Low-phosphorus growth-promoting strains isolated from arabidopsis thaliana rhizosphere microbial community and application thereof

Abstract

The application discloses a low-phosphorus growth promoting strain of an Arabidopsis thaliana root system microbial community and application thereof. The application discloses a Root1280 strain with excellent low-phosphorus growth promoting capacity, and the Root1280 strain establishes a close interaction relationship with plants under low-phosphorus conditions, and can well relieve low-phosphorus stress of the plants and promote growth of the plants when the Root1280 strain is cultured with the plants alone. The Root1280 strain is mixed with other eight strains to obtain an artificial synthetic microbial community, and it is found that, compared with the Root1280 treatment alone, the synthetic microbial community has a very obvious increase in the underground part, including root area, lateral root number and root fresh weight, which indicates that the synthetic microbial community has a better low-phosphorus growth promoting potential than the Root1280. The application not only proves the important role of the interaction between microorganisms and microorganisms on the function of the microbial community, but also provides a reference for trying to mix and apply functional bacteria with different effects and synergistically promote.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to low-phosphorus growth-promoting strains isolated from Arabidopsis root microbial communities and their applications. Background Technology

[0002] Phosphorus, as one of the most abundant elements after nitrogen, is crucial for all stages of plant growth and development. With population growth and the depletion of phosphate rock resources, the scarcity of soluble phosphorus in soil has led to increasingly severe crop yield reductions. Therefore, there is an urgent need to find green, environmentally friendly, and efficient methods to address low-phosphorus stress in agricultural production. In the natural environment, plants are surrounded by a large number of microorganisms; research shows that microorganisms play a vital role in plant growth and development, nutrient absorption, environmental adaptation, and stress resistance. Research on microbial phosphorus solubilization has been conducted for decades. The specific processes of dissolving insoluble phosphorus sources mainly include acid production and chelation. Many phosphorus-solubilizing bacteria with excellent phosphorus-solubilizing capabilities have been identified. However, due to a lack of research on plant-microorganism interactions, the application of microbial fertilizers has had minimal effect on alleviating low-phosphorus stress in agricultural production. "Low retention," "weak effect," and "high cost" are the main problems limiting the application of microbial fertilizers. In recent years, some fungal studies have discovered beneficial interactions between plants and arbuscular mycorrhizal fungi such as Colletotrichumtofieldia, which contribute to the alleviation of low phosphorus stress in plants. However, little is known about bacteria, which make up the vast majority of microorganisms.

[0003] With the development of high-throughput sequencing and microbial isolation and culture technologies, metagenomics-related research has emerged. Plant root microbiomes, analogous to human gut microbiomes, are collectively referred to as the host's "second genome," highlighting their importance. Currently, most studies only describe the changes in plant microbial community composition under different stress conditions; however, the specific biological significance of the regulated components remains largely unknown. Existing literature reports many dynamic regulatory interactions between plants and root bacteria during low phosphorus stress, but whether and how plants actively cooperate with bacteria to absorb insoluble phosphorus sources and promote growth remains unknown. Summary of the Invention

[0004] One object of the present invention is to provide new uses for Acinetobacter guillouiae Root1280 or its bacterial suspension or culture medium or bacterial agents containing it.

[0005] This invention provides the use of Acinetobacter guillouiae Root1280 or its bacterial suspension or culture medium or an agent containing it in any of the following A1)-A3):

[0006] A1) Relieve low phosphorus stress in plants;

[0007] A2) Enhance the ability of plants to alleviate low phosphorus stress;

[0008] A3) Promotes plant growth under low phosphorus stress.

[0009] The present invention also provides the use of Acinetobacter guillouiae Root1280 or its bacterial suspension or culture medium or bacterial agent containing it in any of the following B1)-B3):

[0010] B1) Prepare products to alleviate low phosphorus stress in plants;

[0011] B2) Prepare products that enhance the ability of plants to alleviate low phosphorus stress;

[0012] B3) Prepare products that promote plant growth under low phosphorus stress.

[0013] Another object of the present invention is to provide a compound microbial agent.

[0014] The active ingredients of the compound microbial agent provided by the present invention include Acinetobacter guillouiae Root1280, Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179 and Root670.

[0015] Furthermore, the active ingredients of the compound microbial agent are composed of Acinetobacter guillouiae Root1280, Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179 and Root670.

[0016] Furthermore, the CFU ratio of Acinetobacter guillouiae Root1280, Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179, and Root670 is 1:1:1:1:1:1:1:1:1:1.

[0017] The OD of the compound microbial agent 600 It is 0.5.

[0018] Another objective of this invention is to provide a method for preparing the aforementioned composite microbial agent.

[0019] The preparation method of the above-mentioned composite microbial agent provided by the present invention includes the following steps: mixing Acinetobacter guillouiae Root1280 culture medium, Root1464 culture medium, Root322 culture medium, Root495 culture medium, Root186 culture medium, Root11 culture medium, Root444D2 culture medium, Root179 culture medium and Root670 culture medium to obtain the composite microbial agent.

[0020] Furthermore, the OD values ​​of the Acinetobacter guillouiae Root1280 culture medium, the Root1464 culture medium, the Root322 culture medium, the Root495 culture medium, the Root186 culture medium, the Root11 culture medium, the Root444D2 culture medium, the Root179 culture medium, and the Root670 culture medium are... 600 Both are 0.5.

[0021] The volume ratio of the Acinetobacter guillouiae Root1280 culture medium, the Root1464 culture medium, the Root322 culture medium, the Root495 culture medium, the Root186 culture medium, the Root11 culture medium, the Root444D2 culture medium, the Root179 culture medium, and the Root670 culture medium is 1:1:1:1:1:1:1:1:1:1.

[0022] The mixing process further includes washing with sterile ddH2O and diluting to OD. 600 =0.5 steps.

[0023] Furthermore, the Acinetobacter guillouiae Root1280 culture medium is prepared by culturing Acinetobacter guillouiae Root1280 in 1 / 2 TSB liquid medium until OD. 600The bacterial culture obtained after dilution to 0.5 μL is as follows: Acinetobacter guillouiae Root1280 was inoculated into 600 μL of 1 / 2 TSB liquid medium and cultured at 28°C for 5 days to obtain a 5-day culture; then 20 μL of the 5-day culture was transferred to a new 1 / 2 TSB liquid medium and cultured at 28°C for 2 days to obtain a 2-day culture; the remaining culture was cultured at 28°C for another 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture were combined to obtain the Acinetobacter guillouiae Root1280 culture solution.

[0024] The Root1464 culture medium was prepared by culturing Root1464 in 1 / 2 TSB liquid medium until OD. 600 The bacterial culture obtained after 0.5% concentration is as follows: Root1464 was inoculated into 600 μL of 1 / 2 TSB liquid medium and cultured at 28°C for 5 days to obtain a 5-day culture; then 20 μL of the 5-day culture was transferred to a new 1 / 2 TSB liquid medium and cultured at 28°C for 2 days to obtain a 2-day culture; the remaining culture was cultured at 28°C for another 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture were combined to obtain the Root1464 culture solution.

[0025] The Root322 culture medium is prepared by culturing Root322 in 1 / 2 TSB liquid medium until OD. 600 The bacterial culture obtained after dilution to 0.5 μL is as follows: Root322 is inoculated into 600 μL of 1 / 2 TSB liquid medium and cultured at 28°C for 5 days to obtain a 5-day culture; then 20 μL of the 5-day culture is transferred to a new 1 / 2 TSB liquid medium and cultured at 28°C for 2 days to obtain a 2-day culture; the remaining culture is cultured at 28°C for another 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture are combined to obtain the Root322 culture solution.

[0026] The Root495 culture medium was prepared by culturing Root495 in 1 / 2 TSB liquid medium until OD. 600The bacterial culture obtained after dilution to 0.5 μL is as follows: Root495 is inoculated into 600 μL of 1 / 2 TSB liquid medium and cultured at 28°C for 5 days to obtain a 5-day culture; then 20 μL of the 5-day culture is transferred to a new 1 / 2 TSB liquid medium and cultured at 28°C for 2 days to obtain a 2-day culture; the remaining culture is cultured at 28°C for another 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture are combined to obtain the Root495 culture solution.

[0027] The Root186 culture medium was prepared by culturing Root186 in 1 / 2 TSB liquid medium until OD. 600 The bacterial culture obtained after 0.5% concentration is as follows: Root186 was inoculated into 600 μL of 1 / 2 TSB liquid medium and cultured at 28°C for 5 days to obtain a 5-day culture; then 20 μL of the 5-day culture was transferred to a new 1 / 2 TSB liquid medium and cultured at 28°C for 2 days to obtain a 2-day culture; the remaining culture was cultured at 28°C for another 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture were combined to obtain the Root186 culture solution.

[0028] The Root11 culture medium was prepared by culturing Root11 in 1 / 2 TSB liquid medium until OD. 600 The bacterial culture obtained after 0.5% concentration is as follows: Root11 is inoculated into 600 μL of 1 / 2 TSB liquid medium and cultured at 28°C for 5 days to obtain a 5-day culture; then 20 μL of the 5-day culture is transferred to a new 1 / 2 TSB liquid medium and cultured at 28°C for 2 days to obtain a 2-day culture; the remaining culture is cultured at 28°C for another 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture are combined to obtain the Root11 culture solution.

[0029] The Root444D2 culture medium was prepared by culturing Root444D2 in 1 / 2 TSB liquid medium until OD. 600 The bacterial culture obtained after dilution to 0.5 μL is as follows: Root444D2 is inoculated into 600 μL of 1 / 2 TSB liquid medium and cultured at 28°C for 5 days to obtain a 5-day culture; then 20 μL of the 5-day culture is transferred to a new 1 / 2 TSB liquid medium and cultured at 28°C for 2 days to obtain a 2-day culture; the remaining culture is cultured at 28°C for another 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture are combined to obtain the Root444D2 culture solution.

[0030] The Root179 culture medium was prepared by culturing Root179 in 1 / 2 TSB liquid medium until OD. 600 The bacterial culture obtained after 0.5% concentration is as follows: Root179 was inoculated into 600 μL of 1 / 2 TSB liquid medium and cultured at 28°C for 5 days to obtain a 5-day culture; then 20 μL of the 5-day culture was transferred to a new 1 / 2 TSB liquid medium and cultured at 28°C for 2 days to obtain a 2-day culture; the remaining culture was cultured at 28°C for another 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture were combined to obtain the Root179 culture solution.

[0031] The Root670 culture medium was prepared by culturing Root670 in 1 / 2 TSB liquid medium until OD. 600 The bacterial culture obtained after 0.5% concentration is as follows: Root670 was inoculated into 600 μL of 1 / 2 TSB liquid medium and cultured at 28°C for 5 days to obtain a 5-day culture; then 20 μL of the 5-day culture was transferred to a new 1 / 2 TSB liquid medium and cultured at 28°C for 2 days to obtain a 2-day culture; the remaining culture was cultured at 28°C for another 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture were combined to obtain the Root670 culture solution.

[0032] This invention also provides new uses for the above-mentioned composite microbial agent or the composite microbial agent prepared by the above method;

[0033] This invention provides the application of the above-mentioned composite microbial agent or the composite microbial agent prepared by the above method in any of the following A1)-A3):

[0034] A1) Relieve low phosphorus stress in plants;

[0035] A2) Enhance the ability of plants to alleviate low phosphorus stress;

[0036] A3) Promotes plant growth under low phosphorus stress.

[0037] This invention also provides the application of the above-mentioned composite microbial agent or the composite microbial agent prepared by the above method in any of the following B1)-B3):

[0038] B1) Prepare products to alleviate low phosphorus stress in plants;

[0039] B2) Prepare products that enhance the ability of plants to alleviate low phosphorus stress;

[0040] B3) Prepare products that promote plant growth under low phosphorus stress.

[0041] Another object of the present invention is to provide a product having the function of any one of the following A1)-A3):

[0042] A1) Relieve low phosphorus stress in plants;

[0043] A2) Enhance the ability of plants to alleviate low phosphorus stress;

[0044] A3) Promotes plant growth under low phosphorus stress;

[0045] The product includes Acinetobacter guillouiae Root1280 or its bacterial suspension or culture medium or bacterial agent containing it or the above-mentioned compound microbial agent or compound microbial agent prepared according to the above method.

[0046] The final objective of this invention is to provide a method for alleviating low phosphorus stress in plants, improving the ability of plants to alleviate low phosphorus stress, or promoting plant growth under low phosphorus stress.

[0047] The method provided by the present invention for alleviating low phosphorus stress in plants, improving the ability of plants to alleviate low phosphorus stress, or promoting plant growth under low phosphorus stress includes the step of applying Acinetobacter guillouiae Root1280 or its bacterial suspension or its culture medium or a bacterial agent containing it or the above-mentioned compound microbial agent or a compound microbial agent prepared according to the above method to plants.

[0048] Furthermore, the application method involves culturing the plant in an environment containing Acinetobacter guillouiae Root1280 or its bacterial suspension or culture medium, or a bacterial agent containing it, or the above-mentioned compound microbial agent, or a compound microbial agent prepared according to the above method.

[0049] Furthermore, the method of application involves culturing the plant in a low-phosphorus culture medium containing Acinetobacter guillouiae Root1280 or its bacterial suspension or culture medium, or a bacterial agent containing it, or the above-mentioned compound microbial agent, or a compound microbial agent prepared according to the above method.

[0050] The low-phosphorus culture medium containing Acinetobacter guillouiae Root1280 or its bacterial suspension or culture medium, or its bacterial agent, or the above-mentioned compound microbial agent, or the compound microbial agent prepared according to the above method, is 50 μL OD 600=0.5% of the above-mentioned Acinetobacter guillouiae Root1280 culture medium or the above-mentioned compound microbial agent or the compound microbial agent prepared according to the above method was added to 50 mL P i The culture medium obtained after culturing (625 μM HAP) medium.

[0051] In any of the aforementioned compound microbial communities, applications, products, or methods, Root1280 induces an increase in malic acid expression through the STOP1-ALMT1 signaling pathway under low phosphorus conditions. Malic acid, as a chemotactic molecule, attracts Root1280 to accumulate in the roots of plants and establishes a stable symbiotic relationship with the plants, thereby alleviating low phosphorus stress in plants, improving plant tolerance to low phosphorus stress, and promoting plant growth.

[0052] In any of the aforementioned compound microbial communities, applications, products, or methods, the promotion of plant growth is to increase the fresh weight of the plant, specifically by increasing the fresh weight of the above-ground parts and / or the fresh weight of the underground parts (root fresh weight) and / or the root area and / or the number of lateral roots.

[0053] In any of the aforementioned complex microbial communities, applications, products, or methods, the low phosphorus stress may specifically be 50 μM Pi or 626 μM calcium hydroxyphosphate stress.

[0054] In any of the aforementioned complex microbial communities, applications, products, or methods, the plants include monocotyledons and dicotyledons, and the dicotyledons may specifically be Arabidopsis thaliana.

[0055] This invention is based on a high-quality pure bacterial library isolated from the root microbiome of the model plant Arabidopsis thaliana. Using a reductionist approach, it investigated the ability of various root components to alleviate low-phosphorus stress in plants, identifying approximately 20% of root-related bacteria with potential low-phosphorus growth-promoting capabilities. Next, this invention selected a weakly phosphorus-soluble strain, Acinetobacter guillouiae Root1280, as a representative for detailed study. It was found that Root1280 strain possesses excellent low-phosphorus growth-promoting capabilities and establishes a close interaction with plants under low-phosphorus conditions. When co-cultured alone with plants, it effectively alleviates low-phosphorus stress and promotes plant growth. Furthermore, this invention combined the representative low-phosphorus growth-promoting bacterium Root1280 with eight root-associated bacteria to form another synthetic microbial community (MSC) and studied its function. The results showed that, compared to Root1280 alone, the aboveground fresh weight recovery of plants was comparable in the MSC group, but the underground parts, including root area, number of lateral roots, and root fresh weight, were significantly improved. This indicates that the MSC group has better low-phosphorus growth-promoting potential than Root1280. This invention not only demonstrates the important role of microbial interactions in microbial community function but also provides a reference for exploring the combined application of functional bacteria with different effects for synergistic promotion. Attached Figure Description

[0056] Figure 1 This study aimed to identify low-phosphorus growth-promoting components in the root bacterial microbial community of Arabidopsis thaliana. Figure A shows the identification flowchart. Seven days after germination, PT2-LUC transgenic materials were transferred to a phosphorus-deficient medium containing the test bacteria and hydroxyapatite (HAP) as the sole phosphorus source and grown for 14 days. Plant fresh weight and LUC activity were then recorded. Figure B shows the phenotypic diagrams of representative strains. Red indicates non-functional strains that compete with plants for phosphorus nutrition; blue strains only reduce LUC activity without promoting growth; green strains both reduce LUC activity and promote plant growth. Figures C and D show the relative LUC activity and fresh weight content of each plant in Figure B, respectively. Figure E shows the phylogenetic tree and taxonomic information of all 130 screened root-related bacteria. Yellow triangles indicate that the strain can reduce the expression of plant low-phosphorus response genes, and purple squares indicate that the strain can promote an increase in plant fresh weight. Among these, "0 μM P..." i "625μMHAP" indicates P i -(625μM HAP).

[0057] Figure 2Functional analysis of 26 identified low-phosphorus growth-promoting bacteria. Blue "shoot Pi" indicates the level of phosphorus increase in the aboveground parts of the plant promoted by this strain, in μmol / g; purple "FW" indicates the level of increase in fresh weight promoted by this strain, in mg; green "LUC" indicates the relative expression level of the low-phosphorus response gene PT2, with the PT2 expression level of plants under low-phosphorus aseptic conditions used as a control; yellow "pH" indicates the acid-producing capacity of this strain under single-culture conditions, with the pH of the culture medium (initial medium pH = 7.0) recorded after 3 days of culture; red "PS" indicates the relative solubility of the sparingly soluble phosphorus source HAP by this strain under single-culture conditions, with the soluble phosphorus content in the culture medium under aseptic conditions used as a control (due to significant differences in phosphorus solubility among strains, data in the figure are displayed as log values).

[0058] Figure 3 This study investigated the molecular mechanism of the interaction between Acinetobacter guillouiae Root1280 and plants under low phosphorus conditions. Figure A shows that Root1280 is significantly recruited to plants with low phosphorus, and this recruitment depends on the plant's STOP1-ALMT1 signaling pathway. Figure B shows the chemotactic experiment of Root1280, where malic acid significantly chemotactically attracts Root1280. Figure C shows that the alleviation of plant PSI (Phosphate Starvation Induced) by Root1280 depends on the STOP1-ALMT1 signaling pathway. Figure D is a flowchart of the plate transfer system for detecting the effect of Root1280 on the growth-promoting effect of different mutants under low phosphorus conditions. Figure E shows the growth phenotypes of wild-type Col-0 and mutants almt and stop1 under aseptic conditions under low and high phosphorus conditions, respectively. Figure F shows that the growth-promoting effect of Root1280 on plants depends on the STOP1-ALMT1 signaling pathway.

[0059] Figure 4 This study presents a synthetic microbial community and the low-phosphorus growth-promoting phenotypes of individual strains. Except for Root1280, the other eight strains (Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179, and Root670) showed no low-phosphorus growth-promoting ability and behaved similarly to the sterile control under low-phosphorus conditions. However, the community (MSC, -Root1280) composed of these eight strains significantly promoted plant fresh weight increase under low-phosphorus conditions. A community (MSC) composed of nine strains, including the low-phosphorus growth-promoting bacterium Root1280, maximally promoted plant growth, especially significantly increasing the fresh weight of the underground parts, as well as the number of lateral roots and root area. Among them, "0 μM P" was observed. i "625μM HAP" indicates P i -(625μM HAP). Detailed Implementation

[0060] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0061] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0062] The PT2-LUC transgenic material in the following examples is described in the literature "Karthikeyan et al. Regulated expression of Arabidopsis phosphate transporters. Plant Physiol 130:221-233 (2002)" and is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of this invention and should not be used for other purposes.

[0063] The PSR signaling pathway mutants almt1 (SALK_009629C) and stop1 (SALK_114108) in the following examples are described in the literature "Wang et al. Genetic dissection of Fe-dependent signaling in root developmental responses to phosphate deficiency. Plant Physiol 179:300-316 (2019)". They are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. These biological materials are only used to repeat the relevant experiments of this invention and should not be used for other purposes.

[0064] The PSR signaling pathway mutants phr1phl1 (SALK_067629, SALK_079505) in the following examples are described in the literature "Wang et al. Functional Characterization of Arabidopsis PHL4 in Plant Response to Phosphate Starvation. Front Plant Sci 9:1432 (2018)". They are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of this invention and should not be used for other purposes.

[0065] The Root1280, Root186, Root322, Root1464, Root179, Root444D2, Root670, Root11, and Root495 strains used in the following examples are all described in the literature “Baie et al. Functional overlap of the Arabidopsis leaf and root microbiota. Nature 528:364-369 (2015).”, which are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of this invention and should not be used for other purposes.

[0066] The wild-type Arabidopsis thaliana in the following examples is the Columbia ecotype (Columbia-0, Col-0).

[0067] The culture medium and reagent formulations in the following examples are as follows:

[0068] 1 / 2 MS solid culture medium (1L) was prepared by mixing 2.215g MS powder, 15g sucrose, 3g plant gel with 1L of water, adjusting the pH to 5.8 with KOH, and autoclaving at 121℃ for 15 minutes.

[0069] 1 / 2 TSB bacterial liquid culture medium (1L) was obtained by mixing 2.215g of Tryptone Soya Broth with 1L of water. The mixture was then autoclaved at 121°C for 15 minutes.

[0070] 1 / 2 TSB bacterial solid medium (1L) was prepared by mixing 2.215g Tryptone Soya Broth, 15g agar, and 1L of water. It was then autoclaved at 121°C for 15 minutes.

[0071] NBPIP medium (1L) was prepared by mixing 2.215g of glucose, 15g of MgSO4·7H2O, 3g of KCl, 5g of MgCl2·6H2O, 0.1g of (NH4)2SO4, and 8.1g of HAP with 1L of water, and adjusting the pH to 7.0 with KOH. The mixture was then autoclaved at 121℃ for 15 minutes.

[0072] The formulations of plant culture media with different phosphorus contents are shown in Table 1. All media were adjusted to pH 5.8 with KOH and autoclaved at 121°C for 15 minutes.

[0073] Table 1. Formulations of plant culture media with different phosphorus contents

[0074]

[0075] 40×P i The macro-element stock solution (1L) was obtained by mixing 19g KNO3, 16.5g NH4NO3, and 1.7g KH2PO4 with 1L of water. It was then autoclaved at 121℃ for 15 minutes and stored in a refrigerator at 4℃.

[0076] 40×P i - The macro-element stock solution (1L) was obtained by mixing 19g KNO3, 16.5g NH4NO3, and 2.178g K2SO4 with 1L of water. It was then autoclaved at 121℃ for 15 minutes and stored in a refrigerator at 4℃.

[0077] Vitamin powder 2000× stock solution (5mL) is obtained by mixing 0.5156g of M533 Vitamin Salt with 5mL of water. Filter through a 0.22μm filter membrane, store at 4℃ in a refrigerator, and use within one week.

[0078] The experimental methods and specific operating steps involved in the following embodiments are as follows:

[0079] Plant growth conditions: Arabidopsis thaliana or related materials were cultured vertically in 1 / 2 MS medium in a light incubator with the following conditions: 16 hours of light, 22°C, and 60% humidity; 8 hours of darkness, 20°C, and 60% humidity.

[0080] Seed sterilization and germination: 1) Place an appropriate amount of seeds in a centrifuge tube, surface sterilize with 30% 84 disinfectant for 10 minutes, then rinse 5-6 times with sterile water and place in a 4℃ refrigerator for 2 days. 2) Pour 50mL of the prepared 1 / 2MS solid medium evenly into a 10×10 square petri dish. After solidification, use a pipette tip to inoculate the seeds. Place the seeds vertically in a light incubator for 5-7 days according to experimental needs, under the following conditions: 16h light duration, 22℃ temperature, 60% humidity; 8h darkness duration, 20℃ temperature, 60% humidity. Select seedlings with consistent growth for the experiment.

[0081] Bacterial activation and culture: 1) In a clean bench, use a sterile pipette tip to pick up an appropriate amount of bacteria stored at -80℃ and streak it onto a 1 / 2 TSB plate. Incubate at 28℃ for 1-2 days. 2) Pick bacteria from the 1 / 2 TSB plate and inoculate them into a sterile shake tube containing 2 mL of 1 / 2 TSB liquid culture medium. Incubate overnight at 28℃.

[0082] Bacterial-plant co-culture system: 1) Prepare plant culture media with different phosphorus contents according to the dosage: P i +(625μMP i Culture medium, 50 μM P i The culture medium contains HAP as the sole phosphorus source. i - (625 μM HAP) medium, autoclave at 121℃ for 15 minutes; after sterilization, shake the medium thoroughly to ensure the poorly soluble HAP is fully mixed, and place in a 42℃ water bath for cooling and incubation. 2) Remove the bacteria from the overnight culture, centrifuge at 4000 rpm for 5 minutes to collect the bacteria, and discard the supernatant. 3) Resuspend the bacteria with ddH2O, centrifuge at 4000 rpm for 5 minutes to collect the bacteria, and discard the supernatant; repeat three times. 4) Add 1 mL of sterile water, pipette to mix the bacterial precipitate; aspirate 80 μL of bacterial solution into a centrifuge tube containing 720 μL of ddH2O, and measure the OD value using a spectrophotometer. 600 Measure the bacterial concentration after a 10-fold dilution; calculate the concentration and then adjust the bacterial concentration to OD using ddH2O. 600 0.5.5) Remove the culture medium from the water bath at 42℃ and pour 50mL into a sterile BD tube. Add OD at a 1:1000 ratio. 600 =0.5% bacterial suspension, mix thoroughly and slowly pour into 10×10 square petri dishes to solidify, avoiding air bubbles. Cool and solidify in a clean bench for 30 minutes before use. 6) Remove Arabidopsis seedlings that have grown to the corresponding number of days from the light incubator, and transfer plants with consistent growth stages to the corresponding culture medium containing bacteria using a sterilized yellow pipette tip; change the pipette tip for each plate to prevent contamination. 7) Seal the plates with breathable tape, place them in the light incubator, and grow for the corresponding number of days before experimentation.

[0083] Detection of PT2-LUC expression level and weighing of plant fresh weight: 1) Remove the plate and photograph the seedling growth status. 2) Prepare a 96-well white ELISA plate and add 100 μL of ddH2O to the plate using a multi-channel pipette. 3) Cut off the underground parts of the plants and place them in the ELISA plate, placing one seedling root in each well. 4) Prepare a 100×LUC stock solution: Dissolve 1 g of D-Luciferin Potassium in 31.4 mL of sterile water, aliquot, and store at -20℃. 5) Prepare a 2×LUC sterile aqueous solution: Dilute the prepared 100×LUC stock solution 50 times with ddH2O, add 100 μL of the 2×LUC sterile aqueous solution to each well of the ELISA plate, ensuring the roots are submerged in the liquid, cover with aluminum foil, and let stand in the dark for 5 minutes. 6) Detect the chemiluminescence intensity of the samples using an ELISA reader. 7) Weigh the fresh weight of the above-ground parts and roots of each plant, or the fresh weight of the whole plant, using an analytical balance as needed for the experiment.

[0084] Detection of bacterial in vitro phosphate-solubilizing and acid-producing abilities: 1) Activate and culture bacteria according to the above method. 2) Prepare NBRIP medium according to the formula, autoclave at 121℃ for 15 minutes, cool, and dispense 4 mL into 15 mL sterile BD tubes. Perform at least three replicates for each treatment. 3) Collect the cultured bacteria, centrifuge at 4000 rpm for 5 minutes, resuspend and wash the bacteria with ddH2O, centrifuge at 4000 rpm for 5 minutes, and repeat twice. 4) Add 1 mL of ddH2O, mix well to form a bacterial pellet; aspirate 80 μL of the bacterial solution, add 720 μL of ddH2O to dilute 10-fold, and adjust the bacterial concentration to OD200 using ddH2O. 600 =0.5 for later use. 5) Aspirate half the volume of the diluted bacterial solution and autoclave at 121℃ for 15 minutes as a heat-killed control. 6) Add the bacteria (or inactivated bacteria) at a ratio of 1:1000 to the dispensed NBRIP medium and incubate on a shaker at 28℃ and 200 rpm for 3 days. 7) Fermentation broth collection: Centrifuge at 5000 rpm for 10 minutes, and aspirate 1 mL of supernatant into a 1.5 mL centrifuge tube, avoiding aspiration of the lower layer of insoluble phosphorus; centrifuge at 12,000 rpm for 10 minutes, and aspirate 900 μL of supernatant into a new centrifuge tube; centrifuge again at 12,000 rpm for 10 minutes, and aspirate 100 μL of supernatant into a clear 96-well plate. 8) Preparation of phosphorus reaction solution: The phosphorus reaction solution consists of solution A and solution B; solution A is a 10% (w / v) ascorbic acid solution, which can be stored in a refrigerator at 4℃ for one month, and is best prepared fresh for use; solution B is 4.2g of ammonium molybdate (NH4)6Mo7O 24• 4H₂O is dissolved in 1L of 1N sulfuric acid solution (28.6mL of concentrated sulfuric acid is slowly added to 1000mL of water); Solution B is stable at room temperature; the phosphorus reaction solution is prepared by mixing solution A and solution B in a 1:6 (volume ratio) and is only usable on the same day. 9) Preparation of standard curve: Prepare 1mM KH₂PO₄ standard solution by weighing 0.1361g of KH₂PO₄ and dissolving it in 1L of water; measure 0μL, 5μL, 15μL, ..., 70μL of phosphorus standard solution into different centrifuge tubes, and make up to 300μL with sterile water. Then add 700μL of phosphorus reaction solution to each tube; react at 37℃ for 1 hour or at 45℃ for 20 minutes, and stand at 4℃ for 10 minutes. Measure the absorbance at 820nm using a spectrophotometer to prepare a standard curve and calculate the conversion relationship between the number of micromoles of phosphorus and absorbance. 10) Detection of bacterial in vitro phosphate-solubilizing ability: Add 200 μL of sterile water to the 96-well plate in step 7), then add 700 μL of phosphate reaction solution. Incubate at 37°C for 1 hour or 45°C for 20 minutes, then incubate at 4°C for 10 minutes. Measure the absorbance at 820 nm using a spectrophotometer. Calculate the bacterial in vitro phosphate-solubilizing ability based on the absorbance. 11) Calculation of acid production ability: Measure the pH of the NBRIP medium after co-culturing with the bacteria for 3 days in step 6) using a pH meter.

[0085] Determination of phosphorus content in plant tissues using the malachite green method: 1) Preparation of malachite green solution: 1-1) Weigh 1.2g of boric acid (H3BO3) and amine molybdate [(NH4)6Mo7O 24 ·4H2O] 34.16g dissolved in 350mL distilled water. 1-2) Slowly stir and add 476mL of 5M H2SO4 (5M H2SO4 preparation method: measure about 136mL of concentrated sulfuric acid, slowly add it to 300mL of distilled water, cool and make up to 500mL). 1-3) Add 0.229g of malachite green to the above solution and dissolve. 1-4) Separately measure 100mL of distilled water, add 1g of polyvinyl alcohol (98% can be hydrolyzed, molecular weight 11,000 to 31,000), and continue heating to dissolve; 1-5) After the above solution is cooled, add it to the acidic malachite green solution, and make up to 1L of the mixture; store strictly in the dark at room temperature. 2) Preparation of phosphorus standard solution and plotting of standard curve: 2-1) Dry an appropriate amount of potassium dihydrogen phosphate (analytical grade) in an oven to constant weight. 2-2) Accurately weigh 0.2195 g and dissolve it in 400 mL of distilled water. 2-3) Add 5 mL of concentrated sulfuric acid to prevent mold growth and allow the solution to be stored for a long time. 2-4) Transfer to a 1 L volumetric flask and add water to the mark to obtain a 50 μg / M phosphorus standard stock solution. 2-5) Using the prepared phosphorus standard stock solution and distilled water, prepare 25 mL solutions of P with concentrations of 0 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, and 0.5 μg / mL, respectively. iStandards; 2-6) Take 1.5 mL of each of the above phosphorus standards, add 0.5 mL of malachite green solution, mix well, let stand for 30 minutes, and then measure the absorbance at 650 nm using a spectrophotometer. 2-7) Plot the absorbance value on the x-axis, P i Plot a standard curve in Excel with the content of phosphorus on the ordinate. 3) Determination of inorganic phosphorus content in plant leaves: 3-1) Grind fresh seedling leaves in liquid nitrogen, weigh 30 mg (plus or minus 5 mg) of the ground sample into a 2 mL centrifuge tube, and record the mass. Each sample should be weighed into at least 3 tubes. 3-2) Add 200 μL of 5M sulfuric acid to the centrifuge tube, gently mix, and incubate on ice for 10 minutes. 3-3) Add 1.8 mL of distilled water, mix, and incubate on ice for 10 minutes. 3-4) Centrifuge at 4℃ and 12,000 rpm for 10 minutes, and transfer the supernatant to a new 1.5 mL centrifuge tube for testing. 3-5) After dilution of the sample (P i +Samples generally need to be diluted about 10 times, P i (No dilution required) Mix with malachite green at a 3:1 ratio, let stand for 30 minutes, and then measure the absorbance at 650 nm. 3-6) Calculate the phosphorus content based on the standard curve and dilution factor: Phosphorus content per unit fresh weight (P... i μg / g) = Fresh weight * Volume * Dilution factor * Concentration of the test solution.

[0086] Root bacterial colonization detection: 1) In the root bacterial colonization experiment, to eliminate the influence of HAP on bacteria, the culture medium formulation in Table 1 was adjusted by adding P... i The medium was enriched with 625 μM HAP and P in a high-phosphorus medium containing (625 μM Pi). i - The culture medium is consistent. 2) The seed germination and inoculation system is the same as above. 3) Seedlings with consistent growth status after 5 days of germination are transferred to P culture medium pre-mixed with corresponding root-related bacteria. i + or P i - Corresponding days of growth on plates (1d, 2d, 4d). 4) After cultivation, measure the length of the taproot, cut off the underground part of the plant with sterile scissors, and place the roots of every 3 plants into a centrifuge tube containing 100μL of sterile water and 2 sterile steel balls as one sample. Each treatment should be repeated at least 6 times. 5) Use a Qiagen tissue homogenizer to homogenize the roots at 30Hz / s for 30 seconds. 6) Add 900μL of sterile water to the centrifuge tube to make a 1× bacterial solution. Dilute according to the bacterial growth rate and quantity. Take 20μL of the diluted solution and plate it, and incubate overnight at 28℃. 7) Count the colonies on the second day and calculate the number of bacteria colonizing the roots according to the dilution factor.

[0087] Capillary method for detecting chemotactic response: Root1280 bacteria cultured overnight were washed with sterile water and adjusted to OD. 600To prepare a solution of malic acid and serine at the corresponding concentrations, add 180 μL of the diluted bacterial solution to a 96-well plate. Take a 10 mm diameter medical capillary tube and add 20 μL of the corresponding chemotactic solution. Use sterile water as a control group. Each group should have at least 6 replicates. Immerse the capillary tube in the solution in the 96-well plate, ensuring that the air pressure and water pressure inside the capillary tube are consistent to prevent liquid from flowing into or out of the capillary tube due to pressure differences. After the reaction reaches the corresponding time, remove 20 μL of solution from the capillary tube, serially dilute it, and plate it for counting.

[0088] RNA Extraction: 1) Grind 50 mg of plant sample thoroughly in a mortar with liquid nitrogen. After the liquid nitrogen has evaporated completely, transfer the powder to a centrifuge tube. Add 300 μL of lysis buffer and mix well. For total RNA extraction from roots containing bacteria, grind the sample as in the plant sample, add 100 μL of the corresponding lysozyme solution, and add 200 μL of RNA lysis buffer. Add 300 μL of RNA diluent to both the bacterial lysis buffer and the plant sample, and mix well. 2) Centrifuge at maximum speed for 5 minutes and collect the supernatant. Bacterial RNA samples do not require centrifugation and can be directly used in the next step. 3) Add 0.5 times the volume of anhydrous ethanol to the supernatant and mix by pipetting 3 to 4 times. 4) Remove the centrifuge column, attach it to the collection tube, transfer the mixture to the centrifuge column, centrifuge at 12,000 rpm for 1 minute, and discard the filtrate. 5) Add 600 μL of RNA washing buffer, centrifuge at 12,000 rpm for 45 seconds, and discard the filtrate. 6) Prepare DNase I incubation solution: 5 μL 10×DNase I buffer, 5 μL DNase I, 40 μL nuclease-free water. 7) Add 50 μL DNase I incubation solution to the center of the adsorption membrane and incubate at room temperature for 15 minutes. 8) Add 600 μL RNA washing buffer, centrifuge at 12,000 rpm for 45 seconds, discard the filtrate; repeat once. 9) Centrifuge for 2 minutes to remove air bubbles. 10) Transfer the centrifuge column to an elution tube, add 100 μL nuclease-free water to the center of the column membrane, incubate at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute, and store the RNA at low temperature.

[0089] mRNA reverse transcription to cDNA: 1) RNA to cDNA reverse transcription was performed using the Thermo Reverse Transcription Kit. The system was as follows: 4 μL 5×reaction mix, 2 μL Maxima enzyme, 1 μg RNA template, and water nuclease-free to 20 μL. 2) Reverse transcription program: 20℃ for 10 minutes, 50℃ for 15 minutes, 85℃ for 5 minutes, and stored at 4℃.

[0090] Plant gene real-time quantitative PCR: 1) Real-time quantitative PCR was performed using the Takara TB Green PremixEx Taq kit. The experimental instrument was an Agilent Technologies Stratagene Mx3005P. The internal control was the expression level of Arabidopsis thaliana ACTIN2. Each sample was tested in at least three replicates. The reaction system was as follows: 10 μL SYBR, 0.4 μL ROX, 4.8 μL Water nuclease-free, 0.4 μL Prime-F, 0.4 μL Prime-R, and 4 μL cDNA. 2) Reaction system: 95℃ for 3 minutes; the following steps were repeated 40 times: 95℃ for 5 seconds, 60℃ for 20 seconds, and 72℃ for 20 seconds.

[0091] Aseptic root shading system under laboratory conditions: 1) Fold aluminum foil into rectangles of the appropriate size (to cover a square plate of approximately 10×10 cm). Fold three sides of the rectangle repeatedly to increase thickness, then sterilize with high-temperature steam. 2) Use sterilized tweezers to remove the sterilized aluminum foil. Place the folded side down to adhere to the culture medium, leaving space for root growth. Adhere the remaining three sides to the culture medium, covering the underground parts of the plant and blocking blue light from reaching the roots from all angles. 3) After sealing the plate, tightly cover the back of the plate with multiple layers of black plastic sheeting to ensure the roots are blocked from blue light; place it in an incubator for growth.

[0092] Transfer plate system for studying root bacterial function: Due to the contamination of root-related bacteria in 1 / 2 MS medium, it is difficult to distinguish the increase of P in bacteria in the medium. i To investigate the availability of plant-root bacteria and their interaction with the plant in mitigating PSR, a transfer plate system was designed to study the function of these root bacteria: 1) Plants in the same growth stage were transferred into a P plate containing different root-related bacteria. i 1) Plants were grown in (625 μM HAP) medium for 4 days; used for root recruitment of corresponding bacterial strains. 2) Plants that had recruited root bacteria were then transferred again to P i - (625 μM HAP) number of days for regrowth in sterile plates. 3) Calculate the degree of PSR relief in plants.

[0093] Example 1: Functional identification of root-associated bacteria with low-phosphorus growth-promoting ability

[0094] I. Experimental Methods

[0095] The root microbiome of plants is closely related to the plant itself. It has been reported that the root microbiome plays a strong regulatory role and is considered the plant's "second genome," playing a crucial role in growth, development, nutrient absorption, environmental adaptation, and stress resistance. Therefore, exploring the potential low-phosphorus growth-promoting components of the plant root microbiome is of great significance for studying the interaction between plants and beneficial bacteria under low-phosphorus conditions and for identifying excellent candidates for biological agents. Previous research on phosphate-solubilizing bacteria has mostly focused on the in vitro phosphate-solubilizing capacity of strains outside of plant environments. While many strains with exceptionally high phosphate-solubilizing capabilities have been identified, their effectiveness in practical applications has often been limited. Therefore, this invention combines a plant-bacteria co-culture system and uses the following two indicators of low-phosphorus stress relief in plants to assess the low-phosphorus growth-promoting capacity of root-related bacteria, identifying components in the Arabidopsis root bacterial microbiome with low-phosphorus growth-promoting capabilities: a) decreased expression of the low-phosphorus response gene PT2-LUC; b) the degree of recovery from low-phosphorus-induced plant growth inhibition. The identification flowchart is shown below. Figure 1 As shown in A, the specific steps are as follows:

[0096] 1. Co-culture of bacterial strains and plants

[0097] 130 test strains were mixed in equal amounts into a low-phosphorus medium with calcium hydroxyphosphate as the sole insoluble phosphorus source. PT2-LUC plants with relatively uniform growth status after 7 days of germination were transferred into a low-phosphorus medium containing root bacteria and grown for 14 days.

[0098] 2. Phenotypic Statistics

[0099] Plant phenotypes were statistically analyzed, and plant growth status was recorded proportionally using a camera. Plant roots were sampled and reacted with the substrate Luciferin. The expression level of the PT2 gene was rapidly detected by LUC fluorescence activity. The fresh weight of the plants was recorded using an analytical balance.

[0100] II. Experimental Results and Analysis

[0101] Phenotypic observation results are as follows Figure 1 As shown in Figure B, this figure illustrates the representative phenotypes of 130 strains. The vast majority of root bacteria, like the red strains Root79 and Root83, failed to alleviate PSI gene expression or promote plant growth under low phosphorus conditions. Some strains, like the blue strains Root485 and Root274, only reduced plant PSI gene expression but did not significantly promote plant growth. A small number of green strains, such as Root1280 and Root133, showed excellent results, both reducing plant PSI gene expression and promoting plant growth (PSI gene expression levels and fresh weight data are shown in Figure B). Figure 1 (As shown in CD), these bacteria are the low-phosphorus growth-promoting bacteria identified in this invention.

[0102] Based on the following criteria: a reduction of PSI gene expression by more than 60% and a fresh weight increase of more than 50%, with each functional strain exhibiting at least two similar low-phosphorus growth-promoting phenotypes, this invention ultimately identified 26 strains with potential low-phosphorus growth-promoting abilities from 130 bacteria. Their taxonomic information and phylogenetic trees are shown in Table 2 and [Table data missing]. Figure 1 As shown in E.

[0103] Table 2. Taxonomic information of low-phosphorus growth-promoting bacteria

[0104]

[0105]

[0106] Example 2: Functional Detection of Low-Phosphorus Growth-Promoting Strains

[0107] I. Experimental Methods

[0108] The following five indicators were used to alleviate low phosphorus stress in plants: a) decreased expression levels of low phosphorus response genes ( Figure 2 (green); b. Increased fresh weight ( Figure 2 c. Increase in phosphorus content in the aboveground parts of the plant (purple); Figure 2 (blue); d. Solubility of sparingly soluble phosphorus sources ( Figure 2 (red) and e, acid production capacity ( Figure 2 (Yellow) Analysis of the function of 26 low-phosphorus growth-promoting bacteria identified in Example 1.

[0109] The method for detecting phosphorus content in the aboveground parts includes the following steps: the aboveground parts of the plant are ground in liquid nitrogen, divided into 3 equal parts and weighed, the inorganic phosphorus content in the aboveground parts is determined by the malachite green method, and calculated according to the corresponding standard curve.

[0110] The bacterial in vitro phosphorus-solubilizing and acid-producing abilities were determined using liquid NBRIP medium with an initial pH of 7.0, supplemented with HAP as a poorly soluble inorganic phosphorus source, and inoculated at a 1:1000 ratio into OD. 600 Root bacteria with a pH of 0.5 were incubated on a shaker for 3 days. The acid-producing capacity of the bacteria was determined by measuring the change in pH of the culture medium after 3 days of cultivation; the lower the pH of the culture medium, the stronger the acid-producing capacity of the bacteria.

[0111] II. Experimental Results and Analysis

[0112] The statistical results of various functional indicators of 26 low-phosphorus growth-promoting bacteria are as follows: Figure 2As shown, the darker the color corresponding to each indicator, the better the strain's ability in that area. These five indicators can comprehensively reflect the various capabilities of low-phosphorus growth-promoting bacteria, judging their effectiveness in promoting low-phosphorus growth. They can also be used to select appropriate candidate strains for application based on different conditions. Furthermore, based on the bacteria's in vitro phosphate-solubilizing ability (… Figure 2 The strains (in red) were sorted on the horizontal axis. This shows that although the in vitro phosphate-solubilizing abilities of the 26 strains varied considerably, strains with stronger in vitro phosphate-solubilizing abilities did not necessarily have a better ability to alleviate low-phosphorus stress in plants. Many strains with weaker phosphate-solubilizing abilities, such as Root627 and Root1280, also possessed strong low-phosphorus growth-promoting abilities. Therefore, this indicates that the alleviation of low-phosphorus stress in plants is not solely determined by the phosphate-solubilizing ability of bacteria, and also suggests that active interactions between plants and root-associated bacteria exist under low-phosphorus conditions, contributing to the alleviation of low-phosphorus stress in plants to a certain extent.

[0113] Example 3: Molecular mechanism study of the interaction between representative strain Root1280 and plants under low phosphorus conditions.

[0114] I. Differences in root colonization of strains under different phosphorus contents

[0115] When faced with different environments, plants regulate changes in their metabolites through various signaling pathways. These metabolites can act as signaling molecules in interactions with root microorganisms. To investigate whether the colonization of Acinetobacter guillouiae Root1280 strain in the roots is induced by low phosphorus signals from the plant, the following experiment was conducted:

[0116] 1. In high phosphorus (P) i +(625μM Pi)) and low phosphorus (P) i Under the -(625μM HAP) condition, the colonization of Root1280 strain on Arabidopsis thaliana (Col-0) roots was detected, and the differences between the two were compared.

[0117] The results showed that, compared with high phosphorus conditions, the root colonization of Root1280 was significantly increased under low phosphorus conditions. Figure 3 (WT in A).

[0118] 2. To further investigate whether the low phosphorus accumulation of Root1280 strain is regulated by the plant PSR signaling pathway, this invention separately investigated the low phosphorus accumulation of Root1280 strain under high phosphorus (P) conditions. i +(625μM Pi)) and low phosphorus (P) iThe colonization differences of Root1280 strain in different plant PSR signaling pathway mutants phr1phl1, stop1, and almt1 were detected under -(625μM HAP) conditions. Among them, PHOSPHATESTARVATION 1 (PHR1) is a core transcription factor in plant low phosphorus stress response. When plants are subjected to low phosphorus stress, PHR1 can bind to the promoter sequences of most low phosphorus response genes (such as high-affinity phosphorus transporters PT1 and PT2) and induce their high expression to adapt to the low phosphorus environment. PHR1-Like (PHL1) is a homolog of PHR1. PHR1 and PHL1 are involved in regulating more than 75% of phosphorus starvation response genes. The phr1phl1 double mutant plants showed a significantly weakened response to low phosphorus stress and extremely inhibited growth. Low phosphorus levels can also induce the accumulation of the SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) transcription factor in the cell nucleus. STOP1 can activate some transporter or ion channel proteins, such as ALUMINUM-ACTIVATED MALATETRANSPORTERs (ALMTs), in which ALMT1 plays a major role.

[0119] The results showed that the low-phosphorus-induced recruitment of Root1280 did not depend on the regulation of the PHR1-PHL1 signaling pathway. phr1phl1 exhibited a low-phosphorus enrichment phenotype consistent with wild-type Arabidopsis, but Root1280 did not significantly colonize low-phosphorus roots in the stop1 and almt1 mutants. Figure 3 The phr1phl1, stop1, and almt1 in A showed a bacterial colonization level comparable to that on high-phosphate roots. Under low-phosphate conditions, plants recruited Root1280 by inducing the expression of the STOP1-ALMT1 signaling pathway.

[0120] 3. Since the STOP1-ALMT1 signaling pathway regulates the secretion of the organic acid malate, in order to investigate whether malate is recruited as a signaling molecule chemotactic Root1280, this invention also conducted chemotaxis experiments.

[0121] The results showed that malic acid significantly increased the chemotaxis of Root1280 at both 1 h and 1.5 h compared to the bacterial positive control chemotactic substance serine. This indicates that malic acid has a strong inducing effect on Root1280. In summary, under low phosphorus conditions, plants induce increased malic acid expression through the STOP1-ALMT1 signaling pathway, and malic acid, as a chemotactic molecule, attracts Root1280 to accumulate significantly in the plant roots. Figure 3 B).

[0122] II. PSR gene expression in plants

[0123] To investigate whether the low-phosphorus accumulation of Root1280 contributes to the relief of low-phosphorus stress in plants, this invention observed that under a 4-day treatment condition, low phosphorus induced a significant accumulation of Root1280 in Arabidopsis roots, and examined the expression of the plant low-phosphorus response gene IPS1 after 4 days of treatment. The primer sequences used are as follows:

[0124] Actin2-F: 5'-GACCTTGCTGGACGTGACCTTAC-3';

[0125] Actin2-R: 5'-GTAGTCAACAGCAACAAAGGAGAGC-3';

[0126] IPS1-F: 5'-AGACTGCAGAAGGCTGATTCAGA-3';

[0127] IPS1-R: 5'-TTGCCCAATTTCTAGAGGGAGA-3'.

[0128] The results showed that in wild-type Arabidopsis thaliana, co-culturing with Root1280 for 4 days under low phosphorus conditions significantly reduced the expression of the IPS1 gene. However, in the mutants stop1 and almt1, co-culturing with Root1280 for 4 days not only failed to alleviate the PSR, but also further increased the expression level of the IPS1 gene. Figure 3 C). This suggests that under low phosphorus conditions, early enrichment of Root1280 in plants contributes to reducing the expression of the IPSI gene and alleviating low phosphorus stress in plants; while mutants, due to their inability to enrich Root1280 in the early stages and their failure to establish a stable symbiotic relationship with Root1280, cannot alleviate low phosphorus stress in plants in the early stages (4 days).

[0129] III. Increase in plant fresh weight

[0130] To explore whether the growth-promoting function of Root1280 under low phosphorus conditions depends on the STOP1-ALMT1 pathway in plants, and considering that observing the growth-promoting effect of bacteria on plants requires a relatively long period (10-14 days), the bacteria's reaction with HAP in the culture medium was increased, and the phosphorus content in the culture medium was increased. i Availability also contributes to the alleviation of low phosphorus stress and growth recovery in plants, which may mask the differences in the growth-promoting effects of early recruitment of Root1280 to establish a symbiotic relationship. Therefore, in order to minimize the increase of P by bacteria... i The impact on availability is first addressed by transferring seedlings to P. i Co-cultured with Root1280 on sterile (625 μM HAP) low-phosphorus medium for 4 days, then transferred to fresh P... iFurther growth was carried out for 10 days in (625 μM HAP) low phosphorus medium (sterile), and the transfer procedure was as follows: Figure 3 As shown in D.

[0131] The results showed that all materials exhibited strong growth inhibition when grown on sterile, low-phosphorus medium, but almt1 and stop1 showed weaker growth inhibition compared to Col-0. Figure 3 E) Next, let's look at the co-culture with Root1280 under low phosphorus conditions. After plate transfer, Root1280 best improved the growth inhibition of Col-0 plants, increasing the average fresh weight of the aboveground parts and roots of each Col-0 plant by 12 mg and 5 mg, respectively. However, the increase in fresh weight of stop1 and almt1 plants was lower than that of Col-0 plants, with an average of 8 mg and 1 mg of fresh weight of the aboveground parts and roots, respectively. Figure 3 F). Under low phosphorus conditions, the fresh weights of the aboveground parts of WT, almt1, and stop1 were 2.0 mg, 5.8 mg, and 3.7 mg, respectively, and the fresh weights of the roots of WT, almt1, and stop1 were 2.9 mg, 3.9 mg, and 3.1 mg, respectively. Under high phosphorus conditions, the fresh weights of the aboveground parts of WT, almt1, and stop1 were 30.8 mg, 28.6 mg, and 29.8 mg, respectively, and the fresh weights of the roots of WT, almt1, and stop1 were 12.1 mg, 12.2 mg, and 11.7 mg, respectively. Under low phosphorus conditions, the fresh weights of the aboveground parts of WT, almt1, and stop1 co-cultured with Root1280 were 14.6 mg, 13.0 mg, and 11.7 mg, respectively, and the fresh weights of the roots of WT, almt1, and stop1 co-cultured with Root1280 were 8.5 mg, 5.0 mg, and 4.3 mg, respectively.

[0132] The above results indicate that Root1280 can promote plant growth under low phosphorus conditions, and the STOP1-ALMT1 signaling pathway is also required to promote the maximum recovery of plant growth. This suggests that in the early stage of low phosphorus stress, Root1280 responds to the plant's low phosphorus signal, is recruited to the plant roots, and establishes a stable symbiotic relationship with the plant. Root1280 exerts the best low phosphorus growth-promoting ability, and this process makes an important contribution to alleviating low phosphorus stress in plants and promoting the recovery of plant growth inhibition.

[0133] Example 4: A synthetic community composed of Root1280 and non-functional strains can alleviate low phosphorus stress in plants.

[0134] Under natural conditions, the plant's growth environment is teeming with diverse microorganisms. Besides the interaction between microorganisms and plants, the signal exchange between individual microorganisms and the influence of the microbial community on plant growth and development become particularly important. Therefore, research on the effects of single microorganisms on plants is currently limited to sterile laboratory environments and lacks broader application value. This invention utilizes artificially synthesized microbial communities to further explore the ability of microbial communities to alleviate low phosphorus stress in plants. The specific steps are as follows:

[0135] I. Construction of Synthetic Communities

[0136] Under natural conditions, the proportion of each genera in the root microbiome of plants remains relatively consistent within a certain range. Therefore, in order to better simulate real conditions, this invention selected eight strains with low phosphorus-promoting properties—Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179, and Root670—according to the proportion of each microbial genera in the root microbiome under natural conditions. These strains belong to 3 phyla, 7 families, and 8 genera. The specific numbers and taxonomic information are shown in Table 3.

[0137] Table 3. Taxonomic information of each strain constituting MSC

[0138]

[0139]

[0140] II. Functional Verification of Individual Strains and Communities

[0141] To verify the functions of these root-related bacteria, the eight strains from step one, along with the microbial community (SynCom) composed of the Root1280 strain and the eight strains, were co-cultured with wild-type Arabidopsis thaliana. The growth phenotype and fresh weight increase of the plants were observed. Wild-type Arabidopsis thaliana without inoculation was used as a control group. The specific method for artificially synthesizing the microbial community composed of the Root1280 strain and the eight strains was described in the reference "Finkel et al. A single bacterial genus maintains root growth in a complex microbiome. Nature 587:103-108(2020)", and the specific steps are as follows:

[0142] 1) Activate each strain separately, and inoculate a single colony of each strain into a 96-well plate containing 600 μL of 1 / 2 TSB liquid medium, and grow at 28°C for 5 days.

[0143] 2) Transfer 20 μL of the culture obtained in step 1) to a new 96-well plate containing 500 μL of fresh 1 / 2 TSB liquid medium, and incubate both plates at 28°C for 2 days.

[0144] 3) Combine the culture obtained after 7 days of cultivation with another culture obtained after 2 days of cultivation to obtain cultures for each strain, and measure the OD of each strain culture separately. 600 .

[0145] 4) Add 50 μL OD 600 =0.5% of a single strain of Root1464 culture was added to 50 mL of unsolidified P i A single-strain Root1464 culture system was obtained in (625 μM HAP) medium.

[0146] 50 μL OD 600 =0.5% of a single strain of Root322 culture was added to 50 mL of unsolidified P. i A single-strain Root322 culture system was obtained in (625 μM HAP) medium.

[0147] 50 μL OD 600 =0.5% of a single strain of Root495 culture was added to 50 mL of unsolidified P. i A single-strain Root495 culture system was obtained in (625 μM HAP) medium.

[0148] 50 μL OD 600 =0.5% of a single strain of Root186 culture was added to 50 mL of unsolidified P. i A single-strain Root186 culture system was obtained in (625 μM HAP) medium.

[0149] 50 μL OD 600 =0.5% of a single-strain Root11 culture was added to 50 mL of unsolidified P i A single-strain Root11 culture system was obtained in (625 μM HAP) medium.

[0150] 50 μL OD 600 =0.5% of a single strain Root444D2 culture was added to 50 mL of unsolidified P i A single-strain Root444D2 culture system was obtained in (625 μM HAP) medium.

[0151] 50 μL OD 600 =0.5% of a single strain of Root179 culture was added to 50 mL of unsolidified P. iA single-strain Root179 culture system was obtained in (625 μM HAP) medium.

[0152] 50 μL OD 600 =0.5% of a single-strain Root670 culture was added to 50 mL of unsolidified P. i A single-strain Root670 culture system was obtained in (625 μM HAP) medium.

[0153] 50 μL OD 600 =0.5% of a single strain of Root1280 culture was added to 50 mL of unsolidified P. i A single-strain Root1280 culture system was obtained in (625 μM HAP) medium.

[0154] The concentrations of cultures from each strain (Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179, Root670) were adjusted to be consistent, and equal volumes were mixed to obtain the community culture medium. The community culture medium was then washed twice with sterile ddH2O, followed by washing and dilution with ddH2O to OD200. 600 =0.5, then 50 μL OD 600 =0.5% community culture medium was added to 50 mL of unsolidified P i The community culture system (MSC, -Root1280) was obtained in (625 μM HAP) medium.

[0155] The concentrations of cultures from each strain (Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179, Root670, Root1280) were adjusted to be consistent, and equal volumes were mixed to obtain the community culture medium. The community culture medium was then washed twice with sterile ddH2O, followed by washing and dilution with ddH2O to OD200. 600 =0.5, then 50 μL OD 600 =0.5% community culture medium was added to 50 mL of unsolidified P i The community culture system (MSC) was obtained in (625 μM HAP) medium.

[0156] 5) The effects of individual strains and synthetic microbial communities on the growth of wild-type Arabidopsis thaliana under low phosphorus conditions were investigated. A control group was used, inoculated with no strains.

[0157] The results are as follows Figure 4As shown in Table 4, the results indicate that, compared to the uninoculated control group, none of the eight individual bacterial strains significantly promoted the increase of fresh weight in the aboveground and underground parts of the plants. Furthermore, strain Root179 further inhibited plant growth, manifested as a significant reduction in the fresh weight of the aboveground parts. The microbial community (MSC) composed of eight strains (-Root1280) and the microbial community (MSC) composed of Root1280 and eight other strains significantly alleviated low phosphorus stress in plants, demonstrating significantly better plant growth than the uninoculated control and significantly promoting the increase of fresh weight in both aboveground and underground parts. In particular, the microbial community (MSC), compared to the Root1280 treatment alone, significantly improved the underground parts, including root area, lateral root number, and root fresh weight, indicating that the microbial community (MSC) has better low phosphorus-promoting potential than Root1280. These results suggest a synergistic effect between bacteria, enabling individual bacteria, which themselves do not possess low phosphorus-promoting functions, to work together to help plants alleviate low phosphorus stress after being mixed into a microbial community. This not only indirectly demonstrates the function of plant microbial communities, but also strongly proves the important role of microbial interactions in plant adaptation to the environment.

[0158] Table 4. Effects of individual bacteria and bacterial communities on the increase in plant fresh weight

[0159]

[0160]

[0161] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Acinetobacter guillouiae Application of Root1280 or its bacterial suspension or bacterial agent containing it in any of the following A1)-A2): A1) Alleviating low phosphorus stress in plants; A2) Improve the ability of plants to alleviate low phosphorus stress; The plant in question is Arabidopsis thaliana.

2. Acinetobacter guillouiae Application of Root1280 or its bacterial suspension or bacterial agent containing it in any of the following B1)-B2): B1) Prepare products to alleviate low phosphorus stress in plants; B2) Prepare products that enhance the ability of plants to alleviate low phosphorus stress; The plant in question is Arabidopsis thaliana.

3. A compound microbial agent, the active ingredients of which include Acinetobacter guillouiae Root1280 Agromyces fucosus Root1464 Microbacterium maritypicum Root322 Aeromicrobium sp001426755 Root495 Flavobacterium sp001428525 Root186 Bacillus A thuringiensis S Root11 Paenibacillus E sp001426375 Root444D2, Rhodanobacter sp001427365 Root179 and Bosea robiniae Root670.

4. The compound microbial agent according to claim 3, characterized in that: The Acinetobacter guillouiae Root1280, the aforementioned Agromyces fucosus Root1464, the aforementioned Microbacterium maritypicum Root322, the aforementioned Aeromicrobium sp001426755 Root495, the aforementioned Flavobacterium sp001428525 Root186, the aforementioned Bacillus A thuringiensis S Root11, the aforementioned Paenibacillus E sp001426375 Root444D2, the aforementioned Rhodanobacter sp001427365 Root179 and the aforementioned Bosea robiniae The CFU ratio of Root670 is 1:1:1:1:1:1:1:1:1:

1.

5. A method for preparing the composite microbial agent according to claim 3 or 4, comprising the following steps: [The steps are described in the original text, but are not translated here.] Acinetobacter guillouiae Root1280 culture medium, Agromyces fucosus Root1464 culture medium, Microbacterium maritypicum Root322 culture medium, Aeromicrobium sp001426755 Root495 culture medium, Flavobacterium sp001428525 Root186 culture medium, Bacillus A thuringiensis S Root11 culture medium, Paenibacillus E sp001426375 Root444D2 culture medium, Rhodanobacter sp001427365 Root179 culture medium and Bosea robiniae The Root670 culture medium was mixed thoroughly to obtain the composite microbial agent.

6. The method according to claim 5, characterized in that: The Acinetobacter guillouiae Root1280 culture medium, the Agromyces fucosus Root1464 culture medium, the aforementioned Microbacterium maritypicum Root322 culture medium, the aforementioned Aeromicrobium sp001426755 Root495 culture medium, the aforementioned Flavobacterium sp001428525 Root186 culture medium, the Bacillus A thuringiensis S Root11 culture medium, the Paenibacillus E sp001426375 Root444D2 culture medium, the aforementioned Rhodanobacter sp001427365 Root179 culture medium and the above Bosea robiniae OD of Root670 culture medium 600 Both are 0.5; The Acinetobacter guillouiae Root1280 culture medium, the Agromyces fucosus Root1464 culture medium, the aforementioned Microbacterium maritypicum Root322 culture medium, the aforementioned Aeromicrobium sp001426755 Root495 culture medium, the aforementioned Flavobacterium sp001428525 Root186 culture medium, the Bacillus A thuringiensis S Root11 culture medium, the Paenibacillus E sp001426375 Root444D2 culture medium, the aforementioned Rhodanobacter sp001427365 Root179 culture medium and the above Bosea robiniae The volume ratio of Root670 culture medium was 1:1:1:1:1:1:1:1:1:

1.

7. The application of the composite microbial agent according to claim 3 or 4, or the composite microbial agent prepared according to the method of claim 5 or 6, in any of the following A1)-A3): A1) Alleviating low phosphorus stress in plants; A2) Improve the ability of plants to alleviate low phosphorus stress; A3) Promotes plant growth under low phosphorus stress; The plant in question is Arabidopsis thaliana.

8. The application of the composite microbial agent according to claim 3 or 4, or the composite microbial agent prepared according to the method of claim 5 or 6, in any of the following B1)-B3): B1) Prepare products to alleviate low phosphorus stress in plants; B2) Prepare products that enhance the ability of plants to alleviate low phosphorus stress; B3) Prepare products that promote plant growth under low phosphorus stress; The plant in question is Arabidopsis thaliana.

9. A method for alleviating low phosphorus stress in plants or improving the ability of plants to alleviate low phosphorus stress, comprising: Acinetobacter guillouiae The step of applying Root1280 or its bacterial suspension or bacterial agent containing it to a plant; the plant being Arabidopsis thaliana.

10. A method for alleviating low phosphorus stress in plants or improving their ability to alleviate low phosphorus stress or promoting plant growth under low phosphorus stress, comprising the step of applying the composite microbial agent of claim 3 or 4 or the composite microbial agent prepared according to the method of claim 5 or 6 to the plant; wherein the plant is Arabidopsis thaliana.

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