Preparation method and application of algal-bacterial complex microbial agent against ultraviolet stress

Abstract

The application belongs to the technical field of microbial compound microbial inoculants and fermentation, and particularly relates to a preparation method and application of an algal-bacterial compound microbial inoculant against ultraviolet stress. The algal-bacterial compound microbial inoculant is prepared by mixing Bacillus thuringiensis NBIN-863 and microthamnus with a preservation number of CCTCC NO: M 20251992. The microbial inoculant enhances the ultraviolet resistance through algal-bacterial metabolic interaction, the microthamnus provides physical shielding and antioxidant protection, and the bacillus secretes nematocidal substances, thereby achieving the dual effects of preventing and treating root-knot nematodes and improving soil nutrients. In addition, the microbial inoculant is expected to be used for fermenting and preparing a small-molecule acid 3-methylthiopropionic acid which inhibits pests.

Description

Technical Field

[0001] This invention belongs to the field of microbial compound inoculants and fermentation technology. Specifically, it relates to a method for preparing an algae-bacterial compound inoculant resistant to ultraviolet stress and its application. This compound inoculant can promote the formation of soil micro-aggregates, improve soil pore structure and water and fertilizer retention capacity, and provide a good micro-ecological environment for crop roots. In addition, this compound inoculant can be used to ferment and prepare the small molecule acid 3-methylthiopropionic acid for further use in pest control. Background Technology

[0002] Root-knot nematode diseases, among the most destructive soil-borne diseases in global agricultural production systems, have severely damaged over 3,000 crops, directly causing annual global economic losses exceeding US$70 billion. They are listed by the Food and Agriculture Organization of the United Nations as one of the most threatening plant pathogens. Root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera spp. and Globodera spp.) are the most damaging. Currently, chemical control still accounts for over 80% of the nematode control market. However, the long-term reliance on non-fumigation nematicides (such as abamectin and thiazophos) has led to nematode resistance, soil microecological disturbances, and risks to agricultural product quality and safety, becoming a prominent bottleneck restricting sustainable agricultural development. Therefore, the development of green microbial agents based on functional microorganisms is widely recognized as a key path to breaking through the limitations of chemical control.

[0003] Bacillus strains exhibit unique advantages in the biological control of root-knot nematodes due to their broad-spectrum antibacterial activity. Our team obtained Bacillus thuringiensis NBIN-863 through high-throughput screening. Its secreted small-molecule acid (3-meththiopropionic acid) not only has a high attraction to root-knot nematodes but also effectively inhibits the hatching of nematode egg masses. Under laboratory conditions, the inoculant showed a mortality rate of over 80% against root-knot nematodes and reduced the root disease index of tomato seedlings by over 90%. However, field trials showed that the survival period of this strain was significantly shortened in complex soil environments and it rapidly inactivated under stresses such as high temperature and ultraviolet radiation. Furthermore, its limited functionality restricts its individual application value; in actual production, it often needs to be combined with other inoculants or fertilizers, which may weaken its insecticidal efficacy. The common technical challenges of weak strain colonization and limited functionality have become core pain points restricting the large-scale field application of microbial pesticides.

[0004] In recent years, the synergistic effects of algae-bacteria synergistic technology, generated through metabolic interactions, have been validated in multiple fields. Preparing algae and bacteria into algae-bacteria coupled agents and applying them in combination can not only promote the growth, productivity, and quality of crops such as soybeans and corn, but also significantly increase the content of available nutrients in the soil.

[0005] Therefore, there is an urgent need to construct a multifunctional compound microbial agent that can significantly enhance the stress resistance and soil colonization of Bacillus thuringiensis NBIN-863, so that it can have the dual effects of insecticidal and soil-enriching functions, and is also expected to be used for fermentation to prepare the small molecule acid 3-methylthiopropionic acid, a metabolite that inhibits pests. Summary of the Invention

[0006] One of the objectives of this invention is to provide an algae-bacterial compound agent comprising Bacillus thuringiensis NBIN-863 and microalgae.

[0007] Preferably, the microalgae is Microcoleus subtorulosus.

[0008] Preferably, the preservation number of the microsheath algae is CCTCC NO: M 20251992.

[0009] Preferably, the preparation method of the algae-bacterial compound inoculant includes the following steps: taking 100 mL of a solution with a density of 8×10⁻⁶... 8 A mixture of CFU / mL Bacillus thuringiensis bacterial solution and 6g wet weight microalgae mud was used to obtain a compound bacterial agent.

[0010] Preferably, the 6g wet weight microalgae mud is obtained by concentrating 4L of algal solution with a chlorophyll a concentration of 3.2 mg / L.

[0011] In another preferred embodiment of the present invention, the present invention also provides the application of the algae-bacterial compound agent described herein in the preparation of insecticides.

[0012] Preferably, the insecticide is used to control root-knot nematodes.

[0013] In another preferred embodiment of the present invention, the present invention also provides the application of the algae-bacterial compound agent described herein in improving soil nutrients.

[0014] In another preferred embodiment of the present invention, the present invention also provides a fermentation preparation process for the small molecule acid 3-methylthiopropionic acid, which includes culturing the algae-bacterial composite agent described in the present invention under suitable conditions and collecting the metabolite small molecule acid 3-methylthiopropionic acid.

[0015] Compared with the prior art, the main advantages of this invention are: ① In the application of the algae-bacterial compound agent provided by this invention, microalgae and Bacillus are combined, and the insecticidal effect is significantly improved, indicating that there is a significant synergistic effect between the two. ② The innovation of the algae-bacterial compound microbial agent provided by this invention lies in: compounding Bacillus thuringiensis NBIN-863 with Microcoleina in a specific ratio to form a compound microbial system with both disease and pest prevention and growth promotion functions. Wherein: (a) The Bacillus thuringiensis NBIN-863 described herein can stably colonize in the soil and continuously secrete small molecule organic acids with nematicidal activity, significantly inhibiting the infection and reproduction of root-knot nematodes (Meloidogyne spp.), thereby protecting crop roots; (b) The microsheath algae fix carbon efficiently through photosynthesis and increase soil nitrogen reserves through biological nitrogen fixation. At the same time, they secrete a large amount of extracellular polysaccharides (EPS), which promote the formation of soil micro-aggregates, improve soil pore structure and water and fertilizer retention capacity, and provide a good micro-ecological environment for crop roots. (c) The two types of microorganisms form a synergistic effect in the composite system: the improved soil microenvironment created by the microsheath algae further enhances the colonization rate and activity of NBIN-863, while the metabolites of NBIN-863 can promote the growth of microsheath algae and carbon / nitrogen fixation efficiency, thus achieving multiple functions of "insect prevention + soil fertility" in the same application process, overcoming the shortcomings of traditional microbial agents with single functions.

[0016] ③ This invention further reveals that the algae-bacterial compound agent, through the synergistic interaction between microsheath algae and Bacillus thuringiensis NBIN-863, can maintain the high survival rate and insecticidal activity of NBIN-863 under ultraviolet radiation stress; wherein, microsheath algae effectively alleviate ultraviolet damage by forming physical shielding, secreting ultraviolet absorbing substances and antioxidant components, significantly prolonging the functional retention period of NBIN-863 in the field, overcoming the defect of single agent being easily inactivated by light, resulting in a decrease in control efficacy.

[0017] The algae-bacterial compound of the present invention has a stronger insecticidal effect than NBIN-863. It is speculated that the addition of Microsheath algae further promotes the production of the insecticidal active ingredient, the small molecule acid 3-methylthiopropionic acid. It can be seen that the compound agent can be used for fermentation to prepare 3-methylthiopropionic acid.

[0018] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0019] Figure 1 The effect of adding microsheath algae on the insecticidal efficiency of Bacillus.

[0020] Figure 2 This study investigates the effect of ultraviolet stress on the insecticidal efficiency of algae-bacterial compound agents.

[0021] Figure 3 This refers to the change in the number of viable bacteria in the rhizosphere of tomato seedlings treated with the algae-bacterial compound inoculant NBIN-863. Detailed Implementation

[0022] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0023] Biomaterials: Microsheath algae FK04: From Hubei Provincial Biopesticide Engineering Center, accession number CCTCC NO: M20251992, deposited on September 9, 2025. Bacillus thuringiensis NBIN-863: from Hubei Provincial Biological Pesticide Engineering Center, preservation number CCTCCNO: M 2013612.

[0024] BG-11 culture medium, by weight, comprises: 500-800 parts NaNO3, 8-15 parts K2HPO4, 15-20 parts MgSO4·7H2O, 1-2 parts CaCl2·2H2O, 0.1-0.2 parts C6H8O7, and 0.1-0.2 parts ((NH4)2) x Fe γ (C6H4O7) r 0.1-0.2 parts of EDTA-Na2, 2-3 parts of Na2CO3 and 10-30 parts of A5 culture medium.

[0025] Example 1: Preparation of microalgae The photoautotrophic culture was carried out in BG-11 medium.

[0026] 10 mL of sterile *Microsheatha* (CCTCCM20251992) with a chlorophyll a concentration of 0.05–0.08 mg / L was cultured in 4 L of BG11 liquid medium at 25 °C, with a light intensity of 2200 lux and a photoperiod of 18 h:6 h light-dark ratio. After reaching the logarithmic growth phase, a sterile *Microsheatha* culture solution was obtained. This culture solution was centrifuged at 6000 rpm for 10 min and resuspended three times with ultrapure water to obtain *Microsheatha* algal sludge (6 g wet weight corresponds to 4 L of algal solution with a chlorophyll a concentration of 3.2 mg / L). This process emphasizes aseptic operation to avoid contamination and can be combined with a photobioreactor to achieve high-density cultivation. In addition, Bacillus fermentation waste liquid can be used as a nitrogen source for microalgae cultivation, constructing a production cycle of "Bacillus fermentation - waste liquid resource utilization - microalgae cultivation - high-value utilization of algae". This approach can not only significantly reduce the cultivation cost of microalgae, but also achieve efficient purification of wastewater.

[0027] Example 2 Preparation of functional Bacillus subtilis S1. Slant culture of bacterial strain: Bacillus thuringiensis NBIN-863 was inoculated onto LB solid slant medium containing 5% agar by streaking and cultured at 37°C for 24 h to obtain the bacterial strain; S2. Seed culture: The strain was transferred to seed culture medium and cultured at 37 ℃ and 200 rpm for 24 h by shaking to obtain seed liquid; S3. Fermentation culture: The secondary seed culture was inoculated into LB medium at a volume ratio of 2%, and fermented at 37℃ and 200rpm for 48-72h until the logarithmic growth phase was reached to obtain Bacillus bacterial culture. The LB medium consisted of: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, with a pH of 7.2. The seed culture medium consisted of: 10 g / L anhydrous glucose, 10 g / L peptone, 5 g / L yeast extract, 5 g / L potassium dihydrogen phosphate, 2 g / L calcium carbonate, and 1 g / L ammonium sulfate.

[0028] By controlling the aeration rate (200 rpm) and maintaining pH stability, cell autolysis was prevented, and the cell density was increased to 8 × 10⁸. 8 CFU / mL. This process is suitable for large-scale production. By using soybean flour, corn steep liquor, etc. as basic carbon and nitrogen sources, and by gradually increasing the stirring speed (e.g., 300-600 rpm) and aeration rate (0.8-1.5 vvm), dissolved oxygen is maintained above 20% to ensure high-density cell growth and product synthesis, thereby further improving the yield and achieving large-scale, low-cost fermentation of NBIN-863.

[0029] Example 3: Preparation method of algae-bacterial compound inoculant Take 100 mL of solution with a density of 8 × 10⁻⁶ 8 A mixture of CFU / mL Bacillus thuringiensis NBIN-863 bacterial suspension and 6g wet weight of Microsheath algae mud was shaken at 200 rpm for 2 hours to obtain an algae-bacterial composite microbial agent.

[0030] Example 4: Verification of the synergistic effect between Bacillus and microalgae Nematode egg masses were collected from the roots of tomatoes infected with southern root-knot nematodes. These egg masses were placed in a petri dish containing 15 mL of sterile water and incubated in the dark at 20°C to observe nematode hatching. Nematodes were collected by centrifugation, and their concentration was calculated. The prepared algae-bacterial compound agent was filtered through a membrane (0.25 μm), and the filtrate was added to a 96-well plate. 30-40 nematodes were then added to each well, for a total bioassay volume of 100 μL. Sterile water was used as a negative control, and NBIN-863 alone was used as a positive control. Each group was replicated five times. After 24 h of incubation in the dark at 20°C, the mortality rate of the segmented nematodes was determined by microscopic examination. Figure 1 As shown, the results indicate that the insecticidal efficiency of the algae-bacterial compound agent (84.8%) was significantly higher than that of the single-bacterial group (76.7%).

[0031] Example 5: Verification of the UV stress resistance of the algae-bacterial compound inoculant. The insecticidal activity of the algae-bacterial composite agent prepared in Example 3 under ultraviolet irradiation was tested using the method shown in Example 4, and the results are as follows: Figure 2 As shown, after 105 minutes of UV irradiation (intensity 125 μW / cm²), 2 (At a distance of 15cm), the number of viable bacteria of NBIN-863 in the algae-bacterial compound was 22 times that of the control, and the insecticidal activity of the algae-bacterial compound decreased by only about 10%, which was much lower than the 30% of the control group. This indicates that by mixing Microsheatha (CCTCCM20251992) with Bacillus thuringiensis NBIN-863 as an algae-bacterial compound, the UV resistance of Bacillus thuringiensis NBIN-863 can be effectively improved.

[0032] Example 6: Effect of Algae-Bacterial Compound Inoculant on the Abundance of Root Microbiota in Tomato Seedlings The effect of the algae-bacterial compound inoculant prepared in Example 3 on the abundance of root microbiota in tomato seedlings was investigated, and the results are as follows: Figure 3 As shown, after 50 days of pot cultivation, the abundance of NBIN-863 in the root system of tomato seedlings was analyzed. The results showed that the colony count of NBIN-863 in the algae-bacteria complex group was 3.27 × 10⁻⁶. 8 The CFU / g ratio was 6 times that of the single-strain group, indicating that the algae-bacterial compound agent of the present invention can effectively increase the abundance of NBIN-863 in the root system of tomato seedlings.

[0033] Example 7: Effects of Algae-Bacterial Compound Inoculant on Soil Nutrients The effect of the algae-bacterial compound inoculant prepared in Example 3 on soil nutrients was tested. After 50 days of pot cultivation, compared with the negative control (water) and the positive control (NBIN-863 single bacteria), the addition of the algae-bacterial compound inoculant significantly increased the available carbon and nitrogen nutrient content in the soil, especially the rhizosphere soil. This indicates that during the cultivation of tomato seedlings, the algae-bacterial compound inoculant of the present invention can significantly increase the available carbon and nitrogen nutrient content in the soil.

[0034] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. An algae-bacterial compound inoculant, comprising Bacillus thuringiensis (Bt). Bacillus thuringiensis NBIN-863 and Microcoleina ( Microcoleus subtorulosus FK04, the Bacillus thuringiensis NBIN-863 has the accession number CCTCC NO: M 2013612, and the Microcoleopterus f. FK04 has the accession number CCTCC NO: M 20251992.

2. The algae-bacterial compound agent according to claim 1, characterized in that, The preparation method of the algae-bacterial compound inoculant includes the following steps: take 100 mL of a solution with a density of 8×10 8 A compound bacterial agent of algae and bacteria was obtained by mixing Bacillus thuringiensis NBIN-863 bacterial solution (CFU / mL) with 6g of Microsheatha f. K04 algal mud.

3. The algae-bacterial compound agent according to claim 2, characterized in that, The 6g wet weight of the Microsheatha FK04 algal sludge was obtained by concentrating 4L of Microsheatha FK04 algal solution with a chlorophyll a concentration of 3.2 mg / L.

4. The application of the algae-bacterial compound agent according to any one of claims 1-3 in the preparation of insecticides, characterized in that, The insecticide is used to control southern root-knot nematodes.

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