Preparation method and application of ultraviolet stress resistant phycomycete complex microbial inoculant

Abstract

The invention belongs to the technical field of microbial complex microbial inoculants and fermentation, and particularly relates to a preparation method and application of an ultraviolet stress resistant phycomycetes complex microbial inoculant, and the phycomycetes complex microbial inoculant is prepared by mixing bacillus thuringiensis NBIN-863 and microcoleus with the preservation number of CCTCC NO: M 20251992. According to the microbial agent, the ultraviolet resistance is enhanced through phycomycete metabolism interaction, the microcoleus provides physical shielding and antioxidant protection, and the bacillus secretes nematode killing substances, so that the dual effects of preventing and treating root-knot nematodes and improving soil nutrients are achieved. The microbial agent is expected to be used for preparing metabolite micromolecular acid 3-methylthiopropionic acid for inhibiting pests through fermentation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial complex inoculant and fermentation technology, and particularly relates to a preparation method and application of an algal-bacterial complex inoculant against ultraviolet stress. The complex inoculant can promote the formation of soil micro-aggregates, improve the soil pore structure and water and fertilizer retention capacity, and provide a good micro-ecological environment for crop root systems. In addition, the complex inoculant can be used for fermentation to prepare small-molecule acid 3-methylthiopropionic acid, which can be further used for pest control. BACKGROUND

[0002] Root-knot nematode disease, as one of the most destructive soil-borne diseases in global agricultural production systems, has caused serious harm to more than 3000 crops, directly leading to an annual economic loss of more than 70 billion US dollars worldwide, and has been listed by the Food and Agriculture Organization of the United Nations as one of the most threatening plant pathogenic organisms. Among them, the damage caused by root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera spp. and Globodera spp.) is the most serious. At present, chemical control still accounts for more than 80% of the market share of nematode control, but the evolution of nematode resistance, soil micro-ecological disorder and the risk of agricultural product quality and safety caused by long-term reliance on non-fumigating nematocide (such as abamectin and fosthiazate) have become a prominent bottleneck restricting the sustainable development of agriculture. Therefore, the development of green microbial inoculants based on functional microorganisms is recognized as a key path to break through the dilemma of chemical control.

[0003] Bacillus exhibits unique advantages in the biological control of root-knot nematodes due to its broad-spectrum antibacterial activity. The Bacillus thuringiensis NBIN-863 obtained by high-throughput screening of our team secretes small-molecule acid (3-methylthiopropionic acid) which not only has high attraction to root-knot nematodes but also can effectively inhibit the hatching of root-knot nematode egg masses. Under laboratory conditions, the mortality rate of the nematode caused by the inoculant is more than 80%, and the root disease index of tomato seedlings is reduced by more than 90%. However, field tests show that the survival period of the strain in complex soil environment is significantly shortened, and it rapidly inactivates under stress such as high temperature and ultraviolet light. In addition, the single function limits its value of separate application, and it is often compounded with other microbial inoculants or fertilizers in actual production, which may weaken its insecticidal efficacy. The common technical problems of weak colonization ability and single function have become the core pain points that limit the large-scale application of microbial pesticides in the field.

[0004] In recent years, the synergistic effect produced by algal-bacterial cooperation has been verified in many fields. The preparation of algal-bacterial coupling inoculants and their combined application not only can promote the growth, productivity and quality of crops such as soybean and corn, but also can significantly improve the content of available nutrients in soil.

[0005] Therefore, it is urgent to construct a multifunctional composite microbial preparation capable of significantly improving the stress resistance and soil colonization of Bacillus thuringiensis NBIN-863, so as to have the dual functions of insect killing and soil improvement, and also expected to be used for fermenting to prepare the small molecule acid 3-methylthiopropionic acid for inhibiting pests. SUMMARY

[0006] One of the purposes of the present application is to provide an algae-bacteria composite microbial agent comprising Bacillus thuringiensis NBIN-863 and microalgae.

[0007] Preferably, the microalgae is Microcoleus sp. Microcoleus subtorulosus )。

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

[0009] Preferably, the preparation method of the algae-bacteria composite microbial agent comprises the following steps: mixing 100 mL of Bacillus thuringiensis bacterial liquid with a density of 8 x 10 8 CFU / mL and 6 g of microalgae slurry with a wet weight to obtain the algae-bacteria composite microbial agent.

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

[0011] In another preferable embodiment of the present application, the present application further provides the use of the algae-bacteria composite microbial agent in the preparation of an insecticide.

[0012] Preferably, the insecticide is used for preventing and treating root-knot nematodes.

[0013] In still another preferable embodiment of the present application, the present application further provides the use of the algae-bacteria composite microbial agent in improving soil nutrients.

[0014] In yet another preferable embodiment of the present application, the present application further provides a fermentation preparation process of the small molecule acid 3-methylthiopropionic acid, which comprises culturing the algae-bacteria composite microbial agent under suitable conditions, and collecting the metabolic product small molecule acid 3-methylthiopropionic acid.

[0015] Compared with the prior art, the present application has the following main advantages: ①In the application of the algae-bacteria composite microbial agent, the microalgae and Bacillus are compounded, the insecticidal effect is significantly improved, and there is obvious synergistic effect between the two; ②The algae-bacteria composite microbial agent provided by the present application has the following innovative points: Bacillus thuringiensis NBIN-863 and Microcoleus sp. are compounded in a specific ratio to form a composite microbial system with the functions of preventing pests and promoting growth. (a) The Bacillus thuringiensis NBIN-863 can stably colonize in soil and continuously secrete small-molecule organic acids with nematicidal activity, significantly inhibiting the invasion and reproduction of root-knot nematodes (Meloidogyne spp.), and achieving crop root protection; (b) The microcoleus can efficiently fix carbon through photosynthesis and increase soil nitrogen reserves through biological nitrogen fixation, while secreting a large amount of extracellular polysaccharides (EPS) to promote soil micro-aggregate formation, improve soil pore structure and water and fertilizer retention capacity, and provide a good micro-ecological environment for crop roots; (c) The above two types of microorganisms form a synergistic effect in the composite system: the improved soil microenvironment created by the microcoleus further enhances the colonization rate and activity of NBIN-863, while the metabolic products of NBIN-863 promote the growth and carbon fixation / nitrogen fixation efficiency of the microcoleus, thereby simultaneously achieving the multiple functions of "insect prevention + soil improvement" in the same application process, overcoming the single-function defect of traditional microbial agents.

[0016] ③ The present application further discloses that the algal-bacterial composite microbial agent can maintain high survival rate and insecticidal activity of NBIN-863 under ultraviolet radiation stress through the synergistic interaction of microcoleus and Bacillus thuringiensis NBIN-863; wherein the microcoleus effectively alleviates ultraviolet damage by forming physical shielding, secreting ultraviolet absorbing substances and antioxidant components, significantly prolonging the functional persistence period of NBIN-863 in the field, and overcoming the defect that single microbial agents are prone to inactivation under light and lead to decreased prevention effect.

[0017] The algal-bacterial composite microbial agent of the present application has stronger insecticidal effect than NBIN-863, and it is speculated that the addition of microcoleus further promotes the production of insecticidal effective component small-molecule acid 3-methylthiopropionic acid, so the composite microbial agent can be used for fermentation preparation of 3-methylthiopropionic acid.

[0018] The concept, specific structure and technical effects of the present application will be further described below with reference to the accompanying drawings, so as to fully understand the purpose, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the effect of the addition of microcoleus on the insecticidal efficiency of Bacillus.

[0020] Figure 2 is the effect of ultraviolet stress on the insecticidal efficiency of the algal-bacterial composite microbial agent.

[0021] Figure 3 is the change of the number of viable NBIN-863 in the rhizosphere of tomato seedlings by the algal-bacterial composite microbial agent. DETAILED DESCRIPTION

[0022] The present application will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but should not be understood as limiting the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0023] Biological material: Microsphaeridium FK04: from Hubei Province Biological Pesticide Engineering Center, the preservation number is CCTCC NO: M20251992, preserved in CCTCC on September 9, 2025.

[0024] Bacillus thuringiensis NBIN-863: from Hubei Province Biological Pesticide Engineering Center, the preservation number is CCTCC NO: M 2013612.

[0025] BG-11 culture medium, including 500-800 parts of NaNO3, 8-15 parts of K2HPO4, 15-20 parts of MgSO4·7H2O, 1-2 parts of CaCl2·2H2O, 0.1-0.2 parts of C6H8O7, 0.1-0.2 parts of (NH4) 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 solution.

[0026] Example 1 Preparation of microalgae

[0027] The light autotrophic culture mode is adopted and the culture is carried out in the BG-11 culture medium.

[0028] Take 10 mL of sterile microsphaeridium (CCTCC NO: M 20251992) with chlorophyll a of 0.05-0.08 mg / L, and culture in BG11 liquid medium (4 L) with a culture temperature of 25°C, a light intensity of 2200 lux, and a light cycle of 18h:6h light-dark ratio. After the logarithmic growth phase, sterile microsphaeridium culture solution is obtained, which is centrifuged at 6000 rpm for 10 min and resuspended three times with ultrapure water to obtain microsphaeridium algae mud (wet weight 6 g corresponding to 4 L algae liquid with chlorophyll a concentration of 3.2 mg / L). This process focuses on sterile operation to avoid contamination, and can be combined with a photobioreactor to achieve high-density culture. In addition, Bacillus fermentation waste liquid can also be used as a nitrogen source for microalgae culture to construct a production cycle of "Bacillus fermentation - waste resourceization - microalgae culture - algal high-value utilization", which not only can greatly reduce the culture cost of microalgae, but also can achieve efficient purification of wastewater.

[0029] Example 2 Preparation of functional Bacillus

[0030] S1. Culture of the strain on a slope: Bacillus thuringiensis NBIN-863 was inoculated on a LB solid slope culture medium containing 5% by weight agar by streaking, and cultured at 37°C for 24 h to obtain a strain; S2. Seed culture: the strain was transferred to a seed culture medium and cultured at 37°C at a rotation speed of 200 rpm for 24 h to obtain a seed liquid; S3. Fermentation culture: the secondary seed liquid was inoculated into a LB culture medium at a volume ratio of 2%, and subjected to fermentation culture at 37°C and 200 rpm for 48-72 h, to obtain a Bacillus liquid after logarithmic growth; The components of the LB culture medium are: 10 g / L proteose peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, and the pH of the LB culture medium is 7.2; the components of the seed culture medium are: 10 g / L anhydrous glucose, 10 g / L proteose peptone, 5 g / L yeast extract powder, 5 g / L potassium dihydrogen phosphate, 2 g / L calcium carbonate, and 1 g / L ammonium sulfate.

[0031] By controlling the aeration amount (rotation speed of 200 rpm) and pH stability, autolysis of the bacterial cells is avoided, and the cell density is increased to 8×10 8 CFU / mL. This process is suitable for scale-up. By using soybean powder and corn slurry as the basic carbon and nitrogen source, and by increasing the stirring speed (such as 300-600 rpm) and the aeration amount (0.8-1.5 vvm) in a stepwise manner, the dissolved oxygen is maintained at more than 20% to ensure high-density bacterial growth and product synthesis, further improving the yield, and realizing large-scale low-cost fermentation of NBIN-863.

[0032] Example 3 Preparation method of algal-bacterial complex microbial agent

[0033] 100 mL of Bacillus thuringiensis NBIN-863 liquid with a density of 8×10 8 CFU / mL and 6 g of wet micro-sheath algal paste were mixed and shaken at 200 rpm for 2 h to obtain an algal-bacterial complex microbial agent.

[0034] Example 4 Verification of the synergistic effect of Bacillus and microalgae

[0035] The nematode egg mass was picked from the roots of tomato infected with southern root-knot nematode, the egg mass was placed in a culture dish containing 15 mL sterile water, and the hatching of root-knot nematode was observed by incubation at 20°C in the dark. The nematodes were collected by centrifugation, and the concentration of nematodes was calculated. The prepared algal-bacterial complex microbial agent was filtered (0.25 μm) to obtain a filtrate, which was added to a 96-well plate, and then 30-40 nematodes were added to each well. The total system was 100 μL, and sterile water was used as a negative control, and NBIN-863 alone was used as a positive control. Each group was repeated 5 times. After being placed in a 20°C incubator in the dark for 24 h, the mortality rate of nematodes was observed under a microscope, as shown in Figure 1 The results show that the insecticidal efficiency of the algal-bacterial complex microbial agent (84.8%) is significantly higher than that of the single bacterial group (76.7%).

[0036] Example 5 Verification of the anti-ultraviolet stress ability of the algal-bacterial complex microbial agent

[0037] The insecticidal activity of the algal-bacterial complex microbial agent prepared in Example 3 under ultraviolet irradiation was detected by the method shown in Example 4, and the results are shown in Figure 2 After 105 minutes of ultraviolet irradiation (intensity 125 μW / cm 2 , distance 15 cm), the number of viable bacteria of NBIN-863 in the algal-bacterial complex microbial agent was 22 times that of the control, and the insecticidal activity of the algal-bacterial complex microbial agent decreased by only about 10%, which is much lower than the 30% of the control group. This shows that by mixing Microthamnus (CCTCC NO: M 20251992) and Bacillus thuringiensis NBIN-863 as an algal-bacterial complex microbial agent, the anti-ultraviolet radiation ability of Bacillus thuringiensis NBIN-863 can be effectively improved.

[0038] Example 6 Effect of the algal-bacterial complex microbial agent on the abundance of rhizosphere microbial community of tomato seedlings

[0039] The effect of the algal-bacterial complex microbial agent prepared in Example 3 on the abundance of rhizosphere microbial community of tomato seedlings was detected, and the results are shown in Figure 3 After 50 days of pot culture, the abundance of NBIN-863 in the rhizosphere of tomato seedlings was analyzed, and the results show that the number of colonies of NBIN-863 in the algal-bacterial complex group was 3.27·10 8 (CFU / g), which is 6 times that of the single bacterial group, indicating that the algal-bacterial complex microbial agent of the application can effectively improve the abundance of NBIN-863 in the rhizosphere of tomato seedlings.

[0040] Example 7 Effect of the algal-bacterial complex microbial agent on soil nutrients

[0041] The influence of the algal-bacterial complex microbial agent prepared in Example 3 on soil nutrients is detected, and the results are shown in Table 1. After 50 days of pot culture, compared with the negative control (water) and the positive control (NBIN-863 single bacteria), the addition of the algal-bacterial complex microbial agent can significantly improve the content of available carbon and nitrogen in the soil, especially in the rhizosphere soil, which shows that the algal-bacterial complex microbial agent of the application can significantly improve the content of available carbon and nitrogen in the soil of tomato seedlings during the cultivation process.

[0042] The preferred embodiments of the application are described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and changes without creative work based on the concept of the application. Therefore, any technical solution obtained by logical analysis, reasoning or limited experiment based on the existing technology according to the concept of the application should be within the protection scope determined by the claims.

Claims

1. An algae-bacterial compound agent comprising Bacillus thuringiensis NBIN-863 and microalgae.

2. The algae-bacterial compound agent according to claim 1, characterized in that, The microalgae mentioned are Microcoleus subtorulosus.

3. The algae-bacterial compound agent according to claim 2, characterized in that, The preservation number of the microsheath algae is CCTCCNO: M 20251992.

4. 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 mixture of CFU / mL Bacillus thuringiensis bacterial solution and 6g wet weight microalgae mud was used to obtain a compound bacterial agent.

5. The algae-bacterial compound agent according to claim 1, characterized in that, The 6g wet weight microalgae mud was obtained by concentrating 4L of algal solution with a chlorophyll a concentration of 3.2 mg / L.

6. The use of the algae-bacterial compound agent according to any one of claims 1-5 in the preparation of insecticides.

7. The application according to claim 6, characterized in that, The insecticide is used to control root-knot nematodes.

8. The application of the algae-bacterial compound inoculant according to any one of claims 1-5 in improving soil nutrients.

9. A fermentation preparation process for the small molecule acid 3-methylthiopropionic acid, comprising culturing the algae-bacterial composite agent according to any one of claims 1-5 under suitable conditions and collecting the metabolite small molecule acid 3-methylthiopropionic acid.

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