Screening and Application of Plant Growth-Promoting Paenibacillus

Abstract

The present invention provides a plant growth-promoting Paenibacillus strain and its application. The strain was screened from a corn experimental field in Gongzhuling, Jilin, and its preservation number is CGMCC No. 33517. This strain has the abilities of phosphorus and potassium solubilization and plant growth promotion, and can form an interaction relationship with arbuscular mycorrhizal fungi. Using this strain to prepare a highly efficient microbial inoculant as a fertilizer has the advantages of low production cost, simple operation, and no secondary pollution to the soil.

Description

Technical Field

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

[0002] Since the 21st century, the substantial increase in crop yields has mainly relied on the development and overuse of chemical fertilizers. While achieving yield increases, this has also brought a series of environmental problems, such as soil acidification and compaction, nutrient imbalance, loss of microbial diversity, etc. Therefore, exploring soil native microbial resources to achieve sustainable agricultural production and using the growth-promoting effects of microorganisms in crop nutrient cycling to reduce the use of chemical fertilizers have gradually become the focus and new direction of the development of green fertilizers.

[0003] Beneficial plant rhizosphere microorganisms mainly include various rhizosphere growth-promoting bacteria naturally present around plant roots and in farmland soil. They can help plants absorb nitrogen, phosphorus, potassium, and trace elements, participate in the process of soil nutrient cycling, 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 abundantly present in the soil. They can form a symbiotic relationship with plant roots. On the one hand, they directly promote plant roots to absorb nutrients 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. During the growth process of crops, the efficient operation of the material and energy of the crop-soil-microorganism system is the key to ensuring sustainable crop production. Among them, the interaction between AM fungi and beneficial rhizosphere microorganisms occupies an important position. The level of AM fungal infection rate, whether beneficial microorganisms can exert growth-promoting functions, and whether there is an interaction between the two directly determine whether the crop-soil-microorganism system operates efficiently, and are also important starting points for achieving green, efficient, and sustainable crop production. Therefore, there is a great potential demand to try to screen and isolate plant growth-promoting microbial strains from soil microbial communities that can interact with arbuscular mycorrhiza fungi to partially or completely replace chemical fertilizers as biological fertilizers, while achieving stable production and yield increase without damaging or even improving the soil ecological environment.

[0005] Bacillus cereus is a bacterium widely present in the soil. Because its secondary metabolites contain a variety of antibacterial active substances and can effectively antagonize a variety of pathogenic bacteria, it is often used as a biocontrol bacterium, and related research has more focused on its antibacterial and bacteriostatic activities. The plant growth-promoting ability of Bacillus cereus interacting with AM fungi has not been concerned by anyone, especially the Bacillus cereus found in feed for the first time ( Paenibacillus pabuli), and there is no research or report on it as a plant growth-promoting bacterium. Summary of the Invention

[0006] The purpose of the present invention is to provide a Bacillus cereus forage strain with plant growth-promoting function and its screening and application.

[0007] Another purpose of the present invention is to provide a Bacillus cereus forage strain that can interact with arbuscular mycorrhizal fungi.

[0008] Another purpose of the present invention is to provide a Bacillus cereus forage strain with the function of dissolving phosphorus and potassium.

[0009] Another purpose of the present invention is to provide a Bacillus cereus forage strain that promotes the growth of plants.

[0010] Another purpose of the present invention is to provide a Bacillus cereus forage strain with one or more of the above functions and its related applications.

[0011] To achieve the above invention purposes, the present invention provides the following technical solutions:

[0012] A Bacillus cereus forage strain was deposited on February 12, 2025, at the China General Microbiological Culture Collection Center (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), and its taxonomic name is Paenibacillus pabuli , and the deposit number is CGMCC No. 33517.

[0013] This Bacillus cereus forage strain was obtained through multiple screenings from the surface of AM fungal hyphae outside the roots of corn in Gongzhuling, Jilin. It can grow in a restrictive medium and has the ability to dissolve phosphorus and potassium.

[0014] Proven by greenhouse experiments, this Bacillus cereus forage strain can enhance the infection of arbuscular mycorrhizal fungi in corn roots, increase the nitrogen uptake of corn, and promote the growth of corn at the same time.

[0015] The present invention provides a Bacillus cereus forage strain, and its taxonomic name is Paenibacillus pabuli , and the deposit number is CGMCC No. 33517.

[0016] The present invention provides a microbial inoculant containing the above Bacillus cereus forage strain.

[0017] The present invention also provides the application of the above Bacillus cereus forage strain as a plant growth-promoting bacterium.

[0018] The present invention also provides the use of the above Bacillus cereus forage strain or microbial inoculant in promoting plant growth.

[0019] The present invention also provides the use of the above-mentioned Bacillus cereus forage or microbial inoculum in enhancing the infection of plant roots by arbuscular mycorrhizal fungi.

[0020] The present invention also provides the use of the above-mentioned Bacillus cereus forage or microbial inoculum in increasing soluble phosphorus and soluble potassium in soil.

[0021] The plant is preferably a crop that can form a symbiotic relationship with arbuscular mycorrhizal fungi, and particularly preferably maize with strong mycorrhizal dependence.

[0022] The present invention also provides the use of the above-mentioned Bacillus cereus forage or microbial inoculum, which can increase the dry weight of plants, improve the chlorophyll content, and promote the infection intensity of arbuscular mycorrhizae.

[0023] The present invention provides a method for promoting plant growth, which comprises applying the above-mentioned Bacillus cereus forage or microbial inoculum.

[0024] The present invention provides a method for enhancing the infection of plant roots by arbuscular mycorrhizal fungi, which comprises applying the above-mentioned Bacillus cereus forage or microbial inoculum.

[0025] The present invention provides a method for increasing soluble phosphorus and soluble potassium in soil, which comprises applying the above-mentioned Bacillus cereus forage or microbial inoculum.

[0026] The present invention provides a plant growth promoter, which comprises the above-mentioned Bacillus cereus forage or microbial inoculum.

[0027] The present invention provides a promoter for arbuscular mycorrhizal fungal infection, which comprises the above-mentioned Bacillus cereus forage or microbial inoculum.

[0028] The present invention provides a soil phosphorus and potassium solubilization system, which comprises the above-mentioned Bacillus cereus forage or microbial inoculum.

[0029] The present invention further provides a plant growth-promoting microbial inoculum, which comprises the above-mentioned Bacillus cereus forage strain. The microbial growth-promoting inoculum can be applied as a fertilizer to plants or their seeds. The plant is preferably a crop, especially maize.

[0030] The present invention provides a plant growth promoter, a root development improver, and / or a promoter for arbuscular mycorrhizal fungal infection, which is characterized by comprising the Bacillus cereus forage strain or the microbial inoculum.

[0031] The present invention provides a soil phosphorus and potassium solubilization system, which comprises the Bacillus cereus forage strain or the microbial inoculum.

[0032] In the above-mentioned inoculum, the effective viable count of the Bacillus cereus forage strain is 10 5 ~10 15 CFU / g, preferably 108 ~10 11 CFU / g。

[0033] The present invention further provides a method for preparing a microbial inoculant. After activating the Bacillus cereus strain for feed, a seed solution is prepared, and then it is inoculated into a fermentation medium for enlarged culture until the stationary phase, and the microbial inoculant is obtained after separation.

[0034] The above microbial inoculant can be prepared by conventional inoculant preparation methods in the art, and it can be inoculants in various forms, such as liquid inoculants or solid inoculants, and solid inoculants are preferred.

[0035] The solid inoculant is preferably prepared by the method of fermentation and freeze-drying.

[0036] The present invention provides a method for preparing a solid inoculant. A freeze-drying protectant is added to the fermentation broth of the Bacillus cereus strain for feed, and then bacterial precipitation is obtained by centrifugal separation, and freeze-drying treatment is carried out under aseptic conditions to prepare a freeze-dried powder. Description of the Drawings

[0037] Figure 1 Mycelium collection device buried in the field

[0038] Figure 2 Effect of LWPGPB101 on the growth of maize plants (2 weeks)

[0039] Figure 3 Effect of LWPGPB101 on the growth of maize roots (4 weeks)

[0040] Figure 4 Microscopic examination diagram of the effect of LWPGPB101 on arbuscular mycorrhizal fungi in maize roots Detailed Embodiments

[0041] The present invention will be further described below in conjunction with specific embodiments. All those not indicating specific experimental conditions are conventional conditions well-known to those skilled in the art.

[0042] Example 1 Screening of rhizosphere microorganisms interacting with AM fungi

[0043] (1) In the experimental field of Gongzhuling, Jilin, between maize plants, a mycelium collection device (see Figure 1 ) was used to collect extraradical mycelia of arbuscular mycorrhizal fungi (hereinafter referred to as AM fungi). The mycelial membrane attached with AM fungal mycelia in the collection box was transported to the laboratory at low temperature.

[0044] The hypha collection device mainly consists of a hollow hypha box with openings at both ends, sealing films sealed at the openings at both ends, and a hypha membrane arranged inside the hypha box. The sealing film uses a nylon membrane with a pore size of 30 μm, and the hypha membrane uses a microporous filter membrane with a pore size of 0.45 μm. During use, the two sides of the hypha membrane in the middle of the hypha box are filled with in-situ soil taken from the field to be collected and sterilized, and then the sealing films at both ends are sealed and buried in the soil between the roots of two corn plants in the field. Due to the large difference in the diameters of plant roots (millimeter level) and the external hyphae of AM fungi (micrometer level), the sealing film at the entrance of the hypha box only allows the growth of the smaller-diameter external hyphae of AM fungi to pass through, while the larger-diameter plant roots cannot grow through the sealing film and enter the hypha box. In addition, since bacteria do not have the ability to actively migrate over long distances in the soil, only bacterial strains that can interact with AM fungi and colonize on the hyphae can be carried into the hypha box along with the growth of the hyphae mediated by AM fungi. When the AM fungal hyphae that have passed through the sealing film and entered the hypha box continue to grow and encounter the obstruction of the hypha membrane with a smaller pore size in the middle of the hypha box that cannot pass through, they will grow along the surface of the hypha membrane and accumulate on it, facilitating collection. The bacterial strains that interact with AM fungi are colonized on the surface of the hyphae and are thus collected together.

[0045] (2) Cut the hypha membrane into pieces, place them in a 50-ml centrifuge tube, add sterile water and shake. After shaking, take the supernatant and smear it on the KPM plate of the restrictive medium and culture it at 30 °C for 72 hours.

[0046] Formulation of KPM restrictive medium

[0047]

[0048] (3) Pick the monoclonal strains cultured on the plate in step (2) and streak and purify them again on the KPM plate.

[0049] Since KPM is a restrictive medium with relatively scarce nutrients, the strains that can grow on it indicate good growth vitality and have the potential to be applied as microbial fertilizers to the same soil environment with limited nutrients.

[0050] (4) PCR amplify the 16s rDNA of the purified strains in step (3) and sequence them. After sequence alignment, it is identified that one of the strains numbered LWPGPB101 belongs to Paenibacillus pabuli of the genus Paenibacillus Paenibacillus pabuli , and the 16s rDNA sequence of this strain is shown in SEQ ID NO.1. This strain is preserved in the China General Microbiological Culture Collection Center, and the classification name is Paenibacillus pabuli , and the preservation number is CGMCC No.33517.

[0051] Example 2 Verification of the phosphorus and potassium solubilizing functions of LWPGPB101

[0052] 1. Verification of phosphorus-solubilizing function

[0053] (1) Select the above-screened LWPGPB101 as the experimental strain, and select Klebsiella variicola LWNF004 (preservation number: CGMCC No. 28351) with nitrogen-fixing function disclosed in Patent CN117004540 B as the control strain. Activate and culture the above strains in LB medium.

[0054] (2) Spot inoculate the activated LWPGPB101 and LWNF004 strains onto the NBRIP inorganic phosphorus solid medium respectively, observe the generation of phosphorus-solubilizing circles after culturing at 30 °C for 6 d, and measure the diameter of the phosphorus-solubilizing circle (D) and the colony diameter (d) by the cross method. Preliminarily determine the phosphorus-solubilizing ability of the strains according to the presence or absence of phosphorus-solubilizing circles and the value of D / d.

[0055] NBRIP inorganic phosphorus medium formula

[0056]

[0057] The results are shown in Table 1. LWPGPB101 can produce obvious phosphorus-solubilizing circles on the NBRIP inorganic phosphorus plate, and the phosphorus-solubilizing index can reach 1.31, while there is no obvious phosphorus-solubilizing phenomenon in LWNF004. The above results indicate that LWPGPB101 has a relatively obvious function of dissolving inorganic phosphorus.

[0058] Table 1 Solubility index of strains on phosphorus-solubilizing medium

[0059]

[0060] 2. Verification of potassium-solubilizing function

[0061] (1) Select the above-screened LWPGPB101 as the experimental strain, and select Klebsiella variicola LWNF004 (preservation number: CGMCC No. 28351) with nitrogen-fixing function disclosed in Patent CN117004540 B as the control strain. Activate and culture the above strains in LB medium.

[0062] (2) Spot inoculate the activated LWPGPB101 and LWNF004 onto the Aleksandrov potassium-solubilizing solid medium (Shandong Top Biological Engineering Co., Ltd., product number M2349B) respectively, observe the generation of potassium-solubilizing circles after culturing at 30 °C for 6 d, and measure the diameter of the potassium-solubilizing circle (D) and the colony diameter (d) by the cross method. Preliminarily determine the potassium-solubilizing ability of the strains according to the presence or absence of potassium-solubilizing circles and the value of D / d.

[0063] The results are shown in Table 2. LWPGPB101 can produce an obvious potassium-dissolving zone on the Aleksandrov potassium-dissolving culture medium plate, and the potassium-dissolving index can reach 1.60, which is significantly stronger than LWNF004. The above results indicate that the feed Bacillus sphaericus LWPGPB101 has a strong potassium-dissolving function.

[0064] Table 2 Solubility index of strains on potassium-dissolving culture medium

[0065]

[0066] Example 3 Effects of LWPGPB101 on plant growth

[0067] 1. Experimental method

[0068] The single-factor randomized block method was adopted, and a total of 3 experimental treatments were selected, namely adding LWPGPB101, LWNF004, and no bacteria (CK), with 4 replicates for each treatment.

[0069] (1) After activating LWPGPB101 and Klebsiella variicola LWNF004, they were respectively 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 with sterile PBS to adjust the concentration to 10 8 CFU / ml.

[0070] (2) Corn seeds were sown in the greenhouse, and the culture medium was a mixture of loess and vermiculite (mass ratio 25:1). Before sowing, the soil water content was ensured to be 70%. At 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 designed 3 experimental treatments.

[0071] (3) After sowing, the growth of corn seedlings was observed every day to evaluate the difference in growth promotion effects among treatments. At 2 weeks, 3 weeks, and 4 weeks of cultivation, the plant height, chlorophyll content, nitrogen content, plant fresh weight, plant dry weight, root morphology and other indexes of the plants were measured respectively.

[0072] 2. Experimental results

[0073] (1) Effects on corn plant height

[0074] As can be seen from Table 3 and Figure 2 it can be known that when the corn seedlings were cultured for 2 weeks, 3 weeks, and 4 weeks respectively, the plant heights of the treatment groups applied with LWPGPB101 were 14.05 cm, 27.28 cm, and 34.15 cm respectively, which were increased by 47.89%, 22.06%, and 20.54% respectively compared with the control group (CK) without applying the bacterial liquid; compared with the treatment group applied with LWNF004, they were also increased by 36.67%, 13.20%, and 8.93% respectively.

[0075] All of the above results indicate that Paenibacillus sp. LWPGPB101 in feed can promote the growth of maize seedlings.

[0076] Table 3 Effects on the plant height of maize

[0077]

[0078] (2)Effects on the chlorophyll content and nitrogen concentration of leaves

[0079] As can be seen from Table 4, when the maize seedlings were cultured for 3 weeks, the average chlorophyll content of the leaves in the treatment group applied with LWPGPB101 reached 39.90 SPAD, and the average nitrogen concentration of the leaves reached 15.29 mg / g. Compared with the control group (CK) without applying the bacterial solution, the average chlorophyll content and nitrogen concentration of the leaves increased by 14% and 11.36% respectively. Compared with the treatment group applied with LWNF004, the average chlorophyll content and nitrogen concentration of the leaves increased by 15.02% and 10.32% respectively.

[0080] When the maize seedlings were cultured for 4 weeks, the average chlorophyll content of the leaves in the treatment group applied 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 applying the bacterial solution, the average chlorophyll content and nitrogen content of the leaves increased by 6.6% and 5.23% respectively. Compared with the treatment group applied with LWNF004, the average chlorophyll content and nitrogen content of the leaves increased by 3.2% and 2.56% respectively.

[0081] The above results show that Paenibacillus sp. LWPGPB101 in feed can promote the growth of maize by improving the chlorophyll content and nitrogen content of maize leaves and enhancing leaf photosynthesis.

[0082] Table 4 Effects on the physiological indexes of maize leaves during the growth process

[0083]

[0084] (3)Effects on the dry matter and roots of maize

[0085] From Table 5 and Figure 3It can be seen that when the corn seedlings were cultured for 4 weeks, the fresh weight of plants, dry weight of plants, fresh weight of roots and nitrogen uptake of plants in the treatment group applied with LWPGPB101 were 1.96 g, 0.17 g, 2.78 g and 2.50 mg respectively, which were increased by 45.19%, 30.77%, 84.11% and 42.86% respectively compared with those of the control group (CK) without applying the bacterial solution in terms of fresh weight of plants, dry weight of plants, fresh weight of roots and nitrogen uptake of plants; compared with the treatment group applied with LWNF004, the fresh weight of plants, dry weight of plants, fresh weight of roots and nitrogen uptake of plants were increased by 5.95%, 6.25%, 4.91% and 14.16% respectively.

[0086] The above results indicate that Paenibacillus alimentarius LWPGPB101 can promote the dry matter accumulation of corn seedlings and nitrogen uptake, so as to achieve the growth-promoting effect on corn seedlings.

[0087] Table 5 Effects on the dry and fresh weights of plants and nitrogen uptake during the growth process of corn

[0088]

[0089] Example 4 Effects of LWPGPB101 on arbuscular mycorrhizal fungi in corn roots

[0090] (1) After culturing the corn seedlings in the treatment group applied with LWPGPB101 and the control group (CK) without applying the bacterial solution in Example 3 for 4 weeks, the corn root samples were collected respectively.

[0091] (2) Cut each root sample into 1-cm-long root segments, and water-bath in a 90 °C water bath with 10% KOH for 60 min to remove the cytoplasm and cell nuclei in the roots, making it easy to stain.

[0092] (3) After cooling, discard the KOH solution, wash the roots 3 - 5 times with clear water; then add 2% HCl solution to acidify for 5 min; after washing, add 0.05% trypan blue solution and stain in a 90 °C water bath for 30 min.

[0093] (4) After removing the trypan blue solution, add 1:1 lactic acid glycerol and decolorize at room temperature.

[0094] (5) Use forceps to pick up 15 decolorized root segments, arrange them neatly on the glass slide, make 2 glass slide samples for each treatment, and evaluate and classify the infection situation by microscopy and calculate the arbuscular mycorrhizal infection rate.

[0095] The results are as Figure 4As shown in Table 6, the AM mycorrhizal infection frequency of the maize roots in the treatment group treated with LWPGPB101 was 81.67%, the infection intensity was 27.33%, and the arbuscule abundance was 11.99%. Compared with the control group (CK) without applying the bacterial solution, the AM fungal infection frequency of the maize roots increased by 18%, the infection intensity increased by 3.2 times, and the arbuscule abundance increased by 11 times.

[0096] The above results indicate that the presence of Paenibacillus pabuli LWPGPB101 promotes the infection of plant roots by AM mycorrhiza and increases the number of arbuscules of AM mycorrhizal fungi symbiotic in plant roots, thereby directly or indirectly promoting the growth and development of plants.

[0097] Table 6 Effects on arbuscular mycorrhizal fungi in maize roots

[0098]

[0099] Example 5 Preparation of microbial bacterial fertilizer

[0100] For convenient storage and transportation, the Paenibacillus pabuli LWPGPB101 screened in Example 2 was prepared into a freeze-dried powder bacterial fertilizer. Skim milk powder, sodium glutamate, glycerol, sucrose and water were mixed in a mass ratio of (75:15:70:10:330) to make a freeze-drying protectant, which was added to the LWPGPB101 fermentation broth. Then, the bacterial precipitate was obtained by centrifugal separation and freeze-dried under sterile conditions for standby. The effective viable count of the Paenibacillus pabuli LWPGPB101 was 10 8 ~10 11 CFU / g, and it could be stored at room temperature for 6 months without a decrease in viability.

[0101] Although the present invention has been described in detail above with general descriptions and specific embodiments, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A feed bacillus, characterized in that Classification Name Paenibacillus pabuli , the preservation number is CGMCC No.33517. This feed Bacillus can enhance the infection of arbuscular mycorrhizal fungi in corn roots, increase the nitrogen absorption of corn, promote corn growth, and increase soluble phosphorus and soluble potassium in the soil.

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 corn 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 corn 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 corn 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 solubilizing composition, characterized in that: It comprises the feed Bacillus according to claim 1 or the microbial agent according to claim 2.

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