Quantitative method for distinguishing proton sources of rhizosphere acidification induced by rice root nutrient uptake

Abstract

The application discloses a kind of proton source quantitative differentiation methods of rice root system nutrient absorption induced rhizosphere acidification, comprising the following steps: S1, cultivate rice, S2, set experiment, S3, content determination, S4, numerical calculation, S5, data analysis;The proton quantitative differentiation method established by using the coupling relationship of the nitrogen absorption of crop root system and the induction of proton production and consumption can accurately evaluate the rhizosphere acidification effect induced by the three main processes of crop root system nitrogen absorption, other mineral nutrient absorption and root system organic matter secretion.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of crop rhizosphere acidification research, and particularly relates to a method for quantitatively distinguishing proton sources of rice root system nutrient absorption induced rhizosphere acidification. BACKGROUND

[0002] In the past 30 years, acidification of farmland soil is common in China, and the area of acid soil and the degree of acidification have increased dramatically. The main reason is the unreasonable application of nitrogen fertilizer and the large amount of crop harvesting. A large amount of ammonium nitrogen fertilizer in farmland soil undergoes strong nitrification, not only directly producing protons, but also indirectly acidifying by promoting the leaching of salt base ions carried by nitrate. Studies have found that under the measures of optimized fertilization of farmland management, the contribution of crop harvesting and other agricultural activities such as straw not returning to the field to soil acidification is as high as 70%. Therefore, clarifying the acidification effect of nitrogen in soil and its process of nutrient absorption during crop growth is the key to regulating soil acidification.

[0003] During the growth process, crops will absorb a large amount of inorganic anions and cations from the soil. While plants absorb nutrient ions, they will release an equivalent amount of protons or hydroxyl ions to maintain the charge balance in the body. Due to the synthesis of a large amount of organic compounds in the body by photosynthesis, the absorption of inorganic cation nutrients by plants is usually higher than that of inorganic anions. This unbalanced absorption of nutrients is an important source of protons in soil. In addition, plants will secrete organic matter during growth, among which organic acids can dissociate to release protons and acidify the soil under high pH conditions, and can associate protons to consume hydrogen ions and reduce the acidification effect under low pH conditions. At present, there are still few quantitative studies on the direct contribution of protons by the process of plant nutrient absorption and organic acid release to soil acidification.

[0004] Nitrogen, as the first essential nutrient element, is the component of many compounds and plays an important role in plant life activities. During the growth process, plants mainly absorb NH4 + and NO3 - two inorganic nitrogen forms. When NH4 + is the main nitrogen source, the root system absorbs NH4 + and releases protons, and vice versa, the root system absorbs NO3 - and releases hydroxyl ions, so supplying different nitrogen forms has a direct impact on the net proton release of plant roots. Nitrogen supply form also affects the absorption of other mineral nutrients by plant roots and their proton release. For example, exogenous application of ammonium nitrogen can regulate the absorption of other mineral nutrients by plants through acidification medium environment. Studies have found that ammonium nitrogen can increase the H +ATPase activity significantly enhances the enrichment of plants to phosphorus. In addition, the bioavailability of phosphorus in acidic soil is low, and the secretion of plant root organic acid is induced under phosphorus deficiency, which affects the rhizosphere acid-base environment. Under the condition of phosphorus deficiency, rice roots increase the absorption of NH4 + -N, reduce the absorption of NO3 - -N, and further promote the release of protons. The increase of soil heavy metal activity is also one of the hazards of soil acidification, such as cadmium stress, which affects the absorption, transport and distribution of mineral nutrients of crops on one hand, and changes the membrane composition and fluidity on the other hand, interferes with charge balance and H + flow. The effect of these nutrient supply conditions or environmental conditions on soil acidification has been relatively clear, but there is a lack of quantitative differentiation method for proton flux of different acidification processes.

[0005] Rice is a major food crop in the world, and it has a preference for ammonium nitrogen. The mechanism of rice-induced acidification of paddy soil is not clear. In recent years, researchers have conducted in-depth research on the proton source of crops under different growth stress, crop type, nutrient supply and other factors, but there is less quantitative research on the proton source of rice roots. SUMMARY

[0006] Therefore, the present application provides a method for quantitatively distinguishing the proton source of rice root system induced by rhizosphere acidification under the condition of nutrient absorption, which comprises the following steps:

[0007] S1, cultivating rice:

[0008] Take rice seeds, germinate and cultivate to 2-4 cm in length, then culture with nutrient solution A until the rice seedlings are 21 days old, and obtain experimental seedlings;

[0009] S2, setting up experiments:

[0010] Wash the roots of the experimental seedlings with deionized water several times, prepare multiple groups of nutrient solution B, add 12 experimental seedlings to each group of nutrient solution B for experiment, and the pH of the nutrient solution B is 4.5 and 6.5. Perform water replenishment once a day to the initial mass of the nutrient solution B;

[0011] The multiple groups of nutrient solution B are divided into ammonium-nitrate group, phosphorus level group and cadmium concentration group according to the acidification factors; the ammonium-nitrate group, the phosphorus level group and the cadmium concentration group are respectively divided into multiple subgroups according to the respective component concentration gradient; wherein the content of other nutrients in the nutrient solution B in each subgroup is consistent with that of the nutrient solution A; each subgroup is cultured for 48 h;

[0012] S3, content determination:

[0013] After the end of the cultivation treatment of step S2, each group of nutrient solution B was replenished to the initial weight, and then stirred uniformly to measure the pH change of each group of nutrient solution B, and to measure the net proton release amount of the root system and the content of part of the anion and cation nutrients in each group of nutrient solution B;

[0014] Among them, part of the anion and cation includes NO3 - , H2PO4 - , SO4 2- , NH4 + , K + , Na + , Ca 2+ and Mg 2+ ; the nutrient absorption amount was calculated according to the change of the nutrient content;

[0015] S4, numerical calculation:

[0016] The net proton release amount H + N from rice induced by nitrogen absorption was calculated by using the following formula (1):

[0017] H + N = (NH4 + ) 吸收量 – (NO3 - ) 吸收量 (1)

[0018] The net proton release amount H + U from rice induced by absorption of various inorganic anion and cation nutrients other than nitrogen was calculated by using the following formula (2):

[0019] H + U = H + U阳离子 (K + , Na + , Ca 2+ , Mg 2+ ) – H + U阴离子 (H2PO4 - , SO4 2- ) (2)

[0020] The net proton release amount H + E from the secretion of root organic matter was calculated by using the following formula (3):

[0021] H + E = H + 净 – H +U -H + N (3)

[0022] H + N H + U H + E H + 净 The net proton release amount of each process is collectively described, and the value is positive when the value is positive, indicating the net proton release amount, and negative when the value is negative; (NH4 + ) 吸收量 , (NO3 - ) 吸收量 NH4 + or NO3 - absorbed by rice, which is determined and calculated by step S3, H + U阳离子 (K + , Na + , Ca 2+ , Mg 2+ ), H + U阴离子 (H2PO4 - , SO4 2- ) is the molar equivalent number of anions and cations absorbed by rice for macronutrients and medium nutrients, which is determined and calculated by step S3.

[0023] S5, data analysis:

[0024] The data of each nutrient absorption and each net proton release obtained by steps S3 and S4 are sorted and plotted, single factor variance analysis is carried out by using SPSS 20.0 software, and the significant difference level p<0.05 is tested by Duncan method; the contribution rate of multiple independent variables of rice root system nutrient substance absorption and root system organic acid secretion to net proton release amount is obtained by using hier.part function package of R 4.2.0 software, and the relative importance of multiple independent variables of rice root system nutrient substance absorption and root system organic acid secretion to net proton release amount is evaluated according to the contribution rate of each independent variable to net proton release amount.

[0025] Description: The above method is based on the coupling relationship between crop root nutrient uptake and proton production, and establishes a quantitative differentiation method for proton release of the three main acidification processes of rice roots. The feasibility of the quantitative differentiation method of protons is verified by setting the acidification effect of ammonium-nitrate ratio, phosphorus level and cadmium concentration; the influence of different initial pH conditions on the difference between nutrient uptake and proton release of rice roots is analyzed, which provides a theoretical basis for exploring the mechanism of accelerating soil acidification, reasonable fertilization and safe production of rice planting.

[0026] Further, the step S1 comprises the following steps:

[0027] S1-1, select several full rice seeds, soak in 10% H2O2 for 30 min, wash with deionized water several times, then soak overnight for 12 h, then place the rice seeds on a grid shelf with wet gauze, place in a turnover box with deionized water, avoid light and germinate for 4-6 days, change deionized water every 2 days, until the rice seedlings grow to 2-4 cm;

[0028] S1-2, move the rice seedlings obtained in step S1-1 to the plant culture room for culture, the culture method is: place the rice seedlings in 25% nutrient solution A for 2-3 days, then in 50% nutrient solution A for 7 days, then move the rice seedlings to the planting plate, 1 plant for every 3 plants, use full concentration nutrient solution A to culture until the rice seedlings are 21 days old, then remove, change the nutrient solution A every 2 days during the period.

[0029] Description: Different crop varieties will have differences in nutrient uptake and acidification effect due to differences in seedling growth stage, root development status, physiological characteristics, and nutrient supply, environmental stress and other factors. The quantitative differentiation method of protons established by the above method of rice seedlings can relatively accurately and to a large extent reflect the nutrient uptake and acidification effect, thereby providing an important means for quantitative analysis of acidification effect of other crops.

[0030] Further, the composition of each liter of nutrient solution A is as follows: 1.5 mmol NH4Cl, 1.5 mmol NaNO3, 0.32 mmol NaH2PO4·2H2O, 1.0 mmol KCl, 0.25 mmol MgSO4·7H2O, 0.5 mmol CaCl2, and 9.1 μmol MnCl2·4H2O, 0.16 μmol CuSO4·5H2O, 0.15 μmol ZnSO4·7H2O, 0.07 μmol (NH4)6Mo7O 24• 4H2O, 18.0 μmol H3BO3, 40.0 μmol FeSO4·7H2O-EDTA, the rest is deionized water.

[0031] Note: The setting of nutrient solution A is based on the adjustment of Hoagland nutrient solution. The reason is that the test design contains different ammonium-nitrate ratio treatments, and Cl - and Na + are trace and non-essential nutrients for crops, not observation indicators. NH4NO3 is replaced by NH4Cl and NaNO3; The concentration of N, P and K is adjusted to facilitate weighing, and the amount of salt is slightly adjusted; The adjustment of K is because S is an observation indicator, and the rice seedling age used in this test is 3 weeks, and the culture time is 48 hours. In the pre-test, it was found that the demand of rice seedlings for S was small, and a large amount of S would be input by using K2SO4, which far exceeded the demand of rice. In order to provide a more suitable nutrient environment, and Cl - is not an observation indicator, K2SO4 is replaced by KCl; The adjustment of Ca and Mg is because it is found in the pre-test that the demand of rice seedlings for Ca and Mg is not large, about 10% of the original formula. In order to meet the demand of crops for Ca and Mg during the observation period, reduce the error of index determination and calculation, and reduce the amount of Ca and Mg; Through the above adjustment, the rice seedlings cultivated by nutrient solution B can be more convenient for experimental analysis.

[0032] Further, the culture conditions of the plant culture chamber are: relative humidity is 70%, 14h light at 28℃ per day, 10h dark treatment at 20℃, light intensity is 375 μmol·m -2 ·s -1 .

[0033] Note: Through the above setting of condition parameters, a suitable natural environment can be simulated, which is conducive to the growth of rice seedlings.

[0034] Further, in the ammonium-nitrate group in step S2, the total nitrogen concentration in nutrient solution B is 3 mmol / L; wherein, according to the different concentration ratio of NH4 + -N and NO3 - -N, four groups are divided according to the mass ratio of NH4 + -N:NO3 - -N is 1:3, 2:2, 3:1 and 4:0.

[0035] Note: Through the above setting of ammonium-nitrate ratio, the experiment can be compared for the difference between NH4 + :NO3 - , which is convenient for quantitative analysis of the effect of different ammonium-nitrate ratios on nutrient absorption of rice roots and acid effect; Because nitrogen is the first nutrient element, rice has a high demand for NH4+ -N has a preferential absorption and releases protons by assimilating NH4 + -N releases protons, and the absorption of nitrogen by rice is significantly affected by the ratio of ammonium to nitrate, which causes changes in the amount of proton release and the pH of the nutrient solution, thereby verifying the accuracy of the proton source quantitative differentiation method.

[0036] Further, in step S2, there are three phosphorus concentration gradients in the phosphorus level group, divided into three groups of 0.32 mmol / L, 0.08 mmol / L and 0 mmol / L, representing normal phosphorus level (1P), low phosphorus (1 / 4P) and no phosphorus (0P) level respectively.

[0037] Explanation: Under phosphorus deficiency conditions, rice roots increase the absorption of NH4 + -N, reduce the absorption of NO3 - -N, and further promote the release of protons; at the same time, phosphorus deficiency also induces the secretion of organic matter by roots. By culturing rice under different phosphorus levels, the proton flux of the three acidification processes under different phosphorus levels can be quantitatively differentiated, the proton flux of root organic matter secretion under different phosphorus levels can be analyzed, and the sensitivity of the proton source quantitative differentiation method can be verified.

[0038] Further, in step S2, there are three cadmium concentration gradients in the cadmium concentration group, divided into three groups of 0 μmol / L, 2.5 μmol / L and 5 μmol / L.

[0039] Explanation: The increase in soil heavy metal activity is also one of the hazards of soil acidification. Cadmium stress affects the absorption, transport and distribution of mineral nutrients in crops, changes the membrane composition and fluidity, interferes with charge balance and H + flow, and by setting different cadmium concentrations, the influence of cadmium pollution degree on the proton flux of the three acidification processes can be quantitatively analyzed, thereby further understanding the internal mechanism of the difference in crop acidification effect caused by heavy metal pollution.

[0040] Further, in step S3, the pH of the nutrient solution B is determined by a composite pH electrode; the net proton or hydroxyl release amount of rice roots is determined by an automatic potentiometric titrator; NH4 + and NO3 - in the culture solution are determined by a flow analyzer, H2PO4 - is determined by molybdenum antimony scandium colorimetry, K + and Na + are determined by flame photometry, Ca 2+ and Mg 2+ are determined by atomic absorption spectrophotometry, and SO4 2- is determined by ICP-AES.

[0041] Explanation: Through the above method, the ion concentration can be effectively and accurately determined.

[0042] Further, the net proton or hydroxyl release amount is determined by an automatic potentiometric titrator. One of 0.03 mol / L NaOH or HCl is used to titrate the pH of the nutrient solution B after experimental treatment to the initial value. The amount of NaOH or HCl consumed during titration is the net release amount of protons or hydroxyls (H + 净 ).

[0043] The beneficial effects of the present application are:

[0044] (1) The present application can accurately evaluate the rhizosphere acidification effect induced by crop root nitrogen uptake, other mineral nutrient uptake, and root organic matter secretion, by using the proton quantitative differentiation method established by the coupling relationship between rice root nutrient uptake and induced proton production and consumption.

[0045] (2) The present application establishes a quantitative differentiation method for proton release of three main acidification processes of rice roots, i.e. nitrogen uptake, other mineral nutrient uptake, and root organic matter secretion, by using the coupling relationship between crop root nutrient uptake and induced proton production and consumption. The feasibility of the proton quantitative differentiation method is verified by the acidification effect under the conditions of ammonium-nitrate ratio, phosphorus level, and cadmium concentration. The influence difference of these nutrients or environmental conditions on rice root nutrient uptake and proton release under different initial pH conditions is explored, which provides a theoretical basis for exploring the mechanism of accelerating soil acidification, reasonable fertilization, and safe production of rice planting.

[0046] (3) The present application can relatively accurately and to a large extent reflect nutrient uptake and acidification effect by using the proton quantitative differentiation method established by the cultivated rice seedlings in the experiment, thereby providing an important means for quantitative analysis of acidification effect of other crops. By allocating and preparing the components of the nutrient solution, the growth of rice can be more uniform, and it is more convenient for accurate determination of nutrient uptake and proton source in subsequent experiments. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 The present application is for the difference in the amount of protons released by rice roots through nitrogen uptake, other mineral nutrient uptake, and root organic matter secretion under the conditions of initial pH 4.5 (pHo=4.5) or pH 6.5 (pHo=6.5), and various ammonium-nitrate ratios.

[0048] Figure 2 The present application is for the difference in the amount of protons released by rice roots through nitrogen uptake, other mineral nutrient uptake, and root organic matter secretion under the conditions of initial pH 4.5 (pHo=4.5) or pH 6.5 (pHo=6.5), and various ammonium-nitrate ratios.

[0049] Figure 3 Figure 8 is a diagram showing the difference in the amount of protons released by rice roots through the process of nitrogen absorption, other mineral nutrient absorption and root organic matter secretion at initial pH 4.5 (pHo=4.5) or pH 6.5 (pHo=6.5) and various concentrations of cadmium, wherein Cd0, Cd2.5 and Cd5 represent the concentration of added Cd of 0 μmol / L, 2.5 μmol / L and 5 μmol / L, respectively;

[0050] Figure 4 Figure 9 is a diagram showing the independent effects of various conditions such as different ammonium-nitrate ratios, phosphorus levels and cadmium concentrations on the net amount of protons released by the multi-nutrient substances and root exudates. DETAILED DESCRIPTION

[0051] The application will be described in further detail below with specific embodiments to better reflect the advantages of the application.

[0052] Example 1

[0053] A method for quantitatively distinguishing the proton source of rhizosphere acidification induced by rice root nutrient absorption, comprising the following steps:

[0054] S1, cultivating rice:

[0055] S1-1, select several full rice seeds, soak them in 10% H2O2 for disinfection for 30 min, wash them with deionized water multiple times, then soak them overnight for 12 h, then place the rice seeds on a grid shelf with wet gauze, place them in a turnover box containing deionized water in the dark to germinate for 4-6 days, replace the deionized water every 2 days, until the rice seedlings grow to 2-4 cm;

[0056] S1-2, move the rice seedlings obtained in step S1-1 to a plant culture room for cultivation, the cultivation method is as follows: place the rice seedlings in 25% nutrient solution A for 2-3 days, then place them in 50% nutrient solution A for 7 days, then move the rice seedlings to a planting plate, 3 plants per hill, cultivate them with full-strength nutrient solution A until the rice seedlings reach 21 days of age, then remove them, replace the nutrient solution A every 2 days during the period;

[0057] The relative humidity of the plant culture room is 70%, the light intensity is 375 μmol·m -2 ·s -1 ;

[0058] The composition of each liter of nutrient solution A is: 1.5 mmol NH4Cl, 1.5 mmol NaNO3, 0.32 mmol NaH2PO4·2H2O, 1.0 mmol KCl, 0.25 mmol MgSO4·7H2O, 0.5 mmol CaCl2, and 9.1 μmol MnCl2·4H2O, 0.16 μmol CuSO4·5H2O, 0.15 μmol ZnSO4·7H2O, 0.07 μmol (NH4)6Mo7O 24 ·4H2O, 18.0 μmol H3BO3, 40.0 μmol FeSO4·7H2O-EDTA, and the balance is deionized water;

[0059] S2, setting the experiment:

[0060] The roots of the experimental seedlings are washed with deionized water multiple times, multiple groups of nutrient solution B are prepared, 12 experimental seedlings are added to each group of nutrient solution B for the experiment, the pH of the nutrient solution B is 4.5, and the water is replenished once a day to the initial mass of the nutrient solution B;

[0061] The multiple nutrient solution B is divided into four groups according to the ammonium-nitrate ratio, which is 1:3, 2:2, 3:1 and 4:0; the total nitrogen concentration of the nutrient solution B is 3 mmol / L; wherein the other nutrient contents of the nutrient solution B in each group are consistent with the nutrient solution A; each group is cultured for 48 h;

[0062] S3, content determination:

[0063] After the culture treatment of step S2 is completed, each group of nutrient solution B is replenished to the initial weight and stirred uniformly, the pH change of each group of nutrient solution B is determined, and the proton release amount and the contents of some anions and cations in each group of nutrient solution B are determined;

[0064] Among them, some anions and cations include NO3 - , H2PO4 - , SO4 2- , NH4 + , K + , Na + , Ca 2+ and Mg 2+ ; the nutrient absorption amount is calculated according to the change of the nutrient content;

[0065] The pH of the nutrient solution B is determined by a composite pH electrode; the net proton or hydroxyl release amount is determined by an automatic potential titrator; NH4 + and NO3 - in the culture solution are determined by a flow analyzer, H2PO4 - is determined by a molybdenum-antimony-scan colorimetric method, and K + and Na+ Ca 2+ and Mg 2+ SO4 2- determined by ICP-AES;

[0066] The net proton or hydroxyl release amount was determined by automatic potentiometric titrator. One of 0.03 mol / L NaOH or HCl was used to titrate the pH of the experimental treated nutrient solution B to the initial value. The amount of NaOH or HCl consumed during the titration was the net release amount of hydroxyl or proton H + 净 ;

[0067] S4, Numerical calculation:

[0068] The net proton release amount H from rice induced by nitrogen absorption was calculated by the following formula (1) + N :

[0069] H + N = (NH4 + ) 吸收量 – (NO3 - ) 吸收量 (1)

[0070] The net proton release amount H from rice induced by absorption of various inorganic cation and anion nutrients except nitrogen was calculated by the following formula (2) + U :

[0071] H + U = H + U阳离子 (K + , Na + , Ca 2+ , Mg 2+ ) – H + U阴离子 (H2PO4 - , SO4 2- ) (2)

[0072] The net proton release amount H from organic matter produced by roots was calculated by the following formula (3) + E :

[0073] H + E = H + 净 – H + U矿 – H+ N (3)

[0074] In the formula, H + N H + U H + E and H + 净 The description is uniformly given as the net proton release for each process. In reality, a positive value indicates the net proton release, while a negative value indicates the net hydroxyl release. (NH4) + ) 吸收量 (NO3) - ) 吸收量 Rice's response to NH4 + Or NO3 - The absorption amount is calculated by measuring in step S3, H + U阳离子 (K + Na + Ca 2+ Mg 2+ H + U阴离子 (H2PO4 - SO4 2- ) represents the molar equivalents of cations and anions absorbed by rice for macro- and medium-level nutrients, which are calculated through step S3.

[0075] S5. Data Analysis:

[0076] The data on nutrient uptake and net proton release obtained in steps S3 and S4 were organized and plotted. One-way ANOVA was performed using SPSS 20.0 software, and the significance level of differences (p < 0.05) was tested using Duncan's method. Figures 1 to 3 The contribution rates of multiple independent variables, including the absorption of various nutrients and the secretion of organic acids in rice roots, to net proton release were obtained using the hier.part function package in R 4.2.0 software. Based on the magnitude of the contribution rate of each independent variable to net proton release, the relative importance of the absorption of various nutrients and the secretion of organic acids in rice roots to net proton release was evaluated.

[0077] Example 2

[0078] The difference between this embodiment and Embodiment 1 is that, in step S2, the pH of nutrient solution B is 6.5.

[0079] Example 3

[0080] The difference between this embodiment and embodiment 1 is that in step S2, the phosphorus concentration gradient in the plurality of nutrient solution B has three groups, 0.32 mmol / L, 0.08 mmol / L and 0 mmol / L, representing normal phosphorus level (1P), low phosphorus (1 / 4P) and no phosphorus (0P) level respectively; wherein, the content of other nutrients in each group is consistent with that of nutrient solution A, and the pH of nutrient solution B is 4.5; each group is cultured for 48 hours.

[0081] Embodiment 4

[0082] The difference between this embodiment and embodiment 3 is that in step S2, the pH of nutrient solution B is 6.5.

[0083] Embodiment 5

[0084] The difference between this embodiment and embodiment 1 is that in step S2, the cadmium concentration gradient in the plurality of nutrient solution B has three groups, 0 μmol / L, 2.5 μmol / L and 5 μmol / L; wherein, the content of other nutrients in each group is consistent with that of nutrient solution A, and the pH of nutrient solution B is 4.5; each group is cultured for 48 hours.

[0085] Embodiment 6

[0086] The difference between this embodiment and embodiment 5 is that in step S2, the pH of nutrient solution B is 6.5.

[0087] Experimental example

[0088] I. Explore the differences in nutrient absorption and proton release of rice roots under different ammonium-nitrate ratio supply conditions

[0089] The data and results obtained in embodiment 1 and embodiment 2 are analyzed; as shown in Table 1:

[0090] Table 1 Differences in absorption of main nutrients by rice roots and changes in solution acidity under different ammonium-nitrate ratio conditions

[0091]

[0092] Among them, △pH is the pH change value of the nutrient solution after 48 hours of rice hydroponics, and the negative value represents the decrease of pH, and the positive value represents the increase of pH; * Different lowercase English letters indicate that the same column index difference reaches 5% significant level (p<0.05).

[0093] As shown in Table 1, whether the initial pH is 4.5 or 6.5, when the ammonium-nitrate ratio is 2:2, the absorption amount of NH4 + -N by rice roots is about 1.5 times of NO3 - -N, indicating that rice is an ammonium-loving crop; and the absorption amount of NH4+ The absorption of -N increases with the increase of the ammonium-nitrate ratio, but primarily with NO3-. - When nitrogen (NH4+) is supplied, nitrate nitrogen remains the primary form of nitrogen absorption, indicating that the absorption of nitrogen by rice roots is mainly affected by its supply. Different ammonium-nitrate ratios of nitrogen supply also show significant differences in their effects on the absorption of phosphorus (P) and potassium (K) by rice roots. Rice roots also show a higher absorption rate of H2PO4. - The absorption of K increases with the increase of the ammonium-nitrate ratio, while the absorption of K... + The absorption of NH4+ is the opposite, indicating that the culture medium contains a high concentration of NH4+. + -N is beneficial for rice to absorb H2PO4. - This is not conducive to rice absorbing potassium. + Nitrogen form significantly affected the pH and net proton release of the rice induction culture medium. Under conditions of initial pH 4.5 and an ammonium-to-nitrate ratio of 1:3, the solution pH increased, and rice roots exhibited net hydroxyl release. Under other conditions, the culture medium pH decreased, and rice roots exhibited net proton release. Both the magnitude of pH decrease and the amount of net proton release increased with increasing ammonium-to-nitrate ratio, indicating that nitrogen form is a crucial factor influencing rice growth-induced acidification. When the ammonium-to-nitrate ratio was 1:3 and 2:2, the magnitude of pH decrease and the amount of net proton release at an initial pH of 4.5 were significantly lower than those at an initial pH of 6.5 (p<0.05), indicating that the acidification effect of rice growth on the culture medium is related to the initial pH; the higher the initial pH, the more protons are released by the rice roots, resulting in a stronger acidification effect.

[0094] Linear fitting was performed between net proton release and major nutrient uptake, and it was found that the fit between rice uptake of ammonium nitrogen and net proton release was the best (H). + Net release = 0.89 × NH4 + -0.63, R 2 =0.9631), indicating that the absorption of ammonium nitrogen by rice is an important reason for the acidification of the rhizosphere environment;

[0095] like Figure 1 As shown, nitrogen absorption is characterized by net hydroxyl release at an ammonium-to-nitrate ratio of 1:3. However, at ammonium-to-nitrate ratios of 2:2, 3:1, and 4:0, net proton release occurs after nitrogen absorption by rice roots, with the amount of net proton release increasing with the ammonium-to-nitrate ratio. Specifically, the proton contribution rate of nitrogen absorption is greater than 70% at ammonium-to-nitrate ratios of 3:1 and 4:0. At ammonium-to-nitrate ratios of 1:3, 2:2, and 3:1, other mineral nutrient absorption processes promote net proton release, contributing 4.4%–51.6% of the proton source. However, the amount of net proton release and the proton contribution rate decrease with increasing ammonium-to-nitrate ratio. At an ammonium-to-nitrate ratio of 4:0, net hydroxyl release is dominant.

[0096] In addition, the contribution of root exudates to net protons was negative, indicating that the root exudates were mainly associated with protons, and this contribution increased with the increase of the ratio of ammonium to nitrate, mainly due to the decrease of the system pH with the increase of the ratio of ammonium to nitrate; the change trends of different proton sources were basically the same under the two initial pH values;

[0097] The above results show that the form of nitrogen not only affects the release of protons in rice, but also changes the absorption balance of other inorganic anion and cation nutrients and the amount of proton release; the higher the proportion of ammonium nitrogen added, the higher the net proton release induced by nitrogen absorption and the contribution to the total net protons, but it will reduce the absorption of other mineral nutrients and the proton contribution of root exudates.

[0098] II. Differences in nutrient absorption and proton release of rice roots under different phosphorus supply levels

[0099] The data and results obtained in Example 3 and Example 4 were analyzed; as shown in Table 2:

[0100] Table 2 Differences in main nutrient absorption of rice roots and solution acidity change under different phosphorus levels

[0101]

[0102]

[0103] Among them, △pH is the pH change value of the nutrient solution after 48h of rice hydroponics, negative value represents pH decrease, positive value represents pH increase; * Different lowercase English letters indicate that the difference in the same column index reaches 5% significant level (p<0.05).

[0104] As shown in Table 2, the change of phosphorus concentration has a significant effect on the absorption of H2PO4 - by rice roots, and the change trend is consistent under the two initial pH values. The absorption of H2PO4 - by rice roots increases with the increase of initial phosphorus concentration, indicating that the absorption of H2PO4 - by rice roots is positively correlated with the phosphorus supply level; when the initial pH is 4.5, the absorption of NH4 + -N by rice roots under normal phosphorus level (1P) is significantly increased by 15% compared with that under no phosphorus (0P), indicating that under low initial pH conditions, the increase of phosphorus concentration is beneficial to the absorption of NH4 + -N by rice. When phosphorus is deficient (0P and 1 / 4P), the absorption of NH4 + -N, NO3 - -N, H2PO4 - and K +The difference of nutrient uptake was not significant, and the rice roots under the initial pH of 6.5 had a significantly higher NO3 - -N and K + uptake than under the initial pH of 4.5 at the P level of 1P, which increased by 36% and 41%, respectively, while the H2PO4 - uptake was significantly lower by 10% (p<0.05), indicating that under normal P supply, the higher initial pH promoted the NO3 - -N and K + uptake of rice roots and inhibited the H2PO4 - uptake.

[0105] Under the two initial pH conditions, the pH of the rice culture solution showed a downward trend, and the net proton release of rice roots was positive. Under the same initial pH condition, the pH of the rice culture solution in the P-deficient treatment (0P and 1 / 4P) was slightly higher than that in the normal P treatment (1P). For example, the pH of the 0P nutrient solution B decreased by 0.14 and 0.07 pH units at the initial pH of 4.5 and 6.5, respectively, compared with the 1P treatment. However, there was no significant difference in the net proton release, which was mainly due to the higher acid buffering capacity of the nutrient solution with high P concentration. In addition, the pH reduction range of the nutrient solution and the amount of net proton released by rice roots were higher at the initial pH of 6.5 than at the initial pH of 4.5, indicating that high pH conditions promoted the nutrient uptake of rice roots and the induced net proton release.

[0106] As shown in Figure 2 , under the two initial pH conditions, the rice roots released net protons during the absorption of nitrogen and other mineral nutrients under P-deficient and normal P supply, while the root organic secretions showed net hydroxyl release. The amount of proton release or hydroxyl release induced by rice roots showed a decreasing trend with increasing P concentration. For example, the net proton release of rice inorganic mineral nutrient absorption under 0P, 1 / 4P, and 1P at the initial pH of 4.5 was 0.56, 0.43, and 0.14 mol / L, respectively, indicating that rice mainly promoted net proton release by increasing the relative absorption of inorganic cation mineral nutrients under low P conditions. It was found that crops usually induce the secretion of protons and organic acids under P-deficient conditions to meet their own needs by acidifying and activating soil inactivated phosphorus with organic acids. The more severe the P deficiency, the higher the secretion of protons and organic acids. Therefore, the results of this study also confirmed this phenomenon. The pH of the nutrient solution decreased to 2.5-3.2 after 48 h of rice culture in this experiment, and under such low pH conditions, the functional groups of organic acids were difficult to dissociate, mainly showing the effect of associated protons. Therefore, the more P-deficient, the stronger the effect of associated protons through organic acid secretion, and the stronger the net hydroxyl release. The above results also demonstrated the reliability of the method established in this study to quantitatively distinguish the proton release source of crop roots.

[0107] III. Differences in nutrient uptake and proton release of rice roots under different cadmium stress concentrations

[0108] The data and results obtained in Example 5 and Example 6 were analyzed, as shown in Table 3:

[0109] Table 3 Differences in absorption of main nutrients by rice roots and changes in solution acidity under different cadmium concentrations

[0110]

[0111] Among them, △pH is the pH change value of the nutrient solution after 48h of rice hydroponics, and the negative value represents the decrease of pH, and the positive value represents the increase of pH; * Different lowercase English letters indicate that the same column index difference reaches 5% significant level (p<0.05).

[0112] As can be seen from Table 3, under two initial pH conditions, the absorption of NH4 + -N, NO3 - -N, K + and H2PO4 - by rice roots decreased with the increase of cadmium concentration, for example, when the cadmium addition amount was 5μmol / L, the absorption of NH4 + -N, NO3 - -N and H2PO4 - by rice roots was significantly lower than that without cadmium treatment, indicating that cadmium addition had an inhibitory effect on the growth of rice, and the inhibitory effect on the absorption of nutrients by rice roots was greater with the increase of cadmium concentration; among them, the inhibitory effect of cadmium on nitrogen absorption was the largest, for example, when the cadmium addition amount was 5μmol / L, the absorption of NH4 + -N, NO3 - -N by rice roots decreased by 28%-35% under two initial pH conditions, which was more prominent under high pH condition.

[0113] From the above analysis, it can be seen that the absorption of NH4 Figure 3It is known that the effects of different cadmium concentrations on the pH and net proton release of rice culture solution are significantly different. After the treatment of different cadmium concentrations under two initial pH values, the decrease of pH of rice culture solution decreases with the increase of cadmium concentration, and the decrease of pH under high initial pH condition is higher than that under low initial pH condition. The change trend of net proton release of rice root system is consistent with that of pH, indicating that exogenous addition of cadmium inhibits the release of proton from rice root system. From the effects of different proton sources, cadmium stress mainly significantly reduces the net proton production induced by nitrogen absorption process, and has no significant effect on the proton release induced by other mineral nutrient absorption and organic acid secretion process. With the increase of cadmium concentration, the net proton release induced by nitrogen absorption process decreases, such as the net proton release in 2.5 and 5 umol / L cadmium concentration treatments under initial pH = 4.5 is reduced by 10% and 36% compared with that in no cadmium control treatment, which is consistent with the results under high pH condition. Therefore, cadmium stress mainly reduces the release of proton from rice root system by inhibiting nitrogen absorption.

[0114] Four, result analysis

[0115] From Figure 4 It is known that in the ammonium-nitrate ratio experiments of examples 1 and 2, the contribution of ammonium nitrogen absorption by rice root system to the net proton release is the largest, followed by nitrate nitrogen, and the effects of calcium and sulfur can be ignored. In the phosphorus deficiency experiments of examples 3 and 4, the contribution of ammonium nitrogen absorption by rice root system to the net proton release is 47%, which is much larger than the independent contribution of other factors, followed by root organic matter secretion, which contributes 14%, and the effects of potassium and magnesium can be ignored. In the cadmium stress experiments of examples 5 and 6, the contribution of ammonium nitrogen absorption by rice root system to the net proton release is the largest, followed by nitrate nitrogen, and root organic matter secretion ranks the third, the contributions of phosphorus, potassium and sulfur are relatively smaller, and the effects of sodium, calcium and magnesium can be ignored. It is shown that the explanation rate of ammonium nitrogen absorption by rice root system to the net proton release is the largest, which is the most important factor. The second is the absorption of potassium, and the effect of organic acid secretion on net proton release under stress environment, such as phosphorus deficiency and cadmium pollution, cannot be ignored.

Claims

1. A method for quantitatively differentiating proton sources in rice root nutrient absorption-induced rhizosphere acidification, characterized in that, Includes the following steps: S1. Rice cultivation: Take rice seeds, germinate and cultivate them until the rice seedlings are 2-4 cm long, and then cultivate them with nutrient solution A until the rice seedlings are 21 days old to obtain experimental seedlings. S2. Setting up the experiment: The roots of the experimental seedlings were washed multiple times with deionized water. Multiple groups of nutrient solution B were prepared. Twelve experimental seedlings were added to each group of nutrient solution B for the experiment. The pH of nutrient solution B was 4.5 and 6.

5. Water was added once a day to the initial mass of nutrient solution B. The multiple nutrient solutions B are divided into ammonium nitrate group, phosphorus level group and cadmium concentration group according to the acidification factor; the ammonium nitrate group, phosphorus level group and cadmium concentration group are further divided into multiple subgroups according to their respective component concentration gradients; the content of other nutrients in nutrient solution B in each subgroup is the same as that in nutrient solution A; each subgroup is cultured for 48 hours. S3. Content determination: After the culture treatment in step S2, each group of nutrient solution B was replenished with water to the initial weight and stirred evenly. The pH change of each group of nutrient solution B was measured, and the net proton release from the roots and the content of some anions and cations in each group of nutrient solution B were measured. Among them, some anions and cations include NO3. - H2PO4 - SO4 2- NH4 + K + Na + Ca 2+ and Mg 2+ Calculate the absorption of each nutrient based on changes in nutrient content; S4. Numerical Calculation: The net proton release H induced by nitrogen uptake in rice is calculated using the following formula (1). + N : H + N (NH4) + ) 吸收量 –(NO3 - ) 吸收量 (1) The net proton release H from rice due to the absorption of various inorganic cations and anions other than nitrogen is calculated using the following formula (2). + U : H + U =H + U阳离子 (K + ,Na + ,Ca 2+ ,Mg 2+ )–H + U阴离子 (H2PO4 - ,SO4 2- ) (2) The net proton release H from root organic matter secretion is calculated using the following formula (3). + E : H + E =H + 净 –H + U矿 –H + N (3) In the formula, H + N H + U H + E and H + 净 The value is uniformly described as the net proton release for each process; a positive value indicates the net proton release, and a negative value indicates the net hydroxyl release. (NH4) + ) 吸收量 (NO3) - ) 吸收量 Rice's response to NH4 + Or NO3 - The absorption amount is calculated by measuring in step S3, H + U阳离子 (K + Na + Ca 2+ Mg 2+ H + U阴离子 (H2PO4 - SO4 2- ) represents the molar equivalents of cations and anions absorbed by rice for macro- and medium-level nutrients, which are calculated through step S3. S5. Data Analysis: The data on nutrient uptake and net proton release obtained in steps S3 and S4 were organized and plotted. One-way ANOVA was performed using SPSS 20.0 software, and the significance level of differences was tested using Duncan's method. The contribution rates of multiple independent variables of nutrient uptake and organic acid secretion in rice roots to net proton release were obtained using the hier.part function package of R4.2.0 software. Based on the magnitude of the contribution rate of each independent variable to net proton release, the relative importance of multiple nutrient uptake and organic acid secretion in rice roots to net proton release was evaluated.

2. The method for quantitatively differentiating proton sources in rice root nutrient absorption-induced rhizosphere acidification as described in claim 1, characterized in that, Step S1 includes the following steps: S1-1. Select a number of rice seeds and soak them in 10% H2O2 for 30 minutes for disinfection. Rinse them several times with deionized water and soak them overnight for 12 hours. Then spread the rice seeds flat on a grid rack with a damp gauze and place them in a turnover box containing deionized water to germinate in the dark for 4-6 days. Change the deionized water every 2 days until the rice seedlings grow to 2-4 cm. S1-2. The rice seedlings obtained in step S1-1 are transferred to a plant culture room for cultivation. The cultivation method is as follows: the rice seedlings are placed in nutrient solution A with a mass concentration of 25% for 2-3 days, and then in nutrient solution A with a mass concentration of 50% for 7 days. Then the rice seedlings are transplanted onto a planting board, with 3 seedlings per clump. They are then cultivated in full-concentration nutrient solution A until the rice seedlings reach 21 days of age, and then transplanted. During this period, nutrient solution A is changed every 2 days.

3. The method for quantitatively differentiating proton sources in rice root nutrient absorption-induced rhizosphere acidification as described in claim 1, characterized in that, The composition of nutrient solution A per liter is as follows: 1.5 mmol NH4Cl, 1.5 mmol NaNO3, 0.32 mmol NaH2PO4·2H2O, 1.0 mmol KCl, 0.25 mmol MgSO4·7H2O, 0.5 mmol CaCl2, and 9.1 μmol MnCl2·4H2O, 0.16 μmol CuSO4·5H2O, 0.15 μmol ZnSO4·7H2O, and 0.07 μmol (NH4)6Mo7O 24 The mixture consisted of 4H₂O, 18.0 μmol H₃BO₃, 40.0 μmol FeSO₄·7H₂O-EDTA, with the remainder being deionized water.

4. The method for quantitatively differentiating proton sources in rice root nutrient absorption-induced rhizosphere acidification as described in claim 2, characterized in that, The cultivation conditions in the plant culture chamber were as follows: relative humidity of 70%, daily treatment with 14 hours of light at 28°C followed by 10 hours of darkness at 20°C, and light intensity of 375 μmol·m⁻¹. -2 ·s -1 .

5. The method for quantitatively differentiating proton sources in rice root nutrient absorption-induced rhizosphere acidification as described in claim 1, characterized in that, In step S2, the total nitrogen concentration of nutrient solution B in the ammonium nitrate group is 3 mmol / L; among which, according to NH4 + -N and NO3 - The difference in the concentration ratio of -N is reflected in NH4+. + -N:NO3 - -N is divided into 4 groups with quality scores of 1:3, 2:2, 3:1 and 4:

0.

6. The method for quantitatively differentiating proton sources in rice root nutrient absorption-induced rhizosphere acidification as described in claim 1, characterized in that, In step S2, there are three phosphorus concentration gradients in the phosphorus level group, divided into three groups of 0.32 mmol / L, 0.08 mmol / L and 0 mmol / L, which represent normal phosphorus level (1P), low phosphorus (1 / 4P) and no phosphorus (0P) levels, respectively.

7. The method for quantitatively differentiating proton sources in rice root nutrient absorption-induced rhizosphere acidification as described in claim 1, characterized in that, In step S2, there are three cadmium concentration gradients in the cadmium concentration group, which are divided into three subgroups: 0 μmol / L, 2.5 μmol / L, and 5.0 μmol / L.

8. The method for quantitatively differentiating proton sources in rice root nutrient absorption-induced rhizosphere acidification as described in claim 1, characterized in that, In step S3, the pH of nutrient solution B is measured using a composite pH electrode; the net proton or hydroxyl group release is measured using an automatic potentiometric titrator; and the NH4+ in the culture medium is measured. + and NO3 - H2PO4 was determined using a flow analyzer. - K was determined by the molybdenum-antimony-scandium colorimetric method. + and Na + The Ca content was determined by flame photometry. 2+ and Mg 2+ SO4 was determined by atomic absorption spectrophotometry. 2- ICP-AES was used for determination.

9. The method for quantitatively differentiating proton sources in rice root nutrient absorption-induced rhizosphere acidification as described in claim 8, characterized in that, The method for determining net proton or hydroxyl release using an automatic potentiometric titrator is as follows: The pH of the experimentally treated nutrient solution B is titrated to its initial value using 0.03 mol / L NaOH or HCl. The amount of NaOH or HCl consumed during the titration is the net proton or hydroxyl release H. + 净 .

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