15 June 2023, Volume 43 Issue 3

    

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Research Paper
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  Analysis of Codon Usage Bias and Phylogenesis in the Chloroplast Genome of Ancient Tea Tree Camellia taliensis in Forest-tea Garden

  TONG Yan, HUANG Hui, WANG Yuhua

  Journal of Tea Science. 2023, 43(3): 297-309. https://doi.org/10.13305/j.cnki.jts.2023.03.010

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  Camellia taliensis is one of the important tea plants which is often grown in forest-tea gardens, which participated in the origin and domestication of C. sinensis var. assamica. To determine the codon usage bias pattern and its main influencing factors in the chloroplast genome of C. taliensis, neutral plotting, ENC-plot, PR2-plot analyses were performed and the optimal codons were found. The results show that the GC content of 54 CDS sequences was 37.68%, while GC₁ and GC₂ content were 46.44% and 39.77%, higher than GC₃ (27.67%), indicating that the third base of the codon preferred to end in A/U. The effective codon number (ENC) ranged from 35.64 to 56.67 , with an average value of 46.1, demonstrating weak codon usage bias in chloroplast genome of C. taliensis. Neutral plotting, ENC-plot, PR2-plot analyses show that the main factor affecting the codon usage bias of the C. taliensis chloroplast genome was natural selection. In total, 11 optimal codons were identified in the chloroplast genome of C. taliensis. Although phylogenetic trees constructed by matK gene and CDS sequence display different topological structures, all trees show that C. taliensis and C. gymnogyna are clustered into one branch and are closely related to cultivated tea plant. This study provided the basis for analyses of genetic evolution, phylogeny and improved agronomic traits of C. taliensis.
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  Genetic Analysis and Marker Development for Wuyi Tea (Camellia sinensis, Synonym: Thea bohea L.) Based on GBS-SNP

  LI Li, LUO Shengcai, WANG Feiquan, LI Xiangru, FENG Hua, SHI Yutao, YE Jianghua, LIU Fei, ZHAO Jialin, LI Shuying, ZHANG Bo

  Journal of Tea Science. 2023, 43(3): 310-324. https://doi.org/10.13305/j.cnki.jts.2023.03.001

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  In order to understand the genetic diversity background of Wuyi tea (Camellia sinensis, Synonym: Thea bohea L.), 126 Wuyi tea cultivars/strains and 223 elite tea cultivars/strains (a total of 349 tea germplasm resources) from 12 different regions were collected. Genotyping by sequencing (GBS) technology was used to screen 973 high-quality core SNPs for genetic diversity and background analysis. Model-based structure (Structure), phylogenetic tree construction (NJ-tree) and principal component analysis (PCA) show that the 349 tea resources could be divided into 5 subgroups, and the clustering of subgroups was mainly based on the genetic relationship between tea resources, rather than morphological characteristics such as tree type or leaf shape. Gene flow analysis shows that Wuyi tea might have spread from southern Fujian Province to Wuyi Mountain in northern Fujian Province and from Wuyi Mountain to Zhejiang Province. Genetic similarity analysis shows that among 349 tea cultivars/strains, 136 pairs of cultivars/strains had genetic similarity greater than 0.9, among which 26 pairs involved Wuyi tea. Based on the results of gene flow and genetic similarity, the genetic relationship and background of some representative and controversial Wuyi tea were discussed and analyzed. Furthermore, through the discernibility analysis of pairwise comparison, 21 SNPs were selected from 973 SNP markers that can 100% identify 349 tea cultivars/strains, among which 18 SNPs could 100% identify 126 Wuyi tea cultivars/strains. Genetic fingerprints were established and identification primers were developed. These results provided valuable information for the management and breeding of Wuyi tea in the future.
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  Identification of Transcription Factors Interacting with CsNCED2 Promoter and Their Response to Abiotic Stress

  LI Jiasi, LIU Yingqing, ZHANG Yongheng, ZHANG Ying'ao, XIAO Yezi, LIU Lu, YU Youben

  Journal of Tea Science. 2023, 43(3): 325-334. https://doi.org/10.13305/j.cnki.jts.2023.03.007

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  Nine cis epoxide carotenoid dioxygenase (NCED) is a key rate-limiting enzyme in abscisic acid (ABA) synthesis and widely involved in plant growth and development as well as abiotic stress response. CsNCED2 is involved in the response to drought and salt stress in tea plants, while the transcriptional regulation mechanism involved is still unclear. In this study, two transcription factors, CsDof5.4 and CsERF38, which binded to the CsNCED2 promoter were identified by yeast single hybrid (Y1H) library screening. Subcellular localization, yeast self-activation and luciferase (LUC) assay show that they were located in the cell nucleus and could activate the expression of CsNCED2. RT-qPCR results show that the expressions of CsERF38 and CsDof5.4 were highly correlated with CsNCED2 under salt stress. While under drought stress, only the expression of CsERF38was highly correlated with that of CsNCED2. In this study, two transcription factors (CsDof5.4 and CsERF38) binding to the CsNCED2 promoter were identified. Both drought and salt stresses could induce the expression of CsNCED2, thus participate in the abiotic stress response in tea plants.
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  Study on the Regulation Roles of Plant Hormones on the Growth and Development of Tea Shoots in Spring

  LI Congcong, WANG Haoqian, YE Yufan, CHEN Yao, REN Hengze, LI Yuteng, HAO Xinyuan, WANG Xinchao, CAO Hongli, YUE Chuan

  Journal of Tea Science. 2023, 43(3): 335-348. https://doi.org/10.13305/j.cnki.jts.2023.03.002

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  Hormones play important roles in the regulation of plant growth and development. In order to clarify the effects of different hormones on the growth and development of tea shoots in spring, and to identify the main pathways and key genes involved, tea cultivar ‘Longjing 43’ was used as the experimental materials, and treated with 100 μmol·L^-1 ABA, 100 μmol·L^-1 GA₃ and 100 μmol·L^-1 IAA respectively in the sprouting period. The phenotypic characteristics of buds were determined, and the buds at the 7th day after treatments were investigated using RNA-Seq technique. The results show that exogenous ABA treatment inhibited the germination and growth of shoots, and the length of shoots was significantly shorter than the control after 7 days after treatment. On the other hand, GA₃ and IAA treatments had a promoting effect, and the bud length was significantly extended on the 7th day after GA₃ treatment and on the 14th day after IAA treatment. RNA-Seq analysis indicates differentially expressed genes were mainly enriched in amino acid biosynthesis pathway under ABA treatment, oxidative phosphorylation pathway and photosynthesis pathway under GA₃ treatment, and flavonoid biosynthesis pathway under IAA treatment. GAI, PSBO2, PSBQ-2 and PSBP-1 related to plant hormone and photosynthesis pathways might be the key genes involved in shoot growth and development. The real-time fluorescence quantitative PCR results of some candidate genes were consistent with the RNA-Seq results. The above studies identified the main pathways and key genes involved in the hormone regulation on tea shoot growth and development, which provided a theoretical basis for deeply revealing the regulation mechanism of tea shoot growth and development.
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  Effects of Nitrogen Form and Weak Light Stress on Tea Plant Growth and Metabolism

  SHEN Ruihan, MA Lifeng, YANG Xiangde, FANG Li

  Journal of Tea Science. 2023, 43(3): 349-355. https://doi.org/10.13305/j.cnki.jts.2023.03.01

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  In order to analyze the effects of N form and weak light stress on the tea plant growth and metabolism, the two-year-old cutting seedlings of ‘Longjing 43’ were taken as experimental materials, and two different forms of N sources (NH₄⁺-N and NO₃^--N) and two light intensities (normal light and weak light) were supplied under solution culture. The results show that: (1) the net photosynthetic rate of tea seedlings was mainly affected by the N form under normal light intensity. The net photosynthetic rate of tea seedlings treated with NH₄⁺-N was significantly higher than that treated with NO₃^--N. Under the same nitrogen conditions, compared to normal light, weak light stress had a tendency to reduce the net photosynthetic rate of tea seedlings. (2) Under normal light intensity, the chlorophyll content in young shoots treated with NH₄⁺-N was significantly higher than that treated with NO₃^--N, but the chlorophyll a (Chl a)/chlorophyll b (Chl b) ratio was basically unchanged. The chlorophyll content increased significantly after weak light stress, but Chl a/b ratio under NH₄⁺-N treatment was significantly increased, while Chl a/b ratio under NO₃^--N treatment was slightly decreased. NO₃^--N treated tea plants showed better shade tolerance than that treated with NH₄⁺-N. (3) N uptake by tea seedlings was mainly influenced by both N form and light, and weak light stress helped to improve the accumulation of N in new shoots of tea seedlings. The N content of NH₄⁺-N treated tea seedlings was significantly higher than that treated with NO₃^--N under normal light. Weak light stress significantly increased the above-ground N content in tea plants. (4) Under normal light intensity, the contents of free amino acids and caffeine in young shoots treated with NH₄⁺-N were significantly higher than those treated with NO₃^--N, while the ratio of tea polyphenol to free amino acids was lower. After weak light stress, the contents of free amino acid and caffeine in shoots further increased, while the contents of tea polyphenol and the ratio of tea polyphenol to free amino acids continued to decline. The increase rate of total free amino acid and descent rate of tea polyphenol and tea polyphenol/free amino acids ratio were faster in young shoots treated with NO₃^--N than those treated with NH₄⁺-N. The composition of shoot quality was mainly affected by light intensity, but tea polyphenol-free amino acids ratio was influenced by both light intensity and N form. The research results provide theoretical basis for the analysis of its mechanism.
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  Comparison of Soluble and Membrane-bound Polyphenol Oxidase from Cultivars Suitable to Ninghong Tea Production

  ZHAN Kun, YANG Zhengli, XU Ziyi, LAI Zhangfeng, LI Jun, CHEN Luojun, ZHOU Sixi, LI Mingxi, GAN Yudi

  Journal of Tea Science. 2023, 43(3): 356-366. https://doi.org/10.13305/j.cnki.jts.2023.03.003

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  In order to investigate the enzymatic properties of polyphenol oxidase (PPO) in cultivars suitable for Ninghong tea production, as well as to increase the theaflavin content in Ninghong tea, one bud and two fresh leaves from ‘Ningzhou population’, ‘Ningzhou 2’ and ‘Dayelong’ were used as raw materials to obtain membrane-bound polyphenol oxidase (mPPO) and soluble polyphenol oxidase (sPPO) crude enzymes and to analyze the enzymatic properties. Using catechol as the substrate, the mPPO specific activity of each cultivar was higher than sPPO specific activity. mPPO specific activity of ‘Dayelong’ was the highest (542.59±25.13 U·mg^-1), and sPPO activity of ‘Ningzhou 2’ was the lowest (112.57±14.01 U·mg^-1). The optimum reaction temperature for mPPO was 40-60 ℃, the optimum reaction temperature for sPPO was 30-50 ℃, and the highest optimum reaction temperature for mPPO of ‘Ningzhou 2’ and ‘Dayelong’ was 55 ℃. The lowest sPPO optimum reaction temperature for ‘Ningzhou 2’ and ‘Ningzhou population’ was 35 ℃. The optimum pH of sPPO ranged from 7.00-8.50 with one peak, while the optimum pH of mPPO ranged from 5.00-8.00 with two peaks. The optimum pH of sPPO in ‘Ningzhou population’ was 8.00, and the optimum pH of mPPO in ‘Ningzhou 2’ was 5.50. mPPO and sPPO had strong affinity for dihydroxy phenols, and mPPO in ‘Ningzhou 2’ had the strongest affinity for catechol and the highest catalytic efficiency. Ascorbic acid had the best inhibitory effect on sPPO in ‘Ningzhou population’. While halide inhibitors had no inhibitory effect on both sPPO and mPPO activities in tea cultivars suitable for Ninghong tea production. EDTA had an activating effect on mPPO. The thermal deactivation of sPPO and mPPO in these tea cultivars followed the primary reaction kinetics, with mPPO in ‘Dayelong’ having the best thermal resistance, the worst thermal sensitivity, and the weakest sensitivity to temperature. While sPPO in ‘Ningzhou population’ had the worst thermal resistance. The sPPO of ‘Ningzhou population’ was the worst heat-resistance, the highest heat-sensitivity and temperature-sensitivity. The results show that there were some differences in the sPPO and mPPO enzyme properties among the cultivars. Tea cultivar ‘Dayelong’ had the highest mPPO specific activity and the best heat resistance, which provided a suitable enzyme source for the processing of high theaflavin Ninghong tea. This study also provided a theoretical reference for the actual Ninghong tea production.
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  Effects of Growth and Development on the Visual Ability of Empoasca onukii

  TAN Chang, SHAN Yao, ZHOU Xiaosen, YAO Qiuyi, CAI Xiaoming, BIAN Lei

  Journal of Tea Science. 2023, 43(3): 367-376. https://doi.org/10.13305/j.cnki.jts.2023.03.004

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  Allometry is an important strategy for insects to adapt to the surrounding environment. Visual cues are the key information for host selection and flight movement of Empoasca onukii. With the growth and development of E. onukii, the size and mobility of the leafhopper gradually increase, and the morphology of the compound eye also changes significantly. In this study, histological and behavioral methods were used to determine the morphological structure and visual ability of the compound eye of E. onukii at different ages. The results show that the visual ability gradually increased during the growth and development process of E. onukii. From 1^st instar to 5^th instar, the optical sensitivity of the nymph increased from 0.04 μm²·sr to 0.11 μm²·sr, and the visual acuity increased from 0.086 cycles per degree (cpd) to 0.112 cpd. Both of optical sensitivity and visual acuity were consistent with the allometric growth, but their growth rates were slower than that of the size of nymphs. After the emergence of nymphs, the optical sensitivity of the center region of the compound eye decreased and there was a significant difference between the male and the female. Although the visual acuity kept increasing, the compound eye of E. onukii was still a low-resolution organ with limited effective visual distance. This study confirms that E. onukii slows down the growth rate of visual ability to prioritize the development of body size and motion-related organs, and focuses on the increase of visual field to facilitate to perceive visual cues in a wide range.
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  Preparation of a Cross-linked Polyphenol Oxidase Aggregate Based on Catechins and Its Efficient Catalytic Synthesis of Theaflavin-3,3ʹ-digalate

  ZHOU Jinghui, LIU Changwei, ZHANG Sheng, XU Gang, XU Wei, HUANG Jian′an, LIU Zhonghua

  Journal of Tea Science. 2023, 43(3): 377-388. https://doi.org/10.13305/j.cnki.jts.2023.03.005

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  In order to prepare theaflavin more efficiently and economically, catechins were used as the cross-linking reagent in this study, and a polyphenol oxidase (tyrosinase) crossed-linked aggregate derived from Bacillus megaterium was prepared and used for the synthesis of theaflavin-3,3ʹ-digalate. Through optimization of enzyme cross-linking parameters and the catalytic performance before and after cross-linking, the results show that the optimal enzyme activity recovery rate of cross-linked enzyme (200 U·mL^-1) could be obtained at pH 4.0, EGCG 0.5 mg·mL^-1 and cross-linking time 50 min. Compared with free enzymes, cross-linked enzymes showed better catalytic performance (thermal stability, organic solvent tolerance, substrate tolerance). When the cross-linked enzyme was used to synthesize theaflavin-3,3′-digallate, the concentration of the product could reach 800 μg·mL^-1, and the cross-linked enzyme could be reused for at least three batches. This method could significantly reduce the application cost of theaflavin, which has potential industrial application value.
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  Acid-assisted Improvement of Dispersive Solid Phase Extraction for Rapid Detection of Pesticide Residues in Tea

  WU Zhenghao, ZHENG Qinqin, HAO Zhenxia, WANG Chen, CHEN Hongping, LU Chengyin

  Journal of Tea Science. 2023, 43(3): 389-398. https://doi.org/10.13305/j.cnki.jts.2023.03.008

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  The false-positive results encountered in the rapid detection of pesticide residues in tea samples are primarily caused by matrix interference. However, limited by the physical and chemical properties of tea matrix components (e.g., caffeine), the development of pretreatments suffers major dilemmas in matrix removal effectiveness. In current work, a novel pretreatment method for tea samples was proposed by developing specific removal procedures based on the molecular structure characteristics of the main matrix components. On the basis of conventional pretreatment for tea samples, the novel method introduced acid solutions and polyvinylpolypyrrolidone adsorbents, which reduced the matrix effect of tea sample detection significantly with a high matrix removal rate of more than 99.99%. Under the assistance of the current pretreatment, the rapid detection of methomyl and carbofuran residues in tea samples was successfully achieved accurately. The limits of detection for carbofuran and methomyl in tea were 0.57 mg·kg^-1 and 0.20 mg·kg^-1, respectively.
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  Regulatory Effect and Mechanism of EGCG on Metabolic Disorders in High-fructose Diet Mice

  ZHOU Jihong, CHEN Wei, DING Lejia, WANG Yuefei

  Journal of Tea Science. 2023, 43(3): 399-410. https://doi.org/10.13305/j.cnki.jts.2023.03.012

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  This study investigated the effects and mechanisms of epigallocatechin gallate (EGCG) on high-fructose diet-induced metabolic disorders. Fifteen male SPF C57BL/6 mice were randomly divided into three groups: normal diet group (NCD), high-fructose diet group (HFD), and high-fructose diet supplemented with 1% EGCG group (HFE), with 5 mice in each group. After 8 weeks of feeding, the body weight, energy utilization rate, ALT and AST levels, as well as tissue morphology staining of the mice were measured. Furthermore, hepatic TNF-α, IL-1β, IL-6 and intestinal IL-6 inflammatory cytokine levels were detected by ELISA. The expressions of Srebp-1c, Tlr4, Myd88 in liver and Zo-1, Occludin, Tlr4 and Myd88 in intestine were measured by quantitative real-time PCR. Protein expressions of ZO-1 and Occludin were detected by IHC. The results show that dietary supplementation of EGCG could effectively reduce high-fructose diet-induced body weight gain, fat accumulation, hepatic and intestinal inflammatory responses, and could improve the intestinal barrier function by upregulating the expression of Zo-1 and the protein expressions of ZO-1 and Occludin. It also modulated lipid metabolism by reducing the expression level of Srebp-1c in liver, and downregulated the expression levels of inflammatory-related genes (Tlr4 and Myd88) in colon and liver. The results above suggest that dietary supplementation of EGCG has a preventive effect on high-fructose diet-induced metabolic disorders and inflammatory responses, and its mechanism may be related to the regulation of the gut-liver axis mediated by the TLR4/MyD88 signaling pathway.
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  Data Enhancement Optimization and Class Activation Mapping Quantitative Evaluation for CNN Image Recognition of Multiple Tea Categories

  ZHANG Zhanyi, ZHANG Baoquan, WANG Zhouli, YANG Yao, FAN Dongmei, HE Weizhong, MA Junhui, LIN Jie

  Journal of Tea Science. 2023, 43(3): 411-423. https://doi.org/10.13305/j.cnki.jts.2023.03.006

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  There are many kinds of tea in China, and subjective identification is easy to be confused and very dependent on professional experience. Convolutional Neural Network (CNN) image recognition applied to multi-tea identification has the advantages of objectivity, adaptability to complex image backgrounds and portability to mobile devices. However, the current CNN image recognition of tea lacks data enhancement optimization and objective evaluation of recognition accuracy, which limits the robustness and generalization ability of model recognition. In this study, a total of 6 123 images of 29 common tea categories were collected to construct a dataset, and the ResNet-18 (Residual network-18) training effects of 10 image data enhancement methods were compared. To objectively evaluate the accuracy of the model recognition area, two gradient-weighted class activation mapping (Grad-CAM ) quantitative evaluation indexes (IOB and MPI) were constructed. The results show that grid erasure (Ratio=0.3), resolution perturbation and HSV (Hue, Saturation, Value) color space perturbation are better data enhancement methods, with four indicators of accuracy, loss, IOB and MPI performing better. Furthermore, through the ablation experiment, the optimal combination of data enhancement methods “horizontal mirror flip + grid erasure (Ratio=0.3) + HSV color perturbation” was obtained. The accuracy rate of model test reached 99.82%, with a loss value of only 0.64, and the IOB and MPI indicators also performed better, reflecting good accuracy in image recognition. This study optimized the tea image data enhancement method, and obtained the multi-tea CNN image recognition model with high robustness. The constructed quantization indexes IOB and MPI also solved the problem of accuracy evaluation of CAM recognition region.
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  The Impact of Product Processing Standardization on Tea Firms’ Income from the Perspective of Scale Heterogeneity

  XIONG Yun, CHEN Jianghua, LI Daohe

  Journal of Tea Science. 2023, 43(3): 424-436. https://doi.org/10.13305/j.cnki.jts.2023.03.009

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  Product processing standardization is an important means to promote the quality of tea products, enhance the competitiveness of tea firms. Based on the micro-research data of tea firms in Jiangxi Province, this study analyzed the impact of product processing standardization on tea firms’ income using ordinary least square (OLS), two stage least square (2SLS) and treatment effects model (TEM), and further analyzed the difference of the impact of product processing standardization on tea firms’ income under different operating scales using the threshold effect model. It was found that (1) the product processing standardization had a significant positive impact on tea firms’ income, and the adoption of standardized production could help increase tea firms’ income, among which the blending process and sensory evaluation measurement dimensions had a significant impact, and the sales of tea firms adopting standardized processing methods could be 10.01% higher than the control group. (2) The impact of product processing standardization on tea firms’ income had scale heterogeneity, and the income increasing effect of product processing standardization of tea firms under moderate scale operation was obvious, and the income increasing effect of processing standardization under over-scale operation was not obvious. Therefore, the government should actively guide and promote the promotion and application of processing standardization of tea firms, while encouraging firms to incorporate the blending process into the standardization system. Advocating tea firms in the moderate scale range could expand the operating scale through a variety of ways to fully stimulate the operating income of product processing standardization.
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  Ecological Thoughts in Ancient Chinese Tea Books and Their Contemporary Value

  LIU Xinqiu, DU Xiya

  Journal of Tea Science. 2023, 43(3): 437-446. https://doi.org/10.13305/j.cnki.jts.2023.03.013

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  Chinese traditional ecological thoughts with the core of Confucianism, Buddhism, Taoism culture, in the pursuit of harmony in the process of human and nature, are consisted of a series of environmental protection and respect for life. It is formed by the particular geographical environment, political status, economic conditions and cultural factors, which thereby formed an agriculture production system of intensive cultivation. Under the influence of the ecological thoughts, a special system of tea planting management and tea drinking methods has been established in the production and development of tea industry, which was mostly recorded in the tea books of all dynasties. The ecological thoughts of tea industry were embodied in different periods, including the historical understanding of tea planting ecological environment, the development and evolution of tea garden management technologies and the stage change of tea drinking mode. The ecological concept of ancient tea industry has been inherited and can still be used for reference and implementation today.