sp. nov. (Phaeotremellaceae, Tremellales), a novel yeasts isolated from tea-oil fruits in Jiangxi province China. Current Microbiology. https://doi.org/10.1007/s00284-020-02061-9.
• Wang, Q.M. , & Bai, F.Y. (2004). Four new yeast species of the genus Sporobolomyces from plant leaves. FEMS Yeast Research, 4, 579-586. https://doi.org/10.1016/j.femsyr.2003.11.002
• Wang, Q.M. , Groenewald, M. , Takashima, M. , Theelen, B. , Han P.J. , & Liu, X.Z. et al. (2015a). Phylogeny of yeasts and related filamentous fungi within Pucciniomycotina determined from multigene sequence analyses. Studies in Mycology, 81, 27-54. https://doi.org/10.1016/j.simyco.2015.08.002.
• Wang, Q.M. , Yurkov, A.M. , Göker, M. , Lumbsch, H.T. , Leavitt, S.D. , & Groenewald, M. et al. ( 2015b). Phylogenetic classification of yeasts and related taxa within Pucciniomycotina. Studies in Mycology, 81, 149-189. https://doi.org/10.1016/j.simyco.2015.12.002.
• Yurkov, A.M. , Wehde, T. , Federici, J. , Schäfer, A.M. , Ebinghaus, M. , & Lotze-Engelhard, S. et al. (2016). Yeast diversity and species recovery rates from beech forest soils. Mycological Progress, 15, 845-859. https://doi.org/10.1007/s11557-016-1206-8.