Spruce is an evergreen woody plant that is distributed in cold temperate and subalpine regions. The genus consists of approximately 50 species, including 20 species and five varieties found in China (Chen, 2013). In recent years, pest outbreaks have been accelerated by climate change and increasing international trade. Spruce forests in the Northern Hemisphere, especially in Europe, have been severely affected by bark beetles of genus Ips (Coleoptera: Curculionidae: Scolytinae) (Hlásny et al., 2021). Bark beetles and fungi have formed complex associations during long-term co-evolution, resulting in the ability for the beetles to carry ophiostomatoid fungi (Six, 2012). The combined impact of the association between beetle pests and ophiostomatoid fungi has been studied for a long time (Kirisits and Konrad, 2004; Wingfield et al., 2016) and has received attention in China since the turn of the century, in view of the successful invasion of Dendroctonus valens from North America and the epidemic outbreak of indigenous Ips spp. and Dendroctonus armandii (Lu et al., 2008; Sun et al., 2013; Wang et al., 2022).

Both beetle and fungal partners have developed from occasional and sporadic to relatively stable and intimate relationship during their historical co-occurrence in the same habits (Bleiker and Six, 2009; Biedermann and Vega, 2020; Chakraborty and Roy, 2021; Singh et al., 2021). The benefits of beetle vectors to fungi are evident; for example, beetles can bring the fungi to new hosts and create inoculation holes. However, the benefits of fungi to beetles are unclear and sometimes disputed (Six and Wingfield, 2011). Nevertheless, substantial evidences from studies in nutrition, chemical ecology, and molecular biology have supported that fungi (particularly ophiostomatoid fungi) can enhance the colonization success of beetles in the following ways: by producing ergosterol, which is indispensable for beetle development (Bentz and Six, 2006), improving food quality and availability (Davis et al., 2019; Dzurenko and Hulcr, 2022), detoxifying tree defense compounds (DiGuistini et al., 2011; Wadke et al., 2016; Itoh et al., 2018; Davis et al., 2019; Zhao et al., 2019a), attracting or repelling other individuals through semiochemicals (Zhao et al., 2015, 2019b; Kandasamy et al., 2019), or competitive exclusion of beetle pathogens (Davis et al., 2019). In addition, many members of the ophiostomatoid fungi are pathogens of forests and crops worldwide. Notorious diseases caused by ophiostomatoid fungi include Dutch elm disease, laurel wilt, and oak wilt on broadleaf trees, and stem canker stain and black root disease on coniferous trees, leading to severe ecological and economic losses (Henry, 1944; Brasier, 1986; Harrington et al., 2008; Santini and Faccoli, 2015). Artificial inoculation of certain ophiostomatoid fungi can generate inner bark necrosis and lead to sapwood blue staining and drying within a few months, severely hindering water transportation, affecting tree growth, and even causing the death of host plants (Yamaoka et al., 1998; Davydenko et al., 2017; Devkota and Eckhardt, 2018).

Conifers have evolved a combination of structural and inducible defenses against bark beetles and their associated fungi (Franceschi et al., 2005; Jones and Dangl, 2006; Celedon and Bohlmann, 2019). Studies on the chemical defense of conifers against bark beetle-ophiostomatoid fungal symbionts have shown that terpenes are toxic to the symbionts (Phillips and Croteau, 1999; Raffa et al., 2005; Keeling and Bohlmann, 2006; Pan et al., 2018; Celedon and Bohlmann, 2019; Ullah et al., 2021). The phloem tissue of Pinus spp. contains constitutive monoterpenes that provide immediate resistance to bark beetle attacks (Boone et al., 2011; Raffa et al., 2013); local concentrations of terpenes increase rapidly after beetle attack. When the induced terpene concentration levels exceed the beetle’s physiological tolerance threshold, they inhibit or repel beetles that arrive subsequently and alter the growth of fungi associated with beetles that are already present (Raffa et al., 2005; Erbilgin et al., 2017). In vitro experiments also confirmed that monoterpenes, such as pinene and limonene, can inhibit the growth of fungi (Zhao et al., 2019b; Fang et al., 2020; Wang et al., 2020a). Monoterpenes, such as α-pinene, with stronger growth inhibitory ability against fungi, have a stronger repelling effect on bark beetles (Fang et al., 2020). Long-term production of monoterpenoids consumes a large amount of carbon, which is essential for tree growth (Trapp and Croteau, 2001; Dai et al., 2022), impeding the growth of pest-infested trees. In addition to terpenes, phenolics seem less important in the induced defense against bark beetle-associated fungal infestation (Erbilgin et al., 2017).

RNA-seq facilitates genome-wide assessment of gene expression profiles in plants under various conditions, such as biotic and abiotic stresses (Conesa et al., 2016). At present, more and more studies have used this method to analyze host plant resistance (Nibedita and Jolly, 2017). In a transcriptomic comparison between healthy Gastrodia elata and those infected with Penicillium oxalicum, 10 potential resistance genes were identified, involving plant hormone signal transduction, jasmonic acid signaling, and plant-pathogen interaction pathways, revealing that the immune response mechanism of G. elata to fungal disease is a complex biological process (Wang et al., 2020b). In poplars infected with canker disease in the early stage, most of the carbon metabolism and transportation genes, aquaporin genes, and genes related to secondary metabolites and phenylpropane biosynthesis pathway were inhibited, while the expression of resistance genes was promoted (Li et al., 2019). The transcript abundance of Norway spruce leucoanthocyanidin reductase (LAR) genes increased significantly during Endoconidiophora polonica infection (Hammerbacher et al., 2014). Anthocyanin monomers can mitigate the effects of various abiotic stresses such as ultraviolet radiation and ozone by reducing oxidative stress and inhibiting bacterial growth and fungal spore germination (Jaakola et al., 2004; Karonen et al., 2006).

It is interesting to understand whether the beetle-vectored fungi cause plant disease and whether the plants develop resistance against them. Ophiostoma bicolor is one of the dominant associate fungi with Ips spp. and Dendroctonus micans, which attacked various spruce species in Europe and north China (Giordano et al., 2013; Nève Repe et al., 2018; Chang et al., 2019, 2020; Chakraborty et al., 2020; Wang et al., 2020c,2021). Previous studies have confirmed that the pioneer species in the associated fungal community of Ips, like Endoconidiophora spp., showed strong virulence, which could cause severe host necrosis and induce a drastic resistance response of host conifers. However, O. bicolor could cause a small amount of spruce sapwood blue-staining and drying, and form small necrotic lesions on the inner bark near the inoculation site, accompanied by bits of resin outflow, showing moderate to weak levels of virulence from a series of pathogenicity tests in Europe and Japan (Christiansen and Solheim, 1994; Yamaoka et al., 2000; Sallé et al., 2005; Repe et al., 2015). The pathogenicity of O. bicolor isolated from China is unknown, and whether O. bicolor can induce the host to produce a resistance response like the pioneer species need to be further investigated. In addition, the molecular mechanisms underlying host resistance to ophiostomatoid fungi remain unclear.

In this study, the representative strains of O. bicolor were inoculated on 4-year-old Picea koraiensis to observe the differences in virulence between various strains, given that different strains of the same pathogen species may have different levels of virulence (Lieutier et al., 2003; Sallé et al., 2005). Changes in host defense-related metabolites after inoculation were also analyzed and the defense mechanisms of spruce against O. bicolor were investigated at the molecular level.

With global climate change, forest disasters caused by parasitic and semi-parasitic fungi have become more severe, which was also the case with the beetle-vectored fungi (Kim et al., 2021; Klesse et al., 2021; Morrison et al., 2021; Sitz et al., 2021; Li et al., 2022). However, compared with the former, the diseases caused by beetle-vectored fungi, particularly in gymnosperms, are not well studied. This study combined pathophysiological and transcriptomic analyses to investigate the defense response of P. koraiensis to O. bicolor infection. The results showed that: (1) different O. bicolor strains showed pathogenicity differentiation in P. koraiensis; (2) O. bicolor can induce the release of monoterpenoids in spruce in a short period of time; the release rates are correlated with the fungal virulence. Subsequently, the release rates gradually decrease to a normal level comparable to the control; (3) O. bicolor can significantly change the overall gene expression of P. koraiensis in early infection, and induce the upregulated expression of terpenoid backbone biosynthesis, plant-pathogen interaction, and MAPK signaling pathway.

Some ophiostomatoid fungi associated with I. typographus were pathogenic to spruce, and could induce the host to produce terpenoids, phenols, and other defense substances that were related to disease resistance (Solheim and Safranyik, 1997; Yamaoka et al., 2000; Sallé et al., 2005; Repe et al., 2015). In inoculation experiments by ophiostomatoid fungi, fungal virulence to hosts was not necessarily fatal (Kolb et al., 2019; Stewart et al., 2020). In this study, all five strains of O. bicolor could cause lesions around the inoculation site in P. koraiensis but could not cause wilting or death of the host tree (Figure 1). The elicited lesion areas around the inoculation sites decreased in the order of TS, BH, QH, MX, and LWQ (Table 2). Devkota and Eckhardt (2018) showed that there were differences in host defense responses to different fungal strains inoculation. These results indicated that O. bicolor was a fungus that was weakly virulent to P. koraiensis, and there was pathogenicity differentiation among different strains.

Trees can generate and transfer terpenes, such as monoterpenes, diterpenes, and sesquiterpenes, which are toxic to beetles and fungi; these terpenes could prevent further attacks from beetles and fungi. Raffa and Berryman (1983) reported that lodgepole pines could resist mountain pine beetles by producing high concentrations of two monoterpenes, α-pinene, and limonene, which was further verified in the field by Erbilgin et al. (2017). Meanwhile, the resistance induced by the bark beetle in the host also inhibits the infection of the fungus (Lombardero et al., 2019). The results of this study showed that in addition to the pathogenicity differentiation of different fungi strains, the spruce monoterpenoids synthesis that was induced by O. bicolor, also showed differentiation (Supplementary Table 2). Furthermore, the release rate of monoterpenoids from the host increased at first and peaked at 4 dpi. From 4 to 90 dpi, the release rate of monoterpenoids showed a gradual downward trend (Figure 2). The release rates of monoterpenoids were correlated with the fungal virulence at 30 dpi and almost did not differ with each other under all treatments at 90 dpi. This phenomenon is consistent with the findings of a previous study, which showed that after MeJA was applied to P. abies, the host displayed an induction of terpenes, with concentrations peaking around 16 days after treatment and returning to near-normal levels within 32 days after treatment (Erbilgin et al., 2006). Mageroy et al. (2020a) further verified this phenomenon in Norway spruce, which might be the result of defense priming. Previous studies have shown that host trees inoculated with ophiostomatoid fungi, i.e., E. polonica and Endoconidiophora fujiensis, could generate a rapid and long-term release of monoterpenoids, which possibly deplete the host defense ultimately (Arango-Velez et al., 2018; Fang et al., 2020; Ott et al., 2021). In this study, the release of monoterpenoids did not drastically increase, which was likely due to the different fungal species and inoculation scenarios. The dominant species of ophiostomatoid fungi, which is less virulent, was selected to evaluate host response, whereas in the previous study, the pioneer species, which is more virulent, was selected. This result is in accordance with the conclusion that more virulent fungi induce large changes in host defense terpenoids (Cale et al., 2019; Fang et al., 2020). Therefore, we speculate that O. bicolor stimulates the host to produce defense priming; however, subsequent experiments using MeJA treatment as a positive control of priming defense and challenging stimulus, e.g., E. polonica inoculation or beetle infestation are needed for further verification.

To reveal the regulation of gene expression related to disease resistance and immunity in P. koraiensis, transcriptomic analysis was performed. The results showed that in the early stage of O. bicolor infection of spruce stems (4 dpi), most of the DEGs were upregulated (Figure 3), and these DEGs were mainly related to repair, binding, and signal transduction processes. In the transcriptomic analysis of Grosmannia clavigera hyphae treated with terpenoids for 12 h, it was observed that genes encoding DNA repair, recombination, stabilization, and replication proteins were induced (DiGuistini et al., 2011). This study expands our knowledge of the effects of ophiostomatoid fungi on hosts. Early defense signals in plants often lead to the activation of downstream TF genes, thereby enhancing the expression of defense-related genes (Eulgem and Somssich, 2007). WRKYs play important roles in plant immunity through the ET, JA, and SA pathways in response to various biotic stressors. Several WRKY TFs (mostly upregulated) were involved in the spruce response to O. bicolor. We focused on previously reported pathways involved in plant immune responses, such as plant-pathogen interactions, MAPK signaling, flavonoid biosynthesis, phenylpropane biosynthesis, and terpene biosynthesis and metabolism, to elucidate the molecular mechanisms underlying host defense responses (Gao et al., 2018; Su et al., 2018; Song et al., 2019).

In plants, the biosynthetic precursor of terpenoids can be synthesized via two pathways: the MVA and MEP pathways (Roberts, 2007). In this study, numerous DEGs involved in terpenoid biosynthesis by the MVA and MEP pathways were upregulated (Figure 5A; Supplementary Table 6). On the one hand, the genes of FDPS, which are involved in the synthesis of various monoterpene precursors, and GPP, which comes from DMAPP, were upregulated. On the other hand, the genes of ispS involved in the synthesis of isoprene from DMAPP, were downregulated and thus enhanced the monoterpenoids precursor biosynthesis (Figure 5A). These results suggest that O. bicolor inoculation in P. koraiensis promoted the biosynthesis of host monoterpene precursor, thereby increasing the release of six monoterpenoids. The expression of enzyme genes in the monoterpene synthesis pathway remained nearly unchanged, which is expected and consistent with the findings of previous research (Mageroy et al., 2020b). Moreover, the differences of DEGs related to ispS and HMGCR in TS and LWQ treatments may be the reason for the differences in the release rates of monoterpenoids in the two strain treatments.

Ophiostoma bicolor significantly altered the expression of genes related to plant-pathogen interactions pathways, involving 241 DEGs at 25 nodes. Upregulated genes accounted for 87.97% and were mainly involved in biological processes such as hypersensitive response (HR), cell wall reinforcement, stomatal closure, defense-related gene induction, phytoalexin accumulation, miRNA production, programmed cell death, suppression of plant HR, and defense response (Supplementary Figure 3). These processes are closely related to plant defense responses (Yang et al., 1997). Binding of plant PR proteins to pathogen effector proteins activates ion channels, oxidative bursts, and other signal transductions (Dixon et al., 1994). Ca²⁺ activates the plant early surveillance system to prevent microbial infection in plant defense signaling (Nürnberger and Scheel, 2001). In the present study, the host treated with the two strains (TS and LWQ treatments) showed differential expression pattern of CML, and LWQ treatment showed less downstream calcium-dependent process in defense response.

The upregulated genes in the MAPK signaling pathway accounted for 68.81% (75 of 109) of all upregulated genes and were mainly involved in biological processes such as cell death, accumulation of reactive oxygen species in plant defense, defense responses, stress adaptation, and wounding. The downregulated genes were mainly related to defense responses against pathogens and wounding (Supplementary Figure 4). MAPK cascades play an important role in plant defense against pathogens (Pitzschke et al., 2009). Its activation is one of the earliest responses for sensing pathogen-associated molecular patterns (PAMPs) in plants (Meng and Zhang, 2013). Some DEGs of LWQ treatment were downregulated and enriched in defense response against pathogens, cell death process and active oxygen production process, while these genes were not differentially expressed in TS treatment (Supplementary Table 6), which might indicate weaker induction of host defense response by the weakly virulent strain. These results suggest that P. koraiensis may use different MAPKs to deliver attack signals, and O. bicolor activated the earliest defense via the MAPK cascade, thus inhibiting further infection.

Through the phenylpropanoid pathway, many kinds of lignin (p-hydroxyphenyl lignin, guaiacyl lignin, 5-hydroxy-guaiacly lignin, and syringyl lignin) can be produced by plants. Lignin can help plants resist biotic and abiotic stresses by regulating secondary cell wall development and stomata (Chen et al., 2019). The synthesis process can be roughly divided into two steps: firstly, the lignin monomer is synthesized under catalysis by a series of enzymes including the 4CL and CAD, and secondly the lignin monomer is polymerized into bioactive lignin by a series of chemical reactions catalyzed by POD and other enzymes (Boerjan et al., 2003). Inhibition of CAD activity in vascular plants changed lignin content. For example, a decrease in CAD activity in sorghum results in a decrease in the total amount of lignin (Palmer et al., 2008). In the present study, the key enzymes involved in lignin monomer synthesis, including 4CL, COMT, and CCR, were activated (Supplementary Table 6). However, CAD required for the final step of lignin monomer biosynthesis was inhibited (Supplementary Table 6). In addition, the gene expression of POD was downregulated, which is consistent with the findings of previous studies on poplar responses to canker pathogens (Li et al., 2019). Although our experiment did not measure the change in lignin content in spruce, it was known from the literature that the content of lignin was reduced in hosts infected with ophiostomatoid fungi (Villari et al., 2012). As a result, O. bicolor may inhibit the synthesis and metabolism of lignin in the spruce stem.

The flavonoid biosynthesis pathway produces a variety of phenolic compounds such as procyanidin, catechin, gallocatechin, and epicatechin. When pathogens invade spruce, fluorescent inclusion bodies containing phenolic compounds appear in the phloem parenchyma cells of the bark, suggesting that phenolic compounds may play a key role in defense against herbivores and pathogens (Franceschi et al., 2005). After an E. polonica infection in Norway spruce, the content of flavane-3-ols, catechin, and gallocatechin in the bark increased (Hammerbacher et al., 2014). The results of this study showed that most DEGs (22 of 28) associated with the flavonoid biosynthesis pathway were downregulated at 4 dpi (Supplementary Table 6), and O. bicolor infection inhibited phenolics-related gene expression in spruce, which differed from the expression pattern of the virulent pioneer E. polonica. The downregulated expression pattern in this study was consistent with the transcriptomic results of Norway spruce infected with Chrysomyxa rhododendri at 4 dpi (Trujillo-Moya et al., 2020). The flavonoid contents of spruces inoculated by O. bicolor were not investigated in this study. Thus, further research is needed to clarify the correlation between the contents and gene expressions of flavonoids.

The ophiostomatoid fungal flora of bark beetles is rich and diverse. This study showed that a dominant fungus of the flora, O. bicolor, is a weakly virulent pathogen of spruce, and various strains showed differentiation in pathogenicity. The release of monoterpenoids from the host is positively correlated with the virulence of the inoculated fungus. In terms of amounts of monoterpenoids released by hosts after inoculation, the pioneer species, e.g., Endoconidiophora spp., can induce rapid and long-term defense responses in the host, while O. bicolor is likely to induce the host defense priming phenomenon. The response of spruce to O. bicolor is a complex process involving multiple biological processes, including plant-pathogen interaction process and MAPK metabolic process. The expression patterns of spruce defense-related genes during infection with different O. bicolor strains were different. However, it was still unclear how the host coordinates these different defense responses against different fungal strains. Further studies are necessary to investigate the underlying mechanism of the host defense response against the pathogen fungi. Bark beetles and their associated fungi usually attack adult trees, thus our inoculations conducted on seedlings have certain limitations. The experiments should attempt to conduct under natural conditions in the future.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

QL and YL conceptualized and designed the research. YL, QZ, and GZ performed the experiments. YL and QZ analyzed the data and wrote the manuscript. YL, QZ, ZW, HW, JZ, and QL participated in the discussion for experimental details. QL and JZ revised the manuscript. QL directed the project. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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