Phylogenetic Status of Two Undescribed Zygomycete Species from Korea: Actinomucor elegans and Mucor minutus.

During a survey of fungal diversity of the order Mucorales, three zygomycete isolates, CNUFC-YR113-1, CNUFC-KNU16-7, and CNUFC-BS1-1 were isolated from freshwater and soil samples in Korea. The strains were analyzed both morphologically and phylogenetically based on internal transcribed spacer and 28S rDNA gene sequences. Based on their morphology and phylogeny, the CNUFC-YR113-1 and CNUFC-KNU16-7 isolates were identified as Actinomucor elegans, and CNUFC-BS1-1 was identified as Mucor minutus. To the best of our knowledge, the species A. elegans and M. minutus, belonging to an undiscovered taxon, have not been previously described in Korea.

Actinomucor and Mucor belong to the subphylum Mucoromycotina, order Mucorales, family Mucoraceae [1]. The genus Actinomucor was originally described in 1898 by Schostakowitsch [2]. Although the genus is closely related to Mucor, it differs in having branched stolons that give rise to rhizoids and sporangiophores. It is also distinct from the other two genera Rhizopus and Absidia in its arrangement of the columellae and sporangiophores. The genus originally contained two species, A. elegans (Eidam) C. R. Benj. & Hesselt., and A. taiwanensis S. C. Jong & G. F. Yuan [34]. A. taiwanensis was differentiated from A. elegans by its larger sporangiospore size and by their differing maximum growth temperatures: 37℃ for A. taiwanensis and 32℃ for A. elegans. Later, Zheng and Liu [5] renamed A. taiwanensis to A. elegans var. meitauzae based on morphological characteristics and molecular analyses. Recently, Khan et al. [6] proposed the addition of a new variety, A. elegans var. kuwaitensis. In Index Fungorum (2017; http://www.indexfungorum.org), the genus Actinomucor contains only one species named Actinomucor elegans.

Actinomucor species are found in dung, soil, food, and human sources [567]. Some of them are commonly used for producing popular fermented soybean foods including Sufu and Chao [8]. In addition, A. elegans is also considered a good source of glycine aminopeptidase and glucosamine [910]. A. elegans var. elegans has been reported as a potential biocontrol agent against the chafer beetle [11].

Mucor Fresen. (Mucoraceae, Mucorales) is characterized by the formation of non-apophysate sporangia, producing simple or branched sporangiophores without basal rhizoids. Zygospores have opposed, non-appendaged suspensors [12]. Mucor species have frequently been detected on substrates that support the growth of a fungal host, such as in soil, dung, fruit, and plants [131415]. Several species are able to produce enzymes with biotechnological applications [1617], while some species are considered the causal agent of cutaneous zygomycosis in humans [18]. Although there are more than 300 named species described in the literature, only approximately 50 are known and described [15].

Traditional taxonomy of Mucor species has been determined based on morphological characteristics such as size and shape of sporangia as well as the mode of reproduction (sexual or asexual).

Recently, molecular data have been used to evaluate mucoralean species [1920]. These studies indicated that Mucor is polyphyletic. Based on the phylogeny of internal transcribed spacer (ITS) and large subunit (LSU) rDNA regions of several mucoralean species, Walther et al. [21] observed that some Mucor species with curved sporangiophores were grouped with Backusella Hesselt. & J. J. Ellis. Therefore, these Mucor species were transferred to Backusella.

In Korea, two new Mucor species have been currently reported by authors: Mucor koreanus from tangerine fruit [14] and Mucor stercorarius from rat feces [22]. Only seven species have been recorded: M. circinelloides, M. hiemalis, M. mucedo, M. piriformis, M. racemosus, M. fragilis, and M. irregularis [1523]. To our knowledge, there are no specific published literature records of these species in Korea.

The objective of the present study was to perform morphological and molecular analyses to characterize two unrecorded zygomycete species in Korea: Actinomucor elegans and Mucor minutus.

MATERIALS AND METHODS

Fungal strain isolation from freshwater and soil samples

Freshwater samples were collected from the Yeongsan River located in Gwangju, Korea. Soil samples were collected from the garden of the Chonnam National University located in Gwangju and a field in Gyeongnam, Korea. These samples were transported in sterile 50-mL Falcon tubes, and stored at 4℃ until examination. Fungi were isolated using the serial dilution plating method. In this technique, 1 mL water or 1 g of soil was mixed with 9 mL of sterile distilled water and shaken for 15 min at 25℃; serial dilutions ranging from 10−1 to 10−4 were then made. An aliquot of 0.1 mL from each dilution was transferred to potato dextrose agar (PDA) and incubated at 25℃ for 3–7 days. Individual colonies of fungi that showed varying morphologies were picked up and purely transferred to another PDA plate. All pure isolates, including A. elegans and M. minutus, were maintained in PDA slant tubes and stored in 20% glycerol at −80℃ at the Environmental Microbiology Laboratory Fungarium, Chonnam National University, Gwangju, Korea, as CNUFC-YR113-1, CNUFC-KNU16-7, and CNUFC-BS1-1. CNUFC-KNU16-7 and CNUFC-BS1-1 were also deposited at the Collection of National Institute of Biological Resources (NIBR), Incheon, Korea; CNUFC-YR113-1 deposited at Culture Collection of Nakdonggang National Institute of Biological Resources [NNIBR], Sangju, Gyeongbuk province, Korea.

Morphological studies

For detailed morphological studies, CNUFC-YR113-1 and CNUFC-BS1-1 strains were cultured on synthetic mucor agar (SMA; 40 g dextrose, 2 g asparagine, 0.5 g KH2PO4, 0.25 g MgSO4 · 7H2O, 0.5 g thiamine chloride, and 15 g agar in 1 L of deionized water). The plates were incubated at 10, 20, 25, 30, and 35℃ in the dark for 7 days. Fragments of mycelia were removed from cultures, placed on microscope slides with 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 Solgent Genomic DNA prep Kit (Solgent Co. Ltd., Daejeon, Korea). The ITS region and large subunit of 28S rDNA were amplified with the primer pairs ITS4 and ITS5 [24], and LROR and LR5F [25], respectively. The PCR amplification mixture (total volume, 20 µL) contained fungal DNA template, 5 pmol/µL 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

The fungal sequences obtained from the GenBank database (Table 1) were aligned using Clustal_X v.1.83 [26] and edited with Bioedit v.5.0.9.1 [27]. Phylogenetic analyses were performed using MEGA 6 software [28], and maximum likelihood was constructed by Kimura's two-parameter correction method. The fungus Umbelopsis nana was used as an outgroup. The reliability of internal branches was assessed using the p-distance substitution model with 1,000 bootstrap replications.

Table 1

Taxa, collection numbers, sequences, and GenBank accession numbers used in this study

Bold letters indicate isolates and accession numbers determined in our study.

ITS, internal transcribed spacer; ATCC, American Type Culture Collection, Manassas, VA, USA; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CNUFC, Chonnam National University Fungal Collection, Gwangju, South Korea; EML, Environmental Microbiology Laboratory Fungarium, Chonnam National University, Gwangju, South Korea; NRRL (ARS Culture Collection, Peoria, Illinois); T, ex-type strain.

RESULTS

Phylogenetic analyses of the two sequence datasets (ITS and 28S rDNA) showed that the strains CNUFC-YR113-1, CNUFC-YR113-2, CNUFC-KNU16-7, CNUFC-BS1-1, and CNUFC-BS1-2 were placed within the same clade with species of Actinomucor and Mucor (Figs. 1 and 2).

Fig. 1

Phylogenetic tree based on maximum likelihood analysis of internal transcribed rDNA sequences for Actinomucor elegans CNUFC-YR113-1, A. elegans CNUFC-YR113-2, A. elegans CNUFC-KNU16-7, Mucor minutus CNUFC-BS1-1, and M. minutus CNUFC-BS1-2. Umbelopsis nana was used as an outgroup. Bootstrap support values of ≥ 50% are indicated at the nodes. The bar indicates the number of substitutions per position.

Fig. 2

Phylogenetic tree based on maximum likelihood analysis of 28S rDNA sequences for Actinomucor elegans CNUFC-YR113-1, A. elegans CNUFC-YR113-2, A. elegans CNUFC-KNU16-7, Mucor minutus CNUFC-BS1-1, and M. minutus CNUFC-BS1-2. Umbelopsis nana was used as an outgroup. Bootstrap support values of ≥ 50% are indicated at the nodes. The bar indicates the number of substitutions per position.

In the BLASTn analysis of the ITS sequence, CNUFC-YR113-1 and CNUFC-BS1-1 represented 99.8% (535/536 bp) and 99.4% (613/617 bp) sequence identity values with A. elegans (GenBank accession No. JN205824) and M. minutus (GenBank accession No. JN206048), respectively.

In the BLASTn analysis of the 28S sequence, CNUFC-YR113-1 and CNUFC-BS1-1 strains showed 98.1% (634/644 bp) and 100% (682/682 bp) identity values with A. elegans (GenBank accession No. JN205827) and M. minutus (GenBank accession No. JN206463), respectively.

Taxonomy of CNUFC-YR113-1

Actinomucor elegans (Eidam) C. R. Benj. & Hesselt., Mycologia 49: 241 (1957) (Table 2, Fig. 3).

Table 2

Morphological characteristics of CNUFC-YR113-1 and the reference Actinomucor elegans grown on synthetic mucor agar medium at 25℃

aFrom the description by Benjamin and Hesseltine [3].

Fig. 3

Morphology of Actinomucor elegans CNUFC-YR113-1. A, Colonies on synthetic mucor agar; B–D, Sporangia on branched sporangiophores (observed under stereo-microscope); E–H, Branched sporangiophores forming sporangia and columellae (observed under light microscope); I, Sporangiospores (scale bars: B–D = 200 µm, E–H = 50 µm, I = 20 µm).

= Rhizopus elegans Eidam, Jahresber. Schles. Ges. Vaterl. Kultu. 61: 232 (1884).

= Mucor elegans (Eidam) J. Schröt., Kryptogamen-Flora von Schlesien 3-1: 207 (1886).

= Mucor corymbosus Harz, Bull. Soc. Imp. Nat. Moscou 44: 143 (1871).

= Actinomucor repens Schostak., Ber. Dtsc. Bot. Ges. 16: 155 (1898).

= Glomerula repens Bainier, Bull. Soc. Mycol. Fr. 19: 154 (1903).

= Mucor botryoides Lendn., Bull. Soc. Bot. Genève 2: 79 (1910).

= Mucor botryoides var. minor C.N. Jensen, Bull. Cornell Univ. Agric. Exp. Stn. 315: 457 (1912).

= Mucor cunninghamelloides Pispek, Acta Bot. Inst. Bot. Univ. Zagreb. 4: 91 (1929).

= Actinomucor corymbosus Naumov, Opredelitel Mukorovykh (Mucorales): 56 (1935).

= Actinomucor corymbosus f. palaestinus Rayss, Palestine J. Bot. 3: 162 (1945).

Description: Colonies grew rapidly at 25℃ on SMA, filling the Petri dish after 5 days of incubation. The colony color was initially white, later deep olive-buff. The colony reverse was white. Sporangiophores were 12.2–20.5 µm wide, erect, branched, irregular, and verticillate. Primary sporangia were globose to subglobose, and measured 42.3–83.5 × 39.9–82.1 µm. Secondary sporangia were formed with same shape as the primary sporangia, and measured 29.9–46.2 × 27.5–44.3 µm. Columellae inside the primary sporangia were diverse in shape, oval, pyriform, oblong, and measured 23.3–44.8 × 22.6–42.9 µm. Columellae inside the secondary sporangia were globose, and measured 14.5–26.5 × 17.8–30.4 µm. Sporangiospores were globose to subglobose, and measured 6.1–8.5 × 5.8–8.1 µm. Chlamydospore formations were well-defined on the medium. Zygospores were not observed.

Taxonomy of CNUFC-BS1-1

Mucor minutus (Baijal & B. S. Mehrotra) Schipper, Stud. Mycol. 10: 24 (1975) (Table 3, Fig. 4).

Table 3

Morphological characteristics of CNUFC-BS1-1 and the reference species Mucor minutus grown on synthetic mucor agar medium at 25℃

aFrom the description by Schipper [29].

Fig. 4

Morphology of Mucor minutus CNUFC-BS1-1. A, Colonies on synthetic mucor agar; B, C, Sporangiophores with long and short branches and sporangia (observed under stereo-microscope); D, E, Sporangia and sporangiophores (observed under light microscope); F–J, Columellae with collarette and sporangial septa below the columellae (white arrow); K, Sporangiospores (scale bars: B, C = 200 µm, D–J = 50 µm, K = 20 µm).

= Mucor griseoochraceus var. minuta Baijal & B. S. Mehrotra, Sydowia 19: 206 (1966).

= Mucor saturninus var. minutus (Baijal & B. S. Mehrotra) Milko, Opredeltiel mukoral'nykh gribov. 129 (1974).

Description: Colonies grew rapidly on SMA, attaining a diameter of 70–72 mm after 5 days at 25℃. The colony color was initially white, later turning to smoke gray. Sporangiophores were 9–24.5 µm wide, erect, mostly branched, and irregular. Sporangia were globose, and measured 37.1–109.8 µm × 36.5–103.4 µm. Columellae were globose to ellipsoidal, and measured 27.9–95.2 µm × 24.8–84.5 µm. Sporangiospores were globose, and measured 4.3–5.6 µm × 4.1–5.0 µm. Zygospores were not observed on artificial media.

DISCUSSION

Despite the wide intraspecific variation found among some taxa, the rDNA ITS and D1/D2 regions have been used as critical barcode markers for identifying mucoralean fungi at the species level, including taxa of Actinomucor and Mucor [21].

In the ITS and LSU phylogenetic trees, our strains CNUFC-YR113-1, CNUFC-YR113-2, and CNUFC-KNU16-7 were clustered within the elegans clade including A. elegans, A. elegans var. meitauzae, and var. kuwaitiensis in a well-supported clade. However, our strain CNUFC-YR113-1 differed from A. elegans var. meitauzae and A. elegans var. kuwaitiensis in sporangiospore size; CNUFC-YR113-1 strain exhibited smaller sporangiospores (6.1–8.5 × 5.8–8.1 µm) than A. elegans var. meitauzae (7–19.5 × 6–15 µm) and A. elegans var. kuwaitiensis (5–12 µm). The maximum growth temperature of our strain was 35℃, while A. elegans var. meitauzae and A. elegans var. kuwaitiensis were able to grow under higher temperatures up to 40℃.

Jong and Yuan [4] reported that growth temperature is a criterion for distinguishing between A. elegans and A. taiwanensis. These authors showed that A. taiwanensis has a maximum growth temperature of 37℃, while A. elegans does not grow at this temperature. Contrary to reports by Jong and Yuan [4], maximum growth temperature is less useful for distinguishing between the varieties [56].

The morphological features of our isolates were in line with the description of A. elegans by Benjamin and Hesseltine [3], as the properties including shape, size of the sporangiospores (6–8 µm), and maximum temperature for growth were compared. Under these criteria, our isolate was identified as A. elegans.

In the tree based on D1/D2 sequence analyses, the strains CNUFC-BS1-1 and CNUFC-BS1-2 were placed into the minutus clade within the M. flavus group as presented by Walther et al. [21] including: M. flavus, M. saturninus, M. aligarensis, and M. minutus (Fig. 2), and formed a monophyletic group with M. minutus (type species). The CNUFC-BS1-1 isolate was morphologically most similar to M. minutus as described by Schipper [29], although there were differences in the shape and size of columellae. The size of columellae described by Schipper [29] was larger (110–135 µm) than those (27.9–95.2 × 24.8–84.5 µm) observed in our isolate. According to Schipper [29], the M. minutus species is similar in morphology and closely related to M. flavus because they produce columellae with the same size. However, sporangiospores with different sizes and shapes have been observed. M. minutus has smaller sporangiospores (4–5 µm) than M. flavus (7–12 × 4–6.5 µm). Comparing the colony morphology and culture characteristics of the isolate with previous descriptions [29], the present isolate was similar to M. minutus, with some exceptions. Our M. minutus isolate presented one to three septa below the columella, which were not described by Schipper [29].

Recently, several studies have focused on the increased incidence of mucormycosis in both immunocompromised and immunocompetent patients [30]. Some species belonging to the order Mucorales (subphylum Mucoromycotina) are considered opportunistic pathogens. Particularly, four families, including Cunninghamellaceae, Lichtheimiaceae, Mucoraceae, and Syncephalastraceae, have been described to be responsible for human infections [31].

More recently, A. elegans and A. elegans var. kuwaitiensis have been reported as the agent of mucormycosis in humans in several cases [6732]. Morphological keys are available for identifying Actinomucor. However, it is still difficult to identify taxa to intraspecific rank in Actinomucor. Thus, taxonomic revision and phylogenetic analysis are needed in future studies.

Interestingly, A. elegans has been reported as protease enzyme for generation of small peptides with ACE-inhibitory activity from razor clam Sinonovacula constricta meat [33]. So this finding suggests that the strain CNUFC-YR113-1 may be a useful source for biotechnological applications.