Multi-variety plant fat-soluble root exudates synchronous collection device and application thereof

Abstract

The multi-variety plant fat-soluble root exudates synchronous collecting device comprises a water storage pot and at least one planting module arranged in the water storage pot, each planting module comprises a movable buckle and two fixing plates, water inlet and outlet holes are formed in the lower portions of the fixing plates, and protruding edges are arranged on the inner walls of the upper portions of the fixing plates. The two fixing plates are oppositely spliced and then fixed by a movable buckle to form a hollow structure, and filter paper is clamped between the two fixing plates. According to the device, multiple varieties (dozens or hundreds of crops) can be cultured and fat-soluble root exudates can be collected in a very small space (1 square meter) at the same time, and the collecting efficiency of the fat-soluble root exudates in the dry farming crop seedling stage is greatly improved.

Description

Technical Field

[0001] The invention belongs to the research field of root exudates, and in particular relates to a synchronous collection device for fat-soluble root exudates of multiple varieties of plants and an application thereof. Background Art

[0002] Root exudates refer to a wide variety of substances secreted or released into the growth medium by different parts of the root system during plant growth. Root exudates are considerable in quantity and diverse in function. They are an important component of rhizosphere material circulation and have an important impact on the biological effectiveness of nutrients in the soil and the disease and stress resistance of crops. Among them, fat-soluble root exudates often play a role in chemical signal transduction in plant mineral nutrient absorption and disease resistance. They work at low concentrations and play a "four-two-pound" role in regulating the functions of microorganisms.

[0003] Due to the differences in their genomes, different plant varieties generally have large differences in the biological activity of root secretions. Currently, in the fields of botany, plant nutrition and microbiology, researchers often need to screen high-activity target varieties for follow-up experiments by comparing the activities of multiple plant varieties during the experiment. They can also use the root secretion activity phenotypes of multiple varieties combined with other indicators to conduct genome-wide association analysis to seek key genetic loci for plant root regulation. Collecting multiple varieties at the same time can reduce the error of collecting and comparing a single variety at different time periods, and can significantly increase the collection speed, reduce material loss, and reduce workload.

[0004] At present, the commonly used root secretion collection system is the traditional hydroponic / sand culture system. This system not only has a long cultivation cycle, high cost, and high water consumption, but also has a limited number of plant species (generally less than 5 species) for collecting root secretions at the same time. In addition, the subsequent processing operation is cumbersome and the workload is huge. In recent years, "a high-purity root secretion collection device for xerophytes" (Li Mingjie et al.) has appeared, which reduces the water consumption in the cultivation process of dry crops and simplifies the collection device. For example, "an adsorption-type root secretion collection system" (Yang Xuefang et al.) uses the method of adsorption by the collection unit, and can be collected without digging out the plant roots, avoiding damage to the root system. The collection method is relatively simple and convenient, and can be repeatedly collected in large quantities within a period of time. However, the above methods cannot achieve the simultaneous collection of a large number of varieties (more than 10 varieties) in a small space, and cannot meet the experimental requirements of comparative studies of dozens or hundreds of plant root secretion varieties. Therefore, it is necessary to design a synchronous collection system for fat-soluble root secretions of multiple varieties of crops, which will lay a methodological foundation for the research in the field of plant-microorganism interactions mediated by root secretions. Summary of the invention

[0005] Technical problem to be solved: In order to solve the problems that the roots of dryland crops are in waterlogging conditions for a long time under the traditional hydroponic system, the root secretion collection cycle is long, the simultaneous collection of multiple varieties occupies a large area and has low efficiency, the present invention provides a device and application thereof that can simultaneously cultivate multiple varieties of crops (dozens or hundreds of them) and collect fat-soluble root secretions in a very small space (1 square meter), which greatly improves the collection efficiency of fat-soluble root secretions of dryland crops in the seedling stage.

[0006] Technical solution: A synchronous collection device for fat-soluble root secretions of multiple varieties of plants, including a water storage basin and at least one planting module arranged therein, the planting module including a movable buckle and two holding plates, the lower part of the holding plate is provided with water inlet and outlet holes, and the upper inner wall is provided with ridges. The two holding plates are relatively assembled and fixed by the movable buckle to form a hollow structure, and filter paper is sandwiched between the two holding plates.

[0007] Preferably, the inner wall of the holding plate is provided with a side wall buckle, and the side wall buckles on the two holding plates are correspondingly engaged.

[0008] Preferably, the ridges protrude from the outer surface of the retaining plate toward the inner wall and are slightly narrower than the thickness of the single plate.

[0009] Preferably, the holding plate is made of black opaque plexiglass with a thickness of 0.3-0.5 cm, a length of 20-35 cm, a width of 25-30 cm, and a single-board side wall width of 0.8-1.5 cm.

[0010] Preferably, the upper ridge of the holding plate is arranged 1-2 cm below the top of the plate, and the raised portion protrudes 0.2-0.5 cm to support the seeds.

[0011] Preferably, the bottom of the holding plate is provided with four water inlet and outlet holes equidistantly, with a hole diameter of 1.5-2.5 cm, and located 2-4 cm upward from the bottom of the plate.

[0012] Preferably, the upper and lower water level lines on the inner wall of the water storage basin are flush with both ends of the water inlet and outlet holes, respectively, and the liquid level in the water storage tank needs to be maintained between the upper and lower water level lines.

[0013] The device is used in collecting fat-soluble root secretions of various plant species.

[0014] The specific application steps are: first disinfect the crop seeds according to the conventional method and cultivate them until the root length exceeds 0.5 cm, then transplant them into the device for cultivation, and each module cultivates a single variety of seeds; add nutrient solution into the device and keep the liquid level between the upper and lower water level lines, and each square meter of space can simultaneously meet the cultivation of 100-200 varieties of dryland crops; the cultivated crop roots can be collected after they grow to 15-28 cm, and 5-20g of the middle part of the root of each variety is taken, immersed in 200mL-500mL of dichloromethane and other organic reagents for 1-2 days, and the soaking liquid is evaporated to dryness in a rotary evaporator, methanol is added to dissolve, and stored at -20℃.

[0015] The above device is used to compare the biological nitrification inhibition activity of root exudates of multiple varieties of corn.

[0016] Beneficial effects: The present invention overcomes the problems of the original hydroponic system root secretion collection system, such as the large number of culture devices, large space occupied, long culture cycle, and complex pre-treatment. Compared with the traditional hydroponic system, the present invention (1) adopts the filter paper water absorption method to provide water to the crop root system, better simulates the water demand characteristics of dryland crops, creates a better growth environment for them, significantly reduces the amount of nutrient solution, and saves water resources and costs; (2) adds a convex groove for placing seeds in the plate, which can prevent the seeds from falling off and obtain uniform water, nutrients and light, so that the growth is more consistent and the experimental results are more reliable; (3) can simultaneously The root secretions of dozens or hundreds of varieties are collected to meet the needs of some scientific research on the simultaneous comparison of root secretions of multiple varieties, reducing the previous systematic errors between different batches of varieties; (4) The device can cultivate 40-50 varieties of crops per square meter. Compared with traditional hydroponics, the number of collected crop varieties is increased by 10 times in the same space; (5) The cultivation modules can be increased or decreased, which is convenient to assemble, highly practical, and has a simple manufacturing process. The device specifications can be adjusted according to the plant species and variety quantity to achieve simultaneous high-throughput collection of dozens or hundreds of plant varieties, greatly improving the collection efficiency of fat-soluble root secretions of multiple varieties of dryland crops in the seedling stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a plan view of the culture device holding plate in the collection system of the present invention; 1 plate body, 2 water inlet and outlet holes, 3 convex grooves.

[0018] Figure 2 It is a schematic diagram of the culture device in the collection system of the present invention; 4 is a water storage basin, 5 is a holding plate, and 6 is a movable buckle.

[0019] Figure 3 It is a schematic diagram of the side wall of the holding plate of the culture device in the collection system of the present invention; 7 is the holding plate side wall, 8 is the side wall buckle.

[0020] Figure 4It is a culture device for collecting fat-soluble root secretions of various varieties of corn in the seedling stage.

[0021] Figure 5 It is a schematic diagram comparing the culture device of the present invention and the existing culture device.

[0022] Figure 6 The nitrification inhibition rate curve of Zhengdan 958 root exudates under the new collection system (a) and the traditional hydroponic system (b).

[0023] Figure 7 Comparison of nitrification inhibition rates of root exudates of different varieties of corn under the new collection system (a. first experiment; b. second experiment). DETAILED DESCRIPTION

[0024] Example 1: Structural parameters of the culture device in the synchronous collection system of fat-soluble root exudates of multi-variety corn seedlings

[0025] A device for collecting fat-soluble root secretions of corn seedlings, comprising a water storage tank, a holding plate and a movable buckle, and also comprising a supporting convex groove and a water inlet and outlet hole. The holding plate is a group of two pieces, which are formed by the movable buckle and the buckle inside the plate. The bottom of the plate is provided with a water inlet and outlet hole, the convex groove on the upper part of the plate is slightly narrower than the thickness of a single plate, the inner wall of the plate is sandwiched with filter paper, and the inner wall of the water storage tank is provided with upper and lower water level lines. The holding plate is made of black opaque organic glass with a thickness of 0.5 cm. The length and width of the holding plate are equal, both of which are 30 cm. The width of the single plate side wall is 1 cm. The top convex groove is set 2 cm below the top of the plate, and the convex groove protrudes 0.5 cm. Four water inlet and outlet holes are equidistantly opened at the bottom, with a hole diameter of 1.5 cm, located 2 cm above the bottom of the plate. The upper and lower water level lines on the inner wall of the water storage tank are flush with the two ends of the water inlet and outlet holes, respectively, and the liquid level in the water storage tank needs to be maintained between the upper and lower water level lines.

[0026] Example 2: Cultivation Operation of Collection System

[0027] Lay filter paper on the inner wall of the holding plate, moisten it with pure water so that it can fit the inner wall of the plate, and use the buckle to fit the two plates together. Put corn seeds vertically from the upper part of the plate. The convex groove on the upper part of the plate is slightly narrower than the side wall of the single plate, so the convex groove can support the corn seeds and leave a gap for the roots of the corn seedlings to extend downward. Put several embedded plate modules vertically into the water storage tank, and control the nutrient solution level in the water storage tank between 2.5cm-4.5cm, so that the roots of the corn in the module will not be flooded, but the water transported upward through the filter paper meets the growth needs.

[0028] Example 3: Comparison of the efficiency of the collection system with the traditional hydroponic collection system

[0029] We used the methods of Example 1 and Example 2 and the traditional hydroponic system to collect the fat-soluble root secretions of Zhengdan 958 corn in the seedling stage, and tested the nitrification inhibition ability of its root secretions. The corn seeds were disinfected with 5% sodium hypochlorite solution for 12 minutes, rinsed with distilled water and soaked for 24 hours. The seeds were placed in germination paper and germinated in the dark at 30°C. About one day later, the germinated seeds were placed in a traditional hydroponic collection device and the collection device described in the present invention for culture and collection. Both methods replace the nutrient solution every two days, and the corn roots can be collected when they reach a length of about 25 cm. The two systems have different collection methods, and the specific operation methods are as follows:

[0030] 1. Collection method of traditional hydroponic system

[0031] The roots of corn seedlings that have been cultured for 4 weeks were continuously rinsed with distilled water, washed with distilled water, and transferred to a brown wide-mouth bottle filled with 1L of distilled water. The roots were immersed in water, and the aboveground parts were wrapped and supported on the bottle mouth with a sterilized sponge. The collected liquid was collected for 24 hours. The collected liquid was filtered with a filter membrane and the filtrate was evaporated at 40°C using a rotary evaporator. After adding methanol solution to 2mL, it was stored in a -20°C refrigerator. The methanol solution after the volume was fixed was called the hydroponic methanol extract.

[0032] 2. The root exudate collection method of the present invention

[0033] After the corn plants have grown in the holding plate for 12 days, it can be found that some corn roots have grown to the water inlet and outlet. At this time, the root secretions can be collected: disassemble the holding plate, take out the plant roots wrapped in filter paper in the plate, remove the red root part exposed to light at the top and the part covered by the nutrient solution below the water inlet and outlet at the bottom, weigh 10g of the remaining root fresh weight, soak the remaining part of the root in 250mL of dichloromethane for 2 days to collect its fat-soluble root secretions. The collected dichloromethane rinse or soaking liquid is evaporated to dryness at 40°C by a rotary evaporator, and then 2mL of methanol is added to dissolve and extract. After filtering through a 0.22μm pore size organic phase filter membrane, it is re-diluted to 2mL with methanol and can be stored or used. The methanol solution after dilution is called the new collection system methanol extract.

[0034] Table 1 Comparison of the collection efficiency of corn root exudates under the new collection system and the traditional hydroponic system (300 seedlings)

[0035]

[0036] Note: The area required for traditional hydroponic system to cultivate seeds is 21 turnover boxes with specifications of 0.3m×0.2m, totaling 1.26m 2 The new collection system is a water storage tank with a size of 0.3m×0.5m, with a total area of ​​0.15m 2; The nutrient solution usage of the traditional hydroponic system is 21 culture boxes, each of which requires 10L of nutrient solution, and the nutrient solution is replaced 8 times, totaling 1680L. The multi-variety synchronous collection system is 1 water storage tank, which is replaced 6 times, and 3L of nutrient solution is required each time, totaling 18L. The specific culture mode is as follows Figure 5 .

[0037] The nitrification inhibition rate of the two collected methanol extracts was tested as follows:

[0038] Take 100 μL of methanol extract in a 2 mL centrifuge tube, blow to dryness with nitrogen, and dissolve the residue with 5 μL of dimethyl sulfoxide (DMSO). Collect the European nitrosomonas bacterial solution cultured for 7 days, centrifuge at 7000g for 5 minutes, and resuspend in fresh sterile HEPES medium until OD600≈1.0, and its concentration multiple is 40 to 50 times. Add 195 μL of sterile water, 100 μL of fresh sterile HEPES medium, and 200 μL of resuspended bacterial solution to the centrifuge tube containing the concentrated secretion, and culture in a dark water bath at 25°C for 2 hours. Then add 20 μL of 0.1M allylthiourea (AT) to the mixed system to terminate the nitrosation reaction. Take 400μL of the reaction mixture and add it to a 10mL colorimetric tube, dilute it to about 5mL with deionized water, add 1mL of p-aminobenzenesulfonic acid solution, shake well and let it stand for 7min, then add 1mL of N-(1-naphthyl)-ethylenediamine hydrochloride solution, shake well, dilute it to 10mL with water and let it stand for 15min to wait for the color to develop completely. Use deionized water as a reference and measure the absorbance at a wavelength of 540nm. Use the same method to make a standard curve of NO2- to quantify the NO2 generated in the sample system. - , nitrite inhibition rate = (1-sample NO2 - Amount / Blank NO2 - Generation amount) × 100%.

[0039] The nitrification inhibition rates of root exudates collected by the two collection systems were measured as follows: Figure 6 shown.

[0040] The results showed that under the traditional hydroponic system, the hydroponic methanol extract had a nitrification inhibition rate of about 20% at 80 μL, and the sample inhibition rate reached 50% (ED 50 ) was 125 μL. As the sample volume increased, the nitrification inhibition rate could reach 80%. The root secretions collected by the new collection system also showed obvious nitrification inhibition ability. Under the new collection system, the methanol extract of the new collection system had a nitrification inhibition rate of about 20% at 2 μL, and ED 50The dosage is 2.5μL. As the sample volume increases, the nitrification inhibition rate can also reach 80%. Compared with the traditional hydroponic system, under the same volume, the nitrification inhibition effect of root secretions obtained by the new collection system is significantly higher than that of the traditional hydroponic collection system.

[0041] In summary, compared with the traditional hydroponic system, the new collection system can achieve higher collection efficiency while obtaining a corn root system that is more suitable for this experiment. Therefore, the efficiency of collecting root secretions in the new collection system is significantly higher than that of the traditional hydroponic collection system.

[0042] Example 4: Effect of the new collection system on simultaneous collection of multiple products

[0043] In order to explore the differences in the activity of root exudates of different varieties of corn under the new collection system, we selected 10 typical corn varieties and collected their root exudates in two batches, and measured the biological nitrification inhibition activity respectively. The specific operation method is shown in Example 3. The results are as follows: Figure 7 shown.

[0044] In the first batch, the BNI activity of root exudates of 10 varieties ranged from 26.2% to 88.6%, and the inhibition rate of 6 varieties exceeded 50%, with an average of 58.5%. Among them, Xianyu 335 and Zhengdan 958 exceeded 80%, and the nitrification inhibition rates of Jidan 33, Jinyu 188, Ruipu 909 and Beiyu 851 were 42.1%, 40.1%, 27.7% and 26.9%, respectively. In the second batch, the BNI activity of root exudates of 10 varieties ranged from 29.4% to 78.7%, and the inhibition rate of 5 varieties exceeded 50%, with an average of 51.6%. Among them, Xianyu 335 and Zhengdan 958 still reached more than 75%, and Jidan 33, Jinyu 188, Ruipu 909 and Beiyu 851 were 29.4%, 36.1%, 32.4% and 32.0%, respectively. Therefore, the root exudates of the 10 corn varieties all had high BNI activity, showing obvious differences between different varieties, and the relative standard deviation (RSD) of the BNI activity data of each variety between the two batches was about 10%, with good repeatability, indicating that it can better meet the requirements of multi-variety synchronous collection experiments.

Claims

1. A synchronous collection device for fat-soluble root secretions of multiple varieties of plants, characterized in that: The invention comprises a water storage basin (4) and at least one planting module arranged therein, wherein the planting module comprises a movable buckle and two holding plates (5), the lower part of the holding plate is provided with a water inlet and outlet hole (2), and the upper inner wall is provided with a convex ridge (3), the two holding plates are relatively assembled and fixed by the movable buckle (6) to form a hollow structure, and filter paper is sandwiched between the two holding plates.

2. The synchronous collection device for fat-soluble root secretions of various plants according to claim 1, characterized in that: The inner wall of the holding plate is provided with a side wall buckle (8), and the side wall buckles on the two holding plates are correspondingly engaged.

3. The synchronous collection device for fat-soluble root secretions of various plants according to claim 1, characterized in that: The ridge (3) protrudes from the outer surface of the holding plate toward the inner wall and is slightly narrower than the thickness of the single plate.

4. The synchronous collection device for fat-soluble root secretions of various plants according to claim 1, characterized in that: The holding plate is made of black opaque organic glass with a thickness of 0.3-0.5 cm, a length of 20-35 cm, a width of 25-30 cm, and a width of the single plate side wall of 0.8-1.5 cm.

5. The synchronous collection device for fat-soluble root secretions of various plants according to claim 1, characterized in that: The upper ridge of the holding plate is arranged 1-2 cm downward from the top of the plate, and the raised part protrudes 0.2-0.5 cm to support the seeds.

6. The synchronous collection device for fat-soluble root secretions of various plants according to claim 1, characterized in that: The bottom of the holding plate is provided with 4 water inlet and outlet holes at equal distances, with a hole diameter of 1.5-2.5 cm, and is located 2-4 cm upward from the bottom of the plate.

7. The synchronous collection device for fat-soluble root secretions of various plants according to claim 1, characterized in that: The upper and lower water level lines on the inner wall of the water storage basin are flush with the two ends of the water inlet and outlet holes respectively, and the liquid level in the water storage tank needs to be maintained between the upper and lower water level lines.

8. Use of the device according to any one of claims 1 to 7 for collecting fat-soluble root secretions of various plant species.

9. The use according to claim 8, characterized in that: The specific steps are as follows: first, the crop seeds are sterilized and cultured according to conventional methods until the root length exceeds 0.5 cm, and then they are transplanted into the device for culture, and each module cultures a single variety of seeds; nutrient solution is added into the device and the liquid level is maintained between the upper and lower water level lines, and each square meter of space can simultaneously meet the cultivation needs of 100-200 varieties of dryland crops; the cultured crop roots can be collected after they grow to 15-28 cm, and 5-20 g of the middle part of the root of each variety is taken, and immersed in 200 mL-500 mL of dichloromethane or other organic reagents for 1-2 days, and the immersion solution is evaporated to dryness in a rotary evaporator, and methanol is added to dissolve, and stored at -20°C.

10. Use of the device according to any one of claims 1 to 7 in comparing the biological nitrification inhibition activities of root exudates of multiple varieties of corn.

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