INTRODUCTION
Amos and Barnett introduced the genus Umbelopsis with the type species namely U. versiformis [1]. The members of the subgenus Micromucor were reclassified from Mortierellaceae to the Mucoraceae as the species of the genera Micromucor and Umbelopsis [2]. Later the species of Micromucor and Umbelopsis were combined into the genus Umbelopsis [3]. The genus Umbelopsis comprised two major clades of species, which cannot be differentiated unequivocally by a particular set of morphological characters [4]. Based on molecular evidence these two genera were confirmed to be monophyletic and all these fungi were consequently treated as the members of Umbelopsis [4].
The genus Mucor is part of the family Mucoraceae under the order of Mucorales within the phylum of Mucoromycota [5]. The genus are characterized by fast-growing colonies and the production of simple and/or branched sporangiophores as well as globular sporangia [2,6]. In the genus Mucor, there are more than 50 species that were identified based on the molecular and morphological characteristics [7]. Recently, the numbers of Mucor species were increased with a lot of new taxa worldwide [8]. The various species of Mucor are used in biotechnology applications including bioremediation (remove oil from water) [9], production of biofuel [10], bioproteins [11], pharmaceuticals, industrial enzymes and chemicals [12]. In some rare cases, Mucor species such as M. circinelloides can be involved in human infection [13].
The aim of the current study was to carry out the morphological and molecular analyses of opportunistic fungi from the ecosystems in Korea. Moreover, the accurate identification of the species is very important for further research on antifungal efficacy as well as biotechnology applications.
MATERIALS AND METHODS
Soil sampling and fungal isolation
In 2018 and 2019, the soil samples were collected from Yeongcheon (35°57'56.4"N, 128°59'27.5"E), and grasshoppers were obtained from Daejeon (36°22'33.7"N, 127°22'36.4"E), Korea, respectively. The soil was gathered from a depth of 15 to 30 cm. After air drying, the sample was transferred to polythene zipper bags and then stored at 4℃ before using. For the examination, 1 g of the soil was suspended in 10 mL of sterile distilled water and gently vortexed. The suspension was serially diluted, and 100 μL of each sample was spread on potato dextrose agar plates (PDA; Difco, Detroit, MI, USA). The guts of Chinese rice grasshopper (Oxya chinensis), Chinese grasshopper (Acrida cinerea), and Far eastern devil grasshopper (Oedaleus infernalis) were dissected. The dissected guts were then grinded with double distilled water (DDW), spread on PDA media, and cultured at 25℃ for 3-4 days. The pure cultures of fungal strains were transferred on fresh PDA plates and incubated at 25℃, and then molecular analyses were carried out.
Morphological characterization
To inspect the morphology, the strain KNU-YC-1801B was cultured on three different media, namely, PDA, malt extract agar (MEA; Difco, Detroit, MI, USA), and corn meal agar (CMA; Difco, Detroit, MI, USA), for 5 days and incubated at 25℃ [14]. The strains KNU-20F7, KNU-20F8, and KNU-20F9 were cultured on PDA and MEA and incubated at 25℃ for 3-5 days, after which the cultural and morphological characteristics were examined [15,16]. Then the colony characteristics including color, shape, and size were noted following incubation. A light microscope (BX-50; Olympus, Tokyo, Japan) was utilized to observe the morphological structures of the strain.
Genomic DNA extraction, PCR amplification, and sequencing
The genomic DNA of the strains KNU-YC-1801B, KNU-20F7, KNU-20F8, and KNU-20F9 was extracted with the use of the HiGene Genomic DNA prep kit (Biofact, Daejeon, Korea) following the manufacturer instructions. Polymerase chain reaction (PCR) was carried out to amplify using the primers ITS1F/ITS4 [17,18] for the internal transcribed spacer (ITS) regions, NL1/NL4 [19], and LROR/LR5 [20] for the large subunit (LSU). Then, the amplified PCR products were purified with ExoSAP-IT (Thermo Fisher Scientific, Waltham, USA) and sequenced by Macrogen Co. Ltd. (Daejeon, Korea).
Phylogenetic analyses
Additional sequences were retrieved from the National Center for Biotechnology Information (NCBI) complemented the sequences created in this study (Table 1). The sequences were carried out to show the BLAST search results of the closest members with the taxa. The evolutionary distance matrices were determined with the use of Kimura’s two-parameter model for the neighbor-joining (NJ) algorithm [21]. The tree topology deduced the phylogenetic relationships by using the software program MEGA7.0 with the bootstrap analysis of 1000 replications [22].
RESULTS AND DISCUSSION
Umbelopsis vinacea (Dixon-Stew.) Arx, Sydowia 35:20 (1984) [MB#115505] (Fig. 1)
Fig. 1. Cultural and morphological characteristics of Umbelopsis vinacea KNU-YC-1801B. Colony on potato dextrose agar (A), malt extract agar (B), and corn meal agar (C), correspondingly following 14 days at 25℃; sporangiophores showing branching type (D-F); chlamydospores (G-I); sporangia (J, K); sporangiospores (L). Scale bars: D-L=5 μm.
Specimen examined: Yeongcheon (35°57'56.4"N, 128°59'27.5"E), isolated from soil. The stock culture (NIBRFGC000502246) was deposited in the National Institute of Biological Resources (NIBR) as a metabolically inactive culture.
Morphology of the strain KNU-YC-1801B
On PDA, the colonies 21.0-24.0 mm diam. after a 5-day incubation at 25℃; surface white, non-aerial, and slightly velvety; reverse white, then pale purplish after 2-3 weeks (Fig. 1A). On MEA, colonies 26.0-29.0 mm diam. and cottony felt, slightly reddish in color in the center; reverse brown to yellowish (Fig. 1B). On CMA, colonies 15.0-18.0 mm diam., slow growth, slightly pinkish in the center with age (Fig. 1C). Sporangiophores branched, 33.0-91.6 μm long, one septum, with 2.6-3.4 μm near the base and 2.4-2.8 μm near the tip (Fig. 1D-F). Chlamydospores elliptical, globose to subglobose, single or compact, oil droplets and with a diameter of 8.8-12.9 × 4.8-11.4 μm (Fig. 1G-I). Sporangia globose to subglobose, brown, multi-spored, and brown, with a diameter of 7.2-14.0 μm (Fig. 1J and 1K). Sporangiospores distinctly angular, five to seven edges, hyaline and one-celled containing oil droplet, with a diameter of 3.3-5.2×2.5-4.6 μm (n=100) (Fig. 1L). Zygospores not observed. The cultural and morphological characteristics were the same with previously identified U. vinacea (Table 2).
Molecular phylogeny of the strain KNU-YC-1801B
Genetic sequences of the ITS regions and 28S rDNA were examined to determine the evolutionary relationships with the strains acquired from GenBank (Table 1). The nucleotide sequences, 561 and 868 bp, were obtained from the ITS regions and 28S rDNA, respectively. The BLAST results have shown maximum 99.37% and 99.21% similarities from ITS regions with the strains of Umbelopsis vinacea NEFU37 and U. vinacea CBS 236.82, respectively. The large subunit (LSU) has shown the highest (100% and 99.54%) identities with the strains of U. vinacea (CBS 236.82, CBS 212.32, CGMCC 3.16357), respectively. The combined sequences of ITS regions and 28S rDNA genes were utilized to carry out the phylogenetic analysis. The phylogenetic tree designated that the strain KNU-YC-1801B was grouped together with the previously identified strain U. vinacea (Fig. 2). Therefore, the phylogenetic results firmly support the fact that the strain KNU-YC-1801B is Umbelopsis vinacea. The strain KNU-YC-1801B was deposited in the National Institute of Biological Resources (NIBRFGC000502246).
Lately, Umbelopsis changbaiensis was isolated from amphibian feces and soil samples in Korea [23]. U. dimorpha was isolated from the roots of two terrestrial orchids (Cypripedium japonicum and C. macranthum) during the screening of orchid endophytic fungi in Korea [24]. Furthermore, U. ramanniana was found to be an endophytic fungus that is related to Pinus thunbergii in coastal shelterbelts of Korea [25]. Both U. nana and U. vinacea are isolated from forest soils in Japan [26]. Also, other strains of U. vinacea were isolated from sandy loam soil, forest soil, and soil under bushes in Australia and China, respectively [14]. The strain KNU-YC-1801B was isolated from the soil in Korea. For this reason, further studies are needed to provide an in-depth knowledge as regards this species. In this study, we report Umbelopsis vinacea for the first time in Korea.
Fig. 2. Neighbor-joining phylogenetic tree based on the combined sequences of internal transcribed spacer (ITS) regions and large subunit (LSU), indicating the relationship between Umbelopsis vinacea and the closest Umbelopsis spp. The tree was rooted using Mortierella verticillata NRRL 6337 as an outgroup. The strain isolated in this study is in bold, and the bootstrap values are based on 1000 replications (values smaller than 60% were not shown). Bar, 0.1 substitutions per nucleotide position.
Mucor hiemalis f. corticola (Hagem) Schipper, Studies in Mycology 4:31 (1973) [MB#348494] (Fig. 3)
The strains KNU-20F7, KNU-20F8, and KNU-20F9 were assessed and were found to be the same and clustered together with molecular phylogeny (Fig. 4). Therefore, KNU-20F7, KNU-20F8, and KNU-20F9 strains were recognized as Mucor hiemalis f. corticola. For this reason, one strain (KNU-20F7) was chosen to describe this taxon.
Specimen examined: Daejeon (36°22'33.7"N, 127°22'36.4"E), isolated from grasshoppers. The stock cultures (KNU-20F7, KNU-20F8, and KNU-20F9) were maintained at fungal plant pathology laboratory, Kyungpook National University, Korea as a metabolically inactive culture.
Morphology of the strain KNU-20F7
On PDA, the colonies 90 mm in diam. after a 5-day incubation at 25℃; grew rapidly, filled the petri dish, up to 15 mm in height, white to light gray at first, and then it turned pale yellow (Fig. 3A). On MEA, the colonies 90 mm in diam. also after a 5-day incubation at 25℃; grew quickly, light gray in color, and then turned pale yellow (Fig. 3B). Columellae hyaline to light brown, conical, subglobose to ellipsoidial, diameter of 17.5-35.4×10.5-26.3 μm, with the presence of a clear collar (Fig. 3C-F). Sporangiophores branched monopodially or sympodially, comprising yellowish contents, with a diameter of 4.4-12.6 μm. Sporangia hyaline to light brown at first and then dark brown when they matured, diameter of 14.6-31.0 × 11.2-17.9 μm, with an average diameter of 13.6-19.6 μm (Fig. 3G-I). Sporangiospores oval, ellipsoidal to broadly ellipsoidal, a diameter of 5.1-9.8×3.1-5.0 μm (n=30), with an average diameter of 3.8-6.1 μm (Fig. 3J). The cultural and morphological characteristics were similar to those previously reported M. hiemalis f. corticola (Table 3). This result suggests that the fungal strain KNU-20F7 was closely associated with M. hiemalis f. corticola.
Molecular phylogeny of the strain KNU-20F7
The nucleotide sequences of the ITS (619 bp) and 28S rDNA (643 bp) were acquired and compared to the GenBank database with the use of BLAST to categorize the isolated fungal strain at the species level (Table 1). The BLAST search results of ITS sequences reflected 100% similarity with the strains of Mucor hiemalis f. corticola CBS 362.68 and 99.84% similarities with the different strains of M. hiemalis f. corticola (KCCM60281, CBS 366.68). The large subunit (LSU) gene has shown 98.59-100% similarities with the strains of M. hiemalis f. corticola (CBS 106.09, CBS 366.68, CBS 362.68). The strains KNU-20F8 and KNU-20F9 also manifested a maximum of 99.83-100% similarities from ITS regions (593, 608 bp) with the different strains of M. hiemalis f. corticola (CBS 366.68, CBS 362.68, F95, F96). The 28S rDNA (643, 641 bp) gene has shown 98.29-99.84% similarities with the different strains of M. hiemalis f. corticola (CBS 366.68, CBS 106.09). A combination of ITS regions and 28S rDNA genes sequences was utilized to carry out the phylogenetic analysis with the use of NJ method to know the exact taxonomic position of the strain. The phylogenetic tree has shown that the strains KNU-20F7, KNU-20F8, and KNU-20F9 were grouped together with the previously known M. hiemalis f. corticola (Fig. 4). Therefore, the phylogenetic results support the fact that the strain KNU-20F7 is M. hiemalis f. corticola.
However, the members of the genus Mucor are usually isolated from diversified sources including soil, fruit, vegetables, stored grains, insects, or dung [27]. Recently, there are some Mucor species, namely, M. abundans, M. aligarensis, M. moelleri, and M. heterogamus, that have been reported from freshwater and sediment samples in Korea [28]. Mucor ardhlaengiktus and M. gigasporus were identified from amphibian feces and soil samples in Korea [23]. Furthermore, the zygomycetous fungal strain named M. ramosissimus was also isolated from freshwater samples in Busan, Korea [29]. A fruit soft rot due to M. piriformis occurred on sweet persimmon storages in Gyeongnam Province, Korea [30].
So, further investigation is needed to explore the etiology of M. hiemalis f. corticola and the pathogenicity based on Korean ecological and environmental conditions. In this study, we also firstly report M. hiemalis f. corticola in Korea.
