Global climate change and trade globalization have significantly influenced the proliferation and distribution of forest pests, posing novel challenges for forest management. Bark beetles (Coleoptera, Curculionidae, Scolytinae), a highly diverse group of insects, are prevalent across global forest ecosystems (Morris et al. 2017). These beetles, which primarily target coniferous trees, exhibit strong concealment abilities and can induce extensive tree mortality, thus representing critical forest pests (Ferrenberg et al. 2014). Research indicates a strong correlation between the spread of bark beetles and climate warming (Niemiec et al. 2014). When conditions are favorable, particularly when introduced beyond their native habitats, certain species can transition from minor pests to significant threats to host trees, resulting in substantial economic losses and ecological harm (Ranger et al. 2015; Ranger 2016; Biedermann et al. 2019). In Europe, Ips typographus Linnaeus is regarded as the most significant pest affecting Picea abies (L.) H. Karst. (Schelhaas et al. 2003). Similarly, in China, the invasive alien species Dendroctonus valens LeConte has led to widespread mortality of Pinus tabuliformis Carrière, posing a serious threat to the ecological stability of forestry (Lu et al. 2009; Sun et al. 2013).

In comparison to isolated damage, the combined impact of microbial pathogens and insects can result in severe harm to forest ecosystems (Kenneth F. Raffa and Romme 2008). Consequently, it is imperative to promptly elucidate the novel interactions between forest pests and pathogens (Wingfield et al. 2016; Biedermann and Vega 2020). Ophiostomatoid fungi are primarily associated with bark beetles. These fungi exhibit similar morphological and structural traits, notably the production of adhesive spore masses, which are disseminated by bark beetles to host plants (De Beer 2013). Ophiostomatoid fungi are integral to the successful colonization of bark beetles (Hofstetter et al. 2015), significantly compromising the resistance of host trees and facilitating the invasion by these beetles (Heiniger et al. 2011). Furthermore, these fungi contribute essential nutrients for the growth and development of bark beetles within the nutrient-deficient phloem of coniferous trees (Bentz and Six 2006). Bark beetles associate with a diverse array of ophiostomatoid fungal species, including several highly pathogenic strains (Kgang et al. 2008) and staining fungi (Hýsek et al. 2021), the latter of which can adversely affect wood quality. This group of fungi are regarded as a significant threat to conifers, prompting extensive research of bark beetle fungi globally. In southeastern Australia, fourteen species of Ophiostomatales across six genera, along with one species of Microascales, have been isolated from bark beetles and infected pines (Trollip et al. 2021). In Northeast China, Ips subelongatus Motschulsky is associated with fourteen species of ophiostomatoid fungi spanning four genera (Ceratocystiopsis, Endoconidiophora, Leptographium, and Ophiostoma) (Wang et al. 2020). Notably, Ophiostoma bicolor R.W. Davidson & D.E. Wells, O. brunneo-ciliatum Math.-Käärik, O. ainoae H. Solheim, and Grosmannia piceiperda (Rumbold) Goid are associated with both Ips typographus and Pityogenes chalcographus Linnaeus (Repe et al. 2013).

Hylurgus ligniperda Fabricius (1787), commonly referred to as the red-haired pine bark beetle, is a forest insect that parasitizes the phloem of pine trees (Bedoya et al. 2019). It is presumed to be indigenous to Europe and the Mediterranean Basin, with subsequent introductions into Oceania, North America, and South America. In East Asia, H. ligniperda is considered an invasive species, initially invading Japan and subsequently Korea. Recently, it has successfully invaded and colonized Shandong Province in China (Lin et al. 2021). As a root-dwelling species, H. ligniperda typically enters its host through the soil and feeds on the phloem of sub-healthy pine trees, although it does not directly cause tree mortality. However, in newly invaded regions, the presence of H. ligniperda significantly affects the ecological security of local forestry systems. The invasion of H. ligniperda has resulted in a reduction of the windbreak and sand-fixation capacities of the coastal shelterbelt in Shandong Province, China, and its high dispersal capability poses a substantial threat to the local forest ecosystem.

Numerous studies have documented the isolation of fungi associated with H. ligniperda. In the forests and steppes of eastern Ukraine, H. ligniperda serves as a vector for diverse fungal communities (Davydenko et al. 2014). In Chile, five ophiostomatoid fungi species associated with H. ligniperda and Hylastes ater Paykull have been identified (Zhou et al. 2004c). Eight species of ophiostomatoid fungi have been isolated from H. ligniperda in New Zealand (Ray et al. 2006). In Poland, the ophiostomatoid fungi associated with H. ater, Hylastes opacus Erichson and H. ligniperda on Pinus sylvestris L. was examined, revealing that fungi of the genera Grosmannia and Leptographium were the most prevalent (Jankowiak and Bilański 2013). However, in the newly invaded region of H. ligniperda in Shandong Province, China, research of fungi associated with H. ligniperda remains unexplored.

In this study, we examined the ophiostomatoid fungi community associated with H. ligniperda in the Yanwei region of Shandong Province, China. We employed a combination of morphological observations and multilocus DNA sequence phylogenetic analysis to address existing knowledge gaps in this area. The findings of this research contribute to a deeper understanding of the ophiostomatoid fungi community linked to H. ligniperda in China and establish a foundation for future investigations into the occurrence and management of H. ligniperda.

Currently, H. ligniperda has successfully colonized and caused significant damage to coastal shelter forests in the Yanwei region of Shandong Province, China. In this study, fungi were isolated from adults H. ligniperda obtained from galleries and traps. A total of 425 strains of fungi associated with longhorn beetles were isolated, marking the first investigation into ophiostomatoid communities associated with H. ligniperda in China. Although fungal isolation is influenced by various environmental factors, including individual insect variability and environmental contamination during the isolation process, this study employed extensive sampling and isolation techniques. It is anticipated that the findings will provide a reliable representation of the fungal communities of ophiostomatoid fungi associated with H. ligniperda in Yantai and Weihai, Shandong Province, China. Based on the phylogenetic analysis of multigene fragments and observation of microscopic characteristics, 13 species belonging to six genera were identified. They included seven known species, viz. Gr. translucens, H.w. taylorii, G. huntii, L. koreanum, L. radiaticola, M. pallidulus, and O. ips. And six previously undescribed species, viz. C. pseudoweihaiensis, C. pseudoyantaiensis, Gr. jiuguanense, L. ligniperdae, M. xishanensis, and M. dongshanensis.

The ophiostomatoid fungal communities associated with gallery-derived and trap-captured H. ligniperda adults were significantly different. The symbiotic relationships between bark beetles and ophiostomatoid fungi appear to change with changes in the environmental conditions in which the bark beetles reside. The higher number of ophiostomatoid fungi isolated from bark beetles captured in traps may be attributed to the beetles’ exposure to a broader range of environmental fungi at this stage, rather than solely harboring specific fungi. This finding is consistent with previous studies reporting differences in species composition and isolation frequency of fungi associated with bark beetles across different countries and regions (Taerum et al. 2013; Wang et al. 2020), indicating that beetles acquire and carry environmental fungi. Furthermore, studies have shown that the fungi carried by beetles not only influence nutrient acquisition but also assist in degrading tree defensive compounds, thereby affecting the fitness of the beetles (Ayres et al. 2000; Cale et al. 2016; Six and Elser 2019; Zhao et al. 2019). The limited diversity of ophiostomatoid fungi species observed within the galleries, coupled with a high frequency of isolation, suggests that these fungi may facilitate the colonization of bark beetles in the region.

Currently, O. ips is distributed worldwide and associated with various beetles that infest conifers, contributing to wood bluing and, in conjunction with bark beetles, weakening trees and accelerating forest decline (Raffa and Smalley 1988; Klepzig et al. 1991; Pastirčáková et al. 2018). The fungus has also been isolated from several bark beetle-associated mites in Yunnan, China (Chang et al. 2017). There is significant interest in the relationship between O. ips and Bursaphelenchus xylophilus (Steiner & Bührer) Nickle (Niu et al. 2012; Suh et al. 2013; Zhao et al. 2014; Wang et al. 2018). In this study, O. ips exhibited the highest isolation rate, aligning with findings from previous research conducted on ophiostomatoid fungi associated with pines infected by Cryphalus piceae Ratzeburg (Chang et al. 2021), Bursaphelenchus xylophilus and Monochamus alternatus Hope (Wang et al. 2018) in Shandong, China. Ophiostoma ips was isolated from bark beetles collected across various countries, with the frequency of isolation varying by region (Zhou et al. 2004c; Ray et al. 2006; Davydenko et al. 2014; De Errasti et al. 2018). Ophiostoma ips has been reported from H. ligniperda across all six continents and exhibited the highest isolation frequency in the present study. This pattern may be attributed to its broad global distribution combined with its status as the most frequently isolated ophiostomatoid fungus in Shandong Province, China.

The genus Leptographium accommodates L. radiaticola, L. koreanum, G. huntii, and L. ligniperdae, all of which showed significantly higher isolation frequencies from gallery-derived than trap-captured adults, suggesting their specialized adaptation to the gallery environment. Leptographium radiaticola was the second most abundant ophiostomatoid fungus isolated in this study. Initially it was described as being isolated from P. radiata D. Don imported to Korea (Kim et al. 2005a), but this is not the first time the species has been spotted. Leptographium radiaticola has been previously identified in isolates from South Africa, Chile, Sweden, and California (Zhou et al. 2001; Zhou et al. 2004b; Kim et al. 2011). This is not the first instance of L. radiaticola being recorded in China. It was initially isolated from the gallery of Dendroctonus valens on P. tabuliformis in Shanxi Province, China (Lu et al. 2009). Subsequently, it was isolated again in Yunnan Province, China, during a survey of ophiostomatoid fungi affecting coniferous beetles and beetle-associated mites (Chang et al. 2017). These two reports involved different beetles that harm various conifers. This study reports the first occurrence of L. radiaticola in Shandong Province, China. Additionally, the sequences obtained here were distinct from those of strains previously isolated in the Yunnan region of China (Chang et al. 2017). This fungus is widely distributed globally and is particularly associated with H. ligniperda. The strain isolated in South Africa was obtained from H. ligniperda (Zhou et al. 2001), while those in Chile were isolated from Hylastes ater and H. ligniperda galleries on P. radiata (Zhou et al. 2004c). In California, the fungus was isolated from H. ligniperda, which poses a threat to P. halepensis Mill. and P. pinea L. (Kim et al. 2011). Leptographium radiaticola was isolated from H. ligniperda, an exotic bark beetle, within pine plantations in Argentina. The fungus has also been reported from H. ligniperda on P. taeda L. and P. elliotti Engelm. (De Errasti et al. 2018). Furthermore, during a survey of the fungal community of ophiostomatoid fungi associated with pine and pine bark beetles in southeastern Australia, this fungus was isolated from H. ater and H. ligniperda, both of which are detrimental to P. radiata (Trollip et al. 2021). It is likely that L. radiaticola from the Shandong region of China was introduced alongside H. ligniperda. Research has revealed that multiple beetle species harbor stable symbiotic partners (Lewinsohn et al. 1994; Six and Bentz 2003), and such bark beetle–fungus associations contribute to enhancing host adaptability by assisting the beetles in overcoming tree defense mechanisms (Six 2020). The close association between H. ligniperda and L. radiaticola may be attributed to the fungus’s ability to facilitate the colonization and development of H. ligniperda. This is further supported by the observation that the separation frequency within the adults’ galleries was significantly higher than that of the adults in traps in this study, suggesting that H. ligniperda in the gallery requires more L. radiaticola to withstand the host.

Leptographium koreanum was isolated and described from P. densiflora and P. koraiensis Siebold & Zucc. infested by Tomicus piniperda Linnaeus in Korea (Kim et al. 2005b). In a comprehensive survey of ophiostomatoid fungi associated with Dendroctonus valens in eastern North America, western North America, and China, it was observed that L. koreanum is predominantly confined to Asia. However, this fungus was also isolated in eastern North America, albeit at a lower prevalence compared to China (Taerum et al. 2013). Pathogenicity tests demonstrated that L. koreanum could induce significant necrosis in the bark of P. tabuliformis (Lu et al. 2009). Following its invasion of the Yanwei region of China, H. ligniperda established contact with L. koreanum. Numerous invasive beetles establish novel associations with indigenous fungi at new invasion sites (Jacobs et al. 2003; Hausner et al. 2005), leading to the hypothesis that L. koreanum may facilitate the colonization of H. ligniperda. Grosmannia huntii, a group of fungi well-suited for transmission by bark beetles, exhibits a broad geographical distribution, encompassing regions such as Chile, New Zealand, Argentina, North America, and Australia(Zhou et al. 2004c; Reay et al. 2005; Ray et al. 2006; Taerum et al. 2013; De Errasti et al. 2017; Trollip et al. 2021). Notably, H. ligniperda infestations have been documented in these regions, although G. huntii has not yet been reported in China. The strains isolated in this study displayed high similarity to Australian strains obtained from H. Ater and H. ligniperda (Trollip et al. 2021). It is postulated that G. huntii is closely associated with H. ligniperda and may have been introduced to China alongside H. ligniperda, where it has successfully colonized. Leptographium ligniperdae is a member of the L. olivaceum species complex (De Beer et al. 2022). In this study, it was exclusively isolated from adult H. ligniperda within their galleries. Multigene phylogenetic analysis revealed that it is closely related to L. rhizoidum and L. sagmatosporum. Leptographium rhizoidum was isolated and characterized from H. ater and H. attenuatus Erichson, which infest P. radiata in Spain. Currently known insect vectors include H. ater, H. attenuatus, Hylurgops palliatus Gyllenhal, and Ips sexdentatus Börner (Yin et al. 2019). Leptographium sagmatosporum was initially isolated from bark beetle galleries and fresh wood surfaces on Picea mariana (Mill.) Britton, Sterns & Poggenb., P. resinosa Aiton, and P. strobus L. (Yin et al. 2019). The discovery of L. ligniperdae not only contributes to the diversity of the L. olivaceum species complex but also provides valuable insights into the ophiostomatoid fungal community associated with H. ligniperda.

Species of Masuyamyces isolated in this study include M. pallidulus and two novel species, M. xishanensis and M. dongshanensis (De Beer et al. 2022). In Finland, M. pallidulus was initially isolated from P. sylvestris, which is associated with various beetle species (Linnakoski et al. 2010). In Poland, M. pallidulus was isolated from the roots of dead trees and subsequently identified during a survey of ophiostomatoid fungi linked to root-eating beetles on P. sylvestris in the region (Jankowiak and Bilański 2013). Masuyamyces pallidulus is also present in Australia, including isolates from H. ligniperda (Trollip et al. 2021). In the genus Hawksworthiomyces, H.w. taylorii was first isolated from Eucalyptus poles within the soil layer in South Africa, with an optimal growth temperature of 30 °C (De Beer et al. 2016b). This fungus had not been previously isolated from H. ligniperda. Given that H. ligniperda primarily feeds on the roots of its host and interacts with the soil environment, it is hypothesized that the fungus is a soil-associated organism.

Certain fungi belonging to Ceratocystiopsis and Graphilbum were exclusively isolated from adults of H. ligniperda in traps. In the genus Ceratocystiopsis, C. pseudoweihaiensis is closely related to C. weihaiensis, while C. pseudoyantaiensis is closely related to C. yantaiensis. Both C. weihaiensis and C. yantaiensis have been isolated and reported from the Cryphalus piceae gallery on P. thunbergii in Shandong Province, China (Chang et al. 2021). Within the genus Graphilbum, Gr. translucens, and Gr. jiuguanense are identified. Multilocus phylogenetic analysis indicated that Gr. jiuguanense formed a distinct lineage closely related to Gr. niveum. Furthermore, both Gr. translucens and Gr. niveum were isolated from Cryphalus piceae galleries on P. densiflora and P. thunbergii in Shandong Province, China (Chang et al. 2021). This aligns with the ecological niche of H. ligniperda in China, suggesting that H. ligniperda acquires native fungi from the environment, although some fungi may undergo changes as they adapt to new host insects. The absence of these fungi in adult samples within the gallery may be attributed to their weak association with H. ligniperda.

The significant divergence in ophiostomatoid fungal communities observed across continents demonstrates that regional variation serves as the primary driver of fungal community differentiation in H. ligniperda associations. In this study, six novel fungal species along with several long-term fungal partners were isolated from H. ligniperda adults, indicating that the beetle develops specialized fungal assemblages adapted to local environments across different regions to facilitate its colonization and expansion into new habitats.

Our findings revealed a fungal community of ophiostomatoid fungi associated with H. ligniperda, which has successfully colonization in Yanwei, Shandong Province, China. We identified a total of 13 species across six genera, including six novel species. Ophiostoma ips and L. radiaticola emerged as the dominant species, with isolation frequencies of 36.55% and 14.94%, respectively. Variations in the ophiostomatoid communities associated with H. ligniperda across different regions may be attributed to environmental differences. Nevertheless, the presence of certain fungi, such as O. ips, L. radiaticola, G. huntii, and M. pallidulus, remains consistent. Notably, the L. radiaticola and G. huntii strains isolated in this study clustered into a single clade with high sequence similarity to strains from southeast Australia, also isolated from H. ligniperda. It is hypothesized that the source population of H. ligniperda established in Shandong, China, is the same as that of the Australian population. Furthermore, native fungi and newly identified species closely related to native fungi were isolated from H. ligniperda, suggesting that insects in newly invaded areas establish interactions with native fungi, which in turn adapt to these insects through evolutionary changes. Understanding this novel association formed by H. ligniperda in the Yantai and Weihai regions of China is crucial for future management strategies of this insect. The successful colonization of alien invasive insects in new environments is influenced by numerous factors, among which the fungi associated with bark beetles play a significant role. Consequently, it is imperative to comprehend the relationship between bark beetles and their associated fungi, as this knowledge will facilitate timely control of alien invasive insect populations and help prevent substantial economic losses.