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 . 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 . 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 . 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.
MATERIALS AND METHODS
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℃ . 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.
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 . 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 . 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.
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 . Evolutionary distances were calculated using the maximum likelihood method based on the Tamura-Nei model .
Taxonomy of KNU-GW1901
Plenodomus sinensis Tennakoon, Phookamsak & K.D. Hyde, Phytotaxa 324(1): 76(2017) (Fig. 1).
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 .
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 http://blast.ncbi.nlm.nih.gov/) 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.
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).
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.
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 . 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 . 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 . In addition, P. destruens was isolated from the storage tuber rot on sweet potato in Korea . 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 . To our knowledge, the isolates P. sinensis KNU-GW1901 and P. collinsoniae KNU-AP100C are the first reports of Plenodomus species in Korea.