A complex microbial agent for promoting tobacco plant growth and application thereof
By promoting tobacco growth through compound microbial agents, the nutrient imbalance and soil problems caused by dependence on chemical fertilizers in tobacco cultivation have been solved, resulting in healthy tobacco plant growth, soil improvement, and enhanced tobacco quality and economic benefits.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INNER MONGOLIA KUNMING CIGARETTE CO LTD
- Filing Date
- 2026-03-04
- Publication Date
- 2026-05-29
AI Technical Summary
The long-term reliance on chemical fertilizers in current tobacco cultivation has led to nutrient imbalances in plants, soil compaction, acidification, and microbial imbalances, affecting tobacco yield and quality, and there is a lack of effective biological control methods.
A compound microbial agent, including Bacillus hygroscopicus ZXY01, Bacillus subtilis LGT-2, and Rhodopseudomonas palustris, is fermented to produce a compound fermentation preparation. This preparation is then applied to tobacco plants to promote root development, stem and leaf growth, and synergistically enhance soil nutrient transformation and improve soil quality.
It significantly improves the robustness of tobacco plant stems, the number of leaves, and the supply of soil nutrients, improves the appearance and internal quality of tobacco leaves, reduces dependence on chemical fertilizers, is environmentally friendly, and improves the economic benefits for tobacco farmers.
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Figure CN122104468A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bio-fertilizer technology, specifically, it relates to a compound microbial agent that promotes tobacco plant growth and its application. Background Technology
[0002] Tobacco, as an important economic crop in my country, has always been a focus of attention for its high quality and yield. The final yield and quality are closely related to the growth status of the tobacco plants. In the current planting system, long-term reliance on chemical fertilizers, while providing nutrients in the short term, fails to coordinate the physiological development of the tobacco plants, easily leading to nutrient imbalances. This manifests as agronomical defects such as insufficient plant height, weak stems, and limited leaf expansion, directly affecting the yield and appearance quality of tobacco leaves. Therefore, how to directly promote healthy tobacco plant growth through biological regulation has become a key issue in tobacco cultivation. For a long time, tobacco field planting has relied excessively on chemical fertilizers, leading to a series of problems in the tobacco-growing soil, including soil compaction, acidification, poor aeration, and soil microbial ecological imbalance. This not only restricts the high quality and yield of tobacco but also damages the stability of the tobacco field ecosystem.
[0003] Microbial inoculants are widely used in crop cultivation because they can act on plants directly or indirectly through various physiological pathways. Compared with single-function inoculants, compound microbial inoculants composed of multiple microbial species can more effectively promote crop root development and stem and leaf growth by enhancing nutrient conversion and secreting growth-promoting substances, thus providing a new technical approach to improving agronomic traits. To effectively regulate and enhance key agronomic traits in tobacco, it is necessary to propose a new microbial inoculant and treatment scheme. Summary of the Invention
[0004] The purpose of this invention is to provide a compound microbial inoculant for promoting tobacco plant growth and its application. This compound microbial inoculant is an organic fertilizer with low fermentation costs, improved soil quality, and enhanced tobacco growth and development. The compound microbial inoculant is formulated from a mixture of Bacillus subtilis, Bacillus thuringiensis, and Rhodopseudomonas palustris. This inoculant can effectively reduce reliance on chemical fertilizers, thereby achieving green and efficient development of the tobacco industry.
[0005] In a first aspect, the present invention provides a compound microbial agent comprising Bacillus hygroscopicus ZXY01, Bacillus subtilis LGT-2 and Rhodopseudomonas palustris.
[0006] Furthermore, the effective viable count of the *Bacillus hygroscopicus* ZXY01 is ≥2.0 × 10⁻⁶. 10 cfu / mL.
[0007] Furthermore, the effective viable count of the Bacillus subtilis LGT-2 is ≥2.0 × 10⁻⁶. 10 cfu / mL.
[0008] Furthermore, the effective viable count of the *Rhodopseudomonas palustris* is ≥2.0 × 10⁻⁶. 10 cfu / mL.
[0009] Secondly, the present invention provides the application of any of the above-mentioned compound microbial agents in promoting tobacco growth or improving tobacco-growing soil.
[0010] Furthermore, the application involves preparing seed liquids of the strains of the compound microbial agent separately, then inoculating them into a fermentation medium for expansion to obtain a fermentation broth, and then mixing them to prepare a compound fermentation preparation.
[0011] Furthermore, both Bacillus alpineus ZXY01 and Bacillus subtilis LGT-2 were prepared into seed culture using LB liquid medium, while Rhodopseudomonas palustris was prepared into seed culture using PM liquid medium.
[0012] Furthermore, the volume ratio of the fermentation broth of Bacillus hygroscopicus ZXY01, Bacillus subtilis LGT-2, and Rhodopseudomonas palustris is (1.9~2.1):(0.9~1.1):(0.9~1.1).
[0013] Furthermore, the volume ratio of the fermentation broth of Bacillus hygroscopicus ZXY01, Bacillus subtilis LGT-2, and Rhodopseudomonas palustris is 2.0:1.0:1.0.
[0014] Furthermore, the application also includes applying the compound fermentation agent to plants, with application methods including hole application or ring application. Specifically, the compound fermentation agent can be added to the basic fertilizer at a volume-to-mass ratio of (2~5) mL: 1000 g to prepare a compounded bio-fertilizer, with application methods including hole application or ring application.
[0015] The present invention has at least the following beneficial effects: The compound microbial agent of this invention includes *Bacillus hygroscopicus* ZXY01, which effectively promotes potassium ion release, helps improve the potassium nutritional level of tobacco plants, and provides support for robust stems and nutrient transport; *Rhodopseudomonas palustris*, which has nitrogen-fixing properties, can increase nitrogen supply and provide a material basis for leaf development; and *Bacillus subtilis* LGT-2, which promotes root development and improves nutrient absorption efficiency while synergistically enhancing the growth vitality of tobacco plants. This enhancement increases the environmental tolerance of the entire microbial community, and its metabolites can regulate the growth rate of the other two strains, avoiding the problem of excessive proliferation of a single strain due to the absence of *Bacillus subtilis*, allowing the three bacteria to form a structurally stable microbial community in the rhizosphere. This agent, through multi-level action, specifically promotes thicker stems and increased leaf number in tobacco plants, thereby achieving effective regulation and improvement of key agronomic traits. Simultaneously, this agent reduces dependence on chemical fertilizers, is environmentally friendly, and effectively reduces costs for tobacco farmers while improving the economic benefits of tobacco.
[0016] The preparation method of the present invention is convenient to operate and low in cost.
[0017] The application of this invention can convert mineral potassium in the soil into available potassium and perform nitrogen fixation to increase crop yield. This compound microbial agent can significantly improve the soil condition for tobacco planting and has a positive regulatory effect on tobacco agronomic traits, specifically in promoting the growth and thickness of tobacco leaves, and improving the overall appearance and internal quality of tobacco leaves. Attached Figure Description
[0018] Figure 1 A comparison of tobacco leaf size and root length between the CK and EG groups.
[0019] Figure 2 These are the agronomic traits of tobacco during its vigorous growth period.
[0020] Figure 3 The content of soil physicochemical properties during the vigorous growth period of tobacco leaves.
[0021] Figure 4 This refers to the amount of dry matter accumulated in tobacco plants. Detailed Implementation
[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The embodiments described below are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply.
[0023] A compound microbial agent, comprising Bacillus hygroscopicus (B. hygroscopicus) Bacillus altitudinis ZXY01, Bacillus subtilis ( Bacillus subtilis LGT-2 and Rhodopseudomonas palustris ( Rhodopseudomonas palustris The effective viable count of Bacillus hygroscopicus ZXY01 is ≥2.0 × 10⁻⁶. 10 CFU / mL. Effective viable count of Bacillus subtilis LGT-2 ≥ 2.0 × 10⁻⁶. 10 cfu / mL. Other Bacillus subtilis strains cannot be used in the compound microbial agent of this invention. The effective viable count of *Rhodopseudomonas palustris* is ≥2.0 × 10⁻⁶. 10 cfu / mL. This invention creatively combines the above three bacteria to form a compound microbial agent.
[0024] The application of the aforementioned compound microbial inoculant in promoting tobacco growth or improving tobacco-growing soil includes inoculating the microbial strains preserved at -80℃ onto corresponding solid culture media. After clear single colonies form on the plates, select morphologically regular single colonies free from contamination and inoculate them into the corresponding culture media to activate them into seed culture. Specifically, *Bacillus hygroscopicus* ZXY01 and *Bacillus subtilis* LGT-2 are cultured on LB liquid medium, while *Rhodopseudomonas palustris* is cultured on PM liquid medium. The seed culture is then inoculated into the corresponding fermentation medium for expansion, with the same composition as above, to obtain fermentation broth. The volume ratio of the fermentation broth of *Bacillus hygroscopicus* ZXY01, *Bacillus subtilis* LGT-2, and *Rhodopseudomonas palustris* is (1.9~2.1):(0.9~1.1):(0.9~1.1), preferably 2.0:1.0:1.0. One of the functions of Bacillus subtilis LGT-2 is that its metabolites can regulate the growth rates of the other two strains. If the proportion of Bacillus subtilis LGT-2 is not within the above-mentioned range, the nitrogen fixation, potassium solubilization, and growth-promoting effects will be weakened, and the overall synergistic effect of the compound microbial agent will be significantly reduced. The compound fermentation preparation is then mixed and formulated. After dilution, the compound fermentation preparation is applied to the plants, either by hole application or ring application.
[0025] Example 1 This embodiment provides a compound microbial agent, including Bacillus saltitudinis ZXY01, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36684, deposited on November 20, 2025, with an effective viable count of 2.0 × 10⁻⁶. 10 cfu / mL; Bacillus subtilis LGT-2, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 27295, deposited on May 8, 2023, with a viable count of 2.0 × 10⁻⁶. 10 cfu / mL; Rhodopseudomonas palustris, commercially available, with an effective viable count of 2.0 × 10⁻⁶. 10cfu / mL.
[0026] The process of preparing compound fermentation preparations includes: Bacillus hygroscopicus ZXY01, stored at -80℃, was aseptically inoculated onto LB agar plates and cultured at 37℃ for 48 hours. Single colonies were then transferred to LB liquid medium and cultured at 37℃ and 200 rpm for 24 hours to obtain activated Bacillus hygroscopicus seed culture.
[0027] Bacillus subtilis LGT-2 stored at -80℃ was aseptically inoculated onto LB agar plates and cultured at 37℃ for 48 h. Single colonies were then transferred to LB liquid medium and cultured at 37℃ and 200 r / min for 24 h to obtain activated Bacillus subtilis seed culture.
[0028] Rhodopseudomonas palustris, preserved at -80℃, was aseptically inoculated onto PM agar plates containing a carbon source and cultured at 30℃ for 5-7 days. Single colonies were picked and transferred to PM liquid medium, carbon source was added, and the culture was carried out under light and anaerobic conditions at 30℃ to obtain Rhodopseudomonas palustris seed culture.
[0029] Furthermore, the seed culture of Bacillus hygroscopicus ZXY01 was inoculated into LB liquid medium at a volume ratio of 1:120 to 1:150 and cultured at 35℃ to 37℃ with shaking at 180r / min to 220r / min for 24h to 30h to obtain the fermentation broth of Bacillus hygroscopicus ZXY01.
[0030] Furthermore, the Bacillus subtilis LGT-2 seed culture was inoculated into LB liquid medium at a volume ratio of 1:120 to 1:150 and cultured at 35℃ to 37℃ with shaking at 180r / min to 220r / min for 24h to 30h to obtain Bacillus subtilis LGT-2 fermentation broth.
[0031] Furthermore, the Rhodopseudomonas palustris seed culture was inoculated into PM liquid culture medium at a volume ratio of 1:120 to 1:150 and cultured in a light incubator for 5 to 7 days to obtain Rhodopseudomonas palustris fermentation broth.
[0032] A compound fermentation preparation was prepared by mixing the fermentation broths of Bacillus hygroscopicus ZXY01, Bacillus subtilis LGT-2 and Rhodopseudomonas palustris in a volume ratio of 2.0:1.0:1.0.
[0033] The culture medium used in the examples is as follows: LB liquid medium (1L): 10.0g peptone, 5.0g sodium chloride, 10.0g yeast extract, 1L distilled water, pH 7.2. Sterilize at 0.15MPa and 121℃ for 20min.
[0034] LB solid medium: 1g peptone, 1g sodium chloride, 0.5g yeast extract, 1.8g agar powder, bring to a final volume of 100ml, autoclave at 121℃ for 20min.
[0035] PM liquid culture medium (1L): 1.701125g potassium dihydrogen phosphate, 1g ammonium sulfate, 1.7745g disodium hydrogen phosphate, 1mL concentrate, 100μL 0.1mol / L sodium thiosulfate pentahydrate, 1L ddH₂O. Sterilize at 0.15MPa, 121℃ for 20min. The concentrate (100mL) consists of: 2g aminotriacetic acid, 2.89g anhydrous magnesium sulfate, 5.03g calcium chloride, and 0.0198g ferrous sulfate heptahydrate. Sterilize at 0.15MPa, 121℃ for 20min.
[0036] PM solid medium: Add 1.8g agar powder to every 100mL of PM liquid medium and sterilize at 0.15MPa and 121℃ for 20min.
[0037] Example 2 The compound fermentation preparation prepared in Example 1 was used to conduct an agronomical test on the growth traits of tobacco plants.
[0038] 1. Field Trial Design The selected experimental site was located in Fangyuan Village, Guiyang County, Chenzhou City, Hunan Province, China. The selected tobacco field was a tobacco-rice rotation field, whose soil physicochemical properties and cultivation patterns were well-suited to the field conditions for conventional tobacco cultivation. The experiment consisted of two treatment groups: the experimental group (EG) received conventional tobacco basal fertilizer applied in the field, supplemented with the compound microbial inoculant of this invention; the control group (CK) received only the same amount of conventional tobacco basal fertilizer applied in the field, without the addition of the compound microbial inoculant. Field management practices were consistent across all groups, and no additional functional fertilizers were applied. The study was conducted using a standardized field plot experiment, with the overall experimental layout following the classic randomized block design principle. Each treatment was replicated three times, with each plot consisting of six rows, each row 12m long, and a plot area of 93.6m². 2 A 0.6m wide walkway was designed between the plots to facilitate sampling. Meanwhile, protective rows were set up around the entire experimental plot to isolate it from environmental interference from non-experimental areas at the plot edges, eliminate marginal effects, and ensure the accuracy and reliability of the experimental data.
[0039] 2. Agronomic traits of tobacco leaves Agronomic parameters of tobacco plants during their vigorous growth period, such as stem circumference, number of effective leaves, plant height, and length and width of upper / lower leaves, were measured, as shown in Table 1. Figure 1 and Figure 2 As shown, the regulatory effect of compound microbial agents on the growth of tobacco during its vegetative growth period was investigated.
[0040] Table 1 Agronomic traits of tobacco during its vigorous growth period
[0041] From Table 1 and Figure 2 Data shows that after applying compound microbial agents to the soil, the stem circumference, lower leaf width, and leaf length of the EG tobacco plants in the vigorous growth stage were higher than those in the CK control group, and the number of leaves increased slightly. Specifically, *Bacillus hygroscopicus* ZXY01 promoted stem development and improved nutrient absorption efficiency, increasing the stem circumference from 7.87 cm to 10.33 cm; *Bacillus subtilis* LGT-2 secreted growth-promoting substances, resulting in stable plant height growth; and *Rhodopseudomonas palustris* promoted leaf expansion and improved the overall growth of the tobacco plants. Figure 1 During the vigorous growth period, the root length and leaf size of the EG group were significantly greater than those of the CK group. In conclusion, the application of compound microbial agents can significantly promote root length growth, stem thickening, and leaf number increase during the vigorous growth period of tobacco, and has a positive regulatory effect on the robust growth of tobacco during the vegetative growth period, laying a good agronomic foundation for the subsequent improvement of tobacco yield and quality.
[0042] 3. Physical and chemical properties of soil To clarify the impact of the microbial agent on soil nutrient status, the contents of available potassium, ammonium nitrogen, and nitrate nitrogen in the soil were measured concurrently. The results are shown in Table 2 and... Figure 3 As shown.
[0043] Table 2. Soil physicochemical properties during the peak growing season
[0044] From Table 2 and Figure 3 The data shows that the physicochemical indicators of the soil in the experimental group were improved, with the available potassium content increasing by 14.4% compared to the control group, indicating a significant improvement in soil composition and nutrients. Among these improvements, Bacillus pyriformis ZXY01 enhanced the available potassium in the soil, which helps promote the transport of substances within the tobacco plant and strengthens the stems. The availability of nutrients such as ammonium nitrogen in the soil was also significantly better than that in the control group, indicating that Rhodopseudomonas palustris and Bacillus subtilis LGT-2 can promote soil nitrogen cycling and improve the level of available nitrogen supply, providing a key nutritional basis for leaf differentiation and expansion. The combined effect of these three factors ultimately resulted in a comprehensive optimization of the agronomic traits of the tobacco plant.
[0045] 4. Accumulation of dry matter in tobacco plants The weight of dry matter per plant during the vigorous growth period, such as Figure 4As shown. *Bacillus hygroscopicus* ZXY01 promotes root dry matter accumulation, while *Bacillus subtilis* LGT... 2. To improve the assimilation efficiency of stem and leaf matter, *Rhodopseudomonas palustris* optimizes the microenvironment through nitrogen fixation and other processes. This was achieved by measuring the dry matter weight of individual plants during the vigorous growth period. Figure 4 The data shows that the root dry weight increased by more than 30%, and the leaf dry weight increased steadily by more than 20%. This result indicates that the three components of the inoculant in this invention synergistically improve the rate of dry matter accumulation. By optimizing the supply of soil nutrients, it not only promotes the improvement of agronomic traits such as the thickening of tobacco stem circumference and the number of leaves, but also significantly improves the efficiency of dry matter accumulation, laying a solid material foundation for the final yield and quality.
[0046] In summary, applying the compound microbial agent of this invention to conventional base fertilizer can significantly and positively regulate the agronomic traits of tobacco plants. Compared with existing dual-strain agents, such as those combined with Bacillus and Rhodopseudomonas palustris, Bacillus hygroscopicus ZXY01 and Bacillus subtilis LGT... 2. The combination of Rhodopseudomonas swampus and these three bacteria has a synergistic effect and stronger functional complementarity. It can simultaneously achieve potassium solubilization, growth promotion and nitrogen fixation, effectively overcoming the problems of limited nutrient regulation of dual strain components, weak environmental suitability and excessive proliferation of single strains. By simultaneously increasing the levels of available potassium and available nitrogen in the soil, it promotes the agronomic traits and dry matter accumulation of tobacco during the vigorous growth period, and ultimately achieves the comprehensive effect of improving tobacco leaf quality and improving the quality of tobacco-growing soil.
[0047] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A compound microbial agent, characterized in that, Including Bacillus hygroscopicus ZXY01, Bacillus subtilis LGT-2, and Rhodopseudomonas palustris.
2. The compound microbial agent according to claim 1, characterized in that, The effective viable count of the *Bacillus hygroscopicus* ZXY01 is ≥2.0 × 10⁻⁶. 10 cfu / mL.
3. The compound microbial agent according to claim 1, characterized in that, The effective viable count of Bacillus subtilis LGT-2 is ≥2.0 × 10⁻⁶. 10 cfu / mL.
4. The compound microbial agent according to claim 1, characterized in that, The effective viable count of *Rhodopseudomonas palustris* is ≥2.0 × 10⁻⁶. 10 cfu / mL.
5. The application of the compound microbial agent according to any one of claims 1 to 4 in promoting tobacco growth or improving tobacco-growing soil.
6. The application according to claim 5, characterized in that, This involves preparing seed liquids of different strains of the compound microbial agent, then inoculating them into a fermentation medium for expansion to obtain a fermentation broth, and finally mixing them to prepare a compound fermentation preparation.
7. The application according to claim 6, characterized in that, Both Bacillus alpineus ZXY01 and Bacillus subtilis LGT-2 were prepared into seed culture using LB liquid medium, while Rhodopseudomonas palustris was prepared into seed culture using PM liquid medium.
8. The application according to claim 7, characterized in that, The volume ratio of the fermentation broth of Bacillus hygroscopicus ZXY01, Bacillus subtilis LGT-2, and Rhodopseudomonas palustris is (1.9~2.1):(0.9~1.1):(0.9~1.1).
9. The application according to claim 8, characterized in that, The volume ratio of the fermentation broth of Bacillus hygroscopicus ZXY01, Bacillus subtilis LGT-2, and Rhodopseudomonas palustris is 2.0:1.0:1.
0.
10. The application according to claim 6, characterized in that, It also includes applying the compound fermentation agent to the plants, including hole application or ring application.