A lactiplantibacillus strain lp-14 and applications thereof

Abstract

The application discloses a Lactiplantibacillus plantarum strain LP-14 and application thereof, the Lactiplantibacillus plantarum strain LP-14 is preserved in the China General Microbiological Culture Collection Center on June 3, 2024, and the preservation number is CGMCC No.30841.The application also provides application of the Lactiplantibacillus plantarum strain LP-14 in planting industry.Compared with the prior art, the Lactiplantibacillus plantarum strain LP-14 fermentation liquor can be applied to soil or leaf surface or seed surface, can rapidly reproduce by utilizing nutrients in the environment, can continuously inhibit other pathogenic bacteria and other microorganisms to occupy a microecological position advantage, avoid reproduction of harmful microorganisms, or directly stimulate the immune system of plants and seeds by contact and absorption of leaf surface and seeds, and improve the systemic resistance of plants.

Description

Technical Field

[0001] This invention relates to the field of preservation microbial bacteria technology, and more particularly to a strain of Lactobacillus plantarum LP-14 and its applications. Background Technology

[0002] The excessive use of chemical fertilizers and pesticides is a major cause of soil degradation and non-point source pollution in agriculture. It not only leads to a decline in arable land quality and increasingly severe pests and diseases, but also results in a decrease in the yield and quality of agricultural products. Excessive application of chemical fertilizers causes nitrogen, phosphorus, potassium, and other chemical substances to be bound in the soil, forming various chemical salts, leading to an imbalance in soil nutrient structure, deterioration of physical properties, and, in some areas, excessive levels of harmful metals and pathogens, thus causing soil degradation. Furthermore, water-soluble nutrients in the soil are washed away by rainwater and irrigation water, flowing into groundwater and rivers, causing groundwater and river pollution in some areas. The excessive use of pesticides leads to soil, air, and water pollution, disrupting the ecological balance. Therefore, agriculture needs to vigorously promote technological innovation, develop bio-based farming, and rapidly advance green development and modernization of agriculture.

[0003] To address these issues, China began researching, producing, and applying agricultural microbial products decades ago. Agricultural microbial agents refer to live bacterial preparations made from target microorganisms after propagation. These agents are typically used for seed dressing or root dipping, and have direct or indirect effects such as improving soil, restoring soil fertility, preventing soil-borne diseases, maintaining the balance of rhizosphere microbiota, and degrading toxic substances. However, current domestic agricultural microbial agent production is relatively limited, mainly focusing on traditional microorganisms such as Bacillus (accounting for 98% of production). Manufacturers also focus only on the live count of a single microbial species, neglecting the relationship between the microorganisms and pathogenic microorganisms, their impact on soil microecology, and their actual application effects on crops. The potential of these microorganisms has not been fully realized.

[0004] CN104745510A discloses a *Lactobacillus fermentatus* strain and its application in the control of plant fungal diseases. This strain was isolated from soil used for planting tomatoes and is classified as *Lactobacillus fermentatus*. The *Lactobacillus fermentatus* is fermented to obtain a fermentation broth, which is then concentrated to prepare a lactobacillus concentrate, which is then diluted and sprayed onto the leaves of crops. This strain shows good efficacy in inhibiting fungal diseases in crops and also promotes crop growth, meeting the requirements of ecological agriculture. The fermentation broth of this *Lactobacillus fermentatus* CGMCC No. 8735 strain can effectively inhibit the pathogens of cotton verticillium wilt, tomato gray mold, or rice sheath blight, with the best inhibitory effect on cotton verticillium wilt.

[0005] CN110106106A discloses a compound microbial inoculant and its application in strawberry cultivation, comprising Bacillus licheniformis, Clostridium butyricum, Lactobacillus, Olsenella, Rhodotorula rubra, and Bacillus amyloliquefaciens. It also provides a method for preparing the compound microbial inoculant and its application in strawberry cultivation. This invention's compound microbial inoculant can significantly inhibit and degrade root pathogens in strawberries, while also having a significant positive impact and promoting effect on strawberry growth and metabolism, greatly improving the quality of strawberry fruit.

[0006] CN113403218A discloses a biocontrol strain and its application in controlling soft rot of Fritillaria thunbergii and promoting its growth. The biocontrol strain is Dm97, with the preservation number GDMCC No:61459. The strain Dm97 has been identified as Bacillus sp. Field trials showed that the microbial agent prepared from strain Dm97 achieved a control effect of over 90% against soft rot of Fritillaria thunbergii, a growth-promoting effect of over 70% on disease-free plants, and a yield increase of over 50%. Using this microbial agent for initial soil treatment to prevent soft rot, and subsequent root treatment for better control, demonstrates excellent disease prevention and yield-increasing effects and has significant potential for development into a commercially viable live-bacterial biocontrol agent.

[0007] The patent documents mentioned above can individually address the prevention of cotton Verticillium wilt, strawberry root diseases, and fritillaria diseases, but they cannot simultaneously address multiple plant diseases. In practical applications, it is urgent to provide a microorganism that can simultaneously address multiple plant diseases. Summary of the Invention

[0008] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a strain of Lactobacillus plantarum LP-14 for use in agricultural planting, which can rapidly reproduce by utilizing nutrients in the environment, continuously inhibiting other pathogenic bacteria and other microorganisms from occupying the microecological niche advantage, avoiding the reproduction of harmful microorganisms, or directly stimulating the immune system of plants and seeds through contact and absorption on the leaves and seeds, thereby improving the systemic resistance of plants, including the prevention of cotton Verticillium wilt, strawberry root diseases, and fritillaria diseases.

[0009] To achieve the above objectives, the present invention provides a strain of *Lactobacillus plantarum* LP-14 and its applications.

[0010] A strain of Lactiplantibacillus plantarum, LP-14, with accession number CGMCC No. 30841, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on June 3, 2024.

[0011] The Lactiplantibacillus plantarum strain LP-14 is a Gram-positive bacterium. Physiological and biochemical analysis and 16S rDNA sequencing have identified it as Lactiplantibacillus plantarum. Specifically, the 16S rDNA base sequence of the strain is shown in the sequence listing (SEQ ID NO: 1).

[0012] The source of the plant lactobacillus strain LP-14 of this invention was isolated from natural water bodies and soil in Hangzhou.

[0013] The application of the *Lactobacillus plantarum* strain LP-14 in the planting industry is as follows:

[0014] A. Apply the fermentation broth of Lactobacillus plantarum strain LP-14 to the soil as a base fertilizer or top dressing, with a usage of 3-5L of fermentation broth per acre.

[0015] B. Spray the fermentation broth of Lactobacillus plantarum strain LP-14 as a foliar fertilizer onto the plant leaves (both sides), diluted 100-150 times, until the leaves are covered with water droplets and dripping.

[0016] C. Dilute the fermentation broth of *Lactobacillus plantarum* strain LP-14 by 100-150 times and use it for seed soaking; or dilute the fermentation broth of *Lactobacillus plantarum* strain LP-14 by 100-150 times and use it for root dipping of seedlings or for pulping with soil.

[0017] The soaking time is 1-1.5 hours.

[0018] The root dipping time is 5-10 minutes.

[0019] The fermentation broth of the Lactobacillus plantarum strain LP-14 is prepared as follows, in parts by weight:

[0020] (1) Preparation of culture medium: Add 10-12 parts tryptone, 10-12 parts yeast extract, 4-6 parts glucose, 1-2 parts Tween 80, 1-3 parts dipotassium hydrogen phosphate, 4-6 parts sodium acetate, 1-4 parts triammonium citrate, 0.1-0.4 parts magnesium sulfate, and 0.1-0.4 parts manganese sulfate to 950-1000 parts distilled water, stir and mix evenly, and sterilize under high temperature and high pressure for later use;

[0021] (2) Liquid deep fermentation culture: Inoculate a single colony of Lactobacillus plantarum strain LP-14 into the culture medium of step (1) and culture with shaking at 60-80 r / min to obtain fermentation broth of Lactobacillus plantarum strain LP-14.

[0022] The high-temperature and high-pressure sterilization conditions in step (1) are 115℃ and 0.1MPa.

[0023] The fermentation temperature in step (2) is 30℃-38℃;

[0024] The fermentation time in step (2) is 90-100 hours.

[0025] In step (2), the OD value of the fermentation broth of Lactobacillus plantarum strain LP-14 was measured to be 2.0-2.5.

[0026] The *Lactobacillus plantarum* strain LP-14 of this invention is a lactic acid bacteria microorganism with good biocompatibility and strong decomposition ability, making it suitable for agricultural planting. After being fermented into a liquid, it can be applied to soil, leaves, or seed surfaces, allowing it to rapidly multiply using nutrients in the environment, such as organic matter in the soil and waxy substances on leaves. Simultaneously, by continuously inhibiting pathogenic bacteria and other microorganisms, it occupies a dominant microecological position, preventing the proliferation of harmful microorganisms. Alternatively, through contact and absorption on leaves and seeds, it directly stimulates the immune system of plants and seeds, improving systemic resistance and the growth environment, thus achieving the goals of green agricultural development and modernization. The method of this invention is simple, easy to implement, and has stable effects.

[0027] The beneficial effects of this invention are:

[0028] Compared with existing technologies, the *Lactobacillus plantarum* strain LP-14 of this invention is a lactic acid bacteria microorganism with good biosafety and strong decomposition ability, making it suitable for agricultural planting. After being prepared into a fermentation broth, it can be applied to the soil, leaves, or seed surface for absorption upon contact, directly stimulating the immune system of plants and seeds, improving systemic resistance, including prevention of cotton verticillium wilt, strawberry root diseases, and fritillaria diseases. Detailed Implementation

[0029] Example 1

[0030] A strain of *Lactobacillus plantarum*, LP-14, with accession number CGMCC No. 30841, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on June 3, 2024.

[0031] The preparation method of fermentation broth of Lactobacillus plantarum strain LP-14 is as follows:

[0032] (1) Preparation of culture medium: Add 10g tryptone, 10g yeast extract, 5g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, and 0.1g manganese sulfate to 1000ml distilled water, stir and mix evenly, and sterilize at 115℃ and 0.1MPa for later use;

[0033] (2) Liquid deep fermentation culture: A single colony of *Lactobacillus plantarum* strain LP-14 was inoculated into the culture medium of step (1) above, and cultured by shaking at 60 r / min. The fermentation temperature was 35℃ and the fermentation time was 96 hours. The OD value of the fermentation broth was measured to be 2.2, and the fermentation broth of *Lactobacillus plantarum* strain LP-14 was obtained.

[0034] Example 2

[0035] A strain of *Lactobacillus plantarum*, LP-14, with accession number CGMCC No. 30841, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on June 3, 2024.

[0036] The preparation method of fermentation broth of Lactobacillus plantarum strain LP-14 is as follows:

[0037] (1) Preparation of culture medium: Add 10g tryptone, 10g yeast extract, 5g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, and 0.1g manganese sulfate to 1000ml distilled water, stir and mix evenly, and sterilize at 115℃ and 0.1MPa for later use;

[0038] (2) Liquid deep fermentation culture: A single colony of *Lactobacillus plantarum* strain LP-14 was inoculated into the culture medium of step (1) above, and cultured by shaking at 60 r / min. The fermentation temperature was 35℃ and the fermentation time was 96 hours. The OD value of the fermentation broth was measured to be 2.0, and the fermentation broth of *Lactobacillus plantarum* strain LP-14 was obtained.

[0039] Example 3

[0040] A strain of *Lactobacillus plantarum*, LP-14, with accession number CGMCC No. 30841, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on June 3, 2024.

[0041] The preparation method of fermentation broth of Lactobacillus plantarum strain LP-14 is as follows:

[0042] (1) Preparation of culture medium: Add 10g tryptone, 10g yeast extract, 5g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, and 0.1g manganese sulfate to 1000ml distilled water, stir and mix evenly, and sterilize at 115℃ and 0.1MPa for later use;

[0043] (2) Liquid deep fermentation culture: A single colony of *Lactobacillus plantarum* strain LP-14 was inoculated into the culture medium of step (1) above, and cultured by shaking at 60 r / min. The fermentation temperature was 35℃ and the fermentation time was 96 hours. The OD value of the fermentation broth was measured to be 2.0, and the fermentation broth of *Lactobacillus plantarum* strain LP-14 was obtained.

[0044] Application Example 1

[0045] The fermentation broth of *Lactobacillus plantarum* strain LP-14 from Example 1 was applied to plants via drip irrigation to enhance crop resistance. The specific application is as follows:

[0046] From June to October 2022, a plot experiment was conducted on cotton (Gengye 64) in Shibahu Village, Dafeng Town, Changji Hui Autonomous Prefecture, Xinjiang Uygur Autonomous Region. The *Lactobacillus plantarum* strain LP-14 fermentation broth prepared in Example 1 was diluted 100 times as the experimental group and applied as root fertilization via drip irrigation. A control group was also included, without drip irrigation of the *Lactobacillus plantarum* strain LP-14 fermentation broth. 300 plants were surveyed in each group. The broth was applied four times during the cotton's growth period (June 20th, June 29th, July 8th, and July 18th), with each application using 1 L / mu. Verticillium wilt is the "number one disease" of cotton, causing severe damage and often referred to as the "cancer" of cotton. The results of the Verticillium wilt disease index and disease incidence rate in Test Example 1 are shown in Table 1.

[0047] Table 1. Survey on the preventive effect of cotton Verticillium wilt.

[0048]

[0049] Note: The grading standard is as follows: Grade 0, no disease in the whole plant; Grade 1, less than 5% of the whole plant is diseased; Grade 3, 6%-25% of the whole plant is diseased; Grade 5, 26%-50% of the whole plant is diseased; Grade 7, 51%-75% of the whole plant is diseased; Grade 9, more than 76% of the whole plant is diseased.

[0050] Disease severity index (DSE) = 100 × ∑(number of diseases at each level × representative value at each level) / (total number of surveys × highest representative value).

[0051] Prevention and control efficacy (efficacy, %) = (Control disease index - Treatment disease index) / Control disease index × 100

[0052] As shown in Test Example 1, the experimental group treated with the fermentation broth of the plant lactobacillus strain LP-14 of this invention significantly improved the control effect, alleviated the disease, and reduced the disease index in the control of Verticillium wilt compared with the control group.

[0053] Application Example 2

[0054] The fermentation broth of *Lactobacillus plantarum* strain LP-14 from Example 2 was used as a foliar spray to enhance crop resistance. Specific applications are as follows:

[0055] From September 2021 to March 2022, a strawberry (Yuexiu variety) experiment was conducted at the strawberry base of Senjing Sentai Agricultural Development (Jiande) Co., Ltd., Laocun Village, Datong Town, Jiande City, Hangzhou, Zhejiang Province. The *Lactobacillus plantarum* LP-14 fermentation broth was diluted 100 times as the experimental group and sprayed onto the plant leaves as a foliar fertilizer until the leaves were covered with water droplets and dripped down. A control group was also set up, which was not sprayed with the *Lactobacillus plantarum* LP-14 fermentation broth. The experiment was conducted weekly from the start of cultivation in September, with delays made in cold and humid weather. At the beginning of the Yuexiu strawberry harvest (December 11, 2022), the occurrence of root diseases in the experimental plots was investigated. Five rows (3m each) were randomly selected from each treatment. The incidence rate, disease index, and control efficacy were statistically analyzed and calculated, as shown in Table 2.

[0056] Table 2. Survey on the prevention effect of strawberry root diseases

[0057]

[0058] Note: The grading standard is as follows: Grade 0, no disease in the whole plant; Grade 1, less than 5% of the whole plant is diseased; Grade 3, 6%-25% of the whole plant is diseased; Grade 5, 26%-50% of the whole plant is diseased; Grade 7, 51%-75% of the whole plant is diseased; Grade 9, more than 76% of the whole plant is diseased.

[0059] Disease severity index (DSE) = 100 × ∑(number of diseases at each level × representative value at each level) / (total number of surveys × highest representative value).

[0060] Prevention and control efficacy (efficacy, %) = (Control disease index - Treatment disease index) / Control disease index × 100

[0061] As shown in Test Example 2, compared with the control group, the experimental group sprayed with the fermentation liquid of *Lactobacillus plantarum* strain LP-14 of this invention significantly reduced the incidence and disease index of strawberry root diseases, with a control efficacy of 84.76%. Spraying with the fermentation liquid of *Lactobacillus plantarum* strain LP-14 of this invention can significantly improve the disease-suppressing performance of strawberry rhizosphere and play a role in preventing and controlling strawberry root diseases.

[0062] Application Example 3

[0063] The fermentation broth of *Lactobacillus plantarum* strain LP-14 from Example 3 was used for soaking fritillaria bulbs, and the specific application is as follows:

[0064] In September 2022, a Fritillaria cirrhosa experiment was conducted at the Fritillaria cirrhosa planting base of Minhuifeng Agricultural Technology Co., Ltd. in Pan'an County, Jinhua City, Zhejiang Province. The experimental group used a 100-fold dilution of *Lactobacillus plantarum* strain LP-14 fermentation broth for soaking Fritillaria cirrhosa bulbs for one hour. A control group was also included, without soaking. One hundred seeds were randomly selected from each group. In March 2023, the incidence of gray mold, black spot, soft rot, and dry rot diseases on the above-ground parts of the Fritillaria cirrhosa in the experimental plot was investigated, along with the growth of the Fritillaria cirrhosa. The incidence and control efficacy were statistically analyzed and calculated, as shown in Table 3.

[0065] Table 3. Survey on the prevention effect of Fritillaria diseases

[0066]

[0067] Note: The grading standard is as follows: Grade 0, no disease in the whole plant; Grade 1, less than 5% of the whole plant is diseased; Grade 3, 6%-25% of the whole plant is diseased; Grade 5, 26%-50% of the whole plant is diseased; Grade 7, 51%-75% of the whole plant is diseased; Grade 9, more than 76% of the whole plant is diseased.

[0068] Disease severity index (DSE) = 100 × ∑(number of diseases at each level × representative value at each level) / (total number of surveys × highest representative value).

[0069] Prevention and control efficacy (efficacy, %) = (Control disease index - Treatment disease index) / Control disease index × 100

[0070] As shown in Test Example 2, compared with the control group, the disease incidence rate of the experimental group treated with the fermentation liquid of *Lactobacillus plantarum* strain LP-14 of the present invention was significantly reduced, the control efficacy reached 70.5%, and the seedling emergence rate, average plant height, and leaf width were significantly improved. The experimental group treated with the fermentation liquid of *Lactobacillus plantarum* strain LP-14 of the present invention significantly improved the disease resistance of *Fritillaria cirrhosa* and promoted the healthy growth of *Fritillaria cirrhosa*.

[0071] 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 plant lactobacillus ( Lactiplantibacillus plantarum The strain LP-14 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30841 and deposit date of June 3, 2024.

2. Use of the Lactobacillus plantarum strain LP-14 according to claim 1 in agriculture, characterized by the fact that: The application is the application of Lactiplantibacillus plantarum strain LP-14 in the prevention and treatment of cotton verticillium wilt.

3. Use according to claim 2, wherein the compound is ###0002### The application of Lactiplantibacillus plantarum strain LP-14 in the prevention and treatment of cotton verticillium wilt is as follows: The Lactiplantibacillus plantarum strain LP-14 fermentation liquor is applied to the soil as base fertilizer or topdressing, and the use amount of the fermentation liquor is 3-5 L per mu of land.

4. The use according to claim 3, wherein the compound is ###0002### The preparation method of the Lactiplantibacillus plantarum strain LP-14 fermentation liquor is as follows, and the components are calculated by weight: (1) Preparation of culture medium: 10-12 parts of tryptone, 10-12 parts of yeast extract, 4-6 parts of glucose, 1-2 parts of Tween 80, 1-3 parts of dipotassium hydrogen phosphate, 4-6 parts of sodium acetate, 1-4 parts of triammonium citrate, 0.1-0.4 parts of magnesium sulfate and 0.1-0.4 parts of manganese sulfate are added to 950-1000 parts of distilled water, stirred and mixed uniformly, and then sterilized at high temperature and high pressure for standby; (2) Liquid submerged fermentation culture: the Lactiplantibacillus plantarum strain LP-14 single colony is inoculated into the culture medium of step (1), and cultured at 60-80 r / min, to obtain the Lactiplantibacillus plantarum strain LP-14 fermentation liquor.

5. The use according to claim 4, wherein the compound is ###0002### The high-temperature and high-pressure sterilization condition in step (1) is 115℃, 0.1 MPa.

6. The use according to claim 4, wherein the compound is ###0002### The fermentation temperature in step (2) is 30-38℃.

7. The use according to claim 4, wherein the compound is ###0002### The fermentation time in step (2) is 90-100 hours.

8. The use according to claim 4, wherein The OD value of the Lactiplantibacillus plantarum strain LP-14 fermentation liquor in step (2) is 2.0-2.5.

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