Introduction
The genus Trichocladium, which belongs to the subphylum Pezizomycotina, class Sordariomycetes, order Sordariales, and family Chaetomiaceae, was first described by Harz in 1871 with Trichocladium asperum as the type species [1]. Members of this genus are characterized by the production of darkly pigmented, septate conidia. Species belonging to the genus Trichocladium have been frequently isolated from soil, estuarine sediment, leaf, dung, grass, meal, and Usnea cf. aurantio-atra [2]. Recently, Wang et al. [2] redefined Humicola sensu stricto and related genera in the family Chaetomiaceae based on the phylogenetic analyses of multi-gene sequences combined with morphological observations, and they transferred certain species of Beniowskia, Chaetomium, Chaetomidium, Gilmaniella, Humicola, Monodictys, Thielavia, and Monodictys to the new genus Trichocladium. To date, 12 species belonging to this genus are known [2]. Prior to its renaming, Trichocladium griseum was classified under the name Humicola grisea.
Knowledge of the taxonomy of certain fungi within the genera Trichocladium in Korea is limited. A recent study reported the presence of Trichocladium asperum in soil [3]. A survey of Korean indigenous fungal diversity was conducted in 2017 in Ulsan, South Korea. Fungal isolates possessing various morphologies were isolated from soil samples obtained from paddy fields. Consequently, an unrecorded species belonging to the genus Trichocladium (T. griseum) was discovered. This fungal species has not previously been reported in Korea. In this report, macro-morphological and micro-morphological characteristics of this newly recorded species are presented.
Materials and methods
Isolation of the fungal isolate
Soil samples were collected in 2017 from a paddy field located in Ulsan (35°31'45.36''N, 129°06'23.12''E), South Korea. Crop debris were removed and soil samples were collected at a depth of 10~15 cm. Thesoil samples were dried and then stored at 4°C in a sterile polythene bag prior to use. Morphologically different isolates of fungal species were isolated on potato dextrose agar (PDA, Difco Laboratories, Detroit, Michigan, USA) supplemented with 100 μg L-1 chloramphenicol using conventional soil dilution techniques [4]. The diluted soil suspensions were streaked onto the petri plates and then incubated for 5 days at 25°C. The developing morphologically different colonies were then aseptically transferred to fresh PDA plates to obtain pure cultures. The pure fungal isolates were finally reserved on PDA slants at 4°C for further use.
Morphological examination
For detailed morphological studies, the EML-KNU17-5 strain was cultured on malt extract agar (MEA), oatmeal agar (OA; 30 g of oatmeal and 20 g of agar in 1 L of deionized water), and PDA at 25°C for 7 days. The colonies grown on OA were observed microscopically. Fragments of mycelia were removed from the cultures and placed on microscope slides with lactic acid. An Olympus BX51 microscope possessing differential interference contrast optics (Olympus, Tokyo, Japan) was used to obtain digital images.
Genomic DNA extraction, PCR, sequencing, and phylogenetic analysis
The genomic fungal DNA was isolated from mycelia of the fungal isolate using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The β-tubulin (tub2) gene and the ITS region from EML-KNU17-5 isolates were amplified using the primer pairs Bt2a and Bt2b [5] and ITS1 and ITS4 [6], respectively. The PCR products were sequenced at Macrogen (Seoul, Korea) using an ABI PRISM 3730XL Analyzer (Life Technologies, Gaithersburg, MD, USA). The DNA sequences were subjected to BLAST analysis using the GenBank database. The construction of phylogenetic trees was accomplished by neighbor-joining (NJ) method using Kimuras 2-parameter model [7] implemented in the program MEGA version 6 [8] with 1,000 bootstrap replicates. The EML-KNU17-5 isolate used in this study was deposited at the National Institute of Biological Resources (NIBR, Incheon, Korea), as NIBRFG000499999. The sequences of EML-KNU17-5 were deposited into the NCBI database under the accession numbers indicated in Table 1.
Results
Phylogenetic analysis
For the phylogenetic analysis, ITS and β-tubulin gene sequences were used to analyze the phylogenetic relationships between EML-KNU17-5 and related species. BLASTn search of ITS rDNA indicated that the strain EML-KNU17-5 was most closely related to H. grisea CGMCC 3.13888 (current name: T. griseum) (GenBank accession no. LT993641) with 100% identity. BLASTn analysis of tub2 of EML-KNU17-5 revealed a similarity of 99.6% to H. grisea CBS 119.14 (current name: T. griseum) (Genbank accession no. LT993720). The phylogenetic relationships among the strain EML-KNU17-5 and related species were analyzed using single genes and the combined datasets for ITS and tub2 (Fig. 1-3). The strain EMLKNU17-5 was grouped within the same clade as T. griseum with 90%, 96%, and 99% bootstrap support according to ITS, tub2, and a combination of ITS and tub2 sequences, respectively.
Morphological characterization
Taxonomic descriptions of the morphological structures of the T. griseum EML-KNU17-5 are detailed below.
Fig. 1. Phylogenetic tree based on neighbor-joining analysis of the internal transcribed rDNA sequence obtained from EML-KNU17-5. Microascus trigonosporus was used as outgroup. Bootstrap scores of 60% are indicated at the nodes. The scale bar represents the number of substitutions per site. The new isolate described in this study is shown in bold.
Fig. 2. Phylogenetic tree based on neighbor-joining analysis of β-tubulin gene sequence obtained from EML-KNU17-5. Microascus trigonosporus was used as outgroup. Bootstrap scores of > 60% are indicated at the nodes. The scale bar represents the number of substitutions per site. The new isolate described in this study is shown in bold.
Fig. 3. Phylogenetic tree based on neighbor-joining analysis of the combined datasets for ITS and β-tubulin sequences obtained from EML-KNU17-5. Microascus trigonosporus was used as outgroup. Bootstrap scores of ≥ 60% are indicated at the nodes. The scale bar represents the number of substitutions per site. The new isolate described in this study is shown in bold.
Colony characteristics
Trichocladium griseum (Traaen) X. Wei Wang & Houbraken, Studies in Mycology 93: 141 (2018) [MB#824469] (Table 2, Fig. 4).
Colony characteristics of the fungal isolate EML-KNU17-5 on various agar media are shown in Figure 4. Colonies on PDA exhibited greenish-black centers and grey-white margins, and they were reverse olivaceous black with moderate sporulation and reached 37~40 mm in diameter after 7 days at 25°C. Colonies on MEA were olivaceous grey with a velvety to floccose texture, and they were also reverse greenish black and reached 35~38 mm in diameter after 7 days at 25°C. On OA, colonies were pale olivaceous grey to mouse-grey throughout with greenish-black centers and light grey margins, and they possessed floccose colony texture and reached 40~43 mm in diameter after 7 days at 25°C.
Micromorphology
Conidiophores were hyaline, unbranched, and occasionally exhibited swelling at the tops, and these structures were 3.5~69.0 μm long and 2.5~5.0 μm wide. Conidia were globose or subglobose, brown to dark brown, smooth, and measured 9.5~16.5 × 8.5~15.0 μm. Acremonium-like conidia formed in chains that were obovoid to ellipsoidal.
Discussion
To date, only one species of the genus Trichocladium, T. asperum, has been reported in Korea. Given this, our findings increase the knowledge of the occurrence and distribution of T. griseum in paddy field habitats.
In the β-tubulin phylogenetic tree (Figure 2), our experimental strain (EML-KNU17-5) was clustered with other T. griseum species through well-supported branches. However, the ITS tree of EML-KNU17-5 was grouped with certain species that included T. asperum, T. gilmaniellae, and T. griseum (Figure 1). In the combined datasets for the tub2 and ITS phylogenetic tree, EML-KNU17-5 was clustered into the same clade with Trichocladium griseum CBS 119.14 (Figure 3) through well-supported branches, and this was consistent with a previous study by Wang et al. [2]. There were a few differences observed in the colony diameter of Trichocladium griseum when compared to that of previous descriptions. The colony diameter on OA and MEA media was observed to be different from that of previously described Trichocladium griseum (OA: 42~48 mm; MEA: 41~47 mm). However, the morphological characteristics of our isolate were largely similar to those previously described by Wang et al. [2]. To the best of our knowledge, this study is the first report of Trichocladium griseum in South Korea.
ACKNOWLEDGEMENTS
This research was financed by the Ministry of Environment (MOE) under the Republic of Korea from the National Institute of Biological Resources (NIBR) grant on the survey and discovery of indigenous fungal species of Korea. This research study was also sponsored by the University-Industry Cooperation Foundation of Kangwon National University.
