Phytobacterium pentosus strain LP-22 and application thereof

Abstract

The invention discloses a lactobacillus pentosus strain LP-22 and an application thereof. The lactobacillus pentosus strain LP-22 disclosed by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 12, 2024, and the preservation number of the lactobacillus pentosus strain LP-22 is CGMCC No.31651. The lactobacillus pentosus strain LP-22 disclosed by the invention has the advantages that the lactobacillus pentosus strain LP-22 can be used for preparing the lactobacillus pentosus strain LP-22; the invention further provides application of the lactobacillus pentosus strain LP-22 in the planting industry, compared with the prior art, after being prepared into fermentation liquor, the lactobacillus pentosus strain LP-22 can be applied to soil or leaf surfaces or seed surfaces, breeding of harmful microorganisms is avoided, or immune systems of plants and seeds are directly stimulated through contact and absorption of the leaf surfaces and the seeds, and system resistance of the plants is improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus pentosus strain LP-22 and an application thereof. Background Art

[0002] At present, excessive use of chemical fertilizers and pesticides is the main cause of soil destruction and non-point source pollution in the planting industry, which not only leads to the decline in the quality of cultivated land, the increasing severity of pests and diseases, but also causes the decline in the output and quality of agricultural products. In view of the serious problems such as soil destruction, non-point source pollution in the planting industry, the decline in the quality of cultivated land, the aggravation of pests and diseases, and the decline in the output and quality of agricultural products caused by the current excessive use of chemical fertilizers and pesticides, technological innovation and green development in the agricultural field have become urgent needs. In this context, the importance of biological planting as an environmentally friendly agricultural development model is becoming increasingly prominent. Biological planting not only helps to restore the ecological balance of the soil and reduce the dependence on chemical substances, but also enhances the natural resistance of crops to pests and diseases, and ensures the safety and quality of agricultural products.

[0003] Agricultural microbial products refer to live bacterial preparations made from effective target microorganisms after propagation. They are usually used for seed dressing or root dipping, and have the functions of directly or indirectly improving soil, restoring soil fertility, preventing soil-borne diseases, maintaining the balance of rhizosphere microbial flora, and degrading toxic substances. They are mainly traditional microorganisms, mainly 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 microorganisms and pathogenic microorganisms in the product, the effect on soil microecology, and the actual application effect on crops. The potential of microorganisms has not been fully utilized.

[0004] The application areas of agricultural microbial products are constantly expanding, from the initial grain crop planting to fruit and vegetable planting, soil remediation and other fields. At the same time, as people's awareness of food safety and environmental protection increases, the application of microorganisms in organic agriculture, ecological agriculture and other fields is becoming more and more extensive. However, some companies have insufficient investment in research and development, resulting in weak technological innovation capabilities. This limits the upgrading and performance improvement of microbial products and also affects their competitiveness in the market.

[0005] CN106754469A8 discloses endophytic bacteria HB3S-20 and its application in the prevention and control of cotton Verticillium wilt. The strain HB3S-20 can be used for the biological control of cotton Verticillium wilt. Under greenhouse conditions, the average control effect of the strain HB3S-20 on cotton Verticillium wilt is 62.57%, showing good biocontrol activity, and has no negative impact on the emergence rate and growth of cotton. The plant height and fresh weight of the cotton treated with the HB3S-20 bacterial solution are significantly increased compared with the blank control.

[0006] CN111139199A discloses Bacillus amyloliquefaciens CGMCC No.17843 and its application. The Bacillus amyloliquefaciens can be prepared into a microbial agent, which can be used as a fertilizer or a therapeutic drug in the prevention and treatment of root rot, can prevent and control diseases and insect pests, and increase crop yields. The Bacillus amyloliquefaciens can also be prepared into a composite agent with other microbial agents, and can also be prepared into a fertilizer.

[0007] CN113403218A discloses a biocontrol strain and its application in preventing and controlling soft rot of Fritillaria thunbergii and promoting its growth. 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 a yield increase effect 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.

[0008] 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

[0009] In view of the above-mentioned defects of the prior art, the present invention provides a Lactobacillus pentosus strain LP-22. The fermentation liquid of the Lactobacillus pentosus strain LP-22 is applied to the soil or the leaf surface or the seed surface, and the strain can rapidly reproduce by utilizing the nutrients in the environment, and continuously inhibit other microorganisms (including pathogenic bacteria) from occupying the microecological niche advantage, thereby avoiding the reproduction of harmful microorganisms, or directly stimulating the immune system of plants and seeds through contact and absorption of the leaf surface and seeds, thereby improving the systemic resistance (ISR) of plants.

[0010] A Lactobacillus pentosus strain LP-22, with a deposit number of CGMCC No.31651, and a depository of 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 August 12, 2024.

[0011] The Lactobacillus pentosus strain LP-22 is a Gram-positive bacterium, and is Lactobacillus pentosus (Lactiplantibacillus pentosus) determined by physiological and biochemical analysis and 16rDNA sequence. Specifically, the 16srDNA base sequence of the Lactobacillus pentosus strain LP-22 is shown in the sequence table as SEQ ID NO: 1.

[0012] The source of the Lactobacillus pentosus strain LP-22 of the present invention is that it is separated from natural water and soil in Hangzhou.

[0013] The above-mentioned Lactobacillus pentosus strain LP-22 can be applied in the planting industry, as follows:

[0014] A. Apply the fermentation liquid of Lactobacillus pentosus strain LP-22 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 pentosus strain LP-22 on plant leaves (front and back) in the form of foliar fertilizer, dilute 100-150 times for use, until the leaves are covered with water droplets and drip;

[0016] C. Dilute the fermentation liquid of Lactobacillus pentosaceus strain LP-22 by 100-150 times for soaking seeds; or dilute the fermentation liquid of Lactobacillus pentosaceus strain LP-22 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 3-10 minutes.

[0019] The method for preparing the fermentation broth of the Lactobacillus pentosus strain LP-22 is as follows, in parts by weight:

[0020] (1) preparing a culture medium: adding 10-14 parts of tryptone, 10-14 parts of yeast extract, 4-7 parts of glucose, 0.5-2 parts of Tween 80, 1-3 parts of dipotassium hydrogen phosphate, 4-7 parts of sodium acetate, 1-4 parts of triammonium citrate, 0.1-0.5 parts of magnesium sulfate, and 0.1-0.5 parts of manganese sulfate to 950-1100 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 pentosaceus strain LP-22 was inoculated into the culture medium of step (1), and cultured with shaking at 60-80 rpm to obtain a fermentation broth of Lactobacillus pentosaceus strain LP-22.

[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] The OD value of the fermentation liquid of the Lactobacillus pentosaceus strain LP-22 measured in the step (2) is 1.7-2.0.

[0026] The lactobacillus pentosus strain LP-22 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 fermentation 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 Lactobacillus pentosus strain LP-22 is a lactic acid bacteria microorganism with good biosafety and strong decomposition ability. It can rapidly reproduce by utilizing nutrients in the environment (organic matter in the soil and leaf wax). At the same time, it continuously inhibits other microorganisms (including pathogenic bacteria) from occupying microecological location advantages, avoids the formation of harmful substances, improves the growth environment of plants, and achieves the purpose of green development and modernization of agriculture. The method of the invention is simple and easy to implement, and has a stable effect. DETAILED DESCRIPTION

[0029] 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.

[0030] Application Example 1

[0031] The fermentation liquid of Lactobacillus pentosus strain LP-22 is applied to the soil in the form of basal fertilizer or topdressing by drip application:

[0032] 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, sterilize by high pressure for later use;

[0033] b. Liquid submerged fermentation culture: A single colony of Lactobacillus pentosus strain LP-22 was inoculated into the above culture medium, shaken and cultured at 60r / min, the fermentation temperature was 37°C, the fermentation time was 96h, and the OD value of the fermentation broth was measured to be 2.0;

[0034] c. From June to October 2022, a plot test of cotton (Gengye No. 64) was conducted in Shibahu Village, Dafeng Town, Changji Hui Autonomous Prefecture. Drip application was carried out four times during the whole growth period (June 20, June 29, July 8, and July 18), using 1L / mu each time.

[0035] d. Through the investigation of the disease index and disease rate of Verticillium wilt in cotton fields (the "number one disease" of cotton, which is seriously harmful and is called the "cancer" of cotton), the drip application of the fermentation liquid of Lactobacillus pentosaceus strain LP-22 in the prevention and control of Verticillium wilt greatly improved the prevention and control effect (Table 1) compared with conventional fertilization (ck), alleviated the disease and reduced the disease index.

[0036] Table 1 Preventive effect of the fermentation liquid of Lactobacillus pentosus strain LP-22 on cotton Verticillium wilt

[0037]

[0038] 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%.

[0039] Disease index (disease index) = 100 × ∑ (number of diseases at each level × representative value at each level) / (total number of surveys × highest representative value).

[0040] Control effect (control effect, %) = (control disease index - treatment disease index) / control disease index × 100.

[0041] Embodiment 2:

[0042] Spray the fermentation liquid of Lactobacillus pentosus strain LP-22 on the leaves of plants (front and back) in the form of foliar fertilizer:

[0043] 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, sterilize under high pressure for later use;

[0044] 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 37°C, the fermentation time was 96 h, and the OD value of the fermentation liquid was measured to be 1.7;

[0045] c. From September 2021 to March 2022, a strawberry experiment (Yuexiu variety) was conducted at the strawberry base of Senjing Sentai Agricultural Development (Jiande) Co., Ltd. in Laocun, Datong Town, Jiande City, Hangzhou, Zhejiang Province. The fermentation liquid of Lactobacillus pentosus strain LP-22 was diluted 100 times and sprayed on the leaves of the plants as 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;

[0046] d. 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, with each row being 3m. The incidence rate, disease index and prevention effect were counted and calculated. Compared with the control group, the incidence rate and disease index of strawberry root diseases treated with the fermentation liquid of Lactobacillus pentosus strain LP-22 were significantly reduced, and the prevention effect reached 79.24% (Table 2). The fermentation liquid of Lactobacillus pentosus strain LP-22 significantly improved the disease suppression performance of the strawberry rhizosphere, and played a role in preventing and controlling strawberry root diseases.

[0047] Table 2 Investigation on the efficacy of the fermentation liquid of Lactobacillus pentosus strain LP-22 on strawberry

[0048] Total number of plants surveyed Number of diseased plants Incidence % Disease index Effectiveness% deal with 96 9 9.38 6.25 79.24 Comparison 88 48 54.55 30.11 -

[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] Application Example 3:

[0053] Dilute the fermentation liquid of Lactobacillus pentosus strain LP-22 100-150 times and use it for seed soaking:

[0054] 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, sterilize under high pressure for later use;

[0055] 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 37°C, the fermentation time was 96 h, and the OD value of the fermentation liquid was measured to be 1.9;

[0056] 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 pentosus strain LP-22 was diluted 100 times and the bulbs used as seeds were soaked (1 hour);

[0057] d. In March 2023, the occurrence of aboveground diseases of Fritillaria thunbergii (gray mold, black spot, soft rot, dry rot, etc.) and the growth of Fritillaria thunbergii were investigated in the test plots. The incidence and prevention effect were counted and calculated. Compared with the control group, the incidence of aboveground diseases treated with the fermentation liquid of Lactobacillus pentosus strain LP-22 was significantly reduced, the prevention effect reached 67.7%, and the emergence rate, average plant height, and leaf width were significantly improved (Table 3). The fermentation liquid of Lactobacillus pentosus strain LP-22 significantly improved the disease inhibition performance of Fritillaria thunbergii and promoted the healthy growth of Fritillaria thunbergii.

[0058] Table 3 Investigation on the application of fermentation liquid of Lactobacillus pentosus strain LP-22 in the prevention and treatment of Fritillaria

[0059]

[0060]

[0061] 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%.

[0062] Disease index (disease index) = 100 × ∑ (number of diseases at each level × representative value at each level) / (total number of surveys × highest representative value).

[0063] Control effect (control effect, %) = (control disease index - treatment disease index) / control disease index × 100.

[0064] 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 Lactobacillus pentosus strain LP-22, with a deposit number of CGMCC No.31651, a deposit date of August 12, 2024, and a deposit unit of the General Microbiology Center of the China Microbiological Culture Collection Administration.

2. Use of the Lactobacillus pentosus strain LP-22 as claimed in claim 1 in planting industry.

3. The use according to claim 2, characterized in that The application is specifically: A. Apply the fermentation liquid of Lactobacillus pentosus strain LP-22 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 pentosus strain LP-22 on the front and back of plant leaves as foliar fertilizer, dilute 100-150 times before use, until the leaves are covered with water droplets and dripping; C. Dilute the fermentation liquid of Lactobacillus pentosaceus strain LP-22 by 100-150 times for soaking seeds; or dilute the fermentation liquid of Lactobacillus pentosaceus strain LP-22 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 3-10 minutes.

6. The use according to claim 3, characterized in that The method for preparing the fermentation broth of the Lactobacillus pentosus strain LP-22 is as follows, in parts by weight: (1) preparing a culture medium: adding 10-14 parts of tryptone, 10-14 parts of yeast extract, 4-7 parts of glucose, 0.5-2 parts of Tween 80, 1-3 parts of dipotassium hydrogen phosphate, 4-7 parts of sodium acetate, 1-4 parts of triammonium citrate, 0.1-0.5 parts of magnesium sulfate, and 0.1-0.5 parts of manganese sulfate to 950-1100 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 pentosaceus strain LP-22 was inoculated into the culture medium of step (1), and cultured with shaking at 60-80 rpm to obtain a fermentation broth of Lactobacillus pentosaceus strain LP-22.

7. The use according to claim 3, characterized in that The fermentation temperature in step (2) is 30°C-38°C.

8. The use according to claim 3, characterized in that The fermentation time in step (2) is 90-100 hours.

9. The use according to claim 3, characterized in that The OD value of the fermentation liquid of the Lactobacillus pentosaceus strain LP-22 measured in the step (2) is 1.7-2.0.

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