Complex microbial inoculant for promoting acid sugarcane soil improvement as well as preparation method and application of complex microbial inoculant

Abstract

The invention provides a complex microbial inoculant for promoting acid sugarcane soil improvement as well as a preparation method and application thereof, and belongs to the technical field of agricultural microorganisms. The complex microbial inoculant comprises bacillus amyloliquefaciens N15300, bacillus megatherium 2020055 and bacillus licheniformis 2020049, wherein the ratio of the effective viable count of the bacillus amyloliquefaciens N15300 to the effective viable count of the bacillus megatherium 2020055 to the effective viable count of the bacillus licheniformis 2020049 is (1.8-2.2): (1.8-2.2): (0.8-1.2), and the total effective viable count is greater than or equal to 1 * 10 The complex microbial inoculant can effectively act on acidic sugarcane soil in places such as Guangxi, has multiple functions of dissolving phosphorus, dissolving potassium and secreting cellulase and protease, and can activate fixed nutrients in the soil, improve the utilization rate of a fertilizer, neutralize the acidity of the soil and improve the pH value of the soil. Pot experiments show that the bacterial agent can significantly promote sugarcane root development and plant growth, improve the fertilizer utilization rate, and provide an effective way for realizing high and stable yield of sugarcane and green improvement of acid soil.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, and in particular to a compound microbial agent for improving acidic sugarcane soil, its preparation method, and its application. Background Technology

[0002] Guangxi is my country's largest sugarcane producing region, accounting for over 60% of the country's sugarcane and sugar production. The main soil types in Guangxi's sugarcane-growing areas are red soil and lateritic red soil, characterized by high soil development, poor water and fertilizer retention capacity, strong leaching, and predominantly acidic soils. Due to sugarcane's long growth cycle and high yield, it requires large amounts of nutrients such as nitrogen, phosphorus, and potassium, especially nitrogen, leading to a high dependence on chemical nitrogen fertilizers in sugarcane production. Driven by both high nitrogen fertilizer input and high biomass harvesting, Guangxi's sugarcane soils face a serious risk of acidification. The soil pH in most sugarcane-growing areas of Guangxi is generally weakly acidic (pH 4.5-5.5), with some major producing areas having an average pH below 4.5. This results in aluminum toxicity, nutrient imbalance, and decreased microbial activity, severely hindering the healthy development of the sugarcane industry.

[0003] Soil acidification remediation typically employs chemical amendment methods. Traditional methods primarily involve applying lime, but this method has a short effective period of only 3-6 months, requiring continuous application. Furthermore, large or prolonged application of lime can cause soil compaction and imbalances in the calcium, potassium, and magnesium elements in the soil, disrupting soil aggregate structure. Therefore, developing environmentally friendly biological amendment methods is crucial for the remediation of acidified soils. Utilizing microorganisms to remediate acidic soils is a current hot research topic in biological amendment measures. Applying microbial agents containing functional strains is an effective measure for acidification remediation. Existing microbial amendments mostly use single strains, which have drawbacks such as limited functionality and poor environmental adaptability; single strains also have limited effectiveness in improving multi-factor-related acidified soils. The development of synthetic biology provides new pathways and ideas for using microorganisms to promote soil health. By fully exploring and leveraging the metabolic diversity, functional stability, and environmental adaptability of microbial communities, and rationally constructing synthetic microbial communities, technical support can be provided for the ecological restoration of soils damaged by environmental pollution or degraded arable land. Constructing synthetic microbial communities using multiple functional strains can further enhance the stability and efficacy of the microbial community, demonstrating strong application potential. Summary of the Invention

[0004] In view of this, the present invention provides a compound microbial agent for improving acidic sugarcane soil, its preparation method, and its application, to solve the above-mentioned problems. The present invention constructs a synthetic microbial community composed of multiple strains that can improve the utilization rate of fertilizer by sugarcane and increase the pH value of acidic soil. The compound microbial agent developed from this microbial community achieves a three-in-one improvement of "phosphorus and potassium solubilization, acid reduction, and growth promotion" through the synergistic effect of multiple microbial species. This can significantly alleviate soil acidification, reduce fertilizer application, and promote healthy sugarcane growth.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a compound microbial agent for promoting the improvement of acidic sugarcane soil, comprising Bacillus amyloliquefaciens N15300, Bacillus megaterium 2020055 and Bacillus licheniformis 2020049.

[0007] Preferably, the compound microbial agent is available in powder and liquid forms; the ratio of the effective viable counts of Bacillus amyloliquefaciens N15300, Bacillus megaterium 2020055 and Bacillus licheniformis 2020049 is 1.8~2.2:1.8~2.2:0.8~1.2.

[0008] This invention provides the application of the aforementioned compound microbial agent in at least one of the following:

[0009] (1) Application in improving acidic soils;

[0010] (2) Application in promoting plant growth;

[0011] (3) Application in the preparation of microbial agents for improving acidic soil;

[0012] (4) Application in the preparation of microbial agents that promote plant growth.

[0013] The present invention also provides a method for preparing the compound microbial agent, comprising the following steps:

[0014] (1) Bacillus amyloliquefaciens N15300, Bacillus megaterium 2020055 and Bacillus licheniformis 2020049 were inoculated into TSB solid medium and cultured at 28-30℃ for 18-24h to obtain slant cultures;

[0015] (2) Pick out the slant culture and inoculate it into TSB liquid medium, and incubate at 28~30℃ and 150~200 rpm for 18~24h to obtain seed culture;

[0016] (3) Inoculate the seed culture into TSB liquid fermentation medium at an inoculation rate of 1-3% and culture at 28-30℃ and 150-200 rpm for 18-24h to obtain single bacterial culture;

[0017] If the compound bacterial agent is a liquid, the single bacterial suspensions of the three bacteria are mixed at a volume ratio of 1.8~2.2:1.8~2.2:0.8~1.2, or the single bacterial suspensions of the three bacteria are centrifuged, resuspended, and mixed at a volume ratio of 1.8~2.2:1.8~2.2:0.8~1.2 to obtain the compound bacterial agent.

[0018] If the compound bacterial agent is a powder, centrifuge the single bacterial liquids of the three bacteria to obtain bacterial cells, dry them, and then mix them in a mass ratio of 1.8~2.2:1.8~2.2:0.8~1.2 to obtain the final product.

[0019] Preferably, the effective viable count in the single bacterial culture is 4~6×10⁻⁶. 8 cfu / mL.

[0020] Preferably, the centrifugation speed is 7000~8000 rpm and the centrifugation time is 1~2 min.

[0021] Preferably, the drying temperature is 38~42℃ and the drying time is 24~48h.

[0022] Preferably, the effective viable bacteria count in the bacterial suspension is 4~6×10⁻⁶. 8 cfu / mL.

[0023] The present invention also provides a method for promoting plant growth, wherein the compound microbial agent is applied to the plant roots.

[0024] Preferably, the plant includes sugarcane.

[0025] By adopting the above technical solution, the present invention has the following beneficial effects: The compound microbial agent of the present invention includes Bacillus amyloliquefaciens N15300, Bacillus megaterium 2020055, and Bacillus licheniformis 2020049, and the ratio of their effective viable bacteria counts is 1.8~2.2: 1.8~2.2: 0.8~1.2, with a total effective viable bacteria count ≥ 1×10⁻⁶. 9 CFU / mL. This compound microbial agent is effective in acidic sugarcane soils in Guangxi and other regions. It has multiple functions, including phosphorus solubilization, potassium solubilization, and secretion of cellulase and protease. It can activate nutrients fixed in the soil, improve fertilizer utilization, neutralize soil acidity, and increase soil pH. Pot experiments show that this microbial agent can significantly promote sugarcane root development and plant growth, improve fertilizer utilization, and provide an effective way to achieve high and stable sugarcane yields and green improvement of acidic soils. Attached Figure Description

[0026] Figure 1 The pH changes of the culture system after culturing three strains of bacteria under different acid and alkaline conditions are shown.

[0027] Figure 2 The graph shows the degradation effect of inorganic phosphorus by strains N15300, 2020055, and 2020049.

[0028] Figure 3 The graph shows the degradation effect of strains N15300, 2020055, and 2020049 on insoluble potassium.

[0029] Figure 4 This is a graph showing the enzyme activity analysis of strain 2020049.

[0030] Figure 5 A streak plate plot used to determine whether strains N15300, 2020055, and 2020049 have an antagonistic relationship.

[0031] Biological Preservation Instructions

[0032] The taxonomic name of Bacillus amyloliquefaciens N15300 is Bacillus amyloliquefaciens, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 20426, deposited on August 14, 2020, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0033] The taxonomic name of Bacillus megaterium 2020055 is Bacillus megaterium. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36045, on September 23, 2020, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0034] The taxonomic name of Bacillus licheniformis 2020049 is Bacillus licheniformis. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36045, on June 8, 2021, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1. Acid tolerance of strains N15300, 2020055, and 2020049 and their effect on pH regulation of the growth environment.

[0037] Three bacterial strains were streaked in solid TSB medium and cultured in a 30°C incubator. After single colonies grew, each colony was picked and inoculated into 10 mL of liquid TSB medium and cultured in a shaker at 28°C and 170 rpm for 24 h to obtain the seed culture.

[0038] The seed culture was inoculated at a rate of 1% into liquid TSB medium at pH 4.53, 5.04, and 6.03, respectively, with each bottle containing 50 mL of the medium. The cultures were incubated at 28°C and 170 rpm for 48 h on a shaker. The OD values ​​of each treatment were then measured. 600 The pH changes of the culture system were also studied. Under the same conditions, a blank control without inoculation was used, and each treatment was repeated in triplicate. The results are shown in Table 1.

[0039] Table 1. Results of acid tolerance and pH regulation of the tested strains.

[0040] The results show that ( Figure 1 All three strains exhibited some acid tolerance and could grow normally in a slightly acidic environment with a pH of 5.04. Among them, strains N15300 and 2020049 could grow normally in a culture medium with a pH of 4.53, showing strong acid tolerance. After the culture of all strains was completed, the pH of the culture system increased significantly, with strain N15300 showing the most significant effect: the culture medium with an initial pH of 4.53 reached an average pH of 6.35 after 48 hours of culture with strain N15300, an increase of 1.82 units.

[0041] Example 2: Phosphorus- and potassium-solubilizing characteristics of the tested strains

[0042] (1) Degradation characteristics of inorganic phosphorus by the strain

[0043] After activating the three bacterial strains, they were inoculated into TSB liquid medium and cultured to produce OD. 600 =1.0 seed culture, take 20µL of bacterial suspension of the test strain and spot inoculate it onto a plate containing Pikovaskaia's medium (10g glucose, 5g Ca3(PO4)2, 0.5g (NH4)SO4, 0.2g NaCl, 0.2g KCl, 0.3g MgSO4•7H2O, 0.03g MnSO4, 0.03g FeSO4•7H2O, 0.5g yeast extract, 14g agar, 1000mL distilled water, pH 7.0). After incubation at 28℃ for 3 days, observe the growth of the strain and whether a clear zone forms around the colony. Measure the diameter of the clear zone (D) and the diameter of the colony (d).

[0044] The measurement results show that... Figure 2Strains N15300 and 2020055 produced large phosphate-solubilizing zones on Pikovaskaia's medium, demonstrating that these two strains have good inorganic phosphorus degradation capabilities, while strain 2020049 produced smaller phosphate-solubilizing zones, indicating a slightly weaker phosphorus-solubilizing effect.

[0045] (2) Detection of potassium solubilization characteristics of the strain

[0046] Using the same method described above, 20 µL of bacterial suspension of the test strain was inoculated onto a potassium-solubilizing bacteria selection medium plate (10.0 g glucose, 0.2 g Na2HPO4, 0.2 g MgSO4·7H2O, 0.2 g NaCl, 0.2 g CaSO4·2H2O, 5.0 g CaCO3, 14.0 g agar, 2.5 g potassium feldspar powder (150 mesh), 1000 mL deionized water, pH 7.0). The potassium-solubilizing ability of the strain was determined based on its growth and whether a clear zone formed around the colony.

[0047] The measurement results are shown below. Figure 3 All three strains grew well on a culture medium containing potassium feldspar powder and produced large transparent zones, demonstrating their good potassium-active properties.

[0048] Example 3: Activity characteristics of organic substrate degradation-related enzymes secreted by strain 2020049

[0049] After activating strain 2020049, OD 600 A bacterial suspension with a concentration of 1.0 was inoculated into TSB medium at an inoculum rate of 1%, and cultured with shaking at 170 rpm and 28°C until the bacterial OD reached the target value. 600 =1.0, OD 600 The bacterial culture with a concentration of 1.0 was centrifuged at 7500 rpm for 10 min, the supernatant was discarded, and the culture was resuspended in an equal volume of sterile water. The resuspended culture was inoculated at a 3% inoculum into a medium with soybean meal as the sole nitrogen source (medium formulation: 2% soybean meal, 1% glucose, 0.4% Na₂HPO₄·12H₂O, 0.03% KH₂PO₄, 0.1% CaCl₂, pH=6.0), and cultured in a shaker at 170 rpm and 30℃ until the logarithmic growth phase. After the culture was complete, the fermentation broth was centrifuged at 12000 rpm for 10 min at 4℃, and the supernatant was retained as the crude enzyme solution. The activity of sodium carboxymethyl cellulose enzyme was determined using the dinitrosalicylic acid (DNS) method with sodium carboxymethyl cellulose as the substrate; the activity of the protease was determined using the Folin-Ciocalteu method with a complex protein as the substrate; each treatment was performed in triplicate.

[0050] The results are as follows Figure 4As shown, the activities of protease and cellulase in the fermentation broth were significantly increased, with strain 2020049 exhibiting the strongest enzyme activity, indicating that the strain can efficiently decompose organic matter such as straw and crude protein by synthesizing and secreting cellulase and protease.

[0051] Example 4: Survival rate determination of bacterial strains in acidic soil

[0052] Soil suspension preparation: Acidic red and yellow soil was taken from the top 5-15 cm of sugarcane field in Xinwei Town, Binyang County, Nanning City. It was naturally air-dried, and 5g of soil was added to each Erlenmeyer flask with 25mL of distilled water. After sterilization at 121℃, the natural pH was measured to be 4.46. Another soil suspension of the same type was taken and the pH was adjusted to 7.0 with NaOH as a control.

[0053] OD of 3 test strains 600 =1.0 seed solution was inoculated into the above soil suspension, with 0.3 mL of seed solution inoculated into each bottle. Three bottles were inoculated for each strain and control treatment. After inoculation, the culture was shaken at 28 ℃ and 170 rpm. Samples were taken at 0, 5, 10 and 15 days after inoculation and viable counts were performed using the dilution plate method. The results are shown in Table 2.

[0054] Table 2. Viable bacterial count of the strain in acidic soil

[0055] The results in Table 2 show that all three strains of bacteria can survive stably in acidic soil.

[0056] Example 5: Antagonistic effects among synthetic bacterial strains

[0057] After activation, the strains were streaked in solid TSB medium and incubated at 30°C. Once single colonies were formed, three single colonies were picked up with an inoculation loop and streaked in a crisscross pattern onto solid TSB medium. The cultures were then incubated at 30°C.

[0058] The experimental results are shown in Figure 5 As shown, there is no antagonistic effect between any two strains of N15300, 2020055, and 220049 in the complex bacterial community, indicating that the three strains in the community can coexist.

[0059] Example 6: Preparation of Compound Microbial Agent

[0060] The compound microbial agent of the present invention has three forms: compound microbial powder, compound microbial liquid, and compound microbial suspension, and the preparation methods are as follows:

[0061] 1. Preparation of compound microbial powder

[0062] Strains N15300, 2020055, and 2020049 were streaked in solid TSB medium and cultured in a 30°C incubator. After single colonies grew, a single colony was picked and inoculated into 10 mL of liquid TSB medium and cultured in a shaker at 28°C and 170 rpm for 24 h to obtain the seed culture.

[0063] The seed culture was inoculated into TSB liquid medium at a 1% inoculation rate and cultured at 28℃ and 170 rpm with shaking for 18-24 hours until the effective viable count in the fermentation broth reached 5 × 10⁻⁶. 8 CFU / mL indicates a single bacterial suspension. After centrifuging the suspension at 7500 rpm for 1 min, resuspend it in sterile water to achieve a concentration of 5.0 × 10⁻⁶. 8 CFU / mL indicates a single-strain bacterial suspension. After centrifuging the single-strain suspension at 7500 rpm for 1 min, the resulting bacterial cells are sealed with sterile gauze and dried in a vacuum drying oven at 40℃ for 24–48 h until completely dry. The dried cells are then ground into powder in a mortar to obtain the single-strain bacterial powder. The bacterial powders of strains N15300, 2020055, and 2020049 are mixed at a mass ratio of 2:2:1 to obtain a compound bacterial agent.

[0064] 2. Preparation of compound bacterial solution

[0065] The single bacterial cultures of the three strains were mixed at a volume ratio of 2:2:1 to obtain a composite bacterial culture.

[0066] 3. Preparation of compound bacterial suspension

[0067] The concentration of the single bacterial suspension was adjusted to 5.0 × 10⁻⁶. 8 The single bacterial suspensions of strains N15300, 2020055 and 2020049 were mixed at a volume ratio of 2:2:1 to obtain a composite bacterial solution.

[0068] Example 7: Microbial agents improve sugarcane fertilizer utilization, alleviate soil acidification, and promote sugarcane plant growth.

[0069] Using the sugarcane variety "Yuetang 00236" as the inoculation target, single-bud seedlings were cultivated from sugarcane stalks. Thirty days after sowing, seedlings with similar growth were selected and transplanted into cultivation pots, with one planter per pot. The planting soil was acidified sugarcane soil from Xinwei Town, Binyang County, with a pH of 4.46, and each pot contained 5 kg of soil. Four treatments were implemented for the sugarcane:

[0070] The blank control group CK0 received no fertilizer;

[0071] The conventional fertilization treatment group CK1 was given "2 g of urea (N%=46%) + 3 g of compound fertilizer (N / P2O5 / K2O, 15-15-15)" per pot as base fertilizer and seedling fertilizer according to the fertilization rate during the sugarcane seedling stage;

[0072] Group T0, treated with microbial agents, was inoculated with microbial agents but did not receive chemical fertilizers.

[0073] The microbial agent treatment group T1 was treated with conventional fertilization and microbial agent inoculation at the same time; each treatment consisted of 8 pots.

[0074] The method of applying the bacterial agent is as follows: Dilute the compound bacterial suspension prepared in Example 6 with water to prepare an effective viable count of 1×10⁶. 6 On the day of sugarcane transplanting, the bacterial solution of the bacterial agent treatment group T0 and the bacterial agent treatment group T1 was used to irrigate the roots of each pot with 200 mL of bacterial solution. Fifteen days after transplanting, the roots were irrigated again with the same concentration and amount of bacterial solution. The blank control group CK0 and the conventional fertilization treatment group CK1 were irrigated with the same amount of water.

[0075] Sugarcane plant height was measured 60 days after transplanting; root soil was collected and pH was measured; plants were harvested and separated into above-ground parts (stems and leaves) and underground parts (roots and base of underground stems), and fresh and dry weights were measured separately. The N, P, and K contents of each part were further determined. The fertilizer utilization rate of sugarcane was calculated using the formula "Fertilizer utilization rate % = [(Nutrients absorbed by crop in fertilized area - Nutrients absorbed in blank area) ÷ Total amount of fertilizer applied] * 100". The results are shown in Table 3.

[0076] Table 3. Effects of microbial inoculant treatment on soil pH, plant growth, and fertilizer utilization in sugarcane.

[0077] The results in Table 3 show that, under no fertilization conditions, the rhizosphere soil pH of the microbial agent treatment group T0 was 0.5 units higher than that of the blank control CK0. Under normal fertilization conditions, the rhizosphere soil pH of the microbial agent treatment group T1 was 0.74 units higher than that of the single chemical fertilizer treatment, the plant height increased by 14.84%, and the dry weight increased by 28.54%. The utilization rates of N / P2O5 / K2O in chemical fertilizers by sugarcane plants in the microbial agent treatment group T1 were 57.84%, 45.21%, and 89.74%, respectively, which were significantly higher than those in the control group CK1 (30.28%, 25.27%, and 40.96%).

[0078] As can be seen from the above embodiments, the present invention provides a compound microbial agent for improving acidic sugarcane soil, its preparation method and application. The compound microbial agent of the present invention can effectively increase the pH value of sugarcane soil, promote plant growth and improve fertilizer utilization.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound microbial agent for promoting the improvement of acidic sugarcane soil, characterized in that, This includes Bacillus amyloliquefaciens N15300, Bacillus megaterium 2020055, and Bacillus licheniformis 2020049.

2. The compound microbial agent according to claim 1, wherein the dosage form of the compound microbial agent includes powder and liquid; the ratio of the effective viable counts of Bacillus amyloliquefaciens N15300, Bacillus megaterium 2020055 and Bacillus licheniformis 2020049 is 1.8~2.2:1.8~2.2:0.8~1.2, and the total effective viable count is ≥1×10⁻⁶. 9 cfu / mL.

3. The application of the compound microbial agent according to claim 1 or 2 in at least one of the following: (1) Application in improving acidic soils; (2) Application in promoting plant growth; (3) Application in the preparation of microbial agents for improving acidic soil; (4) Application in the preparation of microbial agents that promote plant growth.

4. The method for preparing the compound microbial agent according to claim 1 or 2, characterized in that, Includes the following steps: (1) Bacillus amyloliquefaciens N15300, Bacillus megaterium 2020055 and Bacillus licheniformis 2020049 were inoculated into TSB solid medium and cultured at 28-30℃ for 18-24h to obtain slant cultures; (2) Pick up the slant culture and inoculate it into TSB liquid medium, and incubate at 28~30℃ and 150~200 rpm for 18~24h to obtain seed culture; (3) Inoculate the seed culture into TSB liquid fermentation medium at an inoculation rate of 1-3% and culture at 28-30℃ and 150-200 rpm for 18-24h to obtain single bacterial culture; If the compound bacterial agent is a liquid, then the single bacterial solutions of the three bacteria are mixed, or the single bacterial solutions of the three bacteria are centrifuged and resuspended, and then mixed to obtain the product. If the compound bacterial agent is a powder, centrifuge the single bacterial cultures of the three bacteria to obtain bacterial cells, dry them, and then mix them to obtain the final product.

5. The method for preparing the compound microbial agent according to claim 4, characterized in that, The effective viable count in the single bacterial culture is 4~6×10⁻⁶. 8 cfu / mL.

6. The method for preparing the compound microbial agent according to claim 4, characterized in that, The centrifugation speed is 7000~8000 rpm, and the centrifugation time is 1~2 min.

7. The method for preparing the compound microbial agent according to claim 4, wherein the drying temperature is 38~42℃ and the drying time is 24~48h.

8. The method for preparing the compound microbial agent according to claim 4, characterized in that, The effective viable bacteria count in the bacterial suspension is 4~6×10⁻⁶. 8 cfu / mL.

9. A method for promoting plant growth, characterized in that, The compound microbial agent according to claim 1 or 2 is applied to the roots of plants.

10. The method according to claim 9, characterized in that, The plant mentioned includes sugarcane.

Images