Empoasca Onukii Research Articles

Transcriptomic and proteomic analysis of putative digestive proteases in the salivary gland and gut of Empoasca (Matsumurasca) onukii Matsuda

BackgroundInfestation by tea green leafhoppers (Empoasca (Matsumurasca) onukii) can cause a series of biochemical changes in tea leaves. As a typical cell-rupture feeder, E. onukii secretes proteases while using its stylet to probe the tender shoots of tea plants (Camellia sinensis). This study identified and analyzed proteases expressed specifically in the salivary gland (SG) and gut of E. onukii through enzymatic activity assays complemented with an integrated analysis of transcriptomic and proteomic data.ResultsIn total, 129 contigs representing seven types of putative proteases were identified. Transcript abundance of digestive proteases and enzymatic activity assays showed that cathepsin B-like protease, cathepsin L-like protease, and serine proteases (trypsin- and chymotrypsin-like protease) were highly abundant in the gut but moderately abundant in the SG. The abundance pattern of digestive proteases in the SG and gut of E. onukii differed from that of other hemipterans, including Nilaparvata lugens, Laodelphax striatellus, Acyrthosiphum pisum, Halyomorpha halys and Nephotettix cincticeps. Phylogenetic analysis showed that aminopeptidase N-like proteins and serine proteases abundant in the SG or gut of hemipterans formed two distinct clusters.ConclusionsAltogether, this study provides insightful information on the digestive system of E. onukii. Compared to five other hemipteran species, we observed different patterns of proteases abundant in the SG and gut of E. onukii. These results will be beneficial in understanding the interaction between tea plants and E. onukii.

Formation of volatiles in response to tea green leafhopper (Empoasca onukii Matsuda) herbivory in tea plants: a multi-omics study.

Combined transcriptome and metabolome analysis of fresh leaf infestation by tea green leafhoppers (Empoasca (Matsumurasca) onukii Matsuda) suggests roles for alternative pre-mRNA splicing and mRNAs in the regulation of aroma formation in tea plants. Oriental Beauty is a high-grade, oolong tea with a pronounced honey-like aroma and rich ripe fruit flavor that develops primarily as a result of the infestation of the fresh leaves by tea green leafhoppers (Empoasca (Matsumurasca) onukii Matsuda). Here, we used PacBio Iso-Seq and RNA-seq analyses to determine the full-length transcripts and gene expression profiles of fresh tea leaves in response to E. (M.) onukii herbivory. We investigated the relationship between RNA-seq, tea metabolites, and aroma response mechanisms in leaves infested by leafhoppers. We found 3644 differentially expressed genes, of which 2552 were up- and 1092 were down-regulated. A total of 49,913 alternative splicing events were predicted, including 324 differential AS events. Moreover, 3105 differentially expressed transcripts were also identified, of which 2295 were up- and 810 were down-regulated. The characterization of expression patterns of the key gene transcript isoforms involved in the aroma formation pathways identified 130 differentially expressed metabolites, 97 of which were up- and 33 were down-regulated. Two key aroma compounds (phenylacetaldehyde and 4-hydroxybenzaldehyde) were highly correlated with genes of the aroma formation pathways. Our results revealed that pre-mRNA AS plays a crucial role in the metabolic regulation surrounding aroma formation under leafhopper herbivory in tea plants.

Differing non‐linear, lagged effects of temperature and precipitation on an insect herbivore and its host plant

1. Multivariate climate change is expected to impact insect densities and plant growth in complex, and potentially different, ways. Tea (Camellia sinensis) is a unique crop system where the increase in quality from chemical defences induced by Empoasca onukii (Homoptera: Cicadellidae) feeding can outweigh reductions in yield and make attack by this leafhopper desirable to tea farmers. Differential impacts of weather attributes on tea and herbivores could impact feasibility of this unique farming strategy in a rapidly changing climate.2. We monitored leafhopper densities and tea shoot growth at a tea farm in Fujian Province, China for 2 months (June and July). We used distributed lag non‐linear models to capture potentially delayed and non‐linear effects of weather attributes on tea growth and leafhopper densities.3. Weather attributes had contrasting effects on leafhopper density and tea shoot growth. Leafhopper densities were highest with low daily mean and maximum temperatures, while warm temperatures favoured tea growth. Effects of temperature on leafhoppers were delayed, while effects on tea growth were immediate. Precipitation reduced tea growth, and had a delayed positive effect on leafhopper density.4. The delayed effects of weather attributes on leafhoppers indicate that earlier, less conspicuous life stages (i.e. eggs and early instar nymphs) may be susceptible to desiccation. Although increasing annual mean temperature is commonly predicted to benefit multivoltine insect pests, our results show that cool, wet conditions benefit E. onukii during summer months when they are most abundant. These results have implications for tea green leafhopper management strategies.

Are Yellow Sticky Cards and Light Traps Effective on Tea Green Leafhoppers and Their Predators in Chinese Tea Plantations?

Simple SummaryLeafhoppers are serious insect pests in agriculture across the world. Both nymphs and adults suck the sap of plant shoots and leaves with their piercing–sucking mouthparts causing damage called “hopperburn”. The tea green leafhopper, Empoasca onukii, is one of most damaging pests in tea plantations in Asia. In China, yellow sticky cards and light traps are increasingly used to control leafhoppers in tea plantations, especially the tea green leafhopper. Visually, several leafhoppers appear to be captured and killed, however, the real control efficiency and the damage to natural enemies remains unclear. In our study, a 16-week open field experiment with daily weather monitoring was designed to test the responses of tea green leafhopper, parasitoids and spiders to yellow sticky cards and light traps (cover with sticky cards) that used different light colours. An exclosure experiment was also designed to further test the influence of the three light systems (without sticky card) on the same groups of species. The results suggested that light, especially green and white, can be useful as a way to control leafhopper populations without affecting parasitoids and spiders too much.In Chinese tea plantations, yellow sticky cards and light traps are increasingly used to control insect pests, especially the tea green leafhopper Empoasca onukii. In this study, a 16-week open-field experiment with daily weather monitoring was designed to test the responses of tea green leafhopper, parasitoids and spiders to yellow sticky cards and three light traps with different wavelengths (covered with sticky cards). An exclosure experiment was also designed to further test the influence of the three light systems (without sticky card) on the same species. The results showed that all three light emitting diode (LED) light traps (white, green and yellow) and yellow sticky cards attracted many more E. onukii male adults than females during the course of the open field experiment, with less than 25% of trapped adults being females. Parasitoids and spiders were also attracted by these systems. Weather variables, especially rainfall, influenced the trapping efficiency. In the exclosure experiment, the population of leafhoppers in the yellow sticky card treatment did not decline significantly, but the number of spiders significantly decreased. The green and white light treatments without sticky cards showed a significant control of E. onukii and no obvious harm to spiders. These results suggest that yellow sticky cards and light traps have limited capacity to control tea green leafhoppers. However, light, especially green light, may be a promising population control measure for tea green leafhoppers, not as killing agents in the traps, but rather as a behavioral control system.

Correlation of tea green leafhopper occurrence with leaf structure and biochemical components in different tea cultivars

Being one of the most disastrous pierce-sucking pests of tea plants [Camellia sinensis (L.) O. Kuntze], tea green leafhopper (Empoasca onukii Matsuda) greatly affects tea yield and quality. In this paper, correlations among leaf structure, biochemical components and tea green leafhopper occurrence in tea cultivars with larger planting area were deeply analyzed, aiming to find the key factor impacting the host selectivity of tea green leafhopper. Tea cultivar ‘Longjing 43’, together with ‘Shuchazao’, showed the most susceptibility to this pest with high tea polyphenols content. And the best resistant tea plant cultivar, ‘Caoxi No.1’, largely relied on the thicker leaf, epidermis (both upper and lower), cuticle and spongy tissue as revealed by principal component analysis. Here, we suggested that tea polyphenols were considered as a biochemical marker for determining the resistance ability to tea green leafhopper of different tea cultivars, along with leaf upper and lower structures as mechanical barriers, which can potentially be used to promote breeding of insect-resistant tea plants.

Development and characterization of novel microsatellite markers for a dominant parasitoid Stethynium empoasca (Hymenoptera: Mymaridae) in tea plantations using high-throughput sequencing

Stethynium empoasca Subba Rao (Hymenoptera: Mymaridae) is an egg parasitoid of the predominant tea pest, Empoasca onukii Matsuda (Hemiptera: Cicadellidae), and is regarded as the most promising candidate for both augmentative and conservative biological control on E. onukii. However, little is presently known about its biology, ecology, and genetics. Here, we developed and characterized novel microsatellite markers for S. empoasca from transcriptome sequences generated using high-throughput sequencing. A total of 54,520 microsatellites were isolated from 117 Mb clean sequences. Fifty primers were developed and used to characterize 98 individuals from five geographically distinct populations, of which 18 loci were demonstrated to be polymorphic, stable, and repetitive. These loci also showed a low frequency of null alleles, suggesting little effect on their use in the estimation of genetic diversity and structure. Additionally, the assessed populations showed low to moderate levels of genetic differentiation, in which individuals clearly grouped into two clusters, indicating that the loci could reveal a pattern of spatial structure and gene flow in S. empoasca populations according to geographic variability. This work provides an important basis for future studies on how these markers can be used in studies of the biology, genetics, and ecology of this important parasitoid.

Evaluation of Botanicals for Management of Piercing-Sucking Pests and the Effect on Beneficial Arthropod Populations in Tea Trees Camellia sinensis (L.) O. Kuntze (Theaceae).

The tea green leafhopper Empoasca onukii Matsuda (Hemiptera: Cicadellidae), the orange spiny whitefly, Aleurocanthus spiniferus (Quaintanca) (Hemiptera: Aleyrodidae), and the green plant bugs Apolygus lucorum Meyer-Dür (Hemiptera: Miridae) are the important piercing–sucking herbivores in tea trees Camellia sinensis (L.) O. Kuntze (Theaceae). The goal of this study was to evaluate the laboratory toxicities and field control efficacies of botanical insecticides including matrine, azadirachtin, veratrine, and pyrethrin to three tea pests. Via leaf-dip bioassay, toxicity tests with botanical insecticides indicated that there were significant differences between the LC50 values for botanical insecticides within the same insect species. Matrine had the highest toxicity to E. onukii, A. spiniferus, and A. lucorum with the LC50 values of 2.35, 13.10, and 44.88 mg/liter, respectively. Field tests showed that, among four botanical insecticides, matrine at dose of 9 g a.i. ha−1 can significantly reduce the numbers of E. onukii and A. spiniferus and the infestation of A. lucorum on the tea plants. Furthermore, botanical insecticides matrine and azadirachtin had no obvious influence on the coccinellids, spiders, and parasitoids densities in tea plantations. The results of this study indicated that use of botanical insecticides, such as matrine, has the potential to manipulate the population of E. onukii, A. spiniferus, and A. lucorum and will be an effective and environmentally compatible strategy for the control of tea pests.

Identifying the Biological Characteristics Associated with Oviposition Behavior of Tea Leafhopper Empoasca onukii Matsuda Using the Blue Light Detection Method.

Simple SummaryThe tea leafhopper (Empoasca onukii Matsuda) is currently one of the most threatening pests in tea gardens in China. To locate and count tea leafhopper eggs, stereomicroscopy is a conventional method by dissecting the tender tissues, which is both time- and labor-consuming. The scarcity of a verified method to directly observe and investigate intact eggs within tea shoots impedes the research efforts to test the oviposition behavior of E. onukii. Herein, comparing against the stereomicroscope detection method (SMDM), we evaluated the blue light detection method (BLDM), a technique recently developed for other species in detecting E. onukii eggs directly and non-destructively within the tender shoot for four tea cultivars. The conclusion indicated that BLDM could correctly measure the egg laying quantity of E. onukii on intact tea shoots, and its accuracy was not affected by both tea cultivars and egg density in the tender shoot. Furthermore, the biological characteristics concerning the oviposition behaviors that have rarely been reported previously for E. onukii were investigated using the BLDM. Our findings provide insights for the basic research and theoretical evidence, and allow the follow-up studies for the strategy and mechanism associated with the egg laying behavior of E. onukii.Tea leafhopper (Empoasca onukii Matsuda) is amongst the key pests in tea plantations around the East Asian region. Stereomicroscopy is a conventional method used for detecting tea leafhopper eggs by dissecting the tender tissues. However, there is a need for a faster and more efficient method to directly observe and investigate intact eggs within tea shoots. The absence of a proven method limits research efforts for determining the oviposition behavior of E. onukii. Herein, we applied the blue light detection method (BLDM), a technique recently developed for other species, in order to detect E. onukii eggs directly and non-destructively within the tender shoot. In addition, we compared BLDM against the traditional stereomicroscope detection method (SMDM) for four tea cultivars. Notably, our results revealed that BLDM was precise and effective in measuring the egg laying quantity of E. onukii on intact tea shoots. Neither tea cultivars nor egg density in the tender shoot significantly affected the accuracy of BLDM. Furthermore, biological characteristics that have rarely been reported previously for E. onukii were investigated using the BLDM, including zygote duration, ovipositional rhythm, egg distribution within the tender shoot, and in different leaf positions, numbers of eggs laid by a single female daily, and laid by the entire generation. Therefore, these findings provide insights into the basic and theoretical evidence for the strategy and mechanism associated with the oviposition behavior of E. onukii.

Foliage Intensity is an Important Cue of Habitat Location for Empoasca onukii.

For many herbivorous insects, vision is more important than olfaction in the prealighting stage of host habitat location. Tea leafhoppers, Empoasca onukii (Hemiptera, Cicadellidae), are serious pests that preferentially inhabit the tender leaves of tea plants across China. Here, we investigated whether tea leafhoppers could distinguish foliage colors associated with different leaf ages and use this visual cue to guide suitable habitat location from short distances. Similar to honeybees, the adult E. onukii has an apposition type of compound eye, and each ommatidium has eight retinular cells, in which three spectral types of photoreceptors are distributed, with peak sensitivities at 356 nm (ultraviolet), 435 nm (blue), and 542 nm (green). Both changes in spectral intensity and hue of reflectance light of the host foliage were correlated with varying leaf age, and the intensity linearly decreased with increasing leaf age. Behavioral responses also showed that adult E. onukii could discriminate between the simulated colors of host foliage at different leaf ages without olfactory stimuli and selected the bright colors that strongly corresponded to those of tender leaves. The results suggest that, compared with the spectral composition (hue), the intensity of light reflectance from leaves at different ages is more important for adult leafhoppers when discriminating host foliage and could guide them to tender leaves at the top of tea shoots.

Susceptibility of Selected Tea Shoots to Oviposition by Empoasca onukii (Hemiptera: Cicadellidae) and Feasibility of Egg Removal with Harvesting.

The Empoasca onukii (Hemiptera: Cicadellidae) female lays its eggs inside the epidermis of the tea plant shoots. This has led to speculation that shoot harvesting could represent a method of egg removal. To verify the validity of this hypothesis, we sought to determine which part of the shoot was used for the oviposition and how the value of the harvested shoot affects the cost of the egg removal. In this study, four tea cultivars were chosen to examine the preferences for the site of oviposition. In addition, a mathematical model was used to describe the correlation between the economic value of the selected shoot and eggs laid within the shoot. Our study revealed that the pest preferred the 3rd and 4th leaf order intervals of the shoot as the oviposition sites, and the oviposition preferences was dependent on the leaf order interval class across all tea cultivars. In addition, a significant negative exponential relationship was found between the economic value of the selected shoot and the percentage of the eggs laid within the shoot, indicating that egg removal through shoot harvesting was limited. The findings of this study could be used to better understand the role of shoot harvesting in egg removal and would provide new insights into the understanding of the incidence of this pest.

Changes in Tea Plant Secondary Metabolite Profiles as a Function of Leafhopper Density and Damage.

Insect herbivores have dramatic effects on the chemical composition of plants. Many of these induced metabolites contribute to the quality (e.g., flavor, human health benefits) of specialty crops such as the tea plant (Camellia sinensis). Induced chemical changes are often studied by comparing plants damaged and undamaged by herbivores. However, when herbivory is quantitative, the relationship between herbivore pressure and induction can be linearly or non-linearly density dependent or density independent, and induction may only occur after some threshold of herbivory. The shape of this relationship can vary among metabolites within plants. The tea green leafhopper (Empoasca onukii) can be a widespread pest on tea, but some tea farmers take advantage of leafhopper-induced metabolites in order to produce high-quality “bug-bitten” teas such as Eastern Beauty oolong. To understand the effects of increasing leafhopper density on tea metabolites important for quality, we conducted a manipulative experiment exposing tea plants to feeding by a range of E. onukii densities. After E. onukii feeding, we measured volatile and non-volatile metabolites, and quantified percent damaged leaf area from scanned leaf images. E. onukii density had a highly significant effect on volatile production, while the effect of leaf damage was only marginally significant. The volatiles most responsive to leafhopper density were mainly terpenes that increased in concentration monotonically with density, while the volatiles most responsive to leaf damage were primarily fatty acid derivatives and volatile phenylpropanoids/benzenoids. In contrast, damage (percent leaf area damaged), but not leafhopper density, significantly reduced total polyphenols, epigallocatechin gallate (EGCG), and theobromine concentrations in a dose-dependent manner. The shape of induced responses varied among metabolites with some changing linearly with herbivore pressure and some responding only after a threshold in herbivore pressure with a threshold around 0.6 insects/leaf being common. This study illustrates the importance of measuring a diversity of metabolites over a range of herbivory to fully understand the effects of herbivores on induced metabolites. Our study also shows that any increases in leafhopper density associated with climate warming, could have dramatic effects on secondary metabolites and tea quality.

(E)-Nerolidol is a volatile signal that induces defenses against insects and pathogens in tea plants

Plants release large amounts of volatile organic compounds (VOCs) in response to attackers. Several VOCs can serve as volatile signals to elicit defense responses in undamaged tissues and neighboring plants, but many questions about the ecological functions of VOCs remain unanswered. Tea plants are impacted by two harmful invaders, the piercing herbivore Empoasca (Matsumurasca) onukii Matsuda and the pathogen Colletotrichum fructicola. To determine the VOC signals in tea, we confirmed CsOPR3 as a marker gene and set up a rapid screening method based on a 1.51 kb CsOPR3 promoter fused with a β-glucuronidase (GUS) reporter construct (OPR3p::GUS) in Arabidopsis. Using this screening system, a terpenoid volatile (E)-nerolidol was identified as a potent signal that elicits plant defenses. The early responses triggered by (E)-nerolidol included the activation of a mitogen-activated protein kinase and WRKY, an H2O2 burst, and the induction of jasmonic acid and abscisic acid signaling. The induced plants accumulated high levels of defense-related chemicals, which possessed broad-spectrum anti-herbivore or anti-pathogen properties, and ultimately triggered resistance against Empoasca onukii and Colletotrichum fructicola in tea. We propose that these findings can supply an environmentally friendly management strategy for controlling an insect pest and a disease of tea plants.

Ecologically controlling insect and mite pests of tea plants with microbial pesticides: a review.

Insect and mite pests are damaging stressors that are threatening the cultivation of tea plants, which result in enormous crop loss. Over the years, the effectiveness of synthetic pesticides has allowed for its prominent application as a control strategy. However, the adverse effects of synthetic pesticides in terms of pesticide residue, environmental contamination and insect pest resistance have necessitated the need for alternative strategies. Meanwhile, microbial pesticides have been applied to tackle the damaging activities of the insect and mite pests of tea plants, and their performances were scientifically adjudged appreciable and environmental friendly. Herein, entomopathogenic microbes that were effective against tea geometrid (Ectropis obliqua Prout), tea green leafhopper (Empoasca onukii Matsuda), paraguay tea ampul (Gyropsylla spegazziniana), tea mosquito bug (Helopeltis theivora Waterhouse) and red spider mite (Oligonychus coffea Nietner) have been reviewed. The current findings revealed that microbial pesticides were effective and showed promising performances against these pests. Overall, this review has provided the basic and integrative information on the integrated pest management (IPM) tool(s) that can be utilized towards successful control of the aforementioned insect and mite pests.

Defensive Responses of Tea Plants (Camellia sinensis) Against Tea Green Leafhopper Attack: A Multi-Omics Study.

Tea green leafhopper [Empoasca (Matsumurasca) onukii Matsuda] is one of the most devastating pests of tea plants (Camellia sinensis), greatly impacting tea yield and quality. A thorough understanding of the interactions between the tea green leafhopper and the tea plant would facilitate a better pest management. To gain more insights into the molecular and biochemical mechanisms behind their interactions, a combined analysis of the global transcriptome and metabolome reconfiguration of the tea plant challenged with tea green leafhoppers was performed for the first time, complemented with phytohormone analysis. Non-targeted metabolomics analysis by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS), together with quantifications by ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-QqQ MS), revealed a marked accumulation of various flavonoid compounds and glycosidically bound volatiles but a great reduction in the level of amino acids and glutathione upon leaf herbivory. RNA-Seq data analysis showed a clear modulation of processes related to plant defense. Genes pertaining to the biosynthesis of phenylpropanoids and flavonoids, plant-pathogen interactions, and the biosynthesis of cuticle wax were significantly up-regulated. In particular, the transcript level for a CER1 homolog involved in cuticular wax alkane formation was most drastically elevated and an increase in C29 alkane levels in tea leaf waxes was observed. The tea green leafhopper attack triggered a significant increase in salicylic acid (SA) and a minor increase in jasmonic acid (JA) in infested tea leaves. Moreover, transcription factors (TFs) constitute a large portion of differentially expressed genes, with several TFs families likely involved in SA and JA signaling being significantly induced by tea green leafhopper feeding. This study presents a valuable resource for uncovering insect-induced genes and metabolites, which can potentially be used to enhance insect resistance in tea plants.

Characterization of Terpene Synthase from Tea Green Leafhopper Being Involved in Formation of Geraniol in Tea (Camellia sinensis) Leaves and Potential Effect of Geraniol on Insect-Derived Endobacteria.

When insects attack plants, insect-derived elicitors and mechanical damage induce the formation and emission of plant volatiles that have important ecological functions and flavor properties. These events have mainly been studied in model plants, rather than crop plants. Our study showed that tea green leafhopper (Empoasca (Matsumurasca) onukii Matsuda), a major pest infesting tea attack significantly induced the emission of geraniol from tea leaves, but did not affect the crude enzyme activity of geraniol synthase in tea leaves. An enzyme extract of E. (M.) onukii specifically produced geraniol from geraniol diphosphate. Furthermore, a terpene synthase (EoTPS) was isolated from E. (M.) onukii. This terpene synthase was able to convert geraniol diphosphate to geraniol in vitro. In addition, geraniol had in vitro ability to inhibit the growth of Acinetobacter johnsonii that is endobacterial isolated from E. (M.) onukii. This information illustrates that elicitors from piercing-sucking insects can induce the formation of volatiles from crop plants and advances our understanding of the roles of plant volatiles in the interaction among crops-insects-microorganisms.

Primary screening and application of repellent plant volatiles to control tea leafhopper, Empoasca onukii Matsuda.

The tea leafhopper, Empoasca onukii Matsuda (Hemiptera: Cicadellidae), is a major pest of tea plants in China. Here, we evaluated the repellent properties of eight volatile chemicals alone and in various combinations as tools for the management of this pest in tea gardens. These chemicals were from the Alliaceae and other aromatic plants, and are known to repel various insect species. Among the eight volatile compounds, dimethyl disulfide (DMDS), 1,8-cineole and allyl methyl sulfide were significantly repellent towards E. onukii adults. DMDS and 1,8-cineole were mixed to formulate a binary repellent. Under field conditions, spraying and slow-release applications of the mixture significantly decreased the density of E. onukii adults. The repelling effect after spraying was very short, only ∼ 2 days, but the slow-release mixture had a longer term repelling effect on E. onukii adults. High emission of the slow-release mixture, which was achieved by increasing the number of slow-release bottles, had a stronger repellent effect than low emission. Moreover, when the amount emitted was sufficient, the slow-release mixture significantly decreased the number of leafhopper nymphs in a treated tea-plant line, and significantly decreased the number of leafhopper adults and nymphs in a tea-plant line adjacent to the treated area. This study demonstrates the repellent action of a mixture of DMDS and 1,8-cineole applied by a slow-release method against E. onukii in a tea plantation. This mixture has potential applications in integrated pest management schemes. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Wound- and pathogen-activated de novo JA synthesis using different ACX isozymes in tea plant (Camellia sinensis)

Acyl-CoA oxidase (ACX; EC 1.3.3.6) plays a vital role in the biosynthesis of jasmonic acid (JA) in plant peroxisomes. We previously identified an herbivore-induced gene CsACX1 in tea plant (Camellia sinensis) and showed CsACX1 was involved in the wound-induced synthesis of jasmonic acid (JA). Here, another ACX gene CsACX3 was isolated from tea plant. CsACX3 was predicted to consist of 684 amino acid residues. CsACX3 can be induced by mechanical wounding, JA application, and infestation by the tea geometrid Ectropis obliqua Prout and the tea green leafhopper Empoasca (Matsumurasca) onukii Matsuda. These expression patterns are consistent with the previously reported expression pattern of CsACX1 under such treatments. Recombinant CsACX3 showed preference for medium-chain acyl-coA oxidase substrates (C8- to C14-CoA). CsACX3 expression could also be induced by the infection of a pathogen Colletotrichum gloeosporioides (Cgl), and the increased ACX activities in tea plants were correlated with the Cgl-induced CsACX3 expression. Cgl could not induce the expression of CsACX1, which showed preference for C12- to C16-CoA substrates. The constitutive expression of CsACX3 rescued wound-induced JA biosynthesis and enhanced the Cgl-induced JA biosynthesis in Arabidopsis mutant atacx1. However, constitutive expression of CsACX1 could not enhance the Cgl-induced JA biosynthesis in atacx1 plant. These results indicate that CsACX1 and CsACX3 functions overlap and have distinct roles in the wound- and pathogen-activated de novo JA synthesis via enzymatic routes that utilize different ACX isozymes in tea plant.

Cloning, phylogenetic research, and prokaryotic expression study of the metabolic detoxification gene EoGSTs1 in Empoasca onukii Matsuda.

Due to the misuse of chemical pesticides, small green leafhoppers (Empoasca onukii Matsuda) have developed resistance to pesticides, thereby posing a serious problem to the tea industry. Glutathione S-transferases (GSTs) are an important family of enzymes that are involved in pesticide resistance in Empoasca onukii Matsuda. Empoasca onukii GST sigma 1 (EoGSTs1, GenBank: MK443501) is a member of the GST family. In this study, the full-length cDNA of EoGSTs1 was cloned by reverse transcription polymerase chain reaction (qPCR), and its taxonomic identity was examined. Furthermore, we performed bioinformatics and phylogenetic analyses of the gene and structural and functional domain prediction of the protein. The results demonstrate that EoGSTS1 belongs to the Sigma family of GSTs; the full-length EoGSTs1 cDNA is 841 bp with a 624-bp coding region that encodes a 23.68932-kDa protein containing 207 amino acids. The theoretical isoelectric point (IEP) was calculated to be 6.00. Phylogenetic analysis indicates that EoGSTS1 is closely related to the Sub psaltriayangi subfamily of the Cicadoidea superfamily in order Hemiptera, whereas it is distantly related to Periplaneta americana of order Blattodea. Amino acid sequence alignment of EoGSTS1 and GSTs from four other insects of order Hemiptera revealed protein sequence conservation. Tertiary structure analysis and structural domain functional predictions of the protein revealed that EoGSTS1 contains nine α helices and two β sheets with one conserved GST domain. The results of enzyme activity assay showed that recombinant EoGSTs1 (rEoGSTs1) protein had catalytic activity for substrate 1-chloro-2,4-dinitrobenzene (CDNB) and exhibited the highest activity at pH 7 and 25 °C. The Michaelis constant Km of rEoGSTs1 protein was 0.07782 ± 0.01990 mmol/L, and the maximum reaction rate Vmax was 12.15 ± 1.673 µmol/min⋅mg. Our study clarified the taxonomic identity of small green leafhopper EoGSTs1 and revealed some properties of the gene and its encoded protein sequence. According to the catalytic activity of the rEoGSTs1 enzyme on the model substrate CDNB, we infer that it functions in the degradation of exogenous substances.

The impact of cover crops on the predatory mite Anystis baccarum (Acari, Anystidae) and the leafhopper pest Empoasca onukii (Hemiptera, Cicadellidae) in a tea plantation.

Empoasca onukii, the tea green leafhopper, is a key pest of tea whose control often requires the extensive use of insecticides. As a predator of the tea green leafhopper, the mite Anystis baccarum is a potential biological control agent worldwide, though little is known about how intercropping cover crops can impact its suppressing effect on E. onukii. Therefore, we conducted a field experiment to investigate how the relationship of the abundance of the predatory mite and its leafhopper prey is influenced by two different cover crops and a manually weeded inter-row treatment as a contrast to naturally growing vegetation in a tea plantation in China. The abundance of A. baccarum was significantly higher in tea canopies of intercropped treatments than in canopies over natural ground cover. Litter samples showed higher abundances of A. baccarum when tea was intercropped with Paspalum notatum than with natural ground cover in the first year of treatment. The abundance of E. onukii in tea canopies was higher over the bare ground treatment in the first year but the opposite was observed in the second year. Results suggest that the abundance of A. baccarum in a tea plantation is influenced by intercropping and it can affect its leafhopper prey, albeit with varying levels of suppression. For informing biological control and suppression of pests, long-term experiments are needed to investigate the interactions of both pest and predator with cover crop treatments. © 2019 Society of Chemical Industry.

Effect of partial pesticide spraying on the number of major pests and damage to new shoots of tea plants

Abstract We studied the effects of partial spraying, targeting tea [Camellia sinensis (L.) O. Kuntze] plants' plucking surfaces, on the annual number of major pests and damage to new shoots. The prevention of feeding damage to shoots caused by Empoasca onukii Matsuda (Hemiptera: Cicadellidae) was more stable when spraying at high volumes. However, the annual number of E. onukii with conventional spraying (200 L/1000 m2) was similar to the control (No pesticide). On the other hand, the prevention effect on shoots and the annual number of Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) in partial spraying (40 L/1000 m2) was similar to conventional spraying. Moreover, although high volumes of spraying (1000 L/1000 m2) are usually needed to control Pseudaulacaspis pentagona (Targioni-Tozetti) (Hemiptera: Diaspididae), the male adult population of P. pentagona in the control and partially sprayed field tended to be lower than that in the conventional spraying. When using pesticides harmful to P. pentagona's natural enemies, partial spraying seems beneficial. Enough pesticide spraying to widely cover the leaf layer was thought necessary to protect the tea plants' new shoots. However, the spraying method targeting only the tea plants' plucking surfaces protects the new shoots with similar efficacy. Besides, partial spraying protects pests' natural enemies, allowing additional pest population control.