Enterococcus lactis WD06 and combined application of enterococcus lactis WD06 and plant extract

Abstract

The invention discloses enterococcus lactis WD06 and combined application of the enterococcus lactis WD06 and a plant extracting solution, and belongs to the technical field of microorganisms. The enterococcus lactis WD06 provided by the invention is preserved in China General Microbiological Culture Collection Center (CGMCC) on March 6, 2025, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.33727. The growth promoting effect is achieved on crops such as cucumbers and wheat; the plant growth promoting agent is combined with a plant extracting solution, and has a growth promoting effect under the environment of non-salt stress and salt and alkali stress. The microbial agent and the plant extract have multiple advantages in the aspects of saline-alkaline tolerance, growth promotion and the like, and are environment-friendly and effective soil amendments which can be used in a combined manner.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain of Enterococcus lactis WD06 and a combined application of the strain and a plant extract. Background Art

[0002] The high salt or alkali content in saline-alkali soil will have an adverse effect on crop growth and is one of the important limiting factors for agricultural production.

[0003] Microbial agents are green, sustainable, and environmentally friendly products. They release numerous active substances that react with soil salt to form insoluble salts, reducing soil alkalinity. Furthermore, as microorganisms decompose soil organic matter, they increase its organic matter content, improve soil structure, and enhance its ability to retain water and nutrients, creating favorable conditions for healthy plant growth. Under saline-alkali stress, microbial agents can also promote the accumulation of osmotic regulators such as proline and soluble sugars in plants, helping to maintain cellular osmotic pressure balance and mitigating damage to plants caused by saline-alkali stress.

[0004] BoChuang Xiran's products contain a variety of plant secretions, which can promote the growth and activity of probiotics in the soil, help maintain the diversity and stability of soil microbial communities, and thus improve soil structure, increase soil fertility and promote plant growth. It is an effective soil microbial awakening agent that can enhance the effect of microbial agents.

[0005] Enterococcus lactis is a Gram-positive, facultatively anaerobic, non-spore-forming lactic acid bacterium. Most patents related to lactic acid bacteria are for applications in food, animal husbandry, straw return to fields, treatment of intestinal diseases, treatment of viral diseases in veterinary and poultry, and regulation of intestinal flora. However, research in the field of plant microorganisms is relatively limited. Lactic acid bacteria can be combined with other microorganisms to create microbial preparations for soil improvement and remediation. A patent by Yi Lanhua et al. demonstrates that lactic acid bacteria can control soft rot in peppers; a patent by Luo Chao et al. demonstrates that the addition of lactic acid bacteria can prevent infection by Bacillus thuringiensis and Aspergillus flavus in hydroponic peanut sprout production; and a patent by Guo Xinnian et al. demonstrates that lactic acid bacteria, fermented with microorganisms such as Candida albicans, Bacillus megaterium, nitrogen-fixing bacteria, and Clostridium butyricum, can produce biofertilizers that can lower the pH of saline-alkali soils and promote crop growth. However, no patents demonstrate that lactic acid bacteria can promote saline-alkali tolerance in wheat or cucumbers. There are also no reports on the combined application of Enterococcus lactis with soil microbial awakening agents. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a strain of Enterococcus lactis ( Enterococcus lactis ) WD06 and its combined application with plant extracts.

[0007] The first aspect of the present invention is to provide a strain of Enterococcus lactis ( Enterococcus lactis ) WD06 was deposited in the General Microbiology Center of China Culture Collection Administration on March 6, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.33727.

[0008] Enterococcus lactis ( Enterococcus lactis ) The 16S rRNA sequence of WD06 is shown in SEQ ID NO.1.

[0009] The second aspect of the present invention is to provide Enterococcus lactis ( Enterococcus lactis ) Application of WD06 in promoting plant growth.

[0010] The plants include wheat or cucumber.

[0011] Further, the present invention provides Enterococcus lactis ( Enterococcus lactis ) Application of WD06 in promoting plant growth under salt or saline-alkali stress conditions.

[0012] The third aspect of the present invention is to provide a bacterial suspension and a preparation method thereof, wherein the strain WD06 is inoculated into an MRS medium and cultured for 12 h to obtain a seed liquid, which is then inoculated into a new MRS medium at a 2% inoculum to obtain a fermentation liquid, which is diluted to prepare a bacterial suspension for later use.

[0013] The fourth aspect of the present invention is to provide the Enterococcus lactis ( Enterococcus lactis ) Combined application of WD06 bacterial agent and plant extract.

[0014] The plant extract is a soil microorganism awakening agent and has a stimulating and awakening function on soil microorganisms.

[0015] In one or more embodiments of the present invention, a soil microbial awakening agent product produced using Bochuang Xirang contains 39.5% deep-sea kelp, 15.9% alfalfa meal, 10.2% barley grain, 6.4% barley straw, 10.2% carbonate, 8.7% liquid fish, 4.5% molasses, 4.3% wheat straw, and 0.3% sulfur, with the content of each ingredient fluctuating within a ±5% range.

[0016] As a fifth aspect of the present invention, there is provided a soil conditioner composition comprising the lactic acid Enterococcus ( Enterococcus lactis ) WD06 and plant extract.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention provides a strain of Enterococcus lactis WD06, which has a significant growth-promoting effect on crops such as cucumber and wheat; when used in combination with plant extracts, it has a good growth-promoting effect under both non-salt stress and saline-alkali stress environments.

[0018] (2) The microbial agent and the plant extract provided by the present invention have multiple advantages in terms of salt-alkali resistance and growth promotion, and are environmentally friendly and effective soil conditioners that can be used in combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 This is the colony morphology of strain WD06.

[0021] Figure 2 These are the functions of strain WD06, where a: solubilizes inorganic phosphorus; b: degrades organic phosphorus; c: degrades cellulose; d: degrades protein; and e: solubilizes potassium.

[0022] Figure 3 Genetic identification phylogenetic tree of strain WD06.

[0023] Figure 4 Effects of different treatments on wheat growth under salt-free conditions, where a: plant height; b: aboveground fresh weight; c: underground fresh weight; d: aboveground dry weight; e: underground dry weight; f: effects of different treatments on wheat growth; where CK: control group; WD06: single bacteria treatment group; Y: plant extract treatment group; WD06Y: strain + plant extract treatment group.

[0024] Figure 5 Effects of different treatments on wheat growth under saline-alkali environment, where a: plant height; b: aboveground fresh weight; c: underground fresh weight; d: aboveground dry weight; e: underground dry weight; f: effects of different treatments on wheat growth; where CK: control group; WD06: single bacteria treatment group; Y: plant extract treatment group; WD06Y: strain + plant extract treatment group.

[0025] Figure 6 Figure 3: Effects of different treatments on cucumber growth under salt-free conditions, where a: plant height; b: aboveground fresh weight; c: underground fresh weight; d: aboveground dry weight; e: underground dry weight; f: leaf area; g: effects of different treatments on cucumber growth; CK: control group; WD06: single bacteria treatment group; Y: plant extract treatment group; WD06Y: strain + plant extract treatment group.

[0026] Figure 7Effects of different treatments on cucumber growth under salt conditions, where a: plant height; b: aboveground fresh weight; c: underground fresh weight; d: aboveground dry weight; e: underground dry weight; f: leaf area; g: effects of different treatments on cucumber growth; where CK: control group; WD06: single bacteria treatment group; Y: plant extract treatment group; WD06Y: strain + plant extract treatment group. DETAILED DESCRIPTION

[0027] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0028] Enterococcus lactis provided in the embodiment ( Enterococcus lactis ) WD06, deposited in the General Microbiology Center of China Culture Collection Administration on March 6, 2025, with the deposit number CGMCC NO.33727; the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0029] Example 1, Enterococcus lactis ( Enterococcus lactis ) Separation of WD06 (1) A strain of Enterococcus lactis was screened from the rhizosphere soil of garlic in Jinxiang and named WD06. The colony morphology is shown in Figure 1 The colonies are spherical, with milky white opaque protrusions, intact edges without thorns, and a smooth and moist surface.

[0030] (2) Molecular biological identification of strain WD06 The 16S rDNA of single colonies was extracted for Blast alignment, and MEGA11 software was used to analyze and construct a phylogenetic tree.

[0031] Through the identification of the growth-promoting ability of the strain, strain WD06 was selected for molecular biological identification, and the 16SrDNA gene sequence of the single colony was extracted for Blast comparison. The MEGA11 software was used to analyze and construct a phylogenetic tree. The analysis results are shown in Figure 3 The results showed that strain WD06 and Enterococcus lactis ( Enterococcus lactis ) with a homology of 97%, the strain WD06 was preliminarily identified as Enterococcus lactis ( E. lactis ).

[0032] The 16S rDNA sequence of strain WD06 is as follows, as shown in SEQ ID NO.1.

[0033]

[0034] Example 2, 1. Materials and Methods 1.1 Culture medium and reagents The strain WD06 was cultured using MRS medium. The culture medium and formula for strain functional identification are shown in Table 1 below.

[0035] Table 1. Screening medium

[0036] 1.2 Experimental methods 1.2.1 Identification of growth-promoting function Validated strain Enterococcus lactis ( Enterococcus lactis ) Whether WD06 has the ability to dissolve phosphate, solubilize phosphate, solubilize potassium, degrade protein and degrade cellulose. The specific steps are shown in Table 2 below.

[0037] Table 2, Verification of the growth-promoting function of the strain

[0038] 1.2.3 Wheat Growth Promotion Potted Plant Verification Wheat seeds were wrapped in soaked gauze and placed in a refrigerator at 4°C for at least 3 days after white buds emerged. Regular soil and vermiculite were mixed in a 2:1 ratio and potted. A 130 mmol / L mixed saline solution containing NaCl, Na₂SO₄, NaHCO₃, and Na₂CO₃ was prepared in a 1:9:9:1 ratio. Two experimental groups were set up: a salt-free group (irrigated with tap water) and a saline-alkali group (irrigated with mixed saline). Each group had four treatments: CK, WD06, Y, and WD06Y. Four germinated wheat seeds were placed in each pot. Strain WD06 was inoculated into MRS medium and cultured at 37°C and 18,000 rpm for 12 hours to obtain seed fluid. A 2% inoculum was then inoculated into fresh MRS medium and cultured at 37°C and 18,000 rpm for 24 hours to obtain a fermentation broth, which was diluted 100-fold to prepare a strain suspension.

[0039] The composition of the plant extract is shown in Table 3 below, which is a soil microbial awakening agent product produced by BoChuang Xiran.

[0040] After the wheat broke through the soil, tap water was added to the CK group; bacterial suspension was added to the WD06 group; the plant extract was diluted 300 times in the Y group; and the bacterial suspension and plant extract dilution were mixed in the WD06Y group. The plants were then cultured in an incubator at 20°C, 50%RH, and 40,000 Lx for 12 days, with equal amounts of tap water added during the incubation period. Plant height was measured every 3 days, and the above- and underground dry and fresh weights of the wheat were weighed and recorded on the 12th day.

[0041] Table 3, Plant extract ingredients

[0042] 1.2.4 Cucumber Growth Promotion Potted Plant Verification Wrap cucumber seeds in soaked gauze and wait for them to germinate before use. Mix regular soil and vermiculite in a 2:1 ratio and pot them. Prepare a 100 mmol / L NaCl solution. Two experiments were conducted: a salt-free treatment with tap water and a salt-containing treatment with NaCl solution. Each experiment consisted of four treatments: CK, WD06, Y, and WD06Y. Four germinated cucumber seeds were placed in each pot. Strain WD06 was inoculated into YPD medium and cultured at 37°C and 18,000 rpm for 12 hours to obtain a seed solution. A 2% inoculum was then inoculated into fresh YPD medium and cultured at 37°C and 18,000 rpm for 24 hours to obtain a fermentation broth. The resulting suspension was diluted 100-fold and used as a suspension. The composition of the plant extract is shown in Table 1. The CK group was added with tap water; the WD06 group was added with bacterial suspension; the Y group used the plant extract diluted 300 times; the WD06Y group used a mixture of bacterial suspension and plant extract dilution. The plants were then cultured in an incubator at 24°C, 70%RH, and 40,000 Lx for 20 days, and regularly watered with an equal amount of tap water. The plant height was measured every 4 days, and the above-ground and underground dry and fresh weights and leaf areas of the cucumbers were measured and recorded on the 20th day.

[0043] 1.2.5 Functional Verification The strain was tested for its ability to dissolve inorganic phosphorus, degrade organic phosphorus, degrade cellulose, degrade protein and solubilize potassium. The results showed that strain WD06 produced a transparent zone in both organic phosphorus bacterial culture medium and inorganic phosphorus bacterial culture medium (see Figure 2 a, b), while no clear ring was produced in carboxymethyl cellulose medium and casein medium, and no oily substance was produced in silicate medium (see Figure 2 ce), indicating that strain WD06 has no ability to degrade cellulose, protein and potassium, and only has the ability to dissolve inorganic phosphorus and degrade organic phosphorus.

[0044] Example 3, Effect on Wheat Growth 3.4.1 Effects of strain WD06 combined with plant extracts on wheat growth under salt-free conditions like Figure 4As shown, the plant height data showed that on the 3rd day, the strain WD06 treatment group increased by 0.45% compared with CK; on the 6th day, the strain WD06 treatment group increased by 13.23%, the plant extract treatment group increased by 4.04%, and the strain and plant extract combination treatment group increased by 3.32% compared with CK; on the 9th day, the strain WD06 treatment group increased by 10.12%, the plant extract treatment group increased by 11.75%, and the strain and plant extract combination treatment group increased by 0.34% compared with CK; on the 12th day, the strain WD06 treatment group increased by 10.40%, the plant extract treatment group increased by 12.79%, and the strain and plant extract combination treatment group increased by 4.93%.

[0045] Aboveground fresh weight refers to the weight of the aboveground part of the plant after it is picked and not processed in any way. Aboveground dry weight refers to the weight of the aboveground part of the plant after it is picked and dried to a constant weight. Belowground fresh and dry weight refers to the weight of the belowground part of the plant, i.e., the rhizosphere, after it is picked and not processed and dried to a constant weight. The aboveground fresh and dry weight data showed that compared with CK, the aboveground fresh weight of the strain WD06 treatment group increased by 71.78% ( P <0.05), the plant extract treatment group increased by 76.17% ( P <0.05), the combined strain and plant extract treatment group increased by 20.18%. Compared to the CK, the WD06-treated group increased its belowground fresh weight by 0.30%. Aboveground dry weight increased by 14.04% in the WD06-treated group and 11.05% in the plant extract-treated group. Compared to the CK, the WD06-treated group increased its belowground dry weight by 35.58%, the plant extract-treated group increased its weight by 11.25%, and the combined strain and plant extract treatment group increased its weight by 3.43%. Under salt-free conditions, plants were unaffected by stress. Adding plant extracts increased soil nutrient content and promoted wheat seedling growth. Adding strain WD06 recruited more beneficial bacteria, increasing soil microbial diversity and promoting wheat root growth. Combining the two, however, resulted in the strain competing with wheat for nutrients in the plant extract, which reduced the effectiveness.

[0046] 3.4.2 Effects of strain WD06 combined with plant extracts on wheat growth under saline-alkali stress like Figure 5As shown, the plant height data showed that on the 3rd day, compared with CK, the strain WD06 treatment group increased by 4.00%, the plant extract treatment group increased by 1.78%, and the strain and plant extract combination treatment group increased by 4.00%; on the 6th day, compared with CK, the strain WD06 treatment group increased by 4.55%, the plant extract treatment group increased by 2.94%, and the strain and plant extract combination treatment group increased by 1.67%; on the 9th day, compared with CK, the strain WD06 treatment group increased by 12.42%, the plant extract treatment group increased by 6.62%, and the strain and plant extract combination treatment group increased by 3.63%; on the 12th day, compared with CK, the strain WD06 treatment group increased by 9.02%, the plant extract treatment group increased by 4.69%, and the strain and plant extract combination treatment group increased by 4.55%.

[0047] The aboveground fresh and dry weight data showed that compared with CK, the aboveground fresh weight of the strain WD06 treatment group increased by 45.15%, and the plant extract treatment group increased by 54.64% ( P <0.05), the strain and plant extract combination treatment group increased by 34.44%; in terms of underground fresh weight, compared with CK, the strain WD06 treatment group increased by 45.37%, the plant extract treatment group increased by 31.89%, and the strain and plant extract combination treatment group increased by 10.70%; in terms of aboveground dry weight, compared with CK, the strain WD06 treatment group increased by 18.65%, the plant extract treatment group increased by 28.07%, and the strain and plant extract combination treatment group increased by 12.90%; in terms of underground dry weight, compared with CK, the strain WD06 treatment group increased by 4.63%, the plant extract treatment group increased by 4.50%, and the strain and plant extract combination treatment group increased by 12.02%. Plant extracts increased soil nutrient content, promoting wheat plant growth. Under saline-alkali stress, elevated soil osmotic pressure affects plant water absorption. Strain WD06 mitigated the effects of this stressful environment and boosted wheat's water absorption capacity. The combined effects of the strain and plant extracts promoted wheat root growth, resulting in higher organic matter content and greater underground dry weight than treatments with either strain alone. The combined application of Enterococcus lactis WD06 and plant extracts enhances wheat root growth and salt tolerance, demonstrating significant practical application value.

[0048] Example 4, Effect on Cucumber Growth 3.4.3 Effects of strain WD06 combined with plant extracts on cucumber growth under salt-free conditions like Figure 6As shown, the plant height data showed that on the 4th day, compared with CK, the strain WD06 treatment group increased by 3.81%; the strain and plant extract treatment group increased by 3.11%; on the 8th day, compared with CK, the strain WD06 treatment group increased by 3.36%, and the strain and plant extract combination treatment group increased by 3.36%; on the 12th day, compared with CK, the strain WD06 treatment group increased by 8.85%, the plant extract treatment group increased by 0.52%, and the strain and plant extract combination treatment group increased by 4.37%; on the 16th day, compared with CK, the strain WD06 treatment group increased by 5.46%, and the strain and plant extract combination treatment group increased by 0.41%; on the 20th day, compared with CK, the strain WD06 treatment group increased by 2.09%, and the strain and plant extract combination treatment group increased by 1.22%.

[0049] Data on aboveground fresh and dry weight showed that the WD06-treated group increased aboveground fresh weight by 17.16% compared to the CK; belowground fresh weight increased by 27.56% in the WD06-treated group and by 37.48% in the WD06-treated group and the combined plant extract treatment group. Aboveground dry weight increased by 21.45% in the WD06-treated group and by 0.85% in the combined plant extract treatment group compared to the CK; belowground dry weight increased by 11.40% in the WD06-treated group and by 20.59% in the combined plant extract treatment group compared to the CK. Leaf area data showed that the WD06-treated group increased by 13.28% and the combined plant extract treatment group increased by 14.15% compared to the CK. Under salt-free conditions, the combined plant extract treatment group exhibited significant advantages in both belowground fresh and dry weight and leaf area.

[0050] 3.4.4 Effects of strain WD06 combined with plant extracts on cucumber growth under saline conditions like Figure 7As shown, the plant height data showed that on the 4th day, compared with CK, the strain WD06 treatment group increased by 2.52%; the plant extract treatment group increased by 6.58%, and the strain and plant extract treatment group increased by 5.81%; on the 8th day, compared with CK, the strain WD06 treatment group increased by 1.38%, the plant extract treatment group increased by 3.69%, and the strain and plant extract combination treatment group increased by 4.61%; on the 12th day, compared with CK, the strain WD06 treatment group increased by 8.95%, the plant extract treatment group increased by 9.47%, and the strain and plant extract combination treatment group increased by 10.09%; on the 16th day, compared with CK, the strain WD06 treatment group increased by 4.44%, the plant extract treatment group increased by 2.02%, and the strain and plant extract combination treatment group increased by 7.26%; on the 20th day, compared with CK, the plant extract treatment group increased by 0.55%, and the strain and plant extract combination treatment group increased by 4.75%.

[0051] Data on aboveground fresh and dry weight showed that, compared with the CK, the WD06-treated group increased aboveground fresh weight by 4.69%, the plant extract-treated group increased by 5.23%, and the combined strain and plant extract-treated group increased by 11.33%. Compared with the CK, the WD06-treated group increased belowground fresh weight by 13.20%, and the combined strain and plant extract-treated group increased by 28.60%. Compared with the CK, the WD06-treated group increased aboveground dry weight by 1.42%, and the combined strain and plant extract-treated group increased by 5.03%. Compared with the CK, the WD06-treated group increased by 3.03%, and the combined strain and plant extract-treated group increased by 18.18%. Data on plant leaf area showed that, compared with the CK, the WD06-treated group increased by 39.36%, the plant extract-treated group increased by 7.76%, and the combined strain and plant extract-treated group increased by 44.49%. Based on the above data, the results showed that the combination of strains and plant extracts had a significantly better growth-promoting effect on cucumber under salt stress than the single strain WD06 combination and the single plant extract combination.

[0052] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A strain of Enterococcus lactis ( Enterococcus lactis ) WD06, characterized in that, It was deposited in the General Microbiology Center of China Culture Collection Administration on March 6, 2025, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.33727.

2. The Enterococcus lactis according to claim 1 ( Enterococcus lactis ) Application of WD06 in promoting plant growth.

3. The use according to claim 3, characterized in that The crops are cucumbers or wheat.

4. The Enterococcus lactis according to claim 1 ( Enterococcus lactis ) Application of WD06 in promoting plant growth under salt or saline-alkali stress environment.

5. A method for preparing a bacterial suspension, characterized in that: The Enterococcus lactis according to claim 1 ( Enterococcus lactis WD06 strain was inoculated into MRS medium to obtain seed solution, which was then inoculated into new MRS medium at a 2% inoculum to obtain fermentation solution, which was diluted to prepare bacterial suspension for later use.

6. The method for preparing the bacterial suspension according to claim 5, wherein MRS medium was as follows: glucose 20 g, peptone 10 g, yeast extract 5 g, beef extract 10 g, diammonium hydrogen citrate 2 g, Tween 80 1 ml, sodium acetate 5 g, dipotassium hydrogen phosphate 2 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, and deionized water 1000 mL.

7. The Enterococcus lactis according to claim 1 ( Enterococcus lactis ) Combined application of WD06 and plant extracts.

8. The combined use according to claim 7, characterized in that: The plant extract is a soil microorganism awakening agent.

9. A soil conditioner composition, characterized in that Containing the Enterococcus lactis according to claim 1 ( Enterococcus lactis ) WD06 and plant extract.

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