Journal of Tea Science

Research Progress in the Residue Analysis and Risk Assessment of Chiral Pesticides in Tea

HU Yue, NING Yating, LI Hongxia, LUO Fengjian, YIN Rongxiu, ZHANG Xinzhong

Journal of Tea Science. 2024, 44(3): 363-385. doi:10.13305/j.cnki.jts.2024.03.013

Abstract ( 210 )

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Chiral pesticides have one or more enantiomers, and their biological activity, toxicity, environmental behavior, degradation and metabolism may be different. Tea, as one of the three major beverages, has attracted much attention in terms of quality and safety. However, more attention is currently paid to the total amount of pesticide racemate residues. With the development of analytical techniques, it is particularly crucial to fully understand the stereoselective behavior of the chiral pesticide enantiomer in tea, as well as to examine residue degradation in tea and to set the maximum residue limits for enantiomers. This would help to improve the efficacies of chiral pesticides and reduce their residues and toxicities to non-target organisms. In this paper, the current status of chiral pesticide separation and residue detection technology in tea were reviewed, such as liquid chromatography, gas chromatography and supercritical fluid chromatography. In addition, it provided a summary of the residue degradation behaviors and risk assessment of chiral pesticide enantiomers in tea, which would be useful as a guide for further in-depth studies on chiral pesticide enantiomers in tea.

Identification and Expression Pattern Analysis of STOP Gene Family in Tea Plants (Camellia sinensis)

LONG Lu, TANG Dandan, CHEN Wei, TAN Liqiang, CHEN Shengxiang, TANG Qian

Journal of Tea Science. 2024, 44(3): 386-398. doi:10.13305/j.cnki.jts.2024.03.001

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STOP (Sensitive to proton rhizotoxicity) is a type of C₂H₂ zinc finger transcription factor, and it plays an important regulatory role in various stress tolerance mechanisms in higher plants. A total of 6 STOP genes were identified based on the whole genome data of tea plant (Camellia sinensis), and analyzed by bioinformatics and real-time fluorescence quantitative PCR. The results show that the six CsSTOP genes encoded 376-505 amino acids, their molecular weights were 42.17-56.36 kDa, and their theoretical isoelectric points were 5.53-8.85, all of which were unstable proteins. Conserved domain analysis of the proteins shows that they all contained zf-C₂H₂ conserved domain. Phylogenetic analysis shows that tea plant has high homology with Arabidopsis, Citrus sinensis and Nicotiana tabacum. Cis-acting element analysis of the promoter shows that CsSTOPs contain many elements related to growth and development, hormone response and abiotic stress. Transcriptome data analysis of different tissues shows that the expression level of CsSTOP1 was the higher in roots, fruits and mature leaves, the expression level of CsSTOP2 was the higher in young leaves, the expression level of CsSTOP3 was the higher in old leaves, and the expression levels of CsSTOP4 and CsSTOP5 were low in all tissues. The expressions of different CsSTOP genes were induced by PEG-induced drought stress, salt stress, methyl jasmonate stress and cold stress, indicating that CsSTOP genes were involved in the regulation of growth and development of tea plants and response to abiotic stress. Fluorescence quantitative PCR detection shows that the expression levels of CsSTOPs, CsGS1s and CsGDHs in leaves and roots of 'Emeiwenchun' treated with high NH₄⁺ concentration (4.5 mmol·L^-1) were higher than those in the control treatment (CK). Particularly, the expression levels of CsSTOPs, CsGS1.1, CsGS1.3 and CsGDH2 were significantly higher than CK in leaves treated with high NH₄⁺ concentration. In this study, the basic characteristics and functions of CsSTOPs were preliminarily analyzed, and it was found that CsSTOPs could coordinate with CsGS1s and CsGDHs genes to regulate the process of tea plant adaptation to high NH₄⁺ environmental availability.

Spatial Transcriptome Sequencing of Shilixiang in Yunnan Province

WANG Dongxue, MAN Jiaxu, WU Simin, ZHAO Xueting, ZHANG Dongying

Journal of Tea Science. 2024, 44(3): 399-410. doi:10.13305/j.cnki.jts.2024.03.003

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In recent years, Yunnan's large leaf tea and ancient tea resources have attracted much attention, while there are relatively few reports on the research of small leaf tea resources. Shilixiang, a distinctive small leaf tea resource in Yunnan, possessed unique quality and a long drinking history. Spatial transcriptome technology, an emerging gene expression analysis technique, has not been previously applied to tea resources according to current literature. The gene characterization and spatial regulation mechanism of the tender buds of Shilixiang were researched by spatial transcriptome sequencing technology in this study. The results show that 13 clusters of different cell types in the tender bud cells were identified by a spot clustering analysis and the spatial transcriptome map was constructed. The expression positions of clusters during the two developmental stages of the bud were different and spatial heterogeneity was observed from this analysis. Further exploration involved the identification of differential genes in various cell type clusters, with a focus on stress response and growth and development regulation. Representative stress responsive genes included LOC114312694, LOC114319171, LOC114320792, LOC114287723, LOC114284011 and LOC114289235. Meanwhile, representative growth and development genes included LOC114263486, LOC114320821, LOC114292779, LOC114321117, and LOC114286858. A spatial distribution map illustrated the high expression of these stress response and growth development genes in young leaves, indicating their crucial role in the early stage of tender bud development. Further GO and KEGG enrichment analysis reveal that the differential genes in the tender buds of Shilixiang are associated with multiple important pathways. These pathways included translation, jasmonic acid signal regulation, calcium ion binding, and plant hormone signal transduction, all of which are closely linked to the growth and development of tea plants. The results of this study provided a solid scientific foundation for understanding the developmental biology of Shilixiang. Additionally, they provided a new perspective for exploring other tea resources.

Genomic Characteristics, Codon Preference, and Phylogenetic Analysis of Chloroplasts of Camellia sinensis cv. ‘Damianbai’

YIN Minghua, ZHANG Jiaxin, LE Yun, HE Fanfan, HUANG Tianhui, ZHANG Mutong

Journal of Tea Science. 2024, 44(3): 411-430. doi:10.13305/j.cnki.jts.2024.03.009

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‘Damianbai’ was approved as a national tea cultivar by the National Crop Variety Approval Committee in 1985, but its origin and evolutionary relationship with other tea resources are still unclear. Using ‘Damianbai’ as the experimental material, high-throughput sequencing technology was used to sequence, assemble and annotate the entire chloroplast genome of ‘Damianbai’. In order to provide a basis for studying its phylogenetic evolutionary relationship, bioinformatics software was used to analyze the characteristics, phylogeny, and codon preference of its chloroplast genome. The results show that the chloroplast genome of the tea cultivar ‘Damianbai’ had a total length of 157 129 bp and was a typical tetrad structure, including 1 LSC region (86 687 bp), 1 SSC region (18 282 bp), and 2 IR regions (including IRa and IRb, both of which were 26 080 bp). A total of 135 functional genes were annotated in the chloroplast genome of ‘Damianbai’, including 90 CDS genes, 8 rRNA genes, and 37 tRNA genes. A total of 52 SSRs and 50 Longrepeat sequences were detected in the chloroplast genome of ‘Damianbai’. The SSRs had only A/T single nucleotide repeat sequences, while Longrepeat sequences had only two types: forward repeat and palindrome repeat. The codon usage bias in the chloroplast genome of tea cultivar ‘Damianbai’ was mainly influenced by natural selection, and was less affected by internal mutation pressure. The chloroplast gene of tea cultivar ‘Damianbai’ had 14 optimal codons (AAU, GAU, UGU, AAA, UAA, GCA, GCU, GGU, CCU, GUA, CGU, CUU, AGU, UCU). The Camellia sinensis cv. ‘Damianbai’ had a close genetic relationship with Camellia sinensis isolate Baiyin cultivar Phoenix Dancong Tea (OL690374). This study analyzed the chloroplast genome sequence characteristics and phylogenetic relationships of ‘Damianbai’, which provided a reference basis for strengthening the identification of tea cultivar ‘Damianbai’ and the development and utilization of its resource diversity.

Effects of Phosphate Solubilizing Bacteriaand Phosphate-solubilizing and Nitrogen-fixing Bacteria on Selenium and Zinc Contents in Selenium-rich Soil and Camellia sinensis Seedlings in Guizhou

ZHANG Shuqing, GUO Jinmei, LI Jianfeng, WU Ling, WANG Xi, ZENG Zhengqun

Journal of Tea Science. 2024, 44(3): 431-442. doi:10.13305/j.cnki.jts.2024.03.011

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In order to investigate the effects of phosphate solubilizing bacteria and phosphate-solubilizing & nitrogen-fixing bacteria on the content of available selenium (Se) and zinc (Zn) in selenium-rich soil and Camellia sinensis, two strains of endogenous phosphate solubilizing bacteria (Paraburkholderia fungorum PSt07, Kluyvera intermedia PSt12) and two endogenous phosphate-solubilizing & nitrogen-fixation bacteria (Paraburkholderia fungorum PMS05, Kluyvera intermedia PCF06) were used as the research object, and 2 year old tea seedlings of ‘Longjing43’ and ‘Huangjinya’ and Se-rich red soil of Kaiyang County, Guizhou Province were used as the test materials. Soil incubation and pot experiment of tea seedlings were carried out. After 60 days of inoculation with each bacterial solution, Se and Zn contents in tea seedlings and available nutrients of nitrogen (N), phosphorus (P), potassium (K), Zn, Se in soil were tested. The results show that the tested strains could increase the content of available P in rhizosphere soil of two tea seedlings, and the content of available N in rhizosphere soil of ‘Longjing43’. Under the treatment of phosphate solubilizing bacteria, the content of available Se and Zn in rhizosphere soil and Se in root tissue of ‘Longjing43’ tea seedlings were increased by 191.83%-573.08%, 37.48%-65.88% and 24.27%-39.73% respectively. The accumulation of Zn in the leaves of the two tea seedlings was increased by 41.23%-247.65% (P<0.05). The contents of available Zn in the rhizosphere soil of potted plants under the treatment of two strains of phosphate solubilizing bacteria were higher than those in the soil incubated with bacteria solution alone without tea plants, indicating that the combined effect of tea plants and phosphate solubilizing bacteria could significantly improve the availability of Zn in soil. The phosphate-solubilizing & nitrogen-fixing bacteria were more beneficial than the phosphate solubilizing bacteria to increase the Se content of ‘Huangjinya’ root tissue and the available Se content of rhizosphere soil, and PMS05 could significantly increase the content of available Se in rhizosphere soil by 602.00% (P<0.05). The accumulation of Se and Zn elements in tea varied greatly from different combinations of strains and tea cultivars. In practical application, the optimal phosphorus solubilizing strain should be selected for different tea cultivars after completing the strain selection and fertilizer efficiency verification experiment.

Antagonistic Activity and Utilization of Bacillus velezensis Strain YJK1 as A Biocontrol Agent Against Anthracnose on Camellia sinensis

TANG Zhaoyang, KONG Liya, HU Qian, SONG Qiujin, HE Luqian, LOU Jun, WANG Zhanqi, HE Yan, ZHANG Liqin, MIN Lijing

Journal of Tea Science. 2024, 44(3): 443-452. doi:10.13305/j.cnki.jts.2024.03.005

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The tea industry is one of China's important agricultural economic resources. However, widespread outbreaks of anthracnose on Camellia sinensis pose a significant threat to the sustainability of the tea industry. Conventional approaches based on chemical pesticides face inherent challenges, including pesticide residue accumulation and environmental pollution. In this study, the biological control effect of Bacillus velezensis YJK1 on tea tree diseases was evaluated. The results demonstrat that the YJK1 isolate exhibited robust antagonistic activity against fungal diseases affecting tea trees, particularly against pathogens such as Colletotrichum camelliae, Colletotrichum fructicola and Fusarium fujikuroi. YJK1 fermentation diluent has a significant inhibitory effect on the spores of C. camelliae and C. fructicola. The inhibitory effects of YJK1 fermented liquid on the diameter of C. camelliae were evaluated with the detached tea leaves. YJK1 fermented liquid effectively prevented tea tree anthracnose with 78.1% control when applied 24 h before C. camelliae inoculation and 61.8% during simultaneous inoculation. However, its efficacy was limited (post-inoculation). The YJK1 fermentation broth has a broad-spectrum of activity against various fungi. The antagonistic activity of the fermented liquid was stable below 80 ℃. After 60 min of UV treatment, the inhibition rate was still above 50%. And after treatment with different pH values, there was no significant difference in the inhibition rate within the pH range of 5-11. These findings suggest that B. velezensis YJK1 is a promising biocontrol agent for the management of tea plant diseases.

TTLD-YOLOv7: An Algorithm for Detecting Tea Diseases in An Unstructured Environment

YU Shuyan, DU Xiaochen, FENG Hailin, LI Yan′e

Journal of Tea Science. 2024, 44(3): 453-468. doi:10.13305/j.cnki.jts.2024.03.012

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Tea diseases have an extremely serious impact on tea plantations and related industries. Traditional methods for disease detection in the dynamic and complex tea plantation environment are inefficient and unsatisfactory. This study proposed that a YOLOv7-tiny-based model enhanced the fine-grained detection of tea tree diseases. By integrating CoordConv and ECA channel attention mechanisms, this model achieved higher spatial recognition capability in convolutional feature maps and reduced the effect of background noise on feature recognition. Further improvements included the use of a normalized Wasserstein distance metric and decoupled heads to improve the detection of small spots. A new anchor frame was generated using the K-means algorithm based on the specificity of tea spots to improve the accuracy and generalizability of the model. Comparative analysis shows that the model outperforms the existing models Faster R-CNN, SSD, YOLOv5s, YOLO-Tea, YOLOv7-tiny, and YOLOv7, with an average accuracy improvement of 5.9 percentage points to 93%. The improved model could be applied to tea disease monitoring.

The Aroma Change of the Enzymatic Processing Stage of Beauty Tea in Different Withering Methods

DING Fengjiao, YUAN Yuwei, LI Yuanchao, LIN Jinlong, YAN Jiawei, LI Pengchun, JIN Shan

Journal of Tea Science. 2024, 44(3): 469-482. doi:10.13305/j.cnki.jts.2024.03.008

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To investigate the aroma changes between the natural withering indoors and sunlight withering outdoors during the enzymatic processing stage of beauty tea, the volatiles collected in the process samples of beauty tea during the enzymatic processing stage were analyzed by gas chromatography-mass spectrometry (GC-MS). Orthogonal partial least squares-discriminant analysis (OPLS-DA) was used to screen for differential volatiles. The results show that the differential volatiles of beauty teas from the two withering methods were similar at different stages of processing, in which the contents of N-hexanol, trans-2-hexen-1-ol, cis-3-hexen-1-ol, geraniol, hexyl isovalerate, cis-3-hexenyl butyrate, hexyl butyrate, N-butyrate (trans-2-hexenyl) ester, linalool oxy-furan-type, and linalool increased with the processing, which was the material basis for the formation of the aroma quality of Beauty Tea. While the contents of 2-hexenal, (E)-2-hexenyl-2-methylbutyrate, (E)-3-hex-enyl butyrate, ethyl caproate, and ethyl acetate-leaf alcohol ester declined continuously with the processing. The results of volatile determination show that there was no significant difference in the types of volatile components in the processing stage of beauty tea between the two withering methods, but mainly in the content, and most of the volatile components were more abundant in the outdoor sunlight withering beauty tea. This study aimed to explore the aroma changing rule of beauty tea during the enzymatic processing stage, in order to provide a theoretical basis for the optimization of beauty tea processing technology, and to improve the flavor quality and economic value of beauty tea.

Effects of Different Temperature Hot Air Withering on Withered Leaves and Tea Quality of Black Tea

ZHANG Xianglin, LING Zhihui, HU Weixia, XIANG Chunhui, CUI Lidan, XU Wei, XIAO Wenjun

Journal of Tea Science. 2024, 44(3): 483-492. doi:10.13305/j.cnki.jts.2024.03.007

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Based on the traditional black tea processing technology, the hot air withering process was used to replace the traditional indoor natural withering process on the basis of the traditional black tea processing technology. The effects of four hot air withering processes of 35 ℃, 45 ℃, 55 ℃ and 65 ℃ on the quality of summer and autumn black tea were studied and analyzed from three aspects of sensory quality, taste quality and aroma quality. The results show that compared with 25 ℃ traditional indoor natural withering black tea, the comprehensive sensory quality of black tea processed by 45 ℃ hot air withering process was better. The color of dry tea was dark brown and moist, the soup color was red and bright, the taste was mellow and refreshing, and the quality characteristics was sweet aroma. At the same time, the contents of tea polyphenols, gallic acid, EGCG, theaflavins, thearubigins and the ratio of the sum contents of theaflavins and thearubigins to the content of theabrownins (TFRB) significantly increased (P<0.05), while the content of soluble sugar decreased significantly (P<0.05). The types of aroma quality components increased, among which alcohols accounted for the highest proportion (70.47%), and the relative contents of alcohols, ketones and esters increased significantly (P<0.05), while pyrroles and aldehydes decreased significantly (P<0.05). And the aroma components with flower and fruit aroma such as trans-3-hexenol, 2,3-dihydro-2,2,6-trimethylbenzaldehyde and α-ionone were detected. This shows that hot air withering with appropriate temperature is conducive to improving the quality of black tea, which can be applied to the production practice of improving the quality of black tea in summer and autumn.

Carbonylation Modification of Epicatechin and Its Activities of UV Absorption and Antioxidant

LEI Xiang, ZHANG Minfeng, LIN Hui, WANG Lili, ZHENG Deyong

Journal of Tea Science. 2024, 44(3): 493-500. doi:10.13305/j.cnki.jts.2024.03.002

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In order to enhance the UV absorption characteristic of epicatechin derivatives and improve their solubility in non-polar solvents such as oils and fats, 2,2'-di(p-hydroxybenzocarbonyl)-epicatechin was synthesized from epicatechin and p-hydroxybenzoic acid using methane sulfonic acid as solvent and catalyst. The target products were separated by column chromatography, and the purity was verified by high performance liquid chromatography (HPLC), and the structure of the target products were verified by UV-vis, FT-IR, ¹H NMR, ¹³C NMR, LC-MS spectroscopy. The UV absorption characteristic of 2,2'-di(p-hydroxybenzocarbonyl)-epicatechin and its ability to scavenge ABTS⁺·, DPPH· and O^2-· were evaluated. The results show that the synthesized 2,2'-di(p-hydroxybenzocarbonyl)-epicatechin had similar UV absorption and comparable molar absorptivity to OMC, the commonly used UV absorber, and is expected to be an excellent UV absorber in the UVB band. 2,2'-di(p-hydroxybenzocarbonyl)-epicatechin had good antioxidant activity in vitro, and its antioxidant capacity in vitro was higher than that of VC and slightly lower than that of epicatechin, which is expected to be used for cosmetic applications.

Study on the Effects of Puerins Ⅴ-Ⅶ on Four Kinds of Breast Cancer Cells

YANG Rui, ZHAO Xingping, HE Mingjie, LIU Min, LUO Rong, CHEN Chuanlong, PAN Shukang, DING Zhanggui

Journal of Tea Science. 2024, 44(3): 501-514. doi:10.13305/j.cnki.jts.2024.03.004

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Puerins, a kind of nitrogen-containing polyphenol compound with unique structure, is a kind of catechin derivative first isolated from ripened Pu-erh tea. In this paper, the effects of puerins Ⅴ-Ⅶ on breast cancer cells were studied. Four breast cancer cell lines with different receptor phenotypes were used as models, and MTT assay, proteomics detection and apoptosis detection were carried out. The results show that all puerins Ⅴ, Ⅵ and Ⅶ had obvious inhibitory effects on the four kinds of breast cancer cells. Proteomics analysis indicates that the differentially expressed proteins were mainly concentrated in the metabolic pathway, followed by cancer pathway and adhesion spot signal pathway under the intervention of puerins Ⅴ-Ⅶ. Apoptosis detection demonstrates that all three puerins compounds could induce the apoptosis of MDA-MB-231 cells. This study first reported the anti-breast cancer activity and preliminary mechanism of puerins Ⅴ-Ⅶ, which provided basic information for studying the anti-cancer active substances of ripened Pu-erh tea.

Determination of Afidopyropen and Metabolite M440I007 in Tea Tissues by Modified QuEChERS Coupled with Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry

QIN Yujie, GUO Mingming, CHEN Yongjing, ZHOU Li

Journal of Tea Science. 2024, 44(3): 515-525. doi:10.13305/j.cnki.jts.2024.03.010

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Afidopyropen, a novel biorational insecticide, was registered in China in 2023 for the control of tea leafhoppers in tea plantation. In this study, an analytical method was developed for the determination of afidopyropen and its metabolite M440I007 in tea tissues by a modified QuEChERS method combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Roots, stems and leaves of tea plants were extracted by water and acetonitrile, and purified with primary secondary amine (PSA), hydroxylated multi-walled carbon nanotubes (MWCNT-OH) and graphitized carbon black (GCB). The target compounds were determined by UPLC-MS/MS in multiple reaction monitoring (MRM) mode, and quantified by external standard method with matrix-matched standard curve. Both targets display excellent linearity (R²>0.999 5) in the range of 0.002-5.000 mg·L^-1. The recoveries of both compounds at the spiked levels of 0.005-2.000 mg·kg^-1 ranged from 78.3% to 106.0% with the relative standard deviations (RSD) ≤ 12.85%. The limits of quantification (LOQs) and detection (LODs) of the method were 0.005 mg·kg^-1and 0.002 mg·kg^-1, respectively. The developed method was applied to detect afidopyrofen and M440I007 in tea seedling tissues cultured in soil treated with afidopyropen in a pot experiment for 7 days. The results show that the distribution of afidopyropen in the tissues was roots (0.102 mg·kg^-1) > stems (0.078 mg·kg^-1) > leaves (0.007 mg·kg^-1). The translocation factors, TF_root-stem and TF_stem-leaf, were both less than 1, and the residue levels of the metabolite M440I007 were below the LOQ. The method established in this study is low-cost, accurate and sensitive, which can serve as a technical basis for further research on the uptake and transport behaviors of afidopyropen and M440I007 in tea plants.

Study on the Impact of Standards Differences in Pesticide Maximum Residue Limits on the Trade Efficiency of RCEP Members Exporting Tea from China: Based on the Stochastic Frontier Gravity Model

HUO Zenghui, LIU Chang, ZHANG Mei, CHEN Fuqiao, LIU Zhonghua

Journal of Tea Science. 2024, 44(3): 526-542. doi:10.13305/j.cnki.jts.2024.03.006

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Currently, tea trade is facing challenges from technical trade barriers. This study used the standards differences in pesticide maximum residue limits (MRLs) as the entry point to construct The Heterogeneity Index of Trade (HIT) to quantify the specific differences in pesticide MRLs between countries. The stochastic frontier gravity model was used to study the impact of standards differences in pesticide MRLs on the trade efficiency of RCEP members exporting tea from China. The results show that there are differences in the pesticide MRLs standards between China and RCEP members, but overall there is a decreasing trend. The negative differences in pesticide MRLs standards between China and RCEP members will lead to a reduction in the trade efficiency of China's tea exports. The inhibitory effect path is manifested by significantly reducing tea export volume and export unit price. China's pesticide MRLs standard system should be further improved to align with international standards, promote mutual recognition of standards among countries in the RCEP region, and reduce the adverse effects of trade barriers.

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