Mycorrhizal helper bacteria for synergistically promoting phosphorus activation and crop growth with AM fungi

Abstract

The invention relates to a mycorrhizal helper bacterium R1-73 for synergistically promoting phosphorus activation and crop growth with AM (arbuscular mycorrhizal) fungi, which is characterized in that the mycorrhizal helper bacterium R1-73 is plantibacter and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.28069. The invention further discloses a preparation method of the mycorrhizal helper bacterium R1-73 for promoting phosphorus activation and crop growth with AM fungi. The strain is harmless to human and livestock, has multiple functions of promoting AM fungus growth, dissolving phosphorus, producing IAA, resisting stress, promoting plant growth and the like through identification, and can be used for preparing products such as fertilizers and agricultural microbial agents.

Description

Technical Field

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

[0002] Phosphorus is an important nutrient element for the growth and development of crops and is known as the "food" of crops. In modern agricultural production, the input of phosphate fertilizer has become a key means to increase crop yields. However, the utilization efficiency of phosphate fertilizer is generally low, and a large amount of phosphate fertilizer accumulates in the soil in the form of effective phosphorus, insoluble inorganic phosphorus and organic phosphorus, resulting in waste of phosphorus resources and environmental pressure. Therefore, improving the utilization efficiency of crops on accumulated phosphorus in the soil is of great significance to improving the utilization rate of phosphate fertilizer and achieving sustainable agricultural development.

[0003] Arbuscular mycorrhizal (AM) fungi are a class of symbiotic microorganisms that are widely present in terrestrial ecosystems and can form symbioses with the roots of most plants. Through this symbiotic relationship, plants can not only directly obtain phosphorus through their own roots, but also absorb phosphorus from the soil through the hyphal network of AM fungi, thereby effectively increasing the plant's absorption of phosphorus. However, although AM fungi have great potential in improving the phosphorus absorption capacity of plants, the effects of AM fungal inoculants currently used in agriculture are not stable, and in some cases even have a negative impact on plant growth.

[0004] Mycorrhizal helper bacteria are a group of microorganisms 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 groups 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, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.28069.

[0006] After blood agar plate culture test, the hemolytic activity was negative, indicating that it is harmless to humans and animals; it was identified to have multiple special functions, mainly including: (1) Phosphorus dissolving: It has the ability to dissolve organic phosphorus, the acid phosphatase activity is 1.49, and the alkaline phosphatase activity is 3.92. (2) IAA production: The IAA synthesis amount is 6.73 mg / kg. (3) Stress resistance: It can tolerate 11% salt and pH 10. (4) Promote plant growth: Increase plant height, aboveground biomass (aboveground dry weight), and promote the activation of soil organic phosphorus.

[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-assisting 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 assisting 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 mycorrhiza-assisting 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 assisting bacteria R1-73 or bacterial agent in the production of IAA.

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

[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 ability to dissolve phosphorus, 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), the strain significantly improves the activation and conversion efficiency of phosphorus in the soil, helping crops to 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 environmental pollution problems it brings.

[0019] (2) Promote crop growth and increase yield: Plantobacillus (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 promotion function. Experiments have shown that crops using this bacterial agent show significant growth in plant height and 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) Enhance crop stress resistance: Plantibacter 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 enables the strain to play its role in dissolving phosphorus and promoting growth even in salinized or barren soil environments, thereby improving crop stress resistance and increasing its application breadth in 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 extraradical hyphae of AM fungi, Plantibacter further enhances the ability of plants to absorb phosphorus through mycorrhizae. This synergistic effect significantly improves the absorption rate of crops to phosphorus in the soil, solving the problem of unstable effect of traditional single bacterial 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 of China Agricultural University to collect extraradical hyphae of AM fungi. In the experiment, 5 mg of fresh mycelium samples were accurately weighed and temporarily stored at 4°C. Subsequently, the high-throughput culture and identification technology system of root microorganisms was used to isolate and culture mycelial bacteria, and the mycelial bacterial strain R1-73 was successfully screened and obtained.

[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 to observe the colony morphology. The strain R1-73 on LB solid medium was 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, and 16S rRNA sequence was amplified by PCR. The forward primer used was 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and the reverse primer was 1492R (5'-TACGACTTAACCCCAATCGC-3', SEQ ID NO.2). Amplification conditions were: 95℃3min; 95℃15s, 60℃15s, 72℃15s, 30 cycles; and finally 72℃ extension for 5min. Qualified PCR amplification products were sent to Beijing Qingke Biotechnology Co., Ltd. (Tsingke Biotechnology Co., Beijing, China).

[0031]

[0032] R1-73 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101, deposit number CGMCC No.28069, 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 organophosphorus 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 phosphorus 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: MgSO4·7H2O1.5g, K2HPO4 15g, glycerol 10mL, peptone 20g, L-tryptophan 0.1g, 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, when the medium is cooled to 50°C, add 5% defibrinated sheep blood, mix well and pour onto a plate.

[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 use it for bacterial culture after sterilization at 121℃ for 20 minutes.

[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 / 100mL), respectively, and sterilize at 121°C for 20 min.

[0042] Salkowski colorimetric solution: Dissolve 4.5g FeCl3 in 300ml distilled water, slowly add 587.4ml 98% concentrated sulfuric acid, and adjust the volume to 1L after cooling. The IAA determination range 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-dissolving (organic and inorganic phosphorus) strains:

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

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

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

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

[0050] The purified strain was inoculated into KB liquid culture medium, and after shaking culture at 180 rpm and 28°C for 15 days, it was 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 at the same time. 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 against the standard curve.

[0051] Safety testing:

[0052] Inoculate strain R1-73 in blood agar medium and culture at 37℃ for 3 days to observe whether there is a hemolysis zone. If there is a hemolysis zone, it means that the strain has hemolytic activity and poses a potential threat to humans and animals. In this case, the strain is prohibited from being used in microbial fertilizers. If there is no hemolysis zone, it means that the strain has no hemolytic activity and is a safe strain, which can be used as a microbial fertilizer.

[0053] Stress resistance test:

[0054] The strain R1-73 was inoculated into the 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.

[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 organic phosphorus and the ability to secrete IAA.

[0058] Table 1 Functional characteristics of 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 stress resistance test results are shown in Table 2. The results show that strain R1-73 has 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 growth of the strain.

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

[0068] (1) Preparation of bacterial 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 bacterial agent. The bacterial agent was centrifuged at 6000 rpm for 10 min, the supernatant was removed, and the precipitate was resuspended with 0.9% sterile saline and centrifuged again. After repeating 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. Sowing was carried out in the greenhouse of the College of Resources and Environment of China Agricultural University on June 23, 2022. The potted plant uses a mesh compartment device that separates the root chamber and the mycelium chamber. 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 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 added to the mycelium chamber.

[0072] (3) Experimental design

[0073] The experimental design was a two-factor experimental design, with factor one being inoculation or not of AM fungi, and factor two being inoculation or not of bacteria in the mycelium chamber, that is, the treatments included: ① the root chamber was not inoculated with AM fungi, and the mycelium chamber was not inoculated with Plant Bacillus (-AMF, -Plantibacter); ② the root chamber was inoculated with AM fungi, and the mycelium chamber was not inoculated with Plant Bacillus (+AMF, -Plantibacter); ③ the root chamber was inoculated with AM fungi, and the mycelium 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 grew for 3-4 weeks, Plantibacter R1-73 was inoculated in the mycelium chamber twice, with 10 bacteria in each inoculation. 8 CFU / kg, 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 the plant roots were not infected by AM fungi without AM fungi inoculation. Compared with +AMF and -Massilia, the mycorrhizal infection rate and hyphae density were significantly increased in +AMF and +Massilia treatments, as shown in Table 3. This shows that strain R1-81 can promote AM fungi to infect plant roots and stimulate the growth of extraradical hyphae.

[0077] Table 3 Effect 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 bacterial agents

[0081] Plantibacter R1-73 was inoculated into 1 / 2 TSB liquid culture medium and cultured on a shaker (28°C, 180 rpm) for 48-72 hours to obtain a liquid bacterial agent. The bacterial agent was centrifuged at 6000 rpm for 10 min, the supernatant was removed, and the precipitate was resuspended with 0.9% sterile saline and centrifuged again, repeated 2-3 times, and adjusted to OD600 = 0.5 with 0.9% saline, that is, the concentration was about 1×106 cfu / mL.

[0082] (2) Experimental Materials and Methods

[0083] The host plant selected was the corn variety Zhengdan 958. The potted experiment was carried out in the greenhouse of 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, such as 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, wherein 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 additionally 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 not of AM fungi, and factor two being inoculation or not of the mycelium 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] AM fungi were inoculated in the root chamber when corn was sown. When the plants grew for 3-4 weeks, Plantibacter R1-73 was inoculated in the mycelium chamber twice, with the number of bacteria inoculated at 108 CFU / kg each time. The corn was harvested when it grew for 8 weeks, and the plant height and aboveground dry weight were counted.

[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, the inoculation of AM fungi significantly increased the biomass of the host plant; compared with the +AMF and -Plantibacter groups, the inoculation of Plantibacter significantly increased the biomass of the host plant, with the biomass increased by 36.9%.

[0089] The above potted experiment results show that Plantibacter R1-73 can significantly promote the activation of organic phosphorus in the soil, 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 cooperate with AM fungi to promote phosphorus activation and crop growth, characterized in that: The mycorrhizal helper bacteria R1-73 is Plantibacter, which is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, with the preservation number of CGMCC No.28069.

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

3. Use of the mycorrhiza-assisting bacteria 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-assisting bacteria 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 bacteria R1-73 according to claim 1 or the bacterial agent according to claim 2 in the preparation of products 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 mycorrhiza-assisting bacteria R1-73 according to claim 1 or the bacterial agent according to claim 2 in the preparation of products that promote soil phosphorus activation and conversion.

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

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