| Guizhou is located in the core area of the karst region of southern China,with a unique ecological environment.The leafhoppers,the largest group in the Hemiptera,poses a threat to crops,vegetables,fruit trees,and trees.They rely on their sensitive and specific olfactory system to identify chemical information substances in the surrounding environment,and complete the positioning,identification and selection of hosts,spouses,prey.Camellia tetracocca,as the ancestor of tea,is an important genetic resource for improvement of tea varieties.The Empoasca onukii Matsuda,1952 is a major pest of the C.tetracocca and seriously affects its growth.Therefore,the interaction mechanism between the tea green leafhopper and C.tetracocca were researched involving in the recognition of odor molecules by the tea green leafhopper,a variety of proteins,such as OBPs,CSPs,IRs,and SNMPs,as well as the response of the C.tetracocca to the feeding of the tea green leafhopper.It can lay a theoretical foundation for the green control of the tea green leafhopper.Therefore,this study focuses on the important agricultural pest（E.onukii）,and conducts a correlation analysis between the secondary metabolites of the host plant and the physicochemical properties of the soil environment it inhabits.The aim is to analyze the effects of various elements in the soil on the host plant.The differences in the number of tea green leafhoppers under different site conditions were analyzed,the olfactory related protein genes were identified to elucidate the role of olfactory related proteins in their chemical communication processes at the transcriptome level.This study investigates the changes in metabolites and differentially expressed genes in the response of C.tetracocca fed by E.onukii,revealing the defense response of C.tetracocca to E.onukii from multiple perspectives such as differential metabolites and genes by the combination of metabolomics and transcriptomics.The fumigation activity of the tea green leafhopper was investigated by using plant essential oils.The above studies are expected to provide a theoretical basis for the breeding of C.tetracocca seedlings and lay a scientific foundation for the green prevention and control of the E.onukii.The main research findings are as follows:（1）The coupling relationship between soil properties and the leaves of C.tetracocca in karst and non karst regions was elucidated.The total potassium（K）in the soil was positively correlated with epicatechin（EC）in C.tetracocca,total phosphorus（P）was negatively correlated with EC,and Mg was negatively correlated with EGC.There are differences between soil and C.tetracocca about organic carbon（SOC）,total nitrogen（N）,total phosphorus（P）,total potassium（K）,total calcium（Ca）,total magnesium（Mg）.Simultaneously,catechins（C）,caffeine（CAFF）,gallic acid（GC）,epicatechin（EC）,epicatechin gallate（ECG）,epicatechin gallate（EGC）,epicatechin gallate（ECGC）,L-theanine,and 17 free amino acids（Asp,Glu,Ser,His,Gly,Thr,Arg,Ala,Tyr,Cys,Val,Met,Phe,Ile,Leu,Lys,Pro）were determined in C.tetracocca.The correlation analysis shows that K in the soil is positively correlated with EC in the leaves of C.tetracocca,P is negatively correlated with EC,and Mg is negatively correlated with EGC.Therefore,increasing the content of K in the soil and reducing the content of Mg and P can improve the secondary metabolites of C.tetracocca.Despite the harsh environment in karst areas,C.tetracocca still grows well and its secondary metabolites are not lower than those in non karst areas.At the same time,the sweeping method was used to collect E.onukii on the C.tetracocca in karst and non karst areas.The results showed that there was no significant difference in the number of tea green leafhoppers.This research result is of great significance for protecting C.tetracocca in karst areas of Guizhou and promoting the cultivation of C.tetracocca in Guizhou.（2）The olfactory organ morphology of the tea green leafhopper in the karst area of Guizhou was clarified,and the olfactory related genes of the tea green leafhopper and the volatile components of the host plant were identified.The binding characteristics of OBPs of the tea green leafhopper and the main volatile components of C.tetracocca were analyzed.Sensors are the smallest structural and functional units on insect sensory organs,and their morphology is closely related to their physiology.Therefore,studying the morphological structure and sensor types of the antennae and leg of the tea green leafhopper is of great significance.The antennaes and legs of tea green leafhoppers have three types of sensory,including Sensilla trichoidea,Sensilla basiconic,microtricchia.There is no significant difference in antennaes and legs between female and male adults.Four major transcriptomes of chemosensory tissue were constructed in the tea green leafhopper,namely female antennae,female leg,male antennae,and male leg.Using the Illumina Hi Seq2500 sequencing platform,12 samples were sequenced,resulting in 79.92 GB of data and splicing of 46,291 Unigenes with a total length of 47,102,395 bp.Unigenes were annotated databases,involving NR（22,104）,Swissprot（16,203）,KEGG（7,652）,KOG（14,597）,egg NOG（19,129）,GO（14,852）,Pfam（15,267）.Finally,33 genes related to olfactory were identified,including 16 OBPs,13 CSPs,3 IRs,and 1SNMP.Using FPKM values to analyze the gene expression profile,multiple genes with gender differences or tissue-specific expression were identified,which provide basic data for the exploration of the olfactory mechanism of tea green leafhopper in the future.The volatiles released by plants play a crucial role in the process of tea green leafhopper searching for the C.tetracocca.The volatile components of C.tetracocca were analyzed by HS-SPEM-GC-MS.The main volatile compounds were alcohols,aldehydes,esters,alkanes,olefins,ketones,and nitriles.Its main components are linalool（14.021%）,（E）-hexenal（2.841%）,α-farnesene（6.75%）.These three compounds serve as the main informational compounds for potential tea green leafhopper searching for C.tetracocca.Molecular docking was performed on these three compounds with OBP3,OBP4,and OBP5 of the tea green leafhopper by MOE2022 software.The results showed that linalool had the best binding performance with OBP5.During the binding process,combined forces of hydrophobic and hydrogen bonding make the protein and ligand more stable.（3）The response mechanism of C.tetracocca in karst areas of Guizhou to tea green leafhopper were discussed,the differential metabolites and genes,as well as changes in volatile components were clarified by metabolomics（GC-MS and LC-MS）,transcriptomics,headspace solid-phase microextraction combined with gas chromatography-mass spectrometry（HS-SPEM-GC-MS）.Plants have diverse morphological,physiological,biochemical,and molecular mechanisms to protect themselves from herbivorous insects.In this study,GC-MS and LC-MS were used to analyze the metabolic profile of tea green leafhopper feeding on C.tetracocca.A total of 56 differential metabolites were identified by GC-MS analysis,of which 24 were upregulated and 32 were downregulated.It mainly includes organic acids and derivatives,heterocyclic compounds,benzene compounds,lipids and lipid compounds,and organic oxygen compounds.A total of 568differential metabolites were identified by LC-MS analysis,of which 280differential metabolites were upregulated and 286 differential metabolites were downregulated.It mainly contains flavonoids,flavanols,flavonoid glycosides,flavonoid glycosides,flavonoid glycosides,amino acids,peptides,phenolic acids,hydrolyzable tannins,glycoside aroma precursors,and proanthocyanidins.The accumulation of many specific metabolites in the leaves of C.tetracocca indicates that they are induced by attacked insects.Most flavonoids are down regulated,while terpenoids are up regulated.Based on the reported literature,we summarize and focus on using differential metabolites such as momordicine I and arabinose B for the prevention and control of tea green leafhopper.The differences gene expression were compared between damgaed leaves and healthy leaves using RNA Seq technology for transcriptome sequencing.Differential expression gene analysis revealed a total of8,172 differentially expressed genes,including 3,074 upregulated genes and 5,098 downregulated genes.Through differential gene expression analysis,it was found that most key enzyme genes in the synthesis pathway of catechins,including phenylalanine aminolysis gene（PAL）,chalcone isomerase gene（CHI）,flavonoid 3’-hydroxylase gene（F3’H）,flavanone 3-hydroxylase gene（F3H）,flavanol synthase gene（FLS）,and dihydroflavonol reductase gene（DFR）,were significantly downregulated after feeding by the tea green leafhopper.The relative content of terpenoids in plant volatiles induced by most pests is significantly higher than that in healthy plant volatiles.Genes related to terpenoids,including（E,E）-α-farnesene synthase and（E,E）-geranyl linalool synthase are upregulated.Especially,The expression level of（E,E）-α-farnesene synthase in leaves damaged by the tea green leafhopper is 70 times higher than that in healthy leaves.（E,E）-α-faniene synthase and（E,E）-linalool synthase,these candidate genes,can be used for genetic improvement of future C.tetracocca to enhance insect resistance.（4）Plant essential oil types with high safety,enviromental friendliness,and can effectively prevent and control tea green leafhopper were selected,and protection strategies for the seedling breeding of C.tetracocca was proposed.Plant essential oils have a wide range of sources,high biological activity against target pests,low resistance to drugs,high safety,and easy degradation.We selected plant essential oils with multiple strong and special odors as the research objects,and found that five essential oils have good fumigation activity.When the concentration of essential oil was 1μL·m L-1and the fumigation time was 48 h,the order of its fumigation activity is Cnidium monnieri（70.00%）,Houttuynia cordata（68.75%）,Zanthoxylum bungeanum（56.92%）,Stemona japonica（54.55%）,and Juniperus sabina（40.30%）.From the LC50values of different plant essential oils,C.monnieri essential oil and H.cordata essential oil can be used as the main essential oils for preventing and controlling tea green leafhopper.However,this study found for the first time that H.cordata can be used for the prevention and control of tea leafhoppers.In addition,as it is commonly used as a vegetable in the southwest region.Therefore,H.cordata can be intercropped with C.tetracocca seedlings to establish isolation zones and prevent the approach of tea green leafhoppers. |
