Application of a composite signal substance in promoting recruitment of rhizosphere beneficial microorganisms and relieving cadmium stress on leaf surface

Abstract

The application discloses application of a composite signal substance in promoting recruitment of rhizosphere beneficial microorganisms and relieving cadmium stress on leaves, and belongs to the technical field of biology, wherein the composite signal substance is L-valine and L-leucine, and the combination is obtained by detecting and identifying rice root exudates under leaf cadmium exposure through metabolomics. L-valine and L-leucine both present high abundance characteristics in indica rice and japonica rice root exudates; both of them can produce significant chemotaxis effect on bacillus subtillus and bacillus pumilus when used alone, and the synergistic effect is stronger after compounding, and the above-mentioned bacillus can be significantly induced to colonize in plant roots. The composite signal substance is applied to the rhizosphere soil of rice, and the relative abundance of bacillus in the soil can be effectively improved; pot experiment shows that the substance can significantly enhance the growth ability of rice under leaf cadmium stress by promoting the colonization of target bacillus, and has good field application and industrialization prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the discovery of a complex signaling substance for recruiting beneficial rhizosphere Bacillus and its application in alleviating cadmium stress on rice leaves. Background Technology

[0002] Cadmium (Cd) is a highly toxic environmental pollutant. In rice-producing areas of my country, heavy metal pollution not only threatens food security but also severely restricts the normal growth and development of rice. Currently, research on cadmium's toxicity to plants focuses primarily on the rhizosphere environment. However, with the acceleration of industrialization, cadmium exposure to leaves through atmospheric deposition has become an undeniable factor affecting crop health. Cadmium deposited on leaves rapidly induces oxidative stress in plants, disrupting the photosynthetic system, inhibiting leaf physiological activity, and consequently leading to stunted growth, reduced biomass, and even yield reduction in rice. Traditional remediation methods for cadmium pollution, such as spraying foliar barrier agents, can alleviate pollution to some extent, but often have limitations such as short-lasting effects and susceptibility to rainfall. In recent years, inducing systemic resistance (ISR) in plants using plant rhizosphere probiotics (PGPR) has become a research hotspot. However, when faced with foliar heavy metal stress, whether plants actively send signals to the rhizosphere, how they recruit beneficial bacteria with specific functions, and which key signaling substances are involved in this process, currently lack in-depth research and effective technical means. In particular, regarding rice's response to cadmium stress, how to precisely trigger a "cry-for-help" response—specifically, how to secrete specific signaling substances to directionally recruit microorganisms with enhanced foliar defense functions to compensate for growth losses caused by foliar heavy metal stress—is a technical bottleneck that urgently needs to be overcome in the field of agricultural biotechnology. Summary of the Invention

[0003] The technical problem to be solved by this invention is: to address the problem of cadmium exposure on leaves causing inhibited growth and decreased physiological activity in rice, to provide a complex that can target and recruit beneficial rhizosphere Bacillus and its application, thereby inducing plant leaf resistance and alleviating rice growth inhibition caused by cadmium exposure on leaves.

[0004] The technical solution of the present invention is: an application of a composite signaling compound, wherein the active ingredient of the composite signaling compound is composed of L-valine and L-leucine; the application is at least one of the following 1)-8):

[0005] 1) Recruit beneficial Bacillus spores from plant roots;

[0006] 2) Preparation of formulations that recruit Bacillus spores from plant roots;

[0007] 3) Promotes the colonization of Bacillus in plant roots;

[0008] 4) Prepare formulations that promote the colonization of Bacillus in plant roots;

[0009] 5) Increase the abundance of Bacillus in the soil;

[0010] 6) Prepare formulations to increase the abundance of Bacillus in soil;

[0011] 7) Synergistically promotes plant environmental adaptability under cadmium stress on leaves with Bacillus subtilis;

[0012] 8) Prepare formulations that synergistically promote plant adaptation to cadmium stress on leaves with Bacillus subtilis;

[0013] Furthermore, the plant is rice;

[0014] Furthermore, the molar ratio of L-valine to L-leucine is 1:1;

[0015] Furthermore, the Bacillus species are Bacillus stratosphericus and Bacillus zanthoxyli.

[0016] Furthermore, when the compound signaling agent is used to promote the colonization of Bacillus on the roots, the application concentration of each amino acid is 50 μg / L;

[0017] When the composite signaling agent is used to increase the abundance of Bacillus in soil, the molar concentration of each amino acid is 10 mmol / L.

[0018] Furthermore, the composite signaling substance is applied via rhizosphere application.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The composite signaling substances of this invention are L-valine and L-leucine, which were obtained by metabolomics analysis of rice root exudates under cadmium exposure. Both L-valine and L-leucine exhibit high abundance in the root exudates of indica and japonica rice. Each substance alone produces a significant chemotactic effect on Bacillus stenothermia and Bacillus zanthoxylum, and the synergistic effect is even stronger when combined, significantly inducing the colonization of these Bacillus species in plant roots. Applying this composite signaling substance to rice rhizosphere soil effectively increases the relative abundance of Bacillus species in the soil. Pot experiments show that this substance significantly enhances the growth capacity of rice under cadmium stress by promoting the colonization of target Bacillus species, demonstrating good prospects for field application and industrialization. Attached Figure Description

[0021] Figure 1Collection and identification of root exudates from rice under cadmium exposure on leaves. Figure a: results of differential metabolite enrichment analysis, b: up-set diagram of differential metabolites, c: relative signal intensity of L-valine and L-leucine.

[0022] Figure 2 The chemotactic effects of L-valine and L-leucine on Bacillus.

[0023] Figure 3 The L-valine-L-leucine complex promotes the colonization of Bacillus in the root system.

[0024] Figure 4 The combined application of L-valine and L-leucine increased the relative abundance of Bacillus in the rice rhizosphere. Figure a: relative abundance of microorganisms; b: Manhattan plot of the top 5 most abundant microorganisms.

[0025] Figure 5 The effect of a compound signaling agent and its recruited Bacillus on mitigating cadmium stress on rice leaves. Figure a: aboveground dry weight of rice after single inoculation and mixed inoculation under cadmium exposure on leaves; b: plant height of rice after single inoculation and mixed inoculation under cadmium exposure on leaves. Detailed Implementation

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0027] Bacillus stratosphericus SRS50 was obtained from the National Microbial Culture Collection, with accession number MCCC 1B00144, and Bacillus zanthoxyli SRS107 was obtained from the China Center for Type Culture Collection, with accession number CCTCC AB 2016326.

[0028] Example 1: Collection and identification of root exudates from rice under cadmium exposure on leaves

[0029] 1. Test materials: Indica rice varieties Yangdao 6 (9311), Zhongjiazao 17, and Wanxiangsimiao, and japonica rice varieties Zhonghua 11 (ZH11), Kitaake, and Nipponbare were selected as test materials; the soil with an available Cd content of 0.02 mg·kg⁻¹ was used. -1 Low cadmium background paddy soil.

[0030] 2. Treatment Setup: During the 28-day experimental period, the cumulative cadmium deposition flux in the foliar cadmium exposure (DD) group was 1.0 mg / m², and the working concentration was uniformly 10 mg / L. CdO was dissolved in 0.05% Tween-80 solution to prepare a 1.0 mg / mL stock solution. After ultrasonic dispersion, the stock solution was prepared and used immediately. After dilution, it was evenly applied to the adaxial surface of the leaves using a precision micro-brush. All treatments were applied 14 times, 0.35 mL per plant, every 2 days. The control group was sprayed with an equal volume of blank Tween-80 solution. The soil surface was strictly sealed to prevent pesticide dripping and contamination.

[0031] 3. Collection of root exudates: The rice roots were removed from the substrate and carefully rinsed with deionized water to avoid physical damage. The whole rice plant was then transferred to a collection container filled with ultrapure water and equilibrated for 24 hours under dark conditions. The collected exudate solution was filtered through a 0.22 μm filter membrane and then concentrated using freeze-drying technology.

[0032] 4. Metabolomics Analysis and Substance Identification: Non-targeted metabolomics technology (LC-MS / MS) was used to detect concentrated secretion samples, and the identified substances and their concentrations were analyzed.

[0033] 5. Experimental Results: Under cadmium exposure (DD) treatment, the composition of rice root exudates changed significantly. Enrichment analysis identified a key metabolic pathway—amino acid metabolism. Further analysis showed that the abundance of L-valine and L-leucine was significantly upregulated in all stressed rice root exudates. Figure 1 ).

[0034] Example 2: Chemotaxis of L-valine and L-leucine on Bacillus subtilis

[0035] 1. Experimental Materials and Reagents Test Strains: Bacillus stratosphericus SRS50, Bacillus zanthoxyli SRS107. Chemotaxis: a. Root exudates (100 μg / L) collected from indica rice (93-11) and japonica rice (ZH11) under foliar treatment (CK control group and DD treatment group); b. Specific metabolites: L-valine (50 μg / L), L-leucine (50 μg / L), and an equimolar mixture of the two (L-Val + L-Leu); c. Negative control: Phosphate-buffered saline (PBS, pH 7.0). Bacterial Suspension Preparation: The bacterial strains were placed in inorganic salt medium (MSM) and the bacterial suspension concentration was adjusted to OD. 600 = 0.1.

[0036] 2. Experimental Methods: Using a micropipette, 10 μL of the aforementioned chemotactic agent was filled into each capillary. The open end of the filled capillary was vertically immersed in a container containing 500 μL of bacterial suspension and incubated at 30°C for 0.5 hours. After incubation, the liquid in the capillary was collected and diluted with 20 μL of phosphate-buffered saline (PBS, pH 7.0). The diluted solution was inoculated onto LB agar plates using the spread plate method and incubated at 30°C for 24 hours. The chemotactic attraction of each component to SRS50 and SRS107 strains was quantitatively assessed by calculating the number of colony forming units (CFUs) on the plates.

[0037] 3. Experimental Results: SRS50 and SRS107 strains showed significantly higher chemotactic responses to rice root exudates under DD treatment compared to the control group. Among the identified specific metabolites, L-valine and L-leucine induced strong chemotaxis in the strains at low concentrations of 50 μg / L. The number of CFUs induced by an equimolar mixture of L-valine and L-leucine was significantly higher than that in the single-component treatment groups. This demonstrates that L-valine and L-leucine are key signaling molecules for recruiting SRS50 and SRS107 strains from the rice rhizosphere, and that their combined application has a significant synergistic chemotactic recruitment effect. Figure 2 ).

[0038] Example 3: L-valine and L-leucine complex promotes Bacillus colonization in roots.

[0039] 1. Tested varieties and strains: Rice variety “Zhonghua 11” (ZH11); Bacillus serotype SRS50 and Bacillus zanthoxylum bungeanum SRS107.

[0040] 2. Treatment settings: a. Wild-type control group: ZH11-CK (cadmium-free control), ZH11-DD (simulated cadmium deposition treatment on leaves), ZH11-composite signal group (root surface immersed in L-Val 50 μg / L + L-Leu 50 μg / L for 30 min).

[0041] 3. Experimental Methods: Rice plants were cultured to the three-leaf stage and then subjected to foliar cadmium treatment or rhizosphere signaling substance addition according to the above-mentioned grouping. Plant seedlings were directly inoculated into the roots. 10 μl of bacterial suspension (single-cell culture, OD600 = 0.2) was pipetted and inoculated under the same conditions as described above in the greenhouse. Seedlings without bacterial inoculation served as the control group. Root samples were collected 5 days after inoculation, and the distribution of bacteria on the root surface was visualized using a double staining method. SYTO 9: nucleic acid staining agent (excitation wavelength 488 nm, emission wavelength 500-540 nm), used to label bacteria colonizing the root surface. Calcofluor White (CFW): cell wall staining agent (excitation wavelength 405 nm, emission wavelength 425-475 nm), used to delineate the outline of rice root cells. The spatial distribution of rice roots was imaged using a Leica STELLARIS STED confocal laser scanning microscopy platform (CLSM).

[0042] 4. Experimental Results: Confocal imaging clearly showed strong green fluorescence in the root system within the composite signal group (exogenous L-Val + L-Leu), and numerous aggregates of SRS50 and SRS107 strains were observed on the surface of rice roots. Exogenous application of the L-valine and L-leucine complex (50 μg / L) effectively simulated the endogenous "distress" signal in rice, significantly promoting the directional aggregation and efficient colonization of *Bacillus stolonifer* and *Bacillus zanthoxylum* in rice roots. Figure 3 ).

[0043] Example 4: The combined application of L-valine and L-leucine increased the relative abundance of Bacillus in the rice rhizosphere.

[0044] 1. Test materials: Rhizosphere soil from 26 rice varieties control groups (CK, untreated with cadmium) was collected, mixed evenly and homogenized as the initial microbial inoculum.

[0045] 2. Reagent preparation: Mix L-valine and L-leucine in an equimolar ratio (1:1) and dissolve them in a 20% methanol solution. Adjust the total carbon concentration of the mixture to 1.3125 g C / L according to the carbon equivalent balance principle.

[0046] 3. Treatment Setup: Treatment group: Added the above-mentioned compound amino acid mixture; Control group: Added an equal volume of 20% methanol solution as a solvent control. Soil was placed in centrifuge tubes for plantless culture. The mixture was applied twice a week, at a rate of 1.20 mL / tube each time, for 6 consecutive weeks. After the culture period, soil samples were collected, total DNA was extracted, and 16S rRNA gene amplicon sequencing analysis was performed.

[0047] 4. Experimental Results: Compared with the control group, the relative abundance of Bacillus spp. in the soil of the compound amino acid treatment group was significantly increased. The combined application of L-valine and L-leucine has the ability to improve the rhizosphere microecology of soil and precisely increase the relative abundance of beneficial Bacillus spp. in complex soil matrix. Figure 4 ).

[0048] Example 5: The alleviating effect of a compound signaling agent and its recruited Bacillus on cadmium stress in rice leaves.

[0049] 1. Experimental materials: Rice varieties “Zhonghua 11” (ZH11) and “Yangdao 6” (9311); Bacillus stratosphericus SRS50 and Bacillus zanthoxyli SRS107; the active ingredient is a complex of L-valine and L-leucine (molar ratio 1:1).

[0050] 2. Experimental Design and Treatment: Pretreated rice seedlings of 'Zhonghua 11' (ZH11) and 'Yangdao 6' (9311) were transplanted into sterilized earthen pots (8.2 cm × 9.2 cm × 8 cm). After 7 days of seedling establishment, the following four treatment groups were set up: Signaling agent group (L-valine + L-leucine group): the same concentration of the complex as in Example 4 was added to the rhizosphere; Mixed bacteria group: the rhizosphere was inoculated with a mixed Bacillus suspension (1.0 mL, equal volume, OD... 600 = 0.5); Mixed bacteria + signaling agent group: The rhizosphere was inoculated with a mixed Bacillus suspension (1.0 mL, OD600 = 0.5) and a complex of the same concentration as in Example 4; the control group was supplemented with an equal volume of methanol solution (buffer). The four treatment groups were simultaneously subjected to simulated atmospheric deposition (DD) treatment. Results determination: After 28 days of continuous culture, the dry weight and height of the plants were measured.

[0051] 3. Experimental Results: Under Cd exposure conditions, the signaling agent group did not significantly affect the plant height and aboveground dry weight of rice. However, the growth of rice treated with the mixed bacteria group and the mixed bacteria + signaling agent group was significantly better than that of the control group; among them, the mixed bacteria + signaling agent treatment group showed the most significant growth-promoting effect, with plant height increasing by 16.7-25.7% and aboveground dry weight increasing by 29.4-33.3% compared to the control group. These results confirm that the composite signaling agent exerts a significant stress-resistant and growth-promoting effect by specifically enhancing the recruitment and efficient colonization of probiotics in the rhizosphere. Figure 5 ).

Claims

1. Use of a composite signaling compound, wherein the active ingredient of the composite signaling compound is composed of L-valine and L-leucine; the use is at least one of the following 1)-8): 1) Recruit beneficial Bacillus spores from plant roots; 2) Preparation of formulations that recruit Bacillus spores from plant roots; 3) Promotes the colonization of Bacillus in plant roots; 4) Prepare formulations that promote the colonization of Bacillus in plant roots; 5) Increase the abundance of Bacillus in the soil; 6) Prepare formulations to increase the abundance of Bacillus in soil; 7) Synergistically promotes plant environmental adaptability under cadmium stress on leaves with Bacillus subtilis; 8) Prepare formulations that synergistically promote plant adaptation to cadmium stress on leaves with Bacillus.

2. The use according to claim 1, characterized in that, The plant in question is rice.

3. The use according to claim 1, characterized in that, The molar ratio of L-valine to L-leucine is 1:

1.

4. The use according to claim 1, characterized in that, The Bacillus species mentioned are Bacillus stratosphericus and Bacillus zanthoxyli.

5. The use according to claim 1, characterized in that, When the compound signaling agent is used to promote Bacillus colonization in the roots, the concentration of each amino acid is 50 μg / L.

6. The use according to claim 1, characterized in that, When the composite signaling agent is used to increase the abundance of Bacillus in soil, the molar concentration of each amino acid is 10 mmol / L.

7. The use according to claim 1, characterized in that, The composite signaling agent is applied via rhizosphere application.

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