Ophiostomatalean Fungi (Ascomycota, Ophiostomatales) Associated with Dendroctonus valens in Liaoning, China

Abstract

The red turpentine beetle Dendroctonus valens is an invasive forest pest that has been rapidly spreading northward since its introduction in China. Some ophiostomatalean associates of D. valens are essential contributing factors for this beetle to become an aggressive pine killer in China. In this study, a survey was conducted in a new invasive area of this beetle’s habitat in Liaoning Province. A total of 126 ophiostomatalean fungal associates of D. valens were isolated. Based on the combination of morphological characteristics and phylogeny, these isolates were identified as Ceratocystiopsis jianpingensis sp. nov., along with two new records, Leptographium terebrantis and Ophiostoma gilletteae. Ophiostoma gilletteae was the dominant species, with an isolation rate of 85.7%. The results of this study show that more shared ophiostomatalean fungi are associated with D. valens in China and North America, and accumulate resources for the development of fungal associate-mediated bark beetle management strategies.

1. Introduction

The red turpentine beetle (Coleoptera: Scolytinae), Dendroctonus valens, is a secondary pine-infesting pest, native to North America [1]. Since its introduction to China, it has spread rapidly to seven provinces, killing more than 10 million healthy Pinus tabuliformis trees and posing a serious threat to the health of native P. bungeana and P. sylvestris var. mongolica [2]. The associate of D. valens, Leptographium procerum, is an essential contributing factor to this beetle becoming an aggressive pine killer in China [3].

Leptographium procerum is an ophiostomatalean fungus (Ascomycota, Ophiostomatales), which is commonly associated with beetles [4]. This fungus has been isolated from more than 20 conifer species, and is associated with a wide variety of bark beetles and weevils [5]. In addition, L. procerum is an agent of pine root disease [6]. The success of symbionts of D. valens and L. procerum in China comes from the facilitation of the invasions by of the both the species [7,8]. Leptographium procerum can deplete the host resistance of P. tabuliformis and induce the production of 3-carene, which attracts D. valens, thereby assisting beetle invasion. Dendroctonus valens benefits L. procerum by producing volatiles that inhibit native fungi reproduction.

To date, 31 ophiostomatalean fungal associates of D. valens have been reported [9]. Of these, 15 and 23 species were isolated from China and North America, respectively. In China, Lu et al. identified seven ophiostomatalean associates of D. valens infesting P. tabuliformis and P. bungeana from Shanxi, Shaanxi, and Henan provinces and found that L. procerum and L. sinoprocerum were the dominant species [10,11]. However, different fungal communities of D. valens were subsequently reported. Ten ophiostomatalean species associated with D. valens have been isolated from P. tabuliformis in Shanxi and Shaanxi Provinces [12]. Although only these two species are common in China, the dominant species is L. procerum. Ophiostomatalean associates of D. valens in North America show higher species diversity and fewer shared species compared to those in China, and are mostly endemic [9,13]. Overall, the interactions between most of the fungal associates and D. valens remain unknown.

Despite various management measures, D. valens has spread northward to Liaoning Province, posing a serious threat to native pine forests in northeast China [14]. In this study, an investigation was conducted in July 2024 in a new invasive area of D. valens, which was Jianping County, Liaoning Province. Fungal associates were isolated from adult beetles infesting P. tabuliformis and identified by combining their morphological characteristics and phylogeny. This study revealed additional shared fungal associates of D. valens in North America and China, contributing resources for the development and implementation of fungus-mediated bark beetle management.

2. Materials and Methods

2.1. Isolation of Fungi

Adults of D. valens were collected from dying P. tabuliformis in Jianping County, Liaoning Province, China in July 2024. The beetles were identified based on morphological characteristics [15]. The vigorous beetles were placed in a sterile centrifuge tube at 4 °C for immediate fungal isolation. Approximately 30 tissue pieces were dismembered from each beetle and placed on the surface of 2% malt extract agar medium (MEA; 10 g malt extract, 10 g agar, and 500 mL distilled water), and incubated at 25 °C. As soon as the mycelia grew from the tissue pieces, a single hyphal tip was cut off and placed in fresh medium. This step was repeated two to three times to obtain pure cultures.

2.2. Morphological and Growth Studies

An Olympus BX43 microscope (Olympus Corporation, Tokyo, Japan) was used to observe the microscopic features of fungal isolates. Typical features were recorded using a BioHD-A20c color digital camera (FluoCa Scientific, Shanghai, China). Optimum and extreme growth temperatures for undescribed species were tested under 2% MEA medium at 5–40 °C in darkness.

2.3. PCR Amplification and Phylogenetic Analysis

Thriving mycelia at the edge of the colony were scraped off the medium for DNA extraction. The Fungal Genomic DNA Extraction Kit (Solarbio Co., Ltd., Beijing, China) was used for DNA extraction, following the protocol outlined by the manufacturer. Four gene regions including the internal transcribed spacer regions 1 and 2 of the nuclear ribosomal DNA operon, including the 5.8S region (ITS); the nuclear ribosomal DNA operon, including the internal transcribed spacer 2 region and part of the large subunit (ITS2-LSU); partial β-tubulin gene region (tub2); and partial transcription elongation factor 1-α gene region (tef1-α) were amplified by the primer pairs listed in

Table 1. Primer pairs and PCR conditions used in this study.

Locus	Primer Pairs	PCR Amplification Procedures	References
ITS	ITS1-F/ITS4	initial denaturation: 94 °C for 3 min; thermal cycles: (94 °C: 1 min, 55 °C: 45 s, and 72 °C: 1 min) × 35 cycles; final chain elongation: 72 °C for 8 min	[16,17]
ITS2-LSU	ITS3/LR3	initial denaturation: 95 °C for 2 min; thermal cycles: (95 °C: 30 s, 54 °C: 30 s, and 72 °C: 1 min) × 35 cycles; final chain elongation: 72 °C for 8 min	[16,18]
tub2	Bt2a/Bt2b	initial denaturation: 95 °C for 2 min; thermal cycles: (95 °C: 30 s, 56 °C: 30 s, and 72 °C: 1 min) × 35 cycles; final chain elongation: 72 °C for 8 min	[19]
tef1-α	EF1F/EF2R	initial denaturation: 95 °C for 2 min; thermal cycles: (95 °C: 30 s, 56 °C: 30 s, and 72 °C: 1 min) × 35 cycles; final chain elongation: 72 °C for 8 min	[20]

. PCR amplification was conducted using 2 × Taq Plus PCR Master Mix (Rui Biotech Co., Ltd., Beijing, China). Sequencing was performed by Rui Biotech Co., Ltd.

The newly generated sequences underwent the BLASTn tool in GenBank database to identify the genus and complex to which they belonged. The reference sequences were obtained from GenBank. All sequences were aligned using the MAFFT v.7 online web server [21]. Alignments were edited using MEGA 7.0.21 [22]. Phylogenies were calculated using Maximum likelihood (ML) and Bayesian Inference (BI) methods. ML phylogenetic inferences were conducted using RAxML-HPC v.8.2.3 [23], the GTR-GAMMA model was selected, and 1000 bootstrap replicates were run. BI phylogenetic analyses were conducted using MrBayes v. 3.1.2 [24]. The best-fit substitution model was selected using jModelTest v.2.1.7 [25]. One to five million generations were run in four Markov chain Monte Carlo chains to guarantee that the average standard deviation of the split frequencies across different runs was less than 0.01.

3. Results

3.1. Isolation of Fungi

In total, 126 ophiostomatalean strains were isolated from 15 adult D. valens infesting P. tabuliformis. Based on microscopic and cultural characteristics, 13 strains were selected for phylogenetic analysis and detailed morphological and growth studies (

Table 2. Strains isolated from Dendroctonus valens infesting Pinus tabuliformis.

Species	Taxa	Isolates	Other Numbers	GenBank Accession Numbers
				ITS/ITS2-LSU	tub2	tef1-α
Ceratocystiopsis jianpingensis sp. nov.	1	CFCC72301	CXY3371	PQ685080	PQ687506	-
		CFCC72302	CXY3372	PQ685081	PQ687507	-
		CFCC72303	CXY3373	PQ685082	PQ687508	-
Leptographium terebrantis	2	CFCC72304	CXY3374	PQ685083	PQ687509	PQ687519
		CFCC72305	CXY3375	PQ685084	PQ687510	PQ687520
		CFCC72306	CXY3376	PQ685085	PQ687511	PQ687521
		CFCC72307	CXY3377	PQ685086	PQ687512	PQ687522
		CFCC72308	CXY3378	PQ685087	PQ687513	PQ687523
Ophiostoma gilletteae	3	CFCC72309	CXY3379	PQ686155	PQ687514	-
		CFCC72310	CXY3380	PQ686156	PQ687515	-
		CFCC72311	CXY3381	PQ686157	PQ687516	-
		CFCC72312	CXY3382	PQ686158	PQ687517	-
		CFCC72313	CXY3383	PQ686159	PQ687518	-

).

3.2. Phylogenetic Analysis

The ITS, tub2, and the combined sequence datasets (containing 659, 435, and 1094 characters, respectively) were used for the phylogenetic analyses of Ceratocystiopsis spp., using the GTR+I+G (ITS sequence dataset) and GTR+G (tub2 and the combined sequence datasets) models, respectively. In three trees, our three isolates formed a robust terminal clade representing an undescribed species, Taxon 1, which was a phylogenetic sister to C. brevicomis and C. ranaculosa (Figure 1, Figure 2 and Figure 3).

The tub2, tef1-α, and the combined sequence datasets (containing 353, 656, and 1009 characters, respectively) were used for phylogenetic analyses of L. clavigerum complex under K80 (tub2 sequence dataset) and GTR+G (tef1-α and the combined sequence datasets) models, respectively. In tub2 tree, five isolates from this study formed a terminal clade with two isolates associated with D. valens from the USA, representing L. terebrantis (Figure 4). Although the type of L. terebrantis was not distinguishable from the two strains of L. aureum in tub2 tree, all eight strains of L. terebrantis grouped a clade distinct from L. aureum in the tef1-α and the combined trees (Figure 4).

The ITS, tub2, and the combined sequence datasets (containing 685, 271, and 956 characters, respectively) were used for phylogenetic analyses of O. ips complex using the GTR+G (ITS and the combined sequence datasets) and HKY+I (tub2 dataset) models, respectively. The five isolates isolated from this study formed a terminal clade, with two isolates associated with D. valens from the USA, representing O. gilletteae, based on phylogenetic analyses of the three datasets (Figure 5).

3.3. Taxonomy

• Ceratocystiopsis jianpingensis Z. Wang and Q. Lu, sp. nov. (Taxon 1, Figure 6).
• Mycobank: 856888
• Etymology: The name refers to Jianping, the county of Liaoning Province, where the fungus was collected.
• Holotype: CXY3371
• Description: Teleomorph unknown. Anamorphs: sporothrix-like and hyalorhinocladiella-like. Conidiophores macronematous or micronematous, simple or branched, erect, occasionally serpentine, arising from the mycelium; conidiogenous cells rough or smooth, hyaline, (5.7–)8.1–13.2(–14.6) × 1.3–1.6(–1.7) μm. Conidia hyaline, smooth, aseptate, obovoid with upper part swollen, (3.1–)3.3–3.9(–4.5) × (1.7–)1.9–2.4(–2.9) μm.

• Culture features: Colonies grew slowly and reached 42.9 mm in 20 days, pure white, occasionally faint yellow. Abundant aerial mycelia, which were either clumpy or spiny, developed on the inoculated plugs. Colonies were dense with irregular margins. Colonies grew fastest at 25 °C and slowest at 5 °C. No growth was observed at 35 °C.
• Associated insects: Dendroctonus valens.
• Hosts: Pinus tabuliformis.
• Material examined: CHINA, Liaoning Province, Chaoyang City, Jianping County, from Dendroctonus valens infesting Pinus tabuliformis, July 2024, Z. Wang and Q. Lu, holotype: CXY3371, ex-type culture CFCC72301, ibid. CFCC72302, and CFCC72303.
• Notes: Ceratocystiopsis jianpingensis was a phylogenetic sister to C. brevicomis and C. ranaculosa (Figure 1, Figure 2 and Figure 3) [26]. Although all three species produce a sporothrix-like asexual morph, C. jianpingensis also produces a hyalorhinocladiella-like asexual morph. Furthermore, C. jianpingensis grew slower than C. brevicomis at 25 °C on 2% MEA (42.9 mm in 20 days vs. 40–50 mm in 13 days). Ceratocystiopsis jianpingensis, C. brevicomis, and C. ranaculosa are associated with pine-infesting Dendroctonus. However, C. jianpingensis is an associate of D. valens in China, and C. brevicomis and C. ranaculosa are associated with D. brevicomis and D. frontalis in North America, respectively [26].

4. Discussion

This study revealed three ophiostomatalean associates of D. valens with P. tabuliformis, including C. jianpingensis sp. nov., and two new records (L. terebrantis and O. gilletteae). Ophiostoma gilletteae was the dominant species, with 108 isolates, and an isolation rate of 85.7%. This species is most frequently isolated from D. valens in western and eastern North America [9,13]. However, O. gilletteae has never been reported in China; therefore, its origin needs to be clarified through population genetics, as was the case with L. procerum [27]. Although we isolated only 14 strains of L. terebrantis (11.1%), this does not prevent it from becoming a species of concern. Similarly to L. procerum, L. terebrantis is associated with a wide variety of weevils and bark beetles in North America, and has been isolated from many dying pine trees [6]. Notably, L. terebrantis has an extremely variable pathogenicity to different hosts [6]. Therefore, the pathogenicity of this species in pine trees native to China needs to be tested urgently. In addition, surveys with larger sample sizes should be conducted to clarify the diversity of ophiostomatalean fungi associated with D. valens.

Leptographium procerum is considered the dominant associate of D. valens in China. However, aside from this species, the three ophiostomatalean fungi we identified in this study are different from the fifteen previously reported in China [9,10,11,12]. The causes of this variation in the fungal assemblages associated with D. valens in different regions may require further investigation using larger sample sizes. Additionally, some associates were neutral or antagonistic to D. valens. For example, O. minus and L. sinoprocerum retard weight gain in larvae of D. valens by depleting the immune defenses of the beetle [28]. Hence, while we are concerned about the pathogenicity of ophiostomatalean fungi to trees, we should also analyze the interactions between these associates and beetles.

Highly heterogeneous forest ecosystems limit the effectiveness of traditional pest control strategies. Innovative biological control programs provide new hope in the fight against forest pests. Dendroctonus valens employs mutualistic microbes to facilitate its invasion success [29]. For instance, gut bacteria are essential for the development of D. valens by regulating glucose transport [30]. Consequently, these mutualistic microbes are expected to be potential targets for beetle control. Furthermore, the early detection of a D. valens infestation using unmanned aerial vehicle (UAV)-based thermal and hyperspectral imaging is conducive to the formulation of effective biological control strategies for this beetle [31]. Some emerging technologies, such as RNA interference (RNAi), have already been applied in pest management [32]. Beetles are generally highly sensitive to RNAi, and feeding and injection of double-stranded RNA (dsRNA) can kill bark beetles [33]. For example, feeding Dendroctonus frontalis dsRNA targeting heat shock protein genes resulted in up to 100% mortality [34]. However, highly heterogeneous forest ecosystems limit the feasibility of delivering dsRNAs to pests. Microbial-mediated dsRNA delivery is increasingly used for pest management [35]. This means that there are prospects for using genetic engineering techniques to modify fungal associates to produce and deliver dsRNA to their vector, D. valens. The discovery of a large number of ophiostomatalean fungi associated with D. valens has led to the accumulation of resources helpful in realizing this vision.

5. Conclusions

We isolated and identified three ophiostomatalean fungal associates of D. valens from P. tabuliformis, including the new species, C. jianpingensis. Leptographium terebrantis and O. gilletteae, which are associates of D. valens in North America, were found in China for the first time. This study provides new understanding of the ophiostomatalean assemblage of D. valens, and enriches the resources for the development of fungal associate-mediated bark beetle management strategies.