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Isolation and Characterization of Four Unrecorded Mucor Species in Korea

Abstract During an investigation of fungi of the order Mucorales from freshwater and sediment samples in Korea, we isolated six strains, NNIBRFG6649, NNIBRFG6255, NNIBRFG1498, CNUFC-YJ13, CNUFC-YR7, and NNIBRFG2739. The morphology and phylogeny of these strains were analyzed. Based on the morphological characteristics and molecular data from internal transcribed spacer (ITS) region, the isolates NNIBRFG6649 and NNIBRFG6255 were identified as Mucor abundans, and M. aligarensis, respectively. The isolates NNIBRFG1498 and CNUFC-YJ13 were identified as M. moelleri, whereas the isolates CNUFC-YR7 and NNIBRFG2739 were identified as M. heterogamus. To the best of our knowledge, M. abundans, M. aligarensis, M. moelleri, and M. heterogamus have not yet been reported in Korea.


Introduction
The genus Mucor was described by Fresenius in 1850 [1]; it belongs to the family Mucoraceae, order Mucorales, subphylum Mucoromycotina, and phylum Mucoromycota, and comprises the largest number of species within Mucorales [2,3]. Mucor species are characterized by the formation of non-apophysate sporangia on simple or branched sporangiophores, and by zygospores that are borne on opposed [4] or tong-like suspensors [5].
More than 50 species have been reported in the genus Mucor by applying a polyphasic approach using molecular and morphological characters [6]. However, this list is rapidly increasing with many new taxa of Mucor being described worldwide recently [7][8][9][10]. Mucor species are commonly isolated from soil, fruit, vegetables, stored grains, insects, or dung [5,11].
Species belonging to this genus are valued in the biotechnology industry due to their ability to produce various secondary metabolites [12,13]. Mucor species are used in biotechnological processes such as bioremediation [14][15][16]. In addition, some Mucor species have been used in the fermentation of traditional Asian foods [17], whereas others are considered to be the causal agents of cutaneous zygomycosis in humans [18].
Mucor species have been identified traditionally by their morphological characteristics such as the size and shape of their sporangia, and their mode of reproduction. Recently, molecular data have revealed the phylogenetic position of Mucor and the relationship among its species [5]. Based on the phylogeny of ITS and large subunit rDNA regions of several mucoralean species, Walther et al. [5] observed that some Mucor species with transitory curved sporangiophores were grouped with Backusella Hesselt. & J.J. Ellis. Therefore, these Mucor species were transferred to the genus Backusella. In Korea, ten Mucor species have been reported as new records and four as new species [7,8,19,20].
During an inventory of fungal species from freshwater and sediment samples, four Mucor species were identified. The objective of the present study was to perform morphological and molecular characterization of the undescribed Mucor species in Korea: M. abundans, M. aligarensis, M. moelleri, and M. heterogamus.

Isolation of fungal strains from freshwater and sediment samples
Freshwater and sediment samples were collected from different geographical regions of the Republic of Korea. Sampling information for the present study is shown in Table 1. The samples were transported in sterile 50-mL tubes and were stored at 4 C until required. The media and method used in this study were based on the protocols described by Nguyen et al. [20] and Mun et al. [21]. The plates were incubated at 20 C and 25 C for 2-5 d. Pure isolates were obtained by picking individual colonies of varied morphologies, transferring them onto potato dextrose agar (PDA; 39 g of potato dextrose agar in 1 L of deionized water) plates, and sub-culturing them until pure mycelia were obtained. For stock storage, pure isolates were maintained in PDA slant tubes with 20% glycerol at À80 C at the Environmental Microbiology Laboratory Fungarium, Chonnam National University, Gwangju, Korea, and were also deposited at the Culture Collection of the Nakdonggang National Institute of Biological Resources (NNIBR, Sangju, Korea).

DNA extraction, polymerase chain reaction, and sequencing
Genomic DNA was extracted directly from the mycelia of fungal isolates using a SolGent Genomic DNA Prep Kit (SolGent Co. Ltd., Daejeon, Korea). The internal transcribed spacer (ITS) region was amplified using the primer pairs ITS1 and ITS4 [22]. The PCR reaction mixture (total volume, 20 mL) contained a fungal DNA template, 5 pmol/mL of each primer, AccuPower PCR PreMix (Bioneer Corp., Daejeon, Korea). The PCR products were purified using an AccuPrep PCR Purification Kit (Bioneer Corp.) according to the manufacturer's instructions. Sequencing was performed using the same PCR primers, and run on an ABI PRISM 3730XL genetic analyzer (Applied Biosystems, Waltham, MA).

Taxonomy of NNIBRFG6649
Mucor abundans Povah, Bulletin of the Torrey Botanical Club 44: 292 (1917) (Figure 2). Description: The strain grew rapidly at 25 C on PDA, and reached 80-82 mm in diameter after 5 d of incubation. The colony was initially white, but subsequently turned brown. The sporangiophores were 163.9-548.6 mm in length and 7.3-17.7 mm in width; they arose from aerial and submerged mycelia, and produced a large number of sporangia. The sporangia were globose to subglobose, 31- (Figure 3).
Description: The strain grew rapidly at 20 C on PDA, and reached 67-70 mm in diameter after 5 d of incubation. The colony was initially white, but subsequently turned brown. The sporangiophores were 957.7-1056.4 mm in length and 8.4-21 mm in width; they arose from aerial and submerged mycelia, septate and produced a large number of sporangia. The sporangia were globose to subglobose, 26.5-102.6 Â 23.0-102 mm, and were borne terminally on the main axes or branches of the sporangiophores. Columellae with large collars were subglobose, obovoid to pyriform, 23.5-55 Â 18.5-41.5 mm. Sporangiospores were hyaline, subglobose to oval, and measured 7.5-10 Â 5.5-8.5 mm. Zygosporangia were not observed.
Description: The strain grew rapidly at 25 C on PDA, spreading completely within the petri dish (90 mm in diameter) after 5 d of incubation. The initial color of the colony was white, which later turned gray. Sporangiophores arose from aerial and submerged mycelia, the formation of numerous zygosporangia was observed. Sporangia were globose to subglobose, and measured 25.6-48.8 Â 24.6-42.3 mm, and were borne terminally on the main axes or branches of sporangiophores. Columellae were conical to globose. Sporangiospores were subglobose, oval, ellipsoidal, and measured 4.3-8.5 Â 3.9-6.8 mm. Zygospores were globose to subglobose, and measured 17.3-41.3 Â 14.3-40.6 mm, reddish-brown when young, and subsequently turned black. The strain was homothallic.

Taxonomy of CNUFC-YR7
Mucor heterogamus Vuill., Bull. S eanc. Soc. Sci. Nancy Description: The strain grew rapidly at 25 C on PDA, and completely filled the petri dish (90 mm in diameter) after 5 d of incubation. The colony was initially white, but subsequently turned pale yellow,   with dark gray in the center. The sporangiophores were 3.5-15 mm in width and variable in length; they arose from aerial and submerged mycelia, and produced a small number of sporangia. The sporangia were globose to subglobose, 18.5-47.5 Â 20.3-50 mm, and were borne terminally on the main axes or branches of the sporangiophores. The zygospores were globose, up to 66.5 mm in diameter, and were formed throughout the colony, but were most abundant just above the substrate; they were reddish-brown when young, and subsequently turned black. The suspensors were globose, pyriform, and unequal. The strain was homothallic.

Discussion
To date, only 14 species of Mucor have been reported in Korea [7,8,19,20]. Therefore, any data pertaining to this genus would increase our understanding of the diversity and geographical distribution of fungal species in Korea. Here, we discuss the phylogeny and morphological characteristics of M. abundans, M. aligarensis, M. moelleri, and M. heterogamus. Walther et al. [5] proposed the use of the ITS region as a standardized marker for the sequence analysis of Mucor species. The phylogenetic tree using ITS region showed that strains NNIBRFG6649, NNIBRFG6255,  NNIBRFG1498 and CNUFC-YJ13, and CNUFC-YR7 and NNIBRFG2739 were grouped with the species M. abundans, M. aligarensis, M. moelleri, and M. heterogamus, respectively (Figure 1).
The morphological characteristics of the M. abundans isolate studied were generally similar to those previously described by Povah [25]. However, differences were observed in the size of sporangia from M. abundans with respect to that from the previous description. Our observations showed that the sporangia in isolate NNIBRFG6649 were smaller than those previously described by Povah [25] (67-78 mm). Previously M. abundans was reported to be isolated from horse dung, sandy tilled soil, decaying tomato, rabbit dung, dog dung, and forest soil [5,25]. To our knowledge, this is the first report of M. abundans isolation from freshwater.
The morphological characteristics of the isolate CNUFC-YJ13 in this study were similar to those previously described by Zheng and Liu [27], as producing globose, multispored sporangia, conical to globose columellae. However, sporangia and zygospore sizes reported in the literature [27] were bigger than those of our isolate. Sporangiospores in isolate CNUFC-YJ13 contains one or two droplets, which was not described by Zheng and Liu [27]. CNUFC-YJ13 was confirmed as M. moelleri based on morphological observations and ITS phylogenetic comparison.
Although the morphological features of isolate CNUFC-YR7 were similar to those of M. heterogamus described by Hesseltine et al. [28], there were differences in the diameters of the sporangia and zygospores. The sizes of sporangia reported in literature ranged from 15-55 mm [28], which are slightly larger than our maximum measurements. The zygospores (30-70) mm [28] were larger than those in our isolates. The molecular data confirmed the morphological identification of CNUFC-YR7 as M. heterogamus.
Several species of Mucor are of great interest to the biotechnology industry because they can produce proteolytic enzymes and secondary metabolites such as phytoalexin elicitor [13,29]. Somkuti [30] showed that M. pusillus and M. miehei produce cellulase enzymes. Andrade et al. [31] demonstrated high protease production by M. circinelloides using glucose as a substrate. Future studies should investigate the abilities of Mucor species to produce extracellular enzymes, and their potential use in biotechnology.
In general, growth temperature plays an important role in determining the physiological characteristics of fungi. Especially, Mucor spp. are able to cause infections in humans. These isolates grow over a wide range of temperatures with varying Fungi play a major role in the decomposition of complex organic compounds in freshwater ecosystems, thereby providing nutrients for other aquatic organisms. In addition, freshwater-derived fungi have been widely studied for their bioactive metabolites. Therefore, further studies are required for a better understanding of the biological role of secondary metabolites, such as extracellular enzyme activity, antibacterial, antifungal, and anticancer activities in fungi, especially in M. abundans, M. aligarensis, M. moelleri, and M. heterogamus.