Mycobiology Research Article
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Mycobiology Research Article Phylogenetic Status of Two Undescribed Zygomycete Species from Korea: Actinomucor elegans and Mucor minutus 1 2 3 4 1, Thuong T. T. Nguyen , Hee-Young Jung , Youn Su Lee , Kerstin Voigt and Hyang Burm Lee * 1 Division of Food Technology, Biotechnology and Agrochemistry, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 61186, Korea 2 School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Daegu 41566, Korea 3 Division of Bioresource Sciences, Kangwon National University, Chuncheon 24341, Korea 4 JMRC at Leibniz Institute for Natural Product Research and Infection Biology e.V. HKI and Friedrich Schiller University Jena, 07745 Jena, Germany Abstract 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. Keywords Actinomucor elegans, Mucor minutus, Undiscovered taxa, Zygomycete fungi Actinomucor and Mucor belong to the subphylum maximum growth temperatures: 37oC for A. taiwanensis Mucoromycotina, order Mucorales, family Mucoraceae [1]. and 32oC for A. elegans. Later, Zheng and Liu [5] renamed The genus Actinomucor was originally described in 1898 A. taiwanensis to A. elegans var. meitauzae based on by Schostakowitsch [2]. Although the genus is closely morphological characteristics and molecular analyses. Recently, related to Mucor, it differs in having branched stolons that Khan et al. [6] proposed the addition of a new variety, A. give rise to rhizoids and sporangiophores. It is also distinct elegans var. kuwaitensis. In Index Fungorum (2017; http:// from the other two genera Rhizopus and Absidia in its www.indexfungorum.org), the genus Actinomucor contains arrangement of the columellae and sporangiophores. The only one species named Actinomucor elegans. genus originally contained two species, A. elegans (Eidam) Actinomucor species are found in dung, soil, food, and C. R. Benj. & Hesselt., and A. taiwanensis S. C. Jong & human sources [5-7]. Some of them are commonly used G. F. Yuan [3, 4]. A. taiwanensis was differentiated from A. for producing popular fermented soybean foods including elegans by its larger sporangiospore size and by their differing Sufu and Chao [8]. In addition, A. elegans is also considered a good source of glycine aminopeptidase and glucosamine [9, 10]. A. elegans var. elegans has been reported as a potential Mycobiology 2017 December, 45(4): 344-352 biocontrol agent against the chafer beetle [11]. https://doi.org/10.5941/MYCO.2017.45.4.344 Mucor Fresen. (Mucoraceae, Mucorales) is characterized pISSN 1229-8093 • eISSN 2092-9323 © The Korean Society of Mycology by the formation of non-apophysate sporangia, producing simple or branched sporangiophores without basal rhizoids. *Corresponding author E-mail: [email protected] Zygospores have opposed, non-appendaged suspensors [12]. Mucor species have frequently been detected on substrates Received October 25, 2017 Revised December 5, 2017 that support the growth of a fungal host, such as in soil, Accepted December 20, 2017 dung, fruit, and plants [13-15]. Several species are able to produce enzymes with biotechnological applications [16, This is an Open Access article distributed under the terms of the 17], while some species are considered the causal agent of Creative Commons Attribution Non-Commercial License (http:// cutaneous zygomycosis in humans [18]. Although there are creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, more than 300 named species described in the literature, provided the original work is properly cited. only approximately 50 are known and described [15]. 344 Two New Records of Zygomycete Species in Korea 345 Traditional taxonomy of Mucor species has been determined to another PDA plate. All pure isolates, including A. elegans based on morphological characteristics such as size and and M. minutus, were maintained in PDA slant tubes shape of sporangia as well as the mode of reproduction and stored in 20% glycerol at −80oC at the Environmental (sexual or asexual). Microbiology Laboratory Fungarium, Chonnam National Recently, molecular data have been used to evaluate University, Gwangju, Korea, as CNUFC-YR113-1, CNUFC- mucoralean species [19, 20]. These studies indicated that KNU16-7, and CNUFC-BS1-1. CNUFC-KNU16-7 and Mucor is polyphyletic. Based on the phylogeny of internal CNUFC-BS1-1 were also deposited at the Collection of transcribed spacer (ITS) and large subunit (LSU) rDNA National Institute of Biological Resources (NIBR), Incheon, regions of several mucoralean species, Walther et al. Korea; CNUFC-YR113-1 deposited at Culture Collection [21] observed that some Mucor species with curved of Nakdonggang National Institute of Biological Resources sporangiophores were grouped with Backusella Hesselt. & [NNIBR], Sangju, Gyeongbuk province, Korea. J. J. Ellis. Therefore, these Mucor species were transferred to Backusella. Morphological studies. For detailed morphological In Korea, two new Mucor species have been currently studies, CNUFC-YR113-1 and CNUFC-BS1-1 strains were reported by authors: Mucor koreanus from tangerine fruit cultured on synthetic mucor agar (SMA; 40 g dextrose, 2 g [14] and Mucor stercorarius from rat feces [22]. Only seven asparagine, 0.5 g KH2PO4, 0.25 g MgSO4 ·7H2O, 0.5 g thiamine species have been recorded: M. circinelloides, M. hiemalis, chloride, and 15 g agar in 1 L of deionized water). The M. mucedo, M. piriformis, M. racemosus, M. fragilis, and M. plates were incubated at 10, 20, 25, 30, and 35oC in the irregularis [15, 23]. To our knowledge, there are no specific dark for 7 days. Fragments of mycelia were removed from published literature records of these species in Korea. cultures, placed on microscope slides with lactophenol The objective of the present study was to perform solution (Junsei Chemical Co. Ltd., Tokyo, Japan) and morphological and molecular analyses to characterize two observed under a light microscope (Olympus, Tokyo, Japan). unrecorded zygomycete species in Korea: Actinomucor elegans and Mucor minutus. DNA extraction, PCR, and sequencing. Genomic DNA was extracted directly from the mycelia of fungal isolates, MATERIALS AND METHODS using the Solgent Genomic DNA prep Kit (Solgent Co. Ltd., Daejeon, Korea). The ITS region and large subunit of Fungal strain isolation from freshwater and soil 28S rDNA were amplified with the primer pairs ITS4 and samples. Freshwater samples were collected from the ITS5 [24], and LROR and LR5F [25], respectively. The Yeongsan River located in Gwangju, Korea. Soil samples PCR amplification mixture (total volume, 20 µL) contained were collected from the garden of the Chonnam National fungal DNA template, 5 pmol/µL of each primer, and University located in Gwangju and a field in Gyeongnam, Accupower PCR Premix (Taq DNA polymerase, dNTPs, Korea. These samples were transported in sterile 50-mL buffer, and a tracking dye; Bioneer Corp., Daejeon, Falcon tubes, and stored at 4oC until examination. Fungi Korea). PCR products were purified using the Accuprep were isolated using the serial dilution plating method. In PCR Purification Kit (Bioneer Corp.) according to the this technique, 1 mL water or 1 g of soil was mixed with manufacturer’s instructions. DNA sequencing was performed 9 mL of sterile distilled water and shaken for 15 min at on an ABI 3700 Automated DNA sequencer (Applied − − 25oC; serial dilutions ranging from 10 1 to 10 4 were then Biosystems Inc., Foster City, CA, USA). made. An aliquot of 0.1 mL from each dilution was transferred to potato dextrose agar (PDA) and incubated at Phylogenetic analysis. The fungal sequences obtained 25oC for 3–7 days. Individual colonies of fungi that showed from the GenBank database (Table 1) were aligned using varying morphologies were picked up and purely transferred Clustal_X v.1.83 [26] and edited with Bioedit v.5.0.9.1 [27]. Table 1. Taxa, collection numbers, sequences, and GenBank accession numbers used in this study Collection No. GenBank accession No. Taxon name (isolate No.) ITS 28S Actinomucor elegans ATCC 46123 AM745430 - A. elegans CBS 338.72 JN205824 - A. elegans CBS111562 AB113009 - A. elegans CBS 100.09 - JN206491 A. elegans CBS154.86 - HM849686 A. elegans CNUFC-YR113-1 MG206066 MG206071 A. elegans CNUFC-YR113-2 MG206067 MG206072 A. elegans CNUFC-KNU16-7 MG206068 MG206073 A. elegans var. elegans ATCC22814T AY492092 - 346 Nguyen et al. Table 1. Continued Collection No. GenBank accession No. Taxon name (isolate No.) ITS 28S A. elegans var. kuwaitiensis CBS117697T JN205823 JN206493 A. elegans var. meitauzae ATCC52370T AM745432 - A. elegans var. meitauzae CBS 111558 - JN206492 Backusella circina CBS 128.70 T - JN206529 B. grandis CBS 186.87 T - JN206527 B. lamprospora CBS 118.08 T - JN206531 Benjaminiella multispora CBS 421.70 - KU561719 Blakeslea sinensis CBS 564.91 - JN206515 Choanephora infundibulifera CBS 153.51 - JN206513 Cokeromyces recurvatus CBS 168.59 - JN206408 C. recurvatus CBS 158.50 - KU561716 Mucor aligarensis CBS 993.70 T - JN206461 M. circinelloides B5-2 KT876701 - M. circinelloides CBS 108.16 JN205954 - M. fragilis CBS 236.35 JN205979 - M. fragilis EML-PUKI06-1 KY047147 - M. fragilis EML-PUKI06-2 KY047150 - M. flavus CBS 230.35 T JN206061 JN206464 M. flavus CBS 681.73 JN206070 - M. flavus CBS 893.73 - JN206465 M. flavus CBS 182.90 - JN206472 M. fuscus CBS 132.22 JF723619 - M. fuscus CBS 230.29 JN206204 - M. genevensis CBS 114.08T HM623318 - M. genevensis CBS 404.71 JN206042 - M. heterogamus CBS 338.74 JN206169 JN206488 M. heterogamus CBS 252.85 - JN206490 M. heterogamus CBS 405.58 JN206167 - M.