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Article

Enhanced Emission of Monoterpene 3-Carene in Pinus densiflora Infected by Pine Wood Nematode and Characterization of 3-Carene Synthase

by
Hwan-Su Hwang
,
Jung-Yeon Han
and
Yong-Eui Choi
*
Department of Forest Resources, College of Forest and Environmental Sciences, Kangwon National University, Chuncheon 200-701, Korea
*
Author to whom correspondence should be addressed.
Forests 2021, 12(5), 514; https://doi.org/10.3390/f12050514
Submission received: 29 March 2021 / Revised: 18 April 2021 / Accepted: 19 April 2021 / Published: 21 April 2021
(This article belongs to the Section Genetics and Molecular Biology)
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Abstract

:
Pinus densiflora is an important pine species in Northeast Asia due to its historical, cultural, and economic values. Pine wood nematode (PWN) seriously damages P. densiflora, causing the pine wilt disease (PWD). Changes of phyto-compounds in resin and monoterpenes in P. densiflora after infection of PWN were studied. The changes were identified by GC-MS in control and infected P. densiflora. Among the resin phytochemicals (in P. densiflora), 3-carene was distinctly enhanced after PWN inoculation. The emitted plant volatile monoterpenes were analyzed by HS-SPME/GC-MS. It was observed that the amount of 3-carene enhanced conspicuously after infection of PWNs in both P. densiflora and P. koraiensis at 9.7 and 54.7 times, respectively. 3-Carene synthase gene (Pd3-cars) of P. densiflora was isolated and functionally characterized by transgenic tobacco overexpressing Pd3-cars. Integration and expression of transgenic tobacco were confirmed by genomic and RT-PCR analysis. The Pd3-cars gene was expressed in transgenic tobacco plants. Furthermore, the production of 3-carene was identified by HS-SPME/GC-MS analysis as the volatile compounds emitted from leaves of transgenic tobacco. Treatment of 3-carene to PWNs showed a mild nematicidal activity with 45.98% mortality at the concentration of 10 mg/mL. The current findings may apply to the early diagnosis of pine wilt disease infected by PWNs through enhanced emission of 3-carene.

1. Introduction

Pinus densiflora is an evergreen conifer that grows in Northeast Asia, specifically Korea, China, Japan, and Russia. This pine tree is widely cultivated for timber plantations and landscaping. However, pine wilt disease (PWD) caused by pine wood nematode (PWN; Bursaphelenchus xylophilus) that extremely damages pine forests in Korea [1], Japan [2,3], China [4], and Portugal, in Europe [5].
Resins are generally a mixture of volatile and non-volatile compounds. It is a representative of a defense substance of conifer, and secreted by reacting to diseases, insects, and wounds [6,7,8]. PWN infection causes a series of physiological and biochemical changes to the pine trees. Since epithelial cells and parenchyma cells in the resin canal are used as food sources for PWNs, the epithelial cells in the pine tree are seriously destroyed, causing PWD symptoms’ development [3]. In addition, PWN infection highly affects the resin secretion of the pine tree [9].
Volatile compounds principally consist of a complex mixture of terpenoid compounds, and act as an important defense mechanism against biotic and abiotic stresses [10,11,12]. Kuroda [13] reported that monoterpenes are significantly enhanced in xylem tissues long before the initiation of tracheid cavitation in pine tree after PWN infection, and eventually, the volatile terpenes induced tracheid cavitation (embolism), which inhibits water transport from soil to leaves.
3-Carene, a bicyclic monoterpene hydrocarbon consisting of a fused cyclohexene and cyclopropane rings, is a common substance in the conifer. Each conifer species has relatively different proportions of 3-carene, and the amount of 3-carene can vary drastically between the same species [14]. The amounts of 3-carene were associated with tolerance or susceptibility to insect and white pine weevil in Sitka spruce (Picea sitchensis) [15,16]. Besides, in Lodgepole pine (Pinus contorta), the high amounts of 3-carene correlated with the resistance to Douglas-fir pitch moths (Synanthedon novaroensis) [17]. Furthermore, in Scots pine (Pinus sylvestris), the high level of 3-carene was found to have a correlation with low larval survival of the Sawfly (Diprion pini) [18]. PWN infection provoked releases of pinene-type monoterpenes and expression of α-pinene synthase, which were temporarily increased in Pinus pinea [19,20]. In addition, a monoterpene, β-myrcene, appeared as the most attractive to propagative larvae of PWNs among the volatile monoterpenes present in pine, and it plays an important role in the dispersal of the of PWNs [21]. 3-Carene synthase genes were characterized enzymatically in some conifer species (Picea abies, Picea sitchensis, P. bankiana, and P. contorta) [14,22]. However, no investigation has been reported for characterization of 3-carene synthase gene and accumulation of 3-carene in P. densiflora and P. koraiensis against PWN infection.
An early diagnosis of the PWD can be an important tool to reduce damage in the pine forest. In this work, we have investigated the changes of monoterpenoids of P. densiflora and P. koraiensis after PWN infection. 3-Carene was highly enhanced in resin and volatile compounds of P. densiflora and P. koraiensis after PWN inoculation. Additionally, phylogenetic analysis of 3-carene synthase (Pd3-cars) of P. densiflora, and functional characterization of Pd3-cars, were performed by tobacco transformation. Nematicidal and antifungal activities of 3-carene were also investigated.

2. Materials and Methods

2.1. Plant Materials, PWNs Culture, and Inoculation

Pine wood nematode (Bursaphelenchus xylophilus) and a fungal species Botrytis cinerea were provided by the Forest Research Institute of Gangwon province. B. cinerea was cultured in potato dextrose agar (PDA) medium for seven days, and then PWNs were inoculated in the same medium. PWNs were cultured at 25 °C in darkness by two-week subculture intervals, periodically. The proliferated PWNs were isolated by the Baermann funnel method [23]. The seedlings of five-year-old P. densiflora and P. koraiensis were infected with PWNs by incising the base of the main stem at 10~15 cm above the ground with a razor blade, according to Yazaki et al. [24]. A total of 2000 mix-staged nematodes in 150 μL sterilized water were inoculated to three saplings and the same amount of the distilled water was used as a control. Plant materials (five years old) used for this study were collected from the individual pots in a greenhouse at least three months before the experiments. The pine trees and soils used in the experiment were incinerated after autoclaving at the end of the experiments.

2.2. Phytochemical Analysis by GC-MS

The resin compounds collected from needle tips of P. densiflora after 20 days of PWN infection were extracted by sonication in 100% methanol for 30 min, and the supernatant after centrifugation was analyzed by gas chromatography-mass spectrometry (GC-MS). To analyze the volatile monoterpenes, the whole infected PWNs plants and non-infected plants (P. densiflora and P. koraiensis) were carefully wrapped up in plastic bags. The headspace solid-phase microextraction (HS-SPME), containing divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) df 50/30 μm, 2 cm length fiber (Supelco Co., Bellefonte, PA, USA), was manually inserted into the sealed plastic bag and exposed to the volatile compounds for one hour at room temperature.
The emission of volatile monoterpenes from two pine species (P. densiflora and P. koraiensis) was measured at 20 days after PWN infection. The analysis of pine compounds was performed by the GC-MS system (Agilent 5975C and 7890A, USA) under the following conditions: 70 °C for 4 min, followed by an increase to 220 °C at a rate of 5 °C min−1 and heated at 4 °C min−1 up to 320 °C, and held for another 5 min. The carrier gas was helium with the 1.2 mL min−1 flow rate. Split/splitless injection was a 10:1 ratio and ionization was performed by electron impact at 70 eV. The standard compounds (α-pinene, camphene, β-pinene, β-myrcene, 3-carene, β-phellandrene, and D-limonene) used in GC-MS analysis were purchased from Sigma-Aldrich Co. (Saint Louis, MO, USA).

2.3. Isolation of 3-Carene Synthase from P. densiflora and Tobacco Transformation

cDNAs were prepared from mRNA isolated from the stem of P. densiflora. Open reading frame of 3-carene synthase was amplified using PCR (35 cycles at 98 °C for 10 s, 55 °C for 5 s, and 72 °C for 2 min) with Pfu DNA polymerase (TaKaRa Bio, Otsu, Shiga, Japan). Primers used to isolate the cDNA were 5′-ATG TCT CTT ATT TCC GCT GTG C-3′ and 5′-TTA CAT AGG CAC AGG TTC AAG A-3′. PCR product was cloned into the binary vector (pCR8/GW/TOPO GATEWAY, Invitrogen, San Diego, CA, USA) for Agrobacterium-mediated transformation and transferred into pK2GW7, the binary destination vector, with 35S Cauliflower mosaic virus (CaMV) promoter. The binary vector was introduced into Agrobacterium tumefaciens GV3101 strain by a heat shock method. Transgenic tobacco was constructed as described by Jo et al. [25] using a different selection marker containing kanamycin.

2.4. Phylogenetic Analysis

Phylogenetic analysis was performed with the MEGA v6.06 software program. The deduced amino acid sequences of monoterpene synthase from other conifer species were obtained from DDBJ/GenBank/EMBL. Multiple sequence alignments were generated using the CLUSTAL W program. The maximum likelihood (ML) method was applied to estimate the strength of nodes with a bootstrap of 1000 replications in the tree [26,27].

2.5. Genomic DNA PCR and RT-PCR Analysis

The PCR analysis of genomic DNA was conducted to identify and select the transgenic tobacco lines. Genomic DNAs of wild‑type and transgenic lines were extracted from the leaf tissues of transgenic lines using the Genomic DNA Extraction Kit Mini (RBC bioscience, New Taipei City, Taiwan). The primers used for amplifying (1881 bp) the Pd3-cars gene were 5′-ATG TCT CTT ATT TCC GCT GT-3′ and 5′-TTA CAT AGG CAC AGG TTC AA-3′, and the primers used for the nptII gene were 5′-TCA GAA GAA CTC GTC AAG AAG G-3′ and 5′-GAG GCT ATT CGG CTA TGA CTG-3′. The PCR analyses were performed in a DNA thermal cycler (Applied Biosystems, CA, USA) under the following conditions: one cycle of 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min, with a final 10 min extension.
Total RNA for reverse transcription polymerase chain reaction (RT-PCR) analysis was isolated from wild-type and transgenic tobacco leaves with the RNeasy Plant Mini Kit (Qiagen, hilden, Germany), and converted to cDNA using the M-MLV reverse transcriptase (Invitrogen, San Diego, CA, USA). RT-PCR was conducted using the first strand DNA as a template and followed a condition: 95 °C for 3 min, 30 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min, and a final extension at 72 °C for 10 min. The primers used for the Pd3-cars gene were 5′-ATG TCT CTT ATT TCC GCT GTG C-3′ and 5′-TTA CAT AGG CAC AGG TTC AAG A-3′. The primers used for the attB1–attB2 region in the overexpression vector were 5′-AAA AAG CAG GCT ATG TCT CTT ATT TCC-3′ and 5′-AGA AAG CTG GGT TTA CAT AGG CAC AGG-3′, and the primers used for β-actin were 5′-ACA GGT ATT GTG TTG GAT TC-3′ and 5′-TGT TGG AAG GTG CTG AGA G-3′. The tobacco β-actin gene was utilized to assess the RNA integrity. RT-PCR analysis was repeated three times, which showed representative data in the figures.

2.6. Nematicidal and Antifungal Activities of 3-Carene

To estimate the nematicidal activity of 3-carene, a test solution of 3-carene was diluted from 0.01 to 10 mg/mL concentration with distilled water containing Triton X-100 (0.5%). Each test solution was treated to approximately 200 nematodes in a 96-well plate. Distilled water–Triton X-100 solution without the test compound was used as a control. The 3-carene solution was treated to the PWNs for two days at 25 °C and then observed by a light microscope. The PWNs were considered as dead if they presented immobilization, even after being stimulated with a fine platinum wire. All treatments were repeated six times. Statistical analysis of nematicidal activity was performed by ANOVA with SPSS software.
Inhibition activity of 3-carene against B. cinerea was measured by an agar dilution method in PDA medium (Difco, Detroit, MI, USA) [28]. 3-Carene standard was mixed with ethyl alcohol and Tween 20 (Sigma Aldrich, St. Louis, MO, USA). The final concentration of each was 5%. Five concentrations of 3-carene (0.05, 0.1, 0.5, 1, and 1.5 mg/mL) were tested. Sterile distilled water was used instead of 3-carene solution in control. The PDA medium was cooled to a temperature of 40–45 °C, then aliquots of the mixture were added and poured into Petri dishes (2.5 cm diameter) [29,30]. Mycelial disc of B. cinerea (0.5 cm) was inoculated at the center of plastic Petri dishes as soon as the medium solidified. Petri dishes were maintained for seven days in the dark under a controlled temperature of 25 °C. The inhibition efficacy of the test solution was investigated by measuring the diameter of the fungal colonies, according to Fraternale et al. [28].

3. Results

3.1. Phytochemical Composition of Resin in P. densiflora

P. densiflora and P. koraiensis plants were observed at 10-day intervals after infection of PWNs. Lower leaves of P. densiflora and P. koraiensis exhibited disease symptoms (wilting with discoloration) after 20 days of PWN infection and showed obvious wilting symptom after 40 days. The two pine trees were completely withered after 70 days of PWN infection (Figure 1). The chemical compositions of resin after 20 days of PWN infection were analyzed by GC-MS in PWN-infected and non-infected P. densiflora plants. A total of eight monoterpenoids (α-pinene, camphene, β-pinene, β-myrcene, 3-carene, β-phellandrene, terpinolene, and L-pinocarveol), five sesquiterpenoids (α-cubebene, copaene, β-caryophyllene, β-cubebene, and δ-cadinene), and diterpenoids (isocembrol and resin acids) were identified in resin of P. densiflora. These compounds were identified by comparison with the mass spectral library of GC-MS (Figure 2). The most abundant compounds were diterpene resin acids followed by β-pinene, α-pinene, and β-phellandrene from P. densiflora. Among the substances investigated, 3-carene was the most markedly changed substance. It was faintly detected in the resin of P. densiflora without PWN infection, whereas the peak area of 3-carene was strongly enhanced (14.75 times) in PWN-infected pine tree (Table 1).

3.2. Volatile Compounds Analysis by HS-SPME-GC/MS

Volatile monoterpenes in pine plants are primarily emitted in response to external stimulation, such as a disease and wound [19,31]. A total of nine volatile compounds were identified in P. densiflora by HS-SPME-GC/MS analysis (Figure 3). The order of abundant monoterpene in P. densiflora without PWNs infection was α-pinene > β-pinene > β-phellandrene > camphene > β-myrcene > 3-carene (Table 2). Among them, five monoterpenes (α-pinene, β-pinene, β-myrcene, 3-carene, and β-phellandrene) were increased by at least two times in the PWN-infected P. densiflora compared with the control trees (Supplementary Table S1). Interestingly, the content of 3-carene was faintly detected in P. densiflora without PWN infection (0.79 μg/g), but highly enhanced (7.65 μg/g) after PWN infection.
Another susceptible pine species, P. koraiensis, had represented the different monoterpenes proportion composed of a total of nine volatile compounds (Figure 4). The order of major monoterpenes in P. koraiensis were in the order α-pinene > β-pinene > 3-carene. The amount of volatile monoterpene of P. koraiensiswas lower than P. densiflora (Table 2). The compositions of volatile monoterpene released from P. densiflora and P. koraiensis were similar, except β-phellandrene and D-limonene. The β-phellandrene was only identified in P. densiflora, but D-limonene was identified only in P. koraiensis (Figure 3 and Figure 4). The 3-carene content (183.27 μg/g, 54.7 times) of P. koraiensis was drastically enhanced at 20 days after PWN inoculation and showed the highest peak among the monoterpenoids (Figure 4). The retention time and fragmentation pattern of seven major compounds (α-pinene, camphene, β-pinene, β-myrcene, 3-carene, β-phellandrene, and D-limonene) were identical to those of authentic standards (Supplementary Figure S1). Overall, the emission of volatile monoterpenes was increased until 20 days from the sampling period and decreased afterward (data not shown).

3.3. Isolation of the 3-Carene Synthase in P. densiflora

Full-length cDNA of 3-carene synthase gene (Pd3-cars) was isolated from P. densiflora. The open reading frame (ORF) of Pd3-cars gene was 1881 bp and the deduced amino acid sequence (626 amino acids with a predicted molecular mass of 71.3 kDa) is 97% identical to that of 3-carene synthase in Lodgepole (P. contorta) and Jack pine (P. banksiana) (Supplementary Figure S2). A phylogenetic tree was constructed based on the deduced amino acid sequences with previously characterized conifer monoterpene synthase (Figure 5). The conifer monoterpene synthases were largely classified into two groups in phylogenetic analysis. Major components of one group were 3-carene, (-)-beta pinene, and (-)-alpha pinene, and another group included myrcene, (-)-beta phellandrene, and (+)-alpha pinene. In alpha pinene, the difference between (+)-alpha and (-)-alpha pinene was noticeable. 3-Carene synthase showed a closer relationship with (-)-alpha pinene than (+)-alpha pinene synthase. It is obvious that 3-carene synthase of P. densiflora is closely related to the other conifer 3-carene synthase and has a higher relationship with Pinus than Picea.

3.4. Expression of 3-Carene Synthase in Transgenic Tobacco

Transgenic tobacco plants overexpressing the Pd3-cars gene were obtained by Agrobacterium-mediated transformation (Figure 6a). Transgenic lines were selected by the resistance of kanamycin. In total, three lines of transgenic tobacco plant were obtained, and the integration of the Pd3-car gene into the tobacco genome was analyzed by genomic PCR. The three transgenic lines showed clearly amplified signals of the Pd3-car gene and nptII gene, but not in the wild-type tobacco (Figure 6b).
The transcription activities of the Pd3-car gene in transgenic tobacco were monitored by RT-PCR. Pd3-car gene and attB1-attB2 vector region were obviously transcribed in the transgenic lines, but not in the wild-type tobacco (Figure 6c). The highest expression of the Pd3-car gene was represented in the transgenic line 8 (Tr8).
The leaf volatile compounds of transgenic and non-transgenic plants were analyzed using SPME/GC-MS. Total ion chromatogram (TIC) indicated a new peak at a retention time of 6.48 min in transgenic plants (Figure 7a), but not in the wild-type tobacco. The retention time of the peak and its MS fragmentation pattern precisely matched those of the 3-carene standard (Figure 7b). The content of 3-carene was the largest at Tr8 (626.56 μg/g), which was consistent with the expression pattern of 3-carene synthase (Figure 7c).

3.5. Nematicidal and Antifungal Activities of 3-Carene

To measure nematicidal and antifungal activities of 3-carene, test solutions containing various concentrations of 3-carene were treated to PWNs for two days. No significant difference was found in PWNs with treatment from 0.01 to 1 mg/mL 3-carene concentration, but 35.25% of PWNs were immobilized at a 2 mg/mL concentration of 3-carene, and 45.98% of PWNs were immobilized at a 10 mg/mL concentration of 3-carene (Figure 8a).
The antifungal activity of 3-carene was measured in Botrytis cinerea, commonly used for propagation of PWN as a feed in the laboratory. Mycelial of B. cinerea in control was fully grown in Petri dish after seven days, whereas growth of the fungus in 3-carene treatment was restricted (Figure 8b). The treatment of 1.5 mg/mL 3-carene concentration inhibited 91% of the fungal growth compared with the control (Figure 8c). In the lowest concentration (0.05 mg/mL), the compound restricted the mycelial growth of B. cinerea at 36% compared with the control.

4. Discussion

The PWD symptoms after PWN infection in P. densiflora and P. koraiensis were investigated. The external symptoms (wilting leaves and discoloration) of pine tree had appeared after 20 days of PWN infection and showed clear wilting of needles after 40 days. Thus, there will be a lot of physiological and chemical changes at around 20 days after PWN infection. PWNs are used parenchyma cells in the resin canal as food sources [3]. PWN infection affected the chemical compositions of oleoresins [9]. We investigated the secreted resin in control and PWN-infected P. densiflora by the GC. The resin acids were highly increased in the resin of PWN-infected plants. 3-Carene is one of the monoterpenoids, which was undetected in control resin but somewhat prominently increased in the resin of PWN-infected plants.
The biosynthesis and emission of terpenoids are rearranged by the abiotic and biotic stress factors such as drought, temperature fluctuations, air and soil pollution, or pathogen attack [32,33]. The PWN migrates to the host tree when insect vectors (Monochamus spp.) feed on the young leaves of pine tree. The insect injury was associated with profile of volatile terpenes of the beetles’ host tree [33]. PWD after PWN infection occurs by interrupted water-conduction in sapwood by cavitation of tracheids [34]. The tracheid cavitation (embolism) is induced by a significant increase in volatile terpenes (monoterpenes) [35]. Gaspar et al. [36] reported that limonene and abietadiene were emitted by PWN-infected P. pinaster branches, and abietadiene emission in PWN-infected branches was interpreted as a result of tree defensive response. The chemical variability of volatile essential oil profiles was found in some pine species, but obvious changes of the essential oil chemicals were showed in the PWN inoculation area [37].
SPME has a number of advantages, including simplicity, high sensitivity, and a relatively non-invasive nature [38]. In HS-SPME/GC-MS analysis of volatile monoterpenes emitted from P. densiflora and P. koraiensis after PWN infection, emission of some monoterpenes was clearly enhanced in PWN-infected pine trees. The amount and proportion of monoterpenes were different between P. densiflora and P. koraiensis. β-Phellandrene and D-limonene were specifically detected in P. densiflora and P. koraiensis, respectively. The other five monoterpenes were commonly detected in both pine species, but the proportion of monoterpenes was different between the two species. Other studies also confirmed that three monoterpenes, α-pinene, β-myrcene, and β-phellandrene, from P. densiflora are emitted dominantly, depending on location or season in the forest of Korea [39,40,41] and Japan [42]. The major monoterpenes in P. koraiensis were α-pinene, camphene, β-myrcene, and D-limonene [43]. Son et al. [44] reported that the terpene emission rates of P. koraiensis were much lower than the other conifers. This result suggested that types and amounts of monoterpene emission are different among Pinus species.
Interestingly, 3-carene was highly correlated to PWN infection among the volatile compounds in P. densiflora and P. koraiensis. After PWN infection, 3-carene was increased clearly, but it was rarely detected without PWN infection in P. densiflora. In P. koraiensis, emission of 3-carene was remarkably enhanced (54.7 times in 20 days) after PWN infection. This result indicates that biosynthesis and emission of 3-carene may be involved in the reaction of PWN infection. It was reported that the specific ratio of monoterpenes influenced PWN behavior [45]. The monoterpenes α-pinene and β-pinene were reported as predominant constituents of Masson pine, whereas their relative amounts and proportions had changed with respect to the progression of PWD [45,46,47]. According to our continuous literature review, no report has been found on the change of 3-carene in pine plant after infection of PWNs. Nonetheless, a few reports expressed the role of 3-carene against insect resistance in pine species [15,16,17,18]. The amounts of 3-carene were associated with the tolerance of Sitka spruce against white pine weevil [15,16]. The high amounts of 3-carene were correlated with the resistance to Douglas-fir pitch moths in Lodgepole pine (Pinus contorta) [17]. In Scots pine (Pinus sylvestris), a high level of 3-carene was correlated with the survival of low larvae of Sawflies (Diprion pini) [18]. In the current study, 3-carene emission was enhanced markedly and detected as the major monoterpene after PWN infection in P. koraiensis. The selective enhance emission of 3-carene might have a relation to the chemical response and/or chemical defense against PWN infection.
Previous studies found that the relative expressions of α-pinene synthase in P. pinea were distinctly enhanced by PWN infection, but no difference was observed between control and PWN infection in P. pinaster [20]. Two terpene synthase genes, α-pinene and longifolene synthase, in P. massoniana, were involved in the oleoresin defense strategy to PWD-resistant Masson pines. Upregulation of these genes in the stem supported their involvement in terpene biosynthesis as part of the defense against PWNs [48]. 3-Carene was characterized enzymatically in some conifer species (Picea abies, Picea sitchensis, P. bankiana, and P. contorta) [14,22]. However, there are no reports on the characterization of monoterpene synthase in P. densiflora and P. koraiensis. 3-Carene synthase gene was isolated from P. densiflora and functionally characterized by tobacco transformation. Transgenic tobacco overexpressing gene (Pd3-car) was isolated from P. densiflora and showed 3-carene production in the transgenic tobacco lines.
Extensive evidence has demonstrated the nematicidal activity of monoterpenoids and essential oils against PWN, although such compounds are not derived from pine species [49,50,51]. Choi et al. [51] reported that thymol and carvacrol have a strong nematicidal activity to juvenile PWN with the LC50 values of 0.096 and 0.099 mg/mL, respectively. Natural ester compounds (isobutyl angelate, 2-methylbutyl angelate, 2-methylbutyl isobutanoate, and linalyl acetate) have been reported for nematicidal activities in the Asteraceae plant essential oils [52]. 3-Carene was also reported to have weak nematicidal activity against B. xylophilus [53].
In one such study, α-pinene, 3-carene, and D-limonene were identified as major constituents in the essential oil of Angelica archangelica roots, which showed antifungal activity against 10 plant pathogenic fungi [26]. Although α-pinene was reported for strong antifungal activity, this compound has no nematicidal activity and even attracts insect vectors of PWD [54,55,56]. In our current experiment, a mild nematicidal activity was exhibited against PWNs by treating 3-carene. However, 3-carene was found as a highly responded compound to P. densiflora and P. koraiensis after PWN infection, but it exhibited strong antifungal properties.

5. Conclusions

In the present work, we investigated resin terpenoid content and the monoterpene emission in P. densiflora and P. koraiensis after PWNs infection. 3-Carene emission was strongly enhanced after PWN infection in the two pine trees. The Pd3-cars gene in P. densiflora was functionally characterized by tobacco transformation. The 3-carene compound showed strong antifungal and weak nematicidal activities. An early diagnosis of PWD is important to reduce the damage in the pine forest. The enhanced emission of volatile 3-carene from P. densiflora and P. koraiensis may be applied to early diagnosis of PWD in pine trees in a non-destructive manner.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/f12050514/s1, Figure S1: Mass fragments of monoterpene peaks detected in volatile organic compounds from Pinus densiflora, P. koraiensis, and those of authentic standards, Figure S2: Amino acid sequences of 3-carene synthase in Pinus and Picea species, Table S1: HS-SPME/GC-MS analysis of comparative peak area of volatile organic compounds in control and pine wood nematode-infected Pinus densiflora and P. koraiensis.

Author Contributions

Y.-E.C. designed the research, and both H.-S.H. and Y.-E.C. wrote the paper. J.-Y.H. performed the analysis of resin compounds by GC/MS. and H.-S.H. performed the analysis of RT-PCR and genetic transformation. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Research Foundation of Korea funded by the Ministry of Education (NRF-2020R1A6A3A01096503), and by R&D Program for Forest Science Technology (Project No. 2021339A00-2123-CD02) provided by Korea Forest Service (Korea Forestry Promotion Institute).

Data Availability Statement

Data available in a publicly accessible repository.

Acknowledgments

We would like to thank M. Saleh-E-In at the Kangwon National University for his assistance with the English language and grammatical editing of the manuscript. We also thank the Forest Research Institute of Gangwon province for providing the pine wood nematode and Botrytis cinerea used in experimental trials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Time-lapsed changes of Pinus densiflora and P. koraiensis after infection of pine wood nematode (PWN): (a) P. densiflora 0, 20, 40, and 70 days after PWN infection, and (b) P. koraiensis 0, 20, 40, and 70 days after PWN infection.
Figure 1. Time-lapsed changes of Pinus densiflora and P. koraiensis after infection of pine wood nematode (PWN): (a) P. densiflora 0, 20, 40, and 70 days after PWN infection, and (b) P. koraiensis 0, 20, 40, and 70 days after PWN infection.
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Figure 2. GC-MS analysis of terpenoids in resin obtained from pine wood nematode (PWN)-infected and non-infected Pinus densiflora plants: (a) Resin compounds of control P. densiflora, and (b) resin compounds of PWN-infected P. densiflora. 1. α-pinene, 2. camphene, 3. β-pinene, 4. β-myrcene, 5. 3-carene, 6. β-phellandrene, 7. terpinolene, 8. L-pinocarveol, 9. methylthymylether, 10. acetic acid, 11. α-cubebene, 12. copaene, 13. β-caryophyllene, 14. β-cubebene, 15. δ-cadinene, 16. isocembrol, 17. resin acids.
Figure 2. GC-MS analysis of terpenoids in resin obtained from pine wood nematode (PWN)-infected and non-infected Pinus densiflora plants: (a) Resin compounds of control P. densiflora, and (b) resin compounds of PWN-infected P. densiflora. 1. α-pinene, 2. camphene, 3. β-pinene, 4. β-myrcene, 5. 3-carene, 6. β-phellandrene, 7. terpinolene, 8. L-pinocarveol, 9. methylthymylether, 10. acetic acid, 11. α-cubebene, 12. copaene, 13. β-caryophyllene, 14. β-cubebene, 15. δ-cadinene, 16. isocembrol, 17. resin acids.
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Figure 3. HS-SPME/GC-MS analysis of volatile organic compounds in pine wood nematode (PWN)-infected and non-infected Pinus densiflora plants: (a) Monoterpenes in total ion chromatograms (TIC) of non-infected P. densiflora, (b) monoterpenes in TIC chromatograms of P. densiflora after PWN infection, and (c) chromatograms of monoterpenes authentic standards. 1. oxime-methoxy-phenyl, 2. tricyclene 3. α-pinene, 4. camphene, 5 β-pinene, 6. β-myrcene, 7. 3-carene, 8. p-cymol, 9. β-phellandrene.
Figure 3. HS-SPME/GC-MS analysis of volatile organic compounds in pine wood nematode (PWN)-infected and non-infected Pinus densiflora plants: (a) Monoterpenes in total ion chromatograms (TIC) of non-infected P. densiflora, (b) monoterpenes in TIC chromatograms of P. densiflora after PWN infection, and (c) chromatograms of monoterpenes authentic standards. 1. oxime-methoxy-phenyl, 2. tricyclene 3. α-pinene, 4. camphene, 5 β-pinene, 6. β-myrcene, 7. 3-carene, 8. p-cymol, 9. β-phellandrene.
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Figure 4. HS-SPME/GC-MS analysis of volatile organic compounds in pine wood nematode (PWN)-infected and non-infected Pinus koraiensis plants: (a) GC chromatograms of volatile monoterpenes in control P. koraiensis, (b) GC chromatograms of volatile monoterpenes in P. koraiensis after PWN infection, and (c) chromatograms of volatile monoterpenes authentic standards. 1. oxime-methoxy-phenyl, 2. tricyclene 3. α-pinene, 4. camphene, 5 β-pinene, 6. β-myrcene, 7. 3-carene, 8. p-cymol, 10. D-limonene.
Figure 4. HS-SPME/GC-MS analysis of volatile organic compounds in pine wood nematode (PWN)-infected and non-infected Pinus koraiensis plants: (a) GC chromatograms of volatile monoterpenes in control P. koraiensis, (b) GC chromatograms of volatile monoterpenes in P. koraiensis after PWN infection, and (c) chromatograms of volatile monoterpenes authentic standards. 1. oxime-methoxy-phenyl, 2. tricyclene 3. α-pinene, 4. camphene, 5 β-pinene, 6. β-myrcene, 7. 3-carene, 8. p-cymol, 10. D-limonene.
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Figure 5. Phylogeny of 3-carene synthase from Pinus densiflora with previously characterized conifer monoterpene synthases based on amino acid sequences. Ag, Abies grandis; Pa, Picea abies; Pb, Pinus banksiana; Pc, Pinus contorta; Pd, Pinus densiflora; Pm, Pseudotsuga menziesii; Ps, Picea sitchensis; Pt, Pinus taeda.
Figure 5. Phylogeny of 3-carene synthase from Pinus densiflora with previously characterized conifer monoterpene synthases based on amino acid sequences. Ag, Abies grandis; Pa, Picea abies; Pb, Pinus banksiana; Pc, Pinus contorta; Pd, Pinus densiflora; Pm, Pseudotsuga menziesii; Ps, Picea sitchensis; Pt, Pinus taeda.
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Figure 6. Construction of transgenic tobacco plants overexpressing Pd3-car gene and RT-PCR analysis: (a) Schematic diagram of the Pd3-car gene overexpression vector plasmid under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. LB represents the left border and RB represents the right border of the T-DNA. NPT, kanamycin resistance gene; P35S, CaMV 35S promoter; 3′35S, CaMV 35S terminator. (b) Genomic PCR of wild-type and transgenic lines overexpressing the Pd3-car gene. PCR products of genomic DNA extracted from the wild-type tobacco and three transgenic lines followed by screening with Pd3-car and nptII gene primers. (c) Expression of the introduced Pd3-car gene and attB1-attB2 vector region by RT-PCR. β-actin was used as a loading control. M = marker (1 kb), Wt = wild type, Tr = selected transgenic tobacco lines.
Figure 6. Construction of transgenic tobacco plants overexpressing Pd3-car gene and RT-PCR analysis: (a) Schematic diagram of the Pd3-car gene overexpression vector plasmid under the control of the Cauliflower mosaic virus (CaMV) 35S promoter. LB represents the left border and RB represents the right border of the T-DNA. NPT, kanamycin resistance gene; P35S, CaMV 35S promoter; 3′35S, CaMV 35S terminator. (b) Genomic PCR of wild-type and transgenic lines overexpressing the Pd3-car gene. PCR products of genomic DNA extracted from the wild-type tobacco and three transgenic lines followed by screening with Pd3-car and nptII gene primers. (c) Expression of the introduced Pd3-car gene and attB1-attB2 vector region by RT-PCR. β-actin was used as a loading control. M = marker (1 kb), Wt = wild type, Tr = selected transgenic tobacco lines.
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Figure 7. Production of 3-carene from wild-type and transgenic tobacco leaves expressing the Pd3-car gene: (a) SPME/GC-MS chromatography of tobacco leaf volatile compound from wild-type and transgenic plants. (b) Mass fraction of the peak in the transgenic plant (line Tr-8) overexpressing the Pd3-car gene and authentic standard. (c) Content of 3-carene in the transgenic line. The data are shown as the means ± standard error obtained from three independent plants.
Figure 7. Production of 3-carene from wild-type and transgenic tobacco leaves expressing the Pd3-car gene: (a) SPME/GC-MS chromatography of tobacco leaf volatile compound from wild-type and transgenic plants. (b) Mass fraction of the peak in the transgenic plant (line Tr-8) overexpressing the Pd3-car gene and authentic standard. (c) Content of 3-carene in the transgenic line. The data are shown as the means ± standard error obtained from three independent plants.
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Figure 8. Nematicidal and antifungal activities of 3-carene: (a) Nematicidal activity of 3-carene against Bursaphelenchus xylophilus, (b) photograph of Botrytis cinerea on potato dextrose agar (PDA) medium with 3-carene test solution, and (c) antifungal activity of 3-carene against B. cinerea.
Figure 8. Nematicidal and antifungal activities of 3-carene: (a) Nematicidal activity of 3-carene against Bursaphelenchus xylophilus, (b) photograph of Botrytis cinerea on potato dextrose agar (PDA) medium with 3-carene test solution, and (c) antifungal activity of 3-carene against B. cinerea.
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Table
Table 1. The comparative peak area of resin compounds of control and pine wood nematode-infected Pinus densiflora.
Table 1. The comparative peak area of resin compounds of control and pine wood nematode-infected Pinus densiflora.
PeakCompoundClassificationRetention Time (min)Peak Area × 10−7
Control Tree (%)Infected Tree (%)
1α-pineneMonoterpene4.416.32 ± 0.07 (100)5.94 ± 0.16 (94.00)
2campheneMonoterpene4.700.33 ± 0.05 (100)0.53 ± 0.03 (160.61)
3β-pineneMonoterpene5.4712.89 ± 0.51 (100)9.83 ± 0.01 (76.26)
4β-myrceneMonoterpene5.772.65 ± 0.57 (100)0.55 ± 0.06 (20.75)
53-careneMonoterpene6.040.08 ± 0.02 (100)1.18 ± 0.15 (1475.00)
6β-phellandreneMonoterpene6.813.14 ± 0.88 (100)6.87 ± 0.03 (218.79)
7terpinoleneMonoterpene8.390.93 ± 0.20 (100)1.52 ± 0.04 (163.44)
8L-pinocarveolMonoterpene9.881.30 ± 0.00 (100)1.57 ± 0.02 (120.77)
9methylthymylether-12.650.35 ± 0.06 (100)0.51 ± 0.01 (145.71)
10acetic acid-14.060.71 ± 0.20 (100)1.69 ± 0.19 (238.02)
11α-cubebeneSesquiterpene15.770.30 ± 0.01 (100)0.58 ± 0.08 (193.33)
12copaeneSesquiterpene16.160.26 ± 0.04 (100)0.70 ± 0.02 (269.23)
13β-caryophylleneSesquiterpene17.630.71 ± 0.14 (100)1.17 ± 0.08 (164.79)
14β-cubebene Sesquiterpene19.170.60 ± 0.05 (100)2.17 ± 0.23 (361.67)
15δ-cadineneSesquiterpene20.190.15 ± 0.03 (100)0.55 ± 0.02 (366.67)
16isocembrolDiterpene31.271.29 ± 0.06 (100)4.87 ± 1.03 (377.52)
17resin acidsDiterpene42.1554.30 ± 1.12 (100)64.27 ± 1.66 (118.36)
Table
Table 2. Content of major monoterpene in volatile compounds of control and pine wood nematode-infected Pinus densiflora and P. koraiensis.
Table 2. Content of major monoterpene in volatile compounds of control and pine wood nematode-infected Pinus densiflora and P. koraiensis.
CompoundP. densifloraP. koraiensis (μg/g)
ControlInfected TreeControlInfected Tree
α-pinene134.47 ± 16.77 269.33 ± 24.14 10.14 ± 1.28 171.84 ± 55.02
camphene22.97 ± 10.53 18.16 ± 0.48 2.52 ± 0.05 27.86 ± 4.06
β-pinene48.40 ± 8.17 141.83 ± 30.78 3.84 ± 1.50 51.37 ± 20.57
β-myrcene2.99 ± 1.45 7.59 ± 1.50 1.16 ± 0.45 9.21 ± 1.04
3-carene0.79 ± 0.09 7.65 ± 2.63 3.34 ± 0.07 183.27 ± 48.99
β-phellandrene24.83 ± 12.09 95.14 ± 26.64——
D-limonene——1.27 ± 0.5036.76 ± 12.45
— indicates not detected.
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Hwang, H.-S.; Han, J.-Y.; Choi, Y.-E. Enhanced Emission of Monoterpene 3-Carene in Pinus densiflora Infected by Pine Wood Nematode and Characterization of 3-Carene Synthase. Forests 2021, 12, 514. https://doi.org/10.3390/f12050514

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Hwang H-S, Han J-Y, Choi Y-E. Enhanced Emission of Monoterpene 3-Carene in Pinus densiflora Infected by Pine Wood Nematode and Characterization of 3-Carene Synthase. Forests. 2021; 12(5):514. https://doi.org/10.3390/f12050514

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Hwang, Hwan-Su, Jung-Yeon Han, and Yong-Eui Choi. 2021. "Enhanced Emission of Monoterpene 3-Carene in Pinus densiflora Infected by Pine Wood Nematode and Characterization of 3-Carene Synthase" Forests 12, no. 5: 514. https://doi.org/10.3390/f12050514

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