CN115606383A - Method for determining step-by-step accurate combined fertilization of compound fertilizer and single fertilizer and application - Google Patents

Method for determining step-by-step accurate combined fertilization of compound fertilizer and single fertilizer and application Download PDF

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CN115606383A
CN115606383A CN202211408713.2A CN202211408713A CN115606383A CN 115606383 A CN115606383 A CN 115606383A CN 202211408713 A CN202211408713 A CN 202211408713A CN 115606383 A CN115606383 A CN 115606383A
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房经贵
王晨
张孜博
刘志龙
上官凌飞
卢素文
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Nanjing Agricultural University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C21/00—Methods of fertilising, sowing or planting
    • A01C21/005—Following a specific plan, e.g. pattern
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
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    • A01C21/007—Determining fertilization requirements
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Abstract

The invention discloses a method for determining step-by-step accurate combined fertilization of a compound fertilizer and a single fertilizer and application thereof.

Description

Method for determining step-by-step accurate combined fertilization of compound fertilizer and single fertilizer and application
Technical Field
The invention relates to the technical field of precise fertilization, in particular to a method for determining step-by-step precise combined fertilization of a compound fertilizer and a single fertilizer and application thereof.
Background
The fertilizer provides a plurality of nutrient components for completing the growth and life cycle of crops. In agricultural production, people apply various fertilizers, especially macroelements, to soil to promote crops to fully grow to obtain high yield and ensure food safety, so that the fertilizers are widely applied to agricultural production. Fertilizer elements are generally divided into macro-elements and micro-elements according to the amount of crop demand. Among them, several nutrients such as nitrogen (N), phosphorus (P), and potassium (K) are classified as macronutrients. Compared with other nutrient elements, the demand of the plants is large, and the deficiency of the plants can limit the growth and development of the plants. At present, with the continuous increase of agricultural production investment, the phenomenon of excessive fertilization for pursuing high yield of crops is very common, which inevitably causes fertilizer waste and environmental pollution. Therefore, the development of an efficient, low-dosage, low-cost and low-pollution fertilization method is an urgent task in current agricultural production.
The use of chemical fertilizers has greatly increased soil productivity and has dramatically improved agricultural development with surprising efficiency, which is estimated to increase the total yield of grain in china by 40% to 60%. The application modes of major elements such as N, P and K or trace elements such as Mo, zn, fe, ca, mg and S can be divided into single fertilizer and compound fertilizer. Compared with single fertilizer, the compound fertilizer can simultaneously obtain various nutrient elements in one-time fertilization, broadens the synchronous interaction period of various elements, improves the fertilizer efficiency through synchronous interaction synergism, and simultaneously saves labor and reduces the dosage so as to reduce pollution. Therefore, in the past decades, the using structure of the fertilizer in China has changed greatly, the application proportion of the compound fertilizer is increased continuously, and the using amount of the compound fertilizer is increased by about 32.3 percent. Although the compound fertilizer brings great benefits to agricultural production, the difference of absorption speeds of different nutrient elements such as N, P, K and the like is ignored, so that different elements are absorbed by plants in different periods, and the fertilizer waste is caused by the reduction of the fertilizer efficiency. If the synchronous period among the elements of the compound fertilizer is widened by accurately combining and fertilizing in different time periods, the interaction synergistic effect of the elements is enhanced, the fertilizer efficiency is greatly improved, the using amount is saved, the environmental pollution is reduced, and the cost is saved. Therefore, the problem of how to use the fertilizer to enhance the synchronous interaction and synergism among different elements so as to achieve the best fertilizer efficiency is receiving much attention. Recently, it has been reported that the 'controlled slow release fertilizer' can effectively regulate the release time of fertilizer efficiency, thereby improving the fertilizer efficiency, and is more beneficial to the growth and development of crops and high yield, but the cost is higher, which limits the wide application of the controlled slow release fertilizer in agricultural production to a certain extent. Therefore, developing a new low-cost, efficient and safe fertilization strategy is one of the urgent needs for developing the simplified agriculture.
The patent aims to provide a new step-by-step accurate fertilization strategy so as to achieve lower fertilizer cost and obtain higher economic benefit.
Disclosure of Invention
The invention provides a method for determining step-by-step accurate combined fertilization of a compound fertilizer and a single fertilizer aiming at the technical problems that the assimilation rate and the absorption speed of N/P elements in an ammonium dihydrogen phosphate compound fertilizer are different to limit the high-efficiency exertion of the fertilizer efficiency and the like, and an application thereof. The invention improves the fertilizer efficiency, reduces the dosage, saves the cost and reduces the pollution by widening the synchronous period among different fertilizer elements. Taking a diammonium hydrogen phosphate compound fertilizer for strawberries as an example, a P fertilizer is additionally applied 7 days before the compound fertilizer is applied (the first flower of strawberries), an N fertilizer is additionally applied 9 days after the compound fertilizer, and the step-by-step precise combined fertilization is carried out, so that the synchronization period between N and P elements is widened by 7-10 days, the utilization efficiency of the compound fertilizer is improved, the using amount is reduced, and the pollution is reduced. The invention establishes a novel efficient and accurate combined fertilization method, has high efficiency, low cost and less pollution, is beneficial to green and safe agricultural production, is also beneficial to the development of simplified fertilization in modern accurate agriculture, and belongs to the category of new fertilization strategies of accurate agriculture.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for determining step-by-step accurate combined fertilization of a compound fertilizer and a single fertilizer comprises the following steps:
(1) And N/P response time determination: after single N/P fertilizer application of plants, collecting leaves every two days, measuring the contents of N and P, and determining the efficient response duration of the NP to N and P according to the difference of the NP absorption speed;
(2) Identifying the expression of response genes in the NP assimilation process by using a transcriptome sequencing result of a sample after N/P fertilizer application, identifying genes with LOG2 values of the expression levels of N/P fertilizer treatment and a reference gene being more than or equal to 2 by comparing and analyzing the expression conditions of different genes of the N/P fertilizer treatment and the reference sample, sequencing the genes, taking the genes with the LOG2 values of the genes being three at the top, and preliminarily determining the genes as candidate biomarkers for N/P element response;
(3) Designing primers aiming at the candidate biomarker genes and the reference genes to detect the expression level of the marker genes, and determining nitrogen assimilation marker genes and phosphorus assimilation marker genes by combining the measurement results of the content of N and the content of P;
(4) Analyzing and identifying the speed and the length of the plants for different fertilizer assimilation rates, and analyzing the speed and the duration of the response of the nitrogen assimilation marker gene and the phosphorus assimilation marker gene to nitrogen and phosphorus fertilization by utilizing a qRT-PCR technology;
(5) And determining the optimal fertilizer efficiency fertilization method by combining phenotypic data and physiological data weighted analysis in the fertilization combination and the detected N/P content and the expression of a nitrogen and phosphorus assimilation marker gene.
Preferably, the plants in step (1) include at least one of strawberry plants and grape plants.
In any of the above schemes, preferably, the N, P single element fertilizer in the step (2) is urea and calcium superphosphate.
In any of the above embodiments, it is preferred that the N/P element-responsive candidate biomarker genes in step (2) include one or more of FaNR (XM _ 004300392), faNIR (XM _ 004294755), faGS (XM _ 011471924.1), faAS (XM _ 004294250.2), faGDH (XM _ 004288369.2), faPHO (XM _ 004296337.2), faPHT1 (XM _ 004301571.2), faPHT2 (XM _ 004301571.2), faPAP (XM _ 004297185.2). The relevant gene sequences can be downloaded from the following website (https:// www.ncbi.nlm.nih.gov. /).
In any of the above embodiments, it is preferable that the step (3) is performed using FaNIR as a nitrogen assimilation marker gene and FaPAP as a phosphorus assimilation marker gene.
The FaNIR gene (Fragaria vesca nitrite reductase) is a key enzyme of nitrogen cycle, and can catalyze nitrite reduction and reduce damage to plant bodies. The FaPAP gene (Fragaria vesca pure acid phosphatase 1) has the function of catalyzing the hydrolysis of phosphate ester to release inorganic phosphorus, and can promote the release of inorganic phosphorus Pi from organic phosphorus at roots.
In any of the above embodiments, it is preferable that the primers for the nitrogen assimilation marker gene farir in step (3) are as follows:
FaNIR-F：
Figure BDA0003935890240000031
FaNIR-R：
Figure BDA0003935890240000032
in any of the above embodiments, preferably, the primers for the phosphorus assimilation marker gene FaPAP in step (3) are as follows:
FaPAP-F：
Figure BDA0003935890240000033
FaPAP-R：
Figure BDA0003935890240000034
preferably in any of the above embodiments, the phenotype data in step (5) includes flowers and fruits.
The invention also discloses a step-by-step accurate combined fertilization method applied to plants, and further the fertilizer efficiency is improved.
Preferably, the plants comprise at least strawberries and/or grapes.
In any scheme, preferably, the single-element phosphate fertilizer is additionally applied 7 days before the compound fertilizer is applied; and (3) applying the single-element nitrogen fertilizer after 9 days of the compound fertilizer.
In any of the above schemes, preferably, the compound fertilizer is ammonium dihydrogen phosphate, the single-element phosphate fertilizer is calcium superphosphate, and the single-element nitrogen fertilizer is urea.
In any of the above schemes, preferably, the step-by-step precise combined fertilizing method for strawberries comprises the following steps:
(1) Applying superphosphate 7 days before the ammonium dihydrogen phosphate of the compound fertilizer is applied;
(2) Applying ammonium dihydrogen phosphate fertilizer when the first flower of the strawberry appears;
(3) And applying urea 9 days after the compound fertilizer is applied.
In any of the above schemes, preferably, the step-by-step precise combined fertilizing method for strawberries specifically comprises the following steps:
(1) Applying calcium superphosphate 7 days before the ammonium dihydrogen phosphate of the compound fertilizer is applied, namely 7 days before the first flower of the strawberry appears, wherein the dosage of the calcium superphosphate is about 1 to 1.5g per plant;
(2) Applying 1.5-2.5g of ammonium dihydrogen phosphate fertilizer per plant when the first flower of the strawberry appears;
(3) The fertilizer amount of urea is applied again by about 0.8-1.2 g/plant 9 days after the compound fertilizer is applied;
(4) The strawberries are watered thoroughly after each fertilization, so that the fertilizer efficiency is fully exerted.
In any of the above schemes, preferably, in step (1), calcium superphosphate is added 7 days before the ammonium dihydrogen phosphate compound fertilizer is applied, namely 7 days before the first flower of the strawberry appears, and the dosage of the calcium phosphate is about 1 g/plant.
In any of the above schemes, preferably, in step (1), calcium superphosphate is added 7 days before the ammonium dihydrogen phosphate compound fertilizer is applied, namely 7 days before the first flower of the strawberry, and the dosage of the calcium phosphate is about 1.25 g/plant.
In any of the above schemes, preferably, in the step (1), calcium superphosphate is added 7 days before the ammonium dihydrogen phosphate compound fertilizer is applied, i.e. 7 days before the first flower of the strawberry appears, and the dosage of the calcium phosphate is about 1.5 g/plant.
In any of the above schemes, preferably, the fertilizer amount of the ammonium dihydrogen phosphate fertilizer applied in the step (2) when the first flower of the strawberry appears is about 1.5 g/plant.
In any of the above embodiments, preferably, the amount of fertilizer applied to the strawberries in step (2) is about 2.0 g/plant of monoammonium phosphate fertilizer when the strawberries flower first.
In any of the above schemes, preferably, the fertilizer amount of the ammonium dihydrogen phosphate fertilizer applied in the step (2) when the first flower of the strawberry appears is about 2.5 g/plant.
In any of the above schemes, preferably, the fertilizer amount of urea applied in step (3) is about 0.8 g/plant after 9 days of compound fertilizer application.
In any of the above schemes, preferably, the fertilizer amount of urea applied in step (3) is about 1.0 g/plant after 9 days of compound fertilizer application.
In any of the above embodiments, preferably, the amount of urea applied in step (3) is about 1.2 g/plant after 9 days of the compound fertilizer application.
Advantageous effects
The invention provides a method for determining step-by-step accurate combined fertilization of a compound fertilizer and a single fertilizer and application thereof.
The invention firstly expands the synchronous interaction and validity increasing period among different elements from the aspect of nutrient element assimilation rate and marker gene expression information, accurately determines the time interval of the step-by-step combined fertilization of different fertilizers, provides a new strategy for improving the compound fertilizer efficiency by the accurate step-by-step combined fertilization of the compound fertilizer, and reduces the fertilizer consumption, saves the cost and reduces the environmental pollution by improving the fertilizer efficiency. The strategy is relatively simple to operate, low in cost, high in sensitivity, and high in precision and fertilizer efficiency. It should be noted that, due to different plant species and environmental conditions, the fertilizer efficiency of the fertilizer nutrient elements is different in the morning and evening and the duration time, thereby affecting the fertilization time interval in the step-by-step combined fertilization strategy.
Drawings
FIG. 1 is a step-by-step precision combined fertilization strategy for strawberries;
FIG. 2 contents of N and P elements in leaves after strawberry fertilization treatment, (A) contents of N in strawberry leaves; (B) P content in strawberry leaves;
FIG. 3 shows the identification of the expression of different nitrogen and phosphorus response genes of strawberries after different N and P fertilization treatments, (A) identification of the FaNR gene of strawberries after N fertilization; (B) identifying the FaPAB gene expression of the strawberries after P fertilization; (C) identifying the FaNR gene expression of the strawberry after N and P fertilization; (D) identifying the FaPAB gene expression of the strawberries after P and P are fertilized;
FIG. 4 morphological characteristics of strawberries after different treatments of N and P fertilization.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1
A method for determining step-by-step accurate combined fertilization of strawberry plants comprises the following steps:
(1) And N/P response time determination: from a single N/P fertilizer (Urea (CH) 4 N 2 O) (0.78 g/strain) and calcium superphosphate [ Ca (H) 2 PO 4 ) 2 ](1.80 g/plant)) every two days after application, collecting each treated leaf, measuring the content of N and P, determining the difference of absorption speed according to the change of the absorption amount of the strawberries to NP in different periods as shown in figure 1, and determining that the high-efficiency response time of the strawberries to N is 3-17 days after application and the high-efficiency response time of the strawberries to P is 7-23 days according to the number of days that the absorption amount of the strawberries to NP in different periods is increased by more than 1 time than that of a control.
(2) And identifying the expression of the response gene in the NP assimilation process by using the transcriptome sequencing result of the sample after the N/P fertilizer is applied, and primarily screening the candidate biomarker gene responded by the N/P element. The expression of response genes of NP assimilation process in strawberry is identified by transcriptome sequencing of strawberry samples after N/P fertilizer application, the differential expression of the N/P fertilizer response genes in treated samples and control samples is analyzed through comparison, genes with LOG2 value more than or equal to 2 of the expression level of the N/P treated genes and the control genes are identified and ranked, the genes with the LOG2 value higher than 3-5 are taken, and candidate biomarker genes FaNR (XM _ 004300392), faGS (XM _ 004472955), faPHT (XM _ 011474.1), faAS (XM _ 004294250.2), faGDH (XM _ 004288369.2), faPHO (XM _ 004296337.2), faPHT1 (XM _ 004301571.2), faPHT2 (XM _ 301. 2) and FaPAP (XM _ 004297185.2) of the N/P element response are preliminarily screened. Gene sequences can be downloaded from the following website (https:// www.ncbi.nlm.nih.gov /).
(3) Designing primers aiming at the candidate biomarker genes and the reference genes to detect the expression level of the marker genes, and determining nitrogen assimilation marker genes and phosphorus assimilation marker genes by combining the measurement results of the content of N and the content of P;
designing primers aiming at the candidate biomarker genes and the reference FaActin gene, and then passing through two single element fertilizers, namely urea (CH) 4 N 2 O) and calcium superphosphate [ Ca (H) ] 2 PO 4 ) 2 ]The strawberry fertilization test of (1) was conducted by detecting the expression level of the marker gene by the fluorescent quantitative PCR technique, and by measuring the N and P contents thereof, when the absorption contents of N and P are higher than those of a control, a gene whose expression level is high in leaf tissue was selected as the marker gene.
The FaNIR gene (Fragaria vesca nitrate reductase) is a key enzyme for nitrogen circulation, can catalyze nitrite reduction, and reduces damage to plant bodies. The FaPAP gene (Fragaria vesca pure acid phosphatase 1) has the function of catalyzing phosphate hydrolysis to release inorganic phosphorus; the release of inorganic phosphorus Pi from organic phosphorus can be promoted at the root.
Wherein, the primers of the marker gene FaNIR of the nitrogen element are as follows:
FaNIR-F：
Figure BDA0003935890240000061
FaNIR-R：
Figure BDA0003935890240000062
primers for marking FaPAP for elemental phosphorus were as follows:
FaPAP-F：
Figure BDA0003935890240000063
FaPAP-R：
Figure BDA0003935890240000064
(4) Analyzing and identifying the speed and the length of assimilation rate of plants to different fertilizers, determining the difference of the absorption speed of the strawberries to NP (nitrogen) absorption amount in unit time according to the change of the absorption amount of the strawberries to NP in comparison with a contrast, increasing the absorption amount of the strawberries to N/P (nitrogen/phosphorus) by more than 1 time for a continuous day according to the absorption amount of the strawberries to N/P in different periods, analyzing the speed of response of the strawberries to N and P in unit time after the treatment of N and P by utilizing qRT-PCR (quantitative reverse transcription-polymerase chain reaction) technology according to the change of Log2 value of gene expression change in comparison with the contrast, and simultaneously indicating the duration time of high-efficiency response of the strawberries to N and P according to the Log2 value of gene expression change in comparison with the contrast which the Log2 value is =2 for a continuous time;
(5) And determining the optimal fertilizer efficiency fertilization method by combining with weighted analysis of phenotype data (the phenotype data comprises flowering number, fruit weight and fruit color) and physiological data (the physiological data specifically comprises anthocyanin, soluble solid and organic acid content in the fruit) in the fertilization combination and simultaneously combining with the detected N/P content and the expression of a nitrogen and phosphorus assimilation marker gene.
Development of a new strategy for improving the fertilizer efficiency of the compound fertilizer by accurate combined fertilization: the phenotype observation and comparison of strawberry flowers and fruits in the 13 fertilization combinations and the weighted analysis of physiological data thereof are combined, and the best fertilization combination of the P-compound fertilizer-N is comprehensively determined by detecting the N/P content and the expression of a marker gene, namely, the calcium superphosphate is applied 7 days before the monoammonium phosphate of the compound fertilizer is added, and then the urea is applied 9 days after the compound fertilizer is applied.
A method for determining step-by-step accurate combined fertilization of strawberry plants comprises the following steps:
(1) When the first strawberry is grown, the 'sweet Charlie' strawberry with consistent growth is selected, the fertilizer is applied to the position 5-8cm away from the root of the strawberry plant according to the set scheme of different fertilizer application combinations (see Table 1), and water is irrigated thoroughly after the fertilizer is applied.
The setting principle is as follows: setting the total fertilizer amount to be 4.25g according to a certain total fertilizer amount, then dividing monocalcium phosphate into four modes of primary fertilizer application, secondary fertilizer application, tertiary fertilizer application and quaternary fertilizer application, and carrying out permutation and combination by adjusting different fertilizer application amounts to obtain 13 treatment combinations.
TABLE 1 strawberry 13 Combined fertilization dressing
Figure BDA0003935890240000071
Figure BDA0003935890240000081
FIG. 2 shows the morphological changes of strawberries in various fertilization treatments, and different treatments can promote the growth and development of strawberry fruits and the ripening and coloring of the fruits to different degrees compared with the control. The T10 treatment (the calcium dihydrogen phosphate is applied once 7 days before the compound fertilizer, and the urea is applied once every 9 days after the compound fertilizer) has the best effect, the strawberry fruits are the largest and the strawberry fruits are colored the best, and the fertilization effect of the strategy of step-by-step accurate combined fertilization of the compound fertilizer and the single fertilizer is the best.
(2) After the first fertilization, each treated leaf sample was collected at 6h, 1d, 3d, 5d, 7d, 9d, 11d, 13d, 15d, 17d, 19d, 21d, 23d, immediately frozen with liquid nitrogen, and stored at-80 ℃ for further use.
(3) Meanwhile, the number of blossoms, the fruit number and the weight of a single fruit of all the strawberries treated are counted once every four days after fertilization. The weight of individual fruits was determined using an analytical balance and repeated 3 times.
(4) The nitrogen content of the different samples was determined by means of a Continuous Flow Analyser (CFA) and the phosphorus content was determined using an ICP-MS instrument, the relevant determination methods being performed according to the instructions. The nitrogen and phosphorus content of strawberries in different treatments are measured as shown in fig. 3.
(5) Total RNA was extracted using a total RNA extraction reagent (Vazyme Biotech, nanjing, china), RNA concentration and A260/A280 ratio were measured before and after DNase I treatment using a Nano-Drop ND-1000 spectrophotometer (Nanodrop Technologies), and electrophoresed on agarose gel or polyacrylamide gel, stained, and electrophorograms of RNA were obtained.
(6) OligodT and Superscript II enzymes (Invitrogen) were used TM ) First strand cDNA was synthesized from 1.0. Mu.g total RNA. cDNA was synthesized using Prime Script RT kit (DRR 047A, takara, dalian, china) with a gDNA eraser.
(7) According to SYBR Premix Ex Taq II (Takara, dalian, china) and CFX96 touch real-time PCR detection system (Bio-Rad, USA), qRT-PCR (real-time quantitative PCR) expression analysis of marker genes of nitrogen and phosphorus elements is carried out by using differently treated sample cDNAs as templates and primers of nitrogen and phosphorus marker genes through the following amplification system (95 ℃ 30s, then 95 ℃ 5s and 60 ℃ 30s 40 cycles) (see Wang et al. Journal of experimental botanic, 2018, 69 (15): 3639-3650). Three replicates of the experimental setup were used.
(8) According to the result analysis in the step (4) and the step (7) and the judgment standard in the step (4), the speed and the length of the assimilation rate of the strawberries to fertilizers with different concentrations are identified (figure 4), the response time of a marker gene is identified, the response time of the marker gene is identified, the time of high-efficiency expression duration is identified, and the phenotype and physiological data and the correlation analysis of the strawberry are combined in different treatments, so that the assimilation of nitrogen by the plants is about 4-6 days earlier than phosphorus, but the higher phosphorus content is about 3-5 days longer than the nitrogen maintenance time, the best combination of the fertilization effect is that calcium superphosphate is added 7 days before the compound fertilizer is applied, and urea is added 9 days after the compound fertilizer is applied.
The invention compares the rule of absorption and assimilation of nitrogen and phosphorus by independent fertilization for two years and the expression characteristics of nitrogen and phosphorus marker genes by combining the characteristics of fast/slow fertilizer efficiency, and accurately determines the interval period of step-by-step combined application of nitrogen and phosphorus elements. Based on the research result, the fertilization amounts and the fertilization efficiencies of 13 different fertilization modes (ammonium dihydrogen phosphate compound fertilizer application, P fertilizer application before compound fertilizer application, N fertilizer application after compound fertilizer application, and fertilization time and dosage consideration) are further compared and analyzed, and finally, the optimal combined fertilization strategy is screened out as shown in table 1.
The method comprehensively determines the P-compound fertilizer-N fertilization combination with the optimal fertilizer efficiency by observing and comparing the phenotypes of strawberry flowers and fruits in 13 fertilization combinations and weighting and analyzing physiological data of the strawberry flowers and the fruits and detecting the N/P content and the expression of a marker gene, namely increasing the calcium superphosphate 7 days before the ammonium dihydrogen phosphate of the compound fertilizer and then applying the urea 9 days after the compound fertilizer. As shown in fig. 3.
Example 2
A high-efficiency strawberry ammonium dihydrogen phosphate accurate combined fertilization method is obtained by the method of the embodiment 1, and the specific fertilization method comprises the following steps:
(1) In the compound fertilizer (NH) 4 ) 2 HPO 4 Ca (H) is added 7 days before the administration, namely 7 days before the first flower of the strawberry appears 2 PO 4 ) 2 The fertilizer amount of the fertilizer is about 1.25g;
(2) Then (NH) is applied when the first flower of the strawberry appears 4 ) 2 HPO 4 The fertilizer amount of the fertilizer is about 2.0 g/plant;
(3) Applying CH again 9 days after the compound fertilizer is applied 4 N 2 The fertilizer amount of the O fertilizer is about 1.0 g/plant;
the three combined fertilizers are adopted, compared with the one-time compound fertilizer (NH) 4 ) 2 HPO 4 The synchronous interaction period of the NP fertilizer is widened by about 7-10, the fertilizer efficiency is obviously improved, compared with a contrast (a T1 group is not fertilized), the average strawberry flower is increased by about 15-20%, the fruit is increased by about 20-25%, and the fruit color change is advanced by about 7-10 days; compared with the single fertilizer application (T2, T3 and T4), the strawberry flowers are increased by about 5 to 10 percent on average, and the fruits are increased by about 10 percent on average.
(4) The strawberries are watered thoroughly after each fertilization, so that the fertilizer efficiency is fully exerted.
The combined fertilization method is also suitable for improving the fertilizer efficiency of the single fertilizer, and the NP single fertilizer with different fertilizer efficiency speeds is applied step by step under the same condition, so that the synchronous interaction period of the NP fertilizer is widened, and the fertilizer efficiency of the single fertilizer is improved.
Example 3
(1) In order to verify the effect of the new fertilization strategy for improving the fertilizer efficiency of the compound fertilizer, similar research is carried out on grapes, and self-rooted seedlings of 'Kyoho' grape varieties are taken as test materials and cultivated in flowerpots, so that the consistency of fertilization treatment and cultivation management conditions is ensured.
(2) And (3) when the grape plants grow to 15-17 leaves, starting single application of nitrogen and phosphate fertilizers and simultaneous combined application of the nitrogen and the phosphate fertilizers, and identifying the response speed and response time of the grapes to the single application of the nitrogen and the phosphate fertilizers as well as the response speed and the response time of the grapes to the simultaneous application of the nitrogen and the phosphate fertilizers.
(3) From the first fertilization, leaves were collected at 0d, 6h, 1d, 3d, 5d, 8d,10d, and 12d, and physiological data were measured.
(4) Measuring the nitrogen and phosphorus contents of different samples, and extracting and preparing RNA, cDNA and the like of the samples, and the steps are the same as the steps of the method in the example 1;
(5) And (5) analyzing and identifying the speed and the length of the assimilation rate of the grapes for different trial fertilizers according to the result in the step (4), and analyzing the speed and the duration of the response of the nitrogen and phosphorus marker genes to the simultaneous fertilization of nitrogen and phosphorus by utilizing a qRT-PCR technology. The determination criteria of the grapes on the N and P absorption speed and the high-efficiency response duration are the same as those in the example 1. Combining different treatments with grape phenotype and physiological data and correlation analysis, finding that a nitrogen marker gene VvNIR (LOC 100253671) starts to respond 6h after fertilization, and high expression lasts for 7-9 days; and the phosphate fertilizer marker gene VvPAP (LOC 100241989) starts to respond 3 days after fertilization, the high expression duration is 11-13 days, and the response speed and duration are faster and longer than those of single application of nitrogen fertilizer or phosphate fertilizer, so that the feasibility of the remarkable synergistic effect of the new stepwise accurate combined fertilization strategy on different crops is proved. Wherein the sequence of the marker gene is downloaded from the following website (https:// www.ncbi.nlm.nih.gov /).
The best application method is to apply the calcium superphosphate 7 days before the ammonium dihydrogen phosphate of the compound fertilizer is applied and apply the urea 9 days after the application. Can obviously increase the leaf number and the branch length of the grapes. Compared with the control, the number of grape leaves is averagely increased by 0.67, and the length of the branch is averagely increased by 10.1cm.
Example 4
A high-efficiency strawberry ammonium dihydrogen phosphate accurate combination application method is similar to example 2, except that in the step (1), calcium superphosphate is additionally applied 7 days before the application of compound fertilizer ammonium dihydrogen phosphate, namely 7 days before the first flower of a strawberry, and the dosage of the calcium superphosphate is about 1 g/plant.
Example 5
A method for precisely combining and applying high-efficiency ammonium dihydrogen phosphate to strawberries is similar to example 2, except that calcium superphosphate is additionally applied 7 days before the application of compound ammonium dihydrogen phosphate in step (1), namely 7 days before the first flowers of strawberries appear, and the dosage of the calcium phosphate is about 1.5 g/plant.
Example 6
A high-efficiency accurate combined application method of ammonium dihydrogen phosphate for strawberries, which is similar to example 2, except that in the step (2), the fertilizer amount of the ammonium dihydrogen phosphate fertilizer is applied when the first flower of a strawberry appears, and is about 1.5 g/plant.
Example 7
A high-efficiency accurate combined application method of ammonium dihydrogen phosphate for strawberries, which is similar to example 2, except that in the step (2), the fertilizer amount of the ammonium dihydrogen phosphate fertilizer is applied when the first flower of a strawberry appears, and is about 2.5 g/plant.
Example 8
A high-efficiency strawberry ammonium dihydrogen phosphate precision combined application method, which is similar to the method in the example 2, except that the fertilizer amount of urea is supplemented and applied in the step (3) 9 days after the compound fertilizer application is about 0.8 g/plant.
Example 9
A high-efficiency strawberry ammonium dihydrogen phosphate accurate combined application method, which is similar to the method in the example 2, except that the fertilizer amount of urea is supplemented and applied for 9 days after the compound fertilizer application in the step (3) is about 1.2 g/plant.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims (10)

1. A method for determining step-by-step accurate combined fertilization of a compound fertilizer and a single fertilizer is characterized by comprising the following steps: the method comprises the following steps:
(1) And determining the N/P response time: after a single N/P fertilizer is applied to a plant, collecting leaves every two days, measuring the contents of N and P, and determining the efficient response duration of the NP to the N and P according to the difference of the NP absorption speed;
(2) Identifying the expression of a response gene in an NP assimilation process by using a transcriptome sequencing result of a sample after N/P fertilizer application, identifying a gene of which the LOG2 value is more than or equal to 2 compared with the expression level of a control gene by comparing and analyzing the expression condition of a difference gene of N/P fertilizer treatment and the control sample, sequencing the gene, taking the gene of which the LOG2 value is three at the top, and preliminarily determining the gene as a candidate biomarker gene responding to an N/P element;
(3) Designing primers aiming at the candidate biomarker genes and the reference genes to detect the expression level of the marker genes, and determining nitrogen assimilation marker genes and phosphorus assimilation marker genes by combining the measurement results of the content of N and the content of P;
(4) Analyzing and identifying the speed and the length of the plants for different fertilizer assimilation rates, and analyzing the speed and the duration of the response of the nitrogen assimilation marker gene and the phosphorus assimilation marker gene to nitrogen and phosphorus fertilization by utilizing a qRT-PCR technology;
(5) And determining the optimal fertilizer efficiency fertilization method by combining phenotypic data and physiological data weighting analysis in the fertilization combination and the detected N/P content and the expression of nitrogen and phosphorus assimilation marker genes.
2. The method for determining the step-by-step accurate combined fertilization of a compound fertilizer and a single fertilizer as claimed in claim 1, wherein: the N/P element-responsive candidate biomarker genes of step (2) include one or more of FaNR, faNIR, faGS, faAS, faGDH, faPHO, faPHT1, faPHT2, faPAP.
3. The method for determining the step-by-step accurate combined fertilization of a compound fertilizer and a single fertilizer as claimed in claim 1, wherein: in the step (3), faNIR is used as a nitrogen assimilation marker gene, and FaPAP is used as a phosphorus assimilation marker gene.
4. The method for determining the step-by-step accurate combined fertilization of a compound fertilizer and a single fertilizer as claimed in claim 3, wherein: the primers of the nitrogen assimilation marker gene FaNIR in the step (3) are as follows:
FaNIR-F：5′-TGTGGGTCTTCACATTCCCG-3′；
FaNIR-R：5′-CACAGTGAGACGGAGTTCCC-3′。
5. the method for determining the step-by-step accurate combined fertilization of a compound fertilizer and a single fertilizer as claimed in claim 3, wherein: the primers of the phosphorus assimilation marker gene FaPAP in the step (3) are as follows:
FaPAP-F：5′-GCCAGGATCCGGTTCTTTCT-3′；
FaP4P-R：5′-AATCGTGTTCTGTGTCCGCT-3′。
6. determination of a method according to any one of claims 1-5 for step-wise accurate combined fertilization of plants to improve fertilizer efficiency.
7. Use according to claim 6, characterized in that: the plants comprise at least strawberry and/or grape.
8. A method for stepwise precision combined fertilization of strawberries determined by the method of any one of claims 1 to 5, characterized in that: applying a single-element phosphate fertilizer 7 days before the compound fertilizer is applied; and supplementing the single-element nitrogen fertilizer 9 days after the compound fertilizer.
9. The method for the step-by-step precise combined fertilization of strawberries as claimed in claim 8, wherein: the compound fertilizer is ammonium dihydrogen phosphate, the single element phosphate fertilizer is calcium superphosphate, and the single element nitrogen fertilizer is urea.
10. The method for the step-by-step precise combined fertilization of strawberries as claimed in claim 8, wherein: the method comprises the following steps:
(1) Applying calcium superphosphate 7 days before the ammonium dihydrogen phosphate of the compound fertilizer is applied;
(2) Applying ammonium dihydrogen phosphate fertilizer when the first flower of the strawberry appears;
(3) And applying urea again 9 days after the compound fertilizer is applied.
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