Biological organic fish guano containing functional bacteria as well as preparation method and application of biological organic fish guano
By fermenting and enzymatically decomposing low-value fish with Bacillus PAPM-H-7, which has anti-disease and proliferation function, the resulting biological organic fish fertilizer not only reduces production costs and increases hydrolysis, but also significantly promotes the Crop growth and prevention and control of pepper blight have solved the problems of high costs and insufficient functions in the prior art.
Patent Information
- Application Number
- CN202411256409.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-09
- Publication Date
- 2024-12-13
AI Technical Summary
Among the existing biological organic fish fertilizer production methods, the enzymatic solution is costly and has low hydrolysis. The microorganisms used in the fermentation method lack the anti-disease and growth function, which affects product quality and crop growth effect.
Bacillus PAPM-H-7, which has antagonistic functions of Phytophthora capsia, protease, lipase and IAA, was isolated and screened from the soil, and the low-value fish was treated by combining fermentation and enzymatic decomposition to produce containing A large amount of Bacillus vegetarian organic fish fertilizer has the functions of improving soil and preventing and treating pests.
It reduces production costs, increases the hydrolysis of fish protein and fat, enhances the promotion of crop growth and the prevention and control of pepper epidemics, and broadens the utilization of low-value fish resources.
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Figure CN119120283A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of agricultural biotechnology, and relates to a biological organic fish fertilizer containing functional bacteria, and a preparation method and application thereof. Background Art
[0002] Low-value fish is a type of fishery resource with abundant stocks, including small fish with low commercial value, as well as by-products such as fish skin, fish fillets, and fish viscera produced during deep processing of fish. In addition to being used as fish meal in the feed industry, low-value fish can also be used to produce new fertilizers with higher added value, such as bio-organic fish fertilizer. Because it is rich in protein, peptides and amino acids, the application effect of bio-organic fish fertilizer is better than that of bio-organic water-soluble fertilizers prepared from other raw materials such as molasses, corn syrup and yeast powder. It has a more significant effect on promoting crop growth and improving crop quality. It is a high-quality bio-organic water-soluble fertilizer with great development potential.
[0003] There are two main methods for producing water-soluble fertilizers using low-value fish as raw materials: enzymatic method and fermentation method. Enzymatic method is to use industrial enzyme preparations (alkaline protease, neutral protease, acidic protease, pancreatin, papain and bromelain, etc.) to hydrolyze low-value fish, hydrolyze macromolecules such as protein into small molecules such as peptides and amino acids, and increase their water solubility. Due to the high price and strong specificity of enzyme preparations, not only the production cost is high, but also the degree of fish protein hydrolysis is low, especially lipase, which is rarely used in production due to its high price. The fat of low-value fish is not hydrolyzed and directly enters the product, affecting the product quality. The fermentation method is to ferment low-value fish with enzyme-producing microorganisms, and use the protease, lipase and other hydrolytic enzymes produced by microorganisms to hydrolyze fish protein and fish fat. Not only is the degree of hydrolysis high, but also the production cost is reduced. It is a more economical and effective production method. However, the fermentation microorganisms currently used are mainly enzyme-producing strains, and strains with beneficial functions such as disease resistance and growth promotion are rarely used. Summary of the invention
[0004] In view of the above problems, the present invention provides a biological organic fish fertilizer containing functional bacteria and its preparation method and application. The present invention first separates and screens a strain of Bacillus velezensis PAPM-H-7 from the soil, which has beneficial functions such as antagonizing pathogens such as pepper blight, producing protease, lipase, extracellular polysaccharides and IAA. It is used to ferment and enzymolyze low-value fish such as fish skin, fish fillets, and fish viscera, which can not only degrade macromolecules such as fish protein and fat into small molecules such as peptides, amino acids and fatty acids, but also produce beneficial components such as extracellular polysaccharides and IAA. At the same time, the prepared biological fish fertilizer also contains a large amount of Bacillus velezensis, which has the functions of fish fertilizer and bacterial fertilizer, and has the effects of improving soil, promoting crop growth, and preventing and controlling pepper blight.
[0005] The present invention firstly isolates a strain of Bacillus velezensis PAPM-H-7 from the soil, which has been deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration Committee on June 21, 2023, with the deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is: CGMCC No. 27682. The strain has beneficial functions such as antagonizing pathogens such as pepper phytophthora, producing IAA and extracellular polysaccharides, and decomposing proteins and fats.
[0006] The invention provides a fermentation and culture method of the above-mentioned Velez Bacillus PAPM-H-7, comprising strain activation, triangular flask seed preparation, liquid seed tank strain preparation and liquid fermentation tank culture, wherein the strain activation adopts LB culture medium test tube slant, the culture temperature is 32-37°C, and the culture time is 20-24h; the triangular flask seed preparation and the liquid seed tank strain preparation both adopt LB liquid culture medium, the culture temperature is 32-37°C, and the culture time is 18-30h; the liquid fermentation tank culture adopts fermentation culture medium, the inoculation amount is 2%-4%, the culture temperature is 32-37°C, and the culture time is 24-36h, so as to obtain Velez Bacillus PAPM-H-7 fermentation liquid.
[0007] The formula of the fermentation medium is: 200g / L low-value fish (fresh weight), 100g / L maltodextrin, 0.5g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate, and an initial pH of 6.5-7.5. The preparation method of the liquid fermentation medium is: after the low-value fish is minced, it is ground into pulp, and the medium is prepared according to the formula amount, and the temperature is raised to 121°C for sterilization for 15-30 minutes.
[0008] The present invention also provides a biological organic fish fertilizer with the above-mentioned Velez Bacillus PAPM-H-7 fermentation liquid as the main component, characterized in that its main raw materials and weight percentages are: 40-60 parts of fermentation liquid, 40-60 parts of low-value fish such as fish skin, fish fillet, fish viscera, etc. Preferably, by weight percentage, the fermentation liquid is 50 parts and the low-value fish is 50 parts.
[0009] The preparation method is as follows: mixing low-value fish with fermentation liquid, mincing, grinding, heating to 50-55°C for enzymatic hydrolysis for 60-120 minutes to obtain a uniform slurry, cooling to below 40°C, adding a preservative, and stirring evenly to obtain the biological organic fish fertilizer of the present invention, wherein the content of the effective ingredient is: the number of live bacteria of Bacillus Velez PAPM-H-7 is ≥1×10 9 cfu / mL, organic matter content ≥100g / L.
[0010] The invention also discloses a method for using the biological organic fish fertilizer: when used for the cultivation of crops such as peppers, the fertilizer is diluted 100-1000 times by weight and then flushed or drip-irrigated, with a dosage of 5-10 kg per mu.
[0011] The advantages of the present invention are:
[0012] 1. The Bacillus Velez PAPM-H-7 obtained by the present invention has multiple beneficial functions such as antagonizing pathogens such as pepper phytophthora, producing IAA and extracellular polysaccharides, decomposing proteins and fats, etc. The biological organic fish fertilizer prepared by the strain through a production process combining fermentation and enzymolysis contains not only fish enzymolytes, but also Bacillus Velez PAPM-H-7 and its metabolites, and has the functions of promoting crop growth, improving soil, preventing and controlling pepper blight, etc.
[0013] 2. The present invention uses low-value fish such as fish skin, fish fillets, and fish viscera as raw materials, and adopts a production process combining fermentation and enzymolysis to prepare bio-organic fish fertilizer, which not only reduces the production cost of bio-organic water-soluble fertilizer, but also broadens the source of raw materials, which is conducive to the resource utilization and high-value utilization of low-value fish. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 This is the colony morphology of Bacillus Velez PAPM-H-7;
[0015] Figure 2 This is the bacterial morphology of Bacillus Velez PAPM-H-7;
[0016] Figure 3 It is a phylogenetic tree constructed based on partial 16S rDNA sequences. DETAILED DESCRIPTION
[0017] The present invention will be further described below in conjunction with the embodiments.
[0018] Example 1: Screening of Bacillus Velez PAPM-H-7
[0019] Separation time: September 2020; Separation location: Ancheng Town, Pingyin County, Shandong Province, Shandong Zuotianshi Biotechnology Co., Ltd.
[0020] The bacteria were isolated from the soil using the plate spreading method. Weigh 10 g of soil, add it to 90 mL of sterile water, and oscillate it on a constant temperature oscillator at 180 rpm for 30 min. Then put it in a clean bench and use sterile water for serial dilution. -3 , 10 -4 and 10 -5Spread the plate with 3 dilutions. Spread 3 plates for each dilution. Use LB medium as the plate culture medium. Invert the plate in a 37℃ constant temperature incubator and culture for 12-24h. Pick different colonies for streaking purification based on the colony size, color, gloss, surface texture and other characteristics. Number the purified strains and store them in a 4℃ refrigerator for further screening of antagonistic bacteria.
[0021] Taking Phytophthora capsici as the target bacteria, the plate confrontation culture method was used to screen its antagonistic bacteria. First, the mycelium of the pathogen was picked up with a sterilized inoculation loop and placed in the center of the PDA plate. The cross lines were marked on the back of the PDA plate for partitioning. Different bacteria screened from the soil were inoculated at an equal distance of 2-2.5 cm from the center of the plate, and each treatment was repeated 3 times. After the plate was placed in a constant temperature incubator at 28℃ for 7 days, the confrontation growth was observed. According to the degree of inhibition of the antagonistic bacteria on the growth of Phytophthora capsici, a strain with the best antagonistic effect was screened, which was the PAPM-H-7 strain.
[0022] Example 2: Identification of Bacillus Velez PAPM-H-7
[0023] (1) Morphological and physiological and biochemical characteristics
[0024] The morphological and physiological and biochemical characteristics of the Velez Bacillus PAPM-H-7 strain are as follows: when cultured on LB medium at 35° C. for 24 hours, the colonies are round, milky white, opaque, with dry, wrinkled and raised surfaces, no pigment is produced, and the color of the medium does not change significantly ( Figure 1 ). Observation under an optical microscope (1000×) revealed that the bacteria were rod-shaped, Gram-positive, and had oval spores ( Figure 2 ). The physiological and biochemical characteristics were (+, positive; -, negative): catalase test +; methyl red test +; starch hydrolysis test +; denitrification test +; ammonia production test +; lactose fermentation test -; sucrose fermentation test +; xylose fermentation test +; arabinose test +; salicin test -; melibiose fermentation test +; rhamnose fermentation test -; mannitol fermentation test +; glucose fermentation test +.
[0025] (2) 16S rDNA sequence analysis
[0026] The PAPM-H-7 strain was inoculated into LB liquid medium and cultured at 37°C and 200r / min shaking for 24h. The bacteria were collected, the total DNA was extracted, and then used as a template to perform PCR amplification of the 16SrDNA gene under the guidance of the universal primers F27:5′-AGA GTT TGATCA TGG CTC AG-3′ and F27:5′-AGA GTT TGA TCA TGG CTC AG-3′ of the prokaryotic 16SrRNA gene. The amplification conditions were: 95°C pre-denaturation for 3min, 94°C denaturation for 1min, 55°C renaturation for 1min, 72°C extension for 1.5min, a total of 30 cycles, and 72°C extension for 10min. The amplified product was separated by 1% agarose gel electrophoresis, recovered using a gel recovery kit, and sequenced. The obtained sequence is shown in the sequence table SEQ No.1. The measured 16SrDNA sequences were compared with the sequences in the GenBank database, multiple sequence homology analysis was performed, and a phylogenetic tree was constructed, such as Figure 3 shown.
[0027] 16S rDNA sequence of Bacillus velez PAPM-H-7 (SEQ No. 1):
[0028]
[0029] Through morphological, physiological and biochemical characteristics and 16SrDNA sequence analysis, strain PAPM-H-7 was identified as Bacillus velezensis. The strain has been deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on June 21, 2023, with the deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number: CGMCC No. 27682.
[0030] Example 3: Determination of beneficial properties of Bacillus Velez PAPM-H-7
[0031] (1) Extracellular polysaccharide production capacity of Bacillus velez PAPM-H-7
[0032] The Bacillus Velez PAPM-H-7 strain was inoculated into a triangular flask containing LB liquid culture medium, placed in a constant temperature shaker for shaking culture for 16 hours, and then transferred to the polysaccharide fermentation medium with an inoculation amount of 2%, and continued to shake culture for 48 hours. The culture temperature was 35℃, and the shaking speed of the shaker was 220r / min. The fermentation broth was centrifuged at 4800r / min for 20 minutes, the precipitate was discarded, the supernatant was taken and 4 times the volume of anhydrous ethanol was added, mixed evenly, and placed at 4℃ for 1 hour to precipitate the polysaccharide. Then centrifuged at 4800r / min for 20 minutes, the supernatant was discarded, and the precipitate was retained. Add an appropriate amount of distilled water to the precipitate to dissolve it, which is the polysaccharide crude extract, and the polysaccharide content was determined by the phenol-sulfuric acid method. The experimental results showed that the polysaccharide content of the fermentation broth of Bacillus Velez PAPM-H-7 strain was 1.78g / L.
[0033] The formula of the polysaccharide-producing fermentation medium is (g / L): sucrose 25, peptone 5, NaNO3 0.5, K2HPO4 0.5, MgSO4 0.5, NaCl 10, pH 7.0-7.5.
[0034] (2) Effects of Bacillus Velezii PAPM-H-7 and its extracellular polysaccharides on soil aggregate formation
[0035] The Bacillus Velez PAPM-H-7 strain was inoculated into a triangular flask containing LB liquid culture medium, placed in a constant temperature shaker for shaking culture for 16 hours, and then transferred to a polysaccharide fermentation medium with an inoculation amount of 2%, and continued to shake culture for 48 hours to obtain a fermentation liquid. The culture temperature was 35°C, and the shaking speed of the shaker was 220r / min. The formula of the polysaccharide fermentation medium was (g / L): sucrose 25, peptone 5, NaNO3 0.5, K2HPO4 0.5, MgSO4 0.5, NaCl 10, pH 7.0-7.5.
[0036] Take 9 500mL triangular flasks and divide them into 3 groups (CK, T1 and T2), 3 bottles in each group, and put 200g soil (passed through a 0.25mm sieve) in each bottle. Add 20mL of polysaccharide fermentation medium to each bottle of the CK group and mix evenly; add 20mL of fermentation liquid of Bacillus Velez PAPM-H-7 strain to each bottle of the T1 group and mix evenly; add 20mL of pre-inactivated fermentation liquid of Bacillus Velez PAPM-H-7 strain (sterilized at 121℃ for 30min) to each bottle of the T2 group and mix evenly. All treatments were cultured in a greenhouse, and water was added regularly to keep the soil surface moist. After 30 days of culture, the proportion of soil water-stable aggregates (>0.25mm) was determined. The determination method refers to the method specified in NY / T 1121.19-2008 "Soil Testing Part 19: Determination of the Composition of Soil Water-Stable Macroaggregates". The determination results are shown in Table 1. The proportion of soil water-stable aggregates in the T1 treatment was the highest, reaching 10.55%, which was 46.3% higher than that in the CK treatment; the proportion of soil water-stable aggregates in the T2 treatment was second, which was 19.6% higher than that in the CK treatment. This shows that Bacillus Velez PAPM-H-7 and its extracellular polysaccharides can significantly promote the formation of soil aggregates.
[0037] Table 1 Proportion of soil water-stable aggregates (>0.25 mm) in different treatment groups
[0038] deal with CK T1 T2 Water-stable aggregates (%) 7.21±0.65a 10.55±1.24c 8.62±0.43b
[0039] Note: Data marked with different letters in the table represent significant differences (P < 0.05).
[0040] (3) Protease and lipase production ability of Bacillus velez PAPM-H-7
[0041] Bacillus Velez PAPM-H-7 was inoculated (inoculation amount 3%) into the liquid enzyme production medium, and cultured at 35°C, 180-220rpm shaking for 48h, and the protease and lipase activities in the fermentation broth were determined. The preparation method of the liquid enzyme production medium is: 300Kg of cod skin, 1Kg of potassium dihydrogen phosphate, 698Kg of water, minced, ground, adjusted to an initial pH of 6.5-7.5, and sterilized at 121°C for 15-30min. The protease activity was determined by the Folin method, and the specific method was referred to the national standard GB / T 23527-2009 "Protease Preparation". The lipase activity was determined by the dynamic titration method, and the specific method was referred to the national standard GB / T23525-2009. The results of the determination showed that the neutral protease activity of the fermentation broth could reach 1260U / mL, and the lipase activity could reach 58U / mL. This indicates that Bacillus Velez PAPM-H-7 has a strong ability to produce protease and lipase, which is beneficial to its decomposition of fish protein and fish fat.
[0042] (4) Determination of IAA production capacity of Bacillus velez PAPM-H-7
[0043] Add L-tryptophan (200 mg / L) to the LB liquid culture medium, sterilize at 121°C for 15 minutes, inoculate Bacillus Velez PAPM-H-7 after cooling, and place in a constant temperature shaker at 35°C and 200r / min for 48 hours. Take 50μL of fermentation liquid and drop it on a white ceramic plate, add 50μL of Salkowski colorimetric solution at the same time, and place it at room temperature and away from light for 30 minutes. The positive control replaces the fermentation liquid with 50μL 50mg / LIAA. The color of the colorimetric solution turns red, indicating that the test strain can secrete IAA. The experimental results show that the treatment of the fermentation liquid of Bacillus Velez PAPM-H-7 is red, which is close to the color of the positive control, indicating that the strain has the ability to secrete IAA. The formula of the Salkowski colorimetric solution is: 35% HClO 450mL, 0.5mol / L FeCl3 1mL.
[0044] Example 4: Preparation of Bacillus Velez PAPM-H-7 fermentation broth
[0045] 1) Activation of strains: Transfer Bacillus Velez PAPM-H-7 to LB slant medium and culture at 35° C. for 20-24 hours. The LB slant medium formula is: 5 g yeast powder, 10 g tryptone, 10 g sodium chloride, 20 g agar powder, 1000 mL water, pH 7.5.
[0046] 2) Preparation of triangular flask strains: The activated Bacillus Velez PAPM-H-7 was transferred to LB liquid culture medium, and cultured at 35°C and 180-200 rpm for 20-24 hours to obtain triangular flask seed solution. The formula of the LB liquid culture medium is: 5g yeast powder, 10g tryptone, 10g sodium chloride, 1000mL water, pH 7.5.
[0047] 3) Preparation of seed tank strains: Transfer the flask seeds to a 20 L seed tank filled with 16 L of LB liquid culture medium and an inoculation amount of 2%. Cultivate at 35 °C, 200 rpm stirring speed, and 16 L / min ventilation for 20-24 h to obtain the seed tank strains.
[0048] 4) Liquid fermentation: Transfer the strain from the seed tank to the fermentation tank with an inoculation volume of 2%. The fermentation tank has a volume of 1000L and contains 700L of fermentation medium. Fermentation is carried out for 36 hours at 35°C, a stirring speed of 200rpm, and a ventilation volume of 600-700L / min to obtain the fermentation liquid of Bacillus Velez PAPM-H-7, with a live bacterial content of about 1.0×10 10 cfu / mL, used in the preparation of biological organic fish fertilizer.
[0049] The formula of the fermentation medium is: 200g / L low-value fish (fresh weight), 100g / L maltodextrin, 0.5g / L potassium dihydrogen phosphate, 0.5g / L magnesium sulfate, and initial pH 6.5-7.5. The preparation method of the liquid fermentation medium is: after the low-value fish is minced, it is ground into pulp, and the medium is prepared according to the formula amount, and the temperature is raised to 121° C. for sterilization for 15-30 minutes.
[0050] Example 5: Preparation of biological organic fish fertilizer
[0051] The fermentation liquid of Bacillus Velez PAPM-H-7 prepared in Example 4 is used to prepare a biological organic fish fertilizer, and the formula thereof is as follows: by weight, 50 parts of low-value fish such as fish skin, fish fillet, and fish viscera, and 50 parts of fermentation liquid. The low-value fish and the fermentation liquid are mixed according to the formula amount, minced, ground, heated to 50-55°C for enzymolysis for 90 minutes, then cooled to below 40°C, added with an appropriate amount of preservatives, and stirred evenly to obtain the biological organic fish fertilizer of the present invention, and the content of the effective ingredient is Bacillus Velez PAPM-H-7 viable bacteria number ≥ 1×10 9 cfu / mL, organic matter content ≥100g / L. Preservatives can be conventional preservatives such as kasone, potassium sorbate, sodium benzoate, etc.
[0052] Application example 1: Application in pepper production
[0053] The potted plant test was conducted in Dongzhuang Village, Ancheng Town, Pingyin County, Shandong Province in 2022. The specifications of the experimental pots are 20 cm inner diameter of the pot mouth, 14 cm inner diameter of the pot bottom, and 16 cm high. The experimental soil is field tillage soil. Soil nutrients: organic matter 17.87 g / kg, available nitrogen 65.2 mg / kg, available phosphorus 75.17 mg / g, available potassium 156.24 mg / g, pH 6.72. There are 3 groups in the experiment: control group (CK), treatment group T1 and treatment group T2, each with 25 pots and 2 peppers per pot. The control group CK applied the pre-sterilized Bacillus Velez PAPM-H-7 fermentation liquid prepared in Example 4; the treatment group T1 applied the Bacillus Velez PAPM-H-7 fermentation liquid prepared in Example 4; the treatment group T2 applied the biological organic fish fertilizer prepared in Example 5. The fertilizer application rate was 30 mL / pot, which was applied with water three times on the 10th, 20th and 30th days after transplanting, 10 mL / pot each time. Plant height and stem diameter were counted on the 60th day after transplanting, and rhizosphere soil enzyme activity was determined. Plant height and stem diameter were measured with a tape measure and vernier caliper, respectively. Determination of rhizosphere soil enzyme activity: urease was determined by sodium phenol colorimetry, catalase was determined by potassium permanganate titration, and sucrase was determined by DNS colorimetry.
[0054] The test results are shown in Tables 2 and 3. Compared with the control group CK, the plant height and stem diameter of the treatment groups T1 and T2 were increased to varying degrees, which shows that the fermentation liquid of the Bacillus Velez subtilis PAPM-H-7 of the present invention and the bio-organic fish fertilizer prepared with it as the main component have a promoting effect on the growth of pepper. Compared with the control group CK, the activities of urease, catalase and sucrase in the treatment groups T1 and T2 were increased to varying degrees, which shows that the fermentation liquid of the Bacillus Velez subtilis PAPM-H-7 of the present invention and the bio-organic fish fertilizer prepared with it as the main component can improve the enzyme activity of the rhizosphere soil of pepper.
[0055] Table 2 Effects on pepper growth
[0056]
[0057]
[0058] Note: Data in the same column marked with different letters represent significant differences (P < 0.05).
[0059] Table 3 Effects on enzyme activities in the rhizosphere soil of pepper
[0060]
[0061] Note: Data in the same column marked with different letters represent significant differences (P < 0.05).
[0062] Application example 2: Prevention and treatment of pepper blight
[0063] The pot experiment was conducted in Dongzhuang Village, Ancheng Town, Pingyin County, Shandong Province in 2022. The specifications of the experimental pots are 20cm inner diameter of the pot mouth, 14cm inner diameter of the pot bottom, and 16cm high. The experimental soil is the field arable layer soil. Soil nutrients: organic matter 17.87g / kg, available nitrogen 65.2mg / kg, available phosphorus 75.17mg / g, available potassium 156.24mg / g, pH6.72. There are 3 groups in the experiment: control group (CK), treatment group T1 and treatment group T2. Each treatment has 25 pots, 2 peppers in each pot, a total of 50 plants.
[0064] The control group CK applied 10 mL / pot of pepper phytophthora suspension on the 10th day after transplanting, and applied it with water; the treatment group T1 applied 10 mL / pot of pepper phytophthora suspension and 10 mL / pot of Bacillus Velez PAPM-H-7 fermentation liquid prepared in Example 4 on the 10th day after transplanting, and applied it with water; the treatment group T2 applied 10 mL / pot of pepper phytophthora suspension and 10 mL / pot of bio-organic fish fertilizer prepared in Example 5 on the 10th day after transplanting. After 15 days of root irrigation treatment, the symptoms were observed and the severity and prevention effect of the disease were calculated. Preparation method of pepper phytophthora suspension: pepper phytophthora was inoculated into a PDA plate, cultured at 28°C for 7-10 days, and after the mycelium was full, sterile water was added to scrape the moss and spores to make a suspension, and the spore concentration was 1×10 6 The PDA medium formula: 200g potato, 20g glucose, 20g agar, 1000mL distilled water, natural pH value.
[0065] The classification standard of pepper blight is as follows: Grade 0: completely disease-free; Grade 1: local symptoms on the outer leaves and stems (dark brown to black, shrunk); Grade 3: less than 1 / 3 of the outer leaves and stems are shrunk and discolored, or a few outer leaves wilt (fall off); Grade 5: more than 1 / 3 of the whole plant is shrunk and discolored, or the leaves wilt (fall off) or fall over; Grade 7: more than 1 / 2 of the whole plant is shrunk and discolored, and the leaves fall off; Grade 9: the whole plant changes color and dies. Disease index (%) = ∑ (grade value × number of plants) / (9 × total number of plants) × 100. Control effect (%) = (control disease index - treatment disease index) / control disease index × 100.
[0066] The experimental results are shown in Table 4. Compared with the control group CK, the incidence rate and disease index of the treatment groups T1 and T2 were significantly reduced, which shows that the fermentation liquid of the Bacillus Velez subtilis PAPM-H-7 of the present invention and the biological organic fish fertilizer prepared with it as the main component have a good preventive and control effect on pepper blight.
[0067] Table 4 The effect of biological organic fish fertilizer on the prevention and control of pepper blight
[0068]
Claims
1. A strain of Bacillus velezensis PAPM-H-7, Its characteristics are: Its deposit number is: CGMCC No.27682.
2. Use of the Bacillus Velez subtilis PAPM-H-7 according to claim 1 in antagonizing Phytophthora capsici.
3. Use of the Bacillus Velez PAPM-H-7 according to claim 1 in producing IAA and exopolysaccharides, and decomposing proteins and fats.
4. Use of the Bacillus Velez subtilis PAPM-H-7 according to claim 1 in preparing biological organic fish fertilizer.
5. The fermentation method of Bacillus Velez subtilis PAPM-H-7 according to claim 1, Its characteristics are: The method comprises strain activation, triangular flask seed preparation, liquid seed tank strain preparation and liquid fermentation tank culture, wherein the liquid fermentation tank culture adopts a fermentation medium with an inoculation amount of 2%-4%, a culture temperature of 32-37°C and a culture time of 24-36h to obtain Bacillus Velez PAPM-H-7 fermentation liquid; the fermentation medium has low-value fish as a main component.
6. The fermentation method of Bacillus Velez PAPM-H-7 according to claim 5, Its characteristics are: The fermentation medium comprises: 200 g / L low-value fish, 100 g / L maltodextrin, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, and an initial pH of 6.5-7.
5.
7. The fermentation liquid of Bacillus Velez PAPM-H-7 prepared by the fermentation method according to claim 5 or 6.
8. A method for preparing a biological organic fish fertilizer containing functional bacteria, Its characteristics are: Its main raw materials and their mass fractions are: 40-60 parts of the fermentation liquid of Bacillus Velez PAPM-H-7 described in claim 7, 40-60 parts of low-value fish; The low-value fish is mixed with the fermentation liquid of Bacillus Velez PAPM-H-7, minced, ground, heated to 50-55°C for enzymatic hydrolysis for 60-120 minutes to obtain a uniform slurry, cooled to below 40°C, added with a preservative, stirred evenly, and obtained a biological organic fish fertilizer containing functional bacteria.
9. The biological organic fish fertilizer containing functional bacteria prepared by the preparation method of claim 8, Its characteristics are: Its active ingredients and content are: Bacillus Velez PAPM-H-7 viable bacteria count ≥ 1×10 9 cfu / mL, organic matter content ≥100g / L.
10. Use of the biological organic fish fertilizer containing functional bacteria according to claim 9 or the fermentation liquid of Bacillus Velez PAPM-H-7 according to claim 7 in promoting the growth of pepper, improving the enzyme activity of pepper rhizosphere soil and preventing and controlling pepper blight.