INTRODUCTION
Dothideomycetes, the largest and most varied class of Ascomycota, consist of 22 orders, 105 families, 178 genera, and over 19,000 species [1], and the genus Leptosphaerulina is classified under this class. The Leptosphaerulina is endemic to North America, South America, South Africa, Asia, Australia and Europe and comprises around 25 described species [2-6]. The anamorphic stage of Leptosphaerulina was reported for the first time in a study in the Karoo region of South Africa by Daniel McAlpine in the year 1902, and Leptosphaerulina australis was designated as the type specimen [6]. It is reported that Leptosphaerulina species are common fungi that can colonize several turf grass species under humid conditions and peanuts [7-8]. The fungus causes damage to 10 different angiosperm host genera belonging to 7 families of dicotyledons [9]. Species of Leptosphaerulina have been classified as saprophytes, senectophyte, has weak pathogenicity in turfgrasses [10]. Recently, L. australis has been reported for the first time in Korea [11].
In this study, a fungal isolate PTT-2 was obtained from the bark of the trunk of a persimmon (Diospyros kaki) tree in Cheongdo, and it was identified as a member of the genus Leptosphaerulina. Based on the morphological and molecular characteristics, the isolate was identified as L. saccharicola which has not been reported in Korea. This is the first report of its isolation and identification in Korea.
MATERIALS AND METHODS
Collection of bark sample and fungal strain isolation
During screening of fungal species in 2018, a fungal strain PTT-2 was isolated from the bark of a persimmon tree in Cheongdo, Korea (35°40′12.6″N, 128°35′52.5″E). A small portion of the bark of the tree was scraped onto potato dextrose agar (PDA) media and then incubated at 25°C. The growth of the colonies was observed for 2~3 days before the colonies were transferred to new PDA media and incubated again at 25°C. Strain PTT-2 was selected from numerous other fungal strains for further morphological and molecular phylogenetic analyses.
Morphological observation
To study growth and morphological characteristics, strain PTT-2 was cultured on four different media: PDA, oatmeal agar (OA), malt extract agar (MEA) and synthetic nutrient poor agar (SNA) [1,11]. After 7 days, colony characteristics such as color, shape, and size were recorded. Colonies on PDA, OA, MEA and SNA media were illuminated with ultraviolet light on a 12 hrs diurnal cycle for 10 days at 25°C to induce sporulation. Morphological characteristics were observed under a light microscope (BX-50; Olympus, Tokyo, Japan).
DNA extraction, PCR amplification, and sequencing analysis
For phylogenetic analysis, DNA from strain PTT-2 was extracted from mycelia on PDA using the HiGene Genomic DNA Prep Kit (BIOFACT, Daejeon, Korea) following manufacturer’s instructions. PCR amplification was performed to amplify the internal transcribed spacer (ITS) rDNA region using primers ITS1F/ITS4, the partial sequence of the translation elongation factor 1-α (TEF1-α) gene region using primers EF1-983F/EF1-2218R, the large subunit (LSU) gene region using primers LROR/LR7 and the RNA polymerase II largest subunit gene (RPB2) using primers fRPB2-5F/fRPB2-7CR [12-16]. Amplified PCR products were purified with ExoSAP-IT (Thermo FisherScientific, Waltham, MA, USA) and sequenced by Solgent (Daejeon, Korea). The obtained sequences of strain PTT-2 were deposited in NCBI GenBank, with accession number of LC465237 for ITS, LC465238 for LSU, LC465240 for TEF1-α and LC465239 for RPB2 gene sequences.
Phylogenetic analysis
Sequences of allied species were retrieved from NCBI GenBank. Phylogenetic trees were constructed based on a concatenated dataset of ITS regions, partial of TEF1-α, LSU and RPB2 gene sequences using the neighbor-joining (NJ) method in the MEGA 6 software program with bootstrap analysis of 1,000 replications [17].
RESULTS AND DISCUSSION
Morphology of the strain PTT-2
Colonies of PTT-2 grew reaching 53.8 mm, 49.8 mm, 48.4 mm, and 28.1 mm in diameter, after 7 days of incubation at 25ºC on PDA, OA, MEA and SNA media, respectively (Fig. 1). The mycelial colonies were circular on PDA, OA and MEA media, but wavy and circular on SNA medium. The morphological characteristics of the colonies were compared with previous description of L. saccharicola (Table 2) [1]. Ascospores of strain PTT-2 were initially hyaline, becoming brown to dark brown on PDA, and reached 29.6 × 11 µm in size. Transverse and longitudinal septa were also observed. The number of longitudinal septa ranged from 0~2, while the transverse septa from 0~1 [1]. Strain PTT-2 produced abundant hyaline ascospores, which had irregular wavy edge, triseriate oblong to ellipsoidal shape and 23.6 × 10 µm diameter (Table 2). The number of longitudinal septa of ascospores ranged from 0~1, while transverse septa numbered 3~4. The asci and ascospores were observed (Fig. 2). The morphological characteristics observed for strain PTT-2 were very similar to the previously described characteristics of L. saccharicola.
Molecular phylogeny of strain PTT-2
After sequencing analysis, sequences of 501 bp, 1,271 bp, 930 bp and 1,048 bp were obtained from ITS regions, LSU, TEF1-α and RPB2 gene, respectively. BLAST search results revealed that strain PTT-2 shared 99% identities with the ITS regions, TEF1-α and RPB2 gene sequences with three other strains of L. saccharicola (MG583749, KF670715, KF670714). For LSU, blast search results showed the 100% similarities with a strain of L. australis (MH871269). A concatenated dataset of ITS, LSU, TEF1-α and RPB2 gene sequences was used to determine the molecular relationship between the present Korean isolate and Leptosphaerulina species retrieved from GenBank. A NJ tree showed that PTT-2 strain clustered in the same clade with the other L. saccharicola strains, indicating that PTT-2 is a strain of L. saccharicola (Fig. 3). Although L. saccharicola associated with leaf disease of Saccharum officinarum, but also as an important pathogen of turf grass [1]. In view of this, the pathogen is very relevant to agricultural production and further studies need to investigate its potential pathogenicity to aid identifying relative cultivars that are susceptible to the pathogen. To our knowledge, this is the first report of Leptosphaerulina saccharicola in Korea and its identity is strongly supported by the morphological and molecular evidence presented here.
Fig. 3. Neighbor-joining phylogenetic tree, based on the concatenated ITS, TEF1-α, LSU and RPB2 gene sequences shows the phylogenetic position of Leptosphaerulina saccharicola PTT-2 among members of the genus Leptosphaerulina. The strain isolated in this study was shown in boldface. Bootstrap values (based on 1,000 replications) are shown at the branch points. Botryosphaeria qingyuanensis CERC 2946 was used an outgroup.
