A Lactobacillus lentus strain LB-09 and its application

Abstract

The present invention relates to the field of microbial technology, and in particular to a Lactobacillus lentus strain LB-09 and its application. The deposit number of the Lactobacillus lentus strain LB-09 of the present invention is CGMCC No.31193, and the deposit date is July 8, 2024. The present invention improves the disease resistance and growth condition of crops by submerging the Lactobacillus lentus strain LB-09 and applying the fermentation liquid to the soil, leaves or seed surface, including soil application, foliar spraying, seed soaking or root dipping, etc., and has the advantages of good biosafety, strong decomposition ability, and easy application, thereby contributing to the green development and modernization of agriculture.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a Lactobacillus brucei strain LB-09 and an application thereof. Background Art

[0002] Currently, excessive use of chemical fertilizers and pesticides is the primary cause of soil damage and non-point source pollution in the agricultural sector. This not only leads to a decline in arable land quality, increasingly severe pests and diseases, but also to a decrease in the yield and quality of agricultural products. Therefore, we need to vigorously promote technological innovation in agriculture, develop bio-based farming, and rapidly advance green agricultural development and modernization.

[0003] To address these issues, China began researching, producing, and applying agricultural microbial products decades ago. However, domestic agricultural microbial agents currently offer a limited range of products, primarily traditional microorganisms based on Bacillus species (98% of production). Manufacturers also focus solely on the viable count of a single bacterial species, neglecting the relationship between the microorganisms and pathogens, their impact on the soil microbiome, and their practical application on crops. Consequently, the potential of microorganisms has not been fully realized. Summary of the Invention

[0004] One of the purposes of the present invention is to solve the above-mentioned deficiencies in the prior art and provide a Lactobacillus lentus.

[0005] The second purpose of the present invention is to solve the deficiencies of the above-mentioned prior art and provide a use of Lactobacillus lentus brockii for application in agricultural planting. The microorganism is applied to the soil, leaf surface or seed surface. The microorganism can rapidly reproduce by utilizing nutrients in the environment, and avoid the reproduction of harmful microorganisms by continuously inhibiting microorganisms including pathogenic bacteria from occupying microecological niche advantages, or directly stimulate the immune system of plants and seeds through contact and absorption by leaf surfaces and seeds, thereby improving the systemic resistance of plants.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A strain of Lactobacillus buchneri, with the strain code LB-09 and the deposit number CGMCC No. 31193, is deposited at the General Microbiology Center of the China Culture Collection Administration (CGMCC). The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is July 8, 2024.

[0008] The strain is a Gram-positive bacterium. According to physiological and biochemical analysis and 16rDNA sequence determination, the Lactobacillus lentus strain LB-09 is a Lactobacillus lentus ( Lentilactobacillus buchneri ).

[0009] The Lactobacillus lentus LB-09 of the present invention is obtained from natural water bodies and soil in Hangzhou.

[0010] The present invention also discloses a method for applying the Lactobacillus brucei strain LB-09 to the planting industry, comprising the following contents:

[0011] 1. Application in the inhibition of pathogenic microorganisms in soil: The fermentation liquid of the Lactobacillus lentus strain LB-09 needs to be applied to the soil in the form of base fertilizer or topdressing, wherein the amount of fermentation liquid used per mu of land is 3-5 L.

[0012] 2. Application of spraying on plant leaves to improve crop resistance: The fermentation liquid of Lactobacillus lentus strain LB-09 needs to be sprayed on the front and back of plant leaves in the form of foliar fertilizer, diluted 100 times and used until the leaves are covered with water droplets and drip.

[0013] 3. Application in seed soaking or root dipping: The fermentation liquid of Lactobacillus lentus strain LB-09 needs to be diluted 100 times and used for seed soaking for 1 hour; or the fermentation liquid of Lactobacillus lentus needs to be diluted 100 times and used for seedling root dipping for 5 minutes or combined with soil for pulping.

[0014] Preferably, the preparation method of the fermentation broth of the Lactobacillus lentus strain LB-09 is: inoculating a single colony of the strain into the above-mentioned culture medium, shaking and culturing at 50-80 r / min, the fermentation temperature is 40°C-45°C, the fermentation time is 84-108h, and the OD value of the fermentation broth is detected to be 1.4±0.3.

[0015] Preferably, the fermentation broth is prepared by inoculating a single colony of the strain into the above culture medium, shaking and culturing at 60 r / min, at a fermentation temperature of 42° C., for 96 h, and measuring the OD value of the fermentation broth to be 1.4.

[0016] The culture medium formula of the Lactobacillus lentus LB-09 is: 8-12g tryptone, 8-12g yeast extract, 3-8g glucose, 1-2mL Tween 80, 1-3g dipotassium hydrogen phosphate, 3-8g sodium acetate, 1-3g triammonium citrate, 0.1-0.2g magnesium sulfate, 0.1-0.2g manganese sulfate, and the volume is adjusted to 1L with distilled water and sterilized under high pressure before use.

[0017] Preferably, the culture medium formula of Lactobacillus lentus LB-09 is: 10g tryptone, 10g yeast extract, 5g glucose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.1g manganese sulfate, and dilute to 1L with distilled water and sterilize under high pressure before use.

[0018] The advantages of the present invention are: the strain LB-09 is a lactic acid bacteria microorganism with good biosafety and strong decomposition ability, and can rapidly reproduce by utilizing nutrients such as organic matter and leaf wax in the soil; at the same time, by continuously inhibiting microorganisms including pathogenic bacteria from occupying microecological location advantages, the formation of harmful substances is avoided, the growth environment of plants is improved, and the purpose of green development and modernization of agriculture is achieved. The method of the present invention is simple to operate and has stable effects. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further illustrated below through specific examples. The reagents and raw materials used in the examples are all commercially available conventional raw materials.

[0020] Example 1:

[0021] Application of a Lactobacillus lentus strain:

[0022] a. Prepare culture medium: 10 g tryptone, 10 g yeast extract, 5 g glucose, 1 mL Tween 80, 2 g potassium dihydrogen phosphate, 5 g sodium acetate, 2 g ammonium citrate tribasic, 0.2 g magnesium sulfate, 0.1 g manganese sulfate, and 1000 mL distilled water. Autoclave and set aside.

[0023] b. Liquid submerged fermentation culture: A single colony of the strain was inoculated into the above culture medium, shaken at 60 r / min, the fermentation temperature was 42 ° C, the fermentation time was 96 h, and the fermentation broth OD value was measured to be 1.4;

[0024] c. From June to October 2022, a plot test of Gengye No. 64 cotton was conducted in Shibahu Village, Dafeng Town, Changji Hui Autonomous Prefecture, Xinjiang Uygur Autonomous Region. Drip application was applied four times during the entire growth period, with 1L / mu used on June 20, June 29, July 8, and July 18.

[0025] Verticillium wilt, the most serious cotton disease, is so devastating it's often called the "cancer" of cotton. The results of a survey of Verticillium wilt disease index and diseased plant rate in cotton fields are shown in Table 1.

[0026] Table 1 Preventive effect of fermentation liquid of Lactobacillus lentus strain LB-09 on cotton Verticillium wilt

[0027]

[0028] Note: The grading standard is level 0, the whole plant is disease-free; level 1, the diseased area of ​​the whole plant is less than 5%; level 3, the diseased area of ​​the whole plant is 6%-25%; level 5, the diseased area of ​​the whole plant is 26%-50%; level 7, the diseased area of ​​the whole plant is 51%-75%; level 9, the diseased area of ​​the whole plant is more than 76%.

[0029] Disease index (disease index) = 100 × ∑ (number of diseases at each level × representative value at each level) / (total number of surveys × highest representative value).

[0030] Control effect (control effect, %) = (control disease index - treatment disease index) / control disease index × 100

[0031] Compared with conventional fertilization in the control group, the treatment of Verticillium wilt with the fermentation liquid of Lactobacillus lentus strain LB-09 can improve the control effect, alleviate the disease and reduce the disease index.

[0032] Example 2:

[0033] Application of a Lactobacillus lentus strain:

[0034] a. Prepare culture medium: 10 g tryptone, 10 g yeast extract, 5 g glucose, 1 mL Tween 80, 2 g potassium dihydrogen phosphate, 5 g sodium acetate, 2 g ammonium citrate tribasic, 0.2 g magnesium sulfate, 0.1 g manganese sulfate, and 1000 mL distilled water. Autoclave and set aside.

[0035] b. Liquid submerged fermentation culture: A single colony of the strain was inoculated into the above culture medium, shaken at 60 r / min, the fermentation temperature was 42 ° C, the fermentation time was 96 h, and the fermentation broth OD value was measured to be 1.7;

[0036] c. From September 2021 to March 2022, a trial was conducted on Yuexiu strawberries at the strawberry base of Senjing Sentai Agricultural Development (Jiande) Co., Ltd. in Lao Village, Datong Town, Jiande City, Hangzhou, Zhejiang Province. The fermentation liquid of CGMCC No. 31193 was diluted 100-fold and applied as a foliar fertilizer to the plant leaves until the leaves were covered with water droplets and dripped. This was done weekly from the start of cultivation in September, with a delay in cold and wet weather.

[0037] d. On December 11, 2022, the first harvest date for Yuexiu strawberries, the incidence of root diseases in strawberries was investigated in the experimental plots. Five rows, each 3 m wide, were randomly selected for each treatment. The incidence rate, disease index, and control efficacy were calculated. The results are shown in Table 2.

[0038] Table 2 Investigation on the strawberry control effect of fermentation broth of Lactobacillus brucei strain LB-09

[0039]

[0040] Note: The grading standard is level 0, the whole plant is disease-free; level 1, the diseased area of ​​the whole plant is less than 5%; level 3, the diseased area of ​​the whole plant is 6%-25%; level 5, the diseased area of ​​the whole plant is 26%-50%; level 7, the diseased area of ​​the whole plant is 51%-75%; level 9, the diseased area of ​​the whole plant is more than 76%.

[0041] Disease index (disease index) = 100 × ∑ (number of diseases at each level × representative value at each level) / (total number of surveys × highest representative value).

[0042] Control effect (control effect, %) = (control disease index - treatment disease index) / control disease index × 100

[0043] Compared with the control group, the incidence rate and disease index of strawberry root diseases caused by the Lactobacillus brasiliensis strain LB-09 were reduced; correspondingly, the disease suppression performance of the strawberry rhizosphere was improved, which played a role in preventing and controlling strawberry root diseases.

[0044] Example 3:

[0045] Application of a Lactobacillus lentus strain:

[0046] a. Prepare culture medium: 10 g tryptone, 10 g yeast extract, 5 g glucose, 1 mL Tween 80, 2 g potassium dihydrogen phosphate, 5 g sodium acetate, 2 g ammonium citrate tribasic, 0.2 g magnesium sulfate, 0.1 g manganese sulfate, and 1000 mL distilled water. Autoclave and set aside.

[0047] b. Liquid submerged fermentation culture: A single colony of the strain was inoculated into the above culture medium, shaken at 60 r / min, the fermentation temperature was 42 ° C, the fermentation time was 96 h, and the fermentation broth OD value was measured to be 1.3;

[0048] c. In September 2022, a Fritillaria cirrhosa experiment was conducted at the Fritillaria cirrhosa cultivation base of Minhuifeng Agricultural Technology Co., Ltd. in Pan'an County, Jinhua City, Zhejiang Province. The fermentation broth of Lactobacillus brucei strain LB-09 was diluted 100-fold and soaked for 1 hour in bulbs used as seeds.

[0049] d. In March 2023, investigate the incidence of some diseases, including gray mold, black spot, soft rot, and dry rot, on Fritillaria cirrhosa in the test plots, as well as the growth of Fritillaria cirrhosa. Calculate the incidence and control efficacy. The results are shown in Table 3.

[0050] Table 3 Investigation on application of fermentation liquid of Lactobacillus lentus strain LB-09 in Fritillaria thunbergii

[0051]

[0052] Note: The grading standard is level 0, the whole plant is disease-free; level 1, the diseased area of ​​the whole plant is less than 5%; level 3, the diseased area of ​​the whole plant is 6%-25%; level 5, the diseased area of ​​the whole plant is 26%-50%; level 7, the diseased area of ​​the whole plant is 51%-75%; level 9, the diseased area of ​​the whole plant is more than 76%.

[0053] Disease index (disease index) = 100 × ∑ (number of diseases at each level × representative value at each level) / (total number of surveys × highest representative value).

[0054] Control effect (control effect, %) = (control disease index - treatment disease index) / control disease index × 100

[0055] Compared with the control group, the incidence of aboveground diseases in the treatment with Lactobacillus brasiliensis strain LB-09 was reduced, and the corresponding germination rate, average plant height and leaf width increased; at the same time, the disease inhibition performance of Fritillaria was improved, and the healthy growth of Fritillaria was promoted.

[0056] The embodiment described above is only a preferred solution of the present invention and does not limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solution described in the claims.

Claims

1. A Lentilactobacillus buchneri strain LB-09, characterized in that: This strain is a Gram-positive bacterium, with a preservation number of CGMCC No. 31193 and a preservation date of July 8, 2024. The preservation unit is the General Microbiology Center of the China Culture Collection Administration.

2. The use of the Lactobacillus lentus strain LB-09 in agricultural planting as claimed in claim 1, characterized in that: Used for the prevention and control of cotton verticillium wilt or strawberry root diseases.

3. The use according to claim 2, characterized in that In agricultural planting, the fermentation liquid of Lactobacillus lentus strain LB-09 is applied to the soil in the form of base fertilizer or topdressing.

4. The use according to claim 2, characterized in that In agricultural planting, the fermentation liquid of Lactobacillus lentus strain LB-09 is diluted and sprayed on plant leaves in the form of foliar fertilizer until the leaves are covered with water droplets and drip.

5. The use according to claim 2, characterized in that In agricultural planting, the fermentation liquid of Lactobacillus lentus strain LB-09 is diluted and used for seed soaking.

6. The use according to claim 2, characterized in that In agricultural planting, the fermentation liquid of Lactobacillus lentus strain LB-09 is diluted and used for root dipping of seedlings.

7. The use according to claim 2, characterized in that In agricultural cultivation, the fermentation liquid of Lactobacillus brucei strain LB-09 is diluted and combined with soil for pulping.

8. The use according to any one of claims 3 to 7, characterized in that The fermentation liquid of the Lactobacillus lentus strain LB-09 is prepared as follows: Prepare S1 culture medium: 8-12 g tryptone, 8-12 g yeast extract, 3-8 g glucose, 1-2 mL Tween 80, 1-3 g potassium phosphate dibasic, 3-8 g sodium acetate, 1-3 g ammonium citrate tribasic, 0.1-0.2 g magnesium sulfate, and 0.1-0.2 g manganese sulfate. Make up to 1 L with distilled water and autoclave until ready to use. S2 Fermentation culture: A single colony of Lactobacillus lentus strain LB-09 was inoculated into the culture medium prepared in step S1, and cultured at 50-80 rpm, a fermentation temperature of 40-45°C, and a fermentation time of 84-108 h to obtain a fermentation broth of the Lactobacillus lentus strain LB-09.

Images