Morphological and phylogeny of Plenodomus sinensis and P. collinsoniae, two unreported species isolated from soil in Korea

Research Article
Than Naing Moe1Kallol Das1In-Kyu Kang2Seung-Yeol Lee1,3*Hee-Young Jung1,3


Two unreported fungal isolates, KNU-GW1901 and KNU-AP100C, were collected from soil sample in Gyeongsangbuk-do, Korea. Their cultural and morphological characteristics were examined after 4 weeks of incubation at 25℃ on potato dextrose agar (PDA), malt extract agar (MEA), and oatmeal agar (OA). The conidial shape of KNU-GW1901 was aseptate, hyaline, globose to ellipsoidal, oblong, and reniform to pyriform and 2.61-4.97×1.93-3.61 µm in size, whereas no conidial structures were observed in KNU-AP100C. The internal transcribed spacer (ITS) regions, large subunit (LSU), and small subunit (SSU) sequences were used to determine the taxonomic positions of the strains using the maximum likelihood phylogenetic tree. The isolate KNU-GW1901 was closely clustered with Plenodomus sinensis MFLUCC 17-0767, and KNU-AP100C was closely matched with P. collinsoniae CBS 120227. Based on the findings of morphological, cultural, and phylogenetic analysis, the isolates KNU-GW1901 and KNU-AP100C were identical to the previously described P. sinensis and P. collinsoniae isolates, respectively, which are first reported in Korea.



Dothideomycetes is the largest class of Ascomycota, comprising more than 100 families and 19,000 species, and Pleosporales represents the largest order in Dothideomycetes, consisting of a quarter of all Dothideomycetous species [1,2]. The family Leptosphaeriaceae was introduced by Barr in 1987, which is an economically important family of the order Pleosporales for plant pathogens, and the genus Plenodomus was included in the family [3,4]. Species of this family are generally saprophytes but also parasitic on herbaceous or woody plants on the land and in water bodies [5]. Members of this family are well known for their immersed perithecial ascomata possessing single papillate ostioles, hyaline to brown cylindrical asci, and their transverse septate ascospores [3,5]. They can also exist as asexual morphs, which are either Coelomycetes or Hyphomycetes [6,7]. The genus Plenodomus was first introduced in 1851 by Preuss with P. rabenhorstii as a type species [3,8]. P. ravenhorstii was replaced by Borerema & Kesteren (1964) with P. lingam (Tode) Hohn, which is a sexual morph because the former material was lost during World War II [3,9]. Currently, there are 100 epithets of Plenodomus listed in Index Fungorum in 2020 [10]. The members of the genus Plenodomus have also been reported in some Asian and European countries while P. biglobosus, P. lindquistii, P. tracheiphilus, and P. wasabiae have been known as important pathogens [11]. In the present study, we aimed to investigate the fungal species in Korea as well as to establish the stable taxonomy and phylogeny for the fungal isolates collected from a field soil sample and to identify the species based on morphological characteristics and phylogenetic analysis. We provide detailed information about the cultural and morphological characteristics of two unreported species for the first time in Korea.


Collection of soil samples

The fungal isolates KNU-GW1901 and KNU-AP100C were isolated from soil, collected from a disused apple orchard in Gyeongsangbuk-do (36°16′32.7″N, 128°27′59.1″E and 36°11′45.9″N, 128°34′10.8″E), Korea. These soil samples were collected from the ground at a depth of approximately 15-30 cm, air-dried, and then placed in a plastic bag at 4℃ till analysis. Each of the soil samples weighed approximately 1 g and were suspended in 10 mL of sterile distilled water. The soil solution was vortexed and diluted until suspended, and then 1 mL was spread on potato dextrose agar (PDA; Difco, Detroit, MI, USA) and incubated for 2-3 days at 25℃ [12]. Then, the individual colonies were transferred to new fresh PDA plates and incubated at 25℃ until the development of mycelium. Based on different cultural characteristics of these two strains, they were selected for further molecular analyses. These isolates, KNU-GW1901 and KNU-AP100C, were maintained in 20% glycerol at -80℃ for further studies.

Morphological characterization

For morphological observations, the strains KNU-GW1901 and KNU-AP100C were transferred onto potato dextrose agar (PDA; Difco, Detroit, MI, USA), oatmeal agar (OA; Difco, MI, USA), and malt extract agar (MEA; Difco, MI, USA) and incubated at 25℃ for 4 weeks [13]. After incubation, the fungal growth of each strain was measured, and colony characteristics such as the shape, color, and size were recorded. Morphological characteristics of the isolates were observed under a light microscope (BX-50, Olympus, Tokyo, Japan).

Genomic DNA extraction, PCR amplification, and sequencing

Fungal mycelia were obtained by scraping from PDA plates for the extraction of genomic DNA. Then, the HiGene Genomic DNA Prep Kit (Biofact, Daejeon, Korea) was used to extract genomic DNA from the two isolates according to the manufacturer’s instructions. For the isolate KNU-GW1901 and KNU-AP100C, the large subunit (LSU), small subunit (SSU), and the internal transcribed spacer (ITS) regions were amplified using the primers LROR/LR5 or LROR/LR7, NS1/NS4, and ITS1F/ITS4 [14-16], respectively. The thermal conditions for PCR amplification were followed as described by a previous report [7]. Then, the amplified PCR products were purified using ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced by Macrogen Co. Ltd. (Daejeon, Korea). Finally, the obtained sequences from KNU-GW1901 and KNU-AP100C isolates were deposited in NCBI GenBank under the accession number of LC550566 and LC550567 for ITS, LC550568 and LC550569 for LSU, LC550570 for SSU.

Phylogenetic analysis

The partial sequences of large subunit (LSU), small subunit (SSU), and ITS regions of the allied species with P. sinensis and P. collinsoniae were retrieved from NCBI for phylogenetic analysis (Table 1). The consent sequences were compared with other allied sequences in the NCBI database using BLAST search results. The MEGA 7 software program was used to construct the phylogenetic tree with the bootstrap analysis of 1,000 replications [17]. Evolutionary distances were calculated using the maximum likelihood method based on the Tamura-Nei model [18].

Table 1. The isolates KNU-GW1901 and KNU-AP100C and culture collection strains of GenBank accession numbers used in this study for phylogenetic analyses.

The species isolated in this study is indicated in bold.

CBS: Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; MFLUCC: Mae Fah Luang University Culture Collection, Chiang Rai, Thailand; KNU: Kyunpook National University, Daegu, South Korea.


Taxonomy of KNU-GW1901

Plenodomus sinensis Tennakoon, Phookamsak & K.D. Hyde, Phytotaxa 324(1): 76(2017) (Fig. 1).

Fig. 1. Cultural and morphological characteristics of Plenodomus sinensis KNU-GW1901. A,colonies on potato dextrose agar; B, colonies on malt extract agar; C, colonies on oatmeal agar; D, pycnidium; E, F, conidia (Scale bars: D-F=10 μm).

Colony characteristics: The isolate KNU-GW1901 had a slow growth rate, with the average fungal growth rates on PDA, MEA, and OA being 48.5, 52.6, and 68.2 mm, respectively, at 25℃ for 4 weeks. The upper surface of the colonies on PDA was white to light gray in color in the center and reverse olivaceous green to black in the center with regular to irregular white margin (Fig. 1A). The colonies on MEA were covered by a gray-white to deep gray, circular margin, fluffy, and reverse olivaceous green to pale black with a light yellowish margin (Fig. 1B). Regarding the colonies on OA surface, the mycelium appeared white to dark gray with iron-gray spots and irregular margins. The elevation of the colonies on all media demonstrated an umbonate type (Fig. 1C).

Micromorphology: The isolate KNU-GW1901 produced pycnidia on the surface of PDA media after 4 weeks. The pycnidia measured 123 µm high and 110 µm wide and were scattered, solitary to gregarious, black and globose to subglobose (Fig. 1D). The conidia were aseptate, hyaline, globose to ellipsoidal, oblong, and reniform to pyriform and measured 2.61-4.97×1.93-3.61 µm (Fig. 1E and 1F). The morphology of the strain KNU-GW1901 was comparable with the previous research finding of P. sinensis [13].

Phylogenetic analysis of KNU-GW1901

Sequences of the LSU, SSU, and ITS regions were used to construct the phylogenetic placement of the isolate KNU-GW1901. BLAST searches in the nucleotide database of GenBank (www were used to obtain the closed taxa of the new isolate. After analyzing the nucleotide sequences, 595, 976, and 731 bp were obtained from the LSU, SSU, and ITS regions, respectively. Based on the BLAST search results, KUN-GW1901 exhibited high similarities in ITS (98.79%), SSU (100%), and LSU (99.59%) with the previously identified P. sinensis (MFLUCC 17-0767). The isolate KNU-GW1901 was closely clustered together with P. sinensis (MFLUCC 17-0767) in the maximum likelihood phylogenetic tree using the combined sequences of LSU, SSU and ITS regions (Fig. 2). Thus, the phylogenetic results support the morphological identification that the strain KNU-GW1901 was P. sinensis.

Fig. 2. Maximum likelihood phylogenetic tree based on the combined dataset of partial sequences of large subunit (LSU), small subunit (SSU), and internal transcribed spacer (ITS) regions. Didymella exigua CBS 183.55 was used as an outgroup. The isolated strain in this study is indicated in bold, and the bootstrap values (based on 1000 replications) higher than 70% is shown at the branch points. Bar, 0.005 substitutions per nucleotide position.

Taxonomy of KNU-AP100C

Plenodomus collinosinae (Dearn. & House) Gruyter, Aveskamp & Verkley, Studies in Mycology 75: 21 (2012) (Figure 3).

Colony characteristics: The isolate KNU-AP100C was cultured on PDA, MEA, and OA to observe the cultural and morphological characteristics. The growth of the colonies reached 57.2 mm on PDA, 58.2 mm on MEA, and 52.5 mm on OA, at 4 weeks of incubation at 25℃. The colonies on PDA were white to light gray color in the center with a circular margin and reverse orange to light black color in the center with a wide white margin (Fig. 3A). The colonies on MEA were white to light gray with black spots near the center and reverse pale yellow to orange with a circular margin (Fig. 3B). The surface of the colonies on OA were initially white to gray becoming brown in the center (Fig. 3C).

Fig. 3. Cultural and morphological characteristics of Plenodomus collinsoniae KNU-AP100C. A, colonies on potato dextrose agar; B, colonies on malt extract agar; C, colonies on oatmeal agar; D, conidiomata on potato dextrose agar (PDA); E, F, chlamydospore-like cells formed in culture. Scale bars: D=200 μm; E, F=10 μm.

Micromorphology: The isolate produced numerous conidiomata on the surface of PDA after 4 weeks of incubation at 25℃. The conidiomata were round to irregular, solitary to aggregate, and dark brown to black on the surface of the medium (Fig. 3D). Wide hyphae, branched mycelium, septate, smooth, brown, and light brown chlamydospores were found on PDA media (Fig. 3E and 3F). No conidial structures were observed in KNU-AP100C despite being cultured on PDA, MEA, and OA media after 4 weeks of incubation at 25℃. These media did not stimulate conidial production, and thus some special methods might be required to induce the production of conidia. Environmental factors such as temperature, humidity, and light should be taken into consideration to assess the production of conidia in KNU-AP100C.

Phylogenetic analysis of KNU-AP100C

Sequence analysis of KNU-AP100C resulted in 589 bp from the ITS regions and 1168 bp from the LSU. The isolate KNU-AP100C demonstrated 99% similarities with P. collinsoniae CBS 120227 in the ITS and LSU. The combined sequences of ITS and LSU were used to construct the maximum likelihood phylogenetic tree, and the isolate KNU-AP100C was matched with P. collinsoniae exhibiting 99% bootstrap values (Fig. 4). Thus, the molecular phylogenetic results supported the identification of the isolate KNU-AP100C as P. collinsoniae.

Fig. 4. Maximum likelihood phylogenetic tree based on the combined dataset of large subunit (LSU) and internal transcribed spacer (ITS) regions. Alternariaster helianthi CBS 327.69 was used as an outgroup. The fungal strain which examined in this study was indicated in bold, and the bootstrap value below 70% is not shown. Bar, 0.005 substitutions per nucleotide position.


In the present study, the two fungal isolates, KNU-GW1901 and KNU-AP100C, were collected and isolated from soil samples in Korea. These isolates were found to be morphologically similar to P. sinensis and P. collinsoniae, respectively, which have been described in previous studies [8,13]. The isolate KNU-AP100C, identified as P. collinosinae, was one of the combined novel species, and its previous name was Leptosphaeria collinsoniae [8]. L. collinsoniae was first reported by Dearn & House from USA who described its morphological characteristics as a sexual stage on the blackened stem of the stone root Collinsonia canadensis L. and asexual data were not determined [19]. It has been reported that P. chrysanthemi infected chrysanthemum plants in Greece, P. biglobosus caused upper stem lesions on Brassica species in France and Netherlands, and P. collinsoniae infected Vitis coignetiae plants in Japan [8]. In addition, P. destruens was isolated from the storage tuber rot on sweet potato in Korea [20]. Moreover, Plenodomus species caused cankers and leaf spots associated with a wide variety of substrates and were reported to be widely distributed from temperate to tropical countries throughout the world [21,22]. Although the specific host of the genus Plenodomus has not yet been confirmed, the species has been recorded from various plant families such as Asteraceae, Fabaceae, Lamiaceae, and Liliaceae [8]. To our knowledge, the isolates P. sinensis KNU-GW1901 and P. collinsoniae KNU-AP100C are the first reports of Plenodomus species in Korea.


This research was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea for the project on survey and discovery of indigenous fungal species (NIBR201902112).


1  1. Crous PW, Verkley GJM, Groenewald JZ. Phytopathogenic Dothideomycetes. Stud Mycol 2013;75:1-6. 

2  2. Kirk PM, Cannon PF, Minter DW, Staplers JA. Dictionary of the fungi. 10th edn. Wallingford: CABI Bioscience; 2008. 

3  3. Ariyawansa HA, Phukhamsakda C, Thambugala KM, Bulgakov TS, Wanasinghe DN, Perera RH, Mapook A, Camporesi E, Kang JC, Jones EG, et al. Revision and phylogeny of Leptosphaeriaceae . Fungal Divers 2015;74:19-51. 

4  4. Hyde KD, Jones EBG, Camporesi E, McKenzie EHC, Hongsanan S, Phookamsak R, Luo ZL, Boonmee S, Li WJ, Dissanayake AJ, et al. Fungal diversity notes 367-500: taxonomic and phylogenetic contributions to fungal taxa. Fungal Divers 2016;80:1-270. 

5  5. Hyde KD, Jones EG, Liu JK, Ariyawansa HA, Boehm E, Boonmee S, Braun U, Chomnunti P, Crous PW, Dai DQ, et al. Families of Dothideomycetes. Fungal Divers 2013;63:1-313. 

6  6. Alves JL, Woudenberg JHC, Duarte LL, Crous PW, Barreto RW. Reappraisal of the genus Alternariaster (Dothideomycetes). Persoonia 2013;31:77-85. 

7  7. Tennakoon DS, Phookamsak R, Wanasinghe DN, Yang JB, Lumyong S, Hyde KD. Morphological and phylogenetic insights resolve Plenodomus sinensis (Leptosphaeriaceae) as a new species. Phytotaxa 2017;324:73-82. 

8  8. de Gruyter J, Woudenberg JHC, Aveskamp MM, Verkley GJM, Groenewald JZ, Crous PW. Redisposition of Phoma -like anamorphs in Pleosporales. Stud Mycol 2012;75:1-36. 

9  9. Torres MS, Bergen M, Singh S, Bischoff J, Sullivan RF, White Jr JE. Plenodomus morganjonesii sp. nov. and a discussion of the genus Plenodomus . Mycotaxon 2005;93:333-44. 

10  10. Index Fungorum. Index Fungorum [Internet]. Kew: Royal Botanic Gardens Kew; 2020 [cited 2020 Apr 20]. Available from: 

11  11. Marin-Felix Y, Groenewold JZ, Cai L, Chen Q, Marincawitz S, Barnes L, Braun U, Camporesi E, Damm U, de Beer ZW, et al. Genera of phytopathogenic fungi: GOPHY 1. Stud Mycol 2017;86:99-216. 

12  12. Paul NC, Mun HY, Lee HW, Yu SH, Lee HB. A new record of Penicillium raphiae isolated from agricultural soil of Ulleung Island, Korea. Mycobiology 2014;42:282-5.  

13  13. Phookamsak R, Hyde KD, Jaewon R, Bhat DJ, Jones EBG, Maharachchikumbura SSN, Rasoe O, Karunarathna SC, Wanasinghe DN, Hongsanan S, et al. Fungal divers notes 929-1035: Taxonomic and phylogenetic contributions on genera and species of fungi. Fungal Divers 2019;95:1-273. 

14  14. Vilgalys R, Hester M. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 1990;172:4238-46. 

15  15. White TJ, Bruns T, Lee S, Taylor JW. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR Protocols: A guide to methods and applications. San Diego: Academic Press; 1990. p. 315-22. 

16  16. Gardes M, Bruns T. ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol Ecol 1993;2:113-8. 

17  17. Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016;33:1870-4. 

18  18. Tamura K, Nei M. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 1993;10:512-26. 

19  19. Dearness J, House HD. New or noteworthy of species fungi II. Bulletin of the York State Museum 1921;233-234:32-43. 

20  20. Paul NC, Nam SS, Park W, Yang JW, Kachroo A. First report of storage tuber rot in sweet potato ( Ipomoea batatas ) caused by Plenodomus destruens in Korea. Plant Dis 2019;103:1020. 

21  21. Wijayawardene NN, Hyde KD, Rajeshkumar KC, Hawksworth DL, Madrid H, Kirk PM, Braun U, Singh RV, Crous PW, Kukwa M. Notes for genera: Ascomycota. Fungal Divers 2017;86:1-594. 

22  22. Farr DF, Rossman AY. Fungal Databases, US National Fungal Collections [Internet]. Washington DC: ARS USDA; 2019 [cited 2020 Apr 20]. Available from: