Bacillus subtilis with algae-lysing effect, composite algae control agent and application of composite algae control agent

Abstract

The invention discloses bacillus subtilis with an algae-lysing effect, a composite algae control agent and application of the composite algae control agent, and belongs to the field of microbial pesticides. Wherein the bacillus subtilis is SJR-02, the strain is preserved in the CGMCC (China General Microbiological Culture Collection Center), and the preservation number of the strain is CGMCC No.32370. The composite algae control agent is obtained by mixing bacillus subtilis SJR-02 and humate, and the humate is one or more of sodium humate, potassium humate and mineral source potassium fulvate. The bacillus subtilis and the composite algae control agent provided by the invention can be used as inhibitors for inhibiting the growth of algae in a water body, are safe to aquatic organisms and aquatic plants in the water body and do not cause deterioration of water quality, so that the bacillus subtilis and the composite algae control agent are very suitable for inhibiting the algae in the aquatic water body, have stable prevention and treatment effects and have relatively good application values.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial pesticides, and in particular relates to a Bacillus subtilis with algae-dissolving effect, a composite algae-control agent and applications thereof. Background Art

[0002] Filamentous algae (mosses), including Spirogyra, Hydroneticula, Transectis, and Cladophora, grow rapidly under favorable temperatures and eutrophic water conditions, quickly covering the water surface. This can cause soil temperatures to drop in rice paddies, hindering tillering and leading to yield reductions. It can also impact rice-shrimp and rice-crab symbioses.

[0003] Currently, the mainstream products for controlling moss in shrimp-rice fields in southern China include moss cleaners, moss control fertilizers, Youcaile, and Tianbaijiu. The best-selling products on the market are mainly moss cleaners (such as Prochloraz and triphenyltin chloride) and peptide control fertilizers. These products have the following main problems: First, their effectiveness is unstable and they are generally sensitive to temperature and rainfall. If it rains soon after application, the control effect is too poor and reapplication is necessary. Second, their safety is worrying. For example, in Hubei's shrimp-rice fields, many "moss cleaners" containing herbicides are prevalent. Although they are effective, they can easily cause water quality deterioration, the death of aquatic plants, and ultimately the death of crayfish or even the extinction of crayfish.

[0004] Therefore, in order to solve the problem of moss in aquaculture water, in addition to the prevention and control effect, it is also necessary to comprehensively consider safety, durability and stability. Summary of the Invention

[0005] In order to solve the problems in the prior art, the present invention proposes a Bacillus subtilis with algae-dissolving effect, a composite algae-control agent and applications thereof.

[0006] According to the first aspect of the present invention, the present invention provides a Bacillus subtilis SJR-02 with algae-lytic effect. The strain has been deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC), the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is October 28, 2024, and the deposit number is CGMCC No. 32370.

[0007] According to the second aspect of the present invention, the present invention also provides an algae-lytic bacteria wettable powder, which is prepared by the following method:

[0008] 1) Fermenting the Bacillus subtilis SJR-02 according to claim 1, wherein the fermentation conditions are: tank pressure 0.2-0.3 MPa, temperature 25-30° C., dissolved oxygen ≥20%, stirring speed 200-240 rpm, and fermentation time 32-40 h; after fermentation, collecting the fermentation broth, adding soluble starch as a protective agent, stirring evenly, and spray drying in a spray dryer to obtain a mother powder;

[0009] 2) Mixing the collected mother powder with a carrier and an adjuvant to obtain SJR-02 wettable powder;

[0010] In the SJR-02 wettable powder, the content of Bacillus subtilis SJR-02 is 1×10 10 ~3×10 10 CFU / g.

[0011] Preferably, the carrier is one or more of kaolin, diatomaceous earth or calcium carbonate, and the mass percentage of the carrier in the wettable powder is 80%; the adjuvants are sodium butylnaphthalene sulfonate and calcium lignin sulfonate, and the mass percentages of sodium butylnaphthalene sulfonate and calcium lignin sulfonate in the wettable powder are both 5%.

[0012] During the fermentation process in step 1), the fermentation medium formula, by weight percentage, is: 0.8% fish peptone, 0.8% yeast powder, 1.5% rice flour, 3% flour, 0.35% magnesium sulfate, 0.3% light calcium carbonate, 0.25% industrial salt, and the balance is water. Preferably, the amount of soluble starch added in step 1) is 10% of the mass of the fermentation liquid.

[0013] According to a third aspect of the present invention, the present invention provides use of the Bacillus subtilis SJR-02 or the algae-lytic bacteria wettable powder as an inhibitor for inhibiting algae growth in water. The water may be natural water, wastewater, or aquaculture water. The algae are preferably Spirogyra, Hydrodiplophyta, Diplophyta, or Cladophora.

[0014] According to a fourth aspect of the present invention, the present invention provides a composite algae control agent, comprising the Bacillus subtilis SJR-02 according to claim 1 and humate, wherein the humate is one or more of sodium humate, potassium humate, and mineral-derived potassium humate.

[0015] According to a preferred embodiment of the present invention, the composite algae control agent further comprises kaolin and chelated trace elements, wherein the kaolin accounts for 5-20% by weight of the composite algae control agent, and the chelated trace elements account for 5-20% by weight of the composite algae control agent. The chelated trace elements are six elements chelated with citric acid, and the weight proportions of each element in the chelated trace elements are: Fe 0.5%, B 0.4%, Zn 0.5%, Mn 0.1%, Cu 1.0%, and Mo 0.01%.

[0016] According to the fifth aspect of the present invention, the present invention also provides the use of the composite algae control agent as an inhibitor in inhibiting the growth of algae in water bodies.

[0017] Further preferably, the water body is aquaculture water. The aquacultured organisms in the aquaculture water body are shrimp, crab, or fish. Preferably, the water body is a shrimp farm, shrimp pond, crab farm, crab pond, fish pond, or the like. The composite algae control agent of the present invention is relatively safe for aquatic products in the aquaculture water body, highly safe for aquatic plants fed thereto, and has excellent control effectiveness against algae such as Spirogyra, Hydrodiplophyta, Diplophyta, and Cladophora.

[0018] The Bacillus subtilis and composite algae control agent provided by the present invention can be used as inhibitors to inhibit the growth of algae in water bodies, and are safe for aquatic organisms and aquatic plants in the water bodies and will not cause water quality deterioration. Therefore, they are very suitable for inhibiting algae in aquaculture water bodies, have stable control effects, and have good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the colony morphology of Bacillus subtilis SJR-02;

[0020] Figure 2 This is a scanning electron micrograph of Bacillus subtilis SJR-02;

[0021] Figure 3 is the phylogenetic tree of Bacillus subtilis SJR-02 strain;

[0022] Figure 4 The growth of Spirogyra after 6 days of treatment with different algicidal bacteria;

[0023] Figure 5 The growth of Spirogyra in different groups after 7 days of treatment in Example 4;

[0024] Figure 6 The growth of Spirogyra after 5 days of treatment in different groups in Example 5;

[0025] Figure 7 The growth of Spirogyra in different groups after 15 days of treatment in Example 5;

[0026] Figure 8 The growth of moss in the control area and the area treated with compound algae control agent after treatment;

[0027] Figure 9 The growth rate of moss 1-2 days after application of the composite algae control agent of the present invention;

[0028] Figure 10 The growth of Spirogyra in shallow water test field before and 4 days after the application of pesticides (experimental temperature 22℃). DETAILED DESCRIPTION

[0029] The present invention will be further described and illustrated below in conjunction with specific embodiments. The embodiments are merely illustrative of the present disclosure and do not limit its scope. The technical features of the various embodiments of the present invention may be combined accordingly, provided that there is no conflict between them.

[0030] Example 1: Isolation and identification of Bacillus subtilis (SJR-02) strain

[0031] (1) Sample collection: The samples were collected from the silt of shrimp aquaculture ponds in Nantou Village, Xianxiang Town, Yinzhou District, Ningbo City, Zhejiang Province. The collected silt was packed in a collection bag, placed in an ice box and brought back to the laboratory for separation.

[0032] (2) Isolation method: 10.0 g of pond sediment was mixed with 90 ml of sterile water, and the mixture was placed in a water bath at 80°C for 10 min. The mixture was then shaken at 30°C and 200 rpm for 30 min. The Bacillus was isolated and purified on LB plates using the dilution coating method and the plate streak method.

[0033] The isolated Bacillus single colony was inoculated into liquid LB medium and cultured at 30°C and 200 r / min for 48 h to prepare a final concentration of 1×10 10 CFU / ml of bacterial solution. Then, perform a gradient dilution, aspirate the gradient dilution solution and spread it onto LB solid medium plates. Place the plate in an incubator at 30°C and incubate it upside down for 48 hours. Pick a single colony and streak it onto the LB solid medium plate to obtain a single colony. The obtained single colony is stored in 40% glycerol and stored in a -80°C ultra-low temperature freezer until use.

[0034] LB medium: peptone 10.0 g, yeast extract powder 5.0 g, NaCl 10.0 g, agar powder 15 g, then add distilled water to make up to 1000 mL; culture at 28°C in the dark for 1-2 days.

[0035] (3) Morphological characteristics of Bacillus subtilis (SJR-02): After culturing on LB medium at 28°C for 24 h, the colonies were medium-sized, milky white, nearly round, with irregular edges, rough and wrinkled. Figure 1 Gram staining is purple and positive; its cells are straight rod-shaped and measure (0.5-1.2 μm) x (1.0-4 μm). Figure 2 Shown is a scanning electron micrograph of Bacillus subtilis (SJR-02).

[0036] (4) Bacillus subtilis (SJR-02) was subjected to physiological and biochemical tests, and the results are shown in Table 1. The physiological and biochemical properties of SJR-02 were searched against the retrieval tables in Bergey's Manual of Bacterial Identification and Manual of Common Bacterial Systematic Identification, confirming that the physiological and biochemical properties of SJR-02 were consistent with those of Bacillus subtilis.

[0037] Table 1 - Physiological and biochemical identification results of Bacillus subtilis (SJR-02)

[0038]

[0039] Note: “+” indicates positive, “-” indicates negative.

[0040] (5) 16S rRNA sequence determination of Bacillus subtilis (SJR-02)

[0041] The bacterial solution was used as a template to directly amplify the 16s rRNA sequence by PCR, and the primers were 27F (5′

[0042] -AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′

[0043] -TACGGCTACCTTGTTACGACTT-3′). PCR amplification used a 25ul system template and the amplification conditions were: 95°C for 2min; 94°C for 30s, 58°C for 30s, 72°C for 2min, 35 cycles; 72°C for 5min, and 4°C for termination. PCR products were detected by 1% agarose gel electrophoresis. The PCR products were sent to Shenzhen BGI Genomics Co., Ltd. for sequencing. The 16S rRNA gene sequence of the Bacillus subtilis (SJR-02) strain is shown in SEQ ID No. 1. After the 16S rRNA gene sequence was determined, it was compared online with known related sequences in the database using the Blast tool. The homology with the genus Bacillus Velezii was as high as 99%.

[0044] The strains of the same genus as Bacillus subtilis were selected and the phylogenetic tree was constructed using MEGA5.0 software. The obtained phylogenetic tree is shown in the figure below. Figure 3 shown.

[0045] Example 2: Determination of the inhibitory effect of Bacillus subtilis (SJR-02) on Spirogyra:

[0046] Spirogyra was used as the target to screen for efficient algae-lytic bacteria. The strains to be evaluated included multiple single colonies isolated and purified by the applicant from the same batch of sludge in Example 1. The physiological and biochemical properties of these single colonies were consistent with those of Bacillus subtilis. They were named SCKC-1 (Bacillus subtilis), SCKC-2 (Bacillus subtilis), SCKC-3 (Bacillus subtilis), SJR-02 (Bacillus subtilis), ZJKC (Bacillus subtilis) and SCZZ (Bacillus subtilis), and a blank group (CK) using only LB medium without bacteria was set as a control. 2 mL of 3×10 10 CFU / mL of the Bacillus isolate was added to 18 mL of Spirogyra algae solution (initial chlorophyll a concentration was 0.8 mg / L) to make the final concentration of the bacterial solution 3.0×10 8CFU / mL, with Spirogyra algae solution added to an equal volume of LB medium as the control, with three replicates per treatment. Culture conditions were 2000 Lx, 25°C, a 14h:10h light-dark cycle, and shaking three times daily. After six days, the chlorophyll a content of Spirogyra gracilis FACHB-354 was determined according to "HJ897-2017 Water Quality - Determination of Chlorophyll a - Spectrophotometric Method," and the algae lysis rate was calculated based on references. The strain with the highest algae lysis rate was designated as the superior strain.

[0047] Test conclusion:

[0048] (1) 6 days after the application, the strains ZJKC and SJR showed significant inhibition on the growth of Spirogyra, mainly manifested by yellowing and a significant reduction in algal biomass. See Table 2 and Figure 4 ; Figure 4 The SJR group in the middle is the experimental result of Bacillus subtilis (SJR-02) of the present invention.

[0049] (2) By measuring the chlorophyll content of Spirogyra after treatment with algae-lytic bacteria, the strain SJR-02 had the highest inhibition rate on Spirogyra, reaching 72.6%, followed by ZJKC, with an inhibition rate of 67.8%.

[0050] Table 2 - Inhibition rate of Spirogyra after 6 days of algicidal treatment

[0051]

[0052]

[0053] Example 3: Preparation of wettable powder of algae-lysing bacteria (Bacillus subtilis SJR-02)

[0054] The algae-lytic bacteria SJR-02 fermentation broth obtained by fermentation (fermentation medium: fish peptone 0.8%, yeast powder 0.8%, rice flour 1.5%, flour 3%, magnesium sulfate 0.35%, light calcium carbonate 0.3%, industrial salt 0.25%), was added with 10% soluble starch as a protective agent, stirred evenly, and spray-dried in a spray dryer to obtain a powder (mother powder).

[0055] Spray drying parameters: air inlet temperature 180℃, air outlet temperature 65℃, pump speed 3000mL / h (adjusted in real time).

[0056] The following weight percentage ratio was used to prepare the following: 10% mother powder, 5% sodium butyl naphthalene sulfonate, 5% calcium lignin sulfonate, and 80% kaolin. The mixture was evenly mixed to obtain the algae-lytic bacteria SJR-02 wettable powder (the content of viable bacteria was determined to be 20 billion / g).

[0057] Example 4: Control effect of algae-lytic bacteria (Bacillus subtilis SJR-02) wettable powder combined with potassium humate on Spirogyra:

[0058] 1. Preparation of composite algae control agent: Mix Bacillus subtilis (SJR-02) wettable powder and potassium humate in a mass ratio of 1:8, add 5% kaolin and 5% chelated trace elements (citric acid chelated six elements: Fe 0.5%, B 0.4%, Zn 0.5%, Mn 0.1%, Cu 1.0%, Mo 0.01%); the agent powder should be evenly adhered to the potassium humate and quickly dispersed in water after dissolution;

[0059] 2. Fill 500 mL of soil nutrient solution into a 1000 mL plastic pot and add 0.5 g of fresh weight of Spirogyra to each pot.

[0060] 3. Set up a blank control group, a Bacillus subtilis (SJR-02) wettable powder, and a Bacillus subtilis (SJR-02) wettable powder + potassium humate group. Add 0.25g of the agent to each treatment, with three replicates per treatment. Observe the color change (yellowing rate) of the Spirogyra every day to determine the control effect of different agents on Spirogyra.

[0061] Test conclusion: The experimental results are shown in Table 3 and Figure 5 As shown in the results, 7 days after the application, the control effect of algae-lytic bacteria SJR-02 on Spirogyra was 69.5%, and the control effect of the combined preparation was 81.7%, indicating that the combination of algae-lytic bacteria and potassium humate can significantly improve the control effect of the bacterial agent on Spirogyra.

[0062] Table 3 - Whitening rate of Spirogyra in each treatment after 7 days

[0063] deal with 0d yellowing rate 7d yellowing rate Increase yellowing rate Actual prevention effect CK 15.5％ 12.7％ -2.8％ —— SJR-02 12.8％ 79.5％ 66.7％ 69.5％ SJR+F 13.6％ 92.5％ 78.9％ 81.7％

[0064] Example 5: Effect of combined algae control agents on Spirogyra control:

[0065] 1. Preparation of a composite algae control agent: Bacillus subtilis (SJR-02) wettable powder and sodium humate were mixed in a mass ratio of 1:8, and 5% kaolin and 5% chelated trace elements (same as in Example 4) were added. The bacterial agent powder was required to adhere evenly to the sodium humate and disperse quickly in water after dissolution. 2. A 12 L acrylic cylinder was filled with 500 g of garden soil and 10 L of tap water. A sinker block was placed in each cylinder, and two Elodea plants with Spirogyra were attached to the sinker block. The culture conditions were 25°C ± 1°C, with a light-dark cycle of 14 h:10 h. The culture was continued for one week before use.

[0066] 3. Set up a blank control treatment group, a sodium humate group, a Bacillus subtilis (SJR-02) wettable powder group (algae-lytic bacteria group in the table), and a compound algae control agent group (compound preparation in the table). Add the agent according to the test requirements and use coarse droplet spray for application. Each treatment is repeated twice. The color change of Spirogyra is observed every day to determine the control effect of different agents on Spirogyra. The results are shown in Tables 4-6 and Figure 6 and Figure 7 shown.

[0067] Table 4 - Treatment ingredients and concentrations

[0068] serial number deal with concentration Dosage 1 CK - 2 Sodium humate 2000 times 5g 3 Bacillus subtilis SJR-02 (20 billion / g) 2000 times 5g 4 Compound algae control agent 2000 times 5g

[0069] Table 5 - Coverage and control effect of each treatment 5 days after application

[0070] Average value (coverage) CK Sodium humate Algae-lytic bacteria combination preparations 5d 92.5％ 55.0％ 0.0％ 0.0％ Relative prevention efficacy — 41％ 100％ 100％

[0071] Table 6 - Coverage and control efficacy of each treatment 15 days after application

[0072] Average value (coverage) CK Sodium humate Algae-lytic bacteria combination preparations 15d 95.7％ 37.2％ 50.6％ 0.0％ Relative prevention efficacy — 61.13％ 46.08％ 100％

[0073] Test conclusion:

[0074] (1) By dripping the compound algae control agent, the surface Spirogyra was quickly removed. After further observation (5 days), the Elodea and Spirogyra on the bottom were also killed.

[0075] (2) After 15 days of treatment, except for the compound algae control agent treatment, the Spirogyra relapsed in all other treatments, indicating that under the experimental conditions, the compound algae control agent composed of algicidal bacteria and sodium humate can control the growth of Spirogyra for a longer period of time, and the control effect reaches 100%;

[0076] Example 6: Effect of the composite algae control agent on the control of moss attached to surface aquatic plants in the field:

[0077] 1. Use the compound algae control agent formula in Example 4; apply the agent by dissolving and then spraying, using 1500g of compound algae control agent per acre of water surface;

[0078] 2. Before applying the drug, take a photo of the moss coverage at the edge of the pond and record the initial coverage rate; on a sunny afternoon at 14:00 when the temperature is highest, dissolve the compound algae control agent sample at 50 times the concentration and spray it along the edge of the pond to the area where the moss occurs. Set the area where the drug is not sprayed as a blank control. The experimental results are shown in Table 7 and Figure 8 shown.

[0079] Table 7-2d and 15d control effects of compound algae control agents

[0080] time 2d 15d Natural growth rate 5.71％ -5.3％ Prevention effect 75.2％ 88.4％ Correction of prevention effect 76.49％ 93.7％

[0081] Control effect (%) = (coverage before treatment - coverage after treatment) / coverage before treatment × 100;

[0082] Natural growth rate = (coverage of CK cell after drug application - coverage of CK cell before drug application) / coverage of CK cell before drug application;

[0083] Corrected control effect (%) = [(control effect + natural growth rate) / (1 + natural growth rate)] × 100.

[0084] Test conclusion:

[0085] Investigation on the second day of the experiment showed that the surface moss in the area where the agent was sprayed turned black and dissolved. Through calculation of coverage, the prevention effect could reach 76.9%, indicating that the compound algae control agent has good rapid effect. 15 days after the application, the compound algae control agent basically completely inhibited the growth of moss, and the prevention effect reached 93.7%.

[0086] Example 7: Safety evaluation of composite algae control agent on crayfish:

[0087] Preparation of composite algae control agent: The composite algae control agent formula in Example 4 was used;

[0088] Test subjects: Whole shrimp (average body length 2-3 cm, weight 0.8 g), collected from farmers' shrimp ponds in Haokou Town, Qianjiang, and acclimated indoors for 1 week;

[0089] Test Method: Following the national standard GB / T31270.21-2014, the test was conducted using a still water bioassay. The test agent was fully dissolved in 4 L of well-aerated water. Ten shrimp fry approximately 3 cm in length were seeded into each replicate for four replicates, with clean water serving as a control. The shrimp were reared in containers at 25°C and examined 24 hours after inoculation. Death was determined by gentle touch with a brush; a control mortality rate of less than 20% was considered valid.

[0090] The test was set at 10ppm, 50ppm, 100ppm, and 250ppm, and a blank control was set at approximately 2, 5, 20, and 50 times the recommended field concentration, respectively;

[0091] Test conclusion: No shrimp fry died in the treatments with any concentration of the agent, indicating that the compound algae control agent is very safe for crayfish.

[0092] Example 8: Effect of compound algae control agent on the control of crayfish in intensive ponds:

[0093] Preparation of composite algae control agent: The composite algae control agent formula in Example 4 was used; the treatment area of ​​each test was 200m 2Each plot was physically isolated by 20 meters, with a blank control group remaining untreated. Each treatment was replicated three times in random order. The water depth was maintained at an average of 50 cm. Pesticides were applied during the afternoon, when sunlight was bright, using a dissolving and then splashing method. 2000 g of compound algaecide was used per mu of water surface. 50 L of water was used per plot. Field management followed conventional practices for shrimp-rice fields, preventing fertilizer and water cross-fertilization. The results are shown in Tables 8 and 9.

[0094] Table 8 - Experimental treatment agents and concentrations

[0095] Serial number Ingredients / Pharmacy Treatment dose 200m2 sample volume (50cm water depth) 1 Compound algae control agent 2kg / mu 2 Blank control —— Equal amount of water

[0096] Table 9 - Moss coverage of each treatment area before the experiment

[0097] Predrug coverage Repeat 1 Repeat 2 Repeat 3 average value Compound algae control agent 98％ 85％ 75％ 86％ Blank control 95％ 90％ 80％ 88％

[0098] Result investigation: Investigate the moss coverage 1, 3, 7 and 15 days after application and calculate the prevention effect.

[0099] Control effect (%) = (coverage before treatment - coverage after treatment) / coverage before treatment × 100;

[0100] Natural growth rate = (coverage of CK cell after drug application - coverage of CK cell before drug application) / coverage of CK cell before drug application;

[0101] Corrected control effect (%) = [(control effect + natural growth rate) / (1 + natural growth rate)] × 100.

[0102] Test results:

[0103] (1) During the test period, the temperature was 20-27℃, and the compound algae control agent was effective quickly. The corrected control efficiency reached 54.8% after 1 day of treatment and 98.2% after 2 days. (2) The agent was also very effective in removing moss on the bottom. The moss attached to the aquatic plants was basically removed after 2 days of treatment. The growth of moss was as follows 1-2 days after the compound algae control agent was applied. Figure 9 As shown, there was no significant effect on the growth of aquatic plants. (3) During the treatment process, the density of crayfish in the farmer's shrimp pond was relatively high. The pesticide was applied in half. The farmer released the water 12 hours after the application. The crayfish showed slight hypoxia. The crayfish were caught normally 2 days after the application, and no adverse reactions were observed. The data are shown in Tables 10 and 11.

[0104] Table 10 - Effect of compound algae control agent on moss control in crayfish ponds 1 day after application

[0105] Coverage 1 day after medication Repeat 1 Repeat 2 Repeat 3 average value Prevention effect Correction of prevention effect Compound algae control agent 45％ 35％ 30％ 37％ 57％ 54.8％ Blank control 85％ 85％ 80％ 83％ Natural growth rate -5.7％

[0106] Table 11 - Effect of compound algae control agent on moss control in crayfish ponds 2 days after application

[0107]

[0108] Example 9: Effect of compound algae control agent on shallow water shrimp and rice fields:

[0109] Preparation of compound algae control agent: Mix Bacillus subtilis (SJR-02) wettable powder and mineral potassium fulvate in a mass ratio of 1:8, add 5% kaolin and 5% chelated trace elements (same as above);

[0110] Each treatment area covered 1-2 mu (approximately 1-2 mu), with 1000 g applied per mu. A separate, untreated area was also established. Each treatment was replicated once, with random placement. The water depth was maintained at an average of 50 cm. Applications were made during the afternoon, when sunlight was bright, with a water rate of 50 L per mu. Areas with moss infestations were sprayed. Field management followed conventional practices for shrimp-rice fields, preventing fertilizer and water intrusion. The experimental treatment agents and concentrations are shown in Table 12. The moss coverage of each treatment plot before the experiment is shown in Table 13.

[0111] Table 12 - Experimental treatment agents and concentrations

[0112]

[0113]

[0114] Table 13 - Moss coverage of each treatment area before the experiment

[0115] Predrug coverage Repeat 1 Repeat 2 Repeat 3 average value Compound algae control agent 98％ 85％ 75％ 86％ Blank control 95％ 90％ 80％ 88％

[0116] Result investigation: Investigate the moss coverage 1, 3, 7 and 15 days after application and calculate the prevention effect.

[0117] Control effect (%) = (coverage before treatment - coverage after treatment) / coverage before treatment × 100

[0118] Natural growth rate = (coverage of CK cell after drug application - coverage of CK cell before drug application) / coverage of CK cell before drug application

[0119] Corrected control effect (%) = [(control effect + natural growth rate) / (1 + natural growth rate)] × 100

[0120] Test results: The moss coverage before the application is shown in Table 14, the control effect of the compound algae control agent 4 days after the application is shown in Table 15, and the growth of Spirogyra in the shallow water test field before and 4 days after the application (experimental temperature 22℃) is shown in Table 14. Figure 1 As shown. From the results we can see:

[0121] (1) In shallow ponds, the compound algae control agent has a significant effect on the initial occurrence of moss (the control effect exceeds 90%). After spraying the moss carried into the fields by aquatic plants, the spread of the moss is well controlled. 30 days after the application, no large amount of moss was seen in the entire pond. The compound algae control agent is highly safe for aquatic plants, and farmers are very satisfied with the effect of the agent.

[0122] (2) A drug immersion test was conducted at farmers' homes. The results showed that the compound algae control agent was relatively safe for crayfish fry at the recommended dose, and no safety risks such as shrimp death and hypoxia occurred after the drug application.

[0123] Table 14 - Moss coverage before medication

[0124] Predrug coverage Repeat 1 Repeat 2 Repeat 3 average value Compound algae control agent 55％ 65％ 80％ 67％ Blank control 50％ 45％ 75％ 57％

[0125] Table 15 - Control effect of compound algae control agent 4 days after application

[0126] Coverage 4 days after medication Repeat 1 Repeat 2 Repeat 3 average value Prevention effect Correction of prevention effect Blank control 45％ 45％ 70％ 53％ Compound algae control agent 1％ 5％ 10％ 5％ 92％ 91.5％ Natural growth rate -5.9％

[0127] The above-described embodiments merely illustrate several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. Persons skilled in the art will readily appreciate that variations and modifications may be made without departing from the scope of the present invention, all of which fall within the scope of protection of the present invention.

Claims

1. A Bacillus subtilis SJR-02 with algae-dissolving effect, characterized in that: The strain has been deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on October 28, 2024, with the deposit number CGMCC No.32370.

2. An algae-dissolving bacteria wettable powder, characterized in that: It is prepared by the following method: 1) Fermenting the Bacillus subtilis SJR-02 according to claim 1, wherein the fermentation conditions are: tank pressure 0.2-0.3 MPa, temperature 25-30° C., dissolved oxygen ≥20%, stirring speed 200-240 rpm, and fermentation time 32-40 h; after fermentation, collecting the fermentation broth, adding soluble starch as a protective agent, stirring evenly, and spray drying in a spray dryer to obtain a mother powder; 2) Mixing the collected mother powder with a carrier and an adjuvant to obtain SJR-02 wettable powder; In the SJR-02 wettable powder, the content of Bacillus subtilis SJR-02 is 1×10 10 ~3×10 10 CFU / g.

3. The algae-lytic bacteria wettable powder according to claim 1, characterized in that The carrier is one or more of kaolin, diatomaceous earth or calcium carbonate, and the mass percentage of the carrier in the wettable powder is 80%; the auxiliary agents are sodium butyl naphthalene sulfonate and calcium lignin sulfonate, and the mass percentage of sodium butyl naphthalene sulfonate and calcium lignin sulfonate in the wettable powder is 5%.

4. The algae-lytic bacteria wettable powder according to claim 1, characterized in that During the fermentation process of step 1), the fermentation medium formula is as follows, by mass percentage: 0.8% fish peptone, 0.8% yeast powder, 1.5% rice flour, 3% flour, 0.35% magnesium sulfate, 0.3% light calcium carbonate, 0.25% industrial salt, and the balance is water.

5. Use of the Bacillus subtilis SJR-02 according to claim 1 or the algae-lytic bacteria wettable powder according to any one of claims 2 to 4 as an inhibitor for inhibiting algae growth in water.

6. A composite algae control agent, characterized in that: The invention comprises the wettable powder of algae-dissolving bacteria according to any one of claims 2 to 4 and humate, wherein the humate is one or more of sodium humate, potassium humate and mineral-derived potassium humate; and the mass ratio of the wettable powder of algae-dissolving bacteria to the humate is 1:

8.

7. The composite algae control agent according to claim 6, characterized in that: The composite algae control agent further comprises kaolin and chelated trace elements, wherein the kaolin accounts for 5-20% of the mass of the composite algae control agent, and the chelated trace elements account for 5-20% of the mass of the composite algae control agent.

8. Use of the composite algae control agent according to claim 6 or 7 as an inhibitor for inhibiting algae growth in water bodies.

9. The use according to claim 7, characterized in that The water body is an aquaculture water body, and the aquaculture organisms in the aquaculture water body are shrimps, crabs or fish.

10. The use according to claim 7, characterized in that The algae are Spirogyra, Hydrodiplophyte, Transodium and Cladophora.

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