Microbial complex microbial agent for promoting growth and yield of pepper and application thereof

Abstract

The application discloses a microbial compound microbial inoculant for promoting growth and yield increase of peppers and application thereof, and the microbial compound microbial inoculant is a fermentation liquor containing Bacillus subtilis, Priestia megaterium, Cytobacillus firmus and Bacillus altitudinis. The application further discloses a preparation method of the compound microbial inoculant and application of the compound microbial inoculant in promoting growth and yield increase of peppers. The compound microbial combination provided by the application has no antagonism among the strains, and the mixed application significantly increases the plant height of the pepper plants, improves the yield per plant, the fruit number per plant and the yield per mu, and can reduce the use intensity of chemical fertilizers, so the compound microbial combination has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, and in particular relates to a microbial compound agent that promotes the growth and yield of chili peppers and its application. Background Technology

[0002] Chili peppers are the most consumed condiment both domestically and internationally, but current chili pepper cultivation suffers from indiscriminate and excessive fertilization. This irrational fertilization pattern has triggered a series of problems, including soil acidification and compaction, decreased fertility, exacerbated ecological pollution, and increased risks to agricultural product quality and safety. Ultimately, this leads to a double decline in both yield (reduction rate of 17%~23%) and quality, hindering sustainable agricultural development. Against this backdrop, reducing fertilizer application while increasing efficiency has become an industry consensus, with significant potential for nutrient reduction in both greenhouse and open-field chili pepper cultivation. Microbial inoculants, derived from beneficial microorganisms in soil or plant materials, are non-toxic and harmless to the environment. Their application can improve soil, increase soil organic matter content, promote crop growth and development, and increase yield and improve quality. Combined with chemical fertilizers, they can also reduce excessive fertilizer use and better protect the ecological environment. Currently, there are few reports on the research and application of chili pepper-specific compound microbial agents. Previous studies have largely focused on the growth-promoting effects of single microorganisms such as Bacillus subtilis, Bacillus tequilensis, Bacillus amyloliquefaciens, and Pseudomonas fluorescens on plants. However, most single-species microbial agents are limited in their growth-promoting function, colonization, and stability due to environmental conditions and interactions between microorganisms. Therefore, the application of single-microorganism-based inoculants remains somewhat limited. Synthetic microbial communities (SynComs) refer to the design and construction of multi-species coexistence systems utilizing interactions between microorganisms. This overcomes the technical barriers to the stable colonization of functional microorganisms, achieving complementary functions among multiple microorganisms and exhibiting greater stability and environmental friendliness.

[0003] Therefore, the development of a compound microbial agent with strong growth-promoting and yield-increasing functions and stable field effects is of great significance for the growth and cultivation of chili peppers. Summary of the Invention

[0004] In view of this, the present invention provides a microbial compound inoculant for promoting the growth and yield of chili peppers and its application, so as to achieve the purpose of reducing chemical fertilizer use and increasing chili pepper yield.

[0005] This invention provides a compound microbial agent for promoting chili pepper growth and yield, comprising equal volume proportions of fermentation broths of Bacillus subtilis, Priestia megaterium, Cytobacillus firmus, and Bacillus altitudinis, all in the logarithmic growth phase; wherein the preservation numbers of Bacillus subtilis, Priestia megaterium, Cytobacillus firmus, and Bacillus altitudinis are CGMCC1.102, CGMCC1.6721, CGMCC1.10560, and CGMCC1.12171, respectively; the compound microbial agent contains a total viable count of 3 × 10⁻⁶ cells. 8 ~3×10 9 CFU / mL.

[0006] This invention also provides a method for preparing a compound microbial agent that promotes the growth and yield of chili peppers, comprising the following steps: a. Strains activation: Bacillus subtilis, Priestella megaterium, Bacillus sturdierium, and Bacillus plateauus strains were inoculated onto solid culture medium and activated overnight; b. Liquid activation: Pick out the bacterial colonies activated by step a and place them in liquid culture medium. Incubate them at 37°C and 150 rpm in a shaker until the logarithmic growth phase. c. Obtain the fermentation broth of each strain: Inoculate the culture broth of each strain in the logarithmic growth phase in step b into the liquid culture medium at a volume ratio of 1%, and culture at 37°C and 150 rpm for 18-36 h to obtain the fermentation broth of Bacillus subtilis, Priestella megaterium, Bacillus sturdierium and Bacillus alpineus strains respectively. d. Preparation of compound microbial agent: The fermentation broths of Bacillus subtilis, Priestella megaterium, Bacillus sturdierium, and Bacillus alpineus strains from step c are mixed evenly in equal volume ratios to obtain the compound microbial agent.

[0007] Furthermore, in step d of the preparation method of the compound microbial agent, the total number of viable bacteria in the compound microbial agent is 3 × 10⁻⁶. 8 ~3×10 9 Between CFU / mL.

[0008] Furthermore, in step a of the preparation method of the compound microbial agent, the solid culture medium is LB solid culture medium.

[0009] Furthermore, in steps b and c of the preparation method of the compound microbial agent, the liquid culture medium is LB liquid culture medium.

[0010] Furthermore, the LB solid culture medium is formulated to contain 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, and 15g of agar per 1000mL of distilled water; the pH value of the LB solid culture medium is 7.0~7.2, and it is used after being sterilized at 121℃ for 20min.

[0011] Furthermore, the LB liquid culture medium formula is as follows: each 1000 mL of distilled water contains 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride; the pH value of the LB liquid culture medium is adjusted to 7.0~7.2, and it is sterilized at 121℃ for 20 min before use.

[0012] The present invention also provides an application of the above-mentioned compound microbial agent in promoting the growth and increasing the yield of chili peppers.

[0013] The present invention also provides a compound microbial agent product for promoting the growth and yield of chili peppers, wherein the active ingredient of the compound microbial agent product comprises the above-mentioned compound microbial agent.

[0014] Furthermore, the compound microbial agent product also includes conventional components of microbial agent products.

[0015] Compared with the prior art, the compound microbial agent strains of the present invention have different nitrogen-fixing, phosphorus-solubilizing and growth-promoting characteristics, and there is no antagonism between the strains. Field application of the compound microbial agent of the present invention shows that, without the use of chemical fertilizers, the application of the compound microbial agent significantly increases the plant height of pepper plants, increases the yield per plant, the number of fruits per plant, and the yield per acre, indicating that the mixed application of the compound microbial agent strains of the present invention can promote pepper growth, increase pepper yield, and reduce the intensity of chemical fertilizer use. Attached Figure Description

[0016] Figure 1 The results show the antagonistic effects between different strains. The bottom of the LB plate is uniformly covered with Bacillus subtilis, A1 is Priestella megaterium, A2 is Bacillus stolonifera, and T4 is Bacillus alpineus. Figure 2 The results of plant height measurements for different treatment groups of chili peppers are shown below. A is the blank control, with no fertilizer or fungicide applied; B is the treatment with manure; C is the treatment with Bacillus subtilis, *Priscilla megaterium*, *Bacillus stolonifer*, and *Bacillus alpineus*; D is the treatment with *Trichoderma harzianum*; E is the treatment with Bacillus subtilis and *Priscilla megaterium*; F is the treatment with *Bacillus stolonifer* and *Bacillus alpineus*; G is the treatment with Bacillus subtilis, *Priscilla megaterium*, *Bacillus stolonifer*, *Bacillus alpineus*, and *Trichoderma harzianum*. Figures 3-9 The meanings of the annotations A through G are all the same. Figure 2 ; Figure 3 The results of fresh weight measurements of chili plants and fruits in different treatment groups; Figure 4 Results of chili pepper fruit count determination in different treatment groups; Figure 5 Soil pH measurement results for different treatment groups; Figure 6 The results of soil organic carbon content determination for different treatment groups; Figure 7 The results of soil available nitrogen content determination for different treatment groups; Figure 8 The results of soil available phosphorus content determination for different treatment groups; Figure 9 The results show the total nitrogen content of soil in different treatment groups. Detailed Implementation

[0017] The embodiments of the present invention will be described in detail below with reference to specific examples. These examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Rather, they should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the invention. Unless otherwise specified, the instruments, reagents, and materials used in the following examples are commercially available, and the experimental methods are conventional methods in the art.

[0018] The strains mainly used in the following examples include: Bacillus subtilis (CGMCC1.102), Priestia megaterium (CGMCC1.6721), Cytobacillus firmus (CGMCC1.10560), Bacillus altitudinis (CGMCC1.12171), and others. ® Trichoderma harzianum fungicide (purchased from Shandong Hairuis Marine Biotechnology Co., Ltd.)

[0019] Example 1: Antagonistic Experiment Between Strains Spread 100 μL of bacterial suspension evenly onto an LB agar plate. Soak a filter paper disc in the bacterial suspension and place it on the plate. Incubate the plate at 37°C upside down for 1–2 days, observing for any antagonistic behavior, such as the appearance of antagonistic zones. Eliminate antagonistic strains and select non-antagonistic strains for preparing the compound bacterial agent.

[0020] Through the above experiments, four strains of *Bacillus subtilis*, *Priscilla megaterium*, *Bacillus stoichioides*, and *Bacillus alpineus* were screened. Each strain showed good growth and normal colony morphology when cultured alone, without any growth inhibition. The filter paper disc method was used to observe whether there was any antagonism among the four strains; the results are shown below. Figure 1In the plate confrontation experiment, no obvious antagonistic effect was observed between the strains. When the strain combinations were co-cultured, the colonies all grew normally, and no clear antagonistic lines or inhibition zones were formed between them. The growth regions of the strains intermingled, and no obvious growth inhibition or repulsion was observed.

[0021] Example 2: Preparation of Compound Microbial Agent The compound microbial agent for promoting growth and increasing yield of chili peppers consists of fermentation broth in equal volumes of Bacillus subtilis, Priestella megaterium, Bacillus sturdierium, and Bacillus alpineus strains.

[0022] The preparation method of the above-mentioned compound microbial agent for promoting growth and increasing yield of chili peppers includes the following steps: a. Slant activation: Bacillus subtilis, Priestella megaterium, Bacillus sturdierium, and Bacillus alpineus strains were inoculated onto LB solid medium and activated overnight. The LB solid medium formula contained 10g tryptone, 5g yeast extract, 10g sodium chloride, and 15g agar per 1000mL of distilled water; the pH value was 7.0~7.2, and it was sterilized at 121℃ for 20min before use. b. Liquid activation: Pick the bacterial colonies activated in step a and place them in LB liquid medium. Incubate at 37°C and 150 rpm for 18-36 hours until the logarithmic growth phase. The LB liquid medium formula is: 10g tryptone, 5g yeast extract, and 10g sodium chloride per 1000mL of distilled water. Adjust the pH of the LB liquid medium to 7.0-7.2 and sterilize at 121°C for 20 minutes before use. c. Obtain the fermentation broth of each strain: Inoculate the culture broth of each strain in the logarithmic growth phase in step b into LB liquid medium at a volume ratio of 1% (the LB liquid medium formula is the same as in step b), and culture at 37°C and 150 rpm for 18-36 h to obtain the fermentation broth of Bacillus subtilis, Priestella megaterium, Bacillus sturdierium, and Bacillus alpineus strains respectively. d. Preparation of the compound microbial agent: The fermentation broths of Bacillus subtilis, Priestella megaterium, Bacillus stolonifera, and Bacillus alpineus strains from step c are mixed evenly in equal proportions (by volume) to obtain the compound microbial agent seed liquid, wherein the total number of viable bacteria in the compound microbial agent is 3 × 10⁻⁶. 8 ~3×10 9 The concentration of seed solution should be between CFU / mL. Apply the resulting seed solution along with the initial watering.

[0023] Example 3: Evaluation of the growth-promoting ability of microbial agents in outdoor field Test site: Tashihe Township, Manas County Test period: May 23, 2025 - August 27, 2025 Experimental crop: chili pepper Experimental variety: Screw pepper Test soil: saline-alkali land Planting method: cuttings Experimental Groups: Treatment group A: Blank control, no fertilizer or fungicide was applied.

[0024] Treatment Group B: Use manure.

[0025] Treatment group C: Bacillus subtilis, Priestella megaterium, Bacillus sturdierium, and Bacillus alpineus (i.e., the compound bacterial agent prepared in Example 2).

[0026] Treatment group D: Use Trichoderma harzianum fungicide.

[0027] Treatment group E: Bacillus subtilis and Priestella megaterium were used.

[0028] Treatment group F: Used Bacillus stolonifer and Bacillus pyriformis.

[0029] Treatment group G: Bacillus subtilis, Priestella megaterium, Bacillus stolonifera, Bacillus alpineus, and Trichoderma harzianum were used as fungicides.

[0030] Application methods for each group of compound microbial agents: The agent is first applied with the transplanting water ten days after transplanting the chili seedlings, followed by applications with the transplanting water at the initial flowering stage (30-40 days after transplanting) and the fruit setting stage (60-70 days after transplanting). Each group of compound microbial agents consists of equal volumes of fermentation broth from the corresponding strains in the logarithmic growth phase, with a total viable count of 3 × 10⁻⁶ cells per application. 8 ~3×10 9 Between CFU / mL.

[0031] Experimental results: 1) Effects on chili plant height and fresh weight: from Figure 2 As can be seen, there are significant differences in plant height under different treatments. Treatment D showed the best plant height, significantly higher than other treatment groups; treatments C and F also had relatively high plant heights, while treatment A had the lowest plant height among all groups. Specifically, treatment D showed the most significant increase in plant height compared to treatment A, directly indicating that the commercial Trichoderma harzianum inoculant had a better effect on promoting plant height; the plant height of treatment C (the compound inoculant of this invention) was also significantly higher than that of treatments A (blank control), B (manure), and G (the compound inoculant of this invention + Trichoderma harzianum inoculant), increasing by 65.52%, 26.32%, and 17.07%, respectively.

[0032] from Figure 3The results show significant differences in plant and fruit fresh weight under different treatments. Treatment C exhibited the most significant differences in both plant and fruit fresh weight, far exceeding all other treatment groups. Treatments D and F also showed relatively high levels, while treatment A had the lowest plant and fruit fresh weight among all groups. Specifically, treatment C showed a highly significant increase in fruit fresh weight compared to treatment A, with plant fresh weight increasing tenfold. Furthermore, treatment C's fruit fresh weight was 55.38% higher than treatment D, clearly demonstrating that the compound microbial agent of this invention has the best effect on promoting fruit fresh weight.

[0033] 2) Effects on the quality and number of chili pepper fruits: from Figure 3 and Figure 4 It can be seen that during the plant growth stage, treatment C significantly increased the number of fruits, far exceeding the other treatment groups; at the same time, treatment C also showed the best fruit fresh weight, with a very significant increase compared to treatment A. Treatments D and F also had high fruit fresh weights, and treatment F had significantly more fruits than treatment A. Overall, different treatments had different effects on fruit number and fruit fresh weight, but treatments C (the compound microbial agent of this invention), D (Trichoderma harzianum agent), and F (Bacillus stolonifera + Bacillus alpineus) all significantly promoted fruit growth, especially in terms of fruit number and fruit fresh weight, with treatment C (the compound microbial agent of this invention) showing the most outstanding improvement.

[0034] 3) Effects of different treatments on soil physicochemical properties: Soil pH was determined using a magnetic pH meter; soil organic matter content was determined using the potassium dichromate external heating method; soil available nitrogen content was determined using the diffusion absorption method; soil total nitrogen content was determined using the semi-micro Kjeldahl nitrogen determination method; soil available phosphorus content was determined using the hydrochloric acid-ammonium fluoride extraction method; and soil available potassium content was determined using the flame photometric method after extraction with 1 mol / L ammonium acetate solution.

[0035] from Figure 5 It can be seen that the soil pH value of treatment A (blank control) is significantly higher than that of other treatment groups. The soil pH value of each treatment ranges from 8.25 to 8.65. Among them, the soil pH value of treatment A is relatively high, while the soil pH value of treatment G (compound microbial agent of the present invention + Trichoderma harzianum agent) is relatively low.

[0036] from Figure 6 It can be seen that the effects of different treatments on soil organic carbon content vary significantly. Compared with treatment A (blank control), treatment F (Bacillus stolonifera + Bacillus alpineus) showed the most significant increase in soil organic carbon content, with a more pronounced increase than treatment A; treatment E (Bacillus subtilis + Priestella megaterium) also showed significantly higher soil organic carbon content than treatment A, and the soil organic carbon content of the other treatments increased to varying degrees compared with treatment A.

[0037] 4) Soil nutrient content: from Figure 7 (Alkaline nitrogen) It can be seen that the alkaline nitrogen content of soil in different treatments differs significantly: the alkaline nitrogen content of treatment A (blank control) is much higher than that of other groups, the alkaline nitrogen content of treatment F (Bacillus stolonifera + Bacillus thunbergii) is also at a high level, while the alkaline nitrogen content of treatments B (manure) and C (the compound microbial agent of this invention) is relatively low.

[0038] from Figure 8 (available phosphorus) It can be seen that the available phosphorus content in the soil treated with F (Bacillus stolonifera + Bacillus thunbergii) was the best, significantly higher than other treatment groups; the available phosphorus content in the treatment with D (Trichoderma harzianum) was also improved to a certain extent, while the available phosphorus content in the treatment with A (blank control) was the lowest among all groups.

[0039] from Figure 9 The total nitrogen content (F) treatment (Bacillus stolonifera + Bacillus alpineus) showed the highest total nitrogen content. The total nitrogen content of the D treatment (Trichoderma harzianum agent) was also significantly higher than most other treatment groups. The total nitrogen content of the E (Bacillus subtilis + Priestella megaterium) and G (the compound microbial agent of this invention + Trichoderma harzianum agent) treatments was relatively lower. Overall, the F (Bacillus stolonifera + Bacillus alpineus) treatment showed the most significant improvement in the three nutrient indicators of available nitrogen, available phosphorus, and total nitrogen. The A (blank control) treatment showed outstanding performance in available nitrogen, while the D (Trichoderma harzianum agent) treatment had a certain promoting effect on available phosphorus and total nitrogen. Furthermore, considering the experimental data from results 1) and 2), it is indicated that the soil treated with this compound microbial agent did not have the highest total nutrient content, but it did have the highest yield.

[0040] Conclusion: Field application of this chili variety showed that, without the use of chemical fertilizers, the application of compound microbial agents significantly increased the plant height, yield per plant, number of fruits per plant, and yield per acre, achieving the effect of reducing chemical fertilizer use and increasing chili yield.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound microbial agent for promoting the growth and yield of chili peppers, characterized in that, The compound microbial agent is composed of equal volume proportions of fermentation broths of Bacillus subtilis, Priestia megaterium, Cytobacillus firmus, and Bacillus altitudinis, all in the logarithmic growth phase; wherein the preservation numbers of Bacillus subtilis, Priestia megaterium, Cytobacillus firmus, and Bacillus altitudinis are CGMCC1.102, CGMCC1.6721, CGMCC1.10560, and CGMCC1.12171, respectively; the total number of viable bacteria in the compound microbial agent is 3 × 10⁻⁶. 8 ~3×10 9 CFU / mL.

2. A method for preparing a compound microbial agent to promote the growth and yield of chili peppers, comprising the following steps: a. Strains activation: Bacillus subtilis, Priestella megaterium, Bacillus sturdierium, and Bacillus alpineus strains were inoculated onto solid culture medium and activated overnight; b. Liquid activation: Pick out the bacterial growth of the activated strain from step a and place it in liquid culture medium. Incubate at 37°C and 150 rpm on a shaker until the logarithmic growth phase. c. Obtain the fermentation broth of each strain: Inoculate the culture broth of each strain in the logarithmic growth phase in step b into the liquid culture medium at a volume ratio of 1%, and culture at 37°C and 150 rpm for 18-36 h to obtain the fermentation broth of Bacillus subtilis, Priestella megaterium, Bacillus sturdierium and Bacillus pyriformis respectively. d. Preparation of compound microbial agent: The fermentation broths of Bacillus subtilis, Priestella megaterium, Bacillus sturdierium, and Bacillus alpineus strains from step c are mixed evenly in equal volume ratios to obtain the compound microbial agent.

3. The method for preparing the compound microbial agent as described in claim 2, characterized in that, In step d, the compound microbial agent contains a total live bacteria count of 3 × 10⁻⁶. 8 ~3×10 9 CFU / mL.

4. The method for preparing the compound microbial agent as described in claim 2, characterized in that, In step a, the solid culture medium is LB solid culture medium.

5. The method for preparing the compound microbial agent as described in claim 2, characterized in that, In steps b and c, the liquid culture medium is LB liquid culture medium.

6. The method for preparing the compound microbial agent as described in claim 4, characterized in that, The LB solid medium is formulated with 10g tryptone, 5g yeast extract, 10g sodium chloride, and 15g agar per 1000mL of distilled water; the pH of the LB solid medium is 7.0~7.2, and it is sterilized at 121℃ for 20min before use.

7. The method for preparing the compound microbial agent as described in claim 5, characterized in that, The LB liquid culture medium formula is as follows: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride per 1000mL of distilled water; the pH of the LB liquid culture medium is adjusted to 7.0~7.2 and sterilized at 121℃ for 20min before use.

8. The application of the compound microbial agent according to claim 1 in promoting the growth and increasing the yield of chili peppers.

9. A compound microbial agent product for promoting the growth and yield of chili peppers, characterized in that, The active ingredient of the compound microbial agent product includes the compound microbial agent as described in claim 1.

10. The compound microbial agent product as described in claim 9, characterized in that, It also includes the conventional ingredients of microbial agents.

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