Lactobacillus plantarum and application thereof
By screening and cultivating Lactobacillus plantarum ASD61, bacteria powder and organic materials were prepared, and the problem of poor adaptability of Trichoderma harziana in the soil was solved, and efficient biological control of ginger white silk disease was achieved.
Patent Information
- Application Number
- CN202510627699.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-15
AI Technical Summary
When preventing and treating ginger white silk disease, the existing biological control agent Trichoderma harziana has problems such as high requirements for soil conditions and sensitivity to ambient temperature, which leads to poor adaptability in the soil and poor results.
A strain of Lactobacillus plantarum ASD61 was used to screen it from the ginger planting base in Weifang City, Shandong Province, and cultured with MRS medium and freeze-dried to prepare bacterial powder. It was used for fermentation of agricultural waste to form organic materials, and it was used to quickly adapt to the soil environment mediated by straw to inhibit the growth of harmful bacteria.
The strain ASD61 can quickly colonize and expand in the soil, effectively inhibit harmful bacteria, reduce crop diseases, and improve prevention and control effects.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microorganisms, and in particular to a plant lactobacillus strain and application thereof. Background Art
[0002] Lactobacillus plantarum is a common probiotic used in food, agriculture, medicine, and health. In the food sector, Lactobacillus plantarum is often used as a fermentation agent, which can not only inhibit the growth of harmful microorganisms, but also be used in the production of probiotic products to help regulate intestinal flora. In the field of medicine and health, by improving the intestinal flora, it can improve digestive problems and enhance immunity, while also having cholesterol-lowering and anti-inflammatory effects. In the agricultural field, it can be used as a feed additive to promote animal digestion and absorption, reducing the use of antibiotics. In the field of environmental protection, it can be used in sewage treatment, decomposing organic matter while reducing odor. It can also be used in agricultural solid waste treatment to prepare biomass organic fertilizer.
[0003] Ginger has edible value, medicinal value and industrial application value, which makes it in huge demand; the growth process of ginger includes germination period, seedling period, growth period and rhizome maturity period; the fertilizer requirements and disease control requirements of each growth stage are different. For example, in the seedling period, nitrogen fertilizer is mainly used to promote stem and leaf growth, and prevent leaf spot, anthracnose, etc.; in the growth period, compound fertilizer is needed to promote branching and rhizome expansion, and prevent white rot, rot, bacterial wilt, etc.; in the maturity period, potassium fertilizer is needed to improve the quality and disease resistance of ginger pieces, and prevent white rot, rot, etc.; among them, white rot begins in the growth period, due to the increase in plant density and the large increase in field humidity, it spreads through the soil, which easily increases the spread of the disease, causing the stems of ginger to be covered with white silky mycelium and the stems and leaves to wilt, while the ginger pieces are covered with mycelium, rot, soften, and smell, making them lose their commercial value.
[0004] White rot is a disease caused by Sclerotium rufin, which has typical white hyphae and brown sclerotia on the rhizomes. Currently, the prevention and control of Sclerotium rufin is mainly chemical control and biological control. The chemical control agents mainly use thiophanate-methyl and cytomegalomycin. The biological control mainly uses Trichoderma harzianum. Among them, biological control has the advantages of being environmentally friendly and pollution-free, and has attracted much attention. However, there are still certain problems when using Trichoderma harzianum, such as high requirements for soil conditions and sensitivity to ambient temperature. The existence of these factors makes it less adaptable after entering the soil, resulting in slightly poor effects and limited applications. Summary of the Invention
[0005] In view of this, the present invention provides a Lactobacillus plantarum strain and its application, the strain numbered ASD61, deposited on July 10, 2024, at the General Microbiology Center of the China Culture Collection Administration, referred to as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Deposit Number: CGMCC No.31254; Latin name: Lactobacillus plantarum Lactobacillus plantarum .
[0006] The technical solutions of the present invention are as follows: A strain of Lactobacillus plantarum, numbered ASD61, was deposited on July 10, 2024, at the General Microbiology Center of the China Culture Collection Administration (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, with a deposit number of CGMCC No. 31254; its Latin name is Lactobacillus plantarum Lactobacillus plants .
[0007] Preferably, the 16S rDNA sequence of the strain ASD61 is shown as SEQ ID NO.1.
[0008] Preferably, the strain ASD61 is screened from the leaf surface of ginger in a ginger planting base in Weifang City, Shandong Province.
[0009] Preferably, the strain ASD61 is cultured in MRS medium at 30±1° C. in a static culture.
[0010] Preferably, MRS medium: 20 g glucose, 10 g tryptone, 5 g yeast powder, 10 g beef extract, 2 g diammonium citrate, 3.12 g sodium acetate, 1.63 g disodium hydrogen phosphate, 2.25 g potassium acetate, 0.58 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate, 801 mL Tween, distilled water to 1000 mL, sterilized at 115°C for 30 min.
[0011] The above strain ASD61 is used to inhibit spoilage bacteria; in a plate test, volatile compounds can inhibit the growth of spoilage bacteria.
[0012] A bacterial powder of strain ASD61 is prepared as follows: (1) Seed culture The preserved strain ASD61 was inoculated into MRS medium and cultured at 30±1°C for 36-48 hours to obtain seed solution. (2) Fermentation Inoculate the seed liquid into the fermentation medium at an inoculum rate of 3-5% (v / v), and ferment statically at 30±1°C for 72-96 hours to obtain a fermentation liquid. (3) Drying The fermentation broth was freeze-dried to obtain bacterial powder, in which the effective viable bacteria count was 1-2×10 9 cfu / g.
[0013] The above strain ASD61 was used in the fermentation of agricultural waste. The process was as follows: SS1: crush the straw to 3-5 cm and adjust the moisture content of the straw to 15-25%; mix 90 parts of straw, 5 parts of glucose, 2 parts of peptone, and 3 parts of yeast powder, sterilize, and obtain a mixture; SS2, mixing the mixed material with ASD61 bacterial powder in a fermentation device, then controlling the fermentation temperature at 30°C and fermenting for 5-10 days to obtain a fermentation material; SS3, freeze-drying the fermented material to obtain organic material; During the fermentation process, strain ASD61 consumes the carbon and nitrogen elements in the mixture to expand; as fermentation proceeds, the element content in the straw decreases, causing its structure to change, such as the disintegration of straw fibers to produce certain pores; these changes provide space for the growth of strain ASD61, allowing strain ASD61 to colonize in the pores; therefore, after the organic material is put into the soil for use, strain ASD61 can quickly adapt to the soil environment under the mediation of straw (organic material), so that it can rapidly reproduce in the soil and occupy an ecological niche, achieving the purpose of inhibiting the growth of harmful bacteria.
[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The strain ASD61 provided by the present invention was deposited on July 10, 2024 at the General Microbiology Center of the China Culture Collection Administration, referred to as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Deposit Number: CGMCC No. 31254; Latin name: Lactobacillus plantarum Lactobacillus plantarum ; Strain ASD61 has the advantage of inhibiting the growth of spoilage bacteria.
[0015] 2. After the organic material prepared by the strain ASD61 of the present invention is used in the soil, the strain ASD61 can quickly take up residence and multiply in the soil, thereby effectively inhibiting harmful bacteria in the soil and reducing the occurrence of crop diseases. DETAILED DESCRIPTION
[0016] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.
[0017] Example 1 Isolation and Identification 1. Strain screening and purification (1) Sample preparation ① Take 10g of ginger leaves, which were collected from the ginger planting base in Weifang City, Shandong Province; add 100ml of sterile water and homogenize to obtain 10 -1 suspension; ② The solution of step ① was diluted 10 times in a gradient manner, namely 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ,stand-by; (2) Preparation of culture medium MRS solid medium: 20 g glucose, 10 g tryptone, 5 g yeast powder, 10 g beef extract, 2 g diammonium citrate, 3.12 g sodium acetate, 1.63 g disodium hydrogen phosphate, 2.25 g potassium acetate, 0.58 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate, 801 mL Tween, 20 g agar, dilute to 1000 mL with distilled water, sterilize at 115°C for 30 min, and let cool for use; (3) Plate culture and selection of single colonies: The gradient solution prepared in step ② was inoculated onto the MRS solid medium in step (2) by coating method, and static cultured at 30±0.1℃ for 48h; single colonies were selected; (4) Separation and purification The selected single colony was inoculated onto the MRS solid culture medium in step (2) by streaking method, and static cultured at 30°C for 40 hours. The single colony was picked and stored in a glycerol tube at -80°C.
[0018] 2. Identification The single colony stored in the glycerol tube was identified, and the 16S rDNA sequence was shown in SEQ ID NO: 1, as follows: SEQ ID NO: 1 After identification, it was determined that the strain was Lactobacillus plantarum Lactobacillus plantarum , numbered ASD61; the strain was deposited on July 10, 2024, at the General Microbiology Center of the China Culture Collection Administration, referred to as CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, Deposit Number: CGMCC No.31254; Latin name: Lactobacillus plantarum Lactobacillus plantarum .
[0019] Example 2: Test on the inhibition of spoilage bacteria by strain ASD61 The plate test was used to verify the inhibitory effect of strain ASD61 on Escherichia coli and Salmonella. The process is as follows: S1, seed culture The strain ASD61 deposited in Example 1 was inoculated into MRS medium and cultured statically at 30° C. for 40 h to obtain a seed solution; S2, fermentation The seed liquid was inoculated into the fermentation medium at an inoculum rate of 4% (v / v), and the fermentation was carried out at 30°C for 80 h to obtain the fermentation liquid. S3, the fermentation broth was centrifuged, the cells were collected, washed with sterile water and centrifuged; then resuspended with sterile water to prepare a viable cell count of 1×10 8 cfu / mL of ASD61 bacterial suspension; Using the existing technology, the viable bacteria count was 1×10 6 cfu / mL of Escherichia coli suspension; Using the existing technology, the viable bacteria count was 1×10 6 cfu / mL of Salmonella suspension; S4, preparing LB agar medium, nutrient agar (NA) medium and MRS solid medium; LB agar medium and nutrient agar (NA) medium are commercially available products, and the preparation method of MRS solid medium is the same as that in Example 1; S5, respectively, by spreading the ASD61 bacterial suspension onto MRS solid medium and marking it as plate A, to prepare two plates A; The E. coli suspension was spread onto LB agar medium and marked as plate B. Two plates B were prepared. The Salmonella suspension was spread onto nutrient agar (NA) medium and labeled as plate C. Two plates C were prepared. S6, aligning the edge of a plate A with the edge of a plate B, and sealing the joint between the plates A and B with plastic wrap, as sample A; Align the edge of another plate A with the edge of plate C, and seal the joint between plates A and C with plastic wrap to serve as sample B; Using MRS solid medium as the control plate, connect the open end of one control plate to the open end of plate B to serve as control A; connect the open end of another control plate to the open end of plate C to serve as control B; then seal the connection points with plastic wrap to serve as controls; S7, incubate the above-treated cells in a 30°C incubator for 48 hours and observe the growth of Escherichia coli and Salmonella; The results showed that compared with the control A and the control B, the volatile substances produced by the strain ASD61 of the present invention can inhibit the growth of Escherichia coli and Salmonella.
[0020] Example 3 Preparation of ASD61 bacterial powder A bacterial powder of strain ASD61 is prepared as follows: (1) Seed culture The strain ASD61 deposited in Example 1 was inoculated into MRS medium and cultured statically at 30° C. for 40 h to obtain a seed solution; (2) Fermentation The seed liquid was inoculated into the fermentation medium at an inoculum rate of 4% (v / v), and the fermentation was carried out at 30°C for 80 h to obtain the fermentation liquid. (3) Drying The fermentation broth was freeze-dried to obtain bacterial powder, and the effective viable bacteria count in the bacterial powder was 1.5×10 9 cfu / g.
[0021] Example 4 Preparation of ASD61 bacterial powder A bacterial powder of strain ASD61 is prepared as follows: (1) Seed culture The strain ASD61 deposited in Example 1 was inoculated into MRS medium and cultured statically at 30° C. for 36 h to obtain a seed solution; (2) Fermentation The seed liquid was inoculated into the fermentation medium at an inoculum rate of 5% (v / v), and the fermentation was carried out at 30°C for 72 h to obtain the fermentation liquid. (3) Drying The fermentation broth was freeze-dried to obtain bacterial powder, in which the effective viable bacteria count was 1×10 9 cfu / g.
[0022] Example 5 Preparation of ASD61 bacterial powder A bacterial powder of strain ASD61 is prepared as follows: (1) Seed culture The strain ASD61 preserved in Example 1 was inoculated into MRS medium and cultured statically at 30° C. for 48 h to obtain a seed solution; (2) Fermentation The seed liquid was inoculated into the fermentation medium at an inoculum rate of 3% (v / v), and the fermentation was carried out at 30°C for 96 h to obtain the fermentation liquid. (3) Drying The fermentation broth was freeze-dried to obtain bacterial powder, and the effective viable bacteria count in the bacterial powder was 2×10 9 cfu / g.
[0023] Example 6 Preparation of organic material containing strain ASD61 The above strain ASD61 was used in the fermentation of agricultural waste. The process was as follows: SS1, crush the straw to 3-5 cm, with a moisture content of 20%; mix 77 parts of straw, 5 parts of glucose, 10 parts of peptone, and 8 parts of yeast powder, sterilize, and obtain a mixture; SS2, mixing the mixed material with the ASD61 bacterial powder provided in Example 3 in a fermentation device at a weight ratio of the mixed material to the ASD61 bacterial powder of 50:1, and then controlling the fermentation temperature to be 30° C. for 8 days to obtain a fermentation material; SS3, freeze-drying the fermented material to obtain organic material.
[0024] Example 7 Preparation of organic material containing strain ASD61 The above strain ASD61 was used in the fermentation of agricultural waste. The process was as follows: SS1, crush the straw to 3-5 cm, with a moisture content of 15%; mix 77 parts of straw, 5 parts of glucose, 10 parts of peptone, and 8 parts of yeast powder, sterilize, and obtain a mixture; SS2, mixing the mixed material with the ASD61 bacterial powder provided in Example 4 in a fermentation device at a weight ratio of the mixed material to the ASD61 bacterial powder of 50:1, and then controlling the fermentation temperature to be 30° C. for 10 days to obtain a fermentation material; SS3, freeze-drying the fermented material to obtain organic material; Example 8 Preparation of organic material containing strain ASD61 The above strain ASD61 was used in the fermentation of agricultural waste. The process was as follows: SS1, crush the straw to 3-5 cm, with a moisture content of 25%; mix 77 parts of straw, 5 parts of glucose, 10 parts of peptone, and 8 parts of yeast powder, sterilize, and obtain a mixture; SS2, mixing the mixed material with the ASD61 bacterial powder provided in Example 5 in a fermentation device at a weight ratio of 50:1, and then controlling the fermentation temperature to be 30° C. for 5 days to obtain a fermentation material; SS3, freeze-drying the fermented material to obtain organic material.
[0025] Field trials Test 1 1.1 Basic soil conditions and experimental plots At the ginger planting base in Weifang, Shandong Province, we selected experimental locations, evenly spread the sclerotia of Sclerotium sclerotiorum on the soil surface, then turned the soil 20-25 cm deep, mixed the sclerotia with the soil, and divided the experimental plots. Each experimental plot was 5m×3m, with 50 ginger trees planted at a plant spacing of 0.25m and a row spacing of 0.55m; 1.2 Experimental Design Control group: no reagent was used; Control group: Trichoderma harzianum powder, effective viable count of 1.5×10 9 cfu / g, the dosage is 3kg / mu; Experimental group A: Example 3 bacterial powder, dosage: 3 kg / mu; Experimental group B: organic material of Example 6, dosage is 2kg / mu; Experimental group C: organic material of Example 7, dosage is 2kg / mu; Experimental group D: organic material of Example 8, dosage is 2kg / mu; Laiwu ginger was selected. Before planting, each plot was treated according to the experimental design. After spreading fungus powder and organic materials, the plot was tilled 20-25 cm to mix thoroughly. Ginger was planted in holes. Three replicates were designed for each treatment. Other fertilization and management were in accordance with planting requirements. 1.3 The growth of ginger was observed during the seedling, growth, and maturity stages, and the yield (fresh weight) after maturity was calculated. The results are shown in Table 1. In the table, plant height, leaf number, and leaf length are all average values. Table 1 Growth status and yield of ginger
[0026] From Table 1, it can be seen that the use of the ASD61 powder of the present invention and the organic material containing the strain ASD61 effectively inhibited the growth of Sclerotium truncatum, and no white mycelium appeared on the rhizomes of ginger, which indicates that the strain ASD61 of the present invention has a significant inhibitory effect on Sclerotium truncatum; and compared with the ASD61 powder (experimental group A), the organic material containing the strain ASD61 (experimental group B, experimental group C and experimental group D) had better effects after use. This may be because as the fermentation proceeds, the element content in the straw decreases, causing its structure to change, such as the presence of certain pores in the straw; these changes provide space for the growth of the strain ASD61, allowing the strain ASD61 to colonize in the space; therefore, after the organic material is put into use, the strain ASD61 can quickly adapt to the soil environment under the mediation of the straw, so that it can rapidly reproduce in the soil and occupy an ecological niche, thereby achieving the purpose of inhibiting the growth of harmful bacteria.
[0027] In the present invention, except for the strain ASD61 provided by the present invention, the other reagents and microorganisms used are commercially available products.
[0028] Test 2 2.1 Basic soil conditions Ginger planting base in Weifang City, Shandong Province; 2.2 Experimental Design Preparation of ASD61 bacterial suspension: The method was the same as in Example 2, and the viable bacterial count was 1×10 8 cfu / mL of ASD61 bacterial suspension; Preparation of Trichoderma harzianum suspension: Using existing technology, the viable count of the suspension was 1×10 8 cfu / mL of Trichoderma harzianum suspension; We selected Laiwu ginger and planted it in holes after stimulating its growth. The ginger pieces were of uniform size. deal with: Before planting, hold the ginger piece close to the ginger sprout and dip it in the ASD61 suspension as the experimental group; dip the ginger piece in the Trichoderma harzianum suspension as the control group; and use the ginger piece not dipped in the suspension as the control group (as the 0-day data). After dipping in the fungus suspension, plant the ginger pieces into the planting holes respectively; Each treatment was designed with three replicates; the rest of the fertilization and management were completely in accordance with the planting requirements; After planting, ginger pieces were taken out at 10 days, 20 days, 30 days, 50 days, and 70 days, and 10 g of ginger was cut and homogenized. The number of viable bacteria was calculated using the plate count method. The results are shown in Table 2, as follows: Table 2 Number of viable bacteria per gram of ginger
[0029] From Table 2, it can be seen that when the initial number of viable bacteria is the same, the colonization amount of the two bacteria on the ginger surface is quite different, especially in the early planting stage of the ginger block, the strain ASD61 of the present invention can reproduce rapidly, which shows that the strain of the present invention can better adapt to the soil environment, thereby being able to respond quickly in the soil with Sclerotium uniformis, thereby reducing the adverse effects of Sclerotium uniformis on ginger.
[0030] Although the present invention has been described in detail with reference to preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any person skilled in the art who can easily conceive of changes or substitutions within the technical scope disclosed in the present invention shall be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope of protection of the claims.
Claims
1. A strain of Lactobacillus plantarum, characterized in that The strain is numbered ASD61 and was deposited on July 10, 2024, at the General Microbiology Center of the China Culture Collection Administration (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101, deposit number: CGMCC No. 31254; Latin name: Lactobacillus plantarum Lactobacillus plantarum .
2. plant lactobacillus according to claim 1, characterized in that, The 16S rDNA sequence of the strain ASD61 is shown as SEQ ID NO.
1.
3. plant lactobacillus according to claim 1, characterized in that, The strain ASD61 was screened from the leaf surface of ginger in a ginger planting base in Weifang City, Shandong Province.
4. plant lactobacillus according to claim 1, characterized in that The strain ASD61 was cultured in MRS medium at 30±1°C in a static culture.
5. plant lactobacillus according to claim 4, characterized in that, MRS medium: 20 g glucose, 10 g tryptone, 5 g yeast powder, 10 g beef extract, 2 g diammonium citrate, 3.12 g sodium acetate, 1.63 g disodium hydrogen phosphate, 2.25 g potassium acetate, 0.58 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate, 1 mL Tween 80, dilute to 1000 mL with distilled water, and sterilize at 115°C for 30 min.
6. Use of the plant lactobacillus according to claim 1 in inhibiting spoilage bacteria.
7. A bacterial powder of Lactobacillus plantarum ASD61 according to claim 1, characterized in that The preparation process is as follows: (1) Seed culture The preserved strain ASD61 was inoculated into MRS medium and cultured at 30±1°C for 36-48 hours to obtain seed solution. (2) Fermentation Inoculate the seed liquid into the fermentation medium at an inoculum rate of 3-5% (v / v), and ferment statically at 30±1°C for 72-96 hours to obtain a fermentation liquid. (3) Drying The fermentation broth was freeze-dried to obtain bacterial powder, in which the effective viable bacteria count was 1-2×10 9 cfu / g.
8. Use of the powder of Lactobacillus plantarum ASD61 according to claim 7 in the fermentation of agricultural waste, characterized in that: The process is as follows: SS1: crush the straw to 3-5 cm and adjust the moisture content of the straw to 15-25%; mix 90 parts of straw, 5 parts of glucose, 2 parts of peptone, and 3 parts of yeast powder, sterilize, and obtain a mixture; SS2, mixing the mixed material with ASD61 bacterial powder in a fermentation device, then controlling the fermentation temperature at 30°C and fermenting for 5-10 days to obtain a fermentation material; SS3, freeze-drying the fermented material to obtain organic material.