Three Ophiostomatalean Fungi Associated with Bark Beetles from Pinus thunbergii Infested by Bursaphelenchus xylophilus in Laoshan Mountain (Shandong, China)

Abstract

Pine wilt disease (PWD) is a devastating disease that occurs worldwide and affects conifers infested by the pine wood nematode (PWN, Bursaphelenchus xylophilus). PWD has caused serious economic and ecological losses in China. The mechanism of disease outbreak is complex, with the associated fungi, specifically ophiostomatoid fungi, thought to play an essential role. However, few ophiostomatoid fungal associates of PWD have been accurately identified. In the present study, we isolated fungi from bark beetles collected from Pinus thunbergii infested by the pine wood nematode on Laoshan Mountain, Shandong province. Three ophiostomatalean fungi were identified and assigned to Graphilbum and Ophiostoma based on phylogenetic analyses and comparison of morphological and cultural features, namely Gra. laoshanense sp. nov., Gra. translucens, and O. ips. This study increases the understanding of the diversity of ophiostomatoid fungi associated with PWD and provides resources for parsing this complex disease.

1. Introduction

Pine wilt disease (PWD), caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is one of the most severe forest biological disasters worldwide, especially in Asia and Europe [1,2]. In China, PWD killed over seven million pine trees in 2023 [3]. More than 60 tree species are thought to be naturally infected with PWD [4]. Moreover, pine trees in areas once thought to be inhospitable to the PWN have become infected [5,6]. Concerns have been raised regarding the ability of PWN populations to gradually adapt to new hosts and natural environments. Furthermore, with global warming and the growing timber trade, PWD is becoming an increasing global threat to coniferous forests.

Fungal associates play a key role in the survival of the PWN and its vectors, as well as in outbreaks of PWD. In particular, some ophiostomatoid fungi, an agent of wood blue stain, are considered crucial for PWN reproduction [7]. The most frequent role reported for these ophiostomatoid fungi is to promote PWN growth, including but not limited to Leptographium, Ophiostoma, and Sporothrix species [7]. For example, ascarosides, produced by nematodes, increase the hyphal mass and spore production of Leptographium pini-densiflorae, thus facilitating a PWD outbreak [8]. In addition, fungal associates of the PWN, Sporothrix sp., enhance the reproduction of nematodes by producing palmitoleic acid [9].

Although over 20 ophiostomatoid fungal species have been reported to associate with PWD (

Table 1. Ophiostomatoid fungi associated with pine wilt disease.

DNA Data 1	Country	Species	Reference
none	China	Ceratocystis sp.	[10]
none	China	Leptographium pini-densiflorae	[8]
none	China	Ophiostoma ips	[8]
none	China	O. minus	[8]
none	China	Sporothrix sp.	[8]
none	Japan	Ceratocystis sp.	[11,12,13]
none	Japan	Leptographium sp.	[11,13]
none	Japan	O. minus	[14]
none	Korea	Ceratocystis sp.	[15]
none	Korea	Ophiostoma sp.	[15]
none	USA	Ceratocystiopsis minima	[16]
none	USA	Jamesreidia tenella	[16]
none	USA	O. allantosporum	[16]
none	USA	O. angusticollis	[16]
none	USA	O. ips	[16]
none	USA	O. minus	[16]
none	USA	Raffaelea deltoideospora	[16]
tub2	Korea	O. ips	[25]
ITS; tub2	China	C. weihaiensis	[21]
ITS	China	Graphilbum cf. rectangulosporium	[22]
ITS; tub2; tef1-α	China	Gra. xianjuensis	[21]
ITS; tub2	China	Masuyamyces massonianae	[22]
ITS; tub2	China	O. album	[22]
ITS; tub2	China	O. ips	[21,22,23]
ITS; tub2	China	O. taizhouense	[21]
ITS	China	Raffaelea cf. deltoideospora	[22]
ITS; tub2	China	S. macroconidia	[21]
ITS; tub2	China	S. zhejiangensis	[22]
ITS	China	Sporothrix sp.	[24]
ITS; tef1-α	Portugal	Graphilbum sp.	[26]
ITS; tub2; tef1-α; cal	Portugal	L. sosnaicola	[26]
ITS; tub2; tef1-α; cal	Portugal	L. terebrantis	[26]
ITS; tub2; tef1-α; cal	Portugal	Leptographium sp.	[26]
ITS; tub2; tef1-α; cal	Portugal	O. ips	[26]
ITS; tub2; tef1-α	Portugal	Sporothrix sp.	[26]

¹ ITS: internal transcribed spacer regions 1 and 2 of the nuclear ribosomal DNA operon, including the 5.8S region; tub2: β-tubulin gene region; tef1-α: transcription elongation factor 1-α gene region; cal: calmodulin gene region.

), many of these fungi were identified based solely on morphological characteristics without DNA data [8,10,11,12,13,14,15,16]. In addition, the subsequent revision of fungal ophiostomatoid species boundaries has led to a reconsideration of their taxonomy [17,18,19,20]. However, reports based on DNA data or phylogenetic analysis in China [21,22,23,24], Korea [25], and Portugal are persuasive [26]. Wang et al. [22] reported six ophiostomatalean species associated with the PWN and Monochamus alternatus infesting Pinus massoniana in Yuyao, Zhejiang Province, and Weihai, Shandong Province. Subsequently, six ophiostomatalean species were reported from M. alternatus and PWNs infesting P. massoniana by Zheng et al. [21]. In Portugal, Vicente et al. [21] isolated six ophiostomatalean species associated with PWD. Given that few ophiostomatoid fungi have been accurately identified and there are many undetermined species, studies are urgently needed to explore ophiostomatoid fungal assemblages associated with PWD.

Recently, large-scale pine tree mortality was caused by PWD, and a large number of bark beetles infected trees on Laoshan Mountain, Shandong province. In this study, we aimed to identify the fungal assemblage associated with this outbreak of PWD. Three ophiostomatalean fungi isolated from Shirahoshizo patruelis and galleries of a Scolytinae bark beetle associated with P. thunbergii infested by PWNs were accurately identified based on integrated morphological features and multilocus phylogenetic analysis.

2. Materials and Methods

2.1. Sample Collection and Fungal Isolation

Vigorous adults of Shirahoshizo patruelis and fresh galleries of a Scolytinae bark beetle were collected from P. thunbergii infested by PWNs on Laoshan Mountain in October 2023. Shirahoshizo patruelis and Scolytinae bark beetle samples were placed in sterile Eppendorf tubes and envelopes, respectively. For fungal isolation, adult beetles were dismembered into approximately 20 pieces and placed onto 2% malt extract agar (MEA, AoBoXing Company Ltd., Beijing, China), whereas fresh galleries were cut into approximately 3 × 3 mm tissue pieces and placed onto 2% MEA. Pure cultures were obtained by mycelium apex isolation and used to select representative strains based on an initial analysis of their culture and microscopic characteristics. All representative strains were preserved in the culture collection of the Forest Pathology Laboratory at the Chinese Academy of Forestry (CXY) and the China Forestry Culture Collection Center (CFCC).

2.2. Morphological and Culture Studies

The microscopic characteristics of the representative strains were observed and recorded using an Olympus BX43 microscope (Olympus Corporation, Tokyo, Japan) and a BioHD-A20c color digital camera (FluoCa Scientific, Shanghai, China). Thirty reproductive structures of the holotype of the new species were measured (such as size of conidiogenous cells and conidia) and presented as the (minimum-) mean minus standard deviation—mean plus standard deviation (-maximum). A 5 mm mycelium plug was cut from an actively growing margin of each culture and placed in the center of a 90 mm Petri dish containing 2% MEA to perform cultural characteristic studies. Five replicate plates were incubated at 5–40 °C with different treatments at 5 °C intervals in darkness. Colony diameters were measured and recorded daily until the mycelium reached the plate margins. All morphological and culture data were deposited in MycoBank (www.MycoBank.org, 856160).

2.3. DNA Extraction, PCR Amplification, and Sequencing

Representative strains were grown in 2% MEA at 25 °C until the mycelium reached the margin of the plates. Actively growing mycelium from the margin of the colony was collected into Eppendorf tubes for DNA extraction using a Fungal Genomic DNA Extraction Kit (Solarbio Co., Ltd., Beijing, China) following the manufacturer’s instructions. Aloquots of 2 × Taq PCR MasterMix (Tiangen Biotech Co., Ltd., Beijing, China) were used to amplify ITS, tub2, and tef1-α regions with the primer pairs ITS1-F/ITS4 [27,28], Bt2a/Bt2b [29], or EF1F/EF2R [30], respectively. PCR and sequencing were conducted following Wang et al. [31].

2.4. Phylogenetic Analysis

We performed preliminary identification of the representative strains based on morphological and cultural characteristics as well as a standard nucleotide BLAST search using ITS or tub2 sequences. After determining the approximate taxonomic status, reference sequences were downloaded from GenBank for alignment, which was performed using the online tool MAFFT v. 7 [32]. Alignments were edited using Molecular Evolutionary Genetic Analyses v. 7.0 [33] for phylogenetic analyses. Maximum likelihood analyses were conducted by RAxML-HPC v.8.2.3 [34] with the GTRGAMMA model. Bootstrap support values of the best trees were evaluated with 1000 replicates. Bayesian inference analyses were conducted using MrBayes v. 3.1.2 [35]. jModelTest v.2.1.7 was used to evaluate the best substitution models [36]. Four Markov chains were run from a random starting tree for 5,000,000 generations with trees sampled every 100 generations. The first 25% of sampled trees were discarded as the burn-in, and posterior probabilities were calculated using the remaining trees.

3. Results

3.1. Fungal Isolation and Phylogenetics

Based on preliminary identification, fungal isolates belonged to Graphilbum spp. and the O. ips complex. Detailed morphological and phylogenetic analyses were based on eight representative strains (

Table 2. Strains from Pinus thunbergii infested by pine wood nematodes.

Species 1	Taxon	Isolate No	Isolate No	Insect Vector 2	Genbank Accession No
					ITS	tub2	tef1-α
Gra. laoshanense sp. nov.	1	CFCC71125	CXY3350	S. patruelis	PQ358763	PQ361736	PQ361742
		CFCC71132	CXY3351	S. patruelis	PQ358764	PQ361737	PQ361743
Gra. translucens	2	CFCC71133	CXY3352	GSBB	PQ358765	-	PQ361744
		CFCC71134	CXY3353	GSBB	PQ358766	-	PQ361745
O. ips	3	CFCC71131	CXY3354	GSBB	PQ358767	PQ361738	-
		CFCC71141	CXY3355	S. patruelis	PQ358768	PQ361739	-
		CFCC71142	CXY3356	S. patruelis	PQ358769	PQ361740	-
		CFCC71140	CXY3357	S. patruelis	PQ358770	PQ361741	-

¹ Gra.: Graphilbum; O.: Ophiostoma. ² S.: Shirahoshizo; GSBB: Galleries of a Scolytinae bark beetle.

). The ITS, tub2, and tef1-α datasets were aligned and used to conduct phylogenetic analyses of Graphilbum. The best substitution models of the ITS (571 characters), tub2 (621 characters), and tef1-α (893 characters) datasets were GTR+G (ITS dataset) and HKY+I+G (tub2 and tef1-α datasets). In the ITS tree, our strains were assigned to two terminal clades, one with Gra. crescericum and Gra. niveum, and the other with Gra. translucens (Figure S1). However, based on the tub2 and tef1-α trees (Figure 1, Figure S2), two of our strains formed a well-supported clade representing an undescribed species (Taxon 1). For the O. ips complex, the ITS, tub2, and concatenated (ITS+tub2) datasets were aligned and used to conduct phylogenetic analyses. The best substitution models of the ITS (623 characters), tub2 (271 characters), and concatenated (894 characters) datasets were GTR+G (ITS and concatenated datasets) and HKY+I (tub2 dataset). Based on phylogenetic inference using the above three datasets, our four strains formed a separate, well-supported, terminal clade with O. ips (Figure 2, Figures S3 and S4).

3.2. Taxonomy

Graphilbum laoshanense Z. Wang and Q. Lu, sp. nov. (Figure 3) MycoBank: 856160.

Etymology. The epithet laoshanense (Latin) refers to Laoshan Mountain, where this fungus was collected.

Sexual state not observed. Asexual state hyalorhinocladiella-like. Conidiophores mononematous, micronematous or macronematous, singly, arising directly from hyphae, hyaline; conidiogenous cells hyaline, (9.3–)12.4–23.0(–27.9) × (1.9–)2.0–2.6(–3.3) μm; conidia hyaline, smooth, 1-celled, clavate to oblong, (6.4–)6.6–8.0(–9.0) × (2.4–)2.6–3.3(–3.9) μm; Conidiophores occasionally curl into a lollipop-like structure and produce conidia from the center.

Culture characteristics: Colonies on 2% malt extract agar reaching 72.5 mm diam in 9 days at 25 °C, hyaline to light greyish white, surface with sparse aerial mycelia, margin thinning radially. No growth at 5 °C and 40 °C, with the optimal growth temperature of 30 °C.

Host tree: Pinus thunbergii.

Distribution: Qingdao City, Shandong Province, China.

Material examined: CHINA, Shandong, Qingdao City, Laoshan Mountain, from Shirahoshizo patruelis infesting Pinus thunbergii, October 2023, Z. Wang & Q. Lu, holotype CXY3350, ex-holotype CFCC71125, ibid. CFCC71132.

Notes: Graphilbum laoshanense is phylogenetically close to Gra. niveum based on ITS, tub2, and tef1-α trees (Figure 1, Figures S1 and S2). Graphilbum niveum is associated with Cryphalus piceae on Pinus densiflora and P. thunbergii from Shandong Province and described by Chang et al. [37]. Although the asexual states of Gra. laoshanense and Gra. niveum are both hyalorhinocladiella-like, Gra. laoshanense differs from Gra. niveum by its larger conidiogenous cell (12.4–23.0 × 2.0–2.6 μm vs. 8.4–13.8 × 0.9–1.3 μm) and conidia (6.6–8.0 × 2.6–3.3 μm vs. 2.6–3.4 × 1.0–1.6 μm). For cultural characteristics, Gra. laoshanense can be distinguished from Gra. niveum by its optimal growth temperature of 30 °C, compared with 25 °C for Gra. niveum. In addition, Gra. niveum grows slowly at 5 °C, whereas Gra. laoshanense cannot grow under the same conditions (

Table 3. Morphological characteristics of Graphilbum laoshanense and Gra. niveum.

Character	Gra. laoshanense	Gra. niveum
Sexual morph	absent	absent
Asexual morphs	hylorhinocladiella-like	hylorhinocladiella-like
Conidiogenous cells	12.4–23.0 × 2.0–2.6 μm	8.4–13.8 × 0.9–1.3 μm
Shape of conidia	clavate to oblong	unicellular oblong to ovoid, with rounded ends
Size of conidia	6.6–8.0 × 2.6–3.3 μm	2.6–3.4 × 1.0–1.6 μm
Colony on 2% MEA	optimal temperature: 30 °C; Radial growth at 25 °C: 72.5 mm in 9 days; no growth: 5 °C and 40 °C; color: hyaline to light greyish white	optimal temperature: 25 °C; Radial growth at 25 °C: 61.0 mm in 8 days; no growth: 0 °C and 40 °C; color: translucent to light brown and turning white
Vector	Shirahoshizo patruelis	Cryphalus piceae
Host	Pinus thunbergii	Pinus densiflora

).

4. Discussion

We isolated three ophiostomatalean fungi from P. thunbergii infested by PWNs and accurately identified these fungi based on phylogenetic analyses and morphological characteristics. Among them, a new species, Gra. laoshanense was associated with S. patruelis, which is the first reported fungal associate of this beetle. Graphilbum translucens was reported as a fungal associate of C. piceae on P. densiflora and P. thunbergii in Shandong Province [37]. We also isolated this species associated with a Scolytinae bark beetle on P. thunbergii. In addition, we found an associate shared by two bark beetles, O. ips, which is one of the most frequently isolated ophiostomatoid fungi associated with bark beetles in China [21,22,23,37,38,39,40]. In summary, this study enhances the understanding of the diversity of ophiostomatoid fungal associates of PWD.

Ophiostomatoid fungal assemblages associated with PWD are different in different countries. In the recognized native country of the PWN, Ceratocystiopsis minima and six Ceratocystis species were reported by Wingfield [16]. According to the latest generic boundaries of Ophiostomatales [20], those six Ceratocystis species are assigned to three genera, namely Jamesreidia (J. tenella), Ophiostoma (O. allantosporum, O. angusticollis, O. ips, and O. minus), and Raffaelea (R. deltoideospora). In countries where PWNs are invasive, many ophiostomatoid fungi are found, including a large number of indeterminate species, viz. five in China (Ceratocystis sp., Gnaphalium cf. rectangulosporium, Raffaelea cf. deltoideospora, and two Sporothrix sp.) [8,10,22,24], three in Portugal (Graphilbum sp., Leptographium sp., and Sporothrix sp.) [26], two in Japan (Ceratocystis sp. and Leptographium sp.) [11,12,13], and two in Korea (Ceratocystis sp. and Ophiostoma sp.) [15]. Excluding these indeterminate species, only two species were shared between different countries: O. minus in China, Japan, and the USA and O. ips in China, the USA, Korea, and Portugal. Whether these two species are beneficial to PWD or can be modified into biocontrol engineering fungi for PWNs requires further analyses.

PWNs can feed on ophiostomatoid fungi and use them as substrates to complete development and reproduction [7]. However, not all ophiostomatoid fungi play a positive role in the growth of PWNs. In addition, although O. ips has been identified as the most suitable species for PWN development [41], intraspecific variability was observed within this species [26]. Therefore, it is necessary to evaluate the interaction of ophiostomatoid fungal strains obtained in this study with their insect vectors and PWNs to determine whether these fungi are actively involved in PWD. In particular, experiments using Gra. laoshanense as substrates to test the propagation of PWNs should be performed. Additional studies are needed to determine the geographic distribution and alternative habitats of the fungi.

To date, only nine species of ophiostomatoid fungi have been reported in Shandong Province, including the four associated with PWD in this study and the study by Wang [22], and seven associated with C. piceae reported by Chang et al. [37]. However, large areas of pine forests in Shandong are threatened by PWD and bark beetles. Therefore, future research to explore the diversity of associated fungi and understand their roles in tree death is crucial. Moreover, further studies are needed to identify the unknown fungi associated with PWD in new endemic areas such as northeast China.

5. Conclusions

We obtained three species of ophiostomatalean fungi associated with beetles from P. thunbergii infested by PWN, including Gra. translucens, O. ips, and the newly described Gra. laoshanense. This study enhances the understanding of the diversity of ophiostomatoid fungi associated with PWD. The geographical distribution of fungi and the interaction between fungi and PWD require further study.