Phytobacterium plantarum strain LP-14 and application thereof

Abstract

The invention discloses a lactobacillus plantarum strain LP-14 and application of the lactobacillus plantarum strain LP-14, the lactobacillus plantarum strain LP-14 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 3, 2024, and the preservation number is CGMCC No.30841. The lactobacillus plantarum strain LP-14 has the advantages that the lactobacillus plantarum strain LP-14 can be used for preparing the lactobacillus plantarum strain LP-14, and the lactobacillus plantarum strain LP-14 can be used for preparing the lactobacillus plantarum strain LP-14; the invention also provides an application of the plant lactobacillus strain LP-14 in the planting industry. Compared with the prior art, the fermentation liquor of the plant lactobacillus strain LP-14 can be applied to soil or leaf surfaces or seed surfaces, can be rapidly propagated by using nutrient substances in the environment, and avoids the propagation of harmful microorganisms by continuously inhibiting the advantage that other pathogenic bacteria and other microorganisms occupy micro-ecological loci, or realizes the rapid propagation through the contact and absorption of leaf surfaces and seeds. Immune systems of plants and seeds are directly stimulated, and systematic resistance of the plants is improved.

Description

Technical Field

[0001] The invention relates to the technical field of preserved microorganisms and bacteria, in particular to a Lactobacillus plantarum strain LP-14 and an application thereof. Background Art

[0002] Excessive use of chemical fertilizers and pesticides is the main cause of soil destruction and non-point source pollution in the planting industry. It not only leads to a decline in the quality of cultivated land and increasingly serious pests and diseases, but also causes a decline in the output and quality of agricultural products. The large-scale application of chemical fertilizers will cause nitrogen, phosphorus, potassium and other chemical substances to be consolidated in the soil, forming various chemical salts, leading to an imbalance in the soil nutrient structure, deterioration of physical properties, and excessive levels of harmful metals and harmful bacteria in some plots, which will lead to deterioration of soil properties; it will also cause the water-soluble nutrients in the soil to be washed away by rainwater and farmland irrigation and flow into groundwater and rivers, causing groundwater and river pollution in some areas. The large-scale use of pesticides will lead to soil, air and water pollution, and destroy the ecological balance. Therefore, it is necessary to vigorously carry out technological innovation in agriculture, develop biological planting, and rapidly promote the green development and modernization of agriculture.

[0003] To solve these problems, China started the research, production and application of agricultural microbial products decades ago. Agricultural microbial agents refer to live bacterial preparations made by expanding the effective bacteria of the target microorganisms. Such agents are usually used for seed dressing or root dipping, and have the functions of directly or indirectly improving the soil, restoring soil fertility, preventing soil-borne diseases, maintaining the balance of rhizosphere microbial flora and degrading toxic substances. However, the current domestic agricultural microbial agent production products are relatively single, mainly traditional microorganisms based on Bacillus (accounting for 98% of the output), and manufacturers only focus on the number of live bacteria of a single strain of the product and neglect the relationship between the microorganisms and pathogenic microorganisms in the product, the effect on the soil microecology and the actual application effect on crops. The potential of microorganisms has not been fully utilized.

[0004] CN104745510A discloses a fermented lactobacillus and its application in the prevention and treatment of plant fungal diseases. The strain is isolated from the soil planted under tomatoes and is classified and named as fermented lactobacillus; fermented lactobacillus is fermented to obtain a fermentation liquid, and then the fermentation liquid is concentrated to prepare a lactobacillus concentrate, which is then diluted and sprayed on the leaves of crops; the strain has a good effect in inhibiting fungal diseases of crops and has the function of promoting the growth of crops, which meets the requirements of ecological agriculture. The fermented lactobacillus CGMCC No.8735 fermentation liquid can effectively inhibit cotton Verticillium wilt, tomato gray mold or rice sheath blight pathogens, among which the inhibitory effect on cotton Verticillium wilt is the best.

[0005] CN110106106A discloses a composite microbial agent and its application in strawberry planting, including Bacillus licheniformis, Clostridium butyricum, Lactobacillus, Olsonella, Red Stink Fungus and Bacillus amyloliquefaciens, and also provides a method for preparing the composite microbial agent and the application of the composite microbial agent in strawberry planting. The composite microbial agent of the invention can achieve the effect of significantly inhibiting and degrading pathogens in the roots of strawberries, and also has a significant positive effect and promotion on the growth and material metabolism of strawberries, which can greatly promote the improvement of the quality of strawberry fruits.

[0006] CN113403218A discloses a biocontrol strain and its application in preventing and controlling soft rot of Fritillaria thunbergii and promoting its growth. The biocontrol strain is Dm97, and its deposit number is GDMCC No: 61459. The strain Dm97 is identified as Bacillus sp. The field test results show that the microbial agent prepared by the strain Dm97 has a control effect of more than 90% on soft rot of Fritillaria thunbergii, a growth promotion effect of more than 70% on disease-free plants, and an increase in yield of more than 50% on Fritillaria thunbergii. The microbial agent is used to prevent soft rot by soil treatment in the early stage, and can be used to better control soft rot by root treatment in the later stage. It has a good disease prevention and yield increase effect, and has great potential to be developed into a commercial live bacterial biocontrol agent.

[0007] The above patent documents report that they can solve the prevention of cotton wilt, strawberry root disease, Fritillaria disease, etc. individually, but cannot solve multiple plant diseases at the same time. In actual use, it is urgent to provide a microorganism that can solve multiple plant diseases at the same time. Summary of the invention

[0008] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a plant lactobacillus strain LP-14 applied to agricultural planting, which can rapidly reproduce by utilizing nutrients in the environment, and avoid the reproduction of harmful microorganisms by continuously suppressing other pathogenic bacteria and other microorganisms from occupying the microecological location advantage, or directly stimulate the immune system of plants and seeds through contact and absorption by leaves and seeds, and improve the systemic resistance of plants, including the prevention of cotton Verticillium wilt, the prevention of strawberry root diseases, the prevention of Fritillaria diseases, etc.

[0009] To achieve the above object, the present invention provides a Lactobacillus plantarum strain LP-14 and application thereof.

[0010] A plant lactobacillus strain LP-14 (Lactiplantibacillus plantarum), with a deposit number of CGMCC No.30841, a depository of the General Microbiology Center of the China Culture Collection Administration (CGMCC), an address of the depository of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and a deposit date of June 3, 2024.

[0011] The Lactobacillus plantarum strain LP-14 is a Gram-positive bacterium, and is identified as Lactobacillus plantarum through physiological and biochemical analysis and 16s rDNA sequence determination. Specifically, the 16s rDNA base sequence of the strain SEQ ID NO: 1 is shown in the sequence table.

[0012] The source of the Lactobacillus plantarum strain LP-14 of the present invention is obtained by separation from natural water and soil in Hangzhou.

[0013] The application of the Lactobacillus plantarum strain LP-14 can be applied to the planting industry, specifically as follows:

[0014] A. Apply the fermentation liquid of Lactobacillus plantarum strain LP-14 to the soil in the form of basal fertilizer or topdressing, wherein the fermentation liquid is used in an amount of 3-5L per mu of land;

[0015] B. Spray the fermentation liquid of Lactobacillus plantarum strain LP-14 on the leaves (front and back) of the plants as foliar fertilizer, dilute it 100-150 times, and use it until the leaves are covered with water droplets and drip;

[0016] C. Dilute the fermentation liquid of Lactobacillus plantarum strain LP-14 by 100-150 times for soaking seeds; or dilute the fermentation liquid of Lactobacillus plantarum strain LP-14 by 100-150 times for dipping roots of seedlings or beating with soil.

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

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

[0019] The preparation method of the fermentation liquid of the plant lactobacillus strain LP-14 is as follows, in parts by weight:

[0020] (1) preparing a culture medium: adding 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 to 950-1000 parts of distilled water, stirring and mixing, sterilizing at high temperature and high pressure for later use;

[0021] (2) Liquid submerged fermentation culture: A single colony of Lactobacillus plantarum strain LP-14 was inoculated into the culture medium of step (1), and cultured with shaking at 60-80 rpm to obtain a fermentation liquid of Lactobacillus plantarum strain LP-14.

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

[0023] In step (2), the fermentation temperature is 30°C-38°C;

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

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

[0026] The plant lactobacillus strain LP-14 of the present invention is a lactic acid bacteria microorganism with good biosafety and strong decomposition ability, and can be applied to agricultural planting. After being made into a fermented liquid, it is applied to the soil or leaf surface or seed surface, and can rapidly reproduce using nutrients in the environment, such as organic matter in the soil, leaf wax, etc.; at the same time, by continuously suppressing pathogenic bacteria and other microorganisms, occupying microecological location advantages, avoiding the reproduction of harmful microorganisms, or directly stimulating the immune system of plants and seeds through contact and absorption of leaf surfaces and seeds, improving the systemic resistance and growth environment of plants, and realizing the purpose of green development and modernization of agriculture, the method of the present invention is simple and easy to implement, and the effect is stable.

[0027] Beneficial effects of the present invention:

[0028] Compared with the prior art, the plant lactobacillus strain LP-14 of the present invention is a lactic acid bacteria microorganism with good biological safety and strong decomposition ability, and can be applied to agricultural planting. After being made into a fermentation liquid, it is applied to the soil or leaf surface or seed surface, and absorbed after contact, which can directly stimulate the immune system of plants and seeds, improve the systemic resistance of plants, including the prevention of cotton wilt, the prevention of strawberry root diseases, the prevention of Fritillaria diseases, etc. DETAILED DESCRIPTION

[0029] Example 1

[0030] A plant lactobacillus strain LP-14, with a deposit number of CGMCC No.30841, the depository is the General Microbiology Center of the China Culture Collection Administration (CGMCC), the depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is June 3, 2024.

[0031] A method for preparing a fermentation liquid of Lactobacillus plantarum strain LP-14 is as follows:

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

[0033] (2) Liquid submerged fermentation culture: A single colony of Lactobacillus plantarum strain LP-14 was inoculated into the culture medium of step (1) above, and cultured at 60 r / min with shaking, a fermentation temperature of 35° C., and a fermentation time of 96 hours. The OD value of the fermentation liquid was measured to be 2.2, thereby obtaining a fermentation liquid of Lactobacillus plantarum strain LP-14.

[0034] Example 2

[0035] A plant lactobacillus strain LP-14, with a deposit number of CGMCC No.30841, the depository is the General Microbiology Center of the China Culture Collection Administration (CGMCC), the depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is June 3, 2024.

[0036] A method for preparing a fermentation liquid of Lactobacillus plantarum strain LP-14 is as follows:

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

[0038] (2) Liquid submerged fermentation culture: A single colony of Lactobacillus plantarum strain LP-14 was inoculated into the culture medium of step (1) above, and cultured at 60 r / min with shaking, a fermentation temperature of 35° C., and a fermentation time of 96 hours. The OD value of the fermentation liquid was measured to be 2.0, thereby obtaining a fermentation liquid of Lactobacillus plantarum strain LP-14.

[0039] Example 3

[0040] A plant lactobacillus strain LP-14, with a deposit number of CGMCC No.30841, the depository is the General Microbiology Center of the China Culture Collection Administration (CGMCC), the depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is June 3, 2024.

[0041] A method for preparing a fermentation liquid of Lactobacillus plantarum strain LP-14 is as follows:

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

[0043] (2) Liquid submerged fermentation culture: A single colony of Lactobacillus plantarum strain LP-14 was inoculated into the culture medium of step (1) above, and cultured at 60 r / min with shaking, a fermentation temperature of 35° C., and a fermentation time of 96 hours. The OD value of the fermentation liquid was measured to be 2.0, thereby obtaining a fermentation liquid of Lactobacillus plantarum strain LP-14.

[0044] Application Example 1

[0045] The fermentation liquid of the plant lactobacillus strain LP-14 of Example 1 is drip-applied to plants to improve crop resistance, and the specific application is as follows:

[0046] From June to October 2022, cotton (No. 64, Gengye) was applied in a plot test in Shibahu Village, Dafeng Town, Changji Hui Autonomous Prefecture, Xinjiang Uygur Autonomous Region. The plant lactobacillus strain LP-14 fermentation liquid prepared in Example 1 was diluted 100 times as a test group, and the root was topdressed in the form of drip application, and a group of control groups were set. The control group did not drip the plant lactobacillus LP-14 fermentation liquid of the present invention, and the test group and the control group investigated 300 strains respectively. Drip application was performed four times (June 20, June 29, July 8, and July 18) during the full growth period of cotton, and 1L / mu was used for each. Verticillium wilt in cotton fields is the "number one disease" of cotton, which is seriously harmful and is called the "cancer" of cotton. Test Example 1 is a survey of the diseased plant rate of Verticillium wilt disease index in cotton fields, as shown in Table 1.

[0047] Table 1 Investigation on the prevention effect of cotton Verticillium wilt

[0048]

[0049] Note: The grading standard is grade 0, the whole plant is disease-free; grade 1, the diseased area of ​​the whole plant is less than 5%; grade 3, the diseased area of ​​the whole plant is 6%-25%; grade 5, the diseased area of ​​the whole plant is 26%-50%; grade 7, the diseased area of ​​the whole plant is 51%-75%; grade 9, the diseased area of ​​the whole plant is more than 76%.

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

[0051] Control effect (control effect, %) = (control disease index - treatment disease index) / control disease index × 100

[0052] It can be seen from Test Example 1 that, in the prevention and treatment of Verticillium wilt, the test group dripping the fermentation liquid of the plant lactobacillus strain LP-14 of the present invention can greatly improve the prevention and treatment effect, alleviate the disease, and reduce the disease index compared with the control group.

[0053] Application Example 2

[0054] The fermentation liquid of Lactobacillus plantarum strain LP-14 of Example 2 is sprayed on plant leaves to improve crop resistance. The specific application is as follows:

[0055] From September 2021 to March 2022, a strawberry (variety is Yuexiu) test was carried out at the strawberry base of Senjing Sentai Agricultural Development (Jiande) Co., Ltd., Laocun, Datong Town, Jiande City, Hangzhou, Zhejiang Province. The plant lactobacillus LP-14 fermentation liquid was diluted 100 times as the test group and sprayed on the plant leaves in the form of foliar fertilizer until the leaves were covered with water droplets and dripped, and a group of control groups were set, and the control group was not sprayed with the plant lactobacillus LP-14 fermentation liquid of the present invention. Since the cultivation started in September, it was carried out once a week, and it was delayed in wet and cold weather. The occurrence of strawberry root diseases in the test plot was investigated at the beginning of the strawberry (Yuexiu) harvest period (December 11, 2022), and 5 rows were randomly selected for each treatment, and each row was 3m. The incidence rate, disease index and prevention effect were counted and calculated, as shown in Table 2.

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

[0057]

[0058] Note: The grading standard is grade 0, the whole plant is disease-free; grade 1, the diseased area of ​​the whole plant is less than 5%; grade 3, the diseased area of ​​the whole plant is 6%-25%; grade 5, the diseased area of ​​the whole plant is 26%-50%; grade 7, the diseased area of ​​the whole plant is 51%-75%; grade 9, the diseased area of ​​the whole plant is more than 76%.

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

[0060] Control effect (control effect, %) = (control disease index - treatment disease index) / control disease index × 100

[0061] It can be seen from Test Example 2 that, compared with the control group, the test group sprayed with the fermentation liquid of the plant lactobacillus strain LP-14 of the present invention can significantly reduce the incidence and disease index of strawberry root diseases, and the prevention effect reaches 84.76%. Spraying the fermentation liquid of the plant lactobacillus strain LP-14 of the present invention can significantly improve the disease suppression performance of the strawberry rhizosphere, and play a role in preventing and controlling strawberry root diseases.

[0062] Application Example 3

[0063] The fermentation liquid of the plant lactobacillus strain LP-14 of Example 3 is used for soaking the seeds and bulbs of Fritillaria thunbergii, and the specific application is as follows:

[0064] In September 2022, a Fritillaria experiment was conducted at the Fritillaria planting base of Minhuifeng Agricultural Technology Co., Ltd. in Pan'an County, Jinhua City, Zhejiang Province. The fermentation liquid of Lactobacillus plantarum strain LP-14 was diluted 100 times as the test group, and the bulbs of Fritillaria seeds were soaked for 1 hour; and a control group was set up. The control group was not soaked, and 100 seeds were randomly selected from the test group and the control group. In March 2023, the occurrence of gray mold, black spot, soft rot, and dry rot on the ground of Fritillaria in the test plot, as well as the growth of Fritillaria, were investigated. The incidence rate and prevention effect were counted and calculated, as shown in Table 3.

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

[0066]

[0067] Note: The grading standard is grade 0, the whole plant is disease-free; grade 1, the diseased area of ​​the whole plant is less than 5%; grade 3, the diseased area of ​​the whole plant is 6%-25%; grade 5, the diseased area of ​​the whole plant is 26%-50%; grade 7, the diseased area of ​​the whole plant is 51%-75%; grade 9, the diseased area of ​​the whole plant is more than 76%.

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

[0069] Control effect (control effect, %) = (control disease index - treatment disease index) / control disease index × 100

[0070] It can be seen from Test Example 2 that compared with the control group, the disease incidence rate of the test group of the plant lactobacillus strain LP-14 fermentation liquid of the present invention is significantly reduced, the prevention effect reaches 70.5%, the emergence rate, average plant height and leaf width are significantly improved, and the test group after soaking the seeds in the plant lactobacillus strain LP-14 fermentation liquid of the present invention can significantly improve the disease inhibition performance of Fritillaria and promote the healthy growth of Fritillaria.

[0071] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A plant lactobacillus strain LP-14, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.30841 and a deposit date of June 3, 2024.

2. Application of the plant lactobacillus strain LP-14 as claimed in claim 1 in planting industry.

3. The use according to claim 2, characterized in that The applications in planting industry are as follows: A. Apply the fermentation liquid of Lactobacillus plantarum strain LP-14 to the soil in the form of basal fertilizer or topdressing, wherein the fermentation liquid is used in an amount of 3-5L per mu of land; B. Spray the fermentation liquid of Lactobacillus plantarum strain LP-14 on the front and back sides of plant leaves as foliar fertilizer, dilute 100-150 times before use, until the leaves are covered with water droplets and drip; C. Dilute the fermentation liquid of Lactobacillus plantarum strain LP-14 by 100-150 times for soaking seeds; or dilute the fermentation liquid of Lactobacillus plantarum strain LP-14 by 100-150 times for dipping roots of seedlings or beating with soil.

4. The use according to claim 3, characterized in that The soaking time is 1-1.5 hours.

5. The use according to claim 3, characterized in that The root dipping time is 5-10 minutes.

6. The use according to claim 3, characterized in that The preparation method of the fermentation liquid of the plant lactobacillus strain LP-14 is as follows, in parts by weight: (1) preparing a culture medium: adding 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 to 950-1000 parts of distilled water, stirring and mixing, sterilizing at high temperature and high pressure for later use; (2) Liquid submerged fermentation culture: A single colony of Lactobacillus plantarum strain LP-14 was inoculated into the culture medium of step (1), and cultured with shaking at 60-80 rpm to obtain a fermentation liquid of Lactobacillus plantarum strain LP-14.

7. The use according to claim 6, characterized in that The high temperature and high pressure sterilization conditions in step (1) are 115° C. and 0.1 MPa.

8. The use according to claim 6, characterized in that The fermentation temperature in step (2) is 30°C-38°C.

9. The use according to claim 6, characterized in that The fermentation time in step (2) is 90-100 hours.

10. The use according to claim 6, characterized in that In the step (2), the OD value of the fermentation liquid of Lactobacillus plantarum strain LP-14 is 2.0-2.5.

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