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Introduction
The walnut Juglans regia L. (1753) (Juglandales: Juglandaceae) is an economically important cultivated trees species. J. regia is widely distributed in China (north to Heilongjiang Province, south to Yunnan Province and Guizhou Province, west to Xinjiang Uygur Autonomous Region, and east to Shandong Province, Liaoning Province). It has nutritional and medicinal value (Ma et al. 2006; Xu et al. 2006; Wang et al. 2006; Zhou and Lv 2006); therefore, the walnut is classified as a strategic species for human nutrition and is included in the FAO priority list of plants (Gandev 2007).
The main herbivore affecting J. regia is Meliboeus ohbayashii primoriensis (Alexeev, 1979) (Coleoptera: Buprestidae) (Wang et al. 2018). M. ohbayashii primoriensis is univoltine and overwinter as larvae on branches, with pupation beginning in mid to late April and adult emergence in early May to early July. Female adults lay eggs on leaf scars of J. regia. The larvae are distributed on the branches of 2- to 3-year-old plants and mainly cause damage to branch bark. Adult J. regia feed on leaves. Although the species does not kill host plants immediately, it can reduce water and nutrient flow, resulting in leaf loss and reduced fruit yields and thereby contributing to tree mortality (Wang and Li 1993; Chen et al. 2015; Wang et al. 2015).
Plant volatiles, used for chemical signaling, affect insect behavior and can contribute to interactions between plants and insects. Specific plant volatiles can help insects locate a plant and select an oviposition site or can serve as repellents (Heil and Ton 2008; Hiltpold et al. 2010; Gish et al. 2015). Many recent studies have focused on the behavioral responses of insects to host volatile emissions; both the quantitative and qualitative characteristics of herbivore-induced changes in the emission of volatiles have attracted increasing attention (Nottingham et al. 1991; Musetti and Neal 1997; Kessler and Baldwin 2001; Mauck et al. 2010; Ramadan et al. 2011; Tamiru et al. 2012). The emerald ash borer locates its host, Agrilus planipennis (Fairmaire, 1888) (Coleoptera: Buprestidae), by plant volatile emissions (Rodriguez-Saona et al. 2006; Grant et al. 2010). It is specifically attracted by (3Z)-lactone and (3Z)-hexenol from leaves of host plants (Groot et al. 2008; Ryall et al. 2012; Ryall et al. 2013). Prior work has evaluated volatiles from healthy walnut leaves, husks, and kernel oils (Farag 2008; Abdallah et al. 2015; Sarles et al. 2017) as well as the behavioral response of pests to volatiles of healthy plants, such as Rhagoletis completa (Cresson, 1929) (Diptera: Tephritidae), Cydia pomonella (L., 1758), (Lepidoptera: Tortricidae), Amyelois transitella (Walker, 1863) (Lepidoptera: Pyralidae), and Batocera horsfieldi Hope, 1839 (Coleoptera: Cerambycidae) (Yang et al. 2011; Román et al. 2015; Sarles et al. 2017). However, studies of the volatiles of J. regia infested with M. ohbayashii primoriensis are lacking. We compared the emission of volatiles from J. regia infested with M. ohbayashii primoriensis and healthy control plants. The objective of this study was to improve our understanding of host plant volatiles and to provide a basis for the development of ecologically sustainable strategies for herbivore control.
Materials and methods
Plant material
An infested J. regia fruit garden of 26.67 hm2, located in Yonghe County, Shanxi, China, was studied. Ten individuals of J. regia were chosen for headspace collection. All plants were 20 years old with an average diameter at breast height of 19.9 cm, tree height of 6-8 m, and crown diameter of 5-6 m.
Herbivore material
J. regia was widely infested with M. ohbayashii primoriensis. Larvae were observed at densities of 10-15 larvae/tree. Volatile compounds were collected from walnuts infested with M. ohbayashii primoriensis.
Headspace collection
At 9:00 am each day, volatiles were collected from healthy and herbivore-infested plants using a dynamic headspace method following a procedure similar to that described by Bäckman et al. (2001). Each aerial part was covered with an oven bag (48.26 cm × 23 cm × 1.27 cm; Reynolds, Richmond, CA, USA). The air was first exhausted from the bag, and the bag was then refilled with air percolated through an activated charcoal filter. Plastic bags were used only once to avoid contamination. For sampling, air was cycled through the oven bag, which was connected to a glass tube (length, 10.60 cm; inner diameter, 0.35 cm; outer diameter 0.85 cm) and filled with 200 mg of Porapak Q (80/100 mesh; Waters Corporation, Milford, MA, USA). Plant volatiles were sampled for 3 hours using a portable battery-operated air sampler with a constant flow of 100 ml/min. Subsequently, the glass tubes were taken to the laboratory and washed five times with 200 μl of dichloromethane to extract samples into 2 ml clear screw vials with patch Borosilicate (9 mm, CNW). The vials were kept at -20 °C until analysis.
GC-MS analysis
A Shimadzu 2010 (Shimadzu, Tokyo, Japan) gas chromatograph was equipped with a split-splitless auto-injector (model AOCi) and an auto sampler (model AOC-20i), and a MS-QP 2010 (Shimadzu) series mass selective detector was used for sample analysis. The analytical conditions were as follows: GC: column, Rtx-5MS (30.0 m × 0.25 mm × 0.25 μm; J&W, Palo Alto, CA, USA); carrier gas, helium; flow rate, 1.01 ml/min; column temperature, 40 °C held for 2 min, then at the rate of 6 °C / min to 180 °C (held for 2 min), and 15 °C/min to 270 °C. MS: analytical mode, full-scan; mass range, m/z 50-400.
Identification of volatile compounds
Retention indices (RI) were calculated based on retention times of n-alkanes, which were injected after volatiles under the same conditions. Relative contents were calculated based on GC peak areas without correction factors. The volatile compounds were identified by comparing mass spectral data with those of authentic samples in a mass spectra library (Turbo Mass ver. 5.4.2, NIST11), and the compounds were confirmed by comparing RI values with published data (Davies 1990; Farag 2008; Abdallah et al. 2016).
Results
Volatile compound detection
The volatile compounds identified from healthy plants and plants infested by J. regia are listed in Table 1. We identified 26 major volatile compounds. With respect to green leaf volatiles, we detected the monoterpenoids β-phellandrene and (E)-β-ocimene, the sesquiterpenoids (-)-β-bourbonene, β-ylangene, (E,E)-α-farnesene, the alcohols linalool, myrtenol, and (E)-(-) -pinocarveol, the ketones (E)-pinocamphone and (Z)-pinocamphone, and the ester methyl salicylate in all herbivore-infested plants.
Table 1 Compositional variation in healthy (H) and buprestid beetle-infestation (AR) Juglans regia.
| No. | Compounds | RIa sample | RI stand. | Relative content ± SE (%) | |
|---|---|---|---|---|---|
| H | AR | ||||
| Monoterpene hydrocarbons | |||||
| 1 | α-Pinene | 939 | 940 | 23.81 ± 0.20 | 20.36 ± 0.22 |
| 2 | Camphene | 947 | 950 | 0.95 ± 0.05 | 0.31 ± 0.04 |
| 3 | Sabinene | 973 | 973 | 8.63 ± 0.22 | 4.52 ± 0.18 |
| 4 | β-Pinene | 976 | 978 | 36.26 ± 0.25 | 37.82 ± 0.20 |
| 5 | β- Myrcene | 991 | 989 | 4.35 ± 0.21 | 2.90 ± 0.20 |
| 6 | D-Limonene | 1027 | 1029 | 12.03 ± 0.08 | 14.71 ± 0.21 |
| 7 | β-Phellandrene | 1031 | 1031 | - b | 3.80 ± 0.19 |
| 8 | (E)-β-Ocimene | 1050 | 1052 | - b | 0.25 ±0 .09 |
| Sesquiterpene hydrocarbons | |||||
| 9 | (-)-β-Bourbonene | 1384 | 1384 | - b | 0.40 ± 0.03 |
| 10 | Longifolene | 1398 | 1399 | 1.22 ± 0.04 | 0.77 ± 0.05 |
| 11 | β-ylangene | 1428 | 1428 | - b | 0.42 ± 0.01 |
| 12 | l-Caryophyllene | 1434 | 1435 | 1.52 ± 0.02 | - b |
| 13 | (E)-β-Farnesene | 1440 | 1440 | 2.13 ± 0.02 | 3.50 ± 0.05 |
| 14 | (E,E)-α-Farnesene | 1458 | 1458 | - b | 0.37 ± 0.03 |
| 15 | Humulene | 1454 | 1454 | 0.38 ± 0.01 | - b |
| 16 | Germacrene D | 1490 | 1491 | 2.26 ± 0.02 | 5.24 ± 0.04 |
| Esters | |||||
| 17 | (Z)-3-Hexen-1-ol acetate | 992 | 993 | 2.62 ± 0.05 | 0.58 ± 0.02 |
| 18 | Methyl salicylate | 1190 | 1190 | - b | 0.12 ± 0.01 |
| Alcohols | |||||
| 19 | Eucalyptol | 1059 | 1060 | 1.43 ± 0.04 | 1.75 ± 0.02 |
| 20 | Linalool | 1082 | 1084 | - b | 0.27 ± 0.02 |
| 21 | (E)-(-)-Pinocarveol | 1131 | 1132 | - b | 0.04 ± 0.06 |
| 22 | Myrtenol | 1191 | 1189 | - b | 0.05 ± 0.03 |
| Ketones | |||||
| 23 | (E)-Pinocamphone | 1164 | 1164 | - b | 0.12 ± 0.08 |
| 24 | (Z)-Pinocamphone | 1165c | 1165c | - b | 0.81 ± 0.06 |
| 25 | m-Ethylacetophenone | 1242c | 1244c | 1.08 ± 0.09 | 0.04 ± 0.08 |
| Aromatic hydrocarbons | |||||
| 26 | Indane | 1047c | 1048c | 0.23 ± 0.02 | 0.03 ± 0.01 |
A: Observed retention index. B: Not detected. C: Compounds for which RI values have not been previously reported in the literature. Compounds were verified by comparison with an authentic standard when possible and compared to internally generated database values. When standards were not available, retention indexes and/or mass spectra were only compared with internally generated data and/or with NIST (NIST02).
Healthy J. regia
The volatile constituents of the aerial parts of J. regia were mainly classified as terpenoids (93.54 %) (monoterpenoids (86.03 %) and sesquiterpenoids (7.51 %), esters (2.62 %), alcohols (1.43 %), ketones (1.08 %), and aromatic hydrocarbons (0.23 %). The most abundant components were β-pinene (36.26 %), α-pinene (23.81 %), D-limonene (12.03%), sabinene (8.63 %), and β-myrcene (4.35 %).
Buprestid beetle-infested (herbivore-infested plants) J. regia
Buprestid beetle-infested volatiles were mainly classified as terpenoids (95.37 %) [monoterpenoids (84.67 %) and sesquiterpenoids (10.70 %)], alcohols (2.11 %), ketones (0.97 %), esters (0.70 %) and aromatic hydrocarbons (0.03 %). The most abundant components were β-pinene (37.82 %), α-pinene (20.36 %), D-limonene (14.71 %), germacrene D (5.24 %), sabinene (4.52 %), and β-phellandrene (3.80 %). Additionally, we detected the monoterpenoids β-phellandrene and trans-β-ocimene, sesquiterpenoids β-ylangene, (-)-β-bourbonene, and (E,E)-α-farnesene, alcohols linalool, myrtenol, and (E)-(-)-pinocarveol, ketones (Z)-pinocamphone and (E)-pinocamphone, and ester methyl salicylate in herbivore-infested plants.
In addition to these qualitative differences, beetle infestation induced quantitative changes in the proportions of terpenoids. In particular, β-pinene, D-limonene, germacrene D, and (E)-β-farnesene contents were higher and α-pinene, sabinene, β-myrcene, camphene, and longifolene contents were lower in infested plants than in healthy plants.
Discussion
Herbivore attacks increase the emission of volatiles (Kessler and Baldwin 2001). The release of volatiles can improve plant resistance by attracting natural enemies (parasitoids and predators) of the herbivore as an indirect defense mechanism (Metcalf and Kogan 1987; Dicke 1994; Turlings and Benrey 1998; Kessler and Baldwin 2001).
Terpenoids are the largest class of natural products. Terpenoid phytoalexins play important roles in attacking herbivores by direct and indirect defense mechanisms (Mumm and Hilker 2006; Gershenzon and Dudareva 2007). The volatiles D-limonene, sabinene, α-pinene, (E,E)-α-farnesene, (E)-β-ocimene, and methyl salicylate, which we detected in our experiments, are very important compounds for the attraction of herbivore enemies (Hardie et al. 1994; Ament et al. 2010).
The relative β-pinene content was higher after herbivore attack than in healthy plants. Inversely, α-pinene contents were lower in infested plants than in healthy plants. The ratio of α-pinene to β-pinene was higher in healthy J. regia than in plants infested with M. ohbayashii primoriensis. Ning et al. (2006) concluded that a low ratio of α-pinene to β-pinene indicates declining host health, consistent with our observations of a relatively low ratio in infested plants. However, further research is needed to verify whether the condition of host plants could be determined by the ratio of α-pinene to β-pinene.
We detected eucalyptol in healthy plants. Eucalyptol, linalool, myrtenol, and (E)-(-)-pinocarveol were present in all herbivore-infested plants. Alcohol compounds play an important role in the attraction of herbivores (Kessler and Baldwin 2001). We detected (Z)-3-hexen-1-ol acetate and methyl salicylate in herbivore-infested plants, both of which contribute to the attraction of herbivore enemies (Hardie et al. 1994; James 2003).
We analyzed the volatiles emitted by healthy J. regia and plants infested with M. ohbayashii primoriensis. We detected qualitative and quantitative differences in the volatile profiles, with particularly notable variation in the terpenoids composition. Dynamic headspace coupled with GC/MS was suitable for the qualitative and quantitative investigation of volatiles in walnut. This approach can be used for further investigations of plant-derived attractants of herbivore enemies, volatile compositions, and ecologically sustainable strategies for the control of pests.
