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Molecular and Morphological Confirmation of Three Undescribed Species of Mortierella from Korea

Abstract Three fungal isolates designated as CNUFC-YR329-1, CNUFC-PTS103-1, and CNUFC-PTS2-1 were discovered during a survey of fungal diversity of the order Mortierellales from freshwater and pine tree rhizosphere soil samples in Korea. The strains were analyzed morphologically and phylogenetically based on the internal transcribed spacer (ITS) and large subunit (LSU) of ribosomal DNA gene sequences. Based on their morphology and phylogeny, the three isolates were identified as Mortierella elongata, M. horticola, and M. humilis, respectively. To the best of our knowledge, M. elongata, M. horticola, and M. humilis, belonging to an undiscovered taxon, have not been previously described in Korea.


Introduction
The genus Mortierella was described by Coemans (1863) with the type species Mortierella polycephala Coem [1]. Currently, Mortierella is a member of the family Mortierellaceae, order Mortierellales, subphylum Mortierellomycotina [2][3][4]. Until now, approximately 100 species of Mortierella have been described [5]. The species belonging to this genus are characterized by the production of a mainly coenocytic but irregularly septate mycelium. Sporangiophores are simple or variously branched, terminating with sporangia and occasionally with a swelling at the base. Sporangia are globose, with multiple or few spores, or a single spore. Species of Mortierella are frequently isolated from soil and dead or dying plant tissues, freshwater, or from animal fecal samples [6][7][8][9][10][11].
Many of the species are potential producers of c-linolenic acid and arachidonic acid [12]. In addition, some members of the genus Mortierella assist crops and mycorrhizal fungi in phosphorus (P) acquisition [13] and also can synthesize and secrete oxalic acid [14]. They also have shown great capacity to decompose plant litter and degrade polyaromatic hydrocarbons [15].
The objective of the present study was to perform morphological and molecular analyses to characterize three undescribed fungal species in Korea: M. elongata, M. horticola, and M. humilis.
in sterile 50 mL conical tubes and stored at 4 C until examination. Fungi were isolated by a serial dilution plating method as described previously [21], and cultured on potato dextrose agar (PDA: 39 g PDA in 1 L of deionized water; Becton, Dickinson and Co., Sparks, MD, USA) supplemented with streptomycin sulfate 0.5 mg/mL, to suppress bacterial growth. After incubation at 25 C for 3-7 days, individual hyphal tips of the developing fungal colonies were removed and placed onto PDA medium. Pure isolates were maintained in PDA slant tubes and stored in 20% glycerol at -80 C at the Environmental Microbiology Laboratory Fungarium, Chonnam National University, Gwangju, Korea, and were designated CNUFC-YR329-1, CNUFC-YR329-2, CNUFC-PTS103-1, CNUFC-PTS103-2, CNUFC-PTS2-1, and CNUFC-PTS2-2. Strain CNUFC-YR329-1 was also deposited at the Culture Collection of the Nakdonggang National Institute of Biological Resources (NNIBR, Sangju, Korea), and strains CNUFC-PTS103-1 and CNUFC-PTS2-1 were deposited at the Culture Collection of the National Institute of Biological Resources (NIBR, Incheon, Korea).

Morphological studies
For detailed morphological studies, CNUFC-YR329-1, CNUFC-PTS103-1, and CNUFC-PTS2-1 strains were cultured on PDA, malt extract agar (MEA: 20 g malt extract, and 20 g agar in 1 L of deionized water), oatmeal agar (OA; 30 g oatmeal extract, 15 g agar in 1 L of deionized water), and water agar (20 g agar in 1 L of deionized water). The plates were incubated at 10, 20, 25, 30, and 35 C in the dark for 7 days. Fragments of mycelia were removed from cultures, placed on microscope slides with distilled water and lactophenol solution (Junsei Chemical Co. Ltd., Tokyo, Japan) and observed under a light microscope (Olympus, Tokyo, Japan).

DNA extraction, PCR, and sequencing
Genomic DNA was extracted directly from the mycelia of fungal isolates, using the Genomic DNA Prep Kit (Solgent Co. Ltd., Daejeon, South Korea). The ITS region and large subunit (LSU) ribosomal RNA were amplified with the primer pairs ITS4 and ITS5 [22], and LROR and LR5F [23], respectively. The PCR amplification mixture (total volume, 20 lL) contained fungal DNA template, 5 pmol/lL of each primer, and Accupower PCR Premix (Taq DNA polymerase, dNTPs, buffer, and a tracking dye; Bioneer Corp., Daejeon, Korea). PCR products were purified using the Accuprep PCR Purification Kit (Bioneer Corp.) according to the manufacturer's instructions. DNA sequencing was performed on an ABI 3700 automated DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA).

Phylogenetic analysis
Fungal sequences (Table 1) were initially aligned using Clustal_X v.2.1 [24] and Bioedit v. 7.2.6 software [25]. Phylogenetic analyses were performed using MEGA 6 with the default settings [26]. Phylogenetic trees were constructed using the Maximum likelihood (ML) analysis with the ITS and LSU sequences. The sequences of Mortierella wolfii and Umbelopsis isabellina were used as the outgroups. The sequence identity was determined using the National Center for Biotechnology Information Basic Local Alignment Search Tool for nucleotides (BLASTn).

Taxonomy
Description: The strain grew rapidly at 20 C on PDA, reaching 62-65 mm in diameter after 5 days incubation, and were white with abundant aerial hyphae. The reverse colony was white and slightly zonate. The colonies exhibited slower growth on PDA and WA than on OA. On OA, mycelial development was better than that on PDA and WA. However, a smaller number of sporangia were produced on OA. On WA, although the mycelial growth was sparse, the sporulation was excellent. Sporangiophores developed from aerial hyphae and were 2.5-4.5 lm at the tip and simple or branched. Sporangia were globose, with multi-spores and a collar after deliquescence. Sporangia were globose and measured 19.5-34.5 Â 18.4-32.5 lm. Sporangiospores were ellipsoidal to short cylindrical, reniform, and measured 8.5-15.7 Â 5.2-8.3 lm. Chlamydospore formation was The thick colored clade lines with the asterisk were constructed by a previous phylogenetic system described by Wagner et al. [5], whereas the clade lines marked by the asterisk were constructed by Wagner et al. [5] and Hyde et al. [10].  Figure 4).
Description: The strain grew rapidly at 20 C on PDA, attaining a diameter of 81 mm after 7 days of incubation. The colony color was initially cotton white and later became slightly beige with a similar flower shape. The reverse colony was also white mixed with slightly yellow and was irregularly zonate. Typical sporangia and sporangiospores were not observed although the PDA medium showed good mycelial growth. Sporangiophores were always unbranched. Sporangia were normally produced on OA after 7 days at 20 C. Sporangia were nearly globose and measured 12.0-20.0 Â 12.0-19.0 lm. Sporangiospores were globose, smooth, and measured 4.9-5.4 Â 4.9-5.8 lm. Zygospores were not observed.
Description: The strain grew rapidly at 20 C on PDA, reaching a diameter of 85 mm after 7 days of incubation and was cottony in the center with a white  margin. The reverse colony was white and irregularly zonate. Growth was rapid on MEA, producing a rosette colony and abundant aerial hyphae reaching 85 mm diameter after 7 days of incubation at 20 C. For colonies grown on WA, aerial hyphae were dispersed on the agar surface reaching a diameter of 82 mm at 20 C after 7 days of incubation. Sporangiophores were simple or branched, with a length of 63.2 lm. Sporangia were globose, finely spinulose, always single-spored, and measured 8.2-13.1 Â 7.9-12.1 lm. Zygospores were not observed.

Discussion
Until now, few studies have reported new and undescribed species belonging to the order Mortierelles in Korea [17][18][19]. Finding of M. elongata, M. horticola, and M. humilis improves our knowledge regarding the occurrence and distribution of zygomycete species within the genus Mortierella known as an undiscovered taxon in Korea.
The phylogenetic analyses of ITS and LSU sequences showed that CNUFC-PTS103-1, CNUFC-PTS103-2, CNUFC-PTS2-1, and CNUFC-PTS2-2 belongs to group 2 "verticillata-humillis" as defined by Wagner et al. [5]. CNUFC-PTS103-1 displayed similar morphological characteristics with M. horticola described by Linnem [6], but is in contradiction to having multispored sporangia resembling M. epicladia. So, there is a possibility that M. horticola produces single-spored as well as multi-spored sporangia. M. horticola and M. epicladia were easily distinguishable in ITS tree, suggesting that the number of spores per sporangium is of no taxonomic relevance due to the lack of fixed spores in this group. M. horticola would have been expected to have evolved multi-spored sporangia as an adaptation to the environmental factors. Additional information regarding suitable morphological criteria and molecular markers is necessary for the correct validation of M. horticola [5].
M. horticola has been isolated from peatland soils [27], from Spagnum fiscum in Canada [31], from a sacred grove and disturbed forest in Northeast India [39], from washed root surfaces [40], as an endophyte [41], from the rhizosphere of Meyna spinosa Roxb [42], and from wheat field soil and agricultural soil from Germany and The Netherlands [5]. The higher amount of docosahexaenoic acid and production of omega-6 and omega-3 fatty acid was also detected in M. horticola [37].
The morphological characteristics of CNUFC-PTS2-1 agree well with the description of M. humilis by Chien et al. [11]. Molecular data revealed that our isolates clustered within one clade with the type species, M. humilis CBS 222. 35.
M. humilis has been isolated from Sphagnum fuscum [40], various soils and roots of herbaceous plants [43], forest soil from North Carolina, a Pinus forest in Mexico, stump bark from South Carolina, soil from The Netherlands, forest soil from China [5], Norway spruce stands on sod-podzolic soil [44], pea rhizosphere soil [45], and on heavily decayed wood [46]. M. humilis is reported to produce useful fatty acids, including arachidonic acid used in medicine, pharmacology, cosmetics, agriculture, and in food industry [36,47], eicosapentaenoic acid [48], and enzymes capable of degrading xylans, sugars (sucrose, galactose, fructose, mannose, maltose), paraffin, and chitin [31]. M. humilis can also degrade cellulose and lignin, which makes plants remnants readily available to other members of the ecological system, which enhances biological activity [49,50].
In comparison to M. elongata, M. horticola, and M. humilis have been a less-studied species. Further studies, including multi-gene analyses and observations of ultrastructure of uni-or multi-spores, are needed to unravel the phylogenetic relationship of the related Mortierella species. In addition, three species obtained from this study may potentially be highly valuable. Thus, the potential biological activities of M. elongata, M. horticola, and M. humilis should be further studied.

Disclosure statement
No potential conflict of interest was reported by the authors.