Screening and application of plant growth-promoting paenibacillus

Abstract

The invention provides a plant growth-promoting paenibacillus and an application of the plant growth-promoting paenibacillus. The strain is screened from a corn experimental field in Jilin Gongzhou Mountain, and the preservation number of the strain is CGMCC No.33517. The strain has the capabilities of dissolving phosphorus and potassium and promoting plant growth, and can form an interaction relationship with arbuscular mycorrhizal fungi. The strain is used for preparing an efficient microbial agent as a fertilizer, and has the advantages of low production cost, simplicity in operation, no secondary pollution to soil and the like.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a strain of feed bacillus Paenibacillus pabuli screening and its application. Background Art

[0002] Since the 21st century, the sharp increase in crop yields has mainly relied on the development and excessive use of chemical fertilizers, which has brought about a series of environmental problems while achieving yield increases, such as soil acidification and compaction, nutrient imbalance, and loss of microbial diversity. Therefore, tapping into soil native microbial resources to achieve sustainable agricultural production and utilizing the growth-promoting role of microorganisms in crop nutrient cycles to reduce the use of chemical fertilizers have gradually become a hot spot and new direction for the development of green fertilizers.

[0003] Beneficial microorganisms in the plant rhizosphere mainly include various rhizosphere growth-promoting bacteria that naturally exist around plant roots and in farmland soil. They can help plants absorb nitrogen, phosphorus, potassium and trace elements, participate in the soil nutrient cycle, and some can also form cooperative relationships with other important farmland microorganisms such as arbuscular mycorrhiza fungi (hereinafter referred to as AM fungi) to better promote crop growth.

[0004] AM fungi are mycorrhizal fungi that exist in large quantities in the soil. They can form a symbiotic relationship with plant roots. On the one hand, they directly promote the absorption of nutrients by plant roots and help plants resist pathogenic microorganisms; on the other hand, they can also indirectly participate in plant nutrient absorption and stress resistance through interactions with rhizosphere microorganisms. In the process of crop growth, the efficient operation of materials and energy in the crop-soil-microorganism system is the key to ensuring sustainable crop production, among which the interaction between AM fungi and beneficial rhizosphere microorganisms occupies an important position. The infection rate of AM fungi, whether beneficial microorganisms can play a growth-promoting function, and whether there is an interaction between the two directly determine whether the crop-soil-microorganism system operates efficiently, and are also an important means for crops to achieve green, efficient and sustainable production. Therefore, attempts to screen and isolate plant growth-promoting microbial strains that can interact with arbuscular mycorrhizal fungi from soil microbial flora, as biofertilizers to partially or completely replace chemical fertilizers, while achieving stable and increased production without destroying or even improving the ecological environment of the soil, have huge demand potential.

[0005] Paenibacillus is a bacterium widely found in soil. Because its secondary metabolites contain a variety of antimicrobial active substances, it can effectively antagonize a variety of pathogens and is often used as a biocontrol bacterium. Related research also focuses more on its antibacterial and antifungal activity. No one has paid attention to the plant growth-promoting ability of Paenibacillus and AM fungi, especially the feed Paenibacillus ( Paenibacillus pabuli), and there has been no research or report on its use as a plant growth-promoting bacterium. Summary of the invention

[0006] The purpose of the present invention is to provide a feed bacillus with plant growth-promoting function and the screening and application thereof.

[0007] Another object of the present invention is to provide a feed Bacillus that can interact with arbuscular mycorrhizal fungi.

[0008] Another object of the present invention is to provide a feed Paenibacillus strain having the function of solubilizing phosphorus and potassium.

[0009] Another object of the present invention is to provide a feed Bacillus having a growth-promoting effect on plants.

[0010] Another object of the present invention is to provide feed spore-forming bacteria having one or more of the above functions and related applications thereof.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: A feed bacillus was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on February 12, 2025 and was classified and named Paenibacillus pabuli , the deposit number is CGMCC No.33517.

[0012] The feed Bacillus was obtained from the surface of AM fungal mycelium outside the corn roots in Gongzhuling, Jilin Province through multiple screening. It can grow in restrictive culture medium and has the ability to solubilize phosphorus and potassium.

[0013] Greenhouse experiments have shown that the feed Bacillus can enhance the infection of arbuscular mycorrhizal fungi in corn roots, increase corn nitrogen absorption, and promote corn growth.

[0014] The present invention provides a feed bacillus, which is taxonomically named Paenibacillus pabuli , the deposit number is CGMCC No.33517.

[0015] The present invention provides a microbial agent comprising the feed Paenibacillus sp.

[0016] The present invention also provides the use of the feed Paenibacillus as plant growth-promoting bacteria.

[0017] The present invention also provides use of the feed Paenibacillus or microbial agent in promoting plant growth.

[0018] The present invention also provides use of the feed Paenibacillus or microbial agent in enhancing the infection of arbuscular mycorrhizal fungi on plant root systems.

[0019] The present invention also provides the use of the feed Paenibacillus or microbial agent in improving the soluble phosphorus and soluble potassium in soil.

[0020] The plant is preferably a crop that can form a symbiotic relationship with arbuscular mycorrhizal fungi, and particularly preferably corn that has a strong mycorrhizal dependence.

[0021] The present invention also provides the use of the feed bacillus or microbial agent, which can increase the dry weight of plants, improve the chlorophyll content, and promote the infection intensity of arbuscular mycorrhizae.

[0022] The invention provides a method for promoting plant growth, which comprises applying the feed Paenibacillus or microbial agent.

[0023] The invention provides a method for enhancing the infection of arbuscular mycorrhizal fungi of plant root system, which comprises applying the feed Paenibacillus or microbial agent.

[0024] The invention provides a method for increasing soluble phosphorus and soluble potassium in soil, which comprises applying the feed Paenibacillus or microbial agent.

[0025] The present invention provides a plant growth promoter, comprising the feed Paenibacillus or microbial agent.

[0026] The invention provides an arbuscular mycorrhizal fungus infection accelerator, comprising the feed Paenibacillus or microbial agent.

[0027] The invention provides a soil phosphorus and potassium solubilization system, comprising the above-mentioned feed bacillus or microbial agent.

[0028] The present invention further provides a plant growth-promoting microbial agent, the microbial agent comprising the above-mentioned feed Bacillus strain. The microbial growth-promoting agent can be applied to plants or their seeds as a fertilizer, and the plants are preferably crops, especially corn.

[0029] The invention provides a plant growth promoter, a root development improver and / or an arbuscular mycorrhizal fungus infection promoter, which is characterized by comprising the feed Paenibacillus strain or the microbial agent.

[0030] The invention provides a soil phosphorus and potassium solubilization system, which comprises the feed Paenibacillus strain or the microbial agent.

[0031] In the above-mentioned bacterial agent, the effective viable count of the feed Bacillus strain is 10 5 ~10 15 CFU / g, preferably 10 8 ~10 11 CFU / g.

[0032] The present invention further provides a method for preparing a microbial agent, wherein the feed Bacillus strain is activated and prepared into a seed liquid, which is then inoculated into a fermentation medium for expansion and culture to a stable period, and the microbial agent is obtained after separation.

[0033] The above-mentioned microbial agent can be prepared by conventional methods for preparing agents in the art, and can be in various forms, such as liquid agents or solid agents, preferably solid agents.

[0034] The solid bacterial agent is preferably prepared by fermentation and freeze-drying.

[0035] The invention provides a method for preparing a solid bacterial agent, comprising the steps of adding a freeze-drying protective agent into the feed bacillus fermentation liquid, then centrifuging to obtain a bacterial precipitate, and freeze-drying the precipitate under aseptic conditions to prepare a freeze-dried powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Mycelium collection device buried in the field Figure 2 Effects of LWPGPB101 on corn plant growth (2 weeks) Figure 3 Effects of LWPGPB101 on root growth of maize (4 weeks) Figure 4 Microscopic examination of the effect of LWPGPB101 on arbuscular mycorrhizal fungi in maize roots DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with specific examples. Where specific experimental conditions are not indicated, they are all based on conventional conditions well known to those skilled in the art.

[0038] Example 1 Screening of hyphae microorganisms that interact with AM fungi (1) Using a hyphae collection device between corn plants in the experimental field of Gongzhuling, Jilin (see Figure 1 ) Collect the extraradical hyphae of arbuscular mycorrhizal fungi (hereinafter referred to as AM fungi), and transport the mycelial film attached with AM fungal hyphae in the collection box to the laboratory at low temperature.

[0039] The mycelium collection device is mainly composed of a hollow mycelium box with openings at both ends, a sealing film sealed at the openings at both ends, and a mycelium membrane arranged inside the mycelium box. The sealing film is a nylon membrane with a pore size of 30 μm, and the mycelium membrane is a microporous filter membrane with a pore size of 0.45 μm. When in use, fill the two sides of the mycelium membrane in the middle of the mycelium box with the in-situ soil taken from the field to be collected and sterilized, and then seal the sealing film at both ends and bury it in the soil between the roots of two corn plants in the field. Due to the large difference between the diameter of plant roots (millimeter level) and the diameter of AM fungal extraradical mycelia (micrometer level), the sealing film at the entrance of the mycelium box only allows the extraradical mycelia of AM fungi with a smaller diameter to grow through, while the plant roots with a larger diameter cannot grow through the sealing film and enter the mycelium box. In addition, since bacteria do not have the ability to actively migrate long distances in the soil, only bacterial strains that can interact with AM fungi and colonize on mycelia can be carried into the mycelium box through the mediation of AM fungi as the mycelium grows. When the AM fungal hyphae that pass through the sealing film and enter the mycelium box continue to grow and encounter the obstruction of the mycelial membrane in the middle of the mycelial box with a smaller pore size and cannot pass through, they will grow along the surface of the mycelial membrane and enrich on it, making it easy to collect. The bacterial strains that interact with the AM fungi are collected together because they colonize on the surface of the mycelium.

[0040] (2) Cut the mycelium into pieces, place them in a 50 ml centrifuge tube, add sterile water and shake, then take the supernatant and spread it on a restrictive culture medium KPM plate and culture at 30°C for 72 hours.

[0041] KPM restricted medium formula

[0042] (3) Pick the monoclonal strain cultured on the plate in step (2) and re-streak it on the KPM plate for purification.

[0043] Since KPM is a restrictive culture medium with relatively poor nutrition, the strains that can grow on it have good growth vitality and have the potential to be applied as microbial fertilizer in soil environments with similar limited nutrition.

[0044] (4) PCR amplification step (3) Purify the 16s rDNA of the strain and sequence it. After sequence comparison, one of the strains, numbered LWPGPB101, was identified as a feed Bacillus of the genus Paenibacillus. Paenibacillus pabuli The 16s rDNA sequence of the strain is shown in SEQ ID NO. 1. The strain is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration and is classified and named Paenibacillus pabuli , the deposit number is CGMCC No.33517.

[0045] Example 2 Verification of the Phosphorus and Potassium Solubilization Functions of LWPGPB101 1. Verification of phosphate solubilization function (1) The above-screened LWPGPB101 was selected as the experimental strain, and the Klebsiella mutans LWNF004 (deposit number: CGMCC No. 28351) with nitrogen fixation function disclosed in patent CN117004540 B was selected as the control strain. The above strains were activated and cultured in LB medium.

[0046] (2) The activated LWPGPB101 and LWNF004 strains were inoculated on NBRIP inorganic phosphorus solid medium, and the formation of phosphate solubilization circles was observed after culturing at 30°C for 6 days. The diameter of the phosphate solubilization circle (D) and the colony diameter (d) were measured by the cross method. The phosphate solubilization ability of the strain was preliminarily determined based on whether the phosphate solubilization circle was formed and the size of the D / d value.

[0047] NBRIP Inorganic Phosphorus Medium Formula

[0048] The results are shown in Table 1. LWPGPB101 can produce an obvious phosphorus solubilization circle on the NBRIP inorganic phosphorus plate, and the phosphorus solubilization index can reach 1.31, while LWNF004 has no obvious phosphorus solubilization phenomenon. The above results clearly show that LWPGPB101 has a relatively obvious function of dissolving inorganic phosphorus.

[0049] Table 1 Solubility index of strains on phosphate-solubilizing medium

[0050] 2. Verification of potassium-dissolving function (1) The above-screened LWPGPB101 was selected as the experimental strain, and the control strain of Klebsiella mutans LWNF004 (deposit number CGMCC No. 28351) with nitrogen fixation function disclosed in patent CN117004540 B was selected. The above strains were activated and cultured in LB medium.

[0051] (2) The activated LWPGPB101 and LWNF004 were inoculated on the Alexander Borov potassium-solubilizing solid medium (Shandong Top Biotechnology Co., Ltd., product number M2349B), and the potassium-solubilizing zone was observed after culturing at 30°C for 6 days. The potassium-solubilizing zone diameter (D) and colony diameter (d) were measured by the cross method. The potassium-solubilizing ability of the strain was preliminarily determined based on whether the potassium-solubilizing zone was generated and the D / d value.

[0052] The results are shown in Table 2. LWPGPB101 can produce an obvious potassium-solubilizing zone on the Alexandrov potassium-solubilizing medium plate, and the potassium-solubilizing index can reach 1.60, which is significantly stronger than LWNF004. The above results show that the feed Bacillus LWPGPB101 has a strong potassium-solubilizing function.

[0053] Table 2 Solubility index of strains on potassium-dissolving medium

[0054] Example 3 Effect of LWPGPB101 on plant growth 1. Experimental methods A single-factor randomized block method was used to select three experimental treatments, including LWPGPB101, LWNF004 and no bacteria (CK), with four replicates for each treatment.

[0055] (1) After activation, LWPGPB101 and Klebsiella mutans LWNF004 were inoculated into LB medium and cultured at 30°C for 16 h. The cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in sterile PBS to adjust the concentration to 10 8 CFU / ml.

[0056] (2) Corn seeds were sown in a greenhouse. The culture medium was a mixture of loess and vermiculite (mass ratio of 25:1). The soil moisture content was maintained at 70% before sowing. During sowing, 1 ml of LWPGPB101 bacterial suspension, 1 ml of LWNF004 bacterial suspension, or 1 ml of sterile PBS (CK) was added around each corn seed according to the three designed experimental treatments.

[0057] (3) After sowing, the growth of corn seedlings was observed every day to evaluate the differences in growth-promoting effects among treatments. At 2, 3, and 4 weeks of cultivation, plant height, chlorophyll content, nitrogen content, plant fresh weight, plant dry weight, root morphology and other indicators were measured.

[0058] 2. Experimental results (1) Impact on corn plant height From Table 3 and Figure 2 It can be seen that when the corn seedlings were cultured for 2 weeks, 3 weeks, and 4 weeks, the plant heights of the groups treated with LWPGPB101 were 14.05 cm, 27.28 cm, and 34.15 cm, respectively, which were 47.89%, 22.06%, and 20.54% higher than those of the control group (CK) without bacterial solution; and 36.67%, 13.20%, and 8.93% higher than those of the group treated with LWNF004.

[0059] The above results all indicate that feed Bacillus LWPGPB101 can promote the growth of corn seedlings.

[0060] Table 3 Effects on corn plant height

[0061] (2) Effects on leaf chlorophyll content and nitrogen concentration As shown in Table 4, when the corn seedlings were cultured for 3 weeks, the average chlorophyll content of the leaves in the treatment group treated with LWPGPB101 reached 39.90 SPAD, and the average nitrogen concentration of the leaves reached 15.29 mg / g, which were 14% and 11.36% higher than those in the control group (CK) without bacterial solution application, and 15.02% and 10.32% higher than those in the treatment group treated with LWNF004.

[0062] When the corn seedlings were cultivated for 4 weeks, the average chlorophyll content of the leaves in the treatment group treated with LWPGPB101 reached 37.33 SPAD, and the average nitrogen concentration of the leaves reached 14.74 mg / g. Compared with the control group (CK) without bacterial solution, the average chlorophyll and nitrogen contents of the leaves increased by 6.6% and 5.23%, respectively, and compared with the treatment group treated with LWNF004, the average chlorophyll and nitrogen contents of the leaves increased by 3.2% and 2.56%, respectively.

[0063] The above results show that feed Bacillus LWPGPB101 can promote corn growth by improving the chlorophyll content and nitrogen content of corn leaves and enhancing leaf photosynthesis.

[0064] Table 4 Effects on leaf physiological parameters during corn growth

[0065] (3) Effects on corn dry matter and root system From Table 5 and Figure 3 It can be seen that when the corn seedlings were cultivated for 4 weeks, the plant fresh weight, plant dry weight, root fresh weight and plant nitrogen absorption of the treatment group treated with LWPGPB101 were 1.96 g, 0.17 g, 2.78 g and 2.50 mg, respectively, which were 45.19%, 30.77%, 84.11% and 42.86% higher than those of the control group (CK) without bacterial solution application; the plant fresh weight, plant dry weight, root fresh weight and plant nitrogen absorption of the treatment group treated with LWNF004 were 5.95%, 6.25%, 4.91% and 14.16% higher than those of the treatment group treated with LWNF004.

[0066] The above results show that feed Bacillus LWPGPB101 can promote the accumulation of dry matter in corn seedlings and promote nitrogen absorption, thereby achieving the growth-promoting effect on corn seedlings.

[0067] Table 5 Effects on plant dry and fresh weight and nitrogen absorption during corn growth

[0068] Example 4 Effect of LWPGPB101 on arbuscular mycorrhizal fungi in maize roots (1) Corn root samples were collected from corn seedlings of the treatment group treated with LWPGPB101 and the control group (CK) not treated with the bacterial solution after culturing for 4 weeks in Example 3.

[0069] (2) Cut each root sample into 1 cm long root segments and bathe them in 10% KOH in a 90°C water bath for 60 min to remove the cytoplasm and nuclei in the roots to make them easier to stain.

[0070] (3) After cooling, discard the KOH solution and wash the roots with clean water for 3-5 times; then add 2% HCl solution to acidify for 5 min; after washing, add 0.05% trypan blue solution and stain in a 90℃ water bath for 30 min.

[0071] (4) After removing the trypan blue solution, add 1:1 lactic acid glycerol to decolorize at room temperature.

[0072] (5) Use tweezers to pick up 15 decolorized root segments and arrange them neatly on a glass slide. Prepare two slide samples for each treatment. Evaluate and grade the infection status under a microscope and calculate the arbuscular mycorrhizal infection rate.

[0073] The results are as follows Figure 4 As shown in Table 6, the frequency of AM mycorrhizal infection in the maize roots of the treatment group treated with LWPGPB101 was 81.67%, the infection intensity was 27.33%, and the arbuscular abundance was 11.99%. Compared with the control group (CK) without application of bacterial solution, the frequency of AM fungal infection in the maize roots increased by 18%, the infection intensity increased by 3.2 times, and the arbuscular abundance increased by 11 times.

[0074] The above results indicate that the presence of feed Bacillus LWPGPB101 promotes the infection of AM mycorrhizae on plant roots and increases the number of arbuscular mycorrhizal fungi symbiotic in plant roots, thereby further directly or indirectly promoting plant growth and development.

[0075] Table 6 Effects on arbuscular mycorrhizal fungi in maize roots

[0076] Example 5 Preparation of microbial fertilizer In order to facilitate storage and transportation, the feed Bacillus LWPGPB101 screened in Example 2 was Prepare freeze-dried powder fertilizer. Mix skim milk powder, sodium glutamate, glycerol, sucrose and water in a mass ratio of (75:15:70:10:330) to prepare freeze-dried protective agent, add it to the fermentation liquid of LWPGPB101, and then obtain bacterial precipitation by centrifugation. Freeze-drying treatment is carried out under sterile conditions to prepare freeze-dried powder for use. The effective viable bacteria count of the feed Bacillus LWPGPB101 is 10 8 ~10 11 CFU / g, and can be stored at room temperature for 6 months without loss of activity.

[0077] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A feed bacillus, characterized in that Classification Name Paenibacillus pabuli , the deposit number is CGMCC No.33517.

2. A microbial agent, characterized in that: The method comprises the feed bacillus as claimed in claim 1.

3. Use of the feed bacillus according to claim 1 or the microbial agent according to claim 2 in promoting plant growth.

4. Use of the feed bacillus according to claim 1 or the microbial agent according to claim 2 in enhancing the infection of arbuscular mycorrhizal fungi on plant roots.

5. A method for promoting plant growth, characterized in that: Apply the feed bacillus according to claim 1 or the microbial agent according to claim 2.

6. A method for enhancing the infection of arbuscular mycorrhizal fungi in plant roots, characterized in that: Apply the feed bacillus according to claim 1 or the microbial agent according to claim 2.

7. A method for increasing soluble phosphorus and soluble potassium in soil, characterized in that: Apply the feed bacillus according to claim 1 or the microbial agent according to claim 2.

8. A plant growth promoter, characterized in that It comprises the feed Bacillus according to claim 1 or the microbial agent according to claim 2.

9. An arbuscular mycorrhizal fungus infection accelerator, characterized in that: It comprises the feed Bacillus according to claim 1 or the microbial agent according to claim 2.

10. A soil phosphorus and potassium solubilization system, characterized in that: It comprises the feed Bacillus according to claim 1 or the microbial agent according to claim 2.

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