CN112730346A - Method for effectively determining nitrogen efficiency of perennial woody plants in field - Google Patents

Method for effectively determining nitrogen efficiency of perennial woody plants in field Download PDF

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CN112730346A
CN112730346A CN202011561118.3A CN202011561118A CN112730346A CN 112730346 A CN112730346 A CN 112730346A CN 202011561118 A CN202011561118 A CN 202011561118A CN 112730346 A CN112730346 A CN 112730346A
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阮丽
王丽鸳
师大亮
成浩
韦康
吴立赟
何梦迪
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Tea Research Institute Chinese Academy of Agricultural Sciences
Hangzhou Institute of Agricultural Sciences
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Abstract

A field effective determination method for nitrogen efficiency of perennial woody plants belongs to the technical field of nitrogen efficiency determination of woody plants. The method comprises the following steps: 1) collecting mature leaves at the top of perennial woody plants; 2) setting a nitrogen concentration gradient, 50% abundance15Urea labeled with N isotope; 3) 500mL of the above 5 concentration gradients were used for each plant15Slowly irrigating the urea marked by N along the circle of the base part; 4) collecting mature leaves at the top of each plant after one week; 5) washing the leaves with pure water, freeze-drying, grinding with a grinder, and placing in a drying dish for storage to be tested; 6) measuring samples with C/N element analyzer15N abundance and total nitrogen content; 7) calculating the nitrogen efficiency of the plant; 8) and (4) fitting in a non-linear mode. The method can directly measure the nitrogen efficiency of the woody plants grown in the fields, the measured result is close to the actual production, the method is not influenced by the age and the site conditions of the plants, the operation is simple, and the method is not easy to be usedIs interfered by the environment.

Description

Method for effectively determining nitrogen efficiency of perennial woody plants in field
Technical Field
The invention belongs to the technical field of woody plant nitrogen efficiency determination, and particularly relates to a field effective determination method for nitrogen efficiency of perennial woody plants.
Background
The nitrogen-efficient variety breeding of perennial woody plants (such as tea trees) is long-lasting, and most of the reasons are due to the lack of an effective nitrogen efficiency measuring method in fields. The existing nitrogen efficiency measuring method for perennial woody plants (such as tea trees) mainly adopts indoor water culture, pot culture and field tests, most of the water culture and the pot culture adopt seedlings, and the difference between the measuring result and the field result after forestation is larger. In field tests, measurement methods such as production, nitrogen content and SPAD value are generally adopted to evaluate the nitrogen efficiency of woody plants, time and labor are consumed, the measured nitrogen efficiency is possibly influenced by site conditions and the age of tea trees, and the measured nitrogen efficiency is possibly influenced by other nutrient elements. The invention aims to develop a field effective nitrogen efficiency measuring method for perennial woody plants (tea trees), the measured nitrogen efficiency is more pertinent, the measured result is close to the actual production, the operation is simple, and the method is not easily interfered by the environment.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to design and provide a method for effectively measuring nitrogen efficiency of perennial woody plants in the field. The method is used for irrigating the woody plants after the forestation in the field15Irrigating with N stable isotope labeled urea solution, and picking mature leaves at the top end for a certain time15N isotopic abundance and nitrogen content were measured to calculate nitrogen efficiency.
A method for effectively measuring nitrogen efficiency of perennial woody plants in the field is characterized by comprising the following steps:
1) blank control sample collection: collecting top mature leaves of perennial woody plants, respectively setting 3 biological repeats, wherein each biological repeat is 3-5 plants, and each plant selects 10 top mature leaves;
2) processing and setting: 5 nitrogen concentration gradients of 0.05, 0.2, 0.5, 2, 5mM were set, in 50% abundance15Urea labeled with N isotope;
3)15and (3) N isotope labeling process: each plant was individually enriched with 500mL of the 50% abundance of the above 5 nitrogen concentration gradients15Slowly irrigating the urea marked by the N isotope along the circle of the base part;
4) collecting samples: collecting mature leaves at the top end of each plant after one week, and collecting 10 leaves of each plant;
5) sample preparation: washing the blank control sample leaves collected in the step 2) and the sample leaves collected in the step 4) with pure water respectively, freezing and drying, grinding by using a grinder, and placing in a drying dish for storage to be detected;
6) sample detection: measuring the 50% abundance of said sample of step 5) by connecting a C/N element analyzer to an isotope mass spectrometer via a continuous flow interface device15N abundance and total nitrogen content, using IAEA-N-1 standard substance to test laboratory steel cylinder N2Calibrating, wherein the nitrogen isotope takes atmospheric nitrogen as a reference standard;
7) calculating the nitrogen efficiency of the plant according to the measured data of the step 6),
the formula is as follows: NE = [ (TN%. Atom%)Treatment-(TN%·Atom%)Blank]·100/15,
Wherein: NE is nitrogen efficiency, TN% is plant total nitrogen content, Atom% is15N atomic percent, (TN%. Atom%)TreatmentAre different from each other15Plants treated with N concentration15N content, (TN%. Atom%)BlankIs not added with15N-labeled Pre-placebo plants15The content of N;
8) carrying out nonlinear fitting on the plant nitrogen efficiency relational expression by adopting Origin or SigmaPlut to obtain a fitting formula:
NE=NEa·C/(A+C),NEb= NEa/A,
wherein: NE is nitrogen efficiency, NEaIs nitrogen under high nitrogen conditionEfficiency potential, C is nitrogen concentration, A is constant, NEbIs the nitrogen efficiency potential under low nitrogen conditions.
The method for effectively measuring nitrogen efficiency of perennial woody plants in the field is characterized in that the perennial woody plants in the step 1) are woody plants after field forestation.
The method for effectively measuring nitrogen efficiency of perennial woody plants in the field is characterized in that the perennial woody plants are tea varieties.
The field effective nitrogen efficiency determination method for perennial woody plants is characterized in that nitrogen efficiency determination in the steps 1) -8) is carried out by selecting a nitrogen efficient variety as a reference variety, and the determination result is compared with the nitrogen efficiency of a sample plant to evaluate the nitrogen efficiency level of the sample plant.
The method for effectively determining the nitrogen efficiency of perennial woody plants in the field is characterized in that the nitrogen-efficient variety plants are Fuding Dabai and Longjing 3.
The method can directly measure the nitrogen efficiency of the woody plants growing into forests in the field, the measured result is close to the actual production, the method is not influenced by the age and the standing conditions of the plants, the operation is simple, and the method is not easily interfered by the environment.
Detailed Description
The present invention will be further illustrated by the following examples.
Example 1:
1) selection of control and reference varieties: taking perennial woody plant tea trees as an example, 3-year-old Longjing 43 and Fuding white are selected as reference varieties, and 3-year-old Zhongming No. 7 and 2807 are selected as reference varieties;
2) selecting a test base: taking a tea research institute Shengzhou base of China academy of agricultural sciences as a test field, and developing research, wherein the physicochemical properties of the foundation soil of the base are shown in Table 1;
TABLE 1 Shengzhou base soil foundation physicochemical properties
Figure 349553DEST_PATH_IMAGE001
3) Blank control setup: in that15Before N marking, collecting the topmost mature leaves of the 4 varieties of tea trees, setting 3 biological repeats (namely samples of 3 cells), repeating 3-5 plants for each biological repeat, and selecting about 10 topmost mature leaves for each plant;
4) processing and setting: 5 nitrogen concentration gradients (0.05, 0.2, 0.5, 2, 5 mM) were set, using 50% abundance15Carrying out an experiment on N isotope labeled urea, setting 3 biological repeats (namely 3 cell samples of the blank control) for each concentration gradient, and repeating 3-5 plants for each biological repeat;
5)15and (3) N isotope labeling process: 500ml of the corresponding concentration is used for each plant15Slowly irrigating the N-marked urea solution along the base part in a circle;
6) collecting samples: collecting the top mature leaves of the plants after 1 week, and collecting about 10 leaves of each plant;
7) sample preparation: collecting leaves, washing with pure water, freeze drying, grinding with a grinder, and storing in a drying dish;
8) sample detection: of samples15The N abundance and total nitrogen content are connected with an isotope mass analyzer by a C/N element analyzer through a continuous flow interface device, and the laboratory steel cylinder N is treated by IAEA-N-1 standard substance2Gas is calibrated, and the nitrogen isotope takes atmospheric nitrogen as a reference standard;
9) the nitrogen efficiency calculation formula of the plants under different concentrations is as follows:
nitrogen Efficiency (NE) = [ (TN%. Atom%)Treatment-(TN%·Atom%)Blank]·100/15
Wherein TN% is the total nitrogen content of the plant, Atom% is15N atomic percent, (TN%. Atom%)TreatmentAre different from each other15Plants treated with N concentration15N content, (TN%. Atom%)BlankIs not added with15N-labeled Pre-placebo plants15The content of N;
10) non-linear fitting was performed in Origin or SigmaPlot:
NE=NEa·C/(A+C),NEb= NEa/A,
wherein: NE is nitrogen efficiency, NEaNitrogen efficiency potential under high nitrogen conditions, C is nitrogen concentration, A is constant, NEbNitrogen efficiency potential under low nitrogen conditions;
11) evaluating the nitrogen efficiency of the field tea trees: mixing Chinese tea No. 7 and 2807 with reference varieties of Longjing 43 and Fuding Dabai NEaAnd NEbThe comparison was carried out and found (table 2): two control varieties, NE of Longjing 43aAnd NEbAll are higher than the great white of the good fortune ancient cooking vessel, which indicates that the dragon well 43 is a double-efficient tea tree variety and is consistent with the production practice. For NEaThe values of Zhongming No. 7 and 2807 are all greater than those of the control varieties Longjing 43 and Fuding Dabai, for NEbThe values of Zhongming No. 7 and 2807 are smaller than those of the control varieties Longjing 43 and Fuding Dabai. Therefore, Zhongming No. 7 and 2807 are high-nitrogen and high-efficiency varieties.
TABLE 2 evaluation of nitrogen efficiency of tea trees grown in Shaoxing Shengzhou 3 years
Figure 807080DEST_PATH_IMAGE002
Example 2:
1) selection of control and reference varieties: taking perennial woody plant tea trees as an example, 5-year-old fuding white is selected as a control variety, and 5-year-old Meifeng, Hangzhou tea 19 and Hangzhou tea 15 are selected as reference varieties;
2) selecting a test base: taking a bottle kiln base land of the Hangzhou city agricultural science institute as a test land, and carrying out research, wherein the physicochemical properties of the soil foundation of the base land are shown in a table 3;
TABLE 3 Shengzhou base soil foundation physicochemical properties
Figure 152610DEST_PATH_IMAGE003
3) Blank control setup: in that15Before N marking, collecting the topmost mature leaves of the 4 varieties of tea trees, setting 3 biological repeats (namely samples of 3 cells), repeating 3-5 plants for each biological repeat, and selecting about 10 topmost mature leaves for each plant;
4) processing and setting: 5 nitrogen concentration gradients (0.05, 0.2, 0.5, 2, 5 mM) were set, using 50% abundance15Carrying out an experiment on N isotope labeled urea, setting 3 biological repeats (namely 3 cell samples of the blank control) for each concentration gradient, and repeating 3-5 plants for each biological repeat;
5)15and (3) N isotope labeling process: 500ml of the corresponding concentration is used for each plant15Slowly irrigating the N-marked urea solution along the base part in a circle;
6) collecting samples: collecting the top mature leaves of the plants after 1 week, and collecting about 10 leaves of each plant;
7) sample preparation: collecting leaves, washing with pure water, freeze drying, grinding with a grinder, and storing in a drying dish;
8) sample detection: of samples15The N abundance and total nitrogen content are connected with an isotope mass analyzer by a C/N element analyzer through a continuous flow interface device, and the laboratory steel cylinder N is treated by IAEA-N-1 standard substance2Gas is calibrated, and the nitrogen isotope takes atmospheric nitrogen as a reference standard;
9) the nitrogen efficiency calculation formula of the plants under different concentrations is as follows:
nitrogen Efficiency (NE) = [ (TN%. Atom%)Treatment-(TN%·Atom%)Blank]·100/15
Wherein TN% is the total nitrogen content of the plant, Atom% is15N atomic percent, (TN%. Atom%)TreatmentAre different from each other15Plants treated with N concentration15N content, (TN%. Atom%)BlankIs not added with15N-labeled Pre-placebo plants15The content of N;
10) non-linear fitting was performed in Origin or SigmaPlot:
NE=NEa·C/(A+C),NEb= NEa/A,
wherein: NE is nitrogen efficiency, NEaNitrogen efficiency potential under high nitrogen conditions, C is nitrogen concentration, A is constant, NEbNitrogen efficiency potential under low nitrogen conditions;
11) evaluating the nitrogen efficiency of the field tea trees: mixing Meifeng tea 19, Hangzhou tea 15 and control varietyNE of Dingda BaiaAnd NEbThe comparison was carried out and found (table 4): NE of comparison variety Fuding whiteaAnd NEbThe results of Hangzhou bottle kiln base and Shaoxing Shengzhou base are very close, which shows that the age and the standing conditions of the tea tree have little influence on the result of the determination method. In addition, for NEaIn value, Hangzhou tea 19 is greater than the reference variety Fuding white and for NEbIn value, Hangzhou tea 19, Hangzhou tea 15 and eyebrow are all larger than the control variety Fuding white, so Hangzhou tea 19 is a double-efficient variety, and Hangzhou tea 15 and eyebrow are low-nitrogen efficient varieties.
TABLE 4 evaluation of nitrogen efficiency of 5-year old tea trees in Hangzhou bottle kiln
Figure 37390DEST_PATH_IMAGE004
The test shows that the nitrogen efficiency evaluation result of the great white of the fuding is little influenced by the conditions of the site and the age of the tea tree by comparing the results of two bases with different ages of the tea tree, and the NEaWithin 10% difference, NEbThe difference was around 5%.

Claims (5)

1. A method for effectively measuring nitrogen efficiency of perennial woody plants in the field is characterized by comprising the following steps:
1) blank control sample collection: collecting top mature leaves of perennial woody plants, respectively setting 3 biological repeats, wherein each biological repeat is 3-5 plants, and each plant selects 10 top mature leaves;
2) processing and setting: 5 nitrogen concentration gradients of 0.05, 0.2, 0.5, 2, 5mM were set, in 50% abundance15Urea labeled with N isotope;
3)15and (3) N isotope labeling process: each plant was individually enriched with 500mL of the 50% abundance of the above 5 nitrogen concentration gradients15Slowly irrigating the urea marked by the N isotope along the circle of the base part;
4) collecting samples: collecting mature leaves at the top end of each plant after one week, and collecting 10 leaves of each plant;
5) sample preparation: washing the blank control sample leaves collected in the step 2) and the sample leaves collected in the step 4) with pure water respectively, freezing and drying, grinding by using a grinder, and placing in a drying dish for storage to be detected;
6) sample detection: measuring the 50% abundance of said sample of step 5) by connecting a C/N element analyzer to an isotope mass spectrometer via a continuous flow interface device15N abundance and total nitrogen content, using IAEA-N-1 standard substance to test laboratory steel cylinder N2Calibrating, wherein the nitrogen isotope takes atmospheric nitrogen as a reference standard;
7) calculating the nitrogen efficiency of the plant according to the measured data of the step 6),
the formula is as follows: NE = [ (TN%. Atom%)Treatment-(TN%·Atom%)Blank]·100/15,
Wherein: NE is nitrogen efficiency, TN% is plant total nitrogen content, Atom% is15N atomic percent, (TN%. Atom%)TreatmentAre different from each other15Plants treated with N concentration15N content, (TN%. Atom%)BlankIs not added with15N-labeled Pre-placebo plants15The content of N;
8) carrying out nonlinear fitting on the plant nitrogen efficiency relational expression by adopting Origin or SigmaPlut to obtain a fitting formula:
NE=NEa·C/(A+C),NEb= NEa/A,
wherein: NE is nitrogen efficiency, NEaNitrogen efficiency potential under high nitrogen conditions, C is nitrogen concentration, A is constant, NEbIs the nitrogen efficiency potential under low nitrogen conditions.
2. The method for effectively measuring nitrogen efficiency of perennial woody plants in the field according to claim 1, wherein the perennial woody plants in the step 1) are woody plants after field forestation.
3. The method for field effective determination of nitrogen efficiency of perennial woody plants of claim 1 or 2, wherein the perennial woody plants are of the species Camellia sinensis.
4. The method for effectively measuring nitrogen efficiency of perennial woody plants in the field as claimed in claim 1, wherein the nitrogen efficiency of the steps 1) -8) is measured by selecting a nitrogen-efficient variety as a control variety, and the measured result is compared with the nitrogen efficiency of the sample plant to evaluate the nitrogen efficiency level of the sample plant.
5. The method for the field effective measurement of nitrogen efficiency of perennial woody plants as claimed in claim 4, wherein said nitrogen-efficient variety plants are Fordingda Bai, Longjing 3.
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