Characterization of Volatile Compounds from Tea Plants (Camellia sinensis (L.) Kuntze) and the Effect of Identified Compounds on Empoasca flavescens Behavior

Abstract

The tea green leafhopper, Empoasca flavescens, is a major pest of tea Camellia sinensis (L.) Kuntze. Until recently, it has mainly been controlled by pesticides, but their use has led to high levels of toxic residues in plants, which threaten both the environment and human health. Therefore, a safer biological control approach is needed. Tea plants produce many volatile compounds, and different tea clones differ in their resistance to the pest. We explored the possibility that volatile compounds influence the resistance of tea. Here, we assessed the resistance of 15 clones of tea plants to the pest, the volatile compounds produced by the clones, and the effects of the compounds on E. flavescens behavior. Six clones were classified as resistant, eight as moderately susceptible, and one as susceptible. Fresh leaf samples from resistant and susceptible clones were analyzed using HS–SPME–GC–MS. Sesquiterpenes and monoterpenes were two major groups characterized, representing 30.15% and 26.98% of the total compounds, respectively. From our analysis, we conclude that 3-hexen-1-ol, 2,6-dimethyleneoct-7-en-3-one, humulene, β-bourbonene, styrene, and benzaldehyde were important for the resistance and susceptibility of the clones. In a bioassay, E. flavescens were attracted to β-ocimene and methyl salicylate, but avoided linalool compounds.

1. Introduction

Tea is a globally popular beverage prepared from the shoots of the tea plant, Camellia sinensis (L.) Kuntze, an evergreen shrub native to Asia. Tea is economically important throughout Southeast Asia and is one of Indonesia’s most important commercial crops. Tea shoots are delicate, and the plants are susceptible to a variety of insects and pathogens. Of particular concern is the tea green leafhopper, Empoasca flavescens (Hemiptera: Cicadellidae), which reduces the tea yield by 15–50% yearly. The adults and nymphs of the leafhopper pierce the shoot and suck the sap from the plant, which causes them to wither [1,2].

Growers have relied on pesticides to control the tea green leafhopper for many decades, but in recent years, as reported in China, the pests have developed resistance to some of the common pesticides, including imidacloprid, bifenthrin, and acetamiprid [3]. The reduced efficacy of these insecticides has caused growers to use more of them. This has led to more toxic residues in the tea and problems in the European tea market, the primary destination of Indonesian tea products. An environmentally sound alternative to control the leafhopper is needed.

Chemicals in the environment are detected by insects and other animals that inhabit the environment. These may function as survival-promoting environments for animals. In particular, an insect’s olfactory system is sensitive to volatile compounds that help it in finding habitat, food sources, mating, determining oviposition sites, and escaping from predators [4,5]. Tea plants produce many aromatic compounds that may attract the leafhopper; understanding the bases of these attractions is a potential avenue to control the pest. Different tea clones produce a plethora of volatile chemical substances, which are likely to affect the behavior of insects. For example, tea plants produce (Z)-3-hexenyl acetate and (Z)-3-hexenol, volatiles that induce oviposition in adult females of E. vitis [3], and they also produce (E)-ocimene, linalool, (E)-2-hexenal, (Z)- 3-hexen-1-ol, (Z)-3-hexenyl acetate, 2-penten-1-ol, (E)-2-pentenal, pentanol, hexanol, and 1-penten-3-ol. The latter group of volatile compounds attracts E. vitis [6].

Here, we sought to identify and classify tea clones that are resistant to the tea green leafhopper, identify the major volatiles produced and emitted by tea shoots, and assess the insect’s behavioral responses to the volatile compounds produced. The study will provide a foundation for the future development of a system, based on the volatile compounds produced by the tea plant, for monitoring, attracting, and trapping this major tea plantation pest.

2. Materials and Methods

2.1. Experimental Sites

The study was conducted from January to December 2021 at the Indonesian Research Institute for Tea and Cinchona (IRITC) experimental garden in Gambung, Bandung Regency, West Java, Indonesia (7” 08’37.3”S’ 107′’30’56.3’’E). The elevation of the site is 1350 m above sea level. The soil type is Andisols and the pH ranges between 4.5 and 6.0. Schmidt and Ferguson [7] classify the rainfall as category B.

2.2. Plant Materials

The materials used in the study were derived from a cross between TRI 2024 with PS 1 series clones, which were seeded in 1980 (Supplementary Table S1). The TRI 2024 clone is easy to propagate vegetatively, produces numerous shoots, and is of high grade; however, it is susceptible to blister blight disease, has a low shoot weight, and does not respond to nitrogenated fertilizers. Clone PS 1 is resistant to blister blight disease. We performed a cross of these clones to obtain clones with high production and resistance to both biotic and abiotic stresses.

Fifteen clones were tested and compared: I.35.8, II.6.10, II.10.11, II.13.2, II.32.15, III.2.15, III.22.15, III.28.4, III.36.15, TPS 17/3, TPS 24/5, TPS 87/1, TPS 87/2, TPS 93/3, and TPS 122/2. These clones were selected based on root development study, yield potential, and resistance to blister blight [8]. Each clone consisted of 12 plants. The plants were grown with a spacing of 120 × 80 cm. We also used one clone of GMB 7, which was known to be resistant to E. flavescens (Supplementary Table S1).

2.3. Resistance Selection in Tea Plants

Resistance tea clones to E. flavescens were selected by observing and assessing the population density of E. flavescens among the clones grown in the IRITC experimental garden in Gambung. Population observations were conducted using the beat bucket method. We used a randomized block design with a single factor for the experiment and replicated it three times.

2.4. Analysis of Tea Leaf Volatile Compounds

Five grams of each leaf sample from resistant and susceptible clones were harvested from the tea plant in the field between 15:00 and 17:00. The shoots were placed in a 22 mL glass vial fitted with a polytetrafluoroethylene/silicone septum (Agilent). The samples were either two leaves and a bud or three leaves and a bud (Figure 1).

The volatile compounds were extracted from the leaves and analyzed with a solid-phase microextraction (SPME)-headspace (HS)/gas chromatograph–mass spectrometer (GC–MS) (Lin et al., 2012). Tissue samples were extracted at 30 °C for 45 min with an SPME fiber coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS). The fiber was immediately inserted into the GC/MS (Agilent 7890A and Agilent 5975C XL EI/CI instruments, respectively) with splitless column effluent. The metabolites were separated using an HP-5MS column (30 m × 250 m × 0.25 m) with helium as the carrier gas, at a flow rate of 1 mL/min.

The oven temperature was programmed from 50 to 250 °C, and the total working time of the GC’s entire operation was 45 min. The MS transfer was conducted at 280 °C, the scanned mass ranged from 29 to 550 amu, the MS source was set to 230 °C, and the MS Quad was set to 150 °C. The oven temperature was held at 50 °C for 5 min and then increased to 220 °C at a rate of 3 °C/min. The retention times of the compounds were compared to those of authentic standards and their mass spectra, as well as to relevant data in the NIST14 Mixture Property Database. The compounds were quantified by comparing their GC total ion current peak areas to the internal standard peak area. The profile of the volatile compounds was analyzed with the principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), and heatmap clustering (MetaboAnalyst version 5.0).

2.5. Y-Tube Olfactometer Test

The behavioral responses of E. flavescens adults were evaluated to odors released from synthetic compounds in a Y-tube olfactometer (10 × 10 × 10 cm arm length, 3 cm diameter, 75° Y angle) (Supplementary Video S1). These bioassays were conducted between 08:00 and 11:00 or 15:00 and 17:00. Adult leafhoppers were collected randomly from the tea plants using the beat bucket technique, placed in cages (30 × 30 × 30 cm), and fed with tea shoots. For the bioassay, the insects were acclimated by transferring them to test tubes without food for an hour.

Insect responses were tested to the identified compounds, specifically β-ocimene, linalool, and methyl salicylate (Sigma-Aldrich Inc., Steinheim, Germany) with liquid paraffin as the control [2,6]. The volatile compounds were dissolved in liquid paraffin (1%), and 20 µL of solution or paraffin alone was applied to a filter paper square (1 × 1 cm). One arm of the olfactometer [6,9] received a filter paper with the sample and the other arm received paraffin only. Charcoal and humidified, filtered air was driven into each tube with a vacuum pump. After acclimation, individual leafhoppers were placed at the downwind end of the main tube. The insect’s ‘choice’ for either the control or compound was assessed based on when it crossed a line 3 cm past the fork of the base tube and remained there for at least 3 min. If a leafhopper did not choose within 10 min, it was considered unresponsive, and its activity was recorded as ‘no choice.’ After testing two leafhoppers, the olfactometer tube was rinsed with 70% ethanol, heated at 100 °C for 5 min, and the sample in each arm of the tube was reversed. At least 30 replicates were performed for each volatile compound tested [3].

2.6. Statistical Analysis

The analysis of variance of the population data was used to determine the significance of differences in the mean size of the pest populations in different clones. The analysis of variance was continued with Duncan’s multiple range test (DMRT) to group clones with similar mean sizes of pest populations. The results of this statistical analysis served as criteria to categorize the level of tea clone resistance to the leafhoppers. A large pest population associated with a clone indicates that the clone is susceptible to the pest [10]. The behavioral responses were analyzed using a non-parametric statistical binomial test. Data analyses were performed with SPSS 16.0 (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Resistance Selection of Tea Plant Resistance

The population density of a pest associated with the host plant is an important factor in the damage it can cause to the host. Here, the population density of E. flavescens was used as a parameter to assess the resistance of the different tea clones to it. The population density of E. flavescens was quite variable for the different clones, as shown in Figure 2, and ranged from 1.63 to 6.99 individuals per plant (Figure 2). We conclude that resistance to E. flavescens infestation in the field varied significantly across the 15 TPS clones tested.

The resistance level of the clones was assessed by comparing the population density of each clone to the known resistant clone GMB 7. Using this comparison, we conclude that the clones TPS 87/2, TPS 87/1, TPS 122/2, TPS 17/3, TPS 93/3, and TPS 24/5 are resistant clones. The clones with a population density of 4.62 to 6.99 individuals/plant were considered susceptible. Clones III.22.15, III.28.4, III.2.15, II.10.11, II.32.15, I.35.8, III.36.15, and II.13.2 were classified as moderately susceptible; only clone II.6.10 was susceptible.

3.2. Profile of Volatile Compounds of Resistant and Susceptible Tea Clones

Sixty-three volatile compounds were detected through the GC–MS analysis. The susceptible and moderately susceptible clones produced 53 different volatile compounds, while the resistant clones produced 47 different compounds. These compounds were classified into 11 groups i.e., alcohols, aldehydes or alkanes, ketones, carboxylic acids, esters, aromatics, monoterpenes, sesquiterpenes, alkaloids, fatty acids, sterols, and phenols (

Table 1. The volatile compounds produced by the tea clones and the chemical groups they belong to. Compounds that are important in the resistance or susceptibility of the clones to the leafhopper E. flavescens are listed.

No	RT (min)	Compound	Area (%)		Group
			Susceptible	Resistant
1	1.4828	acetaldehyde	16.61	19.35	Aldehyde/alkane
2	2.7629	cyclobutanol	4.41	5.03	Alcohol
3	3.7990	hexanal	0.53	1.39	Aldehyde/alkane
4	5.4946	3-hexen-1-ol. (E)-	1.33	-	Alcohol
5	6.6175	styrene	-	1.12	Aromatic
6	9.3340	benzaldehyde	-	0.18	Aromatic
7	10.2260	1-octen-3-ol	0.32	2.72	Alcohol
8	10.6340	β-myrcene	2.58	2.59	Monoterpene
9	12.0731	D-limonene	0.92	0.80	Monoterpene
10	12.5249	trans-β-ocimene	1.17	0.90	Monoterpene
11	12.9299	β-ocimene	10.18	5.24	Monoterpene
12	13.8942	trans-linalool oxide	1.09	0.82	Monoterpene
13	14.4410	terpinolene	0.05	0.05	Monoterpene
14	14.5413	cis-linalool oxide	2.23	1.90	Monoterpene
15	14.7960	4.8-dimethyl-1.3.7-nonatriene	0.35	0.22	Alcohol
16	15.2901	linalool	38.86	32.29	Monoterpene
17	15.6572	2.6-dimethyleneoct-7-en-3-one	-	0.68	Ketone
18	16.1447	allo-ocimene	0.86	0.61	Monoterpene
19	16.6140	neo-allo-ocimene	0.56	0.42	Monoterpene
20	17.1008	2.6-nonadienal. (E.Z)-	0.06	0.08	Aldehyde/alkane
21	17.5362	isoborneol	-	0.06	Monoterpene
22	17.7293	ethyl benzoate	0.10	0.08	Aromatic
23	17.9429	epoxylinalol	0.22	0.20	Monoterpene
24	18.0351	napthalene	0.05	-	Aromatic
25	18.7876	methyl salicylate	14.88	20.00	Carboxylic acid
26	18.8602	2-ethoxybenzoic acid	0.13	-	Carboxylic acid
27	19.2514	methyl aspirin	0.02	0.03	Carboxylic acid
28	19.8077	2-carene	0.03	-	Monoterpene
29	19.9878	cis-3-hexenyl-alpha-methylbutyrate	0.09	0.06	Ester
30	20.1350	cis-3-hexenyl valerate	0.11	0.11	Ester
31	20.3244	hexyl valerate	0.02	0.02	Ester
32	20.4539	isogeraniol	0.09	-	Monoterpene
33	20.8522	geraniol	0.15	0.41	Monoterpene
34	21.3253	ethyl salicylate	0.64	0.63	Carboxylic acid
35	22.2461	indole	0.12	0.18	Aromatic
36	22.8427	3-ethoxy-2-pyridinamine	0.02	-	Alkaloid
37	22.9760	methyl m-methoxymandelate	0.05	0.04	Aromatic
38	23.2036	octane. 2.4.6-trimethyl-	0.02	-	Fatty acid
39	25.1588	4-ethoxybenzaldehyde	-	0.17	Phenol
40	25.1649	β-bourbonene	-	0.01	Sesquiterpene
41	25.4249	β-elemene	-	0.03	Sesquiterpene
42	25.6863	cis-jasmone	0.04	-	Ketone
43	25.8880	isocaryophyllene	0.04	-	Sesquiterpene
44	26.0422	ledene	-	0.04	Sesquiterpene
45	26.2873	caryophyllene	0.49	0.79	Sesquiterpene
46	26.3850	cholestan-2-one oxime	0.01	0.01	Sterol
47	26.6687	β-cubebene	-	0.03	Sesquiterpene
48	27.2546	aromandendrene	0.06	0.03	Sesquiterpene
49	27.3715	humulene	-	0.20	Sesquiterpene
50	27.4949	(E)-β-farnesene	0.01	0.04	Sesquiterpene
51	27.6045	alloaromadendrene	0.01	0.04	Sesquiterpene
52	28.1071	γ-muurolene	0.01	-	Sesquiterpene
53	28.6874	longifolene	0.05	-	Sesquiterpene
54	29.0986	α-farnesene	0.23	0.07	Sesquiterpene
55	29.2642	θ-muurolene	0.01	-	Sesquiterpene
56	29.5492	calamenene	0.06	0.15	Sesquiterpene
57	30.1420	α-calacorene	0.01	-	Sesquiterpene
58	30.6387	trans-farnesol	0.01	-	Sesquiterpene
59	30.7600	E-nerolidol	0.02	0.15	Sesquiterpene
60	30.9116	6-octenal. 7-methyl-3-methylene-	0.01	-	Aldehyde/alkane
61	31.1558	(Z.E)-farnesol	0.04	0.02	Sesquiterpene
62	31.6900	7.9-dimethylhexadecane	0.01	0.01	Fatty acid
63	32.2819	α-patchoulene	0.02	-	Sesquiterpene

). Many of the compounds were mono- and sesquiterpenes; of the 63 compounds detected, 30.15% were monoterpenes and 26.98% sesquiterpenes.

PCA was used to identify the volatile compounds that conferred the characteristics of resistance and susceptibility to the clones. From the analysis, we can determine which characteristics contributed to the overall diversity of volatile chemicals generated. Three principal components (PCs) were used to represent the original volatile compounds, with cumulative variance levels of 74.7% for PC1 (39%), PC2 (19.4%), and PC3 (16.3%) (Figure 3A); we interpret this to mean that the combination of the two PCs accounted for 74.7% of the information obtained from the original. From the PCA analysis, we classified the compounds as belonging to two distinct groups: the resistant clone group (TPS 24/5; TPS 122/2; and TPS 93/3) or the susceptible clone group (II.10.11; II.13.2; and II.06.10). Some compounds were present in both groups; these were detected in the susceptible clone II.12.3 and the resistant clone TPS 93/3 (Figure 3B).

From the PLS-DA analysis, we identified the volatile compounds that distinguished resistant from susceptible clones (Figure 3C). Two major components were apparent, with a total variance of 79.3% (PC1 74.7% and PC3 4.9%). In general, the PC value describes how clones are grouped based on the compounds produced. Variable importance for the projection (VIP) scores greater than 1 identify important volatile substances within groups [1]. Fifteen compounds were detected, specifically 3-hexen-1-ol; 1-octen-3-ol; styrene; methyl salicylate; 2,6-dimethyleneoct-7-en-3-one; hexanal; β-ocimene; humulene; β-bourbonene; linalool; cis-linalool oxide; trans-linalool oxide; caryophyllene; α-farnesene, and benzaldehyde (Figure 3D). The color indicator in Figure 3D shows the concentration of the chemical in each clone. Dark red denotes a very high concentration, whereas dark blue indicates a very low concentration.

A heatmap was constructed to visualize the compounds that may confer resistance and susceptibility. This highlights the variations in the chemicals generated by each group of clones (Figure 4). Resistant clones produced several compounds absent in the susceptible clones, including 2,6-dimethyleneoct-7-en-3-one; humulene; β-bourbonene; styrene; 1-octen-3-ol; and benzaldehyde. In contrast, the susceptible clones produced 3-hexen-1-ol, which was not detected in the resistant clones. The compounds used to test the response of E. flavescens in the bioassay were β-ocimene, linalool, and methyl salicylate; these compounds are produced by all clones and are dominant.

3.3. The Response of E. flavescens Response to the Volatile Compounds Produced by Tea Plants

The significance of the preference of E. flavescens on the treatment was analyzed using the binomial test. E. flavescens showed attraction to methyl salicylate and β-ocimene, and tended to avoid linalool (Figure 5).

4. Discussion

We assessed the population density of E. flavescens associated with each of the 15 TPS clones we studied. From the results, we conclude that the responses of the clones to the pests were variable. The clones were classified based on the population densities of the pests associated with them in the field. Susceptible clones had significantly higher densities than those clones classified as resistant. From the established criteria, six clones were resistant, eight clones were moderately susceptible, and one clone was susceptible. Plant resistance to insect pests can be defined as the ability of plants to withstand pest attacks and minimize damage. Clones that are resistant to a pest generally suppress the development of the pest population, preventing economic losses from the infestation [11].

Plants have evolved various defense mechanisms to protect themselves from insect pests. Some emit volatile compounds that have direct effects on herbivores. These vary between different types of plants and may vary in the same plant in different situations. Plants use volatile compounds as indirect defenses as well. For example, a plant under attack by an herbivore may emit volatile pheromones that attract insect predators of the attacking herbivore. When “summoned” by the plant in this way, the predator destroys the initial pest; the plant’s volatiles may also communicate danger signals to other plants [12]. When tea plants are attacked by their common pests, they may emit numerous herbivore-induced plant volatiles, such as (Z)-3-hexenol, linalool, α-farnesene, benzyl nitrile, indole, nerolidol, and ocimenes at high concentrations [13].

Xin, Li, Bian, and Sun [3] studied the volatile compounds produced in the field by tea varieties that differed in resistance and susceptibility to E. vitis. They compared two resistant varieties, Changxingzisun and Juyan, with two susceptible varieties, Enbiao and Banzhuyuan. The population density of E. vitis associated with the two susceptible varieties was greater than that associated with the resistant varieties, confirming their classification as susceptible and resistant. They also measured the emission levels of (Z)-3-hexenyl in all four varieties. More of this volatile compound was produced by the susceptible varieties Enbiao and Banzhuyuan than by the two resistant varieties. The plant variety is one of the main factors that affect the composition of the volatile organic compound blend released by plants. This background inspired us to characterize the characteristics of volatile compounds emitted by resistant and susceptible tea clones.

GC–MS was used to analyze the volatile compounds produced by the tea clones that have been selected. Most of the detected volatiles were terpenoids, either monoterpenes or sesquiterpenes. In this regard, our results are similar to those of Lin, et al. [14], who observed that the volatile compounds in Longjing tea were dominated by sesquiterpenes. In addition to sesquiterpenes, others have detected monoterpenes, alcoholic terpenes, alcohols, esters, aldehydes, ketones, and alkanes in tea plants [14]. Terpenes in tea plants are important in biological processes unrelated to plant defense and are the main components in the aroma; indeed, the particular terpenes present are determinants of the tea quality [15,16]. Most of the distinctive aromatic compounds in tea are terpenes, specifically geraniol, farnesene, ocimene, linalool, and nerolidol [16]. Monoterpene compounds, such as linalool and sesquiterpene (E)-β-farnesene, have insect-repellent properties [15].

Each of the 15 clones in this study produced a different set of volatile compounds, which may be a distinguishing feature of that clone. The susceptible clones produced 3-hexen-1-ol, which is commonly found in fresh tea leaves. Its concentration increases when the plant is mechanically injured or attacked by pests. Xin, Li, Bian, and Sun [3] reported that adult male and female E. vitis were attracted to 3-hexen-1-ol compounds, and the females preferred the susceptible varieties Enbiao and Banzhuyuan for egg-laying over the resistant varieties. The susceptible varieties produced 3-hexen-1-ol and (Z)-3-hexenyl acetate compounds, while in the resistant varieties, these compounds were detected in much lower amounts. These investigators suggested that these two compounds are signals for adult female insects to detect and choose the location of oviposition.

The resistant clones in this study produced more compounds than the susceptible clones, specifically, humulene, 2,6-dimethyloct-7-en-3-one, β-bourbonene, styrene, and benzaldehyde. Humulene is important in plant resistance to pests. It has antimicrobial, anti-tumor, anti-fungal, and anti-inflammatory properties and is the main component of essential oils from various types of plants. The concentration of humulene varies in different plants but is part of the essential oils of many aromatic plants, notably: Salvia officinalis, Lindera strychnifolia Uyaku, ginseng, Mentha spicata, ginger, Litsea mushaensis, Cordia verbenacea, Vietnamese coriander, Humulus lupulus, pine trees, citrus, tobacco, and sunflowers [17,18]. Humulene has been developed as a biopesticide for several types of pests. For example, extracts of Zingiber officinale, Curcuma longa, and Alpinia galanga contain humulen and are effective for controlling the warehouse pests Sitophilus zeamais and Tribolium castaneum. The compound also inhibits the development of Conopomorpha cramerella Snellen pupae by as much as 49.75% compared to untreated pupae [18]. Humulene essential oil from leaves of Commiphora leptophloeos suppressed the oviposition of the Aedes aegypti mosquito by as much as 31.2% compared to the control [17]. Based on the results of this study, it may be that humulene is important in the defense system of resistant clones of tea to E. flavescens.

The volatile compound 1-octen-3-ol is known as a “fungal alcohol” and is usually associated with the fungi Aspergillus, Cladosporium, Mucor, and Ulocladium. It was produced by both resistant and susceptible clones. Detection of the aroma of 1-octen-3-ol can be an indicator of mold in an area. Application of 1-octen-3-ol suppressed oviposition of Drosophila suzukii on raspberry commodities by up to 55%, impaired locomotion, and caused lipid peroxidation, slow development, cell apoptosis, neurotoxicity, and inflammation [19,20]. Application of 98 L/L 1-octen-3-ol as a fumigant was 100% effective in eradicating the warehouse pest Tribolium castaneum and negatively affected the development and reproduction of adult T. castaneum and its progeny, resulting in decreased survival, reduced pupae weight, and reduced adult insect weight [21].

Beta-ocimene, linalool, and methyl salicylate were produced in the greatest amounts by the 15 clones in this study, compared to the other compounds detected. This was true for both resistant and susceptible clones. In insects, β-ocimene may both attract and repel. In this study, E. flavescens were attracted to methyl salicylate and β-ocimene. In another study, E. vitis and E. onukii were both attracted to (E)-ocimene in a laboratory bioassay using a Y-tube olfactometer [4,6]. Pseudotheraptus wayi recognized its host plant (the cashew) by detecting the ocimene compound, while its natural enemy, Oecophylla longinoda (Latreille) was also attracted to ocimene [22].

This research showed that E. flavescens was attracted not only to β-ocimene, but also to methyl salicylate. Methyl salicylate released by arthropods was attractive to both beneficial insects and insect pests. Methyl salicylate applied to yellow sticky traps attracts herbivores, including leafhoppers, plant bugs (Miridae), weevils, thrips, and soldier beetles in cranberries [23]. Methyl salicylate is also important for the beetle Anthonomus grandis to detect cotton plants, and it was the most effective attractant studied for the spotted lanternfly [24,25]. Linalool is the predominant compound in both fresh and prepared tea leaves. In this study, E. flavescens avoided linalool. Linalool can act as a repellent to female insects and suppress oviposition events and the number of eggs laid by female Ceratitis capitata on citrus fruits [26]. Linalool was also repellent to aphids, Lepidoptera, and thrips in transgenic Arabidopsis and Chrysanthemum plants that overexpressed the linalool synthesis gene [26]. We suggest that linalool might be a promising compound for pest control. The behavioral response shown by E. flavescens to volatile compounds needs to be studied further to determine the response at different doses and mixtures of compounds. Field testing is also needed to identify what risks are posed by these natural pesticides in the environment.

5. Conclusions

We conclude that the volatile compounds β-ocimene, linalool, and methyl salicylate tested have excellent potential to control E. flavescens in tea plants. This approach is environmentally friendly and should lead to reduced use of herbicides.