Lactobacillus buchneri strain LB-09 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a lactobacillus buchneri strain LB-09 and application thereof. The preservation number of the lactobacillus buchneri strain LB-09 is CGMCC (China General Microbiological Culture Collection Center) No.31193, and the preservation date of the lactobacillus buchneri strain LB-09 is July 8, 2024. The lactobacillus buchneri strain LB-09 is subjected to submerged fermentation culture, fermentation liquor is applied to soil, leaf surfaces or seed surfaces, including soil application, leaf surface spraying, seed soaking or root dipping and the like, the disease resistance and the growth condition of crops are improved, and the lactobacillus buchneri strain LB-09 has the advantages of being good in biological safety, high in decomposition capacity, easy to apply and the like, and has a good application prospect. And green development and modernization of agriculture can be realized.

Description

Technical Field

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

[0002] At present, excessive use of fertilizers and pesticides is the main cause of soil damage and non-point source pollution in the planting industry, which not only leads to the decline of arable land quality and the increasing severity of pests and diseases, but also causes the decline of agricultural product output and quality. 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. However, the current domestic agricultural microbial agent production products are relatively single, mainly based on traditional microorganisms such as Bacillus (accounting for 98% of the output), and manufacturers only focus on the number of live bacteria of a single strain of the product, but ignore 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. 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 for application in agricultural planting. The microorganism is applied to the soil or 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 a strain code of LB-09, a deposit number of CGMCC No.31193, 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 July 8, 2024.

[0008] The strain is a Gram-positive bacterium. According to physiological and biochemical analysis and 16s rDNA sequence determination, the Lactobacillus buchneri strain LB-09 is Lentilactobacillus buchneri. The 16s rDNA base sequence of the strain is shown in SEQ ID NO:1.

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

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

[0011] 1. Application in inhibiting 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 basal 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 sides of plant leaves in the form of foliar fertilizer, diluted 100 times for use, until the leaves are covered with water droplets and dripping.

[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 root dipping of seedlings 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 50-80r / 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 method for preparing the fermentation broth is: inoculating a single colony of the strain into the above culture medium, shaking and culturing at 60 r / min, the fermentation temperature is 42° C., the fermentation time is 96 h, and the OD value of the fermentation broth is measured to be 1.4.

[0016] The culture medium formula of the described 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, dilute to 1L with distilled water, and sterilize under high pressure for standby 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, dilute to 1L with distilled water, and sterilize under high pressure for standby use.

[0018] The advantages of the invention are as follows: the strain LB-09 is a lactic acid bacteria microorganism, has good biological safety, strong decomposition ability, and can rapidly reproduce by utilizing nutrients such as organic matter in the soil and leaf wax; 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 invention is simple to operate and has a stable effect. DETAILED DESCRIPTION

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

[0020] Embodiment 1:

[0021] Application of a lactobacillus lentus strain:

[0022] a. Prepare culture medium: 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, 1000mL distilled water, and sterilize by high pressure for later use;

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

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

[0025] d. Verticillium wilt in cotton fields is the "number one disease" of cotton and is known as the "cancer" of cotton. The results of the investigation of the disease index and disease rate of Verticillium wilt 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 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%.

[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] Embodiment 2:

[0033] Application of a lactobacillus lentus strain:

[0034] a. Prepare culture medium: 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, 1000mL distilled water, and sterilize by high pressure for later use;

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

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

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

[0038] Table 2 Investigation on the efficacy of fermentation liquid of Lactobacillus lentus strain LB-09 in preventing strawberries

[0039]

[0040] 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%.

[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 root zone was improved, which played a role in preventing and controlling strawberry root diseases.

[0044] Embodiment 3:

[0045] Application of a lactobacillus lentus strain:

[0046] a. Prepare culture medium: 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, 1000mL distilled water, and sterilize by high pressure for later use;

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

[0048] c. 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 lentus strain LB-09 was diluted 100 times and the bulbs used as seeds were soaked for 1 hour;

[0049] d. In March 2023, investigate the occurrence of some diseases such as gray mold, black spot, soft rot, and dry rot on the ground of Fritillaria thunbergii in the test plot, and the growth of Fritillaria thunbergii. Count and calculate the incidence rate and control effect. The results are shown in Table 3.

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

[0051]

[0052] 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%.

[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 above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A Lactobacillus lentus 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 China National Microbiological Culture Collection Administration.

2. Application of Lactobacillus lentus strain LB-09 as claimed in claim 1 in agricultural planting.

3. The use according to claim 2, characterized in that In agricultural planting, the fermentation liquid of Lactobacillus brasiliensis strain LB-09 is applied to the soil in the form of basal 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 lentus 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: S1 prepares culture medium: 8-12g tryptone, 8-12g yeast extract, 3-8g glucose, 1-2mL Tween 80, 1-3g potassium dihydrogen phosphate, 3-8g sodium acetate, 1-3g ammonium citrate, 0.1-0.2g magnesium sulfate, 0.1-0.2g manganese sulfate, dilute to 1L with distilled water, autoclave and set aside; S2 fermentation culture: a single colony of Lactobacillus lentus strain LB-09 is inoculated into the culture medium prepared in step S1, and cultured at 50-80 r / min with shaking, a fermentation culture temperature of 40° C.-45° C., and a fermentation time of 84-108 h to obtain a fermentation liquid of the Lactobacillus lentus strain LB-09.

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