Clustering Analysis of Volatile Components from the Tea Plants Infested by Tea Aphid (Toxoptera aurantii)

doi:10.11983/CBB21031

Abstract

Abstract: Shuchazao tea (Camellia sinensis cv. ‘shuchazao’) is a newly cultivated tea species in southwest Anhui, China. To investigate the volatile release profiles of tea aphid (Toxoptera aurantii) infested tea shoots, gas chromatography-mass spectrometry (GC-MS) analysis was used to compare the volatile compositions and relative contents in healthy and infested tea shoots. 16 volatile organic compounds (VOCs) were detected in healthy tea shoots with fewer relative contents, whereas more volatiles (24 VOCs) were released with increasing relative contents in tea plants induced by tea aphid feeding. Unsupervised clustering analysis based on the significantly different volatiles indicated that the volatile profiles in healthy and tea aphid infested tea shoots apparently showed clustering characteristics. A model was created by supervised partial least square discrimination analysis (PLS-DA) for volatiles with statistical significance. By experimental verification, the model could clearly discriminate healthy tea shoots from aphid infested ones (R²X=0.903, R²Y=0.875). By calculating the variable importance for the projection (VIP), seven important volatiles (α-pinene, longifolene-(V4), benaldehyde, E-5,9-undecadien-2-one,6,10-dimethyl-, 3-hexenal, camphor and decanal) were identified which collectively contribute to discrimination between healthy and infested tea shoots. The current work has preliminarily demonstrated the changing patterns in tea plant volatiles after tea aphid infestation to provide novel theoretical guidance for tea aphid management.

Key words: tea plant volatiles, GC-MS, PLS-DA, tea aphid, clustering analysis

Tingzhe Sun, Zehua Qi, Kexin Liang, Qin Li, Yuchun Rao, Dan Mu. Clustering Analysis of Volatile Components from the Tea Plants Infested by Tea Aphid (Toxoptera aurantii)[J]. Chinese Bulletin of Botany, 2021, 56(4): 422-432.

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.chinbullbotany.com/EN/10.11983/CBB21031

https://www.chinbullbotany.com/EN/Y2021/V56/I4/422

Figures/Tables 7

Table 1 The relative content of volatile organic compounds (VOCs) from healthy and infested tea shoots by Toxoptera aurantii (means±SE)

No.	Retention time (min)	Volatile organic compounds	Relative content
No.	Retention time (min)	Volatile organic compounds	Healthy	Infested
C1	4.842	3-hexenal	0	1.3204±0.3300**
C2	6.984	Ethylbenzene	0.3174±0.0895	0.8110±0.2546
C3	7.391	Benzene,1,3-dimethy-	0.7028±0.0881	1.5338±0.5370
C4	8.261	p-xylene	0.1596±0.0382	0.5888±0.1525
C5	8.572	2-heptanone	0	0.6570±0.2495**
C6	10.057	α-pinene	0	0.3518±0.0670**
C7	11.485	Benaldehyde	0	0.5694±0.1158**
C8	13.402	Decane	0	0.1562±0.0716**
C9	13.658	Octanal	0.0350±0.0080	0.3076±0.1001
C10	14.796	2-ethyl-1-hexanol	0.4416±0.1631	1.1204±0.2834
C11	16.637	Acetophenone	0.0990±0.0240	0.4676±0.1435
C12	18.471	Undecane	0.0756±0.0166	0.1842±0.0535
C13	18.700	Nonanal	0.1240±0.0379	0.5608±0.2060*
C14	19.071	E-2-butenoic acid, 2-(methylencyclopropyl)prop-2-ylester	0	0.0336±0.0123**
C15	20.682	Camphor	0.1174±0.0318	0.5498±0.1094**
C16	21.912	E-2-nonen-1-ol	0	0.1422±0.0804**
C17	22.387	Naphthalene	0.0552±0.0174	0.2874±0.0868*
C18	23.367	Dodecane	0.0334±0.0092	0.1366±0.0350*
C19	23.666	Decanal	0.1188±0.0332	0.6450±0.3569*
C20	28.033	Tridecane	0.0160±0.0036	0.0384±0.0142
C21	32.427	Tetradecane	0.0358±0.0094	0.1904±0.0688*
C22	32.612	Longifolene-(V4)	0	0.0648±0.0141**
C23	34.363	E-5,9-undecadien-2-one,6,10-dimethyl-	0.0470±0.0118	0.2866±0.1340*
C24	40.538	Hexadecane	0.0408±0.0126	0.2320±0.1007*

Figure 1 The total ion chromatograms of the volatile organic compounds from healthy (A) and infested (B) tea shoots by Toxoptera aurantii C1-C24 are the same as Table 1. IS: Internal standard.

Table 2 Classification of volatile organic compounds from tea plants

Volatile classification	Volatile compounds
GLVs	3-hexenal and 2-ethyl-1-hexanol
Aromatics	Ethylbenzene, benzene,1,3-dimethy-, p-xylene, benaldehyde, acetophenone and naphthalene
Terpenes	α-pinene, E-5,9-undecadien-2-one,6,10-dimethyl-, E-2-butenoic acid, 2-(methylen-cyclopropyl)prop-2-ylester and longifolene-(V4)
Alkanes	Decane, undecane, dodecane, tridecane, tetradecane and hexadecane
Other	2-heptanone, octanal, nonanal, camphor, E-2- nonen-1-ol and decanal

Figure 2 The categories and proportions of volatile organic compounds in healthy and infested tea shoots (A) The relative contents of five kinds of volatiles in healthy and infested tea shoots (**P<0.01); (B) The proportion of five volatile compounds in healthy and infested tea shoots. GLVs is the same as given in Table 2.

Figure 3 Two-dimensional embedding and clustering for volatiles from tea shoots (A) The t-distributed stochastic neighbor embedding (t-SNE) for volatile organic compounds (VOCs) from 5 healthy (No. 1-5) and 5 infested (No.6-10) groups; (B) Clustering analysis for VOCs from 10 groups above

Figure 4 Principal component analysis for volatile organic compounds (VOCs) of tea plants (A) The principal component scores for VOCs from 5 healthy (red points, owing to the partial overlap among points in healthy group, the number was not marked) and 5 infested (No.6-10, blue points) groups; (B) Loading plot for the first (red) and second (blue) principal components (the number of volatiles is the same as Table 1)

Figure 5 The partial least square discriminant analysis for volatile contents of tea plants (A) The fraction of cumulative explained variations for the first two latent variables (LVs) (red: predictor, blue: response); (B) Scores for the healthy and infested groups with respect to the LV1 and LV2 (the eclipse denotes the 95% confidence interval based on Hotelling T2); (C) The biplot in partial least squares discrimination analysis (the radius for solid and dashed circle is 1.0 and 0.5, respectively); (D) Variable importance for the projection (VIPs) for each volatile (the number is the same as Table 1) organic compound (the dashed line is a guideline for 1.0)

References 65

[1]	蔡晓明 (2016). 茶小绿叶蝉与植物间化学通讯物质的鉴定与田间功能验证. 博士论文. 北京: 中国农业科学院. pp. 48-50.
[2]	崔林, 张新亭, 周宁宁, 叶火香, 余继忠, 祝愿, 韩宝瑜 (2015). 茶互利素和蚜性信息素及其组合调控大草蛉行为的效应. 生态学报 35, 1537-1546.
[3]	董燕梅, 张文颖, 凌正一, 李靖锐, 白红彤, 李慧, 石雷 (2020). 转录因子调控植物萜类化合物生物合成研究进展. 植物学报 55, 340-350. DOI
[4]	范培珍, 韩善捷, 韩宝瑜 (2020). 灰茶尺蠖为害诱导茶树释放的互利素的鉴定. 中国生物防治学报 36, 65-71.
[5]	付建玉 (2017). 茶树倍半萜类物质代谢及其对虫害胁迫响应. 博士论文. 北京: 中国农业科学院. pp. 17-20.
[6]	韩宝瑜, 周成松 (2004). 茶梢和茶花信息物引诱有翅茶蚜效应的研究. 茶叶科学 24, 249-254.
[7]	韩善捷, 潘铖, 韩宝瑜 (2016). 假眼小绿叶蝉为害致茶梢挥发物变化及其引诱微小裂骨缨小蜂效应. 中国生物防治学报 32, 142-148.
[8]	焦龙, 蔡晓明, 边磊, 罗宗秀, 李兆群, 辛肇军, 陈宗懋 (2018). 茉莉酸类化合物: 从植物的诱导抗虫防御反应到生长-防御权衡. 应用生态学报 29, 3876-3890.
[9]	林郑和, 钟秋生, 陈常颂, 陈志辉, 游小妹 (2015). 不同香型茶树鲜叶挥发性组分与β-葡萄糖苷酶的相关性分析. 植物学报 50, 713-720.
[10]	苗爱清, 吕海鹏, 孙世利, 王力, 庞式, 赖兆祥, 曾琼, 林智 (2010). 乌龙茶香气的HS-SPME-GC-MS/GC-O研究. 茶叶科学 30, 583-587.
[11]	穆丹 (2011). 茶树挥发性信息素调控假眼小绿叶蝉及叶蝉三棒缨小蜂行为的功效. 博士论文. 北京: 中国农业科学院. pp. 44.
[12]	孙晓玲, 董文霞, 蔡晓明, 桂连友, 陈宗懋 (2016). 外用不同浓度茉莉酸甲酯诱导的茶树挥发物的种类和时序变化. 应用昆虫学报 53, 499-506.
[13]	王国昌 (2010). 三种害虫诱导茶树挥发物的生态功能. 博士论文. 北京: 中国农业科学院. pp. 20-22.
[14]	薛皎亮, 贺珺, 谢映平 (2008). 植物挥发物对天敌昆虫异色瓢虫的引诱效应. 应用与环境生物学报 14, 494-498.
[15]	左照江, 张汝民, 高岩 (2009). 植物间挥发物信号的研究进展. 植物学报 44, 245-252. DOI
[16]	Aartsma Y, Leroy B, van der Werf W, Dicke M, Poelman EH, Bianchi FJJA (2019). Intraspecific variation in herbivore-induced plant volatiles influences the spatial range of plant-parasitoid interactions. Oikos 128, 77-86. DOI URL
[17]	Abdullah ZS, Ficken KJ, Greenfield BPJ, Butt TM (2014). Innate responses to putative ancestral hosts: is the attraction of western flower thrips to pine pollen a result of relict olfactory receptors? J Chem Ecol 40, 534-540. DOI PMID
[18]	Baldwin IT, Kessler A, Halitschke R (2002). Volatile signaling in plant-plant-herbivore interactions: what is real? Curr Opin Plant Biol 5, 351-354. PMID
[19]	Bian L, Sun XL, Cai XM, Chen ZM (2014). Slow release of plant volatiles using sol-gel dispensers. J Econ Entomol 107, 2023-2029. DOI PMID
[20]	Bustos-Segura C, Foley WJ (2018). Foliar terpene chemotypes and herbivory determine variation in plant volatile emissions. J Chem Ecol 44, 51-61. DOI PMID
[21]	D'Alessandro M, Brunner V, von Mérey G, Turlings TCJ (2009). Strong attraction of the parasitoid Cotesia marginiventris towards minor volatile compounds of maize. J Chem Ecol 35, 999-1008. DOI PMID
[22]	De Moraes CM, Lewis WJ, Paré PW, Alborn HT, Tumlinson JH (1998). Herbivore-infested plants selectively attract parasitoids. Nature 393, 570-573. DOI URL
[23]	Gish M, De Moraes CM, Mescher MC (2015). Herbivore- induced plant volatiles in natural and agricultural ecosystems: open questions and future prospects. Curr Opin Insect Sci 9, 1-6.
[24]	Guo H, Wang CZ (2019). The ethological significance and olfactory detection of herbivore-induced plant volatiles in interactions of plants, herbivorous insects, and parasitoids. Arthropod-Plant Interactions 13, 161-179. DOI URL
[25]	Han BY, Chen ZM (2002a). Composition of the volatiles from intact and mechanically pierced tea aphid-tea shoot complexes and their attraction to natural enemies of the tea aphid. J Agric Food Chem 50, 2571-2575. DOI URL
[26]	Han BY, Chen ZM (2002b). Composition of the volatiles from intact and tea aphid-damaged tea shoots and their allurement to several natural enemies of the tea aphid. J Appl Entomol 126, 497-500. DOI URL
[27]	Han BY, Chen ZM (2002c). Behavioral and electrophysiological responses of natural enemies to synomones from tea shoots and kairomones from tea aphids, Toxoptera aurantii. J Chem Ecol 28, 2203-2219. DOI URL
[28]	Han BY, Han BH (2007). EAG and behavioral responses of the wingless tea aphid Toxoptera aurantii (Homoptera: Aphididae) to tea plant volatiles. Acta Ecol Sin 27, 4485-4490. DOI URL
[29]	Han BY, Zhang QH, Byers JA (2012). Attraction of the tea aphid, Toxoptera aurantii, to combinations of volatiles and colors related to tea plants. Entomol Exp Appl 144, 258-269. DOI URL
[30]	Heil M, Land WG (2014). Danger signals-damaged-self recognition across the tree of life. Front Plant Sci 5, 578.
[31]	Heil M, Silva Bueno JC (2007). Within-plant signaling by volatiles leads to induction and priming of an indirect plant defense in nature. Proc Natl Acad Sci USA 104, 5467-5472. DOI URL
[32]	Huffaker A, Pearce G, Veyrat N, Erb M, Turlings TCJ, Sartor R, Shen ZX, Briggs SP, Vaughan MM, Alborn HT, Teal PEA, Schmelz EA (2013). Plant elicitor peptides are conserved signals regulating direct and indirect antiherbivore defense. Proc Natl Acad Sci USA 110, 5707-5712. DOI URL
[33]	Jing TT, Du WK, Gao T, Wu Y, Zhang N, Zhao MY, Jin JY, Wang JM, Schwab W, Wan XC, Song CK (2021). Herbivore-induced DMNT catalyzed by CYP82D47 plays an important role in the induction of JA-dependent herbivore resistance of neighboring tea plants. Plant Cell Environ 44, 1178-1191. DOI URL
[34]	Jolliffe IT, Cadima J (2016). Principal component analysis: a review and recent developments. Philos Trans A Math Phys Eng Sci 374, 20150202.
[35]	Joo Y, Schuman MC, Goldberg JK, Kim SG, Yon F, Brütting C, Baldwin IT (2018). Herbivore-induced volatile blends with both “fast” and “slow” components provide robust indirect defence in nature. Funct Ecol 32, 136-149. DOI URL
[36]	Kitano H (2004). Biological robustness. Nat Rev Genet 5, 826-837.
[37]	Kost C, Heil M (2006). Herbivore-induced plant volatiles induce an indirect defence in neighbouring plants. J Ecol 94, 619-628. DOI URL
[38]	Lee LC, Liong CY, Jemain AA (2018). Partial least squares-discriminant analysis (PLS-DA) for classification of high-dimensional (HD) data: a review of contemporary practice strategies and knowledge gaps. Analyst 143, 3526-3539. DOI URL
[39]	Liu GH, Yang M, Fu JY (2020). Identification and characterization of two sesquiterpene synthase genes involved in volatile-mediated defense in tea plant (Camellia sinensis). Plant Physiol Biochem 155, 650-657. DOI URL
[40]	Matsui K (2006). Green leaf volatiles: hydroperoxide lyase pathway of oxylipin metabolism. Curr Opin Plant Biol 9, 274-280. PMID
[41]	Mu D, Cui L, Ge J, Wang MX, Liu LF, Yu XP, Zhang QH, Han BY (2012). Behavioral responses for evaluating the attractiveness of specific tea shoot volatiles to the tea green leafhopper, Empoaca vitis. Insect Sci 19, 229-238. DOI URL
[42]	Nerio LS, Olivero-Verbel J, Stashenko E (2010). Repellent activity of essential oils: a review. Bioresour Technol 101, 372-378. DOI URL
[43]	Ninkovic V, Markovic D, Rensing M (2021). Plant volatiles as cues and signals in plant communication. Plant Cell Environ 44, 1030-1043. DOI URL
[44]	Pare PW, Tumlinson JH (1999). Plant volatiles as a defense against insect herbivores. Plant Physiol 121, 325-332. PMID
[45]	Park J, Thomasson JA, Gorman Z, Brewer MJ, Rooney WL, Kolomiets MV (2020). Multivariate analysis of sorghum volatiles for the fast screening of sugarcane aphid infestation. J Asia-Pacific Entomol 23, 901-908. DOI URL
[46]	Sanou A, Traoré F, Ba MN, Dabiré-Binso CL, Pittendrigh BR, Sanon A (2019). Effects of volatiles from Clavigralla tomentosicollis Stål.(Hemiptera: Coreidae) adults on the host location behavior of the egg parasitoid Gryon fulviventre (crawford) (Hymenoptera: scelionidae). Int J Insect Sci 11, 1179543318825250.
[47]	Schmelz EA (2015). Impacts of insect oral secretions on defoliation-induced plant defense. Curr Opin Insect Sci 9, 7-15. DOI PMID
[48]	Schröder R, Forstreuter M, Hilker M (2005). A plant notices insect egg deposition and changes its rate of photosynthesis. Plant Physiol 138, 470-477. PMID
[49]	Shiojiri K, Ozawa R, Takabayashi J (2006). Plant volatiles, rather than light, determine the nocturnal behavior of a caterpillar. PLoS Biol 4, e164. DOI URL
[50]	Simpson M, Gurr GM, Simmons AT, Wratten SD, James DG, Leeson G, Nicol HI (2011). Insect attraction to synthetic herbivore-induced plant volatile-treated field crops. Agric Forest Entomol 13, 45-57. DOI URL
[51]	Sobhy IS, Erb M, Turlings TCJ (2015). Plant strengtheners enhance parasitoid attraction to herbivore-damaged cotton via qualitative and quantitative changes in induced volatiles. Pest Manag Sci 71, 686-693. DOI URL
[52]	Sun XL, Wang GC, Cai XM, Jin S, Gao Y, Chen ZM (2010). The tea weevil, Myllocerinus aurolineatus, is attracted to volatiles induced by conspecifics. J Chem Ecol 36, 388-395. DOI URL
[53]	Takabayashi J, Takahashi S, Dicke M, Posthumus MA (1995). Developmental stage of herbivore Pseudaletia separata affects production of herbivore-induced synomone by corn plants. J Chem Ecol 21, 273-287. DOI PMID
[54]	Turlings TCJ, Erb M (2018). Tritrophic interactions mediated by herbivore-induced plant volatiles: mechanisms, ecological relevance, and application potential. Annu Rev Entomol 63, 433-452. DOI PMID
[55]	Turlings TCJ, Tumlinson JH, Lewis WJ (1990). Exploitation of herbivore-induced plant odors by host-seeking parasitic wasps. Science 250, 1251-1253. PMID
[56]	van der Maaten L, Hinton G (2008). Visualizing data using t-SNE. J Mach Learn Res 9, 2579-2605.
[57]	Wang XW, Zeng LT, Liao YY, Li JL, Tang JC, Yang ZY (2019). Formation of α-farnesene in tea ( Camellia sinensis) leaves induced by herbivore-derived wounding and its effect on neighboring tea plants. Int J Mol Sci 20, 4151. DOI URL
[58]	Xiu CL, Zhang W, Xu B, Wyckhuys KAG, Cai XM, Su HH, Lu YH (2019). Volatiles from aphid-infested plants attract adults of the multicolored Asian lady beetle Harmonia axyridis. Biol Control 129, 1-11. DOI URL
[59]	Ye J, Zhang LL, Zhang X, Wu XJ, Fang RX (2021). Plant defense networks against insect-borne pathogens. Trends Plant Sci 26, 272-287. DOI URL
[60]	Ye M, Glauser G, Lou YG, Erb M, Hu LF (2019). Molecular dissection of early defense signaling underlying volatile- mediated defense regulation and herbivore resistance in rice. Plant Cell 31, 687-698. DOI URL
[61]	Yu B, Zhang D, Yan XW, Wang JW, Yao L, Tan LH, Zhao SP, Li N, Cao WG (2016). Comparative evaluation of the chemical composition, antioxidant and antimicrobial activities of the volatile oils of hawk tea from six botanical origins. Chem Biodivers 13, 1573-1583. DOI URL
[62]	Zeng LT, Liao YY, Li JL, Zhou Y, Tang JC, Dong F, Yang ZY (2017). α-farnesene and ocimene induce metabolite changes by volatile signaling in neighboring tea (Camellia sinensis) plants. Plant Sci 264, 29-36. DOI URL
[63]	Zhang ZQ, Bian L, Sun XL, Luo ZX, Xin ZJ, Luo FJ, Chen ZM (2015). Electrophysiological and behavioural responses of the tea geometrid Ectropis obliqua(Lepidoptera: Geometridae) to volatiles from a non-host plant, rosemary, Rosmarinus officinalis (Lamiaceae). Pest Manag Sci 71, 96-104. DOI URL
[64]	Zhang ZQ, Sun XL, Xin ZJ, Luo ZX, Gao Y, Bian L, Chen ZM (2013). Identification and field evaluation of non-host volatiles disturbing host location by the tea geometrid, Ectropis obliqua. J Chem Ecol 39, 1284-1296. DOI URL
[65]	Zhao MY, Wang L, Wang JM, Jin JY, Zhang N, Lei L, Gao T, Jing TT, Zhang SR, Wu Y, Wu B, Hu YQ, Wan XC, Schwab W, Song CK (2020). Induction of priming by cold stress via inducible volatile cues in neighboring tea plants. J Integr Plant Biol 62, 1461-1468. DOI URL