Microbial agent for improving drought resistance of mesona aculeata

Abstract

The present application belongs to the technical field of drought resistance of Mesona chinensis Benth, and particularly relates to a microbial agent for improving drought resistance of Mesona chinensis Benth. The microbial agent for improving drought resistance of Mesona chinensis Benth is composed of Bacillus circulans bacterial liquid and Bacillus mycoides bacterial liquid, and the volume ratio of the Bacillus circulans bacterial liquid to the Bacillus mycoides bacterial liquid is 1:3-5. The microbial agent of the present application is composed of the Bacillus circulans bacterial liquid and the Bacillus mycoides bacterial liquid in a volume ratio of 1:3-5, and the Bacillus circulans bacterial liquid and the Bacillus mycoides bacterial liquid are respectively obtained by culturing corresponding strains in a liquid culture medium. During the planting of Mesona chinensis Benth, the microbial agent of the present application can significantly delay the damage to the Mesona chinensis Benth plants under drought stress, and is helpful to improve the drought resistance of the Mesona chinensis Benth, which is of great significance to the development of the Mesona chinensis Benth industry.

Description

Technical Field

[0001] This invention belongs to the field of stress resistance technology of Mesona chinensis, specifically relating to a microbial agent that improves the drought resistance of Mesona chinensis. Background Technology

[0002] Mesona chinensis, also known as fairy grass, is an annual herb belonging to the genus Mesona in the Lamiaceae family. It has high nutritional and medicinal value and is a plant used for both food and medicine. The branches and leaves of Mesona chinensis can be boiled to extract mesona chinensis gum, which is used to make herbal jelly, grass jelly, grass jelly dew, and grass jelly gel. Currently, 80% of the popular herbal tea beverages on the market use Mesona chinensis as their main ingredient.

[0003] With the intensification of global climate change, the frequency of droughts is increasing. Drought is a significant factor affecting plant growth and physiological and biochemical processes, and excessively low soil moisture can severely impact plant growth and development. The growth and development of *Mesona chinensis* (a type of grass) is also constrained by drought. Therefore, researching how to improve the drought resistance of *Mesona chinensis* is of great significance to the development of the *Mesona chinensis* industry.

[0004] Existing technologies have disclosed that graphene oxide can improve the drought resistance of plants. For example, the study "Effects of Graphene Oxide on the Growth, Development and Drought Resistance of Brassica napus" (Zhang Zishuang et al., 2023) disclosed that soaking Brassica napus seeds in 15 mg / L graphene oxide significantly increased plant water content, proline content, and tetrazolium reducing power on the 10th day of drought stress, thus significantly improving the drought resistance of Brassica napus.

[0005] In addition, existing technologies have also disclosed the use of microbial agents to improve the drought resistance of plants. For example, the study "Effects of Different Microbial Agents on the Heat Tolerance and Drought Resistance of Perennial Ryegrass" (Zhang Zijia et al., 2022) disclosed that a compound agent of Bacillus subtilis (M1), Trichoderma harzianum (M2), Bacillus licheniformis and Streptomyces flavus (M3), and a compound agent of Bacillus erythrophorus and Bacillus subtilis (M4) can all improve the drought resistance of perennial ryegrass. Under drought conditions for 10 days, the effects of agents M1 and M2 on optimizing various physiological indicators were comparable, and both were superior to the treatments with agents M3 and M4. Under drought conditions for 20 days, agent M2 showed the strongest improvement in the drought resistance of perennial ryegrass.

[0006] However, there are currently no reports on using microbial agents to improve the drought resistance of grass jelly. Summary of the Invention

[0007] The purpose of this invention is to provide a microbial agent that enhances the drought resistance of grass jelly, thereby solving the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A microbial agent for improving the drought resistance of grass jelly is composed of a compound of Bacillus circulans bacterial solution and Bacillus mucilaginosus bacterial solution, wherein the volume ratio of Bacillus circulans bacterial solution to Bacillus mucilaginosus bacterial solution is 1:3-5.

[0010] More specifically, the bacterial count of both the circulated Bacillus bacterial suspension and the gelatinous Bacillus bacterial suspension is 1.0 × 10⁻⁶. 7 cfu / mL.

[0011] More specifically, the preparation method of the *Bacillus circulans* bacterial suspension or *Bacillus canolatus* bacterial suspension is as follows: The bacteria are inoculated onto beef extract solid medium and cultured at 30°C for 24 hours. A single colony is picked and inoculated into 5 mL of beef extract liquid medium, and pre-cultured at 30°C and 200 rpm for 12 hours. Then, a 1% inoculum is transferred to fresh beef extract liquid medium and cultured at 30°C and 200 rpm for 12 hours to expand the culture. Finally, fresh beef extract liquid medium is added to the culture medium to adjust the bacterial count to 1.0 × 10⁻⁶. 7 The concentration of cfu / mL yields either a circular Bacillus culture or a gelatinous Bacillus culture.

[0012] More specifically, the formula for the beef extract solid culture medium is: 3.0g beef extract, 10.0g peptone, 5.0g sodium chloride, 1L distilled water, pH 7.3±0.1;

[0013] The formula for the beef extract liquid culture medium is as follows: 3.0g beef extract, 10.0g peptone, 5.0g sodium chloride, 1L distilled water, pH 7.3±0.1.

[0014] The present invention also provides the application of the microbial agent for improving the drought resistance of Mesona chinensis in improving the drought resistance of Mesona chinensis.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The microbial inoculant of this invention is composed of *Bacillus circulans* bacterial suspension and *Bacillus mucilaginosus* bacterial suspension at a volume ratio of 1:3-5. The *Bacillus circulans* and *Bacillus mucilaginosus* bacterial suspensions are respectively obtained by culturing corresponding strains in liquid culture medium. During the cultivation of *Mesona chinensis*, applying the microbial inoculant of this invention can significantly delay the damage to *Mesona chinensis* plants under drought stress, helping to improve the drought resistance of *Mesona chinensis*, and is of great significance to the development of the *Mesona chinensis* industry. Attached Figure Description

[0017] Figure 1 The effect of different compound bacterial solutions on the relative water content of Mesona chinensis leaves under drought stress;

[0018] Figure 2The effects of different compound bacterial solutions on root vigor of Mesona chinensis plants under drought stress;

[0019] Figure 3 The effect of different compound bacterial solutions on proline content in the leaves of Mesona chinensis plants under drought stress;

[0020] Figure 4 The effects of different compound bacterial solutions on malondialdehyde content in the leaves of Mesona chinensis plants under drought stress. Detailed Implementation

[0021] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0022] 1. Materials and Methods

[0023] 1.1 Test Culture Medium

[0024] The formula for beef extract solid culture medium is as follows: 3.0g beef extract, 10.0g peptone, 5.0g sodium chloride, 15g agar, 1L distilled water, pH 7.3±0.1;

[0025] The formula for the beef extract liquid culture medium is as follows: 3.0g beef extract, 10.0g peptone, 5.0g sodium chloride, 1L distilled water, pH 7.3±0.1.

[0026] 1.2 Bacillus circulatory bacteria suspension

[0027] Bacillus circulans (purchased from Beina Biotechnology, product number BNCC186120) was inoculated onto beef extract solid medium and cultured at 30°C for 24 h. Single colonies were then picked and inoculated into 5 mL of beef extract liquid medium, and pre-cultured at 30°C and 200 rpm for 12 h. The culture was then transferred to fresh beef extract liquid medium at a 1% (v / v) inoculation rate and cultured at 30°C and 200 rpm for 12 h. Fresh beef extract liquid medium was added to the culture medium to adjust the bacterial count to 1.0 × 10⁻⁶. 7 The cfu / mL concentration yields the Bacillus circulatory system.

[0028] 1.3 Gelatinous Bacillus Fluid

[0029] Bacillus jellyoides (purchased from Beina Biotechnology, product number BNCC340613) was inoculated onto beef extract solid medium and cultured at 30°C for 24 h. Single colonies were picked and inoculated into 5 mL of beef extract liquid medium and pre-cultured at 30°C and 200 rpm for 12 h. The culture was then transferred to fresh beef extract liquid medium at a 1% (v / v) inoculation rate and cultured at 30°C and 200 rpm for 12 h. Fresh beef extract liquid medium was added to the culture medium to adjust the bacterial count to 1.0 × 10⁻⁶. 7 cfu / mL.

[0030] 1.4 Drought Resistance Experiment

[0031] A compound bacterial solution consisting of Bacillus circulans and Bacillus mucilaginosus was used to study the effect of the compound bacterial solution on the drought resistance of Mesona chinensis.

[0032] 1.4.1 Test Materials

[0033] "Lingshan Large-Leaf" Meadow Jelly Grass

[0034] 1.4.2 Test Setup

[0035] See Table 1.

[0036] Table 1 Different experimental settings

[0037]

[0038] 1.4.3 Test Methods

[0039] Take field soil, air dry it, pass it through a 20-mesh sieve, and mix in 1% organic fertilizer by soil weight (purchased from Hubei Laijing Biotechnology Co., Ltd., organic matter content ≥30%, implementation standard: NY / T252-2021, registration number: E Nong Fei (2023) Zhun Zi 4135). After mixing evenly, fill the pot (diameter 20cm, height 15cm) to 4 / 5 full, and use bottom drip irrigation to make the soil completely moist.

[0040] Seedlings of *Mesona chinensis* reaching 25cm in height were transplanted into pots, the soil around the roots was pressed firmly, and the plants were thoroughly watered. One seedling was planted per pot, with three seedlings per hole. The seedlings were then randomly divided into seven groups of 30 pots each. Seven days after transplanting, the seedlings were treated with different proportions of bacterial solutions (as shown in Table 1 for groups 1-7) at a ratio of 150mL per pot. This was repeated twice after seven days. For the first 20 days after transplanting, the soil was kept moist by watering with 200mL of water every two days. From day 20 onwards, watering was stopped for groups 1-7 to achieve drought stress. Relevant indicators were measured on days 3, 7, and 15 after watering was stopped (i.e., drought stress). All experiments were conducted indoors under natural ventilation conditions.

[0041] 1.4.4 Measurement Indicators

[0042] When measuring the indicators, 10 pots were randomly selected for each treatment, and one plant was selected from each pot. The average value of the measured results was taken.

[0043] a. Relative water content of leaves

[0044] Three fully expanded leaves were selected (the leaves were selected from the same location across different treatment groups, avoiding the top and base), their surfaces were wiped clean, and their fresh weight (m1) was measured. After drying in an oven at 85℃ to constant weight, their dry weight (m2) was measured. The relative water content of the leaves was calculated.

[0045] Relative water content of leaves = (m1-m2) / m1×100%.

[0046] b. Root vitality

[0047] The root vigor of *Gynostemma pentaphyllum* was determined using the triphenyltetrazolium chloride method (Zhang Zijia et al., 2022).

[0048] c. Proline content

[0049] The proline content in the leaves of *Gynostemma pentaphyllum* was determined using the sulfosalicylic acid method (Zhang Zishuang et al., 2023).

[0050] d. Malondialdehyde content

[0051] The malondialdehyde content in the leaves of *Gnaphalium affine* was determined by the thiobarbituric acid method (Zhang Zijia et al., 2022).

[0052] 1.5 Results

[0053] See results Figure 1-4 .

[0054] Depend on Figure 1 It was found that within 3-15 days of drought stress treatment, the relative water content of *Mesona chinensis* leaves gradually decreased with the extension of drought stress duration. The decrease was most significant in study group 7 (blank control), with the relative water content dropping to approximately 45% on day 15 of drought stress. Different bacterial solutions also resulted in a decrease in relative water content. The decrease was smaller with different ratios of compound bacterial solutions compared to single bacterial solutions, especially when *Bacillus circulans* and *Bacillus mucilaginosus* solutions were used in volume ratios of 1:3 and 1:5, resulting in the smallest decrease in relative water content. This indicates that the compound bacterial solution of the present invention can maintain the relative water content of *Mesona chinensis* leaves and mitigate the impact of drought stress on the relative water content of *Mesona chinensis* leaves.

[0055] Depend on Figure 2It was found that during the 3-15 days of drought stress treatment, the root vigor of *Mesona chinensis* gradually decreased with the extension of drought stress duration. Compared with study group 7, the decrease in root vigor was smaller in different bacterial solutions. Among them, when *Bacillus circinus* and *Bacillus mucilaginosus* solutions were used in combination at a volume ratio of 1:1-7, the decrease in root vigor of *Mesona chinensis* was relatively slow, which helped the *Mesona chinensis* plant resist drought stress.

[0056] Depend on Figure 3 It was found that during the 3-15 days of drought stress treatment, the proline content in the leaves of *Mesona chinensis* gradually accumulated with the extension of drought stress duration. Proline accumulation helps regulate osmosis and reduce the impact of drought stress on plants. Compared with study group 7, the proline accumulation in different bacterial solutions was significantly higher than that in study group 7. In particular, when *Bacillus circulans* and *Bacillus mucilaginosus* solutions were used in combination at a volume ratio of 1:3-5, the proline accumulation in *Mesona chinensis* leaves was most significant, indicating that the compound bacterial solution of the present invention can promote proline accumulation in *Mesona chinensis* to resist drought stress.

[0057] Depend on Figure 4 It was found that during the 3-15 days of drought stress treatment, malondialdehyde (MDA) content in the leaves of *Mesona chinensis* gradually accumulated with the extension of drought stress duration. On the 15th day of drought stress treatment, the MDA content in group 7 reached 20 nmol / g, indicating the most severe membrane damage. When different bacterial solutions were used, the MDA accumulation was significantly lower in groups with a composite volume of *Bacillus circulans* and *Bacillus mucilaginosus* solutions of 1:3-5 compared to other groups. This indicates that the composite bacterial solution of this invention can effectively mitigate drought stress-induced membrane damage in *Mesona chinensis*.

[0058] In summary, the microbial agent of the present invention, composed of Bacillus circulans and Bacillus mucilaginosus bacterial solutions, can significantly delay the damage to Mesona chinensis plants under drought stress, thereby helping to improve the drought resistance of Mesona chinensis.

[0059] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. The application of a microbial inoculant in promoting proline accumulation and enhancing the drought resistance of *Mesona chinensis*, characterized in that, The microbial agent is composed of a compound of *Bacillus circulans* bacterial suspension and a *Bacillus cannabinoids* bacterial suspension, with a volume ratio of 1:3-5; the bacterial count of both the *Bacillus circulans* and *Bacillus cannabinoids* bacterial suspensions is 1.0 × 10⁻⁶. 7 cfu / mL.

2. The application according to claim 1, characterized in that, The preparation method of the circular Bacillus bacterial suspension or gelatinous Bacillus bacterial suspension is as follows: Inoculate the bacteria onto beef extract solid medium and incubate at 30℃ for 24 h. Pick a single colony and inoculate it into 5 mL of beef extract liquid medium, pre-culture at 30℃ and 200 rpm for 12 h. Transfer the inoculum to fresh beef extract liquid medium at a 1% inoculation rate and expand the culture at 30℃ and 200 rpm for 12 h. Adjust the bacterial count to 1.0 × 10⁻⁶ by adding fresh beef extract liquid medium to the culture medium. 7 The concentration of cfu / mL yields either a circular Bacillus culture or a gelatinous Bacillus culture.

3. The application according to claim 2, characterized in that, The formula for the solid beef extract culture medium is: 3.0g beef extract, 10.0g peptone, 5.0g sodium chloride, 1L distilled water, pH 7.3±0.1; the formula for the liquid beef extract culture medium is: 3.0g beef extract, 10.0g peptone, 5.0g sodium chloride, 1L distilled water, pH 7.3±0.1.

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