CN112730346A - Method for effectively determining nitrogen efficiency of perennial woody plants in field - Google Patents
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 title claims abstract description 198
- 229910052757 nitrogen Inorganic materials 0.000 title claims abstract description 104
- 238000000034 method Methods 0.000 title claims abstract description 28
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000004202 carbamide Substances 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 6
- 238000005259 measurement Methods 0.000 claims abstract 3
- 241000196324 Embryophyta Species 0.000 claims description 73
- 239000000523 sample Substances 0.000 claims description 18
- 240000003553 Leptospermum scoparium Species 0.000 claims description 17
- 235000009024 Ceanothus sanguineus Nutrition 0.000 claims description 15
- 235000015459 Lycium barbarum Nutrition 0.000 claims description 15
- 238000012360 testing method Methods 0.000 claims description 9
- 235000016299 Canarium odontophyllum Nutrition 0.000 claims description 5
- 244000001582 Canarium odontophyllum Species 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 4
- 238000001948 isotopic labelling Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- 239000013068 control sample Substances 0.000 claims description 3
- 238000000691 measurement method Methods 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 238000002372 labelling Methods 0.000 claims 1
- 239000010421 standard material Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 238000004321 preservation Methods 0.000 abstract 1
- 241001122767 Theaceae Species 0.000 description 12
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 8
- 239000002689 soil Substances 0.000 description 4
- 229910000831 Steel Inorganic materials 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000000227 grinding Methods 0.000 description 3
- 229940068196 placebo Drugs 0.000 description 3
- 239000000902 placebo Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000010959 steel Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 2
- 210000004709 eyebrow Anatomy 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/64—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using wave or particle radiation to ionise a gas, e.g. in an ionisation chamber
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Abstract
一种多年生木本植物氮效率田间有效测定方法，属于木本植物氮效率测定技术领域。该方法包括以下步骤：1）对多年生木本植物进行顶端成熟叶片收集；2）设置氮浓度梯度，50%丰度的15N同位素标记的尿素；3）每株植物分别用500mL上述5个浓度梯度的15N标记的尿素沿基部一圈缓慢浇灌；4）一周后采集每株植物顶端成熟叶片；5）叶片分用纯水洗干净，冷冻干燥后用粉碎机磨碎，放置于干燥皿中保存待测；6）C/N元素分析仪测定样品的15N丰度和全氮含量；7）计算植物氮效率；8）非线性拟合。本方法可对田间成林的木本植物直接进行氮效率测定，测定结果与生产实际贴近，不受植物年龄和立地条件的影响，操作简单不易受环境干扰。The invention discloses a field effective determination method for nitrogen efficiency of perennial woody plants, belonging to the technical field of nitrogen efficiency determination of woody plants. The method includes the following steps: 1) collecting apical mature leaves of perennial woody plants; 2) setting a nitrogen concentration gradient, 50% abundance of 15 N isotope-labeled urea; 3) using 500 mL of the above 5 concentrations for each plant Gradient 15 N-labeled urea was slowly watered along the base in a circle; 4) The mature leaves at the top of each plant were collected after one week; 5) The leaves were washed with pure water, freeze-dried, ground with a pulverizer, and placed in a drying dish for preservation To be tested; 6) C/N elemental analyzer to determine the 15 N abundance and total nitrogen content of the sample; 7) Calculate plant nitrogen efficiency; 8) Non-linear fitting. The method can directly measure the nitrogen efficiency of the woody plants in the field, the measurement results are close to the actual production, not affected by the age of the plants and the site conditions, and the operation is simple and not easily affected by the environment.
Description
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
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
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
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
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%.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117309979A (en) * | 2023-09-28 | 2023-12-29 | 广东工业大学 | Mangrove nitrogen pollution source analysis method based on stable nitrogen isotope composition |
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