Lactiplantibacillus pentosus strain lp-22 and use thereof

Abstract

The application discloses a Lactiplantibacillus pentosus strain LP-22 and application thereof. The Lactiplantibacillus pentosus strain LP-22 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 12, 2024, and the preservation number is CGMCC No. 31651. The application also provides application of the Lactiplantibacillus pentosus strain LP-22 in planting industry. Compared with the prior art, the fermented liquid can be applied to soil or a leaf surface or a seed surface after being prepared, so that the propagation of harmful microorganisms is avoided, or the immune system of plants or seeds is directly stimulated through contact or absorption of the leaf surface or the seed, and the systemic resistance of the plants is improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Pentosacchariformis strain LP-22 and its applications. Background Technology

[0002] Currently, the excessive use of chemical fertilizers and pesticides is a major cause of soil degradation and non-point source pollution in agriculture. This 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. In response to the severe problems caused by the excessive use of chemical fertilizers and pesticides, including soil degradation, non-point source pollution in agriculture, declining arable land quality, intensified pests and diseases, and reduced yields and quality of agricultural products, technological innovation and green development in the agricultural sector have become urgent needs. Against this backdrop, bio-farming, as an environmentally friendly agricultural development model, is increasingly important. Bio-farming not only helps restore the ecological balance of the soil and reduce dependence on chemical substances, but also enhances the natural resistance of crops to pests and diseases, ensuring the safety and quality of agricultural products.

[0003] Agricultural microbial products refer to live bacterial preparations made from target microorganisms after propagation. They 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. These products mainly consist of traditional microorganisms, primarily Bacillus species (accounting for 98% of production). Manufacturers often 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 effects on crops. Consequently, the potential of these microorganisms is not fully realized.

[0004] The application areas of agricultural microbial products are constantly expanding, from initial food crop cultivation to fruit and vegetable planting, soil remediation, and many other fields. Simultaneously, with increasing public awareness of food safety and environmental protection, the application of microorganisms in organic and ecological agriculture is becoming increasingly widespread. However, insufficient investment in research and development by some companies has resulted 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 the endophytic bacterium HB3S-20 and its application in the 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 efficacy of strain HB3S-20 against cotton Verticillium wilt is 62.57%, showing good biocontrol activity. It has no negative impact on cotton emergence rate and cotton growth. Cotton treated with HB3S-20 bacterial solution showed a significant increase in plant height and fresh weight compared with the blank control.

[0006] CN111139199A discloses Bacillus amyloliquefaciens CGMCC No. 17843 and its applications. This Bacillus amyloliquefaciens can be prepared into microbial agents, which can be used as fertilizers or drugs to prevent and control root rot, control pests and diseases, and increase crop yield. It can also be prepared into compound microbial agents with other microbial agents, and can also be prepared into fertilizers.

[0007] CN113403218A discloses a biocontrol strain and its application in controlling soft rot in Fritillaria thunbergii and promoting its growth. Field trial results show that the microbial agent prepared from the aforementioned strain Dm97 achieves a control effect of over 90% against soft rot in Fritillaria thunbergii, promotes growth in disease-free plants by over 70%, and increases yield by over 50%. The above-mentioned microbial agent can be used for early soil treatment to prevent soft rot, and later for root treatment to better control it. It exhibits excellent disease prevention and yield-increasing effects and has great potential for development into a commercially viable live-bacterial biocontrol agent.

[0008] 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

[0009] In view of the above-mentioned deficiencies of the prior art, the present invention provides a *Lactobacillus pentosus* strain LP-22. When the fermentation broth of *Lactobacillus pentosus* strain LP-22 is applied to soil, leaves, or seed surfaces, the strain can rapidly multiply using nutrients in the environment. It continuously inhibits other microorganisms (including pathogens) from occupying a dominant microecological location, thus 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 the plant's systemic resistance (ISR).

[0010] A strain of *Lactiplantibacillus pentosus*, LP-22, with accession number CGMCC No. 31651, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on August 12, 2024.

[0011] The Lactiplantibacillus pentosus strain LP-22 is a Gram-positive bacterium. Physiological and biochemical analysis, as well as 16S rDNA sequencing, have confirmed it to be Lactiplantibacillus pentosus. Specifically, the 16S rDNA sequence of Lactiplantibacillus pentosus strain LP-22, SEQ ID NO: 1, is shown in the sequence listing.

[0012] The source of the Pentosacchariformis strain LP-22 of this invention was isolated from natural water bodies and soil in Hangzhou.

[0013] The aforementioned Lactobacillus pentosaceus strain LP-22 can be used in agriculture, as detailed below:

[0014] A. Apply the fermentation broth of Lactobacillus pentosacchari strain LP-22 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 pentosus strain LP-22 onto the plant leaves (both sides) as a foliar fertilizer, diluted 100-150 times, until the leaves are covered with water droplets and dripping.

[0016] C. Dilute the fermentation broth of Lactobacillus pentosus strain LP-22 by 100-150 times and use it for seed soaking; or dilute the fermentation broth of Lactobacillus pentosus strain LP-22 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 3-10 minutes.

[0019] The fermentation broth preparation method of the *Lactobacillus pentosacchari* strain LP-22 is as follows, in parts by weight:

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

[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 pentosus strain LP-22 was measured to be 1.7-2.0.

[0026] The *Lactobacillus pentosolicus* strain LP-22 of this invention is a lactic acid bacteria microorganism with good biocompatibility and strong decomposition ability, making it suitable for agricultural planting. After being prepared into a fermentation broth, 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 pentosaceus 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 wax on the leaves). At the same time, it continuously inhibits other microorganisms (including pathogens) from occupying the microecological location advantage, avoids the formation of harmful substances, improves the plant growth environment, and achieves the goal of green development and modernization of agriculture. The method of this invention is simple, easy to implement, and has stable effects. Detailed Implementation

[0029] The technical solution of the present invention will be further illustrated below through specific embodiments. The reagents and raw materials used in the embodiments are all commercially available conventional raw materials.

[0030] Application Example 1

[0031] The fermentation broth of Lactobacillus pentosaceus strain LP-22 was applied to the soil as a base fertilizer or top dressing via drip application.

[0032] a. Prepare the 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, autoclave and set aside.

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

[0034] c. From June to October 2022, a plot trial of cotton (Gengye 64) application was conducted in Shibahu Village, Dafeng Town, Changji Hui Autonomous Prefecture. The cotton was drip-dried four times during the entire growth period (June 20, June 29, July 8, and July 18), with 1L / mu applied each time.

[0035] d. Through the investigation of the disease index and disease incidence rate of Verticillium wilt (the "number one disease" of cotton, which is very harmful and is called the "cancer" of cotton), the treatment of drip application of fermentation liquid of Lactobacillus pentosus strain LP-22 significantly improved the control effect of Verticillium wilt compared with conventional fertilization (ck) (Table 1), reduced the disease and lowered the disease index.

[0036] Table 1. Preventive effect of fermentation broth of Lactobacillus pentosaceus strain LP-22 on cotton Verticillium wilt.

[0037]

[0038] 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.

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

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

[0041] Example 2:

[0042] The fermentation broth of Lactobacillus pentosaceus strain LP-22 was sprayed onto the plant leaves (both sides) as a foliar fertilizer:

[0043] a. Prepare the 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, autoclave and set aside.

[0044] b. Liquid deep fermentation culture: A single colony of the strain was inoculated into the above culture medium, and cultured with shaking at 60 r / min. The fermentation temperature was 37℃ and the fermentation time was 96 h. The OD value of the fermentation broth was measured to be 1.7.

[0045] c. 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. in Laocun Village, Datong Town, Jiande City, Hangzhou, Zhejiang Province. The fermentation liquid of Lactobacillus pentosus strain LP-22 was diluted 100 times and used as a foliar fertilizer sprayed on the plant leaves until the leaves were covered with water droplets and dripped down. Starting from the cultivation in September, it was carried out once a week, and the time was delayed in wet and cold weather.

[0046] d. The occurrence of root diseases in strawberry plants in the experimental plots was investigated at the beginning of the strawberry harvest (December 11, 2022) in Yuexiu. Five rows were randomly selected for each treatment, with each row measuring 3 meters. The incidence rate, disease index, and control efficacy were statistically analyzed and calculated. Compared to the control group, the fermentation broth treatment with *Lactobacillus pentosus* strain LP-22 significantly reduced the incidence rate and disease index of strawberry root diseases, achieving a control efficacy of 79.24% (Table 2). The fermentation broth of *Lactobacillus pentosus* strain LP-22 significantly improved the rhizosphere disease suppression performance of strawberries, playing a role in controlling strawberry root diseases.

[0047] Table 2. Investigation on the efficacy of fermentation broth of Lactobacillus pentosaceus strain LP-22 against strawberries.

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

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

[0052] Application Example 3:

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

[0054] a. Prepare the 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, autoclave and set aside.

[0055] b. Liquid deep fermentation culture: A single colony of the strain was inoculated into the above culture medium, and cultured with shaking at 60 r / min. The fermentation temperature was 37℃ and the fermentation time was 96 h. The OD value of the fermentation broth 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 broth of Lactobacillus pentosus strain LP-22 was diluted 100 times and the bulbs used as seeds were soaked for 1 hour.

[0057] d. In March 2023, the incidence of aboveground diseases (gray mold, black spot, soft rot, dry rot, etc.) and the growth of the fritillaria were investigated in the experimental plots. The incidence rate and control efficacy were statistically analyzed and calculated. Compared with the control group, the fermentation broth treatment with *Lactobacillus pentosus* strain LP-22 significantly reduced the incidence of aboveground diseases, achieving a control efficacy of 67.7%, and significantly increased seedling emergence rate, average plant height, and leaf width (Table 3). The fermentation broth of *Lactobacillus pentosus* strain LP-22 significantly improved the disease-suppressing performance of fritillaria and promoted its healthy growth.

[0058] Table 3. Investigation on the application of fermentation broth of Lactobacillus pentosolicus strain LP-22 in the control of Fritillaria cirrhosa.

[0059]

[0060]

[0061] 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.

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

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

[0064] 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 type of pentosacchari lactobacillus ( Lactiplantibacillus pentosus The strain LP-22, with accession number CGMCCNo.31651, was deposited on August 12, 2024, and the depositary institution is the China General Microbiological Culture Collection Center.

2. The application of the Pentosacchariformis Lactobacillus strain LP-22 as described in claim 1 in agriculture, characterized in that: The specific application is the use of Lactobacillus pentosaceus strain LP-22 in the control of Verticillium wilt in cotton.

3. The application as described in claim 2, characterized in that, The application of the Lactobacillus pentosus strain LP-22 in the control of cotton Verticillium wilt is as follows: the fermentation broth of Lactobacillus pentosus strain LP-22 is applied to the soil as a base fertilizer or top dressing, wherein the amount of fermentation broth used per acre is 3-5L.

4. The application as described in claim 3, characterized in that, The fermentation broth preparation method of the *Lactobacillus pentosacchari* strain LP-22 is as follows, in parts by weight: (1) Preparation of culture medium: Add 10-14 parts tryptone, 10-14 parts yeast extract, 4-7 parts glucose, 0.5-2 parts Tween 80, 1-3 parts dipotassium hydrogen phosphate, 4-7 parts sodium acetate, 1-4 parts triammonium citrate, 0.1-0.5 parts magnesium sulfate, and 0.1-0.5 parts manganese sulfate to 950-1100 parts distilled water, stir and mix evenly, and sterilize under high temperature and high pressure for later use; (2) Liquid deep fermentation culture: Inoculate a single colony of Lactobacillus pentosus strain LP-22 into the culture medium of step (1) and culture with shaking at 60-80 r / min to obtain fermentation broth of Lactobacillus pentosus strain LP-22.

5. The application as described in claim 4, characterized in that, The fermentation temperature in step (2) is 30℃-38℃.

6. The application as described in claim 4, characterized in that, The fermentation time in step (2) is 90-100 hours.

7. The application as described in claim 4, characterized in that, In step (2), the OD value of the fermentation broth of Lactobacillus pentosus strain LP-22 was measured to be 1.7-2.0.

Images