Low-phosphorus growth-promoting strain separated from arabidopsis thaliana root microflora and application of low-phosphorus growth-promoting strain

Abstract

The invention discloses a low-phosphorus growth-promoting strain separated from arabidopsis thaliana root microflora and application of the low-phosphorus growth-promoting strain. The invention discloses a Root1280 strain with excellent low-phosphorus growth promoting capability, and the Root1280 strain establishes a close interaction relationship with plants under a low-phosphorus condition, and can well relieve low-phosphorus stress of the plants and promote plant growth when being independently co-cultured with the plants. According to the invention, the Root1280 bacterial strain is mixed with other eight bacterial strains to obtain an artificially synthesized flora, and the result shows that compared with single Root1280 treatment, the synthesized flora has the advantages that the underground parts including root system area, lateral root quantity and root fresh weight are obviously improved, which shows that the synthesized flora has better low-phosphorus growth promoting potential than Root1280. The invention not only proves that the interaction between microorganisms has an important effect on the flora function, but also provides a reference for trying the mixed application and synergistic promotion of functional bacteria with different effects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a low-phosphorus growth-promoting strain separated from a microbial community of Arabidopsis root system and an application thereof. Background Art

[0002] As one of the major elements after nitrogen, phosphorus is crucial to all processes of plant growth and development. With the growth of population and the depletion of phosphate resources, the problem of crop yield reduction caused by the lack of soluble phosphorus in the soil is becoming increasingly serious. Therefore, there is an urgent need to find green, environmentally friendly and efficient methods to deal with low-phosphorus stress in agricultural production. In the natural environment, plants are surrounded by a large number of microorganisms; studies have shown that microorganisms play an important role in plant growth and development, nutrient absorption, environmental adaptation and resistance to stress and pests. Research on microbial phosphorus solubilization has been carried out for decades. Specifically, the process of dissolving insoluble phosphorus sources mainly includes acid production and chelation. Many phosphorus-dissolving bacteria with excellent phosphorus solubilization ability have been identified. However, due to the lack of research on the interaction between plants and microorganisms, the external application of bacterial fertilizers has little effect on alleviating low-phosphorus stress in agricultural production. "Cannot stay", "weak effect" and "high cost" are the main problems restricting the application of bacterial fertilizers. In recent years, some studies on fungi have found that beneficial interactions between arbuscular mycorrhizae and endophytic fungi Colletotrichum tofieldia and plants contribute to the alleviation of plant low-phosphorus stress. However, little is known about bacteria, which account for the vast majority of the total microorganisms.

[0003] With the development of high-throughput sequencing technology and microbial isolation and cultivation technology, metagenomic-related research has emerged. The plant root microbiome is analogous to the human intestinal microbiome and is collectively referred to as the host's "second genome". Its importance is self-evident. At present, most studies only describe the changes in the composition of plant microbial communities under different stress conditions, but little is known about the specific biological significance of the regulated components. It has been reported in the literature that plants have many dynamic regulations with root bacteria during low-phosphorus stress, but whether and how plants actively cooperate with bacteria to absorb insoluble phosphorus sources and promote growth is still unknown. Summary of the invention

[0004] One object of the present invention is to provide a new use of Acinetobacter guillouiae Root 1280 or its bacterial suspension or culture solution or a bacterial agent containing the same.

[0005] The present invention provides the use of Acinetobacter guillouiae Root 1280 or its bacterial suspension or culture solution or a bacterial agent containing the same in any one of the following A1)-A3):

[0006] A1) Alleviate plant low phosphorus stress;

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

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

[0009] The present invention also provides the use of Acinetobacter guillouiae Root 1280 or its bacterial suspension or culture solution or a bacterial agent containing the same in any one of the following B1)-B3):

[0010] B1) preparing a product for alleviating low phosphorus stress in plants;

[0011] B2) preparing products that improve the ability of plants to alleviate low-phosphorus stress;

[0012] B3) Preparation of products for promoting plant growth under low phosphorus stress.

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

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

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

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

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

[0018] Another object of the present invention is to provide a method for preparing the composite microbial agent.

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

[0020] Further, the OD values ​​of the Acinetobacter guillouiae Root1280 culture solution, the Root1464 culture solution, the Root322 culture solution, the Root495 culture solution, the Root186 culture solution, the Root11 culture solution, the Root444D2 culture solution, the Root179 culture solution and the Root670 culture solution are as follows: 600 Both are 0.5.

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

[0022] After mixing, sterile ddH 2 O and diluted to OD 600 =0.5 steps.

[0023] Furthermore, the culture solution of Acinetobacter guillouiae Root1280 is prepared by culturing Acinetobacter guillouiae Root1280 in 1 / 2TSB liquid medium until OD 600The specific preparation method is as follows: inoculate Acinetobacter guillouiae Root1280 into 600 μL 1 / 2TSB liquid culture medium, culture at 28°C for 5 days to obtain a 5-day culture; then transfer 20 μL of the 5-day culture to a new 1 / 2TSB liquid culture medium and culture at 28°C for 2 days to obtain a 2-day culture, and continue to culture the remaining culture at 28°C for 2 days to obtain a 7-day culture; then combine the 2-day culture and the 7-day culture to obtain the Acinetobacter guillouiae Root1280 culture solution.

[0024] The Root1464 culture medium is prepared by culturing Root1464 in 1 / 2TSB liquid medium until OD 600 The specific preparation method is as follows: Root1464 is inoculated into 600 μL 1 / 2TSB liquid culture 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 / 2TSB liquid culture medium and cultured at 28°C for 2 days to obtain a 2-day culture, and the remaining culture is further cultured at 28°C for 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture are combined to obtain the Root1464 culture solution.

[0025] The Root322 culture medium is prepared by culturing Root322 in 1 / 2TSB liquid medium until OD 600 The specific preparation method is as follows: Root322 is inoculated into 600 μL 1 / 2TSB liquid culture 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 / 2TSB liquid culture medium and cultured at 28°C for 2 days to obtain a 2-day culture, and the remaining culture is further cultured at 28°C for 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 is prepared by culturing Root495 in 1 / 2TSB liquid medium until OD 600The specific preparation method is as follows: Root495 is inoculated into 600 μL 1 / 2TSB liquid culture 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 / 2TSB liquid culture medium and cultured at 28°C for 2 days to obtain a 2-day culture, and the remaining culture is further cultured at 28°C for 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 is prepared by culturing Root186 in 1 / 2TSB liquid medium until OD 600 The specific preparation method is as follows: Root186 is inoculated into 600 μL 1 / 2TSB liquid culture medium, cultured at 28°C for 5 days, and a 5-day culture is obtained; then 20 μL of the 5-day culture is transferred to a new 1 / 2TSB liquid culture medium and cultured at 28°C for 2 days to obtain a 2-day culture, and the remaining culture is further cultured at 28°C for 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture are combined to obtain the Root186 culture solution.

[0028] The Root11 culture medium is prepared by culturing Root11 in 1 / 2TSB liquid medium until OD 600 The specific preparation method is as follows: Root11 is inoculated into 600 μL 1 / 2TSB liquid culture medium, cultured at 28°C for 5 days, and a 5-day culture is obtained; then 20 μL of the 5-day culture is transferred to a new 1 / 2TSB liquid culture medium and cultured at 28°C for 2 days to obtain a 2-day culture, and the remaining culture is further cultured at 28°C for 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 is prepared by culturing Root444D2 in 1 / 2TSB liquid medium until OD 600 The specific preparation method is as follows: Root444D2 is inoculated into 600 μL 1 / 2TSB liquid culture 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 / 2TSB liquid culture medium and cultured at 28°C for 2 days to obtain a 2-day culture, and the remaining culture is further cultured at 28°C for 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 is prepared by culturing Root179 in 1 / 2TSB liquid medium until OD 600 The specific preparation method is as follows: inoculate Root179 into 600 μL 1 / 2TSB liquid culture medium, culture at 28°C for 5 days to obtain a 5-day culture; then transfer 20 μL of the 5-day culture to a new 1 / 2TSB liquid culture medium and culture at 28°C for 2 days to obtain a 2-day culture, and continue to culture the remaining culture at 28°C for 2 days to obtain a 7-day culture; then combine the 2-day culture and the 7-day culture to obtain the Root179 culture solution.

[0031] The Root670 culture medium is prepared by culturing Root670 in 1 / 2TSB liquid medium until OD 600 The specific preparation method is as follows: Root670 is inoculated into 600 μL 1 / 2TSB liquid culture 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 / 2TSB liquid culture medium and cultured at 28°C for 2 days to obtain a 2-day culture, and the remaining culture is further cultured at 28°C for 2 days to obtain a 7-day culture; then the 2-day culture and the 7-day culture are combined to obtain the Root670 culture solution.

[0032] The present invention also provides new uses of the composite microbial agent or the composite microbial agent prepared according to the above method;

[0033] The present invention provides the use of the composite microbial agent or the composite microbial agent prepared according to the above method in any of the following A1)-A3):

[0034] A1) Alleviate plant low phosphorus stress;

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

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

[0037] The present invention also provides the use of the composite microbial agent or the composite microbial agent prepared according to the above method in any of the following B1)-B3):

[0038] B1) preparing a product for alleviating low phosphorus stress in plants;

[0039] B2) preparing products that improve the ability of plants to alleviate low-phosphorus stress;

[0040] B3) Preparation of products for promoting plant growth under low phosphorus stress.

[0041] Another object of the present invention is to provide a product, the functions of which are any of the following A1)-A3):

[0042] A1) Alleviate plant low phosphorus stress;

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

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

[0045] The product comprises Acinetobacter guillouiae Root 1280 or its bacterial suspension or its culture solution or a bacterial agent containing it or the above-mentioned composite microbial agent or a composite microbial agent prepared according to the above-mentioned method.

[0046] The last object of the present invention is to provide a method for alleviating low-phosphorus stress in plants or improving the ability of plants to alleviate low-phosphorus stress or promoting plant growth under low-phosphorus stress.

[0047] The method for alleviating plant low-phosphorus stress or improving the ability of plants to alleviate low-phosphorus stress or promoting plant growth under low-phosphorus stress provided by the present invention comprises the step of applying the above-mentioned Acinetobacter guillouiae Root 1280 or its bacterial suspension or its culture solution or a bacterial agent containing it or the above-mentioned composite microbial agent or a composite microbial agent prepared according to the above-mentioned method to plants.

[0048] Furthermore, the application method is to culture the plant in an environment containing the above-mentioned Acinetobacter guillouiae Root 1280 or its bacterial suspension or its culture solution or a bacterial agent containing it or the above-mentioned composite microbial agent or a composite microbial agent prepared according to the above method.

[0049] Furthermore, the application method is to culture the plant in a low-phosphorus culture medium containing the above-mentioned Acinetobacter guillouiae Root 1280 or its bacterial suspension or its culture solution or a bacterial agent containing it or the above-mentioned composite microbial agent or a composite microbial agent prepared according to the above method.

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

[0051] In any of the above-mentioned composite bacterial communities, applications, products or methods, the Root1280 induces increased expression of malic acid 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 plant low-phosphorus stress, improving plant low-phosphorus stress tolerance, and promoting plant growth.

[0052] In any of the above-mentioned composite bacterial communities, applications, products or methods, the promotion of plant growth is to increase the fresh weight of plants, which is specifically embodied in increasing the fresh weight of the above-ground parts of plants and / or the fresh weight of the underground parts (fresh weight of the root system) and / or the root area and / or the number of lateral roots.

[0053] In any of the above-mentioned composite bacterial communities, applications, products or methods, the low-phosphorus stress may specifically be 50 μM Pi or 626 μM hydroxycalcium phosphate stress.

[0054] In any of the above-mentioned composite bacterial communities, applications, products or methods, the plants include monocots and dicots, and the dicots may specifically be Arabidopsis thaliana.

[0055] The present invention is based on a high-quality bacterial pure bacterial library isolated from the root microbiome of the model plant Arabidopsis thaliana. The ability of each root component to alleviate the low-phosphorus stress of plants is studied in a reductionist way, and about 20% of the root-related bacteria are identified to have potential low-phosphorus growth-promoting ability. Next, the present invention selected a weak phosphate-soluble strain Acinetobacter guillouiae Root1280 as a representative for detailed research, and found that the Root1280 strain has excellent low-phosphorus growth-promoting ability, and has established a close interactive relationship with plants under low-phosphorus conditions. When co-cultured with plants alone, it can well alleviate the low-phosphorus stress of plants and promote plant growth. Furthermore, the present invention mixed the representative low-phosphorus growth-promoting bacteria Root1280 and 8 root-related bacteria into another synthetic bacterial community (MSC) and studied the function of this synthetic bacterial community. The results showed that in the synthetic bacterial community MSC, compared with the single Root1280 treatment, the fresh weight of the above-ground part of the plant recovered to a similar degree, but the underground part including the root area, the number of lateral roots, and the fresh weight of the roots were significantly improved, indicating that the synthetic bacterial community MSC has better low-phosphorus growth-promoting potential than Root1280. The present invention not only proves the important role of the interaction between microorganisms and microorganisms in the function of the bacterial community, but also provides a reference for trying to mix and apply functional bacteria with different effects and synergistic promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the identification of low-phosphorus growth-promoting components in the bacterial microbial community of Arabidopsis roots. Figure A is the identification flow chart. After germination for 7 days, the PT2-LUC transgenic material was transferred to a phosphorus-deficient medium mixed with the test bacteria and hydroxycalcium phosphate (HAP) as the only phosphorus source and grown for 14 days. The fresh weight and LUC activity of the plants were counted. Figure B is a phenotypic diagram of representative strains. The red ones are non-functional strains that compete with plants for phosphorus nutrition; the blue strains can only reduce LUC activity but cannot promote growth; the green strains can both reduce LUC activity and promote plant growth. Figures C and D are the relative LUC activity and fresh weight content of each plant in Figure B, respectively. Figure E is the evolutionary tree and taxonomic information of all 130 root-associated bacteria that have been screened. The yellow triangle indicates that the strain can reduce the expression of low-phosphorus response genes in plants, and the purple square indicates that the strain can promote the increase of plant fresh weight. Among them, "0μM P i , 625 μM HAP" indicates P i -(625 μM HAP).

[0057] Figure 2Functional analysis of 26 low-phosphorus growth-promoting bacteria identified. Blue shoot Pi indicates the level of phosphorus content in the aboveground part of the plant promoted by the strain, in μmol / g; purple FW indicates the content of fresh weight increase promoted by the strain, in mg; green LUC indicates the relative expression level of the low-phosphorus response gene PT2 of the strain, and the PT2 expression level of the plant under low-phosphorus sterile conditions is taken as the control; yellow pH indicates the acid production ability of the strain under single culture conditions, and the pH of the culture medium is calculated after 3 days of culture (initial culture medium pH = 7.0); red PS indicates the relative solubility of the strain for the insoluble phosphorus source HAP under single culture conditions, and the soluble phosphorus content in the culture medium under sterile conditions is taken as the control (due to the large difference in the phosphorus solubility ability of each strain, the data in the figure are displayed in log values).

[0058] Figure 3 This is a study of the molecular mechanism of the interaction between Acinetobacter guillouiae Root1280 and plants under low-phosphorus conditions. Figure A shows that Root1280 is significantly recruited on low-phosphorus roots, and this recruitment depends on the plant's STOP1-ALMT1 signaling pathway. Figure B shows the chemotaxis experiment of Root1280, in which malic acid can significantly attract Root1280. Figure C shows that the relief of plant PSI (Phosphate Starvation Induced) by Root1280 depends on the STOP1-ALMT1 signaling pathway. Figure D is a flow chart for the transfer plate system to detect the effect of Root1280 on the growth promotion of different mutants under low-phosphorus conditions. Figure E shows the growth phenotypes of the wild type Col-0 and the mutants almt and stop1 under low-phosphorus and high-phosphorus conditions, respectively, under sterile conditions. Figure F shows that the promotion of plant growth by Root1280 depends on the STOP1-ALMT1 signaling pathway.

[0059] Figure 4 It is an artificially synthesized microbial flora and the low-phosphorus growth-promoting phenotype of each single bacteria. Except for Root1280, the other 8 strains Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179, and Root670 have no low-phosphorus growth-promoting ability, and are similar to the sterile control under low-phosphorus conditions. The community composed of these 8 strains (MSC, -Root1280) can significantly promote the increase in plant fresh weight under low-phosphorus conditions. The community (MSC) composed of 9 strains including the low-phosphorus growth-promoting bacteria Root1280 can promote plant growth to the greatest extent, especially the fresh weight of the underground part increased significantly, and the number of lateral roots and the root area increased significantly. Among them, "0μM P i , 625μM HAP" means P i -(625 μM HAP). DETAILED DESCRIPTION

[0060] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0061] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

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

[0063] The PSR signaling pathway mutant almt1 (SALK_009629C) and stop1 (SALK_114108) mutant materials in the following examples are all recorded 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)". The public can obtain them from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The biological materials are only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0064] The PSR signaling pathway mutant phr1phl1 (SALK_067629, SALK_079505) in the following examples is recorded in the document “Wang et al. Functional Characterization of Arabidopsis PHL4 in Plant Response to Phosphate Starvation. Front Plant Sci 9: 1432 (2018).” and is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0065] The Root1280 strain, Root186 strain, Root322 strain, Root1464 strain, Root179 strain, Root444D2 strain, Root670 strain, Root11 strain, and Root495 strain in the following embodiments are all recorded in the document "Baiet al. Functional overlap of the Arabidopsis leaf and root microbiota. Nature 528: 364-369 (2015)", which can be obtained by the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The biological material is only used for repeating the relevant experiments of the present invention and cannot 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 formulas in the following examples are as follows:

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

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

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

[0071] NBPIP culture medium (1L) is composed of 2.215 g of Glucose, MgSO4 7H 2 O 15g, KCl 3g, MgCl 2 6H 2 O5g, (NH 4 ) 2 SO 4 The product was obtained by mixing 0.1 g of PEG-100 and 8.1 g of HAP with 1 L of water, and adjusting the pH to 7.0 with KOH. Sterilize by high pressure at 121°C 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 sterilized by autoclaving at 121°C for 15 min.

[0073] Table 1. Plant culture medium formula with different phosphorus contents

[0074]

[0075] 40×P i +Majority element mother solution (1L) is KNO 3 19g, NH 4 NO 3 16.5g, KH 2 PO 4 1.7 g was mixed with 1 L of water. Sterilize by high pressure at 121°C for 15 minutes and store in a freezer at 4°C.

[0076] 40×P i -The mother solution of macroelements (1L) is KNO 3 19g, NH 4 NO 3 16.5g, K 2 SO 4 2.178g mixed with 1L water. Autoclave at 121℃ for 15 minutes and store in a freezer at 4℃.

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

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

[0079] Plant growth conditions: Arabidopsis thaliana or its related materials were cultured vertically in 1 / 2MS medium in a light incubator: light duration 16 h, temperature 22°C, humidity 60%; dark duration 8 h, temperature 20°C, humidity 60%.

[0080] Seed sterilization and germination: 1) Take an appropriate amount of seeds and place them in a centrifuge tube. Use 30% 84 disinfectant to disinfect the surface for 10 minutes, then wash with sterile water 5-6 times, and place in a 4°C refrigerator for low-temperature treatment for 2 days. 2) Take 50mL of the prepared 1 / 2MS solid culture medium and evenly pour it into a 10×10 square culture dish. After solidification, use a gun tip to point the seeds. According to the experimental needs, place it in a light incubator and culture it vertically for 5-7 days. The culture conditions are as follows: light duration 16h, temperature 22°C, humidity 60%; dark duration 8h, temperature 20°C, humidity 60%. Take seedlings with the same growth status for the experiment.

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

[0082] Bacteria and plant co-culture system: 1) Prepare plant culture medium with different phosphorus contents according to the dosage: P i +(625μMP i ) medium, 50 μM P i medium, with HAP added as the sole phosphorus source i -(625μM HAP) medium, autoclave at 121℃ for 15 minutes; after sterilization, shake the medium thoroughly to mix the insoluble HAP thoroughly in the medium, and place it in a 42℃ water bath to cool and keep warm. 2) Take out the bacteria cultured overnight, centrifuge at 4000rpm for 5 minutes to collect the bacteria, and discard the supernatant. 3) Use ddH 2 Resuspend and wash the bacteria in 4000 rpm centrifuge for 5 minutes to collect the bacteria and discard the supernatant; repeat three times. 4) Add 1 mL of sterile water and blow the bacterial precipitate with a pipette; aspirate 80 μL of bacterial solution into a 720 μL ddHO solution. 2 O in the centrifuge tube, and the OD 600 Determine the concentration of the bacterial solution after dilution 10 times; after calculation, 2 O to OD 600 5) Take out the culture medium kept at 42℃ in the water bath, pour 50mL into a sterile BD tube, and add OD 600= 0.5 bacterial solution, mix thoroughly and slowly pour into a 10×10 square culture dish to solidify, avoid bubbles, and cool and solidify in a clean bench for 30 minutes before use. 6) Take out the Arabidopsis seedlings that have grown to the corresponding number of days from the light incubator, and use a sterilized yellow pipette tip to transfer plants with the same growth state to the corresponding culture medium mixed with bacteria for cultivation; change the pipette tip for each plate to prevent contamination. 7) Use breathable tape to seal the plate and place it in a light incubator to grow for the corresponding number of days before experiment.

[0083] Detection of PT2-LUC expression level and weighing of plant fresh weight: 1) Take out the plate and take pictures of the growth status of the seedlings with a camera. 2) Prepare a 96-well white ELISA plate and add 100 μL ddH 2 3) Cut the underground part of the plant with scissors and put it into the ELISA plate, and put the root of a seedling in each well. 4) Prepare 100×LUC stock solution: dissolve 1g D-Luciferin Potassium in 31.4mL sterile water, and store it at -20℃. 5) Prepare 2×LUC sterile aqueous solution, add the prepared 100×LUC stock solution into ddH 2 6) Use an ELISA reader to detect the chemiluminescence intensity of the sample. 7) Use an analytical balance to weigh the fresh weight of the aboveground part and roots of each plant, or the fresh weight of the whole plant, according to the experimental needs.

[0084] Detection of bacterial phosphate solubilization and acid production in vitro: 1) Activate and culture bacteria according to the above method. 2) Prepare NBRIP medium according to the formula, sterilize at 121℃ for 15 minutes, and dispense into 15mL sterile BD tubes after cooling, 4mL per tube, and at least 3 replicates for each treatment. 3) Collect the cultured bacteria, centrifuge at 4000rpm for 5 minutes, and wash with ddH 2 Resuspend and wash the bacteria in 5% ddHO and centrifuge at 4000 rpm for 5 minutes. Repeat twice. 4) Add 1 mL of ddHO 2 O, mix the bacterial pellet; aspirate 80 μL of bacterial solution, add 720 μL ddHO 2 Dilute 10 times with ddHO to measure the concentration. 2 Adjust the bacterial concentration to OD 600=0.5 for later use. 5) Aspirate half of the diluted bacterial solution, sterilize at 121℃ for 15 minutes under high pressure as a control for inactivated bacteria (Heat killed). 6) Add bacteria (or inactivated bacteria) to the packaged NBRIP culture medium at a ratio of 1:1000, place in a shaker at 28℃, 200rpm, and culture for 3 days. 7) Fermentation broth collection: Centrifuge at 5000rpm for 10 minutes, aspirate 1mL of supernatant into a 1.5mL centrifuge tube to avoid inhaling the insoluble phosphorus in the lower layer; centrifuge at 12,000rpm for 10 minutes, aspirate 900μL of supernatant into a new centrifuge tube; centrifuge at 12,000rpm for 10 minutes again, aspirate 100μL of supernatant into a transparent 96-well plate. 8) Preparation of phosphorus reaction solution: The phosphorus reaction solution is divided into 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. It is best to prepare it before use; solution B is 4.2g ammonium molybdate (NH 4 ) 6 Mo 7 O 24 ·4H 2 O is dissolved in 1L of 1N sulfuric acid (28.6mL of concentrated sulfuric acid is slowly added to 1000mL of water); Solution B can be stored stably at room temperature; the phosphorus reaction solution is a mixture of Solution A and Solution B at a ratio of 1:6 (volume ratio) and can only be used on the same day. 9) Preparation of standard curve: Prepare 1mM KH 2 PO 4 Standard solution, weigh 0.1361g KH 2 PO 4 Dissolve in 1L water; measure 0μL, 5μL, 15μL, ..., 70μL of phosphorus standard solution into different centrifuge tubes, add sterile water to 300μL, and then add 700μL of phosphorus reaction solution to each tube; react at 37℃ for 1 hour or 45℃ for 20 minutes, leave at 4℃ for 10 minutes, measure the absorbance at 820nm with a spectrophotometer, make a standard curve, and calculate the conversion relationship between the micromolar number of phosphorus and the absorbance. 10) Detection of bacterial in vitro phosphorus solubilization ability: Add 200μL of sterile water to the 96-well plate in step 7), then add 700μL of phosphorus reaction solution, react at 37℃ for 1 hour or 45℃ for 20 minutes, leave at 4℃ for 10 minutes, measure the absorbance at 820nm with a spectrophotometer, and calculate the in vitro phosphorus solubilization ability of bacteria based on the absorbance. 11) Calculation of acid production capacity: The pH value of the NBRIP medium after 3 days of co-culture with bacteria in step 6) was measured using a pH meter.

[0085] Malachite green method for determination of phosphorus content in plant tissues: 1) Preparation of malachite green solution: 1-1) Weigh boric acid (H 3 BO 3 )1.2g, ammonium molybdate [(NH 4 ) 6 Mo 7 O24 ·4H 2 O] 34.16g was dissolved in 350mL distilled water. 1-2) Stir slowly and add 476mL 5MH 2 SO 4 (5M H 2 SO 4 Preparation method: Measure about 136mL of concentrated sulfuric acid, slowly add it to 300mL of distilled water, cool and dilute to 500mL). 1-3) Add 0.229g of malachite green to the above solution to dissolve. 1-4) Measure 100mL of distilled water, add 1g of polyvinyl alcohol (98% can be hydrolyzed, molecular weight 11,000 to 31,000), continue heating to dissolve; 1-5) After the above solution is cooled, add it to the acidic malachite green solution, and the mixed solution is diluted to 1L; store at room temperature in strict light-proof conditions. 2) Preparation of phosphorus standard solution and drawing 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 0.2195g and dissolve it in 400mL of distilled water. 2-3) Add 5mL of concentrated sulfuric acid to prevent mold growth and allow the solution to be stored for a long time. 2-4) Transfer to a 1L volumetric flask and add water to the mark to obtain a 50μg / M phosphorus standard mother solution. 2-5) Use the above prepared phosphorus standard mother solution and distilled water to prepare 25mL of 0μg / mL, 0.1μg / mL, 0.2μg / mL, 0.3μg / mL, 0.4μg / mL, 0.5μg / mL phosphorus standard mother solution. i 2-6) Take 1.5mL of each concentration of phosphorus standard, add 0.5mL of malachite green solution, mix well and let stand for 30 minutes, then use a spectrophotometer to measure the absorbance at 650nm. 2-7) With the absorbance as the horizontal axis, P i The content is the vertical axis, and a standard curve is drawn in Excel. 3) Determination of inorganic phosphorus content in plant leaves: 3-1) Grind fresh seedling leaf samples in liquid nitrogen, weigh 30 mg (plus or minus no more than 5 mg) of the ground sample into a 2 mL centrifuge tube, record the weighed mass, and weigh at least 3 tubes for each sample. 3-2) Add 200 μL of 5 M sulfuric acid to the centrifuge tube, flick to 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°C, 12,000 rpm for 10 minutes, and transfer the supernatant to a new 1.5 mL centrifuge tube for testing. 3-5) After the sample is diluted (P i + The sample generally needs to be diluted about 10 times, P i - no dilution required) mixed with malachite green in a ratio of 3:1, left for 30 minutes and then measured the absorbance at 650nm. 3-6) Calculate the phosphorus content based on the standard curve and dilution multiple: phosphorus content per unit fresh weight (P i μg / g) = fresh weight * volume * dilution factor * concentration of test solution.

[0086] Detection of root bacterial colonization: 1) In the root bacterial colonization experiment, in order to exclude the influence of HAP on bacteria, the culture medium formula in Table 1 was used to i +(625μM Pi) High phosphorus medium was supplemented with 625μM HAP and P i - medium. 2) Seed germination and inoculation system are the same as above. 3) Seedlings with consistent growth status after 5 days of germination are transferred to P mixed with corresponding root-related bacteria in advance. i + or P i -The corresponding number of days (1d, 2d, 4d) of growth on the plate. 4) After the culture is completed, measure the length of the main root of the root system, cut off the underground part of the plant with sterilized scissors, and place the roots of every 3 plants in a centrifuge tube with 100μL sterile water and 2 sterilized small steel balls as a sample. At least 6 samples should be repeated for each group. 5) Use Qiagen tissue disruptor to disrupt the roots, 30Hz / s, and shake for 30 seconds. 6) Add 900μL of sterile water to the centrifuge tube as 1× bacterial solution, dilute according to the bacterial growth amount and speed, take 20μL of the diluted solution to spread on the plate, and culture it at 28℃ overnight. 7) Count the growing colonies on the second day, and calculate the number of root colonizing bacteria according to the dilution multiple.

[0087] Capillary tube method for chemotaxis detection: Take overnight cultured Root1280 bacteria, wash with sterile water and adjust to OD 600 The concentration of chemotactic solution was 0.5, 180 μL of diluted bacterial solution was added to a 96-well plate, malic acid and serine solutions of corresponding concentrations were prepared, a medical capillary with a diameter of 10 mm was taken, and 20 μL of the corresponding chemotactic solution was added; sterile water was used as the control group, and each group was repeated at least 6 times. The capillary was immersed in the 96-well plate solution, and it was noted that the air pressure in the capillary was consistent with the water pressure to prevent the liquid from flowing into or out of the capillary due to the pressure difference; after the reaction to the corresponding time, 20 μL of solution in the capillary was taken out, and the plate was coated with the solution for counting after gradient dilution.

[0088] RNA extraction: 1) Take 50 mg of plant samples and put them into a mortar with liquid nitrogen to grind them thoroughly. After the liquid nitrogen evaporates, put the powder into a centrifuge tube; add 300 μL of lysis solution and mix well; extract total RNA from bacteria in the root system in the same way as plant samples, add the corresponding 100 μL lysozyme solution to react, and add 200 μL of RNA lysis solution; add 300 μL of RNA diluent to both bacterial lysis solution and plant samples and mix well. 2) Centrifuge at maximum speed for 5 minutes and aspirate the supernatant; the RNA of bacterial samples can be directly transferred to the next step without centrifugation. 3) Add 0.5 times the volume of supernatant with anhydrous ethanol, blow 3 to 4 times with a pipette, and mix well. 4) Take out the centrifuge column and install it on 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 RNA washing solution, 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 let it stand at room temperature for 15 minutes. 8) Add 600 μL RNA wash solution, centrifuge at 12,000 rpm for 45 seconds, discard the filtrate; repeat once. 9) Empty and spin dry for 2 minutes. 10) Transfer the centrifuge column to the elution tube, add 100 μL nuclease-free water to the center of the centrifuge column membrane, let it stand 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 was reversed to cDNA using the Thermo reverse transcription kit, the system is as follows: 4μL 5× reaction mix, 2μL Maxima enzyme, 1μg RNA template, Water nuclease free to 20μL. 2) Reverse transcription procedure: 20℃ for 10 minutes, 50℃ for 15 minutes, 85℃ for 5 minutes, and stored at 4℃.

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

[0091] Sterile root shading system under laboratory conditions: 1) Fold the aluminum foil into a rectangle of corresponding size (covering a square plate of about 10×10), pay attention to repeatedly folding the three sides of the rectangle to increase the thickness, and sterilize with high-temperature steam; 2) Use sterilized tweezers to take out the sterilized aluminum foil, place the unfolded side down and bond it to the culture medium to leave space for the growth of the plant roots, and bond the other three sides to the culture medium to cover the underground part of the plant and isolate the blue light from all angles to the roots. 3) After sealing the plate, cover the back of the plate tightly with a black multi-layer plastic sheet to ensure that the roots are isolated from blue light; place it in an incubator for growth.

[0092] Transfer plate system for studying the function of root bacteria: Since root-related bacteria are mixed in 1 / 2MS medium, it is difficult to distinguish the bacteria in the medium that increase P i The availability of root bacteria and plant interactions contribute to the alleviation of plant PSR. Therefore, the following transfer plate system was designed to study the function of root bacteria: 1) Plants with the same growth status were transferred into P plates mixed with different root-associated bacteria. i -(625μM HAP) medium and grown for 4 days; used to recruit the corresponding strains to the root system. 2) The plants that recruited root bacteria were transferred to P i -(625 μM HAP) sterile plates and grown again for the corresponding number of days. 3) Counting the degree of PSR relief of the plants.

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

[0094] 1. Experimental Methods

[0095] The relationship between the root microbiome of plants and the plants is close. It has been reported that the root microbiome of plants has a strong regulatory effect, and the root microbiome is also equivalent to the "second genome" of plants, playing an extremely important role in growth and development, nutrient absorption, adaptation to the environment, resistance to stress and pests, etc. 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 finding excellent candidate materials for biological agents. Because most of the previous studies on phosphate-solubilizing bacteria only studied the in vitro phosphate-solubilizing ability of strains in an environment separated from plants, many strains with extremely excellent phosphate-solubilizing abilities have been identified, but in the process of actual application, they often have little effect. Therefore, the present invention combines the plant and bacterial co-cultivation system, evaluates the low-phosphorus growth-promoting ability of root-related bacteria through the following two low-phosphorus stress relief indicators of plants, and identifies the components with low-phosphorus growth-promoting ability in the root bacterial microbial community of Arabidopsis: a. Reduction in the expression of the low-phosphorus response gene PT2-LUC; b. The degree of recovery of plant growth inhibition caused by low phosphorus. The identification flow chart is as follows Figure 1 As shown in A, the specific steps are as follows:

[0096] 1. Co-cultivation of strains and plants

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

[0098] 2. Phenotypic statistics

[0099] The plant phenotypes were counted and the growth status of the plants was recorded proportionally with a camera. The substrate Luciferin was added to the plant roots for reaction, and the expression of the PT2 gene was quickly detected by LUC fluorescence activity. The fresh weight of the plants was recorded using an analytical balance.

[0100] 2. Experimental Results and Analysis

[0101] Phenotypic observations Figure 1 As shown in Figure B, this figure shows the representative phenotypes of 130 strains. Among them, the vast majority of root bacteria, like the red strains such as Root79 and Root83, cannot alleviate PSI gene expression, nor can they promote plant growth under low phosphorus conditions; some strains, like the blue strains such as Root485 and Root274, can only reduce plant PSI gene expression but cannot significantly promote plant growth; a small number of green strains such as Root1280 and Root133 have excellent effects, which can not only reduce plant PSI gene expression but also promote plant growth (PSI gene expression and fresh weight data are shown in Figure 2). Figure 1 CD), this type of bacteria is the low-phosphate growth-promoting bacteria identified in the present invention.

[0102] According to the following criteria: reducing PSI gene expression by more than 60%, and increasing fresh weight by more than 50%, each functional strain has at least two similar low-phosphorus growth-promoting phenotypes. The present invention finally identified 26 strains with potential low-phosphorus growth-promoting ability from 130 bacteria. Their taxonomic information and evolutionary tree are shown in Table 2 and 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] 1. Experimental Methods

[0108] The following five indicators of alleviating plant low-phosphorus stress were used: a. Decreased expression of low-phosphorus response genes ( Figure 2 green); b. Fresh weight increase ( Figure 2 purple); c. Increase in phosphorus content in the aboveground part of the plant ( Figure 2 blue); d. solubility of sparingly soluble phosphorus sources ( Figure 2 red) and e, acid production capacity ( Figure 2 Yellow) Analysis of the functions of the 26 low-phosphate growth-promoting bacteria identified in Example 1.

[0109] Among them, the method for detecting the phosphorus content in the aboveground part includes the following steps: taking the aboveground part of the plant, putting it into liquid nitrogen and grinding it, dividing it into three equal parts and weighing them, determining the inorganic phosphorus content in the aboveground part by the malachite green method, and calculating it according to the corresponding standard curve.

[0110] The determination of the in vitro phosphorus solubility and acid production capacity of bacteria was carried out using liquid NBRIP medium with an initial pH of 7.0, HAP was added as a poorly soluble inorganic phosphorus source, and the OD 600 = 0.5, and incubate in a shaking table for 3 days. The acid production capacity of the bacteria was determined by measuring the change in the pH value of the culture medium after 3 days of culture. The lower the pH value of the corresponding culture medium, the stronger the acid production capacity of the bacteria.

[0111] 2. Experimental Results and Analysis

[0112] The statistical results of various functional indicators of 26 low-phosphorus growth-promoting bacteria are as follows Figure 2 As shown in the figure, the darker the color of each indicator, the better the ability of the strain. These five indicators can comprehensively reflect the various abilities of low-phosphorus growth-promoting bacteria, judge the effectiveness of low-phosphorus growth-promoting bacteria, and select corresponding candidate strains for application according to different conditions. Figure 2 Red) were sorted on the horizontal axis. It can be observed that although the in vitro phosphorus solubilization ability of the 26 strains is quite different, the strains with stronger in vitro phosphorus solubilization ability are not necessarily better at alleviating low-phosphorus stress in plants. Many strains with weaker phosphorus solubilization ability, such as Root627 and Root1280, also have strong low-phosphorus growth-promoting ability. Therefore, this also shows that the alleviation of low-phosphorus stress in plants is not solely determined by the phosphorus solubilization ability of bacteria, and it also suggests that under low-phosphorus conditions, there is an active interaction between plants and root-related bacteria and contributes to the alleviation of low-phosphorus stress in plants to a certain extent.

[0113] Example 3: Study on the molecular mechanism of interaction between the representative strain Root1280 and plants under low phosphorus conditions

[0114] 1. Differences in colonization of strains on roots under different phosphorus levels

[0115] When plants face different environments, they regulate the changes of metabolites through different signal pathways. These metabolites can act as signal molecules to participate in the interaction process with microorganisms in the root system. In order to study whether the colonization of Acinetobacter guillouiae Root1280 strain in the root is induced by the low phosphorus signal of the plant, the following experiments were conducted:

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

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

[0118] 2. In order to further study whether the low-phosphorus enrichment of Root1280 strain is regulated by the plant PSR signaling pathway, the present invention respectively i + (625 μM Pi)) and low phosphate (P i -(625μM HAP)) state, the colonization differences of Root1280 strain on different plant PSR signaling pathway mutants phr1phl1, stop1, and almt1 were detected. Among them, PHOSPHATESTARVATION 1 (PHR1) is the core transcription factor of 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 homologous protein of PHR1. PHR1 and PHL1 are involved in the regulation of more than 75% of phosphorus starvation response genes; the response of phr1phl1 double mutant plants to low-phosphorus stress is greatly weakened, and growth is extremely inhibited. Low phosphorus also induces the accumulation of SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) transcription factor in the cell nucleus. STOP1 can activate some transporters or ion channel proteins, such as ALUMINUM-ACTIVATED MALATETRANSPORTERs (ALMTs), in which ALMT1 plays a major role.

[0119] The results showed that the recruitment of Root1280 induced by low phosphorus was independent of the regulation of the PHR1-PHL1 signaling pathway. phr1phl1 showed a low phosphorus-enriched phenotype consistent with wild-type Arabidopsis, but Root1280 did not significantly colonize in low phosphorus roots in stop1 and almt1 mutants ( Figure 3 phr1phl1, stop1, almt1 in A, showed comparable bacterial colonization on high-P roots, and plants recruited Root1280 under low-P conditions 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 study whether malate serves as a signal molecule to attract Root1280, the present invention also conducted a chemotaxis experiment.

[0121] The results showed that compared with the positive control chemoattractant serine, malic acid further attracted Root1280 at 1h and 1.5h of treatment. This shows that malic acid has a strong attractant effect on Root1280. In summary, under low phosphorus conditions, plants induce the increase of malic acid expression through the STOP1-ALMT1 signaling pathway. Malic acid, as a chemoattractant molecule, attracts Root1280 and significantly enriches in the roots of plants ( Figure 3 B).

[0122] 2. PSR gene expression in plants

[0123] In order to study whether the low-phosphorus enrichment of Root1280 contributes to the alleviation of low-phosphorus stress in plants, the present invention observed that low-phosphorus induced significant enrichment of Root1280 in Arabidopsis roots under the condition of 4-day treatment, and detected the expression of plant low-phosphorus response gene IPS1 after 4-day 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, co-culture with Root1280 for 4 days under low-phosphorus conditions could significantly reduce the expression of the IPS1 gene in the plant, while in the mutants stop1 and almt1, co-culture with Root1280 for 4 days not only failed to alleviate the PSR of the plant, but the expression of the IPS1 gene was further increased ( Figure 3 C). This suggests that the early enrichment of Root1280 in plants under low-phosphorus conditions contributes to reducing the expression of IPSI genes and alleviating the low-phosphorus stress of plants; however, the mutant cannot enrich Root1280 early and fails to establish a stable symbiotic relationship with Root1280, and cannot alleviate the low-phosphorus stress of plants at an early stage (4 days).

[0129] 3. Increase in plant fresh weight

[0130] To explore whether the growth-promoting function of Root1280 under low-P conditions depends on the STOP1-ALMT1 pathway in plants, considering that a long period (10-14 days) is required to observe the growth-promoting effect of bacteria on plants, the bacteria in the culture medium react with HAP to increase the P content in the culture medium. i The availability rate will also contribute to the relief of plant low-P stress and growth recovery, which may mask the difference in plant growth promotion by early recruitment of Root1280 to establish a symbiotic relationship. i availability, first transfer the seedlings to P i -(625μM HAP) low-phosphate medium (sterile) and Root1280 were co-cultured for 4 days and then transferred to a new P i -(625 μM HAP) low-phosphate medium (sterile) was further grown for 10 days, and the plate transfer process was as follows Figure 3 As shown in D.

[0131] The results showed that the plants of each material showed strong growth inhibition when grown on sterile low-phosphorus medium, but compared with Col-0, the growth inhibition of almt1 and stop1 was weaker ( Figure 3 E), and then look at the situation of co-cultivation with Root1280 under low phosphorus. After transfer, Root1280 can best improve the growth inhibition of Col-0 plants, and promote the average increase of the aboveground and root fresh weight of each Col-0 plant by 12mg and 5mg, respectively. However, the increase of the fresh weight of stop1 and almt1 plants is lower than that of Col-0 plants, and the average fresh weight of the aboveground and root is 8mg and 1mg, respectively ( Figure 3F). Among them, under low phosphorus conditions, the aboveground fresh weights of WT, almt1 and stop1 were 2.0 mg, 5.8 mg and 3.7 mg, respectively, and the root fresh weights of WT, almt1 and stop1 were 2.9 mg, 3.9 mg and 3.1 mg, respectively; under high phosphorus conditions, the aboveground fresh weights of WT, almt1 and stop1 were 30.8 mg, 28.6 mg and 29.8 mg, respectively, and the root fresh weights of WT, almt1 and stop1 were 12.1 mg, 12.2 mg and 11.7 mg, respectively; under low phosphorus conditions, the aboveground fresh weights of WT, almt1 and stop1 co-cultured with Root1280 were 14.6 mg, 13.0 mg and 11.7 mg, respectively, and the root fresh weights 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 show that: under low-phosphorus conditions, Root1280 can promote plant growth, and promoting the maximum recovery of plant growth also requires the participation of the STOP1-ALMT1 signaling pathway; this shows that in the early stage of low-phosphorus stress, Root1280 responds to the plant low-phosphorus signal and is recruited to the plant roots and establishes a stable symbiotic relationship with the plant. Root1280 exerts its best low-phosphorus growth-promoting ability. This process makes an important contribution to alleviating the low-phosphorus stress of plants and promoting the recovery of plant growth inhibition.

[0133] Example 4: Synthetic communities composed of Root1280 and non-functional strains can alleviate plant low-phosphorus stress

[0134] Under natural conditions, the growth environment of plants is full of various microorganisms. In addition to the interaction between microorganisms and plants, the signal exchange between microorganisms and the influence of the microbial community level on plant growth and development become particularly important. Therefore, the study of the effect of a single microorganism on plants is still limited to the sterile environment of the laboratory, lacking more application value. The present invention further explores the ability of the microbial community to alleviate the low-phosphorus stress of plants by using the method of artificially synthesizing microbial communities. The specific steps are as follows:

[0135] 1. Construction of synthetic communities

[0136] Under natural conditions, the proportion of each genus in the community composition of the plant root microbiome remains consistent within a rough range. Therefore, in order to better simulate real conditions, the present invention selected 8 strains with low-phosphorus growth-promoting properties, namely Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179, and Root670, according to the proportion of each microbial genus in the root microbiome under natural conditions. They 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 strains constituting MSC

[0138]

[0139]

[0140] 2. Functional verification of individual bacteria and communities

[0141] In order to verify the functions of these root-associated bacteria themselves, the 8 strains in step 1 and the microbial community (SynCom) composed of the Root1280 strain and the 8 strains were co-cultured with wild-type Arabidopsis to observe the growth phenotype and fresh weight increase of the plants. At the same time, wild-type Arabidopsis without inoculation of strains was used as a control group. Among them, the method for artificial synthesis of the microbial community composed of the Root1280 strain and the 8 strains specifically refers to the method in the document "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, inoculate a single colony of each strain into a 96-well plate containing 600 μL 1 / 2 TSB liquid medium, and grow at 28° C. for 5 days.

[0143] 2) 20 μL of the culture obtained in step 1) was transferred to a new 96-well plate containing 500 μL of fresh 1 / 2 TSB liquid medium, and the two sets of plates were grown 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 the culture of each strain, and measure the OD of each strain culture separately. 600 .

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

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

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

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

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

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

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

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

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

[0154] The concentrations of the cultures of the various strains (Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179, Root670) were adjusted to be consistent, and equal amounts were mixed to obtain the colony culture solution, which was then washed with sterile ddH 2 After washing the colony culture medium twice, ddHO 2 Wash with O and dilute to OD 600 =0.5, then 50 μL OD 600 = 0.5 colony culture solution was added to 50 mL of unsolidified P i -(625μM HAP) culture medium to obtain a colony culture system (MSC, -Root1280).

[0155] The concentrations of the cultures of the various strains (Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179, Root670, Root1280) were adjusted to be consistent, and equal amounts were mixed to obtain the colony culture solution, which was then washed with sterile ddH 2 After washing the colony culture medium twice, ddHO 2 Wash with O and dilute to OD 600 =0.5, then 50 μL OD 600 = 0.5 colony culture solution was added to 50 mL of unsolidified P i -(625 μM HAP) culture medium to obtain a colony culture system (MSC).

[0156] 5) Detect the effects of each single bacteria and synthetic bacterial community on the growth of wild-type Arabidopsis thaliana under low phosphorus conditions. At the same time, no strain was inoculated as a control.

[0157] The results are as follows Figure 4As shown in Table 4, the results show that: compared with the control group without inoculation, the 8 single strains cannot significantly promote the increase of fresh weight of the aboveground and underground parts of the plant, and the Root179 strain also inhibits the growth of the plant, which is manifested as a significant decrease in the fresh weight of the aboveground part. The microbial community composed of 8 strains (MSC, -Root1280) and the microbial community (MSC) composed of Root1280 strain and other 8 strains can significantly alleviate the low-phosphorus stress of the plant, which is manifested as the plant growth state is significantly better than the control without inoculation, and can significantly promote the increase of fresh weight of the aboveground and underground parts of the plant. In particular, the microbial community (MSC), compared with the single Root1280 treatment, has a significant improvement in the underground part including the root area, the number of lateral roots, and the fresh weight of the roots, indicating that the microbial community (MSC) has better low-phosphorus growth-promoting potential than Root1280. These results all show that it is the synergy between bacteria that prompts the single bacteria that do not have the low-phosphorus growth-promoting function to help plants alleviate low-phosphorus stress after mixing into a bacterial community. This not only indirectly demonstrates the function of plant microbial flora, but also strongly proves the important role of interactions between microorganisms in plant adaptation to the environment.

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

[0159]

[0160]

[0161] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of Acinetobacter guillouiae Root 1280 or its bacterial suspension or culture solution or a bacterial agent containing it in any of the following A1)-A3): A1) Alleviate plant low phosphorus stress; A2) Improve the ability of plants to alleviate low phosphorus stress; A3) Promote plant growth under low phosphorus stress.

2. Use of Acinetobacter guillouiae Root 1280 or its bacterial suspension or culture solution or a bacterial agent containing it in any of the following B1)-B3): B1) preparing a product for alleviating low phosphorus stress in plants; B2) preparing products that improve the ability of plants to alleviate low phosphorus stress; B3) Preparation of products for promoting plant growth under low phosphorus stress.

3. A composite microbial agent, whose active ingredients include Acinetobacter guillouiae Root1280, Root1464, Root322, Root495, Root186, Root11, Root444D2, Root179 and Root670.

4. The composite microbial agent according to claim 3, characterized in that: The CFU ratio of the Acinetobacter guillouiae Root1280, the Root1464, the Root322, the Root495, the Root186, the Root11, the Root444D2, the Root179, and the Root670 is 1:1:1:1:1:1:1:1:1:1:

1.

5. The method for preparing the composite microbial agent according to claim 3 or 4, comprising the following steps: mixing Acinetobacter guillouiae Root1280 culture solution, Root1464 culture solution, Root322 culture solution, Root495 culture solution, Root186 culture solution, Root11 culture solution, Root444D2 culture solution, Root179 culture solution and Root670 culture solution to obtain the composite microbial agent.

6. The method according to claim 5, characterized in that: OD of the Acinetobacter guillouiae Root1280 culture solution, the Root1464 culture solution, the Root322 culture solution, the Root495 culture solution, the Root186 culture solution, the Root11 culture solution, the Root444D2 culture solution, the Root179 culture solution and the Root670 culture solution 600 Both are 0.5; The volume ratio of the Acinetobacter guillouiae Root1280 culture solution, the Root1464 culture solution, the Root322 culture solution, the Root495 culture solution, the Root186 culture solution, the Root11 culture solution, the Root444D2 culture solution, the Root179 culture solution and the Root670 culture solution is 1:1:1:1:1:1:1:1:1:1:

1.

7. Use of the composite microbial agent according to claim 3 or 4 or the composite microbial agent prepared by the method according to claim 5 or 6 in any of the following A1)-A3): A1) Alleviate plant low phosphorus stress; A2) Improve the ability of plants to alleviate low phosphorus stress; A3) Promote plant growth under low phosphorus stress.

8. Use of the composite microbial agent according to claim 3 or 4 or the composite microbial agent prepared by the method according to claim 5 or 6 in any of the following B1) to B3): B1) preparing a product for alleviating low phosphorus stress in plants; B2) preparing products that improve the ability of plants to alleviate low phosphorus stress; B3) Preparation of products for promoting plant growth under low phosphorus stress.

9. A product comprising Acinetobacter guillouiae Root 1280 or its bacterial suspension or culture solution or a bacterial agent containing it or the composite microbial agent according to claim 3 or 4 or a composite microbial agent prepared according to the method of claim 5 or 6; The function of the product is any one of the following A1)-A3): A1) Alleviate plant low phosphorus stress; A2) Improve the ability of plants to alleviate low phosphorus stress; A3) Promote plant growth under low phosphorus stress.

10. A method for alleviating low-phosphorus stress in plants or improving the ability of plants to alleviate low-phosphorus stress or promoting plant growth under low-phosphorus stress, comprising the step of applying Acinetobacter guillouiae Root 1280 or its bacterial suspension or culture solution or a bacterial agent containing it or the composite microbial agent according to claim 3 or 4 or a composite microbial agent prepared according to the method of claim 5 or 6 to plants.

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