1. Introduction
Sawflies of the family Diprionidae (Hymenoptera, Symphyta) belong to the most important defoliators of pines, Pinus spp., over a large part of the northern hemisphere [1]. In many countries, notably in Central Europe and China, outbreaks have regularly been controlled by chemical insecticides. Attempts to develop environmentally more friendly control methods have included investigations of diprionid sex pheromones. Moreover, from basic science point of view, the chemical communication in this insect group has received some attention. Diprionid females attract males by pheromones that consist of a unique series of methyl-branched long chain esters, with two to four stereogenic centers [2,3]. Although many stereoisomers of the pheromone can exist, it appears that several species use the same stereoisomers as attractant [2,4]. This led to investigations about species recognition and reproductive isolation, especially among the North American diprionids [5,6].
Until recently only few diprionid species from the Far East were known, but during the last three decades, several new taxa have been described from China [7] and Korea [8]. Many of these are considered severe pests and the target of the present study, Diprion jingyuanensis Xiao and Zhang, is no exception [9]. Large areas planted with Pinus tabulaeformis Carr. in the provinces of Shanxi, Gansu, and Inner Mongolia, P. R. China, have been defoliated by this sawfly since the 1990s, around 10,000 to 200,000 ha per year [10,11] (Biological Disaster Prevention and Control Center, National Forestry and Grassland Administration, P.R. China, unpublished). It has one generation per year and overwinters as prepupae in cocoons in the litter or topsoil beneath its host trees. Adults emerge from late May to early August, with at least one flight peak occurring during mid to late June [12,13]. Calling of virgin females and male responses to traps baited with virgin females occur during the daytime, with a major peak at 9:00–13:00 and a minor peak around 17:00–19:00 [14].
In a first attempt to find an attractant for D. jingyuanensis, several earlier described diprionid pheromone substances were used in a field screening test [14]. It was found that the propionate, but not the acetate, of (2S,3R,7R)-3,7-dimethyl-2-tridecanol attracted males. The acetate and propionate of diprionol (3,7-dimethyl-2-pentadecanol), both as eight-isomer mixtures and as pure (2S,3S,7S)-isomer, were found to be inactive, as was the acetate of (2S,3R,7R)-3,7-dimethyl-pentadecanol. Thus, it was concluded that the propionate of (2S,3R,7R)-3,7-dimethyl-2-tridecanol might be a component of the D. jingyuanensis sex pheromone. The same compound was earlier identified from Diprion pini [15,16]. Traps baited with a four-threo-isomer mixture of 3,7-dimethyl-2-tridecanol propionate (1 to 2 mg/trap) were also attractive to D. jingyuanensis males and have been used to monitor the sawfly adult flight activity and its population dynamics since the late 1990s [12]. We herein report further work to identify the pheromone of D. jingyuanensis, including chemical analysis, electrophysiology, and field tests. Carbon numbers are from here on omitted before the chiral identity.
2. Materials and Methods
2.1. Insects, Extraction, and Liquid Chromatography
Insects were collected as cocoons in the litter in plantations of Pinus tabulaeformis situated in Qingyuan County, Shanxi Province, P.R. China, and stored at room temperature until they started to emerge. When emergence started, male cocoons were put in a refrigerator to delay development and were taken out when needed for electrophysiological studies. Whole bodies of females were extracted in ethyl acetate for 72 h at room temperature—43 females for identification and 118 females for chiral analyses. Initially, the extract was purified by liquid chromatography (LC) on Chromabond (SiOH) columns [16].
2.2. Chemicals
The blends of isomers and samples of highly pure single stereoisomers were synthesized at Mid Sweden University as described by Bergström et al. [16]. The chemical and stereochemical purity of each compound used in the present study is listed in Table 1, and their structural formulas presented in Figure 1.
2.3. Gas Chromatography with Mass Spectrometry Detection (GC-MS)
Analyses of the isolated fractions 7 and 8 from liquid chromatography were performed by combined gas chromatography and mass spectrometry (GC-MS) on a Hewlett-Packard GC-5890 gas chromatograph coupled with a Finnigan TSQ -700 quadrupole mass spectrometer in electron impact (EI) mode. The gas chromatograph was fitted with an analytical fused silica capillary column (30 m × 0.25 mm I.D.) coated with a 0.20 µm layer of CP-WAX 58 (OV-351) as a stationary phase. Helium was used as carrier gas at a flow of 25 cm/s. After sample injection, the temperature was held constant at 50 °C for 5 min, then raised 10 °C/min to 220 °C, and then isothermal 20 min. Injector and transfer line temperatures were 220 °C and 240 °C, respectively.
2.4. Gas Chromatography with Electron Capture Detection (GC-ECD)
Gas chromatographic separations of the natural stereoisomers as well as synthetic 3,7-dimethyl-2-tridecanol as pentafluorobenzoate derivatives were carried out on a HP Agilent 6890 gas chromatograph equipped with an electron capture detector (ECD). A fused silica capillary column (50 m × 0.25 mm I.D.) was coated with the stationary phase CP-Sil-88, df = 0.20 µm [17].
2.5. Electrophysiology
In the electroantennographic (EAG) recordings, antennae were cut off from heads of live pine sawfly males, and each was placed between two silver wire-glass capillary electrodes filled with Beadle-Ephrussi Ringer solution. The antennal base was in connection with the ground electrode, whereas the antennal tip was inserted into the recording electrode that was connected to a high-impedance amplifier. The antennal signals were acquired and stored on a PC equipped with a serial IDAC interface box (Syntech, Hilversum, the Netherlands). The antennal preparation was held 1 cm in front of a humidified and charcoal filtered air stream flowing at 0.5 m/s. The stimulus was injected into the air stream through a hole in the glass tube 20 cm upstream of the antenna. A stimulus was created by a stimulus controller (CS-05; Syntech, Hilversum, the Netherlands), which delivered the stimulus in a 0.5 s puff at a flow rate of 5 mL/s. The stimulus was made of a Pasteur pipette containing a piece of filter paper. Different dosages of the candidate synthetic compounds were diluted in cyclohexane, and then each was added to the filter paper and the solvent was allowed to evaporate. A blank stimulus was prepared using only the solvent. During the recordings, stimulation with a standard stimulus (0.01 μg of the propionate ester of (2S,3R,7R)-3,7-dimethyl-2-tridecanol) was done every third time, including the first and the last one. The EAG data were analyzed using the ‘EAG version 2.2a’ Software (Syntech). Since no pine sawfly species has been shown to use a sex pheromone having a ‘2R’ configuration [2], the EAG recordings were concentrated to the four non-‘2R’ stereoisomers: propionate esters of SRR-, SRS-, SSS-, and SSR-3,7-dimethyl-2-tridecanol, plus the acetate of the SRR-isomer (a pheromone component of D. pini) and the propionate of SRR-3,7-dimethyl-2-pentadecanol (diprionol) (a pheromone component of D. similis).
2.6. Field Experiments
Field tests were made in the same pine plantations as the cocoon sampling took place from 1996 to 1998. The plantations were about 35 years old, and the pine trees were approximately 5 m high with a diameter at breast height of 12 cm. The area is situated 1000–1200 m above sea level. Lund-I cardboard sticky traps [18] were used. Traps within each test set were hung in a line from pine trees (about 1.5 m above ground and at least 30 m apart) with their initial positions randomized. In order to minimize any positional effects, the trap positions within the same set were re-randomized when the traps were checked.
Synthetic pheromone candidates (individuals or blends) were diluted with a known amount of hexane or cyclohexane, and then applied to 1 × 4 cm dental cotton rolls (Celluron® No. 2, Paul Hartmann AG, Heidenheim, Germany) as dispensers. The release rates of the tested compounds decrease exponentially with this type of dispenser [19], but the proportion among stereoisomers should remain constant. Tethered live virgin females, 1–3 d old and one per trap, were also included in the 1996 tests as active controls.
2.7. Statistical Analysis
The EAG data were normalized relative to the average EAG response to the standard stimulus (0.01 μg of the propionate ester of SRR-3,7-dimethyl-2-tridecanol) (% of STD; Figure 2). The normalized EAG data were then analyzed by one-way ANOVA followed by Tukey’s test at α = 0.05. Trap catch data were transformed by log(catch + 1) to improve normality and homogeneity of variances for ANOVA. Means were compared by one-way ANOVA followed by Tukey’s HSD procedure at α = 0.05. Analysis of trap catches from tests performed in 1998 was done by the G-test, due to few replicates (n = 3).
3. Results
3.1. Chemical Analysis
In the female extract, both erythro- and threo-3,7-dimethyl-2-tridecanol were identified using GC retention time and mass spectra for the compounds of interest. For the identification, we used two synthetic reference-mixtures, one consisting of the four stereoisomers of erythro-3,7-dimethyl-2-tridecanol and the second of the four stereoisomers of threo-3,7-dimethyl-2-tridecanol. The ratio of erythro/threo isomers was approximately 1:3 when analyzing the female extract. The two well-resolved peaks in the GC-MS analysis showed that the first eluting peak represents one or more of the erythro-stereoisomers (SSS, RRR, SSR, RRS) and the later eluting peak represents one or more of the threo-stereoisomers (SRR, RSS, SRS, RSR). Although we did not use a tandem system in our analysis, the eluting order of erythro- and threo-stereoisomers was suggested to be the same as for the GC-separation of erythro- and threo-3,7-dimethyl-2-pentadecanol in Högberg et al. [20]. In the GC-analysis, with ECD detector, the two well-resolved peaks had a 1:2 ratio when analyzing the female extract. The first eluting peak now probably represents one or more of the erythro- and threo-isomers: SSS, RRR, SSR, RRS, SRS, RSR. Thus, the second peak now probably represents one or two of the threo-isomers, SRR or RSS. Moreover, in this analysis, the eluting order of erythro- and threo-stereoisomers was suggested to be the same as for the GC-separation of erythro- and threo-3,7-dimethyl-2-pentadecanol in [20]. Our suggestion of which stereoisomers that would be represented by the first and second peaks may, however, not fully explain why the ratio of erythro/threo isomers was 1:3 in the first analysis (GC-MS) and 1:2 in the second (GC-ECD). However, either or both of the two strongly EAG-active threo-isomers, SRR and SRS (see below for details), likely existed in the female extracts.
3.2. Electrophysiology
Two of the propionate threo-isomers of 3,7-dimethyl-2-tridecanol, namely, SRS and SRR, induced the strongest responses from male antennae during the EAG recordings (Figure 2). At the highest tested dose (1 µg), the SRR- and the SRS-isomers elicited significantly higher responses in the male antennae than did the remaining compounds. The SRR-propionate of diprionol (a pheromone component of D. similis) exhibited the third highest antennal activity, whereas the remaining two isomers (propionate esters of SSS- and SSR-3,7-dimethyl-2-tridecanol) plus the acetate of the SRR-isomer (one of the two pheromone components of D. pini) did not elicit any significant increase in antennal response (Figure 2).
3.3. Field Experiments
In the field test in 1996, the propionate threo-blend of 3,7-dimethyl-2-tridecanol was compared with the SRR-isomer alone. The per-isomer amount was kept constant. The SRR-isomer alone at low dosages was inactive and was weakly (but significantly) attractive at the highest dose (1000 µg loading) in at least one set-up (along the stand margin). The threo-blend caught significantly more (>5 times more) males than did the SRR-isomer alone at the highest dose in both set-ups and at the second highest dose in one set-up (Table 2). The trap baited with a virgin female caught approximately as much as the strongest threo-lure, but significantly more males than the single SRR-isomer (Table 2). The dose–response relationship showed that approximately 400 µg of the threo-blend was needed to attract males in 1996 (Table 2) but only around 100 µg in 1997 (Table 3).
The two isomers showing the highest EAG activity, SRS and SRR, were tested alone and combined and with the threo-blend. The SRR-isomer alone was only weakly attractive at 100 µg, whereas the SRS-isomer alone at the same loading was 8–14 times more attractive to D. jingyuanensis males than was the SRR-isomer alone (Table 4). In both set-ups, the combination of the two strongly EAG-active isomers caught significantly more males than the threo-blend or the SRR-isomer alone. This binary blend (SRS and SRR) was also significantly more attractive to the males than was the SRS-isomer alone in Set-Up 1. Thus, this test indicated, first, that the optimal attractant might be a mixture of the SRR- and SRS-isomers, and second, that one or both of the remaining two threo-isomers (RSR and RSS) might have an antagonistic effect. These assumptions were tested in a number of tests during 1998. In three of them (Tests 1–3), different ratios between the SRR- and SRS-isomers were tested, and the results revealed that both isomers are necessary to attract and catch large numbers of males (Table 5). The ratio between the two isomers did not seem to be very critical, and a 1:1 blend caught large numbers in all tests (Figure 3). In Test 4, the effect of adding the remaining threo-isomers (RSR and RSS: not tested in EAG recordings) to the attractive binary (SRR and SRS) blend was investigated. No antagonistic activity of these two isomers was found (Table 5).
4. Discussion
We have shown that the propionate ester of the SRR-isomer of 3,7-dimethyl-2-tridecanol was weakly but significantly attractive to D. jingyuanensis males; thus, it might be part of the pine sawfly sex pheromone system as a synergistic component, whereas the SRS-isomer seemed to be a key/essential pheromone component since it alone was strongly attractive to the males. The blend of all four threo-isomers (2S,3R,7R/S and 2R,3S,7R/S) was also significantly attractive in the current study and seemed to be a good and effective sex-attractant, confirming the results of earlier field screening [14] and monitoring [12].
Chemical analyses of the female whole-body extracts indicated that either or both of the two threo-isomers (SRR/SRS) likely presented in the second peak in the GC-MS analysis, whereas the SRS-isomer might be a part the first peak and SRR-isomer possibly presented in the second peak in the GC-ECD analysis. However, as the amount of insect material was only sufficient for mass spectrometric identification and one analysis of pentafluorobenzoate by electron capture detector, the extensive stereochemical analysis of the precursors for determining the complete isomer composition was not completed.
Together with the SRS-isomer, the SRR-isomer evoked the strongest antennal response in D. jingyuanensis males. These two isomers elicited significantly stronger EAG responses than the other tested compounds. In field experiments, the combinations of these two strongly EAG-active isomers (SRR and SRS; their alcohol precursors likely present in the female whole-body extract) were significantly more attractive to males than either isomer alone, indicating a significant synergism. Females also contained the erythro-isomer(s) SSS/SSR or RRR/RRS (in the first peak in the GC-MS analysis of the female extract), but neither SSS nor SSR were active in the EAG recordings. Since no pine sawflies have been shown to use a sex pheromone having a ‘2R’ configuration [2], RRR/RRS or RSR/RSS isomers will unlikely be a part of the D. jingyuanensis sex pheromone system. However, it is premature to suggest that the sex pheromone of D. jingyuanensis consists of both the SRR- and the SRS-isomers as the exact stereoisomer composition of the female content is still unknown.
Synergism between different steroisomers has earlier been reported in a few species of pine sawflies. In the widely distributed Neodiprion sertifer, Anderbrant et al. [21] examined the attractivity of males to different proportions of either SRR- or SRS-isomers added to the sex pheromone, the acetate ester of SSS-3,7-dimethyl-2-pentadecanol (diprionol), in eight places on the Northern hemisphere from Japan in the East to Canada in the West, via sites in Asia and Europe. Significantly more N. sertifer males were captured only in Siberia when SRR- or SRS- were added to the SSS-isomer at 0.01%–100% of that of the SSS bait.
Males of Neodiprion pratti banksianae were significantly more attracted to SSS + SRR of diprionyl acetate than to the single SSS-stereoisomer, although the catches were quite low [22]. Similarly, males of both Neodiprion rugifrons and Neodiprion dubiosus were more attracted to SRR + SRS diprionyl propionate than to any of the isomers alone [23]. However, again, the catches were low, and these experiments need to be repeated. In both N. rugifrons and N. dubiosus, the male responses in EAG recordings were stronger to SRS than to SRR, although the doses used were relatively high [23].
In all the cases where males of different species were more attracted to combinations of steroisomers rather than to the individual stereoisomers, little information about female content and released amount exists. As no gland responsible for pheromone production has been detected in diprionid females, whole-body extracts are often used, making analysis more difficult. It would be desirable to analyze what the female actually releases instead of what she contains. The first case where synergism in male response between different isomers occurs simultaneously with a female release of these compounds still remains to be identified.
5. Conclusions
The few Diprion species investigated so far all seem to make use of the SRR configuration of their pheromone molecules as the sex attractant. Males of D. similis and D. nipponica were attracted to propionate esters of SRR-3,7-dimethyl-2-pentadecanol and SRR-3,7-dimethyl-2-undecanol, respectively ([2] and references therein). The attraction of D. similis to the SRR-isomer was reported to be synergized by three other isomers, viz., SSS, SSR, and SRS [6], but this could not be confirmed in a later study [24]. In both D. pini and D. jingyuanensis, males responded to the propionate ester of SRR-3,7-dimethyl-2-tridecanol. Males of D. pini also responded to the acetate of this molecule [15,16]. Surprisingly, D. jingyuanensis males in the current study were not only strongly attracted to the propionate ester of SRS-3,7-dimethyl-2-tridecanol (as a key/essential attractant), but also weakly and synergistically to the SRR-isomer (as a weak but synergistic attractant). For maximum trapping of D. jingyuanensis males, a synergistic mixture of SRR- and SRS-isomers (at 1:1 ratio) is recommended. However, due to the difficulty (expensiveness) of the chiral synthesis, the inexpensive propionate threo-blend of 3,7-dimethyl-2-tridecanol seems to be adequate for development of an effective pheromone-based monitoring and/or mass-trapping program against this economically important pine sawfly.
Conceptualization, O.A. and Q.-H.Z.; chemical synthesis, E.H. and H.-E.H.; chemical analysis, G.B. and A.-B.W.; electrophysiology, F.Ö.; field experiments, Q.-H.Z., G.-F.C. and Z.Z.; statistical analysis, O.A. and F.Ö.; original draft preparation, O.A., Q.-H.Z., F.Ö., G.B. and E.H.; funding acquisition, O.A., G.B., E.H. and H.-E.H. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
Not applicable.
The authors declare no conflict of interest.
Footnotes
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Enlarge this image.Figure 1. Structural formulas of all stereoisomers of the propionate esters of 3,7-dimethyl-2-tridecanol, of which six stereoisomers were tested in this work, and also formulas of three other pheromone candidates that also were tested in this work.
Enlarge this image.Figure 2. (a) Mean (±SE) normalized electroantennographic dose–response curves of male D. jingyuanensis antennae (n =10) stimulated with the propionate ester of different 3,7-dimethyl-2-tridecanol isomers and of SRR-diprionol and with the acetate of SRR-3,7-dimethyl-2-tridecanol. The response to a blank was 22 ± 13%, and 0.01 µg of the propionate ester of SRR 3,7-dimethyl-2-tridecanol was used as standard stimulus. (b) The same data as in (a) but in order to improve separation the Y-axis was fitted with a logarithmic scale. Responses with the same letters are not significantly different, ANOVA followed by Tukey’s test (p < 0.05).
Enlarge this image.Figure 3. Relative catch of male D. jingyuanensis, expressed as the proportion of the total (within each test), for different ratios of the SRR- and SRS-isomers of the propionate ester of 3,7-dimethyl-2-tridecanol. Data shown separately for Tests 1–3 in 1998 (see also Table 5).
Chemical and stereoisomeric purity of the synthetic compounds used for electroantennography and in field tests.
| Compound | Chemical Purity (%) | Stereochemical |
Other Isomers Present (%) |
|---|---|---|---|
| Propionate of threo-3,7-dimethyl-2-tridecanol | >99 | >99.9 | 3rythron (<0.05) |
| Propionate of SRR-3,7-dimethyl-2-tridecanol | 99.5 | >99.3 | SRS (0.2), RSR, (<0.4) RRR (<0.03), SSR (<0.03) |
| Propionate of SRS-3,7-dimethyl-2-tridecanol | >96 | >98.9 | SRR (0.6), RSS (<0.4), SSS (<0.03), RRS (<0.03) |
| Propionate of RSS-3,7-dimethyl-2-tridecanol | >96 | >99.2 | RSR (<0.6), SSS (<0.05), RRS (<0.05), SRS (<0.05) |
| Propionate of RSR-3,7-dimethyl-2-tridecanol | >98 | >99.6 | RSS (0.2), RRR (<0.05), SRR (<0.05), SSR (<0.05) |
| Propionate of SSS-3,7-dimethyl-2-tridecanol | >96 | >99.2 | SSR (0.6), RSS (<0.05), RRS (<0.05), RSR (<0.05) |
| Propionate of SSR-3,7-dimethyl-2-tridecanol | >99 | >99.6 | SSS (0.2), RRR (<0.05), SRR (<0.05), RSR (<0.05) |
| Acetate of SRR-3,7-dimethyl-2-tridecanol | >98 | >99.3 | RSR (<0.4), SRS (0.2), RRR (<0.03), SSR (<0.03) |
| Propionate of SRR-3,7-dimethyl-2-pentadecanol | 98.5 | 87 | SRS (13), SSR (0.5), RSR (0.5) |
Catch of male D. jingyuanensis in traps baited with various amounts of the propionate ester of threo-3,7-dimethyl-2-tridecanol or SRR-3,7-dimethyl-2-tridecanol, Shanxi Province, P. R. China, 27 June–4 July, 1996. Means followed by the same letter are not significantly different according to one-way ANOVA of log(catch + 1) transformed data followed by Tukey’s HSD procedure (p < 0.05).
| Bait | Average Catch (±SD) | ||
|---|---|---|---|
| Compound | Amount (µg) | Set-Up 1 |
Set-Up 2 |
| Blank | - | 0.0 ± 0.0 a | 0.0 ± 0.0 a |
| SRR | 1 | 0.0 ± 0.0 a | 0.0 ± 0.0 a |
| SRR | 10 | 0.0 ± 0.0 a | 0.0 ± 0.0 a |
| SRR | 100 | 0.0 ± 0.0 a | 0.0 ± 0.0 a |
| SRR | 1000 | 16.3 ± 16.4 b | 0.4 ± 0.7 ab |
| threo | 4 | 0.0 ± 0.0 a | 0.0 ± 0.0 a |
| threo | 40 | 0.0 ± 0.0 a | 0.0 ± 0.0 a |
| threo | 400 | 3.6 ± 5.4 a | 1.9 ± 1.7 b |
| threo | 4000 | 88.8 ± 20.4 c | 93.6 ± 13.3 d |
| Virgin female | - | 65.7 ± 34.6 c | 62.2 ± 47.3 c |
Catch of male D. jingyuanensis in traps baited with different amounts of the propionate ester of threo-3,7-dimethyl-2-tridecanol, Shanxi Province, P. R. China, 13–23 June 1997. Means followed by the same letter are not significantly different according to one-way ANOVA of log(catch + 1) transformed data followed by Tukey’s HSD procedure (p < 0.05).
| Amount (μg) | Average Catch (±SD) (n = 6) |
|---|---|
| Blank | 0.0 ± 0.0 a |
| 0.1 | 0.2 ± 0.4 a |
| 1 | 0.0 ± 0.0 a |
| 10 | 0.2 ± 0.4 a |
| 100 | 8.3 ± 5.5 b |
| 1000 | 102.3 ± 14.1 c |
Catch of male D. jingyuanensis in traps baited with different isomers or combinations of isomers of propionate ester of 3,7-dimethyl-2-tridecanol, Shanxi Province, P. R. China, 13–18 June 1997. Means followed by the same letter are not significantly different according to one-way ANOVA of log(catch + 1) transformed data followed by Tukey’s HSD procedure (p < 0.05).
| Bait | Average Catch (±SD) | ||
|---|---|---|---|
| Compound | Amount (µg) | Set-Up 1 |
Set-Up 2 |
| Blank | - | 0.0 ± 0.0 a | 0.0 ± 0.0 a |
| threo | 400 | 35.6 ± 24.8 c | 23.5 ± 27.1 b |
| SRR | 100 | 5.1 ± 4.5 b | 4.8 ± 9.4 a |
| SRS | 100 | 40.5 ± 23.4 c | 66.0 ± 39.4 bc |
| SRR + SRS | 100 + 100 | 112.9 ± 17.5 d | 99.8 ± 36.4 c |
Catch of male D. jingyuanensis in traps baited with different combinations of isomers of the propionate ester of 3,7-dimethyl-2-tridecanol, Shanxi Province, P. R. China, 13–21 June 1998. Catch figures within each test having the same letters are not significantly different (p < 0.05). Total catches compared with G-test including Sidák’s compensation for mass significance. Each test is based on three replicates.
| Bait: Isomer, Amount (µg) | Total Catch | ||||
|---|---|---|---|---|---|
| SRR | SRS | RSR | RSS | ||
| Test 1 | Blank | 0 a | |||
| 100 | 0 | 13 a | |||
| 90 | 10 | 10 a | |||
| 50 | 50 | 236 c | |||
| 10 | 90 | 238 c | |||
| 0 | 100 | 170 b | |||
| Test 2 | Blank | 0 a | |||
| 100 | 0 | 1 a | |||
| 100 | 1 | 3 a | |||
| 100 | 11 | 3 a | |||
| 100 | 100 | 546 b | |||
| Test 3 | Blank | 0 a | |||
| 0 | 100 | 18 a | |||
| 1 | 100 | 29 a | |||
| 11 | 100 | 317 b | |||
| 100 | 100 | 693 c | |||
| Test 4 | Blank | 0 a | |||
| 50 | 50 | 181 b | |||
| 50 | 50 | 50 | 232 c | ||
| 50 | 50 | 50 | 280 d | ||
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; Guo-Fa, Chen 3 ; Östrand, Fredrik 1 ; Bergström, Gunnar 4 ; Ann-Britt Wassgren 4 ; Zhang, Zhen 5 ; Hedenström, Erik 6 ; Högberg, Hans-Erik 6 1 Department of Biology, Lund University, Sölvegatan 37, SE-22362 Lund, Sweden
2 Biological Disaster Prevention and Control Center, National Forestry and Grassland Administration, No. 58 Northern Huanghe Street, Shenyang 110034, China;
3 Biological Disaster Prevention and Control Center, National Forestry and Grassland Administration, No. 58 Northern Huanghe Street, Shenyang 110034, China;
4 Chemical Ecology, Göteborg University, SE-40530 Göteborg, Sweden;
5 Key Laboratory of Forest Protection of National Forestry and Grassland Administration, Research Institute of Forest Ecology, Environment and Nature Conservation, Chinese Academy of Forestry, No. 2 Dong Xiao Fu Street, Beijing 100091, China;
6 EcoChemistry, Department of Natural Sciences, Design and Sustainable Development, Mid Sweden University, SE-85170 Sundsvall, Sweden;