Mycorrhizal helper bacteria collaborate with AM fungi to promote phosphorus mobilization and crop growth

Abstract

Mycorrhizal helper bacteria R1-73, which synergizes with amphibian fungi to promote phosphorus activation and crop growth, are characterized by being a strain of Plantobacillus (Plantibacter), deposited with the General Microbiology Center of the China National Center for Microbiological Culture Collection under the deposit number CGMCC No. 28069. This strain is harmless to humans and animals and has been identified as exhibiting multiple functions, including promoting AM fungal growth, phosphate solubilization, IAA production, stress resistance, and plant growth promotion. It can be used to prepare fertilizers, agricultural microbial agents, and other products.

Description

Technical Field

[0001] The present application belongs to the field of agricultural microbial technology. Specifically, the present application provides mycorrhizal helper bacteria that cooperate with AM fungi to promote phosphorus activation and crop growth and their applications. Background Art

[0002] Phosphorus is a vital nutrient for crop growth and development, known as the "food" of crops. In modern agricultural production, the application of phosphate fertilizer has become a key means of increasing crop yields. However, the utilization efficiency of applied phosphate fertilizers is generally low, with large amounts of phosphate fertilizer accumulating in the soil in the form of available phosphorus, insoluble inorganic phosphorus, and organic phosphorus, resulting in waste of phosphorus resources and environmental pressure. Therefore, improving the efficiency of crop utilization of accumulated soil phosphorus is of great significance for improving the utilization rate of phosphate fertilizers and achieving sustainable agricultural development.

[0003] Arbuscular mycorrhizal (AM) fungi are a class of symbiotic microorganisms widely found in terrestrial ecosystems, forming symbiotic associations with the roots of most plants. Through this symbiotic relationship, plants not only obtain phosphorus directly through their own roots, but also absorb phosphorus from the soil through the AM fungal hyphae network, effectively increasing plant phosphorus absorption. However, although AM fungi have great potential in improving plant phosphorus absorption capacity, the effectiveness of AM fungal inoculants currently used in agriculture is unstable and, in some cases, even has a negative impact on plant growth.

[0004] Mycorrhizal helper bacteria are a type of microbial community that can promote the growth and function of AM fungi. They have been shown to help AM fungi in many ways, such as promoting their infection of host plants, stimulating spore germination, and enhancing the expansion of hyphal networks. These bacteria are widely colonized in the mycelial environment of AM fungi and are regarded as the "second genome" of AM fungi. They not only coexist with AM fungi, but also play a key role in them. These bacterial communities have the potential to serve as biofertilizers or bioenhancers. Through synergistic effects with AM fungi, they can significantly improve the efficiency of crop utilization of accumulated phosphorus in the soil and promote the healthy growth of crops. Therefore, studying potential mycorrhizal helper bacteria in mycelial microorganisms will provide new solutions for optimizing phosphorus utilization efficiency in agricultural production and has important application prospects. Summary of the Invention

[0005] To solve the above problems, on the one hand, the present application provides a mycorrhizal helper bacterium R1-73 that synergizes with AM fungi to promote phosphorus activation and crop growth. The mycorrhizal helper bacterium R1-73 is Plantibacter (Plantibacter), which is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with the deposit number CGMCC No. 28069.

[0006] Blood agar plate culture test revealed negative hemolytic activity, indicating it is harmless to humans and animals. It has been identified as possessing several unique functional properties, including: (1) Phosphate solubilization: capable of dissolving organic phosphorus, with an acid phosphatase activity of 1.49 and an alkaline phosphatase activity of 3.92. (2) IAA production: IAA synthesis is 6.73 mg / kg. (3) Stress resistance: tolerant to salt levels up to 11% and alkaline resistance to pH 10. (4) Plant growth promotion: increased plant height and aboveground biomass (aboveground dry weight), and promoted the activation of organic phosphorus in the soil.

[0007] On the other hand, the present application provides a bacterial agent, which contains the above-mentioned mycorrhizal helping bacteria R1-73.

[0008] On the other hand, the present application provides the use of the above-mentioned mycorrhizal helping bacteria R1-73 or bacterial agent in the preparation of products that promote AM fungi to infect plant roots and stimulate the growth of extraradical hyphae.

[0009] On the other hand, the present application provides the use of the above-mentioned mycorrhiza-helping bacteria R1-73 or bacterial agent in the preparation of products that promote plant growth.

[0010] On the other hand, the present application provides the use of the above-mentioned mycorrhizal-helping bacteria R1-73 or bacterial agent in the preparation of products that improve plant stress resistance.

[0011] Furthermore, the stress resistance is salt and alkali resistance.

[0012] Furthermore, the plant is corn, cotton, sorghum, potato or alfalfa.

[0013] Furthermore, the plant is corn.

[0014] On the other hand, the present application provides the use of the above-mentioned mycorrhizal helping bacteria R1-73 or bacterial agent in the preparation of products that promote soil phosphorus activation and conversion.

[0015] On the other hand, the present application provides the use of the above-mentioned mycorrhizal helping bacteria R1-73 or bacterial agent in the production of IAA.

[0016] The products described in this application may be in the form of fertilizers, pesticides, agricultural microbial agents, etc. The products may also contain AM fungal components and other fertilizers, pesticides, and auxiliary ingredients.

[0017] The advantages of the technical solution of this application are:

[0018] (1) Improve the efficiency of phosphate fertilizer utilization: Plantibacter R1-73 strain has a significant phosphate solubility ability and can dissolve organic phosphorus and insoluble inorganic phosphorus accumulated in the soil. Through its acid phosphatase and alkaline phosphatase activities (1.49 and 3.92, respectively), this strain significantly improves the activation and conversion efficiency of phosphorus in the soil, helping crops absorb and utilize phosphorus resources more efficiently. This effect not only reduces the use of phosphate fertilizers and agricultural production costs, but also reduces the waste of phosphorus resources and the resulting environmental pollution problems.

[0019] (2) Promote crop growth and increase yield: Plant Bacillus (Plantibacter) R1-73 strain effectively stimulates plant root development by synthesizing the plant growth hormone IAA (indoleacetic acid), promoting root extension and enhancing absorption capacity. The IAA synthesis amount is 6.73 mg / kg, indicating that the strain has a strong growth-promoting function. Experiments have shown that crops using this microbial agent show significant growth in plant height and increase in aboveground biomass, especially in crops such as corn. This invention significantly improves the yield and quality of crops and has good application prospects.

[0020] (3) Enhanced crop stress resistance: The Plantibacterium R1-73 strain exhibits good salt and alkali tolerance and can survive and function in an alkaline environment with a salt concentration of 11% and a pH of 10. This allows the strain to exert its phosphate-solubilizing and growth-promoting effects even in salinized or infertile soils, improving crop stress resistance and broadening its application across different soil types and climatic conditions.

[0021] (4) Synergistic effect, enhancing the function of AM fungi: Plantibacter R1-73 can synergize with AM fungi to enhance the infection ability of AM fungi. By stimulating the growth of AM fungi's extraradical hyphae, Plantibacter further enhances the plant's ability to absorb phosphorus through the mycorrhizal pathway. This synergistic effect significantly improves the crop's absorption rate of phosphorus in the soil, solving the problem of unstable effects of traditional single microbial agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The growth of R1-73 on blood agar plates.

[0023] Figure 2 Schematic diagram of the experimental device structure. DETAILED DESCRIPTION

[0024] Example 1 Isolation, screening and identification of strain R1-73

[0025] (1) Strain isolation and screening

[0026] In 2021, a potted experiment was conducted in the greenhouse of the College of Resources and Environment at China Agricultural University to collect extraradical mycelia of AM fungi. During the experiment, 5 mg of fresh mycelial samples were precisely weighed and temporarily stored at 4°C. Subsequently, using a high-throughput root microbial culture and identification system, hyphae bacteria were isolated and cultured, successfully screening and obtaining the hyphae bacterial strain R1-73.

[0027] (2) Morphological characteristics identification

[0028] The strain R1-73 in Example 1 was inoculated into LB solid medium and cultured at 37° C. for 3 days, and the colony morphology was observed. The strain R1-73 on LB solid medium had a mucoid colony with neat edges and no capsule.

[0029] (3) 16S rRNA sequence determination and identification

[0030] Bacteria were inoculated into 1 / 2 TSB medium and shaken overnight. 16S rRNA sequences were amplified by PCR using the forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 1) and the reverse primer 1492R (5'-TACGACTTAACCCCAATCGC-3', SEQ ID NO. 2). Amplification conditions were: 95°C for 3 min, followed by 30 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s, followed by a final extension at 72°C for 5 min. Qualified PCR products were sent to Tsingke Biotechnology Co., Ltd. (Beijing, China).

[0031]

[0032] R1-73 was deposited in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, with the deposit number CGMCC No. 28069 and the deposit date July 31, 2023.

[0033] Example 2 Phosphate solubilization, IAA production, salt and alkali tolerance, and safety testing of strain R1-73 (1) Materials and methods

[0034] 1) Preparation of culture medium and reagents

[0035] 1 / 2TSB medium: TSB 15.0 g, agar 10 g (added for solid culture medium), deionized water 1000 mL, sterilized at 121°C for 20 min.

[0036] NBRIP organophosphate medium: glucose 10 g, MgCl2·6H2O 5 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, agar 10 g (added for solid culture medium), calcium phytate 0.5 g, pH 7.0.

[0037] NBRIP inorganic phosphate medium: glucose 10g, MgCl 2· 6H2O 5g, MgSO4·7H2O 0.25g, KCl 0.2g, (NH4)2SO4 0.1g, agar 10g (added for solid culture medium), tricalcium phosphate 0.5g, pH 7.0.

[0038] KB (King B) medium: MgSO4·7H2O 1.5 g, K2HPO4 15 g, glycerol 10 mL, peptone 20 g, L-tryptophan 0.1 g, pH adjusted to 7.2.

[0039] Blood agar medium: 18 g peptone, 1 g yeast powder, 5 g NaCl, 15 g agar, 1000 mL deionized water, pH 6.8-7.2. After sterilization at 121°C for 20 min, add 5% defibrinated sheep blood when the medium cools to 50°C, mix well, and pour onto plates.

[0040] Alkali-resistant culture medium: prepare 7 bottles of LB culture medium, adjust the pH value of the culture medium to 8, 9, 10, and 11 respectively with dilute hydrochloric acid and sodium hydroxide solution, and sterilize at 121℃ for 20 minutes before use for bacterial culture.

[0041] Salt-tolerant culture medium: Prepare 7 bottles of LB culture medium, weigh and add NaCl at salt concentrations of 1%, 3%, 5%, 7%, 9%, 11%, and 13% (g / 100 mL), and sterilize at 121°C for 20 min.

[0042] Salkowski colorimetric solution: Dissolve 4.5g FeCl3 in 300ml distilled water, slowly add 587.4ml of 98% concentrated sulfuric acid, and after cooling, dilute to 1L. The IAA range of the measurement is 5-200mg / L.

[0043] Sterile saline: 0.8 g NaCl was dissolved in 100 mL deionized water and sterilized at 121°C for 20 min.

[0044] 2) Experimental methods

[0045] Qualitative determination of phosphate-solubilizing (organic and inorganic phosphorus) strains:

[0046] The purified strains were inoculated into solid NBRIP organophosphate / inorganic phosphorus medium, with three replicates for each strain. The culture was carried out at 28°C for 7 days. The diameter of the bacterial cell and the diameter of the phosphate-soluble zone (including the bacterial cell) were measured using ImageJ, and the solubility index was calculated according to the following formula: P = D / d (D is the diameter of the phosphate-soluble zone, and d is the colony diameter).

[0047] Quantitative determination of phosphate-solubilizing (organic and inorganic phosphorus) strains:

[0048] The purified strain was inoculated into liquid NBRIP organophosphate / inorganic phosphorus medium and samples were taken after shaking at 180 rpm and 28°C for 8 h, 24 h and 72 h, and the water-soluble phosphorus was determined. At the same time, the phosphatase activity in the organophosphate NBRIP medium was determined after 72 h.

[0049] Quantitative determination of IAA production ability of strains:

[0050] The purified strain was inoculated into KB liquid culture medium, shaken at 180 rpm and 28°C for 15 days, and then centrifuged at 10,000 r / min for 10 min. 2 mL of supernatant was taken and an equal volume of Salkowski colorimetric solution was added. A mixed solution of uninoculated KB culture medium and an equal volume of colorimetric solution was used as a control. After standing in the dark for 30 min, OD530 was measured and the IAA content per unit volume of supernatant was calculated using the standard curve.

[0051] Safety testing:

[0052] Inoculate strain R1-73 onto blood agar and incubate at 37°C for 3 days, observing for the presence of hemolytic zones. The presence of hemolytic zones indicates hemolytic activity, posing a potential threat to humans and animals, and prohibits its use in microbial fertilizers. The absence of hemolytic zones indicates the strain is inactive and safe, allowing its use as a microbial fertilizer.

[0053] Stress resistance test:

[0054] The strain R1-73 was inoculated into salt-tolerance and alkali-tolerance test medium, respectively. After shaking culture at 28°C for 7 days, the OD600 was measured to observe the bacterial growth status.

[0055] 3) Result analysis

[0056] Functional characterization results:

[0057] The results of functional characterization are shown in Table 1. Strain R1-81 has the ability to secrete phosphatase to mineralize organophosphate and the ability to secrete IAA.

[0058] Table 1 Functional characteristics of the strains identified

[0059]

[0060] Safety test results:

[0061] Strain R1-73 was cultured on blood agar plates for 3 days. Figure 2 The results showed that no hemolytic zone appeared and the hemolytic activity was negative.

[0062] Stress resistance test results:

[0063] The results of stress resistance test are shown in Table 2. The results showed that strain R1-73 had good stress resistance, with a salt tolerance of 11% and an alkali tolerance of pH 10.

[0064] Table 2 Salt and alkali resistance of strain R1-81

[0065]

[0066] Note: + indicates strain growth; the more + signs there are, the better the strain growth is.

[0067] Example 3: Effect of R1-73 on AM fungal growth

[0068] (1) Preparation of microbial agents

[0069] Plant bacillus R1-73 was inoculated into 1 / 2 TSB liquid medium and cultured on a shaker (28°C, 180 rpm) for 48-72 hours to obtain a liquid inoculum. The inoculum was centrifuged at 6000 rpm for 10 minutes, the supernatant was removed, and the precipitate was resuspended with 0.9% sterile saline and centrifuged again. After repeating this process 2-3 times, the precipitate was adjusted to OD600 = 0.5 with 0.9% saline, i.e., the concentration was about 1×10 6 cfu / mL.

[0070] (2) Experimental materials and methods

[0071] The host plant is the corn variety Zhengdan 958. Seeding was carried out on June 23, 2022, in the greenhouse of the College of Resources and Environment at China Agricultural University. The potting system used a mesh-type compartment system that separates the root chamber from the mycelial chamber. Before sowing, the soil in both the root and mycelial chambers was supplemented with nitrogen (200 mg / kg), potassium (200 mg / kg), magnesium (50 mg / kg), zinc (5 mg / kg), manganese (5 mg / kg), and copper (2 mg / kg). Phosphorus (20 ppm) in the form of potassium dihydrogen phosphate was added to the root chamber to ensure normal plant growth, and phosphorus (100 mg / kg) in the form of calcium phytate was added to the mycelial chamber.

[0072] (3) Experimental design

[0073] The experimental design was a two-factor experimental design, with factor one being inoculation or non-inoculation of AM fungi, and factor two being inoculation or non-inoculation of bacteria in the mycelial chamber. That is, the treatments included: ① the root chamber was not inoculated with AM fungi, and the mycelial chamber was not inoculated with Plant Bacillus (-AMF, -Plantibacter); ② the root chamber was inoculated with AM fungi, and the mycelial chamber was not inoculated with Plant Bacillus (+AMF, -Plantibacter); ③ the root chamber was inoculated with AM fungi, and the mycelial chamber was inoculated with Plant Bacillus (+AMF, +Plantibacter), with six replicates for each treatment.

[0074] AM fungi were inoculated in the root chamber when corn was sown. When the plants had grown for 3-4 weeks, Plantibacter R1-73 was inoculated in the mycelium chamber twice, with 10 bacteria in each inoculation. 8 CFU / kg, the corn was harvested at 8 weeks of growth, and the mycorrhizal infection rate and hyphae density were tested.

[0075] (4) Test results

[0076] The results showed that plant roots were not infected by AM fungi in the absence of AM fungal inoculation. However, the +AMF and +Massilia treatments significantly increased mycorrhizal infection rates and hyphae density compared to the +AMF and -Massilia treatments, as shown in Table 3. This suggests that strain R1-81 can promote AM fungal infection of plant roots and stimulate extraradical hyphae growth.

[0077] Table 3 Effects of strain R1-81 on the growth of AM fungi

[0078]

[0079] Example 4: Effect of R1-73 on Soil Phosphorus Activation and Plant Growth

[0080] (1) Preparation of microbial agents

[0081] Plant Bacillus strain R1-73 was inoculated into 1 / 2 TSB liquid medium and cultured on a shaker (28°C, 180 rpm) for 48-72 hours to obtain a liquid inoculum. The inoculum was centrifuged at 6000 rpm for 10 minutes, the supernatant removed, and the precipitate resuspended in 0.9% sterile saline and centrifuged again. This was repeated 2-3 times, and the OD600 was adjusted to 0.5 with 0.9% saline, i.e., a concentration of approximately 1×106 cfu / mL.

[0082] (2) Experimental materials and methods

[0083] The host plant was the corn variety Zhengdan 958. A potted experiment was conducted in the greenhouse of the Quzhou Experimental Station in Handan City, Hebei Province on August 31, 2022. The potted device used was a device that separated the root chamber and the mycelium chamber. Figure 2 As shown, N (200 mg / kg), K (200 mg / kg), Mg (50 mg / kg), Zn (5 mg / kg), Mn (5 mg / kg), and Cu (2 mg / kg) were added to the soil in the root chamber and the mycelium chamber before sowing. 20 ppm of phosphorus in the form of potassium dihydrogen phosphate was added to the root chamber to ensure normal plant growth, and 100 mg / kg of P in the form of calcium phytate was also added to the mycelium chamber.

[0084] (3) Experimental design

[0085] The experimental design was a two-factor experimental design, with factor one being inoculation or non-inoculation of AM fungi, and factor two being inoculation or non-inoculation of the mycelial chamber. That is, the treatments included: ① no inoculation of AM fungi and no inoculation of Plantibacter; ② inoculation of AM fungi and no inoculation of Plantibacter; ③ inoculation of AMF and inoculation of Plantibacter, with four replicates for each treatment.

[0086] When corn is sown, AM fungi are inoculated in the root chamber. When the plants have grown for 3-4 weeks, Plantibacter R1-73 is inoculated in the mycelium chamber twice, with the bacterial count of 108 CFU / kg each time. The corn is harvested at 8 weeks of growth, and the plant height and aboveground dry weight are calculated.

[0087] (4) Experimental results

[0088] The results of the pot experiment are shown in Table 4. The results showed that compared with the -AMF and -Plantibacter groups, inoculation with AM fungi significantly increased the biomass of the host plant; compared with the +AMF and -Plantibacter groups, inoculation with Plantibacter significantly increased the biomass of the host plant, with a biomass increase of 36.9%.

[0089] The above potted experiment results show that Plantibacter R1-73 can significantly promote the activation of organic phosphorus in the soil and promote the efficient utilization of phosphorus nutrients by corn, and can be used as a resource for developing stable and efficient microbial fertilizer strains.

[0090] Table 4 Effects of inoculation with AM fungi and Plantibacter on soil organic phosphorus activation and plant growth

[0091]

[0092] Note: + indicates a significant increase or decrease, - indicates no difference; the more + signs there are, the more significant the difference is.

Claims

1. Mycorrhizal helper bacteria R1-73 that synergizes with AM fungi to promote phosphorus activation and crop growth, characterized in that: The mycorrhizal helper bacteria R1-73 is Plantibacter, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 28069.

2. A bacterial agent, characterized in that The bacterial agent comprises the mycorrhiza-helping bacterium R1-73 according to claim 1.

3. Use of the mycorrhizal helping bacterium R1-73 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product that promotes AM fungi to infect plant roots and stimulate the growth of extraradical mycelium.

4. Use of the mycorrhiza-helping bacterium R1-73 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product that promotes plant growth.

5. Use of the mycorrhizal-helping bacterium R1-73 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product for improving plant stress resistance.

6. The use according to claim 5, wherein the stress resistance is salt and alkali resistance.

7. The use according to any one of claims 4 to 6, wherein the plant is corn, cotton, sorghum, potato or alfalfa.

8. The use according to claim 7, wherein the plant is corn.

9. Use of the mycorrhizal-assisting bacterium R1-73 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a product that promotes soil phosphorus activation and conversion.

10. Use of the mycorrhizal helping bacterium R1-73 according to claim 1 or the bacterial agent according to claim 2 in producing IAA.

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