Molecular and Morphological Characterization of Two Novel Species from Soil and Beetles (Dorcus titanus castanicolor) in Korea

Abstract

Two fungal strains belonging to Ascomycota were discovered in Gyeonggi-do, Korea, during this investigation of soil microfungi and microbiota of insects. The strain KNUF-20-047 produced milky white on the back and a milky creamy center to white toward the margin on the front side of colonies. Conversely, the closest Xenoacremonium falcatus displayed a pale luteous to luteous center, white toward margins on the front side, and pale luteous or luteous pigment on the back side, whereas X. recifei produced white colonies. The conidiophores of KNUF-20-047 were slightly larger than those of X. falcatus, and the conidia were distinct from X. recifei. Strain KNUF-20-NI-005 produced light brown to subhyaline conidiophores up to 56.0 μm tall, whereas Rhinocladiella anceps displayed golden to dark brown conidiophores up to 350 μm. Strain KNUF-20-NI-005 also produced larger conidia than R. anceps but smaller than R. coryli and R. fasciculata. Moreover, the molecular phylogeny strongly supports the detailed description and illustration of each proposed species to be designated as Xenoacremonium minutisporum sp. nov. and Rhinocladiella terrigenum sp. nov. in Korea.

Keywords:

• Morphology
• phylogeny
• Rhinocladiella terrigenum
• Xenoacremonium minutisporum

1. Introduction

Among the estimated 2–11 million fungal species, only approximately 150,600 have been adequately characterized [1–3]. The highest estimates of fungal species came from metabarcoding data, with estimates of 11.7–13.2 million species [4]. Fungi are among the most significant microorganisms in the world due to their impact on humans and their activities, as well as the critical roles they play in ecosystem functioning [5]. Despite significant efforts to enhance agricultural productivity, fungi have significantly reduced the postharvest yield of vegetables and fruits globally [6]. The Nectriaceae family is distinguished by the presence of yellow, orange-red, purple, or white ascomata. The MycoBank database (https://www.mycobank.org/) currently lists four members of the genus Xenoacremonium under Nectriaceae. Some members of this genus are facultatively fungicolous or insectivorous, whereas others are soil-borne saprophytes or weak to virulent, facultative, or obligate plant pathogens [7–10]. Additionally, certain species have been identified as critical opportunistic pathogens causing human infections [11], whereas others create mycotoxins of significant scientific value [12].

Chaetothyriales is a fungal order divided into Chaetothyriaceae and Herpotrichiellaceae families, which comprise non-lichenized ascomycetes [13]. The well-defined ascomycete family, Herpotrichiellaceae, includes 16 genera and over 270 species [14,15]. A black yeast-like fungus belonging to the mitosporic Herpotrichiellaceae family was named the Rhinocladiella genus by Swedish botanist John Axel Nannfeldt in 1934. Furthermore, Rhinocladiella atrovirens has been designated as the type species for this fungus [16,17]. The Rhinocladiella genus includes members that cause illnesses in humans and animals, as well as plant pathogens. This widely distributed fungal genus is present in rotting wood, soil, herbaceous substrates, and other environments. Several Rhinocladiella spp. have been reported in Korea. In 2017, a news report stated that Rhinocladiella similis had been newly isolated from agricultural field soils [18].

We aimed to identify and categorize new fungal species native to Korea. Molecular phylogenetic analyses were performed to identify novel species and describe their cultural and morphological characteristics. The isolated fungi were the focus of the current study, in which they were characterized and presented as novel fungal species.

2. Materials and methods

2.1. Sample collection and isolation of fungal strains

In 2018, the fungal isolates used in this investigation were identified in soil samples and beetles collected from Gyeonggi-do (37°28′03.7′′N 127°32′30.3′′E), Korea. The samples were taken from the field at a depth of 10–15 cm, air-dried, and stored at 4 °C in a plastic bag. The fungi were isolated using the traditional dilution plating method [19]. The beetle sample was washed once with 70% ethanol, twice with sterile distilled water, and then ground using a hand grinder. Subsequently, the samples were suspended in 1.0 mL of sterile distilled water, vortexed gently, and diluted serially, followed by the spreading of 100 µL of each sample onto PDA (Difco, Detroit, MI, USA) plates and incubation at 25 °C for 2–3 days [20]. Single colonies were transferred to potato dextrose agar plates and incubated for 4–5 days at 25 °C. The KNUF-20-047 and KNUF-20-NI-005 strains were selected for additional molecular analyses based on various culture parameters. For additional research, the fungal strains were kept at −80 °C in 20% glycerol. The National Institute of Biological Resources (NIBR) received fungal strains KNUF-20-047 (NIBRFGC000507844) and KNUF-20-NI-005 (NIBRFGC000507840).

2.2. Cultural and morphological characterization

The culture characteristics and morphological observations of the KNUF-20-047 and KNUF-20-NI-005 fungal strains were recorded using different media. Each fungal strain was cultured on potato dextrose agar (PDA), 2% malt extract agar (MEA; Difco), and oatmeal agar (OA; Difco) and incubated for 7–21 days at 25 °C [17,21]. Fungal growth and colony characteristics such as color, shape, and size were monitored. Morphological characteristics were examined using a BX-50 light microscope (Olympus, Tokyo, Japan).

2.3. Genomic DNA extraction, PCR amplification, and sequencing

Total genomic DNA from the KNUF-20-047 and KNUF-20-NI-005 strains were extracted from the fungal mycelia grown on the PDA plate using a HiGene Genomic DNA Prep Kit (BIOFACT, Daejeon, Korea) following the manufacturer’s protocol. Polymerase chain reactions (PCR) were performed using the internal transcribed spacer (ITS) regions, 28 s rDNA large subunit (LSU), partial RNA polymerase II subunit (RPB2), and translation elongation factor 1-alpha (TEF1α) genes as targets for molecular identification [22]. The ITS1/ITS4 primer pair was used to amplify the ITS regions of the nuclear rRNA gene [23], fRPB2-5F/fRPB2-7cR primers for the partial RPB2 [24], LROR [25], and LR5 [26] primers for partial gene sequences of the 28S rDNA LSU; and EF1–526F/EF1–986R primers for TEF1α [27]. The RPB2 region was not successfully amplified, resulting in the exclusion of this marker in further analyses of the KNUF-20-047 strain. The quality of the amplified PCR products was checked by 1.2% agarose gel electrophoresis stained with ethidium bromide. The electrophoresed product was purified using ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) and then sent to SolGent for sequencing (Daejeon, Korea).

2.4. Molecular phylogenetic analyses

Phylogenetic analyses were performed using the sequences retrieved from the National Center for Biotechnology Information (NCBI) (Table 1). Ambiguous regions were deleted from the alignments, and evolutionary distance matrices for the neighbor-joining (NJ) algorithm were calculated using Kimura’s two-parameter model [28]. The NJ method [29] was used to infer tree topology using MEGA7.0 software, with bootstrap values based on 1000 replicates [30].

Table 1. GenBank accession numbers used for the phylogenetic analyses in this study.

Species name	Strain numbers	Source	Country	Accession numbers
				ITS	LSU	TEF1α
Rhinocladiella terrigenum	KNUF-20-NI-005^T	Soil	South Korea	OP503368	OP503370
R. anceps	CBS 181.65^T	Soil	Canada	NR111275	EU041862
R. anceps	CBS 538.69	Unknown	Canada	MH859369	MH871137
R. atrovirens	CBS 317.33	Unknown	Sweden	MH855447	MH866906
R. aquaspersa	CBS 313.73^T	Homo sapiens	Mexico	NR119760	MH872396
R. coryli	CPC 26654^T	Corylus avelana	Germany	NR155727	NG059697
R. fasciculata	CBS 132.86^T	Decayed wood	India	NR145356	MH873622
R. phaeophora	CBS 496.78^T	Soil	Colombia	NG057785	MH872933
R. tropicalis	CMRP1307	Skin lesion of human	Brazil	KX434724	KX583710
Cladophialophora carrionii	CBS 160.54^T	Homo sapiens	Australia	MH857278	MH868813
Xenoacremonium minutisporum	KNUF-20-047^T	Dorcus titanus castanicolor	South Korea	OP503369	OP503371	OP515525
X. falcatus	CBS400.85^T	Pinus radiata	New Zealand			KM231967
X. recifei	CBS 541.89	Soil	Brazil			KM231969
X. recifei	CBS 137.35^T	Homo sapiens	Brazil			KM231968
Paracremonium contagium	CBS 110348^T	Homo sapiens	Canada			KM231966
P. inflatum	CBS 485.77^T	Homo sapiens	India			KM231964
P. inflatum	CBS 482.78	Soil	Colombia			KM231965
Stachybotrys chartarum	CBS 129.13	Unknown	Unknown			KM231994

ITS: internal transcribed spacer regions of rDNA; LSU: partial large subunit of 28S rDNA; TEF1α: partial translation elongation factor gene.

The newly generated sequences are indicated in bold.

3. Results

3.1. Taxonomical analysis of Xenoacremonium minutisporum sp. Nov

3.1.1. Taxonomy

The KNUF-20-047 strain exhibited morphological characteristics distinct from those of allied species of Xenoacremonium and was therefore described as a new species.

Xenoacremonium minutisporum S.Y. Lee and H.Y. Jung, sp. nov. (Figure 1)

Figure 1. Cultural and morphological characteristics of KNUF-20-047^T. The colony growth on potato dextrose agar (A), oatmeal agar (B), and malt extract agar (C) after 14 days of incubation at 25 °C. Lateral phialidic pegs (D, E); conidiophores arising laterally from somatic hyphae (F, G); conidia (H, I). Scale bars: D–I = 10 μm.

MycoBank: MB8457434

Etymology: The specific epithet “minutisporum” refers to the small size of conidia.

Typus: Gyeonggi-do (37°28′03.7′′N 127°32′30.3′′E), isolated from the female beetle, Dorcus titanus castanicolor. The stock culture (NIBRFGC000507844) was deposited in the National Institute of Biological Resources (NIBR) as a metabolically inactive culture.

Ecology and distribution: Members of this genus are considered pathogens of plants and humans and are widely dispersed in tropical and temperate ecosystems worldwide. Some species have been isolated from various plants, such as macadamia, Persea sp. (avocado), and pomegranate. Some countries, such as China, New Zealand, Brazil, and the USA, have reported Xenoacremonium spp. mainly from the soil, leaves, and stem/bark of these plants. The proposed novel species, X. minutisporum, was isolated from a female beetle, D. titanus castanicolor, in Korea.

Cultural characteristics: The KNUF-20-047 strain was cultured on MEA, OA, and PDA at 25 °C for 7–14 days to observe its morphological structure with a normal growth rate. Colonies grew moderately, attaining 55–60 mm in diameter on MEA and PDA and 45–55 mm on OA at 25 °C for 10 days of incubation (Figure 1(A–C)). Colonies consisted of semi-immersed aerial mycelium. The surface had a pale luteous to luteous center becoming white toward the margins, and the reverse side of the plate was pale luteous with a pale luteous or luteous pigment throughout the medium. Colonies on OA became buff, planar, and portioned with regular margins (Figure 1(B)). These colonies were initially milky white on the front side of the plate, and over time, they became light yellow-white to dull yellow-white from the center outward. Colonies on the back side of the plate were creamy pale-yellow white on PDA and clear white on MEA and OA from the center outward. The milky white droplets were smooth (Figure 1(A–C)).

Morphological characteristics: The hyphae of strain KNUF-20-047 were a light white color, septate, branched, and were commonly 1.0–4.0 µm wide. Conidiophores that arose laterally from somatic hyphae were either lateral phialidic pegs, 2.0–4.0 × 1.0–2.0 µm, or erect, subcylindrical, unbranched or rarely branched, 0- to 1-septate, up to 80 µm tall, 2.0–4.0 µm in diameter, hyaline, smooth, and terminating in one or two conidiogenous cells (Figure 1(D–G)). Conidia were straight or slightly curved, hyaline, smooth, formed in heads at the apex of conidiogenous cells, subcylindrical, tapering toward the apex with periclinal thinning and inconspicuous collarette, slightly to strongly curved, 2.4–9.6 × 1.0–3.0 µm in size (Figure 1(H,I)). Chlamydospores were not observed.

Note: The KNUF-20-047 strain produced milky white colonies on the back side of the media surfaces and a milky, creamy center turning yellow to white toward the margin on the front side of the colonies. Conversely, Xenoacremonium falcatus displayed a pale luteous to luteous center, turning white toward the margins on the front side of the plate, with the back side displaying a pale luteous or luteous pigment throughout the medium. Furthermore, Xenoacremonium recifei produced white colonies (Table 2). The conidiophores (2.0 × 7.0 µm) of the KNUF-20-047 strain were slightly larger than those of X. falcatus (2.0–4.0 × 1.0–2.0 µm). The conidia of strain KNUF-20-047 (2.4–9.6 × 1.0–3.0 µm) were shorter in length but almost similar in width to those of X. falcatus (4.0–8.0(–10.0) × 1.0–2.0 µm) but were also longer than those of X. recifei (4.0–6.0(–7.5) × 1.0–2.0 µm) (Table 2). Thus, the proposed strain KNUF-20-047 differed from X. falcatus and X. recifei in their sizes of conidiophores, conidia, and cultural characteristics, especially the presence of a milky smooth surface.

Table 2. Morphological comparison between Xenoacremonium minutisporum (KNUF-20-047^T) and the closest species of Xenoacremonium.

Characteristics	KNUF-20-047^a	X. falcatus CBS 400.85^b	X. recifei CBS 137.35^c
Cultural characteristics	Mycelia appeared milky white on the back side of the media surfaces with a milky, creamy center turning yellow to white toward the margin on the front side of colonies. Colonies grew moderately, attaining 55–60 mm in diameter on PDA, 45–55 mm on OA, and 55–60 mm on MEA at 25 °C after 10 days of incubation.	Colonies on PDA reach 55–60 mm in diameter after 7 days at 24 °C; colonies consist of semi-immersed aerial mycelium; surface with pale luteous to luteous center becoming white toward the margins; reverse surface pale luteous with pale luteous or luteous pigment throughout the medium.	White color, slow growth rate.
Conidiophores	Rarely branched, erect, white, and cylindrical to subcylindrical, 2.0 × 7.0 µm.	Lateral phialidic pegs, or arising laterally from somatic hyphae, erect, subcylindrical, unbranched or rarely branched, septate, smooth, terminating, 2.0–4.0 × 1.0–2.0 μm.	Lateral phialidic pegs or arising laterally from somatic hyphae, erect, cylindrical to subcylindrical, unbranched or rarely branched, aseptate or septate, smooth, hyaline.
Conidia	Subcylindrical, tapering toward the apex with periclinal thinning and inconspicuous collarette, 2.4–9.6 × 1.0–3.0 μm.	Terminal, elongate-ampulliform, tapering toward apex with prominent periclinal thickening and inconspicuous collarette, hyaline, smooth, 4.0–8.0(–10.0) × 1.0–2.0 μm.	Terminal, monophialidic, hyaline, smooth, elongate ampulliform or subcylindrical, tapering toward the apex, with periclinal thickening and inconspicuous collarette, 4.0–6.0(–7.5) × 1.0–2.0 μm.

^aFungal strain studied in this research; ^b,cSource of descriptions [22].

3.1.2. Molecular phylogeny of strain KNUF-20-047

The ITS regions (535 bp) demonstrated 99.43, 99.23, and 98.84% sequence similarities to the reported species, X. falcatus isolate 1609, X. falcatus CBS 400.85^T, and X. recifei CBS 541.89, respectively. The closest taxonomic relatives of the isolated strain KNUF-20-047 were identified using the LSU (729 bp) gene sequence, which revealed a high sequence similarity of 99.72–99.86% to the isolates, X. falcatus CBS400.85^T and X. recifei CBS 596.74. TEF1-α (416 bp) displayed 95.00 and 93.87% sequence similarities with X. falcatus CBS400.85^T and X. recifei CBS 541.89, respectively. Phylogenetic analyses using TEF1-α gene sequences were performed for accurate differentiation between isolates. The strain KNUF-20-047 was closely clustered with X. falcatus CBS 400.85^T in the NJ phylogenetic tree using the TEF1-α gene sequences indicated by the node in the neighbor-joining phylogenetic tree along with the filled nodes in the maximum likelihood and maximum parsimony trees. Open circles show nodes created using maximum likelihood or maximum parsimony (Figure 2). The precise taxonomic position of the strain was ascertained by a phylogenetic analysis based on a combination of sequences with maximum parsimony (tree length = 168, consistency index = 0.90, retention index = 0.92, and composite index = 0.87). Therefore, KNUF-20-047 represented a single, novel, and phylogenetically distinct species. Based on the phylogenetic analysis and morphological observations, strain KNUF-20-047 was distinct from the previously identified Xenoacremonium species (Figure 2). Thus, it should be placed in the genus as a novel species, for which we proposed the name X. minutisporum sp. nov.

Figure 2. Neighbor-joining phylogenetic tree of KNUF-20-047^T based on the TEF1α sequences indicating the relationship between Xenoacremonium minutisporum and the closest species. The tree was rooted using Stachybotrys chartarum CBS 129.13 as the outgroup. The neighbor-joining phylogenetic tree, maximum likelihood, and maximum parsimony trees indicated with filled nodes, whereas open circles showed maximum-likelihood or parsimony. The strain isolated in this study is in bold, and the bootstrap values (>50%) are based on 1000 replicates. Bar = 0.1 substitutions per nucleotide position.

3.2. Taxonomical analysis of Rhinocladiella terrigenum sp. nov

3.2.1. Taxonomy

Strain KNUF-20-NI-005 exhibited morphological characteristics different from those of other Rhinocladiella spp. Therefore, it was proposed as a new species.

Rhinocladiella terrigenum H.Y. Jung and S.Y. Lee, sp. nov. (Figure 3)

Figure 3. Cultural and morphological characteristics of KNUF-20-NI-005^T. The colony growth on potato dextrose agar (A), oatmeal agar (B), and malt extract agar (C) for 14 days of incubation at 25 °C. Conidial structure at different stages of development, resulting in semi-micronematous conidiophores and sympodially proliferating conidiogenous cells (D–F); conidiogenous cells (G, H); conidia (I). Scale bars: D–I = 10 μm. Arrows indicate conidial structure.

MycoBank: MB845744

Etymology: This refers to the soil, the substrate from which the type strain was isolated.

Typus: Gyeonggi-do (37°28′03.7′′N 127°32′30.3′′E), isolated from the rhizosphere soil of Robinia pseudoacacia. The stock culture (NIBRFGC000507840) was deposited in the NIBR as a metabolically inactive culture.

Ecology and distribution: Members of this genus are primarily considered human pathogens (for example, they cause chromoblastomycosis) and are present globally, including in soil, herbaceous substrates, and rotting wood. Chromoblastomycosis is common in tropical, subtropical, and temperate climates, with high prevalence rates in Madagascar, South Africa, Brazil, Costa Rica, Australia, and other Asian countries such as India, China, Japan, South Korea, and Malaysia. The proposed novel species, R. terrigenum, was collected from soil in Korea.

Cultural characteristics: After 2 weeks of dark incubation at 25 °C, the colonies of strain KNUF-20-NI-005 developed slowly, with a moist texture, irregular form, and curled margin, with diameters ranging from 19.4–20.7 mm on PDA, 18.8–19.4 mm on MEA, and 18.4–18.9 mm on OA (Figure 3(A–C)). However, this species did not grow at 35 °C. Colonies on PDA were dark olivaceous green on both the front and reverse sides of the plate, with smooth, aerial mycelium with a dark-grey-green circle (Figure 3(A)). The colonies on OA created a dark color on both the front and back sides of the plate, as well as dark-dust grey, smooth aerial mycelium (Figure 3(B)). Additionally, colonies on MEA were dark olivaceous on the front side of the plate and orange-green on the reverse side, with pale yellow aerial hyphae in the inner circle and dark-grey green aerial hyphae in the outer circle (Figure 3(C)).

Morphological characteristics: Strain KNUF-20-NI-005 comprised branched hyphae, which were brown to pale brown, 2.0–3.9 µm wide, and irregularly septate from 10.1–26.5 µm, and conidiophores consisting of conidiogenous cells that were connected with vegetative hyphae. Conidiophores were erect, straight, thick-walled, up to 56.0 µm tall, brown to pale brown, bearing small, pigmented denticles, on which conidia are generated in a sympodial order and are 1.6–3.8 µm wide (Figure 3(D–F)). Furthermore, conidiophores bore multiple conidia, terminal or lateral conidiogenous cells, often intercalary, cylindrical in an apical section with multiple conidia-connected cells, and rachis with scattered and pimple-shaped denticles (Figure 3(G,H)). Single conidia developed budding cells and were unicellular, hyaline, generally 1-septate, and 2.3–4.2 × 3.1–5.7 µm (Figure 3(I)).

Note: The KNUF-20-NI-005 strain produced light brown to subhyaline conidiophores, up to 56.0 µm tall, intercalary cells 4.0–27.0 µm long, whereas the strain, Rhinocladiella anceps, displayed golden to dark brown conidiophores, up to 350 µm tall, and intercalary cells 9.0–14.0 µm long. Lastly, the conidiophores of R. coryli were straight to slightly curved, grey-brown, smooth, subcylindrical with a slight apical taper, unbranched, 0–7-septate, and 10–70 × 2.5–3.0 µm (Table 3). KNUF-20-NI-005 produced larger conidia (2.3–4.2 × 3.1–5.7 µm) than those of R. anceps (2.5–4.0 × 2.0–2.5 µm) but different from those of R. coryli ((3)4–4.5(5) × 2(2.5) µm) and R. fasciculata ((2.5–)4.0–5.0(–6.0) × 2.0–3.0 µm) (Table 3). The proposed strain produced shorter conidiophores than those of R. anceps and different-sized conidia than those of the closest species. Thus, the morphology of strain KNUF-20-NI-005 was distinct from the previously identified species of Rhinocladiella.

Table 3. Morphological comparison between Rhinocladiella terrigenum (KNUF-20-NI-005^T) and the closest species of Rhinocladiella.

Characteristics	KNUF-20-NI-005^a	R. anceps CBS 181.65^b	R. coryli CBS 141407^c	R. fasciculata CBS 132.86^d
Cultural characteristics	Colonies on MEA 18.8–19.4 mm after 14 days at 25 °C, moderate growth rate, moist, dark-olivaceous, pale yellow aerial hyphae in the inner circle and dark-grey green aerial hyphae in the outer circle on the front side and orange-green on the reverse side.	Colonies on MEA reaching 6–12 mm in diameter after 14 days at 24 °C, with entire, smooth, sharp margin; powdery mycelium, becoming hairy at the center.	Colonies with spreading erumpent surface; margins smooth, lobate, and moderate aerial mycelium.	Colonies on MEA reaching 8 mm in diameter after 14 days at 24 °C, with entire, smooth, sharp margin; mycelium velvety, becoming farinose in the center due to abundant sporulation, olivaceous-green to brown, reverse side dark olivaceous.
Hyphae	Brown to pale brown, thick-walled, 2.0–3.9 μm wide, branched, septate.	Smooth, thick-walled, 2.0–2.5 μm wide, branched, aerial hyphae pale brown.	Pale brown, smooth, branched, septate, 2.0–3.0 µm.	Submerged, subhyaline, smooth, thick-walled, 2.0–2.5 µm wide, aerial hyphae pale brown.
Conidiophores	Thick-walled, light brown to subhyaline, up to 56.0 μm tall, unbranched with additional septa, intercalary cells 4.0–27 μm long.	Thick-walled, golden to dark brown, up to 350 μm tall, unbranched, or loosely branched stalks (additional septa, intercalary cells 9.0–14.0 μm long).	Solitary, erect, straight to slightly curved, grey-brown, smooth, subcylindrical with slight apical taper, unbranched, 0–7-septate, 10–70 × 2.5–3 µm.	Arising vertically from ascending hyphae in loose fascicles, unbranched or loosely branched at acute angles, cylindrical, smooth, brown, and thick-walled at the base, up to 220 µm long and 2.0–3.0 µm wide, with 0–5.0 thin additional septa.
Conidia	Unicellular, thick-walled, ovoid, 2.3–4.2 ×  3.1–5.7 μm with budding cell.	Thin-walled, smooth, subglobose to ellipsoidal, 2.5–4.0 × 2–2.5 μm, with a less conspicuous hilum.	Solitary, aseptate, pale brown, smooth, ellipsoidal with obtuse apex, tapering to truncate base, hilum 0.5 µm in diameter, (3)4–4.5(5) × 2(2.5) µm.	Solitary, smooth, thin-walled, subhyaline, ellipsoidal, (2.5–)4.0–5.0(–6.0) × 2.0–3.0 µm, with truncate, slightly pigmented hilum, approximately 0.5 µm.

^aFungal strain studied in this research; ^b–dSources of descriptions [17,31,32].

3.2.2. Molecular phylogeny of strain KNUF-20-NI-005

Based on the sequencing analyses, the lengths of the sequences were 639 bp and 837 bp for the ITS regions and 28S rDNA of LSU, respectively. The NCBI BLAST tool was used to investigate the novel species in this genus, and gene markers for the ITS regions and 28S rDNA were used to analyze the sequences. Strain KNUF-20-NI-005 had 86.7% and 86.6% sequence similarity to R. anceps (CBS 538.69; CBS 157.54) for the ITS regions. For the 28S rDNA, the isolated strain demonstrated sequence similarities of 98.2% to R. atrovirens (CBS 317.33; CBS 264.49) and 97.8% to R. aquaspera (CBS 127261; CBS 127260; CBS 313.73), R. anceps (MUCL 7792), and R. aquaspera (CBS 122635). The phylogenetic analysis was built using a combination of ITS regions and partial 28S rDNA sequences, as indicated by the node in the neighbor-joining phylogenetic tree, as well as the filled nodes in the maximum likelihood and maximum parsimony trees. The maximum likelihood or maximum parsimony was also used to make the corresponding nodes, as shown by the open circles (Figure 4). A phylogenetic analysis was also conducted based on a combination of sequences with maximum parsimony (tree length = 345, consistency index = 0.64, retention index = 0.61, and composite index = 0.45) to determine the exact taxonomic position of the strain. The phylogenetic tree revealed that KNUF-20-NI-005 was distinctly clustered in a different clade with respect to the previously identified strains of Rhinocladiella (Figure 4). Thus, strain KNUF-20-NI-005 was phylogenetically distinct from other Rhinocladiella spp.

Figure 4. Neighbor-joining phylogenetic tree of KNUF-20-NI-005^T based on ITS and LSU sequences, depicting the phylogenetic position among the related strains in the genus Rhinocladiella. Cladophialophora carrionii CBS 160.54^T was selected as the outgroup. The neighbor-joining tree, maximum likelihood, and maximum parsimony trees indicated with filled nodes and open circles were made using maximum likelihood or parsimony. The strain isolated in this study is in bold, and the numbers above the branches represent the bootstrap values obtained for 1000 replicates. Bar = 0.01 substitutions per nucleotide position.

4. Discussion

Morphologically distinct strains, KNUF-20-047 and KNUF-20-NI-005, were isolated from soil and female beetles, D. titanus castanicolor, obtained from Gyeonggi-do, Korea. The strains were morphologically different from each of the identified closely related species (Tables 2 and 3).

According to Ko and Kunimoto [33], Xenoacremonium spp. are widely distributed from temperate to tropical regions worldwide and cause infected trees to perish within 2–3 months of the onset of early foliar symptoms, such as fading color and yellowing. Although the exact hosts of the genus Xenoacremonium have not yet been determined, this species has been detected in several plants, including macadamia, Persea sp. (avocado), and pomegranate [34]. Several nations, including China, New Zealand, Brazil, and the USA, have documented the presence of Xenoacremonium spp. in soil, plant leaves, stems, and bark. The first sexual species in this genus, X. brunneosporum, was identified in a marine habitat on Rhizophora mucronata in Thailand and is the most well-known species in the genus, along with X. recifei and X. falcatus (plant and human pathogens) [35]. However, the genus Xenoacremonium was established with a group of fungi previously treated as Acremonium recifei [36], including the ex-type of A. recifei (CBS 137.35) [22]. Members of the genus Acremonium, such as A. zeylanicum, have been isolated from infected citrus cottony scale (Pulvinaria aurantia), which belongs to the family Coccidae in the order Hemiptera [37]. The pathogenic effect of A. zeylanicum has previously been reported on the sugarcane wooly aphid (Ceratovacuna lanigera) in India [38] and the two-spotted spider mite (Tetranychus urticae Koch) [39]. Currently, four species of Xenoacremonium have been identified so far, including a sexual morph from different countries with diverse hosts and none of the species was not recorded in a freshwater environment. However, no sexual or asexual morph has been recorded under this genus in Korea until now. Although members of the genus Xenoacremonium occur in many habitats worldwide, the identified strain, KNUF-20-047, was isolated from the female beetle, D. titanus castanicolor, in Korea.

The Rhinocladiella genus comprises a black yeast-like fungus present in various environments, including soil, herbaceous substrates, and decaying wood [17,40]. The close relatives of black filamentous fungi R. similis were isolated from both groundwater and surface water [41]. Chromoblastomycosis is a disease that can affect humans and is caused by the genera of melanized fungi. Chromoblastomycosis and/or phaeohyphomycosis are caused by the genera Exophialia, Cladophialophora, Fonsecaea, and Phialophora [42,43]. Chromoblastomycosis is most prevalent in tropical, subtropical, and temperate regions, with significant incidence rates in Madagascar, South Africa, Brazil, Costa Rica, and other Asian countries. Additionally, this infection has been observed on the limbs of agricultural farmers and laborers because the causative agents are primarily located in the soil and decaying vegetation [44–46]. The Rhinocladiella genus contains several clinically relevant species such as R. mackenziei, R. basitonum, R. fasciculatum, R. anceps, R. atrovirens, R. phaeophora, R. aquaspersa, and R. similis. Some of these species are also responsible for human infection. R. mackenziei is one of the most well-known causes of cerebral phaeohyphomycosis, with cases frequently appearing in healthy individuals. R. aquaspersa is also frequently linked to chromoblastomycosis lesions, and R. basitona and R. similis are two of the most common skin pathogens [11,17,47,48].

In conclusion, each species analyzed in this study had a variety of hosts, including both plants and humans, and caused various diseases. Based on cultural, morphological, and phylogenetic analyses, the strains were identified as X. minutisporum and R. terrigenum. These analyses suggested that the strains notably differed from the previously described strains of the genera. Further research is required to explore the etiology of these species as well as their pathogenicity, along with their ecological importance based on Korean soils, environmental conditions, and relationships in agricultural production, thereby considering all the aspects of these two new species.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work 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 [NIBR202002104].