Method for in-situ determination of crop rhizosphere glucose secretion characteristics in soil

Abstract

The invention discloses a method for in-situ determination of crop rhizosphere glucose secretion characteristics in soil, and belongs to the technical field of plant rhizosphere nutrition determination. The method comprises the following steps: taking field soil, removing impurities, sieving, adding the sieved soil into a root box, placing germinated seeds of crops on the transparent side of the root box, and culturing at constant temperature; after the constant-temperature culture is finished, a microporous filter membrane fully infiltrated with a glucose fluorescent developing solution is flatly laid on the surface of the soil on the transparent side of the root box, so that the filter membrane is tightly attached to the surface of the soil, and an image of the filter membrane is obtained under ultraviolet light after standing; and counting the secretion characteristics of the crop rhizosphere glucose according to the development condition of the glucose in the image. According to the method, the defect of destructively obtaining a soil sample in the past is avoided, the spatial distribution characteristics of the crop rhizosphere glucose in the soil are visually displayed, and technical support is provided for exploring the influence of different preceding crops on the rhizosphere glucose range and the hot spot area of succeeding crops.

Description

Technical Field

[0001] The present invention relates to the technical field of plant rhizosphere nutrition determination, and particularly to a method for in-situ determination of the glucose secretion characteristics of crop rhizosphere in soil. Background Art

[0002] In a rotation system, the previous crop can affect the abiotic and biotic properties of the soil through root exudates and straw returning, etc., and thus affect the growth of the subsequent crop. Leguminous crops can increase the yield of the subsequent wheat by 20%. The yield increase effect of legume crop rotation is mainly attributed to the nitrogen effect and the non-nitrogen effect. Legumes fix atmospheric nitrogen through symbiosis with rhizobia. This biological nitrogen fixation can not only replace part of the inorganic nitrogen fertilizer on the premise of ensuring high crop yields, reduce the input of inorganic nitrogen fertilizer, but also improve the soil nitrogen level to promote the absorption and utilization of the subsequent crop.

[0003] Leguminous crops affect the soil carbon and nitrogen cycling processes through positive soil residue effects. Compared with gramineous crops, leguminous root debris, decaying roots and rhizosphere exudates release nitrogen-containing organic matter with a lower C:N ratio, which can enhance the activity of soil rhizosphere microorganisms, stimulate the secretion of soil enzymes, promote the degradation of soil organic carbon and release nitrogen, and thus increase the soil net nitrogen mineralization rate, promising to synchronize the soil nitrogen supply with the nitrogen demand of legume subsequent crops, thereby promoting crop yield increase. However, the specific process of how leguminous crops affect the soil and then regulate the rhizosphere of subsequent wheat is still unclear.

[0004] Current research has explored the impact of the soil legacy effect of previous crops on the soil of subsequent wheat by measuring soil enzyme activity, soil microbial diversity, and soil physical and chemical properties. However, it is only limited to a certain soil physical and chemical factor, and data is obtained through destructive sampling, making it impossible to monitor the in-situ soil of wheat. Glucose around the rhizosphere is a key carbon source for nutrient acquisition in the rhizosphere. In the study of soil carbon and nitrogen cycles, crop rhizosphere glucose is an important energy source that drives microorganisms to obtain energy, affects the composition and diversity of rhizosphere microbial communities, and can also reflect the intensity of rhizosphere organic carbon degradation. Among the existing methods for measuring rhizosphere glucose secretion, methods such as hydroponic extraction, soil culture collection, and substrate culture collection are commonly used to quantitatively analyze the glucose secreted around the plant rhizosphere. Hydroponic extraction means that the plant seedlings are placed in a pre-treated solution to grow for a period of time, and then the culture solution is collected to measure the glucose content in it. This method has a long collection time, and there are significant differences between solution culture and the real soil environment, so it cannot truly reflect the secretion situation of plants in the soil. The soil culture collection method is to plant the plant in soil with known components. After a period of time, the soil around the plant rhizosphere is collected and sieved. After removing obvious roots, residues, etc., the soil solution is extracted to measure the concentration of glucose in the plant rhizosphere. This method can reflect the real secretion situation of plants in the soil, but it cannot measure the glucose secretion characteristics of specific parts of the root system. The substrate culture collection method is to plant the plant in a selected substrate and add nutrient solution regularly. After the plant grows for a period of time, the substrate is soaked in an organic solvent to extract the organic matter adhering to the substrate, and its soaking solution is collected and then concentrated and filtered to obtain root exudates. This method has a more complicated operation process, and the cultivation environment is easily contaminated, affecting the experimental results. Therefore, the above methods cannot achieve in-situ monitoring of rhizosphere glucose secretion, and cannot effectively avoid disturbing the natural growth of roots and destroying the in-situ collection of the rhizosphere. Therefore, how to accurately display the distribution of rhizosphere glucose along the root system of subsequent wheat and its spatio-temporal changes to accurately explore the coupling effect of the residual effect of leguminous crops promoting the yield increase and efficiency improvement of the above-ground part and triggering soil changes in the underground part is still an urgent problem to be solved. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for in-situ determination of the secretion characteristics of rhizosphere glucose of crops in soil to solve the problems existing in the above-mentioned prior art. By adopting fluorescence color development and image processing methods, the present invention can avoid the defects of previous destructive acquisition of soil samples, intuitively display the spatial distribution characteristics of rhizosphere glucose of crops in soil, and provide technical support for exploring the influence of different previous crops on the rhizosphere glucose range and hot spot area of subsequent crops.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides a method for in-situ determination of the glucose secretion characteristics in the rhizosphere of crops in soil, comprising the following steps:

[0008] Take field soil, remove impurities and sieve it, add the sieved soil to a rhizobox, place the germinated seeds of the crop on the transparent side of the rhizobox, and incubate at a constant temperature.

[0009] After the incubation at a constant temperature is completed, lay a microporous filter membrane fully soaked with a glucose fluorescence developer solution flat on the soil surface on the transparent side of the rhizobox, make the filter membrane closely adhere to the soil surface, let it stand, and obtain an image of the filter membrane under ultraviolet light. According to the development of glucose in the image, statistically analyze the glucose secretion characteristics in the rhizosphere of the crop.

[0010] Optionally, the field soil is the field soil under the crop planting mode, and the field soil is the soil in the surface layer of 0 - 20 cm in the field.

[0011] Optionally, the glucose secretion characteristics include glucose secretion concentration and spatial distribution characteristics.

[0012] In the past, when measuring the glucose concentration in the rhizosphere of crops, a destructive method was used to obtain rhizosphere soil samples of crops. After mixing the soil samples, the glucose concentration around the rhizosphere was measured. The data obtained could roughly reflect the average concentration of glucose, but could not reflect the true concentration and spatial distribution characteristics of glucose around the rhizosphere. Compared with the prior art, the present invention realizes the in-situ determination of glucose secretion in the rhizosphere of crops, which is more accurate and intuitive.

[0013] Optionally, the diameter of the sieve holes for sieving is 2 mm.

[0014] Optionally, the bud length of the germinated seeds is 1 cm.

[0015] Optionally, the temperature for incubation at a constant temperature is 21 °C, the relative humidity is 60%, the daily light exposure time is 12 hours, and the light intensity is 10000 Lux.

[0016] Optionally, the incubation time at a constant temperature is 21 days.

[0017] Optionally, the pore diameter of the microporous filter membrane is 0.45 μm.

[0018] Optionally, the standing time is 30 min.

[0019] Generally, the areas where glucose is secreted, such as the root tips, will show a pink color. Since glucose diffuses from a region of higher concentration to a region of lower concentration along the root to the surrounding of the root, when the standing time exceeds 30 min, the pink color around the root becomes diffuse over time, and the spatial variability of glucose release is not obvious.

[0020] Optionally, the standing is carried out in the dark.

[0021] The present invention discloses the following technical effects:

[0022] Compared with the prior art, the present invention realizes the in-situ determination of glucose secretion in the rhizosphere of crops, which is more accurate and intuitive. By adopting the methods of fluorescence color development and image processing, the present invention can avoid the defects of obtaining soil samples destructively in the past, and intuitively display the spatial distribution characteristics of glucose in the rhizosphere of crops in the soil, providing technical support for exploring the influence of different previous crops on the rhizosphere glucose range and hot spot area of subsequent crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is a schematic diagram of wheat after growing in a rhizobox for three weeks;

[0025] Figure 2 It is a developed image of a microporous filter membrane (A) and a developed image of glucose after being processed by ImageJ software (B);

[0026] Figure 3 It is the spatial distribution characteristics of glucose secretion in the rhizosphere of wheat. Among them, A is the developed image of glucose secretion in the rhizosphere of wheat in different treatment groups; B is the statistical chart of the rhizosphere range of glucose secretion in the rhizosphere of wheat in different treatment groups; C is the hot spot area map of glucose secretion in the rhizosphere of wheat in different treatment groups. Different lowercase letters indicate significant differences between different treatments (p < 0.05). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The various exemplary embodiments of the present invention will be described in detail below. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention has been described only in terms of preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to those documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of this invention, various modifications and variations to the specific embodiments of the description of this invention will be apparent to those skilled in the art. Other embodiments derived from the description of this invention will be apparent to those skilled in the art. The description and examples of this invention are merely exemplary.

[0031] Regarding the use of "comprising", "including", "having", "containing", etc. in this text, they are all open-ended terms, meaning including but not limited to.

[0032] Examples

[0033] 1. Soil sample collection and rhizobox preparation

[0034] Soil samples were taken from a long-term wheat-soybean rotation field that had been rotated for 7 years. Soil samples (0 - 20 cm) were taken when wheat was at the seedling stage in the field. Animal and plant residues and impurities were removed, and the samples were passed through a 2 mm sieve for later use.

[0035] A two-factor experimental design was adopted. The design included two planting patterns: maize-wheat mode (MW) and soybean-wheat mode (SW), and two nitrogen fertilizer addition methods: no nitrogen application (N0) and nitrogen fertilizer addition (N1). The nitrogen fertilizer type was urea [CO(NH 2 ) 2 , and the addition amount was 66 mg N / kg soil (~158 kg hm -2 ). There were a total of 4 treatments, and each treatment was replicated 4 times.

[0036] Winter wheat (Jimai 22) was used to carry out seed germination experiments in an artificial climate chamber. When the seed buds were about 1 cm long, they were transferred to the root box (the length and width of the root box were 12 cm and the thickness was 2 cm), with a total of 16 root boxes. 350 g (dry soil weight) of sieved soil samples were added to each root box, and a germinated wheat seed was placed on the transparent cover of the root box. The soil layer of the root box was wrapped with tin foil, and then the root box was placed on a bracket with an inclination angle of 60° and placed together in a constant temperature incubator. The ambient temperature was set to 21°C, the relative humidity was 60%, the daylight time was 12 hours, and the light intensity was 10000 Lux. According to the growth status of wheat, water was poured every 2-3 days to keep the soil moisture at 60% of the field water holding capacity. In order to ensure that the wheat roots grow towards the surface soil as much as possible, the root box wheat root side was always tilted downward at 60° and cultured for 21 days.

[0037] 2. Glucose development and UV imaging

[0038] Wheat seedlings grown in root boxes for three weeks ( Figure 1 ) and then perform glucose development. Measure 100 mL of phosphate buffer solution (0.05 M, pH 7.4). Weigh 0.003 g of peroxidase (P8375, Sigma, USA) and 0.00107 g of glucose oxidase (G7141, Sigma, UK) respectively and add them to the buffer solution to obtain a first mixed solution. Add 0.005144 g of Ampliflu Red (C 14 H 11 NO 4 , 90101-5MG-F, Sigma, Switzerland) was added with 60 μL DMSO (CH 3 ) 2 SO, use a pipette to mix, and transfer it to the first mixed solution after it is fully dissolved. Pour the reaction solution into a tray and cover it with tin foil to avoid light. Then, spread the microporous filter membrane (13×13cm, 0.45μm, Taoyuan) into the reaction solution to make it fully infiltrated. Then, use tweezers to clamp the two corners of the filter membrane and spread it on the soil surface on the transparent side of the root box. Cover it with tin foil and sponge pad to make the filter membrane fit tightly with the soil surface. After 30 minutes, immediately transfer the filter membrane to a dark room and use a camera (Canon 750D) in the dark room to take pictures under ultraviolet light (wavelength 365nm), keeping the camera lens at the same distance from the microporous filter membrane to be photographed, with a straight-line distance of 40cm.

[0039] Usually, the areas where glucose is secreted, such as the root tips, will show a pink color. The preliminary experiment confirmed that the optimal development time for wheat is 30 minutes. When the experiment lasts for more than 30 minutes, the pink color around the roots becomes diffuse over time, and the spatial variability of glucose release is not as obvious as at the earlier time points. This may be because glucose diffuses from the area of higher concentration to the area of lower concentration along the root to the surrounding of the root.

[0040] Preparation of the standard curve: Use a 2×2 cm 2 membrane and immerse it into glucose solutions with concentrations of 0, 0.2, 0.5, 2, 4, and 10 mmol·L -1 respectively. Take out the filter membrane and evenly drop the reaction solution on its surface as much as possible. Take pictures and form images under uniform ultraviolet light irradiation (wavelength 365 nm) using a fixed camera.

[0041] 3. Image processing and data analysis

[0042] Use ImageJ software to process the zymogram and glucose images. First, establish a standard curve between enzyme activity and gray value: Take the actual concentration of the standard fluorescent substance as the X-axis and the actual gray value (average gray value of the saturated fluorescence area - background gray value) as the Y-axis to obtain the standard curve. Set the gray value when the glucose concentration is 0 as the background value of the image, and set the gray value of 255 as the upper limit value of glucose concentration. Definition of rhizosphere glucose "hot spots": Calculate the standard deviation (SD) and average value of glucose concentration. Glucose concentration higher than the average value + 2SD is regarded as a "hot spot". Definition of rhizosphere range: From the center of the rhizosphere to the non-rhizosphere, the glucose concentration will show a rapid decrease to a stable change trend, and this distance is regarded as the rhizosphere range. Then import the development photos of different treatments ( Figure 2 A) into the software. After converting them into 8-bit grayscale images, according to the standard curve values, convert different gray values into different colors to represent the glucose secretion concentration ( Figure 2 B). The analysis results are as Figure 3 shown.

[0043] Figure 3 The results show that the glucose secretion in the wheat root zone is strongly affected by nitrogen fertilizer addition and planting patterns. From the perspective of the spatial distribution characteristics of glucose secretion in wheat roots, it is mainly manifested that without nitrogen fertilizer addition, the glucose secretion amount of the main wheat roots is relatively high. After nitrogen fertilizer addition, the glucose secretion levels of the main wheat roots and the soil around the roots are similar. By calculating the rhizosphere secretion range and total hot spot area of glucose, it can be seen that compared with no nitrogen fertilizer application, nitrogen fertilizer addition significantly reduces the rhizosphere secretion range of glucose (p < 0.05), but has no significant effect on the total hot spot percentage. Compared with the wheat-maize rotation, the wheat-soybean rotation significantly reduces the rhizosphere range and hot spot area of glucose by 35.2% and 39.1% respectively (p < 0.05).

[0044] Comparative Example 1 Hydroponic Extraction Method

[0045] Select well - grown winter wheat (Jimai 22) seedlings in the field. Wash the soil on the roots of the seedlings with deionized water, put them in a complete nutrient solution for cultivation for 2 months. Then take out the wheat plants from the nutrient solution, wash the roots with deionized water, and then place the wheat plants in deionized water for 24 h to collect root exudates. After the cultivation is over, remove the wheat. Place the deionized water containing root exudates in a vacuum rotary evaporator and evaporate to dryness at 35 °C. Add ethanol for ultrasonic elution. After the ethanol volatilizes, obtain the root exudate sample. After the sample is collected, use gas chromatography - mass spectrometry (GC - MS) and ultra - performance liquid chromatography - mass spectrometry (UPLC - MS) to analyze the glucose content in the root exudates.

[0046] Comparative Example 2 Soil Extraction Method

[0047] Select well - grown winter wheat seedlings in the field. Take out the wheat seedlings intact from the soil, wash the rhizosphere soil on the surface of the seedlings with distilled water, transfer the washing solution to a centrifuge tube for shaking and centrifugation, and take the supernatant as the root exudates. After the sample is collected, use gas chromatography - mass spectrometry (GC - MS) and ultra - performance liquid chromatography - mass spectrometry (UPLC - MS) to analyze the glucose content in the root exudates.

[0048] Effect Comparison

[0049] Comprehensively compare the in - situ determination method of the examples with the methods of Comparative Examples 1 - 2. The results are shown in Table 1.

[0050] Table 1 Effect Comparison between Examples and Comparative Examples 1 - 2

[0051]

[0052] The above - described examples are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for in situ determination of glucose secretion characteristics of crop rhizosphere in soil, characterized in that: The following steps are involved: Taking field soil, removing impurities and then sieving, adding the sifted soil into a root box, placing the germinated seeds of the crop on the transparent side of the root box, and culturing at a constant temperature; After the constant temperature cultivation is completed, the microporous filter membrane fully soaked in glucose fluorescent developer is spread on the soil surface of the transparent side of the root box so that the filter membrane is closely attached to the soil surface. After standing, the image of the filter membrane is obtained under ultraviolet light, and the secretion characteristics of glucose in the rhizosphere of the crop are counted according to the development of glucose in the image.

2. The method according to claim 1, characterized in that The field soil is the field soil in the crop planting mode, and the field soil is the soil in the field surface layer of 0-20 cm.

3. The method according to claim 1, characterized in that The glucose secretion characteristics include glucose secretion concentration and spatial distribution characteristics.

4. The method according to claim 1, characterized in that: The diameter of the sieve holes is 2 mm.

5. The method according to claim 1, characterized in that The sprout length of the germinated seeds is 1 cm.

6. The method according to claim 1, characterized in that The constant temperature culture temperature is 21° C., the relative humidity is 60%, the daylighting time is 12 hours, and the light intensity is 10000 Lux.

7. The method according to claim 1, characterized in that The constant temperature culture time is 21 days.

8. The method according to claim 1, characterized in that The pore size of the microporous filter membrane is 0.45 μm.

9. The method according to claim 1, characterized in that: The standing time is 30 min.

10. The method according to claim 1, characterized in that The described standing still is standing still in the dark.

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