The genus Mortierella (Mortierellaceae, Mortierellales) was described by Coemans (1863) with the type species Mortierella polycephala Coem [1]. To date, nearly 100 spe- cies of Mortierella have been described [2]. The species belonging to this genus are characterized by the produc- tion of primarily coenocytic but irregularly septate myce- lium. Sporangiophores are simple or variously branched terminating with sporangia and occasionally with a swell- ing at the base. Sporangia are globose, multi-, few- or uni-spored. Mortierella species typically exhibit rapid growth at temperatures ranging from 15°C to 25°C. They are frequently isolated from the soil and dead or dying plant, and tissues or from animal fecal samples [3, 4]. Many of them show potential as producers of polyunsat- urated fatty acids [5, 6]. In addition, several species of Mortierella have been used as a pesticide degrading agent, suggesting that they might have potential for the biore- mediation of sites contaminated with organochlorine pesticides [7].
Recently, molecular data have been used to evaluate the diversity of the genus Mortierella [2, 8]. Six species of the genus Mortierella have been reported in Korea [9, 10]. A new Mortierella species, M. fluviae, was isolated from a reshwater sample from Yeongsan River located in Gwangju, Korea in 2016 [10].
The objective of the present study was to perform the morphological and molecular analyses to characterize an unrecorded zygomycete species−M. minutissima in Korea.
Freshwater samples were collected from the Yeongsan River located in Gwangju (35°10'N, 126°55'E), Korea in February 2016. These samples were transported in sterile Falcon tubes, and stored at 4°C until use. A serial dilu- tion technique was used to isolate fungi. In this techni- que, 1 mL of water sample was mixed with 9 mL of ster- ilized water. The water sample was serially diluted with sterilized water to obtain a concentration range from 10 -1 to 10-4. Subsequently, 0.1 mL of each dilution was trans- ferred to potato dextrose agar (PDA; BD Diagnostics, Sparks, MD, USA) and incubated at 25°C for 3~7 days. Individual colonies of fungi that showed varying morpho- logies were picked up and purely transferred to another PDA plate. All pure isolates, including M. minutissima 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.
Genomic DNA was directly extracted from mycelia using the HiGene Genomic DNA prep kit for fungi (BIO-FACT, Daejeon, Korea). The internal transcribed spacers (ITS1 and ITS2) and 5.8S gene were amplified using the primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') fol lowing the method described by White et al. [11]. The sequences were initially aligned using CLUSTAL X [12], and edited manually [13]. Phylogenetic analyses were performed using MEGA 6 [14] with the default settings. Phylogenetic trees were constructed from the data using maximum likelihood (ML). The ITS sequences of EML-YR717-1 and EML-YR717-2 were deposited in the GenBank database with the accession numbers (KY056587 and KY056588,respectively). A BLASTN search revealed that the rDNA ITS homology of EML-YR717-1 and EML-YR717-2 repre- sented 100% (584/584 bp) sequence identity value with M. minutissima (GenBank accession no. AB476417). Based on analysis of the ITS region, isolates were identified as M. minutissima (Fig. 1). To confirm the molecular species identification, we examined morphological features of the isolate EML-YR717-1. Cultural features were observed on PDA and water agar (WA). The plates were incubated at 20°C in the dark for 7 days. Samples were mounted on lactophenol solution (Junsei Chemical, Tokyo, Japan) and observed under a light microscope (DFC 290; Leica Microsystems, Wetzlar, Germany).
Fig. 1
Phylogenetic tree of Mortierella minutissima EML-YS717-1 and EML-YS717-2 and related species based on maximum likelihood analysis of internal transcribed spacer (ITS) rDNA sequences. Sequence of Umbelopsis isabellina was used as the outgroup. Bootstrap support values of ≥50% are indicated at the nodes. The bar indicates the number of substitutions per position. The group and clades on tree was named based on the classification system constructed by Wagner et al. [2].
Mortierella minutissima Tiegh., Annales des Sciences Naturelles Botanique 4: 385 (1878) (Table 1, Fig. 2).
Table 1. Morphological characteristics of the Mortierella minutissima EML-YS717-1 and other closely related species on water
agar medium at 20°C
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| *From description of Van Tieghem [15]. |
Description: Colonies grew fast on PDA, cotton in the center with a white margin, the reverse white and irreg- ularly zonate, reaching 56~59 mm diameter after 7 days of incubation at 20°C. For colonies grown on WA, aerial hyphae were dispersed on the agar surface. Sporangio- phores were short and grew to a width of 2.0~3.5 μm. Sporangia measured 13.5~20.5 × 13.0~5.0 μm and were globose, having deliquescent walls. Spores were globose to subglobose and measured 4.0~5.5 × 3.5~5.0 μm. Zygo- spores were not observed. The isolate showed the best growth and abundant sporulation when grown on WA agar. Morphology of the present isolate was most similar to that of the previously described of M. minutissima [15].
Currently, Mortierellales contains only one family (Mor- tierellaceae) and six genera: AquaMortierella, Dissophora, Modicella, Lobosporangium, Gamsiella, and Mortierella [2]. The sporangial morphology of Mortierella is quite variable. Based on the morphological characteristics, Gams divided the subgenus Mortierella into nine sections: Actinomorti- erella, Alpina, Haplosporangium, Hygrophila, Mortierella, Schmuckeri, Simplex, Spinosa, and Stylospora [3]. Based on the sequences of the ITS rDNA regions, Wagner et al. [2] demonstrated that the genus Mortierella contains 7 groups:selenospora and parvispora; verticillata-humilis; lignicola; mutabilis, globulifera and angusta; strangulata and wolfii; alpina and polycephala; and gamsii. In the ITS tree, our strains, EML-YR717-1 and EML-YR717-2, belonged to group 2 (verticillata-humilis) as presented by Wagner et al. [2]. Furthermore, this fungus is morphologically most similar to M. minutissima placed in the minutissima clade. M. minutissima has been reported as a novel psychrotro- phic fungus for biotransformation D-limonene and as a producer of arachidonic acid and dihomo-gamma-linole- nic acid [16, 17]. This finding suggests that the strain EML-YR717-1 may be a useful source for biotransforma- tions and biotechnological applications. Thus, potential application of M. minutissima EML-YR717-1 should be studied further.
