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Arthropod-Plant Interactions

, Volume 2, Issue 1, pp 31–41 | Cite as

Infestation by a Nalepella species induces emissions of α- and β-farnesenes, (−)-linalool and aromatic compounds in Norway spruce clones of different susceptibility to the large pine weevil

  • Astrid Kännaste
  • Namphung Vongvanich
  • Anna-Karin Borg-KarlsonEmail author
Original Paper

Abstract

The emissions of spruce grafts (Picea abies), caused by infestation of an acarid species of the genus Nalepella were investigated. Volatiles of three clones, both healthy and infested, with different susceptibility to the large pine weevil Hylobius abietis were collected by solid phase micro extraction (SPME) and analyzed by gas chromatograph coupled to mass-spectrometry (GC-MS). In addition, enantiomers of the main chiral compounds were separated by a two dimensional-gas chromatograph (2D-GC). In the characteristic flower-like fragrances emitted by the infested grafts large amounts of E-β-farnesene, E,E-α-farnesene, (−)-linalool, methyl salicylate and minute amounts of benzyl alcohol, E-anethole, methyl benzoate, neral and geranial were found. All together, these compounds could explain the characteristic scent emitted by the infested seedlings. Large differences in the emissions of E-β-farnesene, E,E-α-farnesene and methyl salicylate were found between but not within the clones.

Keywords

Nalepella Picea Infestation Farnesene Methyl salicylate Linalool Neral Stress 

Introduction

During our search for chemical markers for resistance in conifers we analysed the volatiles of spruce (Picea abies L.) graphs infested by the tiny mite tentatively identified as Nalepella haarlovi Boczek var. Picea abietis (Acarina, Ereophyidae) (Phytoptidae) (Löyttyniemi 1973). The mites were sucking the sap of the needles and the infested specimens emitted a flower-like scent different from healthy speciments. Outbreaks of N. haarlovi causing serious damage are rare but a potential risk in nurseries (Ehnstrom et al. 1974; Gibbs and Evans 1998; Poteri et al. 2005). Normally the plants recover if placed outdoors, but in severe cases the plants can die. At a high level of infestation, the needles on the shoots become grey and turn to reddish brown. The biology of N. haarlovi is not well known. Eggs can be found on the needles and up to five generations per year has been observed in related species (Löyttyniemi 1973). Induced chemical defence reactions to herbivory by mites are well studied in angiosperms (Arimura et al. 2004a, b; Arimura et al. 2005; Bounwmeester et al. 1999; Dicke et al. 1990; Himanen et al. 2005; Takabayashi et al. 1994, 2000), as well as in conifers (Kielkiewicz et al. 2005). Furthermore the production of volatiles released by attacked plants is shown to be herbivore species specific (Takabayashi et al. 1991; Delphia et al. 2007).

In this study our aim was to evaluate if the tiny mite N. haarlovi, could be used to test the resistance in spruce graphs with known susceptibility to the large pine weevil Hylobius abietis. We identified the chemical responses of the whole infested grafts, elicited by the mites, and compared them with non infested grafts of the same clones. By using SPME-GC-MS we collected the volatiles without disturbing the plants and mites.

Materials and methods

Biological material

Four years old spruce grafts (45 cm in height, 5–8 branches) of Picea abies originating from the clone archives of Skogforsk (the Forestry Research Institute of Sweden), with documented low (clone number 1091; 74% eaten), medium (72; 55% eaten) and high (1090; 10% eaten) resistance to pine weevil gnaw (Weslien 2004) were used in this study. The grafts were initially developed in greenhouse and kept outdoors from the age of 1 year, but were kept inside at room temperature for two months prior the experiments. The grafts were divided into infested (5–10 mites/needle) and healthy plants (no mites detected on the aeral parts of the plants). The defence reactions of three infested specimens of each clone investigated were compared to the emissions of one healthy spruce of each clone.

Collection of volatiles

Each tree was enclosed in an oven proof plastic bag (polyethylene terephthalate, Toppits Melitta 35 cm× 43 cm). Headspace collections were carried out at room temperature in static conditions by inserting the fibre of SPME into the bag. Fiber coated with 65 μm of polydimethylsiloxane/divinylbenzene (Supelco, Bellefonte, PA, USA) was used to trap the plant volatiles. For GC-MS the collection time of compounds was 15 h. The enantiomeric composition of chiral monoterpenes in the stem phloem (the food source of the feeding pine weevil) was evaluated by trapping the terpenes for 30 min upon piercing the phloem 3 times by a metal needle.

Analysis and identification

A Finnigan SSQ 7000 mass spectrometer (70 eV, ion source at 150°C) coupled to a GC of Varian 3400 equipped with a CB1 column (30 m, 0.25 mm id and 0.25 μm film thickness) was used for analyzing the emissions. Sufficient separation of compounds was achieved with the following temperature program: 40°C (2 min) at 4°C/min to 180°C (0.01 min), followed by an increase of 20°C/min to 220°C (1 min). SPME fibres were desorbed in the GC-injector at a temperature of 215°C (30 s splitless injection).

Identifications of spruce volatiles were made by comparing the mass spectra obtained with available standards and the calculated retention indexes (RI) to the published RIs (Davies 1990; Ruther 2000). In addition the reference libraries of NIST (National Institute of Standards and Technology) and MassFinder 3 (Hochmuth Devtler) were used. Commercially available references of highest purity available (>98%) were purchased from the companies of Lancaster Synthesis and Sigma-Aldrich.

The chiral compounds were separated by using a two-dimensional-GC (2D-GC) (Borg-Karlson et al. 1993). In the first GC a DB-wax column (J&W Scientific™, 30 m, 0.25 mm id and 0.25 μm film thickness) with the following temperature program was used: 40°C (2 min), with the rate of 4°C/min increase to 130°C, after 12 min temperature continued to rise with 4°C/min to 150°C(0.01), final conditions were obtained by an increase from 150°C to 220°C at 10°C/min for 10 min. Injections were performed with injector temperature of 220°C in 30 s splitless mode. In the second GC a HP Chiral-20 β 19091G-B233 (J&W, 30 m × 0.25 mm × 0.25 μm, 20%) was used for separation of the chiral constituents. Temperature program: 55°C for 11 min, followed by an increase of 1°C/min to 75°C (65 min), final conditions were obtained by the increase of temperature at 1°C/min from 75 to 90°C (60 min). Helium was used as the carrier gas from both GCs at a pressure of 34 psi for the first GC and a pressure of 22 psi for the second GC.

Multivariate data analysis

Distribution of compounds and spruce grafts was evaluated by multivariate data analysis and software Canoco (version 4.5, developed by Cajo JF ter Braak and Petr Smilauer, Biometris Plant Research International, The Netherlands). On the plots the attention on the relationships among substances was achieved by centering the data by compounds. In addition dividing the scores of compounds with their standard deviations gave equal weight to each substance. After square-root transformation of relative amounts—and logarithmic transformation of absolute amounts of compounds the data were subjected to Principal Components Analysis (PCA). Clone specific emissions of selected volatiles from N. haarlovi infested grafts were evaluated by T-test assuming equal or unequal data variance.

Correlation analysis

Correlation analysis, where the absolute amounts of the selected volatiles were plotted against each other, were accomplished to estimate the biosynthetic routes involved in the production of volatiles. Correlation coefficient R2 is expressed as square of Pearson correlation coefficient r calculated with the help of Excel (Microsoft).

Results

Emissions of healthy and infested spruces

Emission of healthy grafts contained mainly monoterpene hydrocarbons, oxygenated monoterpenes and sesquiterpene hydrocarbons (Table 1). The emissions of the infested grafts were dominated by large amounts of E,E-α- and E-β-farnesene, (−)-linalool and methyl salicylate (Fig. 1, Table 1).
Table 1

Relative amounts of substances emitted by healthy spruces and spruces infested by Nalepella haarlovi and sp. mites S = susceptible, M = medium, R = resistant

No.

Compunds

RI CPB-1a

Healthy spruces, clones 72, 1090 and 1091, n = 3

Spruces infested by Nalepella haarlovi

Lit.

Calc.

Clone 1091 S n = 3

Clone 72 M n = 3

Clone 1090 R n = 3

1

(3Z)-Hexenol

851

836

0.01 ± 0.01

0.03 ± 0.05

2

1-Hexanol

837

854

0.01 ± 0.02

3

α-Pinene

936

930

3.58 ± 2.94

0.02 ± 0.00

4

Camphene

950

956

0.67 ± 0.60

5

Sabinene

973

974

0.14 ± 0.15

6

β-Pinene

978

990

1.53 ± 1.11

0.01 ± 0.01

7

Myrcene

987

990

2.91 ± 1.05

0.06 ± 0.01

0.08 ± 0.07

0.06 ± 0.02

8

(3Z)-Hexenyl acetate

1002

995

0.14 ± 0.11

0.03 ± 0.03

0.18 ± 0.29

9

Unknown HC, MW 150

1002

0.01 ± 0.01

0.01 ± 0.01

10

Hexyl acetate

1006

1003

0.16 ± 0.12

0.05 ± 0.02

0.05 ± 0.05

11

3-Carene

1010

1008

3.05 ± 3.31

0.01 ± 0.01

0.01 ± 0.01

12

Benzyl alcohol

1006

1012

0.04 ± 0.07

0.10 ± 0.02

0.05 ± 0.03

0.13 ± 0.14

13

p-Cymene

1015

1017

2.30 ± 2.18

14

β-Phellandrene

1023

1025

1.52 ± 2.22

0.01 ± 0.02

0.02 ± 0.02

0.02 ± 0.02

15

Limonene

1025

1027

13.30 ± 5.22

0.04 ± 0.03

0.03 ± 0.00

0.05 ± 0.01

16

Z-β-Ocimene

1029

1037

0.03 ± 0.00

0.05 ± 0.03

0.03 ± 0.02

17

2,6-Dimethyl-2,6-octadiene

 

1038

0.76 ± 0.18

0.57 ± 0.34

0.60 ± 0.24

18

E-β-Ocimene

1041

1048

0.25 ± 0.23

0.04 ± 0.00

0.03 ± 0.02

0.03 ± 0.01

19

γ-Terpinene

1051

1043

0.39 ± 0.55

20

1,1′-Methylene-dipyrrolidine

1054

0.06 ± 0.01

0.06 ± 0.04

0.04 ± 0.04

21

trans-Linalooloxide (furanoid)

1058

1066

0.03 ± 0.01

0.02 ± 0.01

0.02 ± 0.02

22

p-Cymenene

1075

1067

0.97 ± 1.17

23

Terpinolene

1082

1072

0.27 ± 0.26

24

Methyl benzoate

1072

1076

0.55 ± 0.78

0.16 ± 0.05

0.05 ± 0.02

0.13 ± 0.10

25

cis-Linalooloxide (furanoid)

1072

1079

0.01 ± 0.00

0.01 ± 0.01

0.01 ± 0.01

26

Linalool

1086

1093

0.15 ± 0.18

1.39 ± 0.67

0.31 ± 0.22

0.11 ± 0.06

27

Unknown MT, MW 136

1100

 

0.48 ± 0.28

0.05 ± 0.03

0.19 ± 0.12

28

Camphor

1123

1110

0.69 ± 0.40

29

4-Acetyl-1-methylcyclohexene

1114

0.03 ± 0.01

0.06 ± 0.02

0.16 ± 0.12

30

4,8-Dimetylnona-1,3,7-triene (DMNT)

1103

1115

0.13 ± 0.13

0.01 ± 0.01

0.01 ± 0.00

0.02 ± 0.02

31

β-Terpineol

1137

1122

0.27 ± 0.26

32

Citronellalb

1129

1128

0.04 ± 0.02

0.06 ± 0.05

0.06 ± 0.08

33

Benzyl acetate

1134

1144

0.07 ± 0.05

0.07 ± 0.02

34

Isoborneol

1142

1151

0.81 ± 0.70

0.02 ± 0.00

0.01 ± 0.01

0.02 ± 0.01

35

Benzoic acid

1160

1165

0.42 ± 0.15

0.05 ± 0.01

0.19 ± 0.18

36

Methyl salicylate

1171

1178

9.91 ± 6.47

9.05 ± 2.30

1.48 ± 0.66

6.68 ± 7.51

37

α-Terpineol

1176

1198

0.62 ± 0.60

0.01 ± 0.02

0.02 ± 0.00

0.12 ± 0.14

38

Neralc

1215

1226

0.29 ± 0.45a

0.01 ± 0.01

0.01 ± 0.01

0.03 ± 0.01

39

Piperitone

1226

1236

0.21 ± 0.36

0.02 ± 0.02

40

Geraniol

1235

1247

0.03 ± 0.00

0.04 ± 0.02

0.03 ± 0.01

41

trans-Myrtanold

1240

1249

0.06 ± 0.04

0.02 ± 0.02

0.03 ± 0.01

42

Geranial

1244

1255

0.05 ± 0.01

0.04 ± 0.03

0.05 ± 0.02

43

Bornyl acetate

1270

1263

3.80 ± 2.90

0.01 ± 0.01

0.01 ± 0.00

0.02 ± 0.00

44

E-Anethole

1262

1270

0.03 ± 0.06

0.47 ± 0.07

45

γ-Nonanolide

1318

1327

0.13 ± 0.09

0.02 ± 0.00

0.09 ± 0.08

46

Eugenol

1331

1339

0.02 ± 0.02

0.03 ± 0.05

47

α-Terpinyl acetate

1335

1339

0.41 ± 0.37

0.01 ± 0.00

0.14 ± 0.17

48

Unknown SqT, MW 204

1339

11.79 ± 10.47

49

Methyl-2,4-dihydroxybenzoate

1349

0.01 ± 0.01

50

α-Longipinene

1360

1351

2.12 ± 1.90

51

α-Ylangene

1376

1354

0.47 ± 0.82

52

α-Copaene

1379

1359

1.47 ± 1.18

53

Unknown SqT, MW 204

1370

0.08 ± 0.14

54

cis-β-Elemene

1381

1370

0.25 ± 0.43

55

Longifolene

1411

1388

2.64 ± 2.06

56

Nopyl acetate

1400

1.58 ± 2.62

57

Methyl 4-methoxysalicylate

1400

0.01 ± 0.02

0.01 ± 0.00

58

cis-α-Bergamotene

1411

1407

0.21 ± 0.22

59

Isocaryophyllene

1409

1411

0.17 ± 0.29

0.03 ± 0.02

0.01 ± 0.00

0.01 ± 0.02

60

E-β-Caryophyllene

1421

1418

0.02 ± 0.01

0.03 ± 0.03

61

Geranylacetone

1430

1420

1.03 ± 1.78

62

Z-β-Farnesene

1420

1435

0.07 ± 0.03

0.02 ± 0.01

0.07 ± 0.03

63

trans-α-Bergamotene

1434

1443

0.19 ± 0.12

0.08 ± 0.02

0.25 ± 0.17

64

Sesquisabinene A

1435

1446

0.05 ± 0.01

0.06 ± 0.02

0.12 ± 0.03

65

Unknown SqT

1458

0.35 ± 0.48

66

E-β-Farnesene

1446

1467

10.41 ± 10.87

49.56 ± 7.30

24.49 ± 2.92

38.81 ± 3.64

67

trans-β-Bergamotene

1480

1490

0.12 ± 0.04

0.06 ± 0.02

0.10 ± 0.04

68

(3Z,6E)-α-Farnesene

1480

1496

0.88 ± 0.76

2.43 ± 0.86

3.26 ± 1.87

3.21 ± 0.97

69

α-Muurolene

1496

1481

0.24 ± 0.22

70

δ-Amorphene

1499

1505

0.55 ± 0.60

71

E,E-α-Farnesene

1498

1516

16.82 ± 13.04

26.22 ± 9.96

58.95 ± 4.44

38.21 ± 14.00

72

β-Sesquiphellandrene

1516

1530

0.13 ± 0.01

0.05 ± 0.02

0.12 ± 0.02

73

E-α-Bisabolene

1530

1547

0.19 ± 0.33

1.47 ± 0.42

8.35 ± 4.54

7.50 ± 5.37

74

Unknown SqT, MW 220

1557

0.86 ± 0.46

0.19 ± 0.10

0.46 ± 0.28

75

Unknown SqT

 

1607

0.90 ± 0.55

0.15 ± 0.07

0.43 ± 0.29

76

Unknown SqT, MW 220

1610

0.42 ± 0.26

0.47 ± 0.26

0.45 ± 0.13

77

Unknown, MW 220

1613

0.16 ± 0.07

0.23 ± 0.11

0.20 ± 0.07

78

Unknown SqT, MW 218

1660

0.28 ± 0.14

0.25 ± 0.16

0.25 ± 0.11

79

Unknown HC, MW 236

1680

1.22 ± 0.81

0.17 ± 0.29

80

Unknown HC, MW 234

1683

1.74 ± 1.21

0.37 ± 0.65

81

Unknown HC, MW 238

 

1689

0.63 ± 0.57

0.09 ± 0.15

aHealthy spruces released mostly methyl thymyl ether

bCitronellal co-eluting with trans-limonene oxide

cNeral co-eluted with methyl thymyl ether

d trans-myrtanol co-eluted with (2E)-decenal

Fig. 1

Representative SPME-GC-MS analyses of three years old spruce graphs from clone 1091. Oven temperature: 40°C (2 min) at 4°C/min to 180°C (0.01 min), followed by an increase of 20°C/min to 220°C (1 min). a—Healthy spruce, bNalepella haarlovi infested spruce. Identified compounds—1: nonanal, 2: decanal, 3: 2-(2-butoxyethoxy)ethyl acetate (from the air of laboratory), 4: 6-methyl-5-hepten-2-one, 5: (3Z)-hexenyl acetate, 6: 2,6-dimethyl-2,6-octadiene, 7: methyl benzoate, 8: linalool, 9: unknown MT (MW 136), 10: methyl salicylate (MeSA), 11: E-anethole, 12: E-β-farnesene, 13:(3Z,6E)- α-farnesene , 14: E,E-α-farnesene, 15: E-α-bisabolene, 16: unknown SqT (MW 220), 17: unknown (MW 218), 18: unknown HC (MW 236), 19: unknown HC (MW 234), 20: unknown HC (MW 238)

The volatiles released by healthy and N. haarlovi infested spruces are presented in Table 1. When comparing the relative and absolute amounts of the compounds of attacked grafts by N. haarlovi with healthy grafts, several terpenes detected in the headspace of healthy spruces were absent or present at trace levels in the emissions of infested spruces. Infestation by N. haarlovi decreased the relative amounts of myrcene, 3-carene, β-phellandrene and E-β-ocimene. In clone 1090, the absolute amounts of limonene, the main monoterpene of healthy grafts, decreased from 7.50 ng/15 h (n = 1) to 1.29 ± 0.62 ng/15 h (n = 3). For the same clone the emission of bornyl acetate, the main oxygenated monoterpene, decreased from 4.96 ng/15 h (n = 1) to 0.29 ± 0.11 ng/15 h (n = 3).

Figure 2 presents a PCA plot based on the absolute amounts of compounds of the healthy and infested spruces and shows clearly, that although MeSA together with farnesenes were present in minor amounts in the headspace of healthy grafts, infestation of N. haarlovi increased their amounts tremendously. Also, the emissions of spruces with feeding mites increased. During the constant collection time of 15 hours the absolute amount of methyl salicylate increased from 3.82 ± 5.33 ng (healthy spruces, n = 3) to 100.32 ± 50.55 ng (infested spruces of three clones, n = 9), E-β-farnesene increased from 9.75 ± 15.59 ng (healthy spruces, n = 3) to 1.63 ± 0.72 μg (infested spruces, n = 9) and; the absolute amount of E,E-α-farnesene increased from 12.76 ± 18.63 ng (healthy spruces, n = 3) to 2.46 ± 2.05 μg (infested spruces, n = 9. The headspace of the susceptible clone 1091 was dominated by E-β-farnesene and MeSA. Infested spruces of clone 72 emitted mostly E,E-α-farnesene.
Fig. 2

A PCA plot based on the absolute amounts of volatiles released by healthy and N. haarlovi infested spruces. Circle: clone 1090, x-mark: clone 72 and up-triangle: clone 1091. Direction of arrows points out to the distribution between compounds and spruce grafts A total 83% of variance in the data is explained by the first and second principal components (PC 1:70%, PC 2:13%)

The emission of stress compounds from infested spruces was clone specific according to T-test (Table 2). Between the grafts of clones 1090 and 1091 significant difference in absolute scale was found only for E-α-bergamotene. The relative amounts of E-β-farnesene and β-sesquiphellandrene released by clones 72 and 1090 differed considerably, whereas their absolute amounts were equal. With linalool and E,E-α-farnesene, if absolute amounts were evaluated to differ remarkably, upon the normalization of data no difference was found for their relative proportions. Between the grafts of clones 72 and 1091 the relative proportions of MeSA, E-β-farnesene, E,E-α-farnesene and β-sesquiphellandrene were different. The emission of other stress compounds e.g. E-α-bisabolene, (3Z,6E)-α-farnesene, Z-β -farnesene and E-β-bergamotene was not related to a specific clone.
Table 2

P-Values of selected stress compounds released by N. haarlovi infested spruces

Clones

Stress compounds

Linalool

Methyl salicylate

E-α-bergamotene

E-β-farnesene

E,E-α-farnesene

β-sesqui-phellandrene

Based on relative amounts

1090–1091

0.08

0.64

0.70

0.07

0.29

0.47

72–1090

0.22

0.30

0.25

<0.01

0.07

0.02

72–1091

0.06

<0.01

0.26

<0.01

<0.01

<0.01

Based on absolute amounts

1090–1091

0.10

0.45

0.01

0.91

0.37

0.65

72–1090

0.04

0.62

0.89

0.29

0.01

0.81

72–1091

0.45

0.17

0.19

0.27

0.01

0.72

The largest proportion of linalool was released by the most susceptible clone 1091 as shown in Table 1. Separation of enantiomers showed R-(−)-linalool to be the main component to be present (95%).

Compounds present in minor amounts were 2,6-dimethyl-2,6-octadiene, cis- and trans-linalool oxide, citronellal, geraniol, geranial, neral, benzyl acetate, benzoic acid, E-anethole and methyl-2,4-dihydroxybenzoate. A good correlation was found between geraniol and geranial (R 2 = 0.93, n = 9). In addition, the absolute amount of neral showed to be correlated to geraniol and geranial (correlation coefficients R 2 = 0.91 and R 2 = 0.94 respectively, n = 8).

The susceptible clone 1091 was found to emit the highest amounts of benzoic acid, methyl benzoate and MeSA, whereas E-anethole was mostly released by the clone 1090. The emissions of MeSA and the aromatic compounds methyl benzoate, benzyl alcohol and benzoic acid showed a good correlation both within and between the clones, indicating a biosynthetic relationship (Fig. 3). In addition, grafts of clone 1091 released methyl-2,4-dihydroxybenzoate and its methylated product methyl-4-methoxysalicylate. Small amounts of γ-nonalactone were also present in the emissions. None of these compounds were found in the emission of the healthy grafts.
Fig. 3

Correlation plots between aromatic compounds released by N. haarlovi infested spruces. Positive correlation coefficients are R 2  = 0.73 for benzoic acid and MeSA, R = 0.67 for benzyl alcohol and methyl benzoate, R = 0.29 for MeSA and methyl benzoate and r = 0.22 for benzoic acid and methyl benzoate

Emission of wounded phloem

Large differences in the emission of monoterpenes of the wounded phloem were found between the clones (Fig. 4). No changes in the relative amounts of terpenes in the phloem between the healthy and three infested spruces of each clone were observed, as they were grouped together in the PCA plot (Fig. 5). This is also shown by the small standard deviations for infested grafts in Fig. 6. The susceptible clone 1091 had the highest proportion of 3-carene, while for the other two clones the content of 3-carene remained <5% (Fig. 4). The highest amount of (−)-limonene known as repellent for the large pine weevil Hylobius abietis was detected for the resistant clone 1090 (Nordlander 1991; Wibe et al. 1998), 56% of all monoterpenes from the healthy and 49% from the infested grafts.
Fig. 4

Composition of chiral monoterpenes (mean ± SD) in the emission released upon piercing the stem of N. haarlovi infested grafts. Relative amounts were calculated by normalization of peak areas of (−)-α-pinene, (+)-α-pinene, (−)-β-pinene, (+)-β-pinene, (+)-3-carene, (−)-limonene and (+)-limonene. Three replicates of each clone were tested

Fig. 5

A PCA plot based on the relative amounts of (−)-α-pinene, (+)-α-pinene, (−)-β-pinene, (+)-β-pinene, (+)-3-carene, (−)-limonene and (+)-limonene released upon piercing the stem base of spruces. Circle: clone 1090; x-mark: clone 72; up-triangle: clone 1091. Arrows present the distribution between selected monoterpenes and spruce grafts. A total 99% of variance in the data is explained by the first and second principal components (PC 1:84%, PC 2:15%)

Fig. 6

Enantiomeric composition (mean ± SD) of α-pinene, β-pinene and limonene released upon piercing the stem of healthy and N. haarlovi infested spruces. Proportion of enantiomers were calculated by dividing the peak area of (−)-enantiomer with sum of peak areas of (−)- and (+)-enantiomers (normalized to 100%)

Enantiomeric compositions of α-pinene, β-pinene and limonene are shown in Fig. 6. The bars of healthy clones represent mean values of one spruce per each clone. Based on enantiomeric analysis the healthy plants released 98% of (−)-β-pinene, 89% of (−)-α-pinene and 89% of (−)-limonene. By comparing the data of uninfested and infested spruces no changes in the enantiomeric compositions were observed.

Discussion

Stress induced volatiles from plants have been the focus for numerous investigations, especially stress reactions induced by spraying the plants with MeJA (Huber et al. 2005; Martin et al. 2003). Spraying the conifers with MeJA and feeding of the white pine weevils Pissodes strobi on the stem of conifers increases the activity of the enzymes involved in the biosynthesis of terpenes and the accumulation of terpenes in the tissues (Miller et al. 2005). Zeneli and co-workers showed in 2006 that with Norway spruce MeJA caused minor changes among the terpenes in newly formed ducts. Sitca spruce attacked by feeding white pine weevils Pissodes strobi have been shown to release α- and β-farnesenes (Miller et al. 2005).

In our study all three infested clones emitted large amounts of E,E-α- and E-β-farnesene. N. haarlovi feeding seems to induce a similar defence reaction in conifers, with regard to the major compounds, as for MeJA treatments (Huber et al. 2005; Martin et al. 2003; Miller et al. 2005; Zeneli et al. 2006), or pine weevil feeding (Miller et al. 2005). In fact MeJA has been used to identify the enzymes producing E,E-α- and E-β-farnesene; and E-α-bisabolene as single product of enzymes (Bohlmann et al. 1998; Huber et al. 2005; Martin et al. 2004).

The signals released by wounding, seem to be tissue selective as a micro size painting of MeJA or wounding of the bark induces the emission of farnesenes in the needle above and closest to the injury (Pettersson 2007). This is confirmed by studies on Norway spruce, where the effects of insect attacks or MeJA treatments upregulated the foliar TPS gene expression and increased enzyme activities for the main compounds E,E-α- and E-β-farnesene (Miller et al. 2005). Only trace amounts of DMNT were present in the emissions of Norway spruce, which is different from abiotically stressed or mite infested Salix spp emitting DMNT and E,E-α-farnesene, indicating a species specific regulation of terpenoid defences (Borg-Karlson et al. unpublished).

Farnesenes are known to affect the behaviour of several insect species (Bengtsson et al. 2001; Binder et al. 1995; Svensson and Bergström 1977; Tasin et al. 2006). The egg parasitoid Chrysonotomyia ruforum responded to E-β-farnesene only in the presence of wounded Pinus sylvestris twigs (Mumm and Hilker 2005). The large pine weevil, Hylobius abietis, was attracted to E-β-farnesene alone, and in multi-choice test; thus, a seedling that emits high amounts of farnesenes, might easily be detected by the feeding weevils (Kännaste et al. manuscript).

A higher amounts of (−)-linalool in the emission from the most susceptible clone, might be explained as a result of indirect defence resulting in herbivore pressure on these genotypes. Linalool in a high dose is known as antifeedant for the pine weevil, (Kännaste et al. manuscript). The presence of a few percent of the (S)-(+)-enantiomer of linalool was also found in enzyme products obtained by treatment of MeJA (Martin et al. 2004). The two enantiomers of linalool elicited similar antennal receptor responses to Colletes bees (Borg-Karlson et al. 2003), but by using single cell recordings, different types of enantioselective linalool receptors were found in the strawberry weevil Anthonomus rubi (Bichao et al. 2006) and in the cabbage moth Mamestra brassicae (Ulland et al. 2007). Phenolic compounds have an important role in conifer defence. Stress induces the biosynthesis of phenylalanine and may via a series of enzymes increase the amounts of ceratin biosynthetically related aromatic compounds. Thus depending on the concentration, the combination of benzylalcohol, (−)- and (+)-linalool, E-anethole, methyl benzoate and MeSA, as a fragrant flower-like blend, may function as an insect repellent in high concentrations and insect/predator attractant in low concentration.

The enantiomeric composition of 3-carene was not measured in this study. In previous investigations, only the (+)-enantiomer has been present (Borg-Karlson et al. 1993; Persson et al. 1996). Feeding behaviour of N. haarlovi did not to change the relative amounts or the enantiomeric composition of the monoterpene hydrocarbons in the phloem (Figs. 46). Fungal infestations might induce other mechanisms as in our results from analyses of fungi inoculated Scots pines (Fäldt et al. 2006), where an increase in the amount of (−)-β-pinene was observed.

In the headspace of N. haarlovi infested spruces both, geranial and neral were present in small but in characteristic amounts. They are identified in the essential oils of spruce needles (Kolesnikova 1985; Mardarowicz et al. 2004), but their exact biosynthetic pathway is still unknown. The biosynthetic route of geraniol has been investigated in basil (Iijima et al. 2006). They claimed nerol to be produced through the oxidation of geraniol to geranial, which due to tautomerization will be turned to neral. In the final step neral will be reduced to nerol (Iijima et al. 2006). Although geranial and neral have been detected in the oil gland exudates of mites (Sakata et al. 2003), high correlation between geraniol and neral in the emission of infested grafts suggests their production to be similar to the pathway suggested by Iijima et al (2006). For several mite species, neral and geranial act as alarm pheromones, e.g. the acarid mites Histiogaster rotundus Woodring (Acari: Acaridae), whose prey Southern pine beetle releases geranial and neral along with alarm pheromone neryl formate (Hiraoka et al. 2003). However, in astigmata mites (Schwiebea elongate) neral acts as an attractant (Nishimura et al. 2002).

Methyl salicylate was emitted together with the minor constituent methyl benzoate and showed a good correlation (Fig. 3). This was also valid for benzoic acid and benzylalcohol. Benzoic acid and benzyl alcohol have been found in the aquatic extracts of the needles of conifers (Isidorov et al. 2006). Aromatic esters have earlier been identified in the scent of spruce flowers (Borg-Karlson et al. 1985) which might indicate the presence of functionally similar enzymes both during stress responses and flowering. The emission of methyl benzoate, benzylalcohol, benzyl acetate and benzoic acid in the needles of N. haarlovi infested grafts could be explained by their biosynthesis occurring in parallel with the general phenylpropanoid pathway and their derivation from trans-cinnamic acid identified in the needles of conifers (Lebedeva et al. 1982; Dudareva et al. 2006; Isidorov et al. 2006). Thus the release of aromatic compounds might be the result of increased lignification, as induced by a fungus (Campbell and Ellis 1992).

Benzylalcohol and methyl salicylate are known to affect the behaviour of bark beetles (Huber et al. 2000). In 2002 Grant and Langevin showed benzoic acid to increase the oviposition of the females of spruce budworm in high doses (Grant and Langevin 2002). With GC coupled to electrophysiological recordings E-anethole was shown to activate the pine weevil antennal receptor neurones (Hylobius abietis) (Wibe et al. 1997 and references therein). E-anethole, methyl benzoate and methyl salicylate are pine weevil antifeedants in 50 mM concentration (Borg-Karlson et al. 2006; Unelius et al. 2006) and the scent of MeSA in a combination with the conifer twig scent seems to act as a repellent to the large pine weevil (Kännaste et al. manuscript). Thus, we might speculate that certain infested spruce grafts with high amounts of MeSA might be less attractive to the weevils than the healthy ones. Similar effects have been observed while examining the behaviour of predatory mites towards the volatiles released by lima bean plants infested by spider mites Tetranychus urticae. Methyl salicylate alone as the volatiles of uninfested plants did not attract the predatory mites, while their combination instead turned out to be attractive to mites (de Boer and Dicke 2004).

This study was a part in a larger project with the aim to identify chemical markers for resistance in conifers. Here, with the help of a biological stressor, Nalepella haarlovi, we were able to show, that spruce grafts with highly different susceptibility to the large pine weevil responded chemically differently. Our results indicated, that the defence reactions differ between the clones: the most preferred (susceptible) clone by feeding pine weevils is the one emitting the largest amounts of (−)-linalool, E-β-farnesene and MeSA during N.h. infestation. We also found that the most resistant clone produced the lowest amounts of (−)-linalool. The number of clones in our investigation was low, so further research is needed. The acarid N. haarlovi seems to overcome the induced defence in the spruce; all of the plants displayed a similar degree of infestation. Thus we suggest that the acarid N. haarlovi can be used as a biological tool to improve the search for resistance markers in spruce. The induced clone specific emissions were easy to measure by SPME. The non healthy conifer (alien) fragrance that is induced by infestation might have several biological functions that we plan to further investigate.

Notes

Acknowledgements

Prof. Bo Långström tentatively identified the damage to be made by a Nalepella-species, probably Nalepella haarlovi. This study was financially supported by FORMAS, Carl Trygger Foundation and an Archimedes stipend from Estonia to Astrid Kännaste.

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Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Astrid Kännaste
    • 1
  • Namphung Vongvanich
    • 1
  • Anna-Karin Borg-Karlson
    • 1
    Email author
  1. 1.Department of ChemistryEcological Chemistry Group, KTH, Royal Institute of TechnologyStockholmSweden

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