Morphological and Phylogenetic Characterization of Raffaelea xyleboricola sp. nov. from Xyleborus Beetles in Korea

Abstract

A fungal isolate was obtained from ambrosia beetles (Xyleborus sp.) collected using beetle traps placed in an apple orchard in Gunwi-gun, Daegu, Republic of Korea. Cytochrome oxidase I (COI) gene sequencing confirmed that the beetles belonged to the genus Xyleborus. The fungal isolate, designated ARI-25-A12, was subjected to morphological and molecular identification and characterization. On malt extract agar (MEA), colonies exhibited a white, fur-like surface, and hyphae penetrated the medium along the margins. As the colony matured, the center became dull yellow, and after 20 days of incubation, the colony diameter reached 49.6–56.0 mm. Morphologically, conidiophores were hyaline, simple or occasionally branched, and conidia were hyaline, thin-walled, unicellular, and globose. Conidia produced yeast-like sprout cells through a budding-like process, and the average conidial size was 5.2 × 5.2 μm (n = 100). Molecular phylogenetic analyses based on ITS, LSU, SSU, and β-TUB gene sequences indicated that ARI-25-A12 is classified within the genus Raffaelea. Phylogenetic trees constructed from ITS sequences and a combined dataset of LSU, SSU, and β-TUB gene sequences consistently classified the isolate as a distinct lineage, clearly separated from previously reported Raffaelea species, with additional morphological differences supporting its distinct classification. Based on these results, ARI-25-A12 is described herein as Raffaelea xyleboricola sp. nov.

1. Introduction

Ambrosia beetles, members of the weevil family (Curculionidae) and particularly of the subfamilies Scolytinae and Platypodinae, engage in mutualistic relationships with fungi, which serve as their principal food source while providing the fungi with shelter, dispersal, and upkeep in return [1–3]. Most of these fungal partners belong to the Ascomycota, particularly the orders Ophiostomatales and Microascales, although representatives from Hypocreales and the Basidiomycota have also been documented [4–7]. Within the Ophiostomatales, Affroraffaelea [8], Aureovirgo [9], and Raffaelea sensu lato [10] are recognized as major ambrosia fungal symbionts.

Traditionally, obligate insect fungus mutualisms have been regarded as highly specific, often involving one-to-one associations [11]. Among ambrosia beetles, Ambrosiella (Microascales: Ceratocystidaceae) is predominantly associated with females of the Xylosandrus and Euwallacea species complexes, whereas Raffaelea (Ophiostomatales: Ophiostomataceae) is strictly linked to females of Xyleborinus and Xyleborus, as confirmed by several studies [12–15]. This pattern suggests host fidelity toward specific fungal taxa. However, Batra [16] challenged this hypothesis, and certain studies have reported that ambrosia beetles are not limited to a single symbiont but may associate with multiple auxiliary fungi [17]. The exact contributions of these auxiliary fungi to the symbiosis are unclear, though hypotheses include suppression of host plant defenses and provision of alternative nutritional sources [18,19].

The genus Raffaelea [20], a major fungal symbiont of ambrosia beetles, is predominantly asexual. Colonies are confluent and mucilaginous, and conidiophores are hyaline, simple or branched, and sometimes monilioid in structure. The conidiogenous cells proliferate percurrently or sympodially and occasionally form denticles or annellations. Conidia are generally hyaline and unicellular, varying in shape from ellipsoidal to globose with rare irregular variants, and may also produce secondary conidia through budding [21].

In addition to its morphological characteristics, recent molecular phylogenetic studies have led to substantial revisions in the taxonomy of this genus. Although Raffaelea was once considered monophyletic, Musvuugwa et al. [22] divided it into three major clades: Raffaelea sensu stricto, the R. lauricola complex, and the R. sulphurea complex. Subsequent taxonomic revisions reclassified the R. lauricola complex as the genus Harringtonia, and the R. sulphurea complex as the genus Dryadomyces [21]. According to the taxonomic revision of the genus Raffaelea by De Beer et al. [21], a total of 21 species are currently recognized, among which two species, R. cyclorhipidii and R. subfusca, have been documented in Korea [23].

In this study, fungal isolates obtained from ambrosia beetles collected in apple orchards in Korea were subjected to detailed morphological examination and characterization and rDNA-based molecular phylogenetic analyses to accurately determine their taxonomic identity and elucidate their phylogenetic position.

2. Materials and methods

2.1. Collection of ambrosia beetles and fungal isolation

Insect traps positioned within apple orchards at the Apple Research Center, Gunwi-gun (Daegu, Republic of Korea), were utilized to capture ambrosia beetles. For surface sterilization, the beetles collected from the traps were immersed in 70% ethanol and subsequently allowed to air-dry for roughly 10 min. The whole body of each beetle was placed in a 1.5 mL tube containing 1 mL of sterile double-distilled water (DDW), homogenized, and serially diluted to 10⁻¹ and 10⁻². For fungal isolation, aliquots from the serial dilutions were spread onto potato dextrose agar (PDA; Difco, Detroit, MI, USA) plates and then incubated at 25 °C for 3 days. Newly developed mycelia were re-isolated on PDA and cultured for 20 additional days under identical and stable incubation conditions. Among the pure isolates obtained through subculturing, the strain with potential novelty was selected based on cultural and morphological characteristics, designated as ARI-25-A12, and cryopreserved for long-term storage.

2.2. Morphological characterization

The collected ambrosia beetles were photographed using a stereomicroscope (SZX-16, Olympus, Japan) prior to processing. The isolated strain ARI-25-A12 was then cultured on potato dextrose agar (PDA; Difco, Detroit, MI, USA) and malt extract agar (MEA; Difco, Detroit, MI, USA) at 25 °C for 20 days to examine its cultural and morphological characteristics. Colony traits, including diameter, pigmentation, and texture, were recorded. The principal morphological features and dimensions of the isolate were examined and documented using a light microscope (CX-43, Olympus, Japan).

2.3. DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted using the HiGene Genomic DNA Prep Kit (BIOFACT, Daejeon, Republic of Korea). For the ambrosia beetles, the Genomic DNA Preparation for Animal Tissue protocol was followed, while for the isolated strain ARI-25-A12, the Genomic DNA Preparation for Fungus protocol was used.

From the insect DNA extracts, the mitochondrial cytochrome oxidase I (COI) gene was amplified using the primers LCO1490/HCO2198 [24]. For fungal DNA, the nuclear ribosomal internal transcribed spacer (ITS) region was amplified using the primers ITS1F/ITS4 [25]. Furthermore, partial fragments of the large subunit ribosomal RNA (LSU), small subunit ribosomal RNA (SSU), and β-tubulin (β-TUB) genes were PCR amplified. Specifically, the LSU region was amplified with the primer set LR0R/LR5 [26], the SSU region with NS1/NS4 [25], and the β-TUB region with Bt2a/Bt2b [27].

PCR amplicons were visualized on 1% agarose gels stained with ethidium bromide, purified using EXOSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA), and subsequently sequenced by Solgent Co., Ltd. (Daejeon, Republic of Korea). The resulting sequences were assembled and processed using the SeqMan module in Lasergene (DNAStar Inc., Madison, WI, USA).

The final sequences were deposited in GenBank under the accession numbers COI (PX113196), ITS (PX118595), LSU (PX118597), SSU (PX118596), and β-TUB (PX115488).

2.4. Molecular phylogenetic analysis

The phylogenetic position of ARI-25-A12 was examined using reference sequences of related taxa retrieved from the National Center for Biotechnology Information (NCBI) (Table 1). Sequence alignments were performed with Clustal X 2.0 in MEGA 12 [28]. Phylogenetic trees were constructed based on the ITS region as well as a concatenated dataset of large subunit ribosomal RNA (LSU), small subunit ribosomal RNA (SSU), and β-tubulin (β-TUB) sequences. Analyses were conducted under Kimura’s two-parameter substitution model [29] with the nearest neighbor interchange algorithm, excluding gaps. Neighbor joining (NJ) [30], maximum likelihood (ML) [31], and maximum parsimony (MP) [32] analyses were performed, with branch support assessed using 1,000 bootstrap replicates.

Table 1.

A list of species included in the phylogenetic analyses, along with their corresponding GenBank accession numbers.

Species	Strain	Isolation sources	GenBank accession numbers
			ITS	LSU	SSU	β-TUB
Ceratocystiopsis minuta-bicolor	CBS 635.66	Ips sp.	—	MH870571	EU984268	EU977482
Ceratocystiopsis Pallidobrunnea	WIN(M)51	Populus tremuloides	—	EU913682	HQ634842	MN901013
Dryadomyces amasae	CBS 116694T	Amasa concitatus	—	MF399174	MF398177	MT880112
Dryadomyces montetyi	CBS 451.94T	Platypus cylindrus	—	EU984301	AY497520	EU977475
Dryadomyces quercus-mongolicae	KACC 44405T	Quercus mongolica	—	NG_074466	GQ225700	GQ225688
Dryadomyces sulphureus	ARI-24-A9	Ambrosia beetle	—	PV465507	PV465508	PV476179
Esteya vermicola	CBS 115803	Scolytus intricatus	—	EU668903	MZ827948	FJ490552
Harringtonia Aguacate	213	N/A	—	MG673961	MG674027	MG674053
Harringtonia brunnea	CBS 378.68	Monarthrum sp.	—	EU984284	AY858654	EU977460
Raffaelea albimanens	CBS 271.70T	Platypus externedentatus	MH859603	NG_064077	NG_062680	MT644111
Raffaelea ambrosiae	CBS 185.64T	Platypus cylindrus	NR_160096	NG_074464	NG_070903	MT644094
Raffaelea arxii	CBS 273.70T	Xyleborus torquatus	MH859604	NG_074465	NG_062649	MW066753
Raffaelea borbonica	CMW 51724T	Leucaena leucocephala	MT633061	—	—	—
Raffaelea canadensis	CBS 168.66T	Platypus wilsonii	GQ225699	EU984299	AY858665	EU977473
Raffaelea crossotarsi	Hulcr 7182T	Crossotarsus emancipatus	KX267135	KX267103	KX267129	KX267114
Raffaelea cyclorhipidii	Hulcr 7168T	Cyclorhipidion ohnoi	KX267136	KX267104	KX267130	KX267115
Raffaelea ellipticospora	CMW 38056T	Xyleborus glabratus	MT633070	—	—	—
Raffaelea fusca	90p2	Xyleborus glabratus	—	KR018415	KR018399	KR018441
Raffaelea promiscua	CMW 55899T	Xyleborinus saxesenii	MW028176	—	—	—
Raffaelea rapaneae	CMW 40357T	Platypodinae sp.	KT192596	—	—	—
Raffaelea santoroi	CBS 399.67T	Monarthrum mutatus	MH859006	NG_064067	EU984261	EU977476
Raffaelea scolytodis	CBS 124461	N/A	NR_160234	—	—	—
Raffaelea seticollis	CMW 1031T	Tsuga canadensis	MT633076	—	—	—
Raffaelea subalba	C2401	Xyleborus glabratus	—	EU177443	KJ909304	KJ909305
Raffaelea subalba	uffeSlant18331	Euplatypus compositus	OP221769	—	—	—
Raffaelea subfusca	195	N/A	—	MG673963	MG674029	MG674055
Raffaelea subfusca	Hulcr 4520	Xyleborus glabratus	KX267137	—	—	—
Raffaelea sulcati	CBS 806.70T	Gnatotrichus sulcatus	MH859951	NG_064084	NG_062681	EU977477
Raffaelea tritirachium	CBS 726.69T	Monarthrum mali	NR_160119	MH871169	NG_063092	EU977478
Raffaelea xyleboricola	ARI-25-A12	Xyleborus sp.	PX118595	PX118597	PX118596	PX115488
Raffaelea xyleborini	CMW 45859T	Xyleborinus andrewesii	MT633078	—	—	—
Taphrina populina	CBS 337.55T	Populus nigra	PQ013134	AF492050	D14165	AF170968

^T(ex-type).

The isolated strain is shown in bold.

3. Results

3.1. Ambrosia beetles identification

Molecular identification of the collected ambrosia beetle (stain ARI-25-AmH) (Figure 1(C–D)) was based on the COI gene, yielding a 624 bp sequence. The obtained sequence was compared and analyzed against other ambrosia beetle sequences registered in the NCBI database. High sequence similarity was observed with Xyleborus pfeili SAX375 (Accession no. MN620051), X. pfeili SAX371 (MN620050), and X. pfeili SAX466 (OP617785), with similarities of 99.0%, 98.4%, and 98.0%, respectively. In contrast, lower similarities were observed with Euwallacea similis VN12 (OL704763) and Coptoborus pseudotenuis 316 (HM064071), at 85.3% and 85.1%, respectively. Therefore, ARI-25-AmH was identified as belonging to the genus Xyleborus, exhibiting over 98% similarity with X. pfeili.

Figure 1.

Cultural and morphological characteristics of ARI-25-A12 (Raffaelea xyleboricola sp. nov.). (A) Front and reverse sides of the colony grown on PDA for 20 days at 25 °C, (B) Front and reverse sides of the colony grown on MEA for 20 days at 25 °C, (C) Ambrosia beetle (Xyleborus sp.), (D) Lateral habit of ambrosia beetle (Xyleborus sp.), (E) Branched conidiophores, (F) Conidiogenous cells producing conidia, (G) Swelling was observed in some conidiogenous cells, (H–I) Conidia, (J–L) Conidia produce yeast-like sprout cells. Scale bars: C–D = 2 mm., E–L = 10 μm.

3.2. Taxonomy

Raffaelea xyleboricola J.H. Lee, Y. Kim & H.Y. Jung, sp. nov. ( Figure 1 ).

MycoBank No.: 860535

Etymology: Derived from the beetle genus Xyleborus with the Latin suffix “-cola,” meaning “inhabiting” or “dwelling in,” to indicate its close association with ambrosia beetles of this group.

Typus: This culture was isolated from ambrosia beetles (Xyleborus sp.) collected in Gunwi-gun, Gyeongbuk, Republic of Korea (36°29′68.9″N, 128°46′56.1″E). The stock culture is preserved in a metabolically inactive state in the Korean Agricultural Culture Collection (Holotype: KACC 411077) and the Korean Collection for Type Cultures (KCTC 56993).

Habitat: This fungus is associated with ambrosia beetles (Xyleborus sp.).

Cultural characteristics: When cultured on PDA at 25 °C for 20 days, the colony initially appeared entirely white, with hyphae penetrating into the medium along the margins. As the colony matured, the colony color gradually darkened; parts of the center turned black, surrounded by a yellow band and a wavy black pattern. The colony diameter reached 26.9–28.9 mm after 10 days and 51.0–51.7 mm after 20 days (Figure 1(A)). Under the same conditions on MEA, the colony was entirely white, with a surface resembling animal fur, with hyphae penetrating into the medium at the margins. As the colony matured, the center became dull yellow and developed scattered black spots. The colony diameter reached 25.7–26.6 mm after 10 days and 49.6–56.0 mm after 20 days (Figure 1(B)).

Mycological characteristics:

Hyphae are hyaline, branched, and densely interwoven. Septa are present but occur at relatively wide intervals. Conidiophores are hyaline, simple or occasionally branched, typically erect, and septate, sometimes bearing small surface protuberances (Figure 1(E)). Conidiogenous cells are hyaline, blastic, monoblastic, and cylindrical, rarely exhibiting an apical swelling, measuring 8.7–14.4 × 2.2–3.1 μm (n = 30) (Figure 1(F–G)). Conidia are hyaline, thin-walled, and unicellular, with a smooth surface and a nearly globose shape (Figure 1(H–I)). Occasionally, clearly visible intracellular organelles are present. Through a budding-like process similar to that of yeast, they produce sprout cells; although secondary conidia are not formed, these sprout cells elongate to develop into hyphae-like structures (Figure 1(J–L)). Conidia measure 3.9–6.3 × 3.8–6.2 μm, with an average size of 5.2 × 5.2 μm (n = 100).

Notes: Strain ARI-25-A12 exhibited clear differences in both cultural and morphological characteristics compared to previously described species in the genus Raffaelea. Unlike most Raffaelea species, which typically produce mucilaginous colonies, ARI-25-A12 lacks this feature. Additionally, while ellipsoidal conidia are common in the genus, ARI-25-A12 produces globose conidia, and its conidiophores display a characteristic apical swelling. Compared with R. arxii, the colony color was similar, being white in both species, but the conidial morphology differed. Conidia of R. arxii are oblong to ovoid, whereas those of ARI-25-A12 are globose. Furthermore, root-like hyphae at the base of conidiophores, present in R. arxii, were absent in ARI-25-A12. In comparison with R. ambrosiae, differences were found in both conidial morphology and size. R. ambrosiae has reniform conidia measuring 5.2–6.0 × 3.0–4.0 μm, and the terminal aleuriospores measure 10–16 μm in diameter. In contrast, ARI-25-A12 has conidia measuring 3.9–6.3 × 3.8–6.2 μm, which differ in both size and shape. R. fusca has globose conidia that are similar in size (3.5–6.5 × 3.5–5.0 μm) to those of ARI-25-A12, but the colony color on MEA is distinctly different. Colonies of R. fusca are dark brown to olive brown, whereas those of ARI-25-A12 are white. Finally, R. scolytodis differs markedly in conidial morphology, having ellipsoidal to clavate conidia that are much larger (15–30 × 2.5–6 μm) than those of ARI-25-A12. These morphological and cultural distinctions from related taxa provide strong evidence that strain ARI-25-A12 represents a novel species within the genus Raffaelea (Table 2).

Table 2.

Morphological comparison between strain ARI-25-A12 and phylogenetically related species within the genus Raffaelea.

Characteristics		R. xyleboricola sp. nov.a (ARI-25-A12)	R. arxii b		R. ambrosiae c	R. scolytodis d	R. fusca e
Colony	Color, Shape	On MEA, colonies are entirely white at first, with mycelia resembling animal fur. Later, the center turns dull yellow, with scattered areas becoming black.	On MEA, colonies are white and tenacious, with centers often raised over 1 mm above the agar and a dark brown pigment zone forming near the margin.		On YEME, colonies are white to tan with a ripened apple odor, centrally raised, dull, felty to cerebriform or lacunose and frequently sectoring into grayish brown to dark olive gray.	On MEA, colonies are flat with a narrow margin, initially hyaline and later becoming dull brown, covered with slimy masses or aerial mycelium.	On MEA, colonies have a dark brown to brownish-olive center, a faint white margin, and an edge that becomes mucilaginous, later forming a concentric mat with a fuscous to mouse gray center.
	Size(mm)	PDA: 28.0 (25 °C, 10 days) MEA: 26.2 (25 °C, 10 days)	MEA: grow slowly		YMEA: 50.0 (25 °C, 14 days)	MEA: 20.0 (25 °C, 7 days)	MEA: 13.0 (25 °C, 14 days)
Conidiophores	Color	hyaline	Subhyaline		hyaline	N/A	hyaline
	Shape	Simple or occasionally branched, typically erect, and septate, sometimes bearing small surface protuberances.	Erect in dense fascicles, cylindrical, mostly branched with dichotomous or sympodial branching, reaching up to 750 μm in length and 3–4 μm in width.		Tapering toward the apex, typically unbranched or rarely branched	Simple or branched, densely arranged in palisades	Micronematous, mononematous, erect, cylindrical, fasciculate, aseptate, and scattered.
Conidiogenous cells	Color	hyaline	N/A		N/A	N/A	N/A
	Shape	Blastic, monoblastic, Cylindrical, with an apical swelling rarely observed, 8.7–14.4 × 2.2–3.1 μm.	Tapering slightly toward the fertile apex with faintly visible conidial scars, 15.0–20.0 × 3.0–4.0  μm		N/A	Cylindrical, often with an apical swelling, 10.0–20.0 × 3.0–4.0 μm	N/A
Conidia	Color	hyaline	hyaline		hyaline to subhyaline	N/A	Hyaline
	Shape	Thin-walled, unicellular, smooth surface, globose, and producing yeast-like sprout cells upon germination.	Thin-walled, unicellular or rarely 1-septate, obovate to pyriform-ellipsoidal and germinating into budding yeast cells and pseudohyphae.		Blastosporic, turbinate, acrogenous, and sympodial with smooth walls, producing yeast-like globose to subglobose sprout cells upon germination.	Ellipsoidal to clavate, mostly single-celled, though larger two-celled forms also occur, germinating into conidia or thin-walled, vacuolated ‘sprout’ cells.	Produced singly, ovate to obovoid, sometimes pyriform, smooth-walled.
	Size (μm)	3.9–6.3 × 3.8–6.2	8.0–12.0 × 4.0–6.0		5.2–6.0 × 3.0–4.0	15.0–30.0 × 2.5–6.0	3.5–6.5 × 3.5–5.0

^a Fungal strain studied in this paper.

^b Source of description [38].

^c Source of description [16].

^d Source of description [4].

^e Source of description [39].

3.3. Phylogenetic analysis

For the elucidation of the molecular and evolutionary position of ARI-25-A12, analyses were conducted using four genetic loci: ITS, LSU, SSU, and β-TUB. The lengths of the obtained sequences were 560 bp for ITS, 1,292 bp for LSU, 1,258 bp for SSU, and 537 bp for β-TUB. BLAST comparisons against NCBI-registered fungal sequences revealed low similarity to known Raffaelea species. In the ITS region, ARI-25-A12 showed 93.0% similarity to Raffaelea arxii CBS 273.70, 91.4% to R. xyleborini LESF1119, and 91.0% to R. canadensis CBS 326.70, indicating generally low similarity to other Raffaelea species. LSU sequences exhibited 96.3% similarity to R. arxii CBS 273.70% and 95.2% to R. albimanens CBS 271.70, overall showing relatively low similarity, similar to the ITS results. In contrast, SSU gene sequences showed higher similarity, with 99.0% to R. scolytodis C2747, R. gnathotrichi C2219, and R. arxii CBS 273.70, and 98.0% to R. canadensis C2233. Regarding the β-TUB gene, no closely related species were retrieved due to very low similarity with other Raffaelea species, suggesting considerable nucleotide sequence divergence. Furthermore, even in the ITS region, the highest similarity was only 93.0%, confirming substantial sequence differences from other Raffaelea species. Nevertheless, considering the highest similarities observed in ITS (93.0%), LSU (96.3%), and SSU (98.0%) gene sequences, R. arxii was identified as the closest related species to ARI-25-A12.

In addition, to clarify the phylogenetic placement, two trees were generated based on the ITS and a concatenated dataset of LSU, SSU, and β-TUB gene sequences (total length of 3,087 bp), respectively, and the phylogenetic position in each analysis was assessed. Taphrina populina CBS 337.55 served as the outgroup for phylogenetic tree construction. ARI-25-A11 formed a distinct clade in both the ITS region-based phylogenetic tree (Figure 2) and the concatenated LSU, SSU, and β-TUB phylogenetic tree (Figure 3), indicating its phylogenetic distinction.

Figure 2.

The phylogenetic tree was constructed using the Maximum Likelihood (ML) method based on the nucleotide sequence of the ITS region. Bootstrap values greater than 70% from 1,000 replicates are indicated on the corresponding branches. This tree shows the phylogenetic placement of strain ARI-25-A12 among species of the genus Raffaelea, and the strain isolated in this study is highlighted in bold. Taphrina populina (CBS 337.55) was used as the outgroup. The scale bar represents 0.05 nucleotide substitutions per site. ‘T’ indicates the type strain.

Figure 3.

A phylogenetic tree was constructed using the maximum-likelihood method based on concatenated nucleotide sequences of the LSU, SSU, and β-TUB genes, showing the phylogenetic placement of strain ARI-25-A12 within the genus Raffaelea and its related genera (Ceratocystiopsis, Dryadomyces, Esteya, and Harringtonia). Bootstrap values greater than 70% from 1,000 replicates are indicated on the corresponding branches. Filled circles represent nodes that were recovered by both NJ (Neighbor-Joining) and MP (Maximum Parsimony) analyses, while open circles indicate nodes supported by either method. Strains isolated in this study are shown in bold. Taphrina populina (CBS 337.55) was used as the outgroup. The scale bar represents 0.02 nucleotide substitutions per site. ‘T’ indicates the type strain.

4. Discussion

In this study, we isolated fungal symbionts from ambrosia beetles (Xyleborus sp.) collected in domestic apple orchards with the objective of precise species-level identification. Based on morphological and phylogenetic characteristics, the isolate was confirmed to be a novel species belonging to the genus Raffaelea, designated as Raffaelea xyleboricola.

In beetles, the mycangia are independently evolved structures that store, transport, and enable the propagation of fungal spores. The fungi inhabiting these mycangia were historically classified under the genus Raffaelea, but recent studies have reclassified them into the genera Raffaelea, Harringtonia, and Dryadomyces. Notably, Raffaelea lauricola, a major symbiont of Xyleborus species and a tree pathogen, has also been reclassified as Harringtonia lauricola [33]. Several of these reassigned symbionts have demonstrated pathogenicity toward host trees. A representative example is Har. lauricola, the primary symbiont of Xyleborus glabratus in Florida and the causal agent of laurel wilt. This fungus is also known to associate with multiple ambrosia beetle species outside of Xyleborus, and the ecological implications of such “promiscuity” (non-selective symbiosis with multiple vectors) have been discussed [34].

The genus Raffaelea was first redefined in 2014 by Dreaden et al. [10] through a comprehensive multilocus phylogenetic analysis based on sequences of three genes: LSU, SSU, and TUB. As a result, Raffaelea sensu stricto was circumscribed to include species such as R. brunnea, R. lauricola, R. scolytodis, R. arxii, and several others (e.g. R. gnathotrichi, R. fusca, R. subfusca, R. ellipticospora, R. ambrosiae, R. canadensis, R. albimanens, R. subalba, R. tritirachium, R. santoroi, and R. sulcati). In contrast, R. amasae, R. sulphurea, R. quercus-mongolicae, R. quercivora, and R. montetyi were excluded from Raffaelea but provisionally retained due to unresolved taxonomic placement.

Subsequently, De Beer et al. [21] conducted a comprehensive reevaluation of genera within the order Ophiostomatales through phylogenetic analyses using four commonly employed genetic markers: ITS, LSU, TEF1-α, and RPBII. This analysis led to the reclassification of the former Raffaelea lauricola complex (comprising R. lauricola, R. brunnea, and R. aguacate) into the newly established genus Harringtonia, and the R. sulphurea complex (R. amasae, R. montetyi, R. sulphurea, R. quercus-mongolicae, and R. quercivora) was reassigned to Dryadomyces.

The genus Raffaelea was originally established to accommodate mutualistic fungi of wood-boring ambrosia beetles. Morphologically, Raffaelea and Ambrosiella were initially distinguished based on differences in conidial development. However, due to many morphological similarities, the generic boundaries between the two remained ambiguous until DNA sequence data became available. It is now established that Ambrosiella belongs to the family Ceratocystidaceae (order Microascales), whereas Raffaelea is placed within the order Ophiostomatales [21]. In addition, Raffaelea species are characterized by reduced conidiophore structures, which distinguishes them from other genera in the Ophiostomatales [35].

The COI gene sequence of the ambrosia beetle collected in this study exhibited >99% similarity to Xyleborus pfeili in the NCBI database. However, due to the absence of morphological observations, it was classified as Xyleborus sp. In addition, isolate ARI-25-A12, obtained from Xyleborus sp., was predicted to belong to the genus Raffaelea based on its morphological characteristics and ITS sequence analysis. Molecular phylogenetic identification following the protocol of De Beer et al. was attempted; however, due to the unavailability of TEF1-α and RPBII gene sequences for most Raffaelea species, these markers were deemed unsuitable for phylogenetic inference. Therefore, in this study, we employed ITS, LSU, SSU, and TUB gene sequences previously utilized for Raffaelea identification in taxonomic studies [10,36,37]. Given the limited number of isolates possessing all four markers, we constructed a phylogenetic tree based on concatenated LSU, SSU, and TUB sequences to enable the broadest comparative analysis among species of Raffaelea, Harringtonia, and Dryadomyces. Additionally, a separate ITS-based phylogenetic tree was constructed, including only Raffaelea species, to confirm the phylogenetic placement of ARI-25-A12 in both analyses.

Phylogenetic analyses revealed that ARI-25-A12 clustered within the main clade of Raffaelea species, yet it occupied a distinct phylogenetic position separate from existing species. Morphological differences from closely related species further supported its novelty. Therefore, we determined ARI-25-A12 to be a new species in the genus Raffaelea, designated  Raffaelea xyleboricola. Given that R. xyleboricola, isolated from ambrosia beetles collected in apple orchards, may possess pathogenic potential, future investigations into its pathogenicity and geographic distribution in Korea will be essential for effective tree disease management and quarantine efforts.

Funding Statement

This work was performed with the support of the “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ017183)” funded by the Rural Development Administration, Republic of Korea.

Conflict of interests

The authors declare no conflicts of interest.