Yield of exudate

Exudates of Pinaceae and C. macrolepis were light-yellow liquid, Araucariaceae and genus Cunninghamia were milky white liquid, genus Chamaecyparis and L. formosana were light-yellow solid. Exudates of Araucariaceae, genus Cunninghamia and Pinus could be collected more than 1000 mg/day, Cupressaceae and P. wilsoniana were 300–500 mg/day.

Volatile compounds identification in exudates

Table 2 shows the analysis results of volatiles emitted from the conifer exudates. Although contents and compositions of volatiles were various in different conifers, the dominant compounds were monoterpenoids and sesquiterpenoids. Based on the results obtained in this study, the most abundant volatiles in conifer exudates were α-pinene, β-ocimene, β-pinene, sabinene, and caryophyllene. The abundant volatiles in Pinaceae were α-pinene and sabinene; however, α-pinene and β-pinene were the abundant compounds in Cupressaceae. Regarding Araucariaceae, the molecular weight of volatiles emitted from A. heterophylla was higher than others; most of them were sesquiterpenoids. However, the dominant volatiles in A. cunninghamii and A. dammara were α-pinene and β-ocimene. Caryophyllene was abundant in genus Cunninghamia. Multivariate statistical analysis was performed to compare the degrees of similarity of the volatile composition from conifer (Fig. 2). Exception was C. obtusa, which was classified into the Araucaria group by volatiles, because it had higher β-ocimene. Results from other tree species analysis are consistent with the traditional (morphology) taxonomy results. The results obtained from this study show that chemotaxonomy has considerable reference value.

Table 2 The volatile organic compounds of exudates
Fig. 2
figure 2

Epigenetics of conifers relationship of 13 conifers grown in Taiwan by GC–MS analysis of volatile constituents from exudates

Identification of nonvolatile compounds in exudates

To understand the skeletons of the main constituents in the exudates, four conifers, including A. cunninghamii, C. macrolepis, C. formosensis, and P. taiwanensis., with higher among of exudates were selected for the separation and identification of the compounds. After spectral analysis, 22 compounds (Fig. 3) were identified from exudates of A. cunninghamii, C. macrolepis, C. formosensis, and P. taiwanensis. All of these compounds of exudates were diterpenoids, including isocupressic acid (1), acetyl isocupressic acid (2), 15-hydroxy-8,13-labdadien (3) [42], 15-hydroxy-8,13-labdadien-19-carbonsaeure (4), 15-acetoxy-8,13-labdadien-19-oic acid (5), 8,13-labdadien-15,19-diol (6), 15-hydroxy-8,13-labdadien-19-ol (7), 15-hydroxy-8,13-labdadien-19-al (8), 15-acetoxy-8,13-labdadien-19-al (9) [43], ferruginol (10) [44], 6α-hydroxysugiol (11) [45], trans-communic acid (12) [44], isopimarol (13) [46], agathadiol (14) [44], 13-epi-cupressic acid (15) [47], 8,15-isopimaradien-19-al (16), 8,15-isopimaradien-19-oic acid (17), 8,15-isopimaradien-19-ol (18) [48], trans-communal (19), trans-communol (20), dehydroabietic acid (21) [44], isopimaric acid (22), respectively. The 1H-NMR spectra of compounds 1 to 11 are shown in Additional file 1: Figs. S1–S22. Regarding the skeletons of the compounds identified from exudates, they could be classified into three types, namely abietane-, labdane-, and pimarane-types. The compound 19 were isolated from the exudates of A.c (Araucariaceae); compounds 1013 were isolated from the exudate of C. macrolepis (Cupressaceae); compounds 1, 2, 5, 14, and 15 were isolated from the exudate of C. formosensis (Cupressaceae); compounds 12 and 1620 were isolated from the exudate of C. lanceolate (Cupressaceae); compounds 21 and 22 isolated from the exudate of P. taiwanensis (Pinaceae).

Fig. 3
figure 3

Compounds identified from exudates of conifers in this study

HSQC analysis of exudates of conifer

The main compounds of conifer exudates are diterpenoids [3,4,5,6,7], and most of them belong to abietane-, labdane-, and pimarane-types diterpenoids. The diterpenoids of the above three types of skeletons have their own special signals in the NMR spectrum. For the abietane-type diterpenoid, the NMR signals are at δC 120–150 ppm and δH 6.0–8.0 ppm, represented the carbon and proton characteristic signals at benzene ring; and signals at δC 20–30 ppm and δH 3.0–3.5 ppm is an isopropyl absorption peaks at C15 of the benzene ring. According to the above information, special cross-peak signals of abietane-type in HSQC spectrum were at M3-4, N3-4 or O3-4 coupled with C7. Consideration of labdane-type diterpenoids, two double bonds are at C-8 and C-13, which NMR signals were at δC 100–150 ppm and δH 4.0–7.0 ppm. According to the above information, cross-peak signals of labdane-type were shown at E-F6 coupled with L-M5 or N5 coupled with O4, and sometimes the signals were presented at K-L6. The third skeleton, pimarane-type diterpenoids, an end double bond is at C-16, the NMR signal are at δC 100–120, 140–150 ppm and δH 4.0–6.0 ppm. According to the above information, characteristic cross-peak signals of pimarane-type diterpenoids are at K-L6 coupled with O5.

The HSQC analysis spectra are shown in Additional file 1: Figs. S23–S36. The results revealed that the exudates of P. elliottii, P. insularis, P. morrisonicola, P. taiwanensis, P. wilsoniana, C. obtusa, C. formosensis, and C. macrolepis contained the abietane-type diterpenoids; the exudates of P. elliottii, P. insularis, C. obtusa, C. formosensis, C. macrolepis, C. lanceolate, C. konishii, A. cunninghamii, A. heterophylla, and A. dammara contained the labdane-type diterpenoids; the exudates of P. elliottii, P. insularis, P. taiwanensis, C. obtuse, C. formosensis, C. macrolepis, C. lanceolate, C. konishii, A. cunninghamii and A. heterophylla contained the pimarane-type diterpenoids. Moreover, cross peaks in the HSQC spectra of exudates from 13 conifers were the useful chemotaxonomic index for conifers classification. The cross peaks at C8, D8, K6, M3, M4, and M5 were presented in family Pinaceae (Fig. 4); H3, K4, K6, M5, N3, N5, O4, and O5 were in Cupressaceae (Fig. 5); and C8, F7, K6, L5, L6, M5, N4, N5, and O4 were presented in Araucariaceae (Fig. 6). PCA and cluster analysis were performed to detect the degrees of similarity of the compositions of the exudates analyzed. Three different groups can be identified in the loading plots of PCA 1 and PCA 2 (Fig. 7), i.e., Pinaceae (blue triangle), Cupressaceae (green inverted triangle), and Araucariaceae (light blue square); an angiosperm species, L. formosana (Hamamelidaceae), was used as an outer group. Figure 8 shows the results of cluster analysis, it is obviously that all Pinus species were closer than others. Both species of Araucaria were similarly, and they were close to A. dammara, all of them are Araucariaceae. C. obtusa, C. formosensis, and C. macrolepis. were very close to each other. C. lanceolate and C. konishii were similarly, but they were closer to Araucariaceae than C. obtusa, C. formosensis, and C. macrolepis. (Cupressaceae). It might be the abietane type of diterpenoids were in C. obtusa, C. formosensis, C. macrolepis, but not in C. lanceolate, C. konishii. Besides, only P. elliottii and P. insularis (Pinaceae) had labdane-type diterpenoids, both of them were similar in cluster analysis.

Fig. 4
figure 4

Cross-peaks region of Pinaceae exudate in HSQC spectrum

Fig. 5
figure 5

Cross-peaks region of Cupressaceae exudate in HSQC spectrum

Fig. 6
figure 6

Cross-peaks region of HSQC Araucariaceae exudate in HSQC spectrum

Fig. 7
figure 7

PCA score plot based on signal regions of HSQC and grouped by families

Fig. 8
figure 8

Epigenetics of conifers relationship of 13 conifers grown in Taiwan by HSQC analysis of exudates

Evaluation of antifungal activities

In the growth test of the white-rot fungus, T. versicolor, the mycelial growth period was 7 days after the inoculum. As the results show in Table 3, C. obtusa, P. taiwanensis, and P. elliottii exhibited the stronger antifungal activity. On the other hand, the exudates of P. wilsoniana, P. elliottii, and C. formosensis presented better antifungal activity against L. sulphureus (brown-rot fungus) than others. The results indicated Pinaceae and Cupressaceae had better antifungal activities, and according to HSQC analysis this two families had abietane-type diterpenoids, but not in others. The conclusion that abietane-type diterpenoids have good antifungal activities is supported by some studies [49,50,51,52].

Table 3 Antifungal index of exudates of conifers grown in Taiwan

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