Lactobacillus muciapparius mucosae lm-04 and application thereof

Abstract

This invention discloses a type of *Lactobacillus mucosa* LM-04 and its applications. When the fermentation broth of *Lactobacillus mucosa* LM-04 is applied to soil, the *Lactobacillus mucosa* LM-04 exhibits strong decomposition capabilities, enabling it to rapidly multiply using environmental nutrients, improve soil fertility, and secrete metabolic products (biostimulants) that promote crop growth, increasing yield and quality. Simultaneously, it continuously inhibits other microorganisms (including pathogens) from occupying their microecological niche, preventing the formation of harmful substances, improving the plant growth environment, and achieving green agricultural development.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a mucosal lactobacillus LM-04 and its applications. Background Technology

[0002] The core of soil problems lies in the soil microecology, thus the importance of microbial technology in agricultural production is undeniable. However, currently, there are very few agricultural microbial products that can overcome the harm of chemical fertilizers and pesticides, solve continuous cropping obstacles, and are suitable for modern farming management. Effective, economical, combined prevention and control, and comprehensive soil remediation agricultural microbial technologies are even rarer. Domestic research on microbial fertilizers mostly focuses on the isolation and screening of easily isolated and cultured strains. The main functions of these strains include nitrogen fixation (mainly rhizobia, which have increased significantly since the 1960s), biodegradation, and promotion of crop growth (mainly Bacillus, which have grown rapidly since the 1990s). Among these, the microbial strains applicable to production are relatively limited, and there is a general lack of new and highly efficient strains. Moreover, many microbial strains show good results in laboratory research, but often fail to achieve ideal results in field application. Summary of the Invention

[0003] One of the objectives of this invention is to overcome the shortcomings of the prior art and provide a mucosal lactobacillus LM-04.

[0004] The second objective of this invention is to overcome the shortcomings of the prior art and provide a use for *Lactobacillus mucosa* LM-04 in agricultural planting. When the fermentation broth of *Lactobacillus mucosa* LM-04 is applied to the soil, the bacterium, with its strong decomposition ability, can rapidly multiply using environmental nutrients, improving soil fertility. It also secretes metabolic products (biostimulants) to promote crop growth, increasing yield and quality. Simultaneously, by continuously inhibiting other microorganisms (including pathogens) from occupying their microecological niche, it avoids the formation of harmful substances, improves the plant growth environment, and achieves green development and modernization in agriculture.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A type of mucosal lactobacillus, with the Latin name Limosilactobacillus mucosae, strain code LM-04, accession number CGMCC No. 30844, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on June 3, 2024.

[0007] The *Limosilactobacillus mucosa* strain LM-04 has rod-shaped, Gram-positive cells measuring 0.5–0.6 μm × 1.3–2.5 μm, and does not form spores. Physiological and biochemical analysis, 16S rDNA sequencing, and whole-genome ANI and dDDH analysis identified it as *Limosilactobacillus mucosa*. The 16S rDNA sequence of this strain is shown in SEQ ID NO: 1.

[0008] SEQ ID NO: 1

[0009]

[0010] This invention also discloses the application of *Lactobacillus mucosa* LM-04 in agricultural planting. Specifically, the fermentation broth of *Lactobacillus mucosa* LM-04 is applied to the soil as a base fertilizer or top dressing, wherein the amount of fermentation broth used per acre is 3-6L.

[0011] More specifically, in rice cultivation, the fermentation broth of *Lactobacillus mucosa* LM-04 is applied to the soil as a base fertilizer and the soil is immediately tilled. After successful strawberry planting, the fermentation broth of *Lactobacillus mucosa* LM-04 is applied to the strawberry soil as a top dressing via drip irrigation. In cotton cultivation, the fermentation broth of *Lactobacillus mucosa* LM-04 is dripped onto the cotton roots.

[0012] Preferably, the method for preparing the fermentation broth of *Lactobacillus mucosa* LM-04 includes the following:

[0013] Step 1: Prepare the culture medium: 10g beef extract, 10g egg white, 10g yeast extract, 10g glucose, 10g lactose, 1mL Tween 80, 10g sodium acetate, 4g triammonium citrate, 4g dipotassium hydrogen phosphate, 0.4g magnesium sulfate and 0.1g manganese sulfate, add distilled water and stir to dissolve, then bring the volume to 1000mL; dispense into containers, autoclave at 115℃ for 20 minutes, and cool to room temperature for later use;

[0014] Step 2: Liquid deep fermentation culture: Inoculate a single colony of *Lactobacillus mucosa* LM-04 into the culture medium prepared in Step 1, and culture with shaking at 80-120 r / min. The fermentation temperature is 35℃-38℃, and the fermentation time is 48-72 h. The OD value of the fermentation broth is measured to be 4-7; thus, the fermentation broth of *Lactobacillus mucosa* LM-04 is obtained.

[0015] The beneficial effects of this invention are:

[0016] Lactobacillus mucosa LM-04 is a lactic acid bacterium that does not produce dormant bodies (spores). It can tolerate high osmotic pressure and low temperature environments and can maintain metabolic activity under harsh conditions.

[0017] Lactobacillus mucosa LM-04 is a facultative anaerobic bacterium with low oxygen dependence, and can still perform biological functions in the absence of oxygen.

[0018] Lactobacillus mucosa LM-04 does not secrete antibiotics and has good biosafety. Its antibacterial substances are mainly short- and medium-chain fatty acids, which are green and safe and will not affect the ecological safety of microorganisms in the soil.

[0019] Mucosal lactobacillus LM-04 possesses strong decomposition capabilities, enabling it to rapidly multiply using environmental nutrients, improve soil fertility, and secrete metabolic products (biostimulants) that promote crop growth, increasing yield and quality. Simultaneously, it continuously inhibits other microorganisms (including pathogens) from occupying their microecological niche, preventing the formation of harmful substances, improving the plant growth environment, and achieving green development and modernization in agriculture. The method of this invention is simple, easy to implement, and has stable effects. Attached Figure Description

[0020] Figure 1 The rice growth-promoting effect of Lactobacillus mucosa LM-04 is shown in the comparison chart of tillering normalization index and yield between the fermentation broth treatment group and the control group of Lactobacillus mucosa LM-04.

[0021] Figure 2 The images show field photos of rice treated with Lactobacillus mucosa LM-04, specifically comparing the growth of the fermentation broth treatment group and the control group. Detailed Implementation

[0022] Example 1

[0023] Application of *Lactobacillus mucosa* LM-04 in rice cultivation:

[0024] a. Preparation of culture medium: 10g beef extract, 10g egg white, 10g yeast extract, 10g glucose, 10g lactose, 1mL Tween 80, 10g sodium acetate, 4g triammonium citrate, 4g dipotassium hydrogen phosphate, 0.4g magnesium sulfate and 0.1g manganese sulfate, add distilled water and stir to dissolve, then bring the volume to 1000mL; dispense into containers, autoclave at 115℃ for 20 minutes, and cool to room temperature for later use;

[0025] b. Liquid deep fermentation culture: A single colony of *Lactobacillus mucosa* LM-04 was inoculated into the above culture medium and cultured with shaking at 100 r / min. The fermentation temperature was 36℃ and the fermentation time was 72 h. The OD value of the fermentation broth was measured to be 7; thus, the fermentation broth of *Lactobacillus mucosa* LM-04 was obtained.

[0026] c. In May 2020, a rice growth-promoting experiment was conducted at the China National Rice Research Institute experimental site in Caoqiao Town, Pinghu City, Jiaxing, Zhejiang Province. The experiment was a large-scale regional trial, with each treatment covering approximately 300 square meters. Fermentation broth of *Lactobacillus mucosa* LM-04 was applied to the soil as basal fertilizer, followed by immediate tilling. 3L of fermentation broth was used per mu (approximately 0.067 hectares). Transplanting (seedling variety: Yongyou 1540, machine transplanting) began one week after application. The fertilizer ratio was NPK (pure): 11.31-4.8-6; pure nitrogen was 11.31 kg N / mu, with a basal-tillering ratio of 8:2. Phosphorus fertilizer was entirely used as basal fertilizer, and potassium fertilizer was configured at a basal-early panicle ratio of 5:5. Other water management and pest and disease management followed the local field cultivation practices (mainstream cultivation model in the Yangtze River Basin). The control group did not receive fermentation broth of *Lactobacillus mucosa* LM-04. At the tillering stage, 50 rice hills were sampled to determine the number of tillers. During the ripening period, rice is harvested uniformly, threshed and impurities are removed, weighed, and the real-time grain moisture content is measured. The actual yield is then calculated based on the standard moisture content of 14%.

[0027] d. Experimental results are as follows Figure 1 As shown. From Figure 1 The results clearly show that the fermentation broth of *Lactobacillus mucosa* LM-04 significantly increased tillering in the treatment group. The normalized index of tillering in the control group was 85.1, while that in the treatment group was 93.0. The yield per mu (667 square meters) also increased significantly, from 585.0 kg / mu in the control group to 651.2 kg / mu in the treatment group, representing an increase of 11.31%. (See on-site inspection) Figure 2 It was found that the mucosal lactobacillus treatment group had green stems that ripened to yellow color and had well-developed root systems compared to the control group.

[0028] Example 2

[0029] Application of Lactobacillus mucosa LM-04 in strawberry cultivation

[0030] a. Preparation of culture medium: 10g beef extract, 10g egg white, 10g yeast extract, 10g glucose, 10g lactose, 1mL Tween 80, 10g sodium acetate, 4g triammonium citrate, 4g dipotassium hydrogen phosphate, 0.4g magnesium sulfate and 0.1g manganese sulfate, add distilled water and stir to dissolve, then bring the volume to 1000mL; dispense into containers, autoclave at 115℃ for 20 minutes, and cool to room temperature for later use;

[0031] b. Liquid deep fermentation culture: A single colony of *Lactobacillus mucosa* LM-04 was inoculated into the above culture medium and cultured with shaking at 100 r / min. The fermentation temperature was 36℃ and the fermentation time was 48 h. The OD value of the fermentation broth was measured to be 4; the fermentation broth of *Lactobacillus mucosa* LM-04 was obtained.

[0032] c. In September 2021, a strawberry planting (off-season) experiment was conducted at the Zhejiang Agricultural and Forestry University experimental site in the 1,000-mu Xixiang Strawberry Industrial Park in Laocun Village, Datong Town, Jiande City, Hangzhou, Zhejiang Province. The experiment used a greenhouse plot, with a single treatment area of ​​approximately 250 square meters, and the variety was Yuexiu. After successful strawberry planting, fermentation broth of *Lactobacillus mucosa* LM-04 was applied to the soil as top dressing (drip irrigation), with 1L of fermentation broth used per mu per month. Other water and fertilizer management, and pest and disease management were the same as in local facility cultivation. The control group did not receive fermentation broth of *Lactobacillus mucosa* LM-04. Forty-five days after strawberry planting (October 19, 2021), five random sampling points were surveyed, with 10 strawberry plants selected at each point, for a total of 50 plants. Plant height, stem diameter, leaf size, and petiole length were measured. Strawberry yield was recorded from the beginning of the strawberry harvest (December 2021) until February 28, 2022.

[0033] d. The experimental results are shown in Tables 1 and 2. Table 1 clearly shows that, compared to the control group, the fermentation broth treatment group with *Lactobacillus mucosa* LM-04 effectively reduced initial excessive growth, with plant height decreasing by 5.0% and petiole length decreasing by 7.2%. However, the plants were more robust (stem diameter and leaf area are key indicators in strawberry development), with stem diameter increasing significantly by 25.2% and leaf size increasing by 12.8%-22.4%. Comparing strawberry yields (Table 2), it was found that the fermentation broth treatment group with *Lactobacillus mucosa* LM-04 increased yield by 226 kg / mu compared to the control group, representing a yield increase of 24.7%.

[0034] Table 1. Various growth indicators of strawberries

[0035]

[0036] Table 2. Strawberry Harvest Statistics

[0037]

[0038] Example 3

[0039] Application of Lactobacillus mucosa LM-04 in cotton cultivation

[0040] a. Preparation of culture medium: 10g beef extract, 10g egg white, 10g yeast extract, 10g glucose, 10g lactose, 1mL Tween 80, 10g sodium acetate, 4g triammonium citrate, 4g dipotassium hydrogen phosphate, 0.4g magnesium sulfate and 0.1g manganese sulfate, add distilled water and stir to dissolve, then bring the volume to 1000mL; dispense into containers, autoclave at 115℃ for 20 minutes, and cool to room temperature for later use;

[0041] b. Liquid deep fermentation culture: A single colony of the strain was inoculated into the above culture medium and cultured with shaking at 80-120 r / min. The fermentation temperature was 36℃ and the fermentation time was 72h. The OD value of the fermentation broth was measured to be 7; thus, the fermentation broth of Lactobacillus mucosa LM-04 was obtained.

[0042] c. In April 2022, an application experiment of cotton in saline-alkali land was conducted at the Xinjiang Agricultural University-Xinjiang Academy of Agricultural Sciences Experimental Base in Dafeng Town, Hutubi County, Changji Prefecture, Xinjiang. The experiment was a plot-based comparative trial with a randomized block design. Each treatment was replicated three times, resulting in a total of six plots, each with an area of ​​54 m². 2 (A single-film, 6-row machine-harvested cotton planting pattern was adopted, with a row spacing of (66+10) cm and a plant spacing of 10 cm. The cotton variety was Xinluzao 82, sown on April 15, 2022, and seedlings were watered on April 28. The treatment group received 1 L / mu of fermentation broth of *Lactobacillus mucosa* LM-04 via drip irrigation on June 2, 18, 28, July 12, 28, and August 7 (applied to the roots, ensuring the same amount of irrigation per plant, and applied with the irrigation water). Other water and fertilizer management, and pest and disease management were the same as in local field cultivation. The control group did not receive fermentation broth of *Lactobacillus mucosa* LM-04. Cotton agronomic traits, cotton yield, and its components were statistically analyzed.

[0043] d. Experimental results are shown in Tables 3 and 4. Table 3 clearly shows that treatment with the fermentation broth of *Lactobacillus mucosa* LM-04 helped increase cotton plant height and total boll count, while reducing the number of empty fruit branches. There was no significant difference in the number of harvested plants among the treatments. The drip application treatment resulted in 6.37 bolls per plant, a significant increase of 6.9% compared to the conventional fertilization (control). The average boll weight reached 6.17 g, a significant increase of 0.52 g (9.2%) compared to the control. Seed cotton yield results showed that the treatment increased yield by 910.68 kg / hm² compared to the control. 2 This represents an increase of 13.33%.

[0044] Table 3 Effects of *Lactobacillus mucosa* on agronomic traits of cotton.

[0045]

[0046] Table 4. Effects of *Lactobacillus mucosa* on cotton yield and its components.

[0047]

[0048] 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. A type of *Limosilactobacillus mucosae* LM-04, with accession number CGMCC No. 30844, deposited on June 3, 2024, at the China General Microbiological Culture Collection Center.

2. The application of *Lactobacillus mucosa* LM-04 as described in claim 1 in agricultural planting, characterized in that: The agricultural planting method is as follows: In rice planting, the fermentation liquid of Lactobacillus mucosa LM-04 is applied to the soil of rice planting as a base fertilizer and the land is immediately turned over. Alternatively, the agricultural planting method is as follows: after the strawberry plants are successfully planted, the fermentation liquid of Lactobacillus mucosa LM-04 is applied to the soil for strawberry planting in the form of top dressing and drip irrigation. Alternatively, the agricultural planting method is as follows: in cotton planting, the fermentation liquid of Lactobacillus mucosa LM-04 is dripped onto the roots of cotton.

3. The application as described in claim 2, characterized in that: The method for preparing the fermentation broth of *Lactobacillus mucosa* LM-04 includes the following steps: Step 1: Prepare the culture medium: 10g beef extract, 10g egg white, 10g yeast extract, 10g glucose, 10g lactose, 1mL Tween 80, 10g sodium acetate, 4g triammonium citrate, 4g dipotassium hydrogen phosphate, 0.4g magnesium sulfate and 0.1g manganese sulfate, add distilled water and stir to dissolve, then bring the volume to 1000mL; dispense into containers, autoclave at 115℃ for 20 minutes, and cool to room temperature for later use; Step 2: Liquid deep fermentation culture: Inoculate a single colony of *Lactobacillus mucosa* LM-04 into the culture medium prepared in Step 1, and culture with shaking at 80-120 r / min. The fermentation temperature is 35℃-38℃, and the fermentation time is 48-72h. The OD value of the fermentation broth is measured to be 4-7; thus, the fermentation broth of *Lactobacillus mucosa* LM-04 is obtained.

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