A sampling method for accurately evaluating the nutrient status of tea trees and its application

Abstract

This invention discloses a sampling method and application for accurately evaluating the nutrient status of tea trees, and for the first time specifies the sampling time and leaf position for mature leaves and new shoots when evaluating the nutrient status of tea trees. The method involves selecting previous-year-old vegetative branches from the same growth position on the tea tree canopy surface, and topping these branches at the end of January, removing the terminal buds. In mid-to-late February, while the tea trees are still dormant, the first mature leaf at the top of the pruning cut is selected. When the dormant buds sprout and develop to one bud and two leaves, new shoot samples corresponding to the marked mature leaves are taken; mature leaves are not collected at this stage. Validation was conducted in tea gardens with 10-year and 20-year long-term nitrogen fertilization sites and with 2000 population samples. The method of this invention ensures the correlation between the nutrient status of tea trees across years, and shows a highly significant positive correlation between tea garden fertilization levels and tea tree nutrient status; it also ensures a highly significant positive correlation between the amino acid composition of new shoots and fertilization levels, and a highly significant negative correlation between the phenol-amino acid ratio and fertilization levels.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tea tree cultivation and breeding phenotype identification, and relates to a sampling method for accurately evaluating the nutrient state of tea trees and application thereof. BACKGROUND

[0002] Tea trees are perennial woody plants, and the mature leaf nutrient element contents of different leaf positions on the vegetative branches are quite different. During the spring tea picking period, the element contents of the top shoots in the tea tree canopy are different from those of the shoots below the canopy, and the nutrient element contents of the shoots in different development stages, such as one-bud two-leaf stage and one-bud three / four-leaf stage, also differ. During field test sampling, how to ensure that the samples taken can best represent the nutrient state of tea trees and reflect the fertilization level of the tea garden, and how to achieve good repeatability between years without being affected by the climate and environment. SUMMARY

[0003] In order to solve the above problems, the application designs a sampling method for accurately evaluating the nutrient state of tea trees and application thereof, which is verified by a long-term positioning test of nitrogen fertilizer in a tea garden and nearly 2000 samples of 221 single plants and parent and offspring of tea tree F1 hybrid population from 2023 to 2025 for three consecutive years. The method can accurately evaluate the nutrient state of tea trees and better reflect the nitrogen fertilizer application level of the tea garden, has good repeatability between years and is not affected by environmental factors such as climate, and has important significance for the accurate identification of the nutrient state of tea trees and the phenotype of tea tree nutrient-efficient varieties.

[0004] The application provides a sampling method for accurately evaluating the nutrient state of tea trees, which comprises three stages of pretreatment before sampling, mature leaf sampling and shoot sampling.

[0005] Pretreatment before sampling: from January 20 to 28, the overwintering vegetative branches on the shed surface are topped, the top buds are removed, and the positions are marked.

[0006] Mature leaf sampling: from February 20 to 28, the first mature leaf L1 at the top of the marked branch is taken.

[0007] Shoot sampling: after the marked dormant buds germinate and develop to one-bud two-leaf stage, the shoots S1 corresponding to the marked mature leaves are taken, and the mature leaves are not taken at this time.

[0008] Measurement index: the nitrogen concentrations of the first mature leaf L1 and the shoot S1 are NL1 and NS1, respectively, with a unit of mg / g and a dry weight; the amino acid A1 of the shoot S1 sample, with a unit of mg / g and a dry weight, and the total amount of tea polyphenols are measured, and the phenolic amino acid ratio P1 is calculated.

[0009] The mature leaf L1 in the dormant period and the shoot S1 in the one-bud two-leaf period of the long-term positioning site of nitrogen fertilizer are collected to measure the above indexes, a single linear regression analysis in statistics is adopted, the regression coefficient is determined by the least square method, and finally the nutrient state formula index is obtained, wherein N is the fertilization level:

[0010] NL1=0.03433 x N + 24.79;

[0011] NS1=0.005839 x N + 51.98;

[0012] P1=-0.004544 x N + 12.85;

[0013] A1=0.009215 x N + 27.01;

[0014] In combination with the results of long-term experiments in multiple sites for many years, the nitrogen fertilizer application rate in tea garden is low when it is 0-200 kg / ha, at this time, NL1 value is 24.79-31.656, NS1 value is 51.98-53.1478, P1 value is 11.9414-12.85, A1 value is 27.01-28.853, therefore, when 25<NL1<32, 52<NS1<53, 12<P1<13, 27<A1<29, it is determined that the fertilization level in tea garden is low, and tea tree is in the state of poor nutrients;

[0015] In combination with the results of long-term experiments in multiple sites for many years, the nitrogen fertilizer application rate in tea garden is high when it is more than 600 kg / ha, at this time, NL1 value is 45.388, NS1 value is 55.4834, P1 value is 10.1236, A1 value is 32.539, therefore, when NL1>45, NS1>55, P1<10, A1>32, it is determined that the fertilization level in tea garden is high, and tea tree is in the state of nutrient surplus.

[0016] In the process of tea garden management, the tree shape is maintained by pruning regularly every year, and the tea yield and quality of the next year are ensured. The nutrient branches at the top of the canopy are all the nutrient branches of the previous year, which can represent the nutrient status of tea tree in the current year. Previous studies have found that most of the nutrients required for new shoot germination are derived from the corresponding mature leaves. Therefore, the first mature leaf at the top of the canopy of tea tree best represents the nutrient status of tea tree. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The total nitrogen content of early spring new shoots, the total amount of amino acids, the phenolic amino acid ratio and the correlation with the nitrogen application level in tea garden are shown; A: the nitrogen content in early spring new shoots is significantly positively correlated with the nitrogen fertilizer application level in tea garden; B: the content of free amino acids in new shoots is significantly positively correlated with the nitrogen fertilizer application level in tea garden; C: the phenolic amino acid ratio in new shoots is significantly negatively correlated with the nitrogen fertilizer application level in tea garden.

[0018] Figure 2 The total nitrogen content of mature leaves at the end of February, the total nitrogen content of new shoots at one bud and two leaves stage, 15N content, nitrogen reutilization efficiency and tea garden nitrogen application level correlation; A: mature leaf L1 N content in February at the end of dormancy period and tea garden nitrogen application level significantly positively correlated; B: new shoot N content at one bud and two leaves stage and tea garden nitrogen application level significantly positively correlated; C: total N content in each new shoot and tea garden nitrogen application level significantly positively correlated; D: nitrogen reutilization efficiency and tea garden nitrogen application level significantly positively correlated. 15 N content, nitrogen reutilization efficiency and tea garden nitrogen application level correlation; A: mature leaf L1 N content in February at the end of dormancy period and tea garden nitrogen application level significantly positively correlated; B: new shoot N content at one bud and two leaves stage and tea garden nitrogen application level significantly positively correlated; C: total N content in each new shoot and tea garden nitrogen application level significantly positively correlated; D: nitrogen reutilization efficiency and tea garden nitrogen application level significantly positively correlated.

[0019] Figure 3 N content, nitrogen reutilization efficiency and tea garden nitrogen application level correlation; A: mature leaf L1 N content in February at the end of dormancy period and tea garden nitrogen application level significantly positively correlated; B: new shoot N content at one bud and two leaves stage and tea garden nitrogen application level significantly positively correlated; C: total N content in each new shoot and tea garden nitrogen application level significantly positively correlated; D: nitrogen reutilization efficiency and tea garden nitrogen application level significantly positively correlated.

[0020] Figure 4 N content, nitrogen reutilization efficiency and tea garden nitrogen application level correlation; A: mature leaf L1 N content in February at the end of dormancy period and tea garden nitrogen application level significantly positively correlated; B: new shoot N content at one bud and two leaves stage and tea garden nitrogen application level significantly positively correlated; C: total N content in each new shoot and tea garden nitrogen application level significantly positively correlated; D: nitrogen reutilization efficiency and tea garden nitrogen application level significantly positively correlated.

[0021] Figure 5 N content, nitrogen reutilization efficiency and tea garden nitrogen application level correlation; A: mature leaf L1 N content in February at the end of dormancy period and tea garden nitrogen application level significantly positively correlated; B: new shoot N content at one bud and two leaves stage and tea garden nitrogen application level significantly positively correlated; C: total N content in each new shoot and tea garden nitrogen application level significantly positively correlated; D: nitrogen reutilization efficiency and tea garden nitrogen application level significantly positively correlated. T2 N content, nitrogen reutilization efficiency and tea garden nitrogen application level correlation; A: mature leaf L1 N content in February at the end of dormancy period and tea garden nitrogen application level significantly positively correlated; B: new shoot N content at one bud and two leaves stage and tea garden nitrogen application level significantly positively correlated; C: total N content in each new shoot and tea garden nitrogen application level significantly positively correlated; D: nitrogen reutilization efficiency and tea garden nitrogen application level significantly positively correlated. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0023] Please refer to Figures 1-5 The present application provides a sampling method for accurately evaluating the nutrient status of tea plants.

[0024] Select the consistent growth site of the previous year's nutrient branches on the tea tree canopy, and cut the branches at the end of January to remove the top buds.

[0025] In mid-to-late February, when the tea tree is still in the dormant period, select the first mature leaf at the top of the pruning mouth. When the dormant bud germinates and develops to one bud and two leaves, take the new shoot sample corresponding to the marked mature leaf. At this time, do not take the mature leaf. Example 1

[0026] In the long-term nitrogen positioning test site (20 years), the method described in the application is used to verify whether the method in the application can accurately reflect the fertilization level of the tea garden.

[0027] Test materials and methods: The long-term nitrogen positioning site is located in the Tea Research Institute of Chinese Academy of Agricultural Sciences (30°10′48.83″N, 120°05′20.49″E). The tea variety is "Longjing 43". Since 2005, six nitrogen application levels have been set, and the annual application of chemical fertilizer nitrogen is 0, 119, 285, 474, 569, and 712 kg / ha, respectively. Only early spring one-bud two-leaf shoots are collected, and the total nitrogen content of the shoots, the amino acid content of the shoots, and the total tea polyphenol content are analyzed, and the phenolic amino acid ratio is calculated.

[0028] Result analysis: single linear regression analysis in statistics is used, the regression coefficient is determined by the least square method, and finally the new shoot formula index for determining the nutrient state is obtained, where N is the fertilization level:

[0029] NS1=0.005839×N+51.98;

[0030] P1=-0.004544×N+12.85;

[0031] A1=0.009215×N+27.01;

[0032] According to the results of the long-term positioning experiment, when the nitrogen fertilizer use amount of the tea garden is 0-200 kg / ha, the fertilization level is low, at this time the NS1 value is 51.98-53.1478, the P1 value is 11.9414-12.85, and the A1 value is 27.01-28.853, therefore when 52<NS1<53, 12<P1<13, 27<A1<29, it is determined that the fertilization level of the tea garden is low, and the tea tree is in a poor nutrient state.

[0033] According to the results of the long-term positioning experiment, when the nitrogen fertilizer use amount of the tea garden exceeds 600 kg / ha, the fertilization level of the tea garden is high, at this time the NS1 value is 55.4834, the P1 value is 10.1236, and the A1 value is 32.539, therefore when NS1>55, P1<10, A1>32, it is determined that the fertilization level of the tea garden is high, and the tea tree is in a state of nutrient surplus.

[0034] According to the method of the present invention, in early spring, take the new shoot corresponding to the first mature leaf at the top of the tree crown. The total nitrogen content of the new shoot is significantly positively correlated with the nitrogen application level in the tea garden (A in Figure 1 ), the total amino acid content of the new shoot is significantly positively correlated with the nitrogen application level in the tea garden (B in Figure 1 ), and the phenol-ammonia ratio of the new shoot is significantly negatively correlated with the nitrogen application level in the tea garden (C in Figure 1 ). It shows that the sampling method in the present invention can better reflect the fertilization level of the tea garden. Example 2

[0035] A long-term fixed-position test field (10 years) for the coupling of tea tree varieties and nitrogen fertilizers is used to further verify whether the method of the present invention can indicate the nutritional status of tea trees and reflect the fertilization level of the tea garden.

[0036] Materials and methods: The long-term fixed-position test field for the coupling of tea tree varieties and nitrogen fertilizers is located at the Shengzhou Base of the Tea Research Institute, Chinese Academy of Agricultural Sciences (120°82′53″ E, 29° 74′83″ N). The tea tree varieties are "Longjing 43" and "Zhongcha 108". Since 2015, three nitrogen application levels have been set, and the annual nitrogen application amounts are 150, 300, and 450 kg / ha respectively.

[0037] At the end of January, use pruning shears to cut off the apical dormant buds, and apply 15 N tracer to some leaves. In the middle and late February, collect the first mature leaf under the pruning cut. The one-bud-two-leaf new shoot is the one-bud-two-leaf new shoot germinated from the dormant bud corresponding to the first mature leaf under the pruning cut. Analyze the total nitrogen content and 15 N content of the mature leaf L1 in the dormant period and the one-bud-two-leaf new shoot, and calculate the nitrogen recycling efficiency, that is, the 15 N content in the new shoot divided by the total amount of 15 N applied.

[0038] Result analysis: Use simple linear regression analysis in statistics to determine the regression coefficient by the least squares method, and finally obtain the formula index for determining the nutrient status of the mature leaf in the dormant period, where N is the fertilization level: NL1 = 0.03433×N + 24.79;

[0039] Combined with the experimental results of Example 1 and Example 2 in two long-term fixed-position test fields for many years, when the nitrogen fertilizer application amount in the tea garden is 0 - 200 kg / ha, the fertilization level is low. At this time, the NL1 value is 24.79 - 31.656. Therefore, when 25 < NL1 < 32, it is determined that the fertilization level of the tea garden is low and the tea tree is in a nutrient-poor state; when the nitrogen fertilizer application amount in the tea garden exceeds 600 kg / ha, the fertilization level of the tea garden is high. At this time, the NL1 value is 45.388. Therefore, when NL1 > 45, it is determined that the fertilization level of the tea garden is high and the tea tree is nutritionally sufficient.

[0040] The total nitrogen content of the mature leaf collected at the end of February, that is, the mature leaf in the dormant period, is extremely significantly positively correlated with the fertilization level of the tea garden (Figure 2 In the A section, the total nitrogen content of new shoots with one bud and two leaves is significantly positively correlated with the fertilization level in tea gardens. Figure 2 (B) The mature leaves of tea trees during their dormant period at the end of January are processed. 15 N-labeling can indicate the redistribution of nitrogen from mature leaves to new shoots. New shoots 15 N content was significantly positively correlated with nitrogen application levels in tea gardens. Figure 2 The nitrogen reuse efficiency corresponding to C in the text is significantly positively correlated with the nitrogen application level in tea gardens. Figure 2 (D in the text). Additionally, analysis of the correlation between nitrogen content and fertilization level in mature leaves at the one bud and two leaves stage after new shoot emergence revealed that the nitrogen nutrient status of mature leaves at this stage was unrelated to fertilization level. Figure 3 The above results demonstrate that the sampling method of the present invention can accurately reflect the nutrient status of tea trees and its correlation with the fertilization level of tea gardens. Example 3

[0041] The sampling method of this invention was used to sample 223 individual plants and parent plants of the F1 generation hybrid population of tea trees for three consecutive years to verify that the sampling method of this invention can ensure strong correlation between the years of sampling and is not affected by environmental and climatic factors.

[0042] According to the method of the present invention, the first mature leaf at the top of the canopy of 223 individual plants was collected in mid-to-late February of 2023, 2024 and 2025, and the total nitrogen content and biomass were measured. Figure 4 It can be seen that the dry weight of each mature leaf during the dormant period of a single plant in the population was significantly positively correlated between 2023 and 2024. Figure 4 A) shows a significant positive correlation between the two years 2023 and 2025. Figure 4 C) shows a significant positive correlation between 2024 and 2025. Figure 4 The nitrogen content in L1 of mature leaves during dormancy was significantly positively correlated between 2023 and 2024. Figure 4 (B in the equation) shows a significant positive correlation between 2023 and 2025. Figure 4 (D in the original text); significantly positively correlated between the two years 2024 and 2025 ( ). Figure 4 (F in the text). Therefore, the biomass and total nitrogen of the 2023, 2024, and 2025 samples were significantly correlated across years, and the nutrient status of the samples showed good repeatability. Simultaneously, samples of all new shoots in the tea canopy and mature leaves after shoot emergence were collected. The nutrient status of samples collected during this period could not be reproduced across years. The L1 level of each mature leaf at the one-bud-two-leaf stage on a single plant in the population was [not specified]. T2 There was no correlation between dry weight in 2023 and 2024. Figure 5 In section A), the nitrogen content of all new shoots in the canopy mixed sample showed no correlation between the two years of 2023 and 2024. Figure 5B), i.e. no correlation between nutrient contents of samples between years Figure 5 Therefore, only the dormant period mature leaves described in the present application can be used for accurate identification of population nutrient utilization trait phenotypes.

[0043] Therefore, using the method in the present application can accurately evaluate the nutrient status of tea plants, providing a fast, efficient and accurate method for the field of tea plant cultivation and breeding phenotype identification.

[0044] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is limited by the appended claims and their equivalents.

Claims

1. A method of accurately assessing the nutrient status of tea plants, characterised in that: It includes three stages: pretreatment, mature leaf sampling and new shoot sampling. Pretreatment: from January 20 to 28, the overwintering vegetative branches on the shed were topped, the apical buds were removed, and the positions were marked. Mature leaf sampling: from February 20 to 28, the first mature leaf L1 at the top of the marked branches was taken. New shoot sampling: after the marked dormant buds germinated and developed to one bud and two leaves, the new shoots S1 corresponding to the marked mature leaves were taken, and the mature leaves were not taken at this time. Determination index: the nitrogen concentration of the first mature leaf L1 and the new shoot S1 samples was determined as NL1 and NS1, respectively, with the unit of mg / g, dry weight; the amino acid A1 of the new shoot S1 sample was determined with the unit of mg / g, dry weight, and the total amount of tea polyphenols was determined, and the phenolic acid ratio P1 was calculated. The mature leaf L1 and new shoot S1 samples from the long-term nitrogen fertilizer positioning site were collected, the single linear regression analysis in statistics was used, the regression coefficient was determined by the least square method, and finally the nutrient state formula index was obtained, wherein N is the fertilization level: NL1=0.03433×N+24.79; NS1=0.005839×N+51.98; P1=-0.004544×N+12.85; A1=0.009215×N+27.01; When 25<NL1<32, 52<NS1<53, 12<P1<13 and 27<A1<29, it is determined that the fertilization level of the tea garden is low, and the tea tree is in a poor nutrient state. When NL1>45, NS1>55, P1<10 and A1>32, it is determined that the fertilization level of the tea garden is high, and the tea tree is in a nutrient surplus state.

2. The application of the method for accurately evaluating the nutrient state of tea trees according to claim 1, characterized in that: The mature leaves during the dormancy period are used for accurate identification of the phenotypic traits of population nutrient utilization.

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