INTRODUCTION
The genus Phoma, since it was first identified over 170 years ago, has been considered one of the largest fungal genera with over 3,000 infrageneric taxa [1,2]. Moreover, it is very well-known because it includes several fungal pathogens associated with plants. Most species in this genus cause mainly leaf and stem spots on plants and usually live in the environment as saprobic soil organisms, but become pathogenic when the conditions are suitable [3,4]. In addition, other species of the genus Phoma are pathogenic to humans, cattle, and fish [5,6]. They are also responsible for production of toxic secondary metabolites that may indirectly affect human and animal health [7]. Epicoccum draconis (syn. Phyllosticta draconis, Phoma draconis) was first acknowledged by Chen et al. [8]. It is distributed worldwide as pathogenic fungi causing diseases in plants and can thrive in the soil, air, animals, and humans [9,10]. E. draconis was first reported in Rwanda, Africa, from the wild Dracaena spp. and later found in the cultivated species of the Dracaena in Europe, India, and North America. This species is known as causing agent of leaf spot and dieback in Cordyline spp. [11]. The main objective of the study was to study the morphological and molecular characteristics of the isolate US-18-11 and compare it with the previously reported E. draconis.
MATERIALS AND METHODS
Soil sample collection and fungal isolation
Soil samples were collected from a tobacco (Nicotiana tabacum L.) field in Uiseong (N36°25'14.7", E128°45'34.1"), Korea. The soil sample was collected whenever all the tobacco were harvested. A sterile spatula was used to collect the soil sample at a depth of 15-30 cm. The soil sample was placed in a polythene zipper bag, transferred to the laboratory, and stored at 4℃ until analysis. For analysis, one gram of the soil was suspended in 10 mL of sterile distilled water. The suspension was vortexed, serially diluted, and spread on potato dextrose agar (PDA; Difco, MI, USA). Individual colonies that developed on the agar were purified by subculture on fresh PDA and incubated at 25℃ until mycelium development. The pure cultures were preserved on PDA slants at 4℃. The isolate US-18-11 was selected for subsequent morphological and molecular analysis.
Morphological and cultural characterization
The colony characteristics of isolate US-18-11 such as color, shape and size were recorded after 7 days of incubation at 22℃ [12] on malt extract agar (MEA; Difco, MI, USA) and oatmeal agar (OA; Difco, MI, USA) while morphological characteristics were studied after 14 days. A light microscope (BX-50 light microscope-Olympus, Tokyo, Japan) was used to observe the morphological characteristics of the isolate.
Genomic DNA extraction, PCR amplification and phylogenetic analysis
For molecular identification based on multiple gene sequences, we extracted the genomic DNA of the isolate US-18-11 from fungal mycelium growing on MEA medium following the manufacturer’s instructions for the HiGene Genomic DNA prep kit (BIOFACT, Daejeon, Korea). PCR amplification was performed to amplify four molecular markers. The primers ITS1F and ITS4 for the internal transcribed spacer (ITS) region, LROR and LR7 for the 28S rDNA (LSU) gene, Btub2Fd and Btub4Rd for the partial beta-tubulin (TUB2) gene and rpb2-5F2 and rpb2AM-7cR for the second largest subunit of DNA-directed RNA polymerase II (rpb2) gene region were used for the amplification [13-19]. Then, the amplified PCR products were purified with ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced (Macrogen, Daejeon, Korea). The similarities of obtained sequences were analyzed using basic local alignment search tool (BLAST) in the National Center for Biotechnology Information (NCBI) and Genetyx-Win ver. 10.0 (Genetyx Corporation, Tokyo, Japan). The partial sequences of ITS region, LSU, rpb2 and TUB2 genes of allied species of E. draconis were retrieved from NCBI to perform the phylogenetic analysis (Table 1). The obtained sequences of US-18-11 were deposited in NCBI GenBank under the accession number of LC496555 for ITS region, LC500288 for LSU, LC500353 for rpb2 and LC500354 for TUB2 genes. The sequences of our isolate were compared with reference sequences from the NCBI GenBank database by using BLAST. The fungal strains used for a construction of the phylogenetic tree are summarized in Table 1. Based on the Kimura’s neighbor-joining algorithm model, the evolutionary distance matrices were generated using the program MEGA 7 with bootstrap values based on 1,000 replications [20].
RESULTS AND DISCUSSION
Morphology of the isolate US-18-11
The isolate US-18-11 was observed on MEA, and OA media after 14 days at 22℃. The diameters of the colonies on MEA and OA were 42-43 mm and 41-44 mm, respectively. These characteristics were compared with those of previously described E. draconis CBS 186.83 (Table 2). On MEA, colonies formed white-to-dull green aerial mycelium, floccose texture, and regular margins and appeared olivaceous black with leaden grey patches on the reverse side. On OA, colonies showed regular margins with white-to-grey olivaceous aerial mycelium, floccose texture, and were tufted (Fig. 1A-D). Morphological characters were described after 14 days of incubation (Fig. 1E-H). Hyphae were hyaline-to-light brown, thin-walled, smooth, and septate. Pycnidia were immersed, globose to subglobose or obpyriform, and pale brown-to-dark brown with a size of 167-173 μm. Conidia were hyaline-to-brown in color, ellipsoidal to ovoid, and guttulate conidia were abundant, globose, solitary, or confluent with a size of 3.2-7.2×1.1-2.3 μm (n=50). Comparing to E. draconis CBS 186.83, the colony on MEA was dull green-to-grey olivaceous, greenish, with olivaceous margins, and the reverse side was olivaceous black with leaden grey patches with olivaceous to greenish olivaceous margins.
Table 2. Comparison of morphological characteristics of the isolate US-18-11 with the reference strain of E picoccum draconis.
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a Fungal isolate studied in this paper, bSource of description [11]. |
Molecular phylogeny of the isolate US-18-11
A phylogenetic tree of the isolate US-18-11 was constructed to determine the phylogenetic relation with its allied species. Amplification of the LSU, ITS, rpb2 and TUB2 loci yielded fragments of 1,268, 501, 884, and 830 bp, respectively. The BLAST search results revealed that the LSU, ITS region, rpb2 and TUB2 showed 99.7, 100, 97.1 and 99.3% similarities to that of E. draconis CBS 186.83 (GU238070, GU237795, KT389628 and GU237607). To check the phylogenetic relationship, the LSU, ITS, rpb2 and TUB2 sequences of allied Epicoccum spp. were retrieved from GenBank (Table 1). The phylogenetic tree based on a concatenated dataset of LSU, ITS, rpb2 and TUB2 sequences revealed that the isolate US-18-11 clustered in the same clade with E. draconis CBS 186.83 (Fig. 2). The isolate US-18-11 was deposited at the National Institute of Biological Resources (NIBRFGC000502241). Currently, several species belonging to the genus Epicoccum have been reported as quarantine pathogens and concern a lot of serious problems to organizations that are involved in the plant health quarantine activities. According to Animal and Plant Quarantine Agency in Korea, the pathogen Phoma draconis (syn. E. draconis, Phyllosticta draconis) has been regulated as a quarantine pathogen causing leaf spot diseases of plants [21]. P. andigena and P. tracheiphila are considered as quarantine organisms in the European and Mediterranean regions [22], respectively, as well as P. exigua var. foveata in Southern America [23] and P. macdonaldii in Australia [24]. On the other side, some Phoma species are regarded as very helpful by acting as biocontrol agents of weeds. The Phoma species such P. macrostoma and P. herbarum are very effective in a control of broadleaf weeds [25]. In this study, E. draconis KNU-1811 was also isolated from a tobacco cultivated soil in Korea. Further researches on Epicoccum species have proved to affect vital aspects of the agricultural field and therefore a further research needs to be done to deepen our understanding of this fungal species.
