Bacillus atrophae DG-19 and application thereof in lily yellowing and / or continuous cropping obstacle

Abstract

This invention provides a strain of Bacillus atrophicus DG-19 and its application in lily etiolation and / or continuous cropping obstacles, belonging to the field of functional bacterial strain technology. This invention provides a strain of Bacillus atrophicus DG-19 and a bacterial agent containing Bacillus atrophicus DG-19. It mainly works by increasing chlorophyll content, jasmonic acid content, and antioxidant properties; promoting the recycling of nitrogen and phosphorus; improving potassium absorption; degrading autotoxic substances; increasing the abundance of beneficial bacteria and fungi; and reducing the abundance of pathogenic bacteria and fungi. This effectively alters the physiological and biochemical state of lilies and the rhizosphere microbial community, alleviating lily etiolation and reducing continuous cropping obstacles.

Description

Technical Field

[0001] This invention belongs to the field of functional strain technology, specifically relating to a strain of Bacillus atrophus DG-19 and its application in lily etiolation and / or continuous cropping obstacles. Background Technology

[0002] Lanzhou lily (Lilium davidii var. unicolor salisb.) is a variety of Lilium davidii belonging to the genus Lilium in the family Liliaceae. It is a perennial bulbous herbaceous plant. Lanzhou lily mainly reproduces asexually. When harvesting commercial lilies, small mother seeds attached to the underground stems are picked for propagation. This process generally takes 3 years. Commercial lilies are harvested 3 years after transplanting the mother seeds. Continuous cropping leads to varietal degeneration, severe viral, bacterial, and fungal diseases, soil nutrient imbalance, and yellowing of the entire plant shortly after emergence in mid-May each year. Growth is weakened, photosynthetic efficiency is significantly reduced, and bulb yield and quality decline, seriously hindering the sustainable development of the planting industry.

[0003] The causes of continuous cropping obstacles in lilies are complex, mainly including the following: soil nutrient imbalance, as lilies have high requirements for certain elements, and long-term continuous cropping leads to deficiencies in specific nutrients; deterioration of soil physical and chemical properties, such as decreased pH, reduced organic matter content, soil compaction, and poor aeration; rampant soil-borne diseases and pests, as continuous cropping causes a large proliferation of pathogenic microorganisms and pests in the soil, such as lily blight and lily rhizoctonia; accumulation of plant allelopathic substances, as lily roots secrete autotoxic substances that inhibit their own growth, ultimately leading to a decline in yield and quality. Currently, crop rotation can balance soil nutrients, improve soil physical and chemical properties, and reduce diseases and pests, but it is limited by land resources and the number of rotation years; rational fertilization, through increasing the application of organic fertilizer and soil testing-based fertilizer application, can improve soil fertility; soil disinfection, using methods such as dazomet fumigation, can effectively kill diseases, pests, weeds, and underground pests in the soil. Although some results have been achieved, there are still shortcomings. For example, the long crop rotation cycle is difficult for producers to accept; chemical soil disinfection causes environmental pollution, and long-term use can easily lead to drug resistance in pathogens. Summary of the Invention

[0004] This invention provides a strain of Bacillus atrophus DG-19 and its application in lily yellowing and / or continuous cropping obstacles. The strain DG-19 can alleviate lily yellowing and / or reduce lily continuous cropping obstacles by foliar spraying or seed soaking.

[0005] This invention provides a strain of Bacillus atrophaeus DG-19, which has been biodeposited with accession number CGMCC No. 28907.

[0006] The present invention provides a biocontrol agent containing the above-mentioned Bacillus atrophicus DG-19 cells.

[0007] In a preferred embodiment of the present invention, the effective viable concentration of Bacillus atrophus DG-19 in the biocontrol agent is 2 × (10⁻⁶)⁻¹. 8 ~10 9 ) CFU / mL or 2×(10 8 ~10 9 )CFU / g.

[0008] The present invention also provides the application of the above-mentioned Bacillus atrophus DG-19 or the above-mentioned biocontrol agent in alleviating lily yellowing and / or overcoming lily continuous cropping obstacles.

[0009] In a preferred embodiment of the present invention, the process of alleviating lily yellowing and / or overcoming lily continuous cropping obstacles includes at least one of the following:

[0010] (1) Significantly increases chlorophyll content and decreases flavonoid content;

[0011] (2) Promotes lily growth and enhances resistance to pathogenic fungi, bacteria and viruses;

[0012] (3) Increase the content of endogenous jasmonic acid in lilies;

[0013] (4) Improve the degradation capacity of soil organic matter and aromatic autotoxic substances, promote the recycling of nitrogen and phosphorus, and promote the activation and absorption of potassium.

[0014] (5) Increase the abundance of probiotics and fungi;

[0015] (6) Reduce the abundance of pathogenic bacteria and fungi.

[0016] The present invention also provides a method for cultivating lilies, which includes soaking lily seedlings in a diluted solution of the above-mentioned biocontrol agent before sowing.

[0017] The present invention also provides a method for cultivating lilies, which includes soaking commercial lily bulbs in a diluted solution of the above-mentioned biocontrol agent and then transplanting them.

[0018] In a preferred embodiment of the present invention, the effective viable concentration of Bacillus atrophus DG-19 in the diluted solution of the biocontrol agent is 5 × (10⁻⁶)⁻¹. 6 ~10 7 CFU / mL; the soaking time is 30-60 min.

[0019] The present invention also provides a method for cultivating lilies, including spraying the lily leaves with the above-mentioned biocontrol agent.

[0020] In a preferred embodiment of the present invention, the spraying is carried out 3 to 4 times, and the detection time between two consecutive sprayings is 4 weeks.

[0021] Based on the viable count of the aforementioned Bacillus atrophus DG-19, the application rate for each spraying is 4 × (10⁻⁶)⁻¹. 11 ~10 12 )CFU / mu.

[0022] Beneficial effects: This invention provides a strain of Bacillus atrophus, DG-19. Examples have confirmed that strain DG-19 can significantly increase chlorophyll content, decrease flavonoid content, and alleviate lily yellowing; promote lily growth, enhance resistance to pathogenic fungi, bacteria, and viruses, and reduce continuous cropping obstacles in lilies; increase the content of endogenous jasmonic acid in lilies, improving resistance to oxidative stress; enhance the degradation capacity of soil organic matter and aromatic autotoxins, promote nitrogen and phosphorus recycling, and promote potassium activation and absorption; increase the abundance of beneficial bacteria and fungi, especially Bacillus and Chaetomium; and decrease the abundance of pathogenic bacteria and fungi, especially pathogenic fungi such as Cladosporium, Alternaria, Globosa, Fusarium, Alternaria nepalensis, Fusarium, Unclassified Caudataaceae, and Diplostomum tonghuaense.

[0023] This invention also provides a biocontrol agent with the Bacillus atrophus DG-19 bacterial cells as the active ingredient. The biocontrol agent can be used by soaking seeds, followed by normal sowing or transplanting; it can also be applied as a foliar spray after sowing or transplanting. This solves the problems of current solid root application microbial products on the market being affected by environmental factors, soil type, and indigenous microorganisms in the soil, as well as the difficulty of later operations due to the use of mulch film in lily planting. Examples have confirmed that the biocontrol agent can alleviate lily yellowing and continuous cropping obstacles.

[0024] Biological Preservation Information

[0025] Bacillus atrophaeus DG-19 was deposited on November 8, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 28907. Attached Figure Description

[0026] Figure 1 An evolutionary branching diagram for fungal LEfSe analysis;

[0027] Figure 2 An evolutionary branching diagram for the bacterial LEfSe analysis. Detailed Implementation

[0028] This invention provides a strain of Bacillus atrophaeus DG-19, which has been biodeposited with accession number CGMCC No. 28907.

[0029] The *Bacillus atrophus* DG-19 of this invention was isolated from *Rheum palmatum*, a medicinal plant grown in the cold and humid region of Minxian County, Dingxi City, Gansu Province, China. After isolation, screening, and purification, its colony characteristics on LB solid medium are: black, opaque, wrinkled surface, irregular edges, and cloud-like diffusion, and its 16S rDNA sequence is shown in SEQ ID No. 1.

[0030]

[0031] Verification by embodiments of the present invention shows that the *Bacillus atrophicus* DG-19 possesses phosphorus solubilization, nitrogen fixation, citrate utilization, and nitrification capabilities, with a nitrification power of 0.838 ± 0.01%. The *Bacillus atrophicus* DG-19 also exhibits ACC deaminase activity, peroxidase activity, cellulase activity, and laccase activity. Furthermore, the *Bacillus atrophicus* DG-19 demonstrates a strong ability to decompose glucose and produce acid, a strong ammonification capacity, and the ability to degrade autotoxic substances such as ferulic acid and anthraquinones. Antagonistic experiments revealed that the *Bacillus atrophicus* DG-19 exhibits strong antagonistic activity against pathogenic fungi such as *Fusarium solani* and *Alternaria alternata*.

[0032] The present invention provides a biocontrol agent containing the above-mentioned Bacillus atrophicus DG-19 cells.

[0033] The biocontrol agent of this invention uses Bacillus atrophicus DG-19 cells as the active ingredient. It can be obtained by collecting the cells after centrifugation of the fermentation broth of Bacillus atrophicus DG-19 and resuspending the cells in a solvent. Alternatively, the fermentation broth of Bacillus atrophicus DG-19 can be used directly as the biocontrol agent, ensuring that the effective viable concentration of Bacillus atrophicus DG-19 in the biocontrol agent is 2 × (10⁻⁶)⁻¹. 8 ~10 9 ) CFU / mL or 2×(10 8 ~10 9 CFU / g is sufficient.

[0034] In one embodiment of the present invention, a fermentation broth is used as the biocontrol agent. The preparation method of the fermentation broth includes inoculating *Bacillus atrophicus* DG-19 into a liquid fermentation medium for fermentation culture to obtain the fermentation broth of *Bacillus atrophicus* DG-19. The liquid fermentation medium of the present invention can be PDB medium, and the fermentation temperature can be 25–30°C, such as 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C. In one embodiment, the fermentation is carried out at 28°C. The fermentation time of the present invention can be 1–3 days, such as 1 day, 2 days, or 3 days. In one embodiment, the fermentation is carried out for 2 days. The fermentation process of the present invention is accompanied by shaking, and the shaking frequency can be 150–200 r / min, such as 150 r / min, 160 r / min, 170 r / min, 180 r / min, 190 r / min, or 200 r / min. In one embodiment, the shaking frequency is 180 r / min.

[0035] The present invention also provides the application of the above-mentioned Bacillus atrophus DG-19 or the above-mentioned biocontrol agent in alleviating lily yellowing and / or overcoming lily continuous cropping obstacles.

[0036] The biocontrol agent of this invention can increase chlorophyll and jasmonic acid content, improve antioxidant properties, promote the recycling of nitrogen and phosphorus, promote potassium absorption, degrade autotoxic substances, increase the abundance of beneficial bacteria and fungi, and reduce the abundance of pathogenic bacteria and fungi, thereby effectively changing the physiological and biochemical state of lilies and the rhizosphere microbial community, alleviating lily yellowing, and reducing lily continuous cropping obstacles. Based on the advantages of the biocontrol agent, it can be applied to lily yellowing and continuous cropping obstacles. Specifically, in lily yellowing and continuous cropping obstacles, the biocontrol agent can play at least one of the following roles:

[0037] (1) Significantly increases chlorophyll content, reduces flavonoid content, and alleviates lily yellowing;

[0038] (2) Promotes lily growth, enhances resistance to pathogenic fungi, bacteria and viruses, and reduces obstacles to continuous lily cultivation;

[0039] (3) Increase the content of endogenous jasmonic acid in lilies; improve resistance to oxidative stress;

[0040] (4) Improve the degradation capacity of soil organic matter and aromatic autotoxic substances, promote the recycling of nitrogen and phosphorus, and promote the activation and absorption of potassium.

[0041] (5) Increase the abundance of probiotics and fungi, especially the abundance of probiotics Bacillus and probiotics Chaetomium;

[0042] (6) Reduce the abundance of pathogenic bacteria and fungi, especially the abundance of pathogenic fungi such as Cladosporium, Alternaria, Globosa, Fusarium, Alternaria nepalensis, Fusarium, Unclassified Cactusaceae and Fusarium tonghuaense.

[0043] The present invention also provides a method for cultivating lilies, which includes soaking lily seedlings in a diluted solution of the above-mentioned biocontrol agent before sowing.

[0044] The lily cultivation method described in this invention can alleviate lily yellowing and the obstacle of continuous cropping of lilies. When soaking the small lily seeds, the effective viable bacteria concentration of the inoculant is 5 × (10⁻⁶)⁻¹. 6 ~10 7 The concentration of CFU / mL is used, and the soaking time is 30–60 min, or 30–45 min. This invention does not impose any special limitations on post-sowing management. For example, in this embodiment, the planting and management process of Lanzhou lilies is not particularly limited; it can be carried out according to conventional planting and management methods in the field, such as conventional ground mulching.

[0045] The present invention also provides a method for cultivating lilies, which includes soaking commercial lily bulbs in a diluted solution of the above-mentioned biocontrol agent and then transplanting them.

[0046] The lily cultivation method described in this invention can alleviate lily yellowing and the obstacle of continuous cropping of lilies. When soaking the commercial lily bulbs, the effective viable bacteria concentration of the inoculant is 5 × (10⁻⁶)⁻¹. 6 ~10 7 The concentration of CFU / mL is used, and the soaking time is 30–60 min, or 30–45 min. This invention does not impose any special limitations on post-sowing management. For example, in this embodiment, the planting and management process of Lanzhou lilies is not particularly limited; it can be carried out according to conventional planting and management methods in the field, such as conventional ground mulching.

[0047] The present invention also provides a method for cultivating lilies, including spraying the lily leaves with the above-mentioned biocontrol agent.

[0048] The lily described in this invention can be obtained by soaking seeds in the above-mentioned biocontrol agent and then sowing or transplanting them. Alternatively, it can be obtained by foliar spraying on lilies that have been directly sown or transplanted. The spraying is performed 3 to 4 times, with a 4-week interval between consecutive sprayings. Based on the viable count of Bacillus atrophus DG-19, the spraying amount for each application is 4 × (10⁻⁶)⁻¹⁹. 11 ~10 12 )CFU / mu.

[0049] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, describes a strain of Bacillus atrophus DG-19 provided by the present invention and its application in lily etiolation and / or continuous cropping obstacles, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0050] Unless otherwise specified, the test materials used in the embodiments of this invention are all conventional commercially available products in the art.

[0051] In this embodiment of the invention, the small female lily seeds were harvested from the lily plants during the harvesting of commercial lilies. The experiment was conducted in 2024 in Mapo Town, Yuzhong County, Gansu Province, using mountain chestnut-calcareous soil with a pH of 7.3. Base fertilizer consisted of 1 ton / mu of organic fertilizer (organic matter ≥40%, N+P2O5+K2O ≥4%) and 20 kg / mu of balanced compound fertilizer (17-17-17).

[0052] All reagents and culture media used in the experiment were chemically pure. Kits for detecting the activities of cellulase, laccase, xylanase, peroxidase, ACC deaminase, glutamine synthase, glutamate synthase, nitrite reductase, nitrate reductase, and phenylalanine ammonia-lyase, as well as kits for detecting the content of ABTS, DPPH, ferrophosphate, malondialdehyde, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugars, chlorophyll, flavonoids, nitrite nitrogen, and nitrate nitrogen, were purchased from Beijing Box Biotechnology Co., Ltd. The plant total RNA extraction kit, reverse transcription kit, and SYBR Green quantitative PCR kit were purchased from Tiangen Biotech Co., Ltd.

[0053] The composition of the culture medium used in the embodiments of the present invention is as follows:

[0054] PDB medium: 200 g / L potato and 20 g / L glucose, natural pH.

[0055] LB medium: tryptic peptone 10 g / L, yeast extract 5 g / L and NaCl 10 g / L, pH adjusted to 7.0 ± 0.2.

[0056] Ashby medium: dipotassium hydrogen phosphate 0.2 g / L, magnesium sulfate heptahydrate 0.2 g / L, calcium sulfate dihydrate 0.1 g / L, sodium chloride 0.2 g / L, calcium carbonate 5.0 g / L, mannitol 10.0 g / L, agar 18.0 g / L, pH 6.8-7.0.

[0057] Modified Stephenson medium: ammonium sulfate 2 g / L, manganese sulfate 0.01 g / L, sodium dihydrogen phosphate 0.25 g / L, magnesium sulfate 0.03 g / L, calcium carbonate 0.5 g / L, dipotassium hydrogen phosphate 0.75 g / L, pH adjusted to 8.2.

[0058] NBRIP solid medium: glucose 10 g / L, calcium phosphate 5 g / L, magnesium chloride 5 g / L, magnesium sulfate heptahydrate 0.25 g / L, potassium chloride 0.2 g / L, ammonium sulfate 0.1 g / L, agar 15 g / L, pH 7.0 ± 0.2.

[0059] Simon's citrate agar medium: sodium chloride 5.0 g / L, magnesium sulfate 0.2 g / L, ammonium dihydrogen phosphate 1.0 g / L, dipotassium hydrogen phosphate 1.0 g / L, sodium citrate 5.0 g / L, agar 20 g / L, 0.2% bromothymol blue solution 40 mL / L, adjust pH to 6.8 ± 0.2.

[0060] Glucose-peptone aqueous medium: glucose 0.5 g / L, peptone 0.5 g / L, dipotassium hydrogen phosphate 0.2 g / L, adjust the pH to 7.2-7.4.

[0061] Peptone amination medium: 5 g / L peptone, 0.5 g / L dipotassium hydrogen phosphate, 0.25 g / L sodium chloride, 0.5 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate, adjusted to pH 7.2.

[0062] DF medium: potassium dihydrogen phosphate 4.0 g / L, disodium hydrogen phosphate 6.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, ferrous sulfate heptahydrate 0.1 g / L, calcium chloride dihydrate 0.02 g / L, ammonium sulfate 2.0 g / L, boric acid 0.3 g / L, copper sulfate pentahydrate 0.04 g / L, manganese sulfate monohydrate 0.04 g / L, zinc sulfate heptahydrate 0.12 g / L, sodium molybdate dihydrate 0.025 g / L, and glucose 2.0 g / L.

[0063] ADF medium: potassium dihydrogen phosphate 4.0 g / L, disodium hydrogen phosphate 6.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, ferrous sulfate heptahydrate 0.1 g / L, calcium chloride dihydrate 0.02 g / L, boric acid 0.3 g / L, copper sulfate pentahydrate 0.04 g / L, manganese sulfate monohydrate 0.04 g / L, zinc sulfate heptahydrate 0.12 g / L, sodium molybdate dihydrate 0.025 g / L, glucose 2.0 g / L, and ACC (1-aminocyclopropane-1-carboxylic acid) 3.0 mM.

[0064] M9 medium: disodium hydrogen sulfate heptahydrate 12.8 g / L, potassium dihydrogen phosphate 3.0 g / L, sodium chloride 0.5 g / L, ammonium chloride 1 g / L, magnesium sulfate heptahydrate 0.492 g / L, calcium chloride hexahydrate 0.022 g / L and glucose 4.0 g / L.

[0065] Example 1

[0066] The isolation and identification of Bacillus atrophus DG-19 were performed as follows:

[0067] Bacillus atrophus DG-19 was isolated from rhubarb, a medicinal plant that grows in the cold and humid region of Minxian County, Dingxi City, Gansu Province, China.

[0068] The colony characteristics of the strain are as follows: The isolated strain was inoculated into LB solid medium and cultured at 30°C for 2 days. The colony morphology of the strain was: black, opaque, wrinkled on the surface, with irregular edges and a cloud-like diffusion.

[0069] Genomic DNA was extracted from the strain and amplified. Its 16S rDNA sequence was determined as shown in SEQ ID NO:1.

[0070] According to biocloud comparison, the strain has 99.25% similarity to the standard strain Bacillus atrophaeus strain JCM 9070. Based on morphology and function, it was confirmed to be Bacillus atrophaeus and named Bacillus atrophaeus DG-19. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 28907.

[0071] Example 2

[0072] The steps for functional testing of Bacillus atrophus DG-19 are as follows:

[0073] 1) Determination of phosphorus solubilization characteristics: An appropriate amount of Bacillus atrophicus DG-19 strain was inoculated into LB medium and cultured at 28℃ and 180 r / min for 2 days. 10 μL of Bacillus atrophicus DG-19 bacterial culture was inoculated onto NBRIP solid medium and incubated at 30℃. The presence and size of phosphorus-solubilizing zones on the medium were observed periodically over 7 days. The phosphorus solubilization capacity of the strain was determined based on the size of the phosphorus-solubilizing zones.

[0074] The results showed that Bacillus atrophus DG-19 has phosphorus solubilizing ability.

[0075] 2) Determination of nitrogen fixation characteristics: An appropriate amount of Bacillus atrophus DG-19 strain was inoculated into LB medium and cultured at 30℃ and 180r / min for 2 days with shaking. An equal amount of 30μL of bacterial solution was inoculated into sterile Ashby medium with 0.2% Congo red chromogenic agent and cultured at 30℃ for 3-5 days. The growth of colonies and color changes on the medium were observed for qualitative detection.

[0076] The results showed that Bacillus atrophus DG-19 has nitrogen-fixing ability.

[0077] 3) Citrate utilization: Take an appropriate amount of Bacillus atrophus DG-19 and inoculate it into the center of Simon's citrate agar medium using an inoculation loop. Repeat the inoculation three times to ensure even colony distribution. Invert the inoculated plate in a 30°C incubator and incubate for 24–48 hours. Observe whether a color change occurs around the colonies on the plate; a blue color indicates a positive result.

[0078] The results showed that Bacillus atrophus DG-19 can utilize citrate.

[0079] 4) Nitrification power determination: Bacillus atrophus DG-19 was inoculated into LB medium and cultured at 30℃ with shaking at 180 r / min for 2 days. The bacterial cells were collected by centrifugation and resuspended in sterile water to OD. 600 The concentration was set to 1.0, and then inoculated with sterilized modified Stephenson's medium. The mixture was incubated at 30°C with shaking at 180 rpm for 2 days, and the OD was measured. 600 The nitrification power was determined according to the methods described in the nitrite nitrogen and nitrate nitrogen kits. The nitrification power was calculated using formula (I), and the result was divided by the turbidity of the corresponding bacterial solution to normalize it to the nitrification power of the turbidity bacterial solution. Each strain was repeated 3 times, and the average value was calculated.

[0080]

[0081] The results showed that the nitrification power of Bacillus atrophus DG-19 was 0.838±0.01%.

[0082] 5) Determination of relative content of ferrophile: Bacillus atrophus DG-19 was inoculated into LB medium and centrifuged to obtain ferrophile fermentation supernatant (SCS). SCS and cerazurin S (CAS) solutions were mixed in a 1:1 volume ratio and incubated in a 37°C water bath in the dark for 0.5 h before measuring the OD of the mixture. 630 The relative content of ferrophile in the strain was obtained by formula (II).

[0083] The relative content of the ferrophile in the strain = (Ar-As) / Ar×100%, formula (II);

[0084] Where Ar is the OD of the reference. 630 (A mixture of blank control and CAS); As is the OD of the sample. 630 (A mixture of strains SCS and CAS). The calculated result is divided by the turbidity of the corresponding bacterial solution and standardized to the relative content of ferrophile in the bacterial solution per unit turbidity.

[0085] The results showed that the relative content of siderophile in Bacillus atrophus DG-19 was 58.5%.

[0086] 6) Determination of ACC deaminase activity of the strain: Bacillus atrophus DG-19 was cultured in LB medium at 30℃ and 180 r / min for 1 day with shaking. Then it was centrifuged at 4℃ and 8000 r / min for 10 min, the supernatant was discarded, the bacterial cells were washed twice with ADF medium without (NH4)2SO4, centrifuged again, and the bacterial cells were resuspended in ADF medium and cultured at 30℃ and 180 r / min for 1 day. After centrifugation at 8000 rpm for 10 min at 4 °C, the supernatant was discarded and the bacterial cells were collected. The cells were washed twice with 0.1 mol / L Tris-HCl buffer (pH 7.6), centrifuged again, and resuspended in 0.2 mL of Tris-HCl buffer (pH 8.5). A small amount of toluene was added, and the cells were sonicated to disrupt the cell structure. 20 μL of 0.5 mol / L LaCz was added, and the mixture was incubated at 30 °C for 15 min. Then, 0.3 mL of dinitrophenylhydrazine was added, the container was capped, and the mixture was incubated at 30 °C for 0.5 h. The reaction was then terminated by adding 2 mL of 2 mol / L NaOH, and the absorbance at 540 nm was measured. The enzyme activity of ACC deaminase was the amount of α-butanone produced per minute (μmol / min).

[0087] The results showed that the ACC deaminase activity of Bacillus atrophus DG-19 was 0.360 μmol / min.

[0088] 7) Determination of enzyme activity of the strain: Bacillus atrophus DG-19 was inoculated into 50 mL of sterile LB medium and cultured at 30 °C with shaking at 180 r / min for 2 days. After filtration, the bacterial cells were collected and resuspended in sterile water to a turbidity OD value.600 The bacterial culture to be tested was obtained by setting the bacterial culture to enzyme extract at a volume ratio of 1:10. Cells were disrupted by sonication in an ice bath, followed by centrifugation at 8000 rpm for 10 min at 4°C. The supernatant was placed on ice for testing. The activity of peroxidase, cellulase, neutral xylanase, and laccase was determined using spectrophotometry according to the instructions of the kits, with three replicates. The results were divided by the turbidity of the corresponding bacterial culture and standardized to the enzyme activity per unit turbidity.

[0089] The results showed that the peroxidase activity of Bacillus atrophus DG-19 was 1.217±0.032 U / mL, the cellulase activity was 2.59±0.018 U / mL, and the laccase activity was 1.004±0.226 U / mL.

[0090] 8) Methyl red test: Use a sterile inoculation loop to inoculate Bacillus atrophus DG-19 into sterile glucose peptone water medium, with no medium inoculated as a control. Incubate at 30°C for 2-5 days, add 5 drops of methyl red reagent to the medium and observe the color change. A red color indicates a positive result.

[0091] The results showed that Bacillus atrophus DG-19 has a strong ability to decompose glucose and produce acid.

[0092] 9) Ammoniation test: Inoculate Bacillus atrophicus DG-19 fermentation broth into peptone ammoniation medium, and inoculate 6 dilutions (10⁻⁶). -2 10 -3 10 -4 10 -5 10 -6 10 -7 Three tubes were inoculated for each dilution, and one tube of culture medium was inoculated with sterile water as a control. The cultures were incubated at 30°C. The turbidity of the culture medium was checked on days 3 and 5. On day 7, five drops of culture medium were placed on a white porcelain colorimetric plate, two drops of Nessler's reagent were added, and the presence of a brownish color was checked to determine if ammonia had been produced.

[0093] The results showed that Bacillus atrophus DG-19 had a strong ammonification ability.

[0094] 10) Catalase test: Pick a colony of Bacillus atrophus DG-19, place it on a clean glass slide, and add an appropriate amount of 3% hydrogen peroxide solution. A positive result is indicated by the generation of a large number of bubbles within 1 minute.

[0095] The results showed that Bacillus atrophus DG-19 had strong catalase activity.

[0096] 11) Degradation of autotoxic substances: Sugar-free M9 medium with added acid-base indicators was used as the basal medium, with 50 mg / L ferulic acid or anthraquinone added as the sole carbon source. *Bacillus atrophus* DG-19 was inoculated into the above medium. The inoculated medium was cultured at 30℃ and 180 r / min for 3–5 days, and the color change of the medium was observed and the OD was measured. 600 value.

[0097] The results showed that Bacillus atrophus DG-19 can degrade ferulic acid and anthraquinones.

[0098] 12) Determination of antagonistic characteristics of strains: The antagonistic activity of Bacillus atrophicus DG-19 against the pathogenic fungi Fusarium solani and Alternaria alternifolia was determined using the plate confrontation culture method. Pathogen fungal discs were inoculated in the center of PDA medium, and an equal amount of the co-tested strain was inoculated at a distance of 2 cm from the pathogen using a cross-cross method. The medium was incubated at 30℃ for 5-7 days, and the colony growth and inhibition zone size were observed. The inhibition rate was calculated according to formula (III).

[0099] Inhibition rate = (inhibition zone radius - antagonistic bacteria radius) / antagonistic bacteria radius × 100%, Equation (III).

[0100] The results showed that Bacillus atrophus DG-19 could inhibit Fusarium solani and Alternaria solanacearum, and the inhibition rates were relatively strong at 31.96±0.04% and 35.18±0.09%, respectively.

[0101] Example 3

[0102] The efficacy of Bacillus atrophus DG-19 in reducing continuous cropping obstacles in lilies was verified through the following steps:

[0103] The experimental group is set up as follows:

[0104] Treatment group (T): Bacillus atrophicus DG-19 fermentation broth cultured in PDB medium at 28℃ and 180r / min for 2 days was diluted 40 times with water to achieve an effective viable cell count of 5×(10⁻⁶). 6 ~10 7 CFU / mL. Apply 40L per acre.

[0105] Positive control group (CK): PDB medium without bacteria was diluted by the same factor as the treatment group and sprayed at 40L per acre.

[0106] The experiment employed a single-factor, completely randomized design, using plots that had previously yielded commercial lilies (i.e., planted for 3 years) for continued commercial seed breeding. Two treatments, T and CK, were implemented, with each treatment replicated three times. The plot area was 30 m². 2(4m×7.5m). Small mother seeds were sown in late April 2024. Foliar spraying was applied 4 times from May to September, once every 4 weeks. The volume of spraying was the same for different plots.

[0107] In late April 2024, before transplanting lily seedlings, the corresponding seedlings were soaked in treatment group T and positive control group CK for 30 minutes, respectively. After draining the surface moisture, they were transplanted. After the seedlings emerged uniformly, the above treatment was applied to the leaves from May to September, once every 4 weeks for a total of 4 times. The spray volume was equal for different plots. Field management was carried out according to conventional measures.

[0108] In late August, 3 days after the fourth treatment, 100 lily seedlings were randomly selected from each plot, leaves were removed, and rhizosphere soil of the corresponding lilies was collected, mixed separately, and brought back to the laboratory for later use.

[0109] At the end of September, lilies were harvested, and the number and length of roots were counted. The roots were removed, the surface soil of the lilies was washed off, and they were dried. The weight of 100 plants was then measured. The weight of 100 plants and growth indicators after treatment with Bacillus atrophus DG-19 are shown in Table 1 (student T test analysis).

[0110] Table 1. Average weight per 100 lilies, average number of roots, and root length (x±se, n=3) under different treatments

[0111]

[0112] In this invention, in the provided table, different lowercase letters indicate significant differences (P < 0.05).

[0113] Compared with the positive control group CK, the T group treatment described in this invention resulted in 1.43 times the weight of 100 lilies, 1.58 times the average number of leaves, 1.37 times the average number of roots, and 1.24 times the average root length, indicating that the T treatment group described in this invention effectively promoted the growth of lilies and alleviated the obstacle of continuous cropping of lilies.

[0114] Example 4

[0115] The effects of Bacillus atrophus DG-19 on endogenous hormone levels in lily leaves were investigated using the following steps:

[0116] The test setup and test plan are the same as in Example 3.

[0117] (1) Detection of endogenous hormones: The endogenous hormone content of lily seedlings was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS) (see Table 2). Leaves from different samples were sent to a third party for detection. The indicators were: 109 substances in 9 major categories of plant hormones and intermediate metabolites, including auxin, cytokinins (CKs), abscisic acid (ABA), jasmonates (JAs), salicylic acid (SA), gibberellins (GAs), 1-aminocyclopropanecarboxylic acid (ACC), strigolactones (SLs), and melatonin (MLT). The most important substances that effectively distinguished the differences between the T and CK groups (VIP>1) were screened using OPLS-DA (a supervised pattern recognition multivariate statistical analysis). The FC value was used to compare the degree of difference between different substances in the two groups.

[0118] Table 2. Endogenous hormones in lily leaves (ng / g)

[0119] L-Tryptophan 831.643 2602.163 1.16 0.00 -1.65 Indole 875.955 2394.274 1.13 0.00 -1.45 dihydrozein nucleoside 0 0.361 1.16 0.00 -Inf Isoprene adenine nucleoside 0 0.141 1.16 0.00 -Inf Gibberellin 7 0.352 0.979 1.16 0.00 -1.48 Jasmonic acid 24.218 6.879 1.15 0.00 1.82 Jasmonic acid-valine 0.240 0.064 1.13 0.00 1.91 Methyl jasmonate 1.120 0.000 1.16 0.00 Inf salicylic acid 29.723 60.644 1.14 0.00 -1.03 Cinnamon acid 0.000 92.983 1.16 0.00 -Inf

[0120] In Table 2, Log2FC represents the logarithm of the difference factor to the base 2, -Inf represents negative infinity, and Inf represents positive infinity, and so on.

[0121] As shown in Table 2, among the 10 significantly different metabolites, 3 were significantly upregulated and 7 were significantly downregulated. The T-group treatment described in this invention significantly increased jasmonic acid content and decreased the content of auxin, cytokinin, gibberellin, and salicylic acid hormones, thus promoting lily bulb enlargement.

[0122] (2) Determination of physiological and biochemical indicators of leaves: The equivalent quantitative values ​​of ABTS scavenging capacity, DPPH scavenging capacity, total phenols, peroxidase, malondialdehyde, glutamine synthase, glutamate synthase, nitrite reductase, nitrate reductase, ammonium nitrogen, soluble sugar, chlorophyll, phenylalanine ammonia-lyase (PAL) activity, and flavonoids in lily leaves were determined by spectrophotometry according to the kit instructions. Each treatment was repeated three times, and the results showing significant differences are shown in Table 3.

[0123] Table 3. Detection of physiological and biochemical indicators of leaves (x±se, n=3)

[0124] Equivalent quantification of ABTS scavenging power (mmol / L) 2.355±0.032a 2.21±0.035b 0.092 Total phenols (mg / g) 6.687±0.076a 6.21±0.258b 0.107 Peroxidase (U / g) 1500±58.878a 1033.33±329.98b 0.538 Soluble sugars (mg / g) 15.54±0.129a 12.626±0.084b 0.300 Chlorophyll (mg / g) 2.537±0.015a 1.028±0.154b 1.303 PAL(U / g) 42.385±0.907a 30.362±4.442b 0.481 Flavonoids (mg / g) 1.445±0.251b 3.054±0.221a -1.080

[0125] As shown in Table 3, the indicators with an absolute value of log2FC greater than 1 are chlorophyll and flavonoids. Chlorophyll was significantly increased and flavonoids were significantly decreased, indicating that the treatment group T described in this invention significantly alleviated lily yellowing. Peroxidase activity, total phenols, and soluble sugar content were significantly increased, indicating that the treatment group T described in this invention significantly improved antioxidant capacity and promoted growth. The content of phenylalanine ammonia-lyase (PAL) was significantly increased, indicating improved disease resistance.

[0126] Example 5

[0127] The effects of Bacillus atrophus DG-19 on soil nutrients and enzyme activity in lilies were investigated using the following steps:

[0128] The test setup and test plan are the same as in Example 3.

[0129] The activities of sucrase, urease, β-glucosidase, xylanase, alkaline phosphatase, and catalase in soil were determined by spectrophotometry using a kit; the contents of ammonium nitrogen and nitrate nitrogen were also measured; alkaline available nitrogen was determined by alkaline hydrolysis-diffusion method; available potassium was determined by flame photometry after ammonium acetate dissolution and extraction; total iron was determined by tetraacid digestion-inductively coupled plasma atomic emission spectrometry; and available phosphorus was determined by molybdenum-antimony colorimetric method after sodium bicarbonate solution extraction. T-tests were used to analyze statistically significant indicators (P<0.05), as shown in Table 4. FC values ​​were used to compare the degree of difference between different substances in the two groups. Each treatment was repeated three times, and the results are shown in Table 4.

[0130] Table 4. Detection of soil physiological and biochemical indicators (x±se, n=3)

[0131]

[0132]

[0133] Table 4 shows that the index with an absolute value of log2FC greater than 1 is soil alkaline xylanase. After treatment group T described in this invention, the activities of β-glucosidase and alkaline xylanase significantly increased, indicating improved soil organic matter degradation capacity; urease activity and alkaline nitrogen content increased, indicating that treatment group T promoted nitrogen cycling; soil alkaline phosphatase activity increased, and the absolute value was large, and available phosphorus content increased, indicating that treatment group T promoted phosphorus cycling; available potassium and total phenolic acid content decreased, indicating that treatment group T promoted potassium absorption, degraded autotoxic substances, and contributed to bulb growth.

[0134] Example 5

[0135] The effects of Bacillus atrophus DG-19 on the diversity of rhizosphere microorganisms in lily were investigated using the following steps:

[0136] The test setup and test plan are the same as in Example 3.

[0137] Rhizosphere soil microbial diversity assessment: After sample collection, samples were kept cold with dry ice and sent to a third-party sequencing provider for 16S, ITS amplicon sequencing analysis. Small fragment libraries were constructed and sequenced using the Illumina NovaSeq sequencing platform and the paired-end sequencing method. Bioinformatics analysis: LEFSE biomarker analysis (see...) Figure 1 and Figure 2 ), species analysis of significant differences at the fungal level (see Table 5), bacterial function prediction analysis, etc. (see Tables 6, 7 and 8).

[0138] Depend on Figure 1 It is known that there are 15 types of characteristic fungi in the rhizosphere soil of treatment group T described in this invention. Four of them are annotated at the genus level: Penicillium, unclassified Helotiales, Brunneocylindrosporum, and unclassified Chaetomiaceae; and four of them are annotated at the species level: unclassified Penicillium, unclassified Helotiales, Brunneocylindrosporum nepalense, and unclassified Chaetomiaceae. The rhizosphere soil of the control group (CK) contained 19 types of characteristic fungi, with 5 genera annotated at the genus level: *Cladosporium*, *Alternaria*, *Plectosphaerella*, *Fusarium*, and *Mortierella*; and 5 species annotated at the species level: *Alternaria nepalensis*, *Plectosphaerella niemeijerae*, *Fusarium domesticum*, unclassified *Filobasidium*, and unclassified *Mortierella*. This indicates that the treatment group T described in this invention is mainly enriched with the probiotic *Chaetoceros*; the positive control group (CK) is mainly enriched with pathogens of the genera *Cladosporium*, *Alternaria*, *Plectosphaerella*, and *Fusarium*.

[0139] Depend on Figure 2It can be seen that there are 6 types of marker microorganisms in the rhizosphere soil of treatment group T described in this invention, with 1 marker at the genus level (unclassified Gemmatimonadaceae) and 1 marker at the species level (unclassified JG30 KF CM45).

[0140] Table 5 shows five fungi with significant differences in levels.

[0141]

[0142] Note: Log2FC: Logarithm of the difference factor to the base 2.

[0143] Compared with the positive control group CK, the treatment group T described in this invention significantly increased the abundance of the probiotic Unclassified Chaetomiaceae and significantly decreased the abundance of the pathogens Alternarianepalensis, Fusarium domesticum, Unclassified Ceratobasidiaceae, and [Bisifusarium] tonghuanum.

[0144] The indicators showing significant differences in bacterial functional abundance, based on PICRUSt2 annotations in the KEGG and COG databases, are as follows.

[0145] Table 6. Prediction of bacterial KEGG function

[0146] Cellular communityprokaryotes 1.782091 1.704088 0.040405 0.0646 Infectious diseases: Viral 0.053387 0.062372 0.042536 -0.2244 Membranetransport 3.693772 3.610384 0.048325 0.03294 Signal Transduction 2.371044 2.411013 0.048525 -0.0241

[0147] Compared with the positive control group CK, the treatment group T described in this invention significantly improved prokaryotic community immunity and membrane transport function; and reduced viral infection and signal transduction function. This indicates that the treatment group T described in this invention can reduce the occurrence of bacterial and viral diseases.

[0148] Table 7. Bacterial COG Function Prediction

[0149] Aminoacidtransportandmetabolism 10.3629 10.17137 0.045456 0.0269 Cytoskeleton 0.011688 0.013863 0.008009 -0.2462

[0150] Compared with the positive control group CK, the treatment group T described in this invention significantly improved amino acid transport and metabolism.

[0151] FAPROTAX uses published and validated literature on culturable microorganisms to perform functional annotation predictions on biogeochemical cycling processes (especially elemental cycles such as carbon, hydrogen, nitrogen, phosphorus, and sulfur) in environmental samples. The results are shown in Table 8.

[0152] Table 8 Prediction of the ecological function of bacterial FAPROTAX

[0153]

[0154]

[0155] Compared with the positive control group CK, the treatment group T described in this invention significantly improved cellulose decomposition, aromatic compound degradation, and fermentation functions. This indicates that the treatment group T described in this invention can degrade organic matter, degrade aromatic autotoxic substances, and activate mineral elements through fermentation acid production.

[0156] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Bacillus atrophus ( Bacillus atrophaeus DG-19, characterized in that, The Bacillus atrophus DG-19 has been bio-conserved, with accession number CGMCC No. 28907.

2. A biocontrol agent comprising Bacillus atrophus DG-19 cells as described in claim 1.

3. The biocontrol agent according to claim 2, characterized in that, The effective viable cell concentration of Bacillus atrophaeus DG-19 in the biocontrol agent is 2×(10 8 ~10 9 ) CFU / mL or 2×(10 8 ~10 9 ) CFU / g.

4. The use of Bacillus atrophus DG-19 as described in claim 1 or the biocontrol agent as described in claim 2 or 3 in alleviating lily yellowing and / or overcoming lily continuous cropping obstacles.

5. The application according to claim 4, characterized in that, The relief of lily yellowing and / or overcoming lily continuous cropping obstacles includes at least one of the following: (1) Significantly increases chlorophyll content and decreases flavonoid content; (2) Promotes lily growth and enhances resistance to pathogenic fungi, bacteria and viruses; (3) Increase the content of endogenous jasmonic acid substances in lilies; the jasmonic acid substances are jasmonic acid, jasmonic acid-valine and methyl jasmonic acid; (4) Improve the degradation capacity of soil organic matter and aromatic autotoxic substances, promote the recycling of nitrogen and phosphorus, and promote the activation and absorption of potassium; the aromatic autotoxic substance is ferulic acid; (5) Increase the abundance of probiotics and fungi; (6) Reduce the abundance of pathogenic bacteria and fungi.

6. A method for cultivating lilies, characterized in that, This includes soaking lily buds in a diluted solution of the biocontrol agent described in claim 2 or 3 before sowing.

7. A method for cultivating lilies, characterized in that, This includes soaking commercial lily bulbs in a diluted solution of the biocontrol agent described in claim 2 or 3 before transplanting.

8. The lily cultivation method according to claim 6 or 7, characterized in that, The effective viable concentration of Bacillus atrophus DG-19 in the diluted solution of the biocontrol agent is 5 × (10⁻⁶)⁻¹. 6 ~10 7 The concentration of CFU / mL was 30-60 min.

9. A method for cultivating lilies, characterized in that, This includes spraying the leaves of lilies with the biocontrol agent described in claim 2 or 3.

10. The lily cultivation method according to claim 9, characterized in that, The spraying is carried out 3 to 4 times, with an interval of 4 weeks between two consecutive sprayings; Based on the viable count of the aforementioned Bacillus atrophus DG-19, the application rate for each spraying is 4 × (10⁻⁶)⁻¹. 11 ~10 12 )CFU / mu.

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