Discovery and application of a signal substance for recruiting nitrogen-fixing microorganisms
By discovering and applying the signal substance benzoic acid in sugarcane and corn root tip mucus, the problem of recruiting nitrogen-fixing microorganisms in plants under low nitrogen conditions was solved, and the plants' absorption capacity and growth performance of nitrogen were significantly improved.
Patent Information
- Application Number
- CN202310312220.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-28
- Publication Date
- 2025-02-25
- Estimated Expiration
- 2043-03-28
AI Technical Summary
It is not yet clear how sugarcane and corn recruit nitrogen-fixing microorganisms in soil, and their nitrogen acquisition capacity is insufficient under low nitrogen conditions.
A signal substance benzoic acid was discovered and used. It was identified in the mucus at the root tip of sugarcane at low nitrogen by metabolomic analysis. It can recruit nitrogen-fixing microorganisms through chemotaxis, inhibit the growth of other bacteria, and promote the enrichment and concentration of nitrogen-fixing microorganisms. Farming.
By adding benzoic acid, the abundance of nitrogen-fixing microorganisms and the copy number of nitrogen-fixing genes in the soil are enhanced, the absorption capacity of plants is enhanced, and the growth of plants is promoted, especially under low nitrogen conditions, which significantly increases the biomass of corn and Nitrogen absorption.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and specifically relates to the discovery and application of a signal substance for recruiting nitrogen-fixing microorganisms. Background Art
[0002] Sugarcane and corn are both important grasses. Both are C4 photosynthetic crops with efficient photosynthetic carbon fixation ability. Their growth requires a large amount of nitrogen. Current research suggests that in addition to absorbing nitrogen from the soil, sugarcane and corn can also interact with nitrogen-fixing microorganisms in the soil to perform combined nitrogen fixation, providing a certain amount of nitrogen for their growth and development. However, it is still not very clear how sugarcane and corn recruit nitrogen-fixing microorganisms in the soil. The present invention discovers and provides a signal substance that can recruit nitrogen-fixing microorganisms in sugarcane and corn. The substance recruits combined nitrogen-fixing microorganisms through chemotaxis, while inhibiting the growth of other bacteria, etc., promotes the enrichment of nitrogen-fixing microorganisms, and enhances the colonization of nitrogen-fixing microorganisms on the root system of host plants. The application of this substance in potted plants promotes the ability of corn to obtain nitrogen, promotes the accumulation of corn biomass, and ultimately improves the adaptation of corn to low nitrogen, and has application potential. Summary of the invention
[0003] The present invention aims to provide a discovery and application of a signal substance for recruiting nitrogen-fixing microorganisms.
[0004] To achieve the above object, the present invention adopts the following technical solutions:
[0005] An application of a signal substance for recruiting nitrogen-fixing microorganisms, wherein the signal substance for recruiting nitrogen-fixing microorganisms is benzoic acid, which is identified in the mucus of sugarcane root tips under low nitrogen conditions using a metabolome analysis method; the application is at least one of the following 1)-10):
[0006] 1) Recruit nitrogen-fixing microorganisms from plant roots;
[0007] 2) preparing a preparation for recruiting nitrogen-fixing microorganisms to plant roots;
[0008] 3) Promote the colonization of nitrogen-fixing microorganisms on plant roots;
[0009] 4) preparing a preparation for promoting the colonization of nitrogen-fixing microorganisms on plant roots;
[0010] 5) Increase the abundance of nitrogen-fixing microorganisms in the soil;
[0011] 6) preparing a preparation for increasing the abundance of nitrogen-fixing microorganisms in soil;
[0012] 7) Promote nitrogen absorption by plants;
[0013] 8) Preparation of preparations for promoting nitrogen absorption by plants;
[0014] 9) Promote plant growth;
[0015] 10) Preparation of preparations for promoting plant growth;
[0016] wherein the plants include sugarcane and corn;
[0017] Wherein, the nitrogen-fixing microorganism is Burkholderia parasitica;
[0018] Wherein, the application method is to mix benzoic acid into a substrate for plant cultivation;
[0019] Further, the concentration of the benzoic acid is 500 μmol / L;
[0020] Furthermore, the amount of benzoic acid used is 500 μL / g substrate.
[0021] The advantages of the present invention are:
[0022] The discovery and application of benzoic acid, a signal substance for recruiting nitrogen-fixing microorganisms described in the present invention, was discovered by using metabolomics methods in the detection and identification of mucus at the root tip of sugarcane under low nitrogen. Benzoic acid has a high content in sugarcane mucus; the substance can inhibit the growth of other bacteria, but has no obvious inhibitory effect on nitrogen-fixing microorganisms, and has the function of recruiting nitrogen-fixing microorganisms to colonize the root system. The substance is added to the soil to provide the relative abundance of soil nitrogen-fixing microorganisms Burkholderia and increase the copy number of the nitrogen-fixing gene nifH. The application of the substance in potted plants promotes the nitrogen acquisition ability of corn and the growth of sugarcane and corn under low nitrogen, and has application potential. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 :RBCS17 colonizes in the grass crops sugarcane and corn and promotes their growth.
[0024] Figure 2 : Collection of sugarcane mucilage and identification of mucilage substances.
[0025] Figure 3 :Analysis of chemotaxis of nitrogen-fixing bacteria to benzoic acid.
[0026] Figure 4 : Benzoic acid increases the relative abundance of (para) Burkholderia and increases the copy number of nifH.
[0027] Figure 5 : Application of benzoic acid increases nitrogen acquisition of corn and promotes corn growth. DETAILED DESCRIPTION
[0028] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.
[0029] In this application, the nutrient solution formula is as follows:
[0030] Table 1 Total nitrogen nutrient solution (5300 μmol / LN) formula
[0031]
[0032]
[0033] Table 2 Low nitrogen nutrient solution (530 μmol / LN) formula
[0034]
[0035]
[0036] Table 3 Formula of low nitrogen nutrient solution (1060 μmol / LN)
[0037]
[0038] In this application, the nitrogen-fixing bacterium Paraburkholderia sp. RBCS17, classified and named as Paraburkholderia sp. RBCS17, has been deposited in the China Center for Type Culture Collection on October 20, 2022, with a deposit number of CCTCC NO: M 20221610, and the deposit address is Wuhan University, Wuhan, China. Paraburkholderia sp. RBCS17 contains the nifH gene and has the ability to fix nitrogen biologically. The suspension of nitrogen-fixing bacteria RBCS17 is inoculated into the substrate to culture corn or sugarcane. RBCS17 can colonize in sugarcane and corn, a gramineous crop, and promote the growth of sugarcane and corn ( Figure 1 ). Specifically, GUS-tagged RBCS17 can be expressed in sugarcane ( Figure 1 A) and corn ( Figure 1 B) colonization on the root system; and after inoculation with RBCS17, sugarcane ( Figure 1 C, 1D) and maize root nitrogenase activity ( Figure 1 E, 1F).
[0039] Example 1
[0040] (1) Collection of sugarcane mucus and identification of mucus substances
[0041] After transplanting sugarcane tissue culture seedlings and hydroponically planting them in a full nitrogen nutrient solution (5300μmol / LN) for 3 months, they were cultured with a low nitrogen nutrient solution (530μmol / LN). After the sugarcane root tips secreted mucus, a gun was gently placed against the sugarcane root tips to absorb the mucus. The mucus samples were collected every two days and stored in a -80℃ refrigerator for later use. The collected sugarcane mucus was freeze-dried and sent to Beijing Novogene Technology Co., Ltd., where the metabolites contained in the mucus were identified using liquid chromatography-mass spectrometry (LC-MS-MS), and the identified substances and their contents were analyzed. The results are as follows: Figure 2 As shown in the figure, LC-MS-MS identified that the mucilage of sugarcane root tips contained a variety of substances, including some unknown substances that could not be identified; among the identified substances, benzoic acid was found to have the highest relative content among the known substances. The chemical structure of benzoic acid is as follows:
[0042]
[0043] Example 2
[0044] (1) Analysis of chemotaxis of nitrogen-fixing bacteria to benzoic acid
[0045] Since the previous analysis showed that the root tip mucus formed under low nitrogen conditions in sugarcane contains a large amount of benzoic acid, this study further used the agarose plug method to explore the role of benzoic acid in enriching nitrogen-fixing microorganisms. First, benzoic acid was mixed with 0.4% heated and dissolved liquid agarose at a ratio of 1:10 w / w and placed on a microscope slide (two strips of thick tape were glued to both sides of the slide in advance). A cover glass was placed on the benzoic acid agarose mixture to form a space between the slide and the cover glass. After the benzoic acid agarose mixture solidified, the bacterial solution (OD 2.0) of the nitrogen-fixing bacterium Burkholderia parasitica RBCS17 labeled with GFP fluorescence was placed on the surface of the microscope slide. 600 = 0.2) was gently injected into the gap, and the chemotaxis characteristics of the bacteria were observed under the GFP fluorescence channel. Photos were taken at 0 min and 30 min using a GFP green fluorescence microscope (Axio ImagerA2m) to record the positions of the bacteria at the starting and ending points. The experiment was designed to use agarose gel without benzoic acid as a control. Figure 3 As shown, the fluorescent photos taken at the starting point (0min) show that the strains are distributed more evenly in the field of view, and after 30min of bacterial movement, the fluorescent photos taken show that a large amount of fluorescent signals are enriched around the agar gel containing benzoic acid, and the fluorescent signals are significantly stronger than the agar gel without benzoic acid. This shows that the tested nitrogen-fixing Paraburkholderia RBCS17 has a chemotactic effect on benzoic acid, and conversely, benzoic acid can recruit and enrich nitrogen-fixing Paraburkholderia RBCS17.
[0046] Example 3
[0047] (1) Benzoic acid increases the relative abundance of (para) Burkholderia
[0048] Soil was collected from the sugarcane experimental field of Fujian Agriculture and Forestry University. After sieving, 5 g of soil was weighed and placed in a 5 mL centrifuge tube, and 500 μL of 500 μmol / L benzoic acid was added. The bottle mouth was sealed with a breathable tape and replenished every two days. During the treatment period, the culture was carried out at 26 ° C, and 5 biological replicates were set. The water treatment with an equal amount of benzoic acid was used as a control. Appropriate amounts of soil were taken and placed in 2 mL centrifuge tubes at 15 days and 30 days of culture. The samples were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd., and total DNA was extracted as a PCR template. Two-step PCR was performed using the V5-V7 segment PCR primers of the 16S rDNA gene hypervariable region (799f: 5'-AACMGGATTAGATACCCKG-3', 1193r: 5'-ACGTCATCCCCACCTTCC-3', 1392r: 5'-ACGGGCGGTGTGTRC-3'), and the soil microbial community composition was sequenced and analyzed using the Illumina Miseq PE300 platform (Illumina, USA). The results are shown in Figure 2 . Figure 4 As shown in A, the addition of benzoic acid caused the relative abundance of (para)Burkholderia in the soil to increase from 0.0098% to 0.09% (15 days) and 0.12% (30 days), which indicates that benzoic acid can change the composition of soil microbial communities and increase the relative abundance of (para)Burkholderia in the soil.
[0049] Note: (Para)Burkholderia means ParaBurkholderia + Burkholderia.
[0050] (2) Benzoic acid increases nifH copy number
[0051] Soil was collected from the sugarcane experimental field of Fujian Agriculture and Forestry University. After sieving, 5g of soil was placed in a sterile EP tube and treated with 500μmol / L benzoic acid. 100μL 500μmol / L benzoic acid was added to 5g of soil in the tube every 2 days and cultured at 26℃. The control group added an equal amount of sterile water. Soil sampling was performed 25 days after the addition of benzoic acid. Then it was handed over to Jizhen Biology for gene quantitative analysis. The total microbial DNA in the soil sample was extracted using the silicon matrix adsorption column method. First, a 2mL thick-walled EP tube was prepared and 0.5g of grinding beads were added. Then 0.25-0.5g of soil was weighed and added to the EP tube, and then 0.8mL Buffer SOL (suspension) was added, and vortexed at high speed for 5min on a vortex instrument. Then 80μL Buffer SDS (lysate) was added, and after vortexing at high speed for 3min on a vortex instrument, it was placed in a 70℃ water bath for 10min. After cooling, centrifuged at 13000rpm for 1min. Transfer 600μL of supernatant to a 1.5mL EP tube, add 150μL Buffer PS (neutralizer), vortex mix for 15s, centrifuge at 13000rpm for 5min, take 700μL of supernatant to a new 2mL EP tube, add an equal volume of Buffer GWP (precipitation aid), invert and mix, transfer to a silica-based adsorption column, and centrifuge at 13000rpm for 30-60s. Pour out the filtrate in the collection tube, put the column back into the collection tube, add 500μL Buffer DW1 (deproteinization solution) to the column, and centrifuge at 13000rpm for 30-60s. Pour out the filtrate, add 600μL BufferGW2 (containing ethanol, rinsing solution), centrifuge at 13000rpm for 30-60s, and repeat twice. Discard the effluent, put the column back into the collection tube, and centrifuge at 13000rpm for 2min. Place the column in a 1.5mL centrifuge tube. Add 30-50μL of BufferAE (elution solution) preheated to 70℃ to the center of the column membrane, let it stand for 3 minutes, and centrifuge at 13000rpm for 1min. Add another 30-50μL of BufferAE preheated to 70℃ to the center of the column membrane, let it stand for 3 minutes, and centrifuge at 12000rpm for 1min. Discard the DNA binding column to obtain the extracted soil total DNA. Use nifH-F: 5'-AAAGGYGGWATCGGYAARTCCACCAC-3'; nifH-R: 5'-TTGTTSGCSGCRTACATSGCCATCAT-3') as primers for quantitative PCR, perform a 10-fold gradient dilution of the plasmid standard, and then draw the nifH standard curve (a total of 6 gradients, 2μL sample loading per well), and the gradient copy number order of magnitude is 10 -7 , 10 -6 , 10 -5 , 10 -4 , 10-3 , 10 -2 Then, the total nifH gene in the sample was absolutely quantified by fluorescent quantitative PCR. The PCR reaction system used the SYBR Green method system: 2×SYBR Green Mix 7.5μL, Primer_F+R (10μM) 0.7μL each, gDNA 1μL, and the rest was supplemented with ddH2O to make up 15μL system. PCR reaction program: 95℃ pre-denaturation for 5min, 95℃ denaturation for 20s, 53℃ annealing for 20s, 72℃ extension for 40s, after 40 cycles, 95℃ 10s, 60℃ 60s, 95℃ 30s, 95℃ 15s to collect the melting curve, and finally substituted the Ct value into the standard curve equation to calculate the nitrogen-fixing nifH copy number. The results are as follows Figure 4 As shown in B, the application of benzoic acid significantly increased the copy number of the nifH gene in the soil, which increased by 35.05% relative to the control, indicating that benzoic acid can increase the number of nitrogen-fixing bacteria in the soil, thereby improving the nitrogen fixation capacity.
[0052] Example 4
[0053] (1) Effect of benzoic acid application on the growth of maize nitrogen acquisition
[0054] Soil was collected from the experimental field of Fujian Agriculture and Forestry University, sieved, and mixed with vermiculite and perlite in a volume ratio of 1:2:7. 100mL of 500μmol / L benzoic acid was added to 1L (200g in total) of the matrix and mixed evenly. The soil was pretreated for 15 days. At the same time, a treatment with an equal volume of water was set as a control group. After 15 days of pretreatment, the soil was put into pots with a specification of 7*7*15, and 4 corn seeds (Mintian 6855) were sown in each pot. After the first leaf of the corn is fully unfolded, thinning is carried out to ensure that one corn seedling with relatively consistent growth is left in each pot. During the growth period of corn, 1060μmol / LN low-nitrogen nutrient solution was used for irrigation. The corn was planted in a growth room for three weeks, and the phenotype of the corn was recorded and the nutrients of the plants were measured. The results are as follows Figure 5 As shown in the results, under low nitrogen conditions, corn planted in soil pretreated with benzoic acid grew significantly better than the control group. Further comparison of corn biomass and total nitrogen absorption showed that the biomass of plants in the benzoic acid pretreated group increased by 25.9%, and the total nitrogen absorption of plants increased by 19.4%, indicating that benzoic acid pretreatment of soil may promote corn growth by increasing the abundance of nitrogen-fixing microorganisms in the soil and promoting corn to obtain nitrogen through combined nitrogen fixation, which has potential application potential.
[0055] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. An application of a signal substance for recruiting nitrogen-fixing microorganisms, characterized in that: The signal substance for recruiting nitrogen-fixing microorganisms is benzoic acid, and the application is at least one of the following 1)-2): 1) Recruit nitrogen-fixing microorganisms from plant roots; 2) Preparation of preparations for recruiting nitrogen-fixing microorganisms to plant roots; The nitrogen-fixing microorganism is Burkholderia parabacterium.
2. The use according to claim 1, characterized in that: Such plants include sugar cane and corn.
3. The use according to claim 1, characterized in that: Benzoic acid is mixed into the substrate for plant cultivation.
4. The use according to claim 3, characterized in that: The concentration of the benzoic acid is 500 μmol / L.
5. The use according to claim 4, characterized in that: The amount of benzoic acid used is 500 μL / g substrate.