Introduction
As initially described, the fungal family Roussoellaceae Liu et al. comprised the three genera Neoroussoella Liu et al., Roussoella Sacc., and Roussoellopsis Hino and Katum [1]. Subsequently, based on the multigene analysis of limited taxa, Jaklitsch and Voglmayr [2] synonymized Roussoellaceae under Thyridariaceae. However, the taxonomic status of Roussoellaceae was later re-established by Tibpromma et al. [3], who considered Roussoellaceae and Thyridariaceae to be separate families within the Pleosporales. More recent studies employing additional taxa and combining morphological and phylogenetic analyses have validated this familial distinction [4-8]. Currently, the family Roussoellaceae comprises the following 12 genera: Appendispora Hyde; Cytoplea Bizz. and Sacc.; Elongatopedicellata Zhang et al.; Immotthia Barr; Neoroussoella, Pararoussoella, and Pseudoneoconiothyrium Wanas et al.; Pseudoroussoella Mapook and Hyde; Roussoella Sacc.; Roussoellopsis Hino and Katum.; Setoarthopyrenia Mapook and Hyde; and Xenoroussoella Mapook and Hyde [9]. Among these, the genus Xenoroussoella was initially proposed by Mapook et al. in 2020 [7]. Phylogenetic analysis based on a combined dataset of internal transcribed spacer (ITS) regions, small subunit rDNA (SSU), large subunit rDNA (LSU), translation elongation factor 1-α (TEF1α), and RNA polymerase II second largest subunit (RPB2) genes revealed that Xenoroussoella forms a separate branch and groups with Pseudoroussoella species. The genus differs, however, from Pseudoroussoella in having smaller ascomata and asci, a thin peridium, and larger ellipsoid to obovoid bi-seriate ascospores that are 3-septate with irregular longitudinal striations and lacking a gelatinous sheath. In contrast, Pseudoroussoella produces oval to ellipsoid uniseriate ascospores that are 1-septate, have reticulate spore wall ornamentation, and are encased in a hyaline gelatinous sheath. The name Xenoroussoella is derived from the Greek terms Xeno, meaning separate, and Roussoella, meaning roussoella-like [7].
Since the original description of Xenoroussoella in 2020 [7], there has been relatively little research conducted on this genus. In Korea, among species in the family Roussoellaceae, Roussoella doimaesalongensis has recently been isolated and reported [10]. Given the unknown virulence of Xenoroussoella species, it is important to determine the suitable growth media, optimal pH, and temperature for these fungi. In this study, we describe a fungal strain isolated from soil collected from Gunsan-si, Jeollabuk-do, Korea, based on its cultural characteristics on three different media, asexual morphs, and the effect of temperature and pH on its mycelial growth. Finally, on basis of phylogenetic analysis, we identify the fungus as a strain of Xenoroussoella triseptata (designated KNUF-20-NI009), which is reported in Korea for the first time.
MATERIALS AND METHODS
Collection of soil samples
The fungal strain KNUF-20-NI009 was isolated from soil collected from Gunsan-si, Jeollabuk-do (35°49ʹ02.2ʹʹN 126°23ʹ36.4ʹʹE), Korea. Soil samples were extracted from the soil at a depth of approximately 15-30 cm. Before analysis, the samples were air-dried and then stored in a plastic bag at 4℃. Fungi were isolated using the serial dilution technique. Soil suspensions were prepared by the addition of 1 g of the soil sample to 9 mL of sterile distilled water, followed by vortexing and serial dilution to give dilations of 10-3, 10-4, 10-5, and 10-6. Aliquots (1 mL) of each dilution were spread on potato dextrose agar (PDA; Difco, Detroit, MI, USA), followed by incubation for 2 to 3 days at 25℃ [11]. Individual colonies developing on the agar were subcultured on fresh PDA plates and incubated at 25℃ until the development of mycelium. Based on different cultural characteristics, isolated fungal strains were selected for further molecular analyses. In particular, an isolate designated KNUF-20-NI009 was maintained in 20% glycerol at -80℃ for further studies. This strain has been deposited at the National Institute of Biological Resources (NIBR), Incheon, South Korea, with the accession number NIBRFGC000507836.
Morphological characterization
For morphological observations, strain KNUF-20-NI009 was transferred onto PDA, malt extract agar (MEA), and oatmeal agar (OA) and incubated at 25℃ for 14 days [12], after which, we measured growth, recorded colony characteristics, such as shape, color, and size, and observed conidiomata under a Dimis-M stereo microscope (Siwon Optical Technology Co Ltd., Anyang, Korea). For an examination of micro-morphological characteristics, the isolate was observed under a BX-50 light microscope (Olympus, Tokyo, Japan).
Effects of temperature and pH on growth
To determine the mycelial growth of the isolate at different temperatures and pH, 6-mm-diameter mycelial plugs were extracted from the edge of 4-days-old PDA cultures using a sterile cork-borer and transferred to the center of 90-mm Petri dishes containing PDA. For an assessment of the effects of temperature, cultures were incubated at 5, 10, 15, 20, 25, and 30℃ with three replicate plates per isolate being assessed at each temperature. The effect of pH on the growth rate of KNUF-20-NI009 was evaluated by adjusting the culture medium (PDA) to pH 4, 5, 6, 7, 8, and 9 with HCl or NaOH and incubating at 25℃. To determine growth rates, colony diameters were measured after incubating for 14 days.
Genomic DNA extraction, PCR amplification, and sequencing
Genomic DNA was extracted from fungal mycelia scraped from PDA plates using a HiGeneTM Genomic DNA Prep Kit (Biofact, Daejeon, Korea) according to the manufacturer’s instructions. The isolated DNA was amplified for the detection of five gene markers, namely, internal transcribed spacer (ITS) regions, small subunit rDNA (SSU), large subunit rDNA (LSU), translation elongation factor 1-α (TEF1α), and RNA polymerase II second largest subunit (RPB2) genes, using the primer pairs ITS1F/ITS4, NS1/NS4, LROR/LR5, EF1-983F/EF1-1567R, and fRPB2-5f/fRPB2-7cr, respectively [13-17]. The thermal conditions used for PCR amplification have been described previously [13,17]. The amplified PCR products thus obtained were purified using ExoSAP-IT reagent (Thermo Fisher Scientific, Waltham, MA, USA) and sequenced commercially by Macrogen Co., Ltd. (Daejeon, Korea). Sequences obtained from the KNUF-20-NI009 isolate have been deposited in the National Center for Biotechnology Information (NCBI) GenBank and the DNA Databank of Japan (DDBJ) databases, with the accession numbers LC719282, LC723530, LC719283, LC723531, and LC723532 for ITS, SSU, LSU, TEF1α, and RPB2 respectively.
Phylogenetic analysis
Datasets of the ITS, SSU, LSU, TEF1α, and RPB2 sequences were used to search for similar sequences in the NCBI GenBank database, and comparable sequences from related species were retrieved for phylogenetic analysis (Table 1). Phylogenetic tree was constructed based on a combined alignment of multi-genes using the neighbor-joining (NJ) method with the Kimura 2-parameter model [18] in the MEGA 7 software program, with bootstrap analysis of 1,000 replications [19].
RESULTS AND DISCUSSION
Morphology of the KNUF-20-NI009 strain
To determine the morphological characteristics of strain KNUF-20-NI009 colonies, we cultured spore suspensions on PDA, MEA, and OA at 25℃ for 14 days. Colony diameters on these media after the 2-week incubation were 55.9-68.0, 45.3-46.2, and 67.8-68.9 mm, respectively. On PDA, the color in the upper surface of the colonies was olivaceous gray, whereas the lower surface was olivaceous green to black in the center with a pale white margin (Figs. 1A and D). The colonies on MEA were olivaceous-brown to pale olivaceous-white at the margin on the upper surface, with undersides of dark olivaceous to olivaceous-brown and a white margin. (Figs. 1B and E). The colonies on OA were characterized by a rugged olivaceous-gray surface with a white to olivaceous margin (Figs. 1C and F). After 2 to 3 weeks on PDA medium, the isolate KNUF-20-NI009 showed pycnidial conidiomata, solitary or gregarious, scattered, globose to subglobose, dark brown to black. The pycnidial wall was 13-17 µm wide (Figs. 2A and B) and the conidiophores were reduced to conidiogenous cells. These cells were 4-7.9×2.6-4.8 µm (x̅=5.5×3.6 µm, n=20) in size, ampulliform to doliiform, proliferating percurrently at the apex, integrated, discrete, hyaline, and smooth (Figs. 2C-G). Conidia were aseptate, globoid to ellipsoid, with hebetate apex, thick-walled, hyaline when immature, becoming light brown to golden-brown when mature, with 1 or 2 guttules, and measured 2.7-5.1×1.6-3.3 µm (x̅=3.6×2.6 µm, n=50) (Fig. 2H). However, we were unable to compare the asexual morphological characteristics of KNUF-20-NI009 with those of Xenoroussoella triseptata MFLUCC 17-1438, as these features have yet to be reported for this species. Based on asexual morphology, we compared the isolate with other phylogenetically close reference strains [7]. The conidia diameters of Roussoella euonymi (=Pseudoneoconiothyrium euonymi) and Pseudoneoconiothyrium rosae are (6-)7(-8)×(4-)5-6 µm and 7-10×4-8 µm, respectively [20-22]. Strain KNUF-20-NI009 can accordingly be distinguished from R. euonymi and P. rosae by its comparatively smaller conidia. Morphological comparisons with the previously reported closely related species are shown in Table 2.
Temperature and pH are important factors affecting the growth and survival of living organisms. To determine the effects of the two factors on KNUF-20-NI009 growth, we cultured the isolate in a PDA medium, which is generally and widely used for the cultivation of fungi. We accordingly established that the isolate grows optimally at a temperature of 25℃, reaching a colony diameter of 59.3±2.1 mm. No growth was observed at 5℃, whereas the colonies obtained at 10℃ were characterized by a white upper surface and olivaceous black to the light beige lower surface. At 15℃ and 20℃, the colonies developed a rugged upper surface, olivaceous in the center with white margins, whereas the lower surface was olivaceous to iron-gray with a pale white margin. When grown at 25℃, the upper surface of colonies was olivaceous gray in the center and the lower surface was olivaceous green to black with a pale white margin. Contrastingly, at 30℃, we observed colonies with a cottony pale white surface colored olivaceous beneath, with the lower surface being olivaceous green to iron-gray with a white margin. Generally, we found that incubation at temperatures below 20℃ and above 30℃ resulted in an increase of white mycelia and colonies with a pale white margin.
With respect to pH, we established that growth of the isolate was maximal at pH 7 (63.3±0.6 mm) and minimal at pH 4 (35±2.6 mm), with the optimum pH for growth being neutral or slightly alkaline. Under optimum pH conditions, colonies were mostly olivaceous green to pale white with a white margin, whereas, at pH 9, the colonies were olivaceous brown to pale white with a light brown to pale white margin. Details of colony diameters at different temperatures and pH values are presented in Table 3.
Phylogenetic analysis
The taxonomic status of isolate KNUF-20-NI009 was determined based on an analysis of a combined dataset of the ITS region and LSU, SSU, RPB2, and TEF1α gene sequences [7,22,23]. Amplification of the ITS, LSU, SSU, RPB2, and TEF1α loci of the strain yielded fragments of 586, 837, 1,008, 1,041, and 921 bp, respectively. A BLAST search of the NCBI database revealed similarities of 99.41% between the ITS regions of KNUF-20-NI009 and Xenoroussoella triseptata MFLUCC 17-1438 (MT214343). Similarly, analysis based on the partial LSU gene sequence revealed a 100% similarity with Xe. triseptata MFLUCC 17-1438 (MT214437) and 99.40% similarity with Roussoella euonymi CBS 143426 (MH107961) and Pseudoneoconiothyrium rosae MFLU 18-0117 (NG_059868). Comparisons based on the SSU sequence indicated that KNUF-20-NI009 has identities of 100, 99.70, and 99.60% with Xe. triseptata DC9 (OL700219), Roussoella intermedia CBS 170.96 (KF43390), and Roussoella padinae MUT <ITA >:5503 (MN556315), respectively, whereas the RPB2 gene sequence of KNUF-20-NI009 showed 99.83% similarity with Xe. triseptata MFLUCC 17-1438 (MT235804), but only 85.77% similarity with P. rosae IT3796 (MN814846). Moreover, based on TEF1α region sequence similarity, we identified Xe. triseptata MFLUCC 17-1438 (MT235767), with 99.73% similarity, as the closest relative to KNUF-20-NI009.
However, despite Xe. triseptata being identified as taxonomically closest to strain KNUF-20-NI009 based on all of the molecular markers assessed in this study, it is clear that the sequence of any one of these loci does not enable us to unambiguously identify the novel fungal strain. Accordingly, to attain a more precise identification, we performed multi-locus sequence analysis using the concatenated sequences of the ITS regions, and LSU, SSU, RPB2, and TEF1α genes of strain KNUF-20-NI009 (Table 1). This combination of five molecular markers has previously been shown to be highly effective in resolving species in the family Roussoellaceae [7]. The neighbor-joining phylogenetic tree (Fig. 3) constructed based on analysis of these concatenated sequences demonstrated that strain KNUF-20-NI009 maps to a location distinct from those of Roussoella, Pseudoroussoella, and Pseudoneoconiothyrium species and that its phylogenetically closest neighbor is Xe. triseptata, as indicated by the fact that both the KNUF-20-NI009 and MFLUCC 17-1438 strains clustered together with a high bootstrap value of 100%.
Fig. 3.Neighbor-joining phylogenetic tree based on combined dataset (ITS+LSU+SSU+RPB2 and TEF1α) showing the phylogenetic position of strain KNUF-20-NI009 among members of the family Roussoellaceae and its closest relationship with Xenoroussoella triseptata . The tree was rooted using Occultibambusa bambusae MFLUCC 13-0855T as an outgroup. Bootstrap values greater than 70% (based on 1,000 replications) are shown at the branching points. The isolated strain is indicated in bold. Bar=0.01 substitutions per nucleotide position. ITS, internal transcribed spacer regions; LSU, large subunit rDNA; SSU, small subunit rDNA; RPB2, RNA polymerase II largest subunit; TEF1α, translation elongation factor 1-alpha.
Previous studies have reported that species of Roussoella and Roussoellopsis can typically be found on or in the vicinity of a diverse range of plants, including monocotyledons, tall grasses, bamboos, and palms [1]. In particular, the single representative of the genus Xenoroussoella, Xe. triseptata was initially isolated from dead stems of the invasive weed Chromolaena odorata [7]. Contrastingly, in the present study, we isolated the KNUF-20-NI009 strain of this species from soil samples collected in Korea, thereby highlighting the wide geographical distribution and diverse habitats of this species. Although the cultural characteristics of Xe. triseptata on MEA medium and the morphological characteristics of its sexual stage have been described previously [7], until the present study, its asexual features had remained undetermined. By providing a detailed description of the asexual morphology of the KNUF-20-NI009 strain, the findings of this study complement our existing knowledge of this species. Moreover, we have established the cultural characteristics of the strain grown on three different media, determined the optimal temperature and pH for its cultivation, and evaluated the effects of various temperatures on its cultural characteristics. However, further investigations are needed to gain more in-depth insights into its ecological, etiological, and biological roles, particularly under the environmental conditions found in Korea.
In conclusion, our morphological and phylogenetic analyses in this study have revealed KNUF-20-NI009 to be a strain of the fungal species Xenoroussoella triseptata. To the best of our knowledge, this study is the first to report the isolation of this species in Korea.
