Introduction
Arthrinium Kunze (=Apiospora Sacc.) is a fungal genus which contains about 40 species (Index Fungorum: http:// www.indexfungorum.org). They are known as plant-asso- ciated fungi, but also easily isolated from various substra- tes such as soil, bamboo, air and marine habitats [1-4] Marine environment and seaweed are supposed major habitat of Arthrinium because four Arthrinium species were isolated from same seaweed at the same time [4].
Remarkable biological activities of some Arthrinium strains had been reported. A. saccharicola KUC21221 showed strong cellulolytic enzyme, antifungal activity and antioxidant capacity [4]. Other Arthrinium spp. showed high antifungal activities as well.
In Korea, three Artrinium species, A. arundinis (Corda) Dyko & B. Sutton, A. phaeospermum (Corda) M.B. Ellis and A. saccharicola F. Stevens, were previously reported, but have been not fully described [1, 4, 5]. Thus, we have explored the diversity of marine Arthrinium from Korea. In this study, a total of seven cultures were collected from marine brown algae Sargassum sp. and identified by phy- logenetic analysis [4]. They were classified into four cla- des. Among them, two clades were identified with A. arun- dinis and A. saccharicola, respectively. These Arthrinium species are described based on macro- and micro-scopic observation. A phylogenetic tree of Arthrinium species based on translation elongation factor 1-alpha (EF1) and beta-tubulin (TUB) sequences was also offered.
Materials and Methods
Arthrinium Cultures and analysis of phenotype
Arthrinium cultures were collected previously [4]. They were obtained from the Korea University Culture Collec- ion (KUC). Among them, three strains, KUC21221, KUC 21229 and KUC21261, were analyzed to be new to Korea. Cultures were grown on 9 cm-diam plastic petri dishes ontained 20 mL of oatmeal agar (OA; oatmeal agar 72.5 g, DW 1 L; Difco, Detroit, MI, USA), potato dextrose gar (PDA; potato dextrose agar 39 g, DW 1 L; Difco) or malt extract agar (MEA; malt extract 20 g, agar 15 g, DW 1 L; Difco). The cultures were grown in room tem- perature. After a week, colonies were observed and pic- ures were taken using NEX-5R digital camera (Sony, To- kyo, Japan). Observation of colonies had been performed until two weeks grown.
Morphological analyses of microscopic characters were observed with Olympus BX51 light microscope (Olympus, Tokyo, Japan). Pictures of conidiophore and conidia were taken using the same microscope mounted with Olympus DP20 microscopic camera (Olympus). Measurements were made from DW mounts. At least 30 units of each para- meter were measured for each collection. To make relia- ble data, 5% of the measurements from each end of the range were removed. The isolates were deposited at the National institute of Biological Resources (NIBR), Incheon, South Korea.
Phylogenetic analysis
Genomic DNAs of Arthrinium spp. from KUC were previously extracted [4]. Genomic DNAs extraction from cultures was performed using Accuprep Genomic DNA extraction kit (Bioneer, Daejeon, Korea) according to the manufacturer’s protocol. PCR reaction of EF1 region was carried out according to the previously described method [6]. DNA sequencing was performed using Sanger method with 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA) by Macrogen (Seoul, Korea). Sequences of TUB region were obtained by previous study [4].
The obtained EF1 and TUB sequences were proofread and aligned with the selected reference sequences from GenBank using MAFFT 7.130 [7], and modified manually with MacClade 4.08 [8], respectively. Both EF1 and TUB datasets contained 588 characters. Datasets were tested by MrModeltest 2.3 using the AIC criteria with default options [9], respectively. The HKY+I+G model or GTR+I+G model were chosen for EF1 or TUB dataset, respec- tively, under the AIC criteria as a result of the test. Baye- sian analysis was performed with MrBayes 3.2.1 [10]. Phy- logenetic tree was created according to previous study [4].
Results and Discussion
Taxonomy
Arthrinium arundinis (Corda) Dyko & B. Sutton, Mycotaxon 8: 119 (1979) (Fig. 1A~1D)
Fig. 1
Arthrinium arundinis (A~D) and Arthrinium saccharicola (E~H). A, conidiogenous cells and conidia; B, colonies on MEA; C, colonies on OA; D, colonies on PDA; E, conidiogenous cells and conidia; F, colonies on MEA; G, colonies on OA; H, colonies on PDA. MEA, malt extract agar; OA, oatmeal agar; PDA, potato dextrose agar (scale bars = 10 μm).
Teleomorphic synonym: Apiospora montagnei Sacc.
Colonies flat, spreading, aerial mycelium abundant, white to grey colored. On MEA, conidia forming in abundant, scattered; no distinctive odor or diffusing pigment. On PDA and OA, aerial mycelium abundant and cottony. Mycelium consisting of smooth, hyaline, branched, septate, hyphae measured 1.5~4 μm diam. Conidiophores redu- ced to conidiogenous cells. Conidiogenous cells aggregated in clusters on hyphae, pale brown, smooth, ampulliform, 5~12 × 2~4 μm, apical neck 3~5 μm long, basal part 4~ 5.5 μm long. Conidia brown, smooth, globose, 5.5~7.5 × (4~)5~6 μm.
Note: This species is easily isolated from bamboos, such as Arundinaria spp. like its epithet: ‘arundinis’ [1]. Our strains and other A. arundinis were monophyletic with a high posterior probability (100%, Fig. 2).
Fig. 2
The 50% majority-rule consensus tree which contains 34 taxa and 1,153 characters obtained from the Bayesian analysis based on combined translation elongation factor 1-α (EF1), and β-tubulin (TUB) sequence alignment. Numbers above branches indicate posterior probabilities. Fungal cultures examined in this study are in bold. The scale bar indicates nucleotide substitutions per position.
Cultures examined: KOREA, Jeju Island, N33°23'39.3" E126°14'23.2", isolated from Sargacium sp., 10 January 2015, Seokyoon Jang (Culture KUC21229, GenBank KT 207645; KUC21261, GenBank KT207677).
Arthrinium saccharicola F. Stevens, J. Dept. Agric. Porto Rico 1(4): 223 (1917) (Fig. 1E~1H)
Colonies flat, spreading, with sparse aerial mycelium, conidia forming in few, white to grey colored with dark brown patches on agar media. On OA and PDA at first white colored, sometimes becoming pinkish; brown pig- ment diffused in media. Mycelium consisting of smooth, hyaline, branched, septate, hyphae measured 2.5~4 μm diam. Conidiogenous cells forming rare, aggregated in clusters on hyphae, ampulliform, 6~10 × 2.5~4 μm, apical neck 2~4 μm long, basal part 3~5 μm long. Conidia brown, smooth, granular, globose to ellipsoid, (7.5)8~9.5 (~10) × (7~)7.5~8(~8.5) μm.
Note: KUC21221 showed high activities of cellulolytic enzymes, antifungal metabolites and antioxidant capacity[4]. This strain often altered irregular form which grow slowly. KUC21221 and other A. saccharicola were mono phyletic with high support (100% of PP, Fig. 2).
Culture examined: KOREA, Jeju Island, N33°23'39.3" E126°14'23.2", isolated from Sargacium sp. 10 January 2015, Seokyoon Jang (culture KUC21221, GenBank KT 207637).
Phylogenetic analysis
Some species of Arthrinium formed species complexes in a phylogenetic analysis based on internal translated spacer (ITS) or 28s ribosomal RNA, large subunit (LSU) region [3]. To avoid this problem, EF1 and/or TUB with higher resolution power were recommended [3, 4]. Arth- rinium cultures examined in this study were classified into two clades. Morphology of the tree was in line with other studies [3, 4]. Three cultures were identified with A. arundinis or A. saccharicola. We suppose more than 10 species of Arthrinium present in Korea. Bamboos and marine environments are major habitats of Arthrinium [1-4]. Thus, further study with fungi from these habitats is needed to reveal bio-diversity of Korean Arthrinium.
