Bio-organic water-soluble fertilizer, and preparation method and application thereof

Abstract

The application discloses a kind of bio-organic water-soluble fertilizer and its preparation method and application.First, a strain of compost Bacillus stercoris (Bacillus stercoris) PAPM-Y1-2 is isolated from kanamycin fermentation residues, and the preservation number is CGMCC No.27681.The strain has the beneficial characteristics of producing IAA, protein degradation, degrading kanamycin and antagonizing tomato soft rot pathogen, etc.The strain is fermented and enzymatically hydrolyzed with kanamycin fermentation residues, and is compounded with low-value fish enzymatic hydrolysate to prepare bio-organic water-soluble fertilizer, which has the effects of promoting crop growth, improving soil and preventing tomato soft rot, etc.The application uses kanamycin fermentation residues and low-value fish enzymatic hydrolysate as main raw materials to prepare bio-organic water-soluble fertilizer, which not only helps to reduce production cost and broaden raw material sources, but also helps to harmless and resource utilization of kanamycin fermentation residues.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a bio-organic water-soluble fertilizer, its preparation method, and its application. Background Technology

[0002] With the rapid development of biotechnology and fertigation technology, the application of bio-fertilizers is becoming more and more widespread. There is an increasing demand for new bio-fertilizers that can be applied with drip irrigation or fertigation, integrate microorganisms and soluble organic matter, and have multiple functions such as soil improvement, growth promotion and disease resistance. The development of bio-organic water-soluble fertilizers suitable for fertigation has also become a research hotspot.

[0003] Currently, bio-organic water-soluble fertilizers are mainly produced using water-soluble organic raw materials. Fulvic acid, amino acids, humic acid, molasses, and wood vinegar are the most commonly used raw materials, but their large-scale application is limited by the availability and price of these materials. Using fermentation and enzymatic hydrolysis to convert insoluble organic raw materials into water-soluble substances for the production of bio-organic water-soluble fertilizers is a promising production method, but current research is limited, with only reports of using tannery by-products and low-value fish by-products in the preparation of water-soluble organic fertilizers.

[0004] Kanamycin fermentation waste is a fermentation residue produced in the antibiotic fermentation industry. It is rich in protein, amino acids, carbohydrates, trace elements, and residual mycelium, making it an abundant and inexpensive resource and a good raw material for producing water-soluble bio-organic fertilizer using fermentation and enzymatic methods. However, the presence of residual kanamycin in the waste presents challenges for its comprehensive utilization. Enzymatic hydrolysis leaves kanamycin residue in the hydrolysate as it cannot be degraded, posing a risk. Fermentation hydrolysis is difficult due to kanamycin's inhibitory effect on fermenting microorganisms; if kanamycin cannot be degraded by microorganisms, it will also remain in the product. Therefore, it is essential to select microorganisms capable of degrading kanamycin for fermentation.

[0005] The applicant previously obtained a strain of *Bacillus sphaeroides* PAPM-A1 (CN202211697422X), with the accession number CGMCC No. 25969. This strain exhibits good application results in degrading kanamycin, producing extracellular polysaccharides, degrading proteins, producing IAA, promoting crop growth, and accelerating the composting of organic materials. However, this strain lacks significant antagonistic ability against pathogens such as tomato soft rot pathogens, and its application in disease prevention and control in agricultural production is not significant. To address this issue, this invention provides a composting Bacillus that can degrade kanamycin and decompose proteins. It can be used for the fermentation and hydrolysis of kanamycin fermentation waste. Compared to *Bacillus sphaeroides* PAPM-A1, it has stronger pathogen antagonistic ability, especially against tomato soft rot pathogens. When used in the production of bio-organic water-soluble fertilizer, it not only improves soil and promotes crop growth but also has a preventive effect against crop diseases such as tomato soft rot. Summary of the Invention

[0006] The purpose of this invention is to provide a bio-organic water-soluble fertilizer, its preparation method and application. This bio-organic water-soluble fertilizer is prepared by fermenting Bacillus stercoris PAPM-Y1-2 as an effective strain and enzymatically hydrolyzing kanamycin fermentation waste to obtain fermentation broth, and then compounding it with low-value fish enzymatic hydrolysate dry powder. It has the functions of improving soil, promoting crop growth and preventing tomato soft rot.

[0007] This invention first isolated a strain of Bacillus stercoris PAPM-Y1-2 from kanamycin fermentation waste compost. This strain was deposited on June 21, 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. 27681. This strain exhibits functions including degrading kanamycin, protein breakdown, IAA production, and antagonism against pathogens causing tomato soft rot.

[0008] This invention also provides a fermentation method for the aforementioned Bacillus composting strain PAPM-Y1-2, including steps such as strain activation, preparation of seed culture in Erlenmeyer flasks, preparation of strain in a liquid seed tank, and fermentation in a liquid fermenter. Specifically, strain activation is performed using LB medium slant culture in test tubes at a temperature of 35-37℃ for 20-24 hours; both Erlenmeyer flask seed preparation and liquid seed tank strain preparation use LB liquid medium at a temperature of 32-37℃, with shaking or stirring at 180-220 rpm for 18-30 hours; the inoculum size for liquid fermentation is 2%-4% (volume ratio), and the culture is performed at 32-37℃ for 36-48 hours to obtain the Bacillus composting strain PAPM-Y1-2 fermentation broth.

[0009] The above-mentioned liquid fermenter uses a composite culture medium with the following formula: 100-150 g / L kanamycin fermentation waste powder, 0.3-0.8 g / L potassium dihydrogen phosphate, 0.3-0.8 g / L magnesium sulfate, and 0.8-1.2 g / L neutral protease. The medium is enzymatically hydrolyzed at 50-55℃ for 60-90 min, then sterilized, with an initial pH of 6.5-7.5. A preferred formula is: 120 g / L kanamycin fermentation waste powder, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, and 1.0 g / L neutral protease, with an initial pH of 6.5-7.5.

[0010] This invention also provides a bio-organic water-soluble fertilizer with the above-mentioned Bacillus composting spores PAPM-Y1-2 fermentation broth as the main component, comprising: 60-80 parts by weight of Bacillus composting spores PAPM-Y1-2 fermentation broth, 19.85-39.95 parts by weight of low-value fish enzymatic hydrolysate dry powder, and 0.05-0.15 parts by weight of Kathon. Preferably, the composition is: 70 parts by weight of Bacillus composting spores PAPM-Y1-2 fermentation broth, 29.9 parts by weight of low-value fish enzymatic hydrolysate dry powder, and 0.1 parts by weight of Kathon.

[0011] The above-mentioned bio-organic water-soluble fertilizer is prepared by slowly adding low-value fish enzymatic hydrolysate dry powder and Kathon into the fermentation liquid while stirring until it is uniform and free of lumps. This is the bio-organic water-soluble fertilizer of the present invention, and its effective ingredient content is: Bacillus compostii PAPM-Y1-2 viable bacteria count ≥2×10⁻⁶. 9 cfu / mL, organic matter content ≥300g / L.

[0012] The preparation method of the low-value fish enzymatic hydrolysate powder is as follows: Low-value fish meal is prepared into a substrate solution with a concentration of 8-12%, and the protein is moderately denatured by heating. After cooling the solution, a complex protease is added at a concentration of 2.5-3.5% (percentage of substrate). Enzymatic hydrolysis is carried out at 50-60℃ for 5-8 hours, followed by enzyme inactivation by heating and rapid cooling to below 50℃. Solid-liquid separation is performed using a plate and frame filter press to obtain a clear filtrate. The filtrate is then concentrated under vacuum and spray-dried. The complex protease contains an alkaline protease activity ≥50,000 U / g and a neutral protease activity ≥30,000 U / g.

[0013] The present invention also discloses the method of using the above-mentioned biological organic water-soluble fertilizer, characterized in that, when used for crop cultivation, it is diluted with water 100-1000 times by weight and then applied by irrigation, with a dosage of 5-10 kg per acre each time, and applied 1-3 times throughout the crop's growth period.

[0014] The advantages of this invention are:

[0015] 1. The composting Bacillus PAPM-Y1-2 of the present invention has the functions of degrading kanamycin, decomposing proteins, producing IAA, and antagonizing the pathogens of tomato soft rot. The bio-organic water-soluble fertilizer produced by using this strain as a functional bacteria has the functions of improving soil, promoting crop growth, and preventing tomato soft rot.

[0016] 2. This invention uses kanamycin fermentation waste residue and low-value fish enzymatic hydrolysate as the main raw materials to prepare bio-organic water-soluble fertilizer, which not only helps to reduce production costs and broaden the sources of raw materials, but also helps to harmlessly and resourcefully utilize kanamycin fermentation waste residue. Attached Figure Description

[0017] Figure 1 The colony morphology of Bacillus compostii PAPM-Y1-2;

[0018] Figure 2 The cell morphology of Bacillus compostii PAPM-Y1-2;

[0019] Figure 3 A phylogenetic tree constructed based on a partial 16S rDNA sequence. Detailed Implementation

[0020] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0021] Example 1: Screening of Bacillus compostii PAPM-Y1-2

[0022] Bacillus subtilis PAPM-Y1-2 was isolated from kanamycin fermentation waste compost. The kanamycin fermentation waste compost samples were obtained from the composting workshop of Shandong Zuotian Biotechnology Co., Ltd.

[0023] Weigh 10g of compost sample, add 100mL of sterile water, place in a shaker and extract at 180r / min and 35℃ for 30min. Let stand, collect the suspension, and inoculate into LB liquid medium containing kanamycin for enrichment culture. Enrichment culture method: First, incubate in LB medium containing 300mg / L kanamycin at 35℃ and 180r / min for 3-5 days with constant temperature shaking; after the culture medium becomes turbid, transfer to LB medium with a kanamycin concentration of 600mg / L, and continue incubation at 35℃ and 180r / min for 3-5 days with constant temperature shaking; after the culture medium becomes turbid again, transfer to LB medium containing 900mg / L kanamycin, and continue incubation at 35℃ and 180r / min for 3-5 days with constant temperature shaking; finally, transfer to LB medium containing 1200mg / L kanamycin, and continue incubation at 35℃ and 180r / min for 3-5 days with constant temperature shaking to obtain the enriched culture. The LB liquid culture medium formula is as follows: 10g peptone, 5g yeast extract, 10g NaCl, 1000mL distilled water, pH 7.0, sterilized at 121℃ for 20min.

[0024] Enriched cultures were serially diluted with sterile water and spread onto 1 / 10 concentration LB agar plates. They were incubated at 35°C. After colony growth, single colonies were isolated to obtain strains with different colony morphologies. Based on differences in colony and cell morphology, different strains were transferred to LB agar slants and incubated at 35°C for 2-3 days. After confluent growth, they were stored at 4°C for later use. The 1 / 10 concentration LB agar is a medium with nutrient concentrations 1 / 10 of the standard LB agar, containing 1000 mg / L kanamycin. The obtained strains with different colony morphologies were inoculated into a medium with kanamycin as the sole carbon source and incubated at 35°C with constant temperature shaking at 180 rpm for 24-72 hours. Strains that caused turbidity in the medium were selected; these were the kanamycin-degrading strains and stored for later use. The culture medium formula with kanamycin as the sole carbon source is as follows: 2g (NH4)2SO4, 0.2g MgSO4, 0.5g NaH2PO4, 0.1g CaCl2, 0.5g K2HPO4, 1000mg kanamycin, and distilled water to 1L.

[0025] Antagonistic strains of the pathogen causing tomato soft rot were screened using the inhibition zone method. 200 ml of LB medium was melted and cooled to approximately 45°C. Then, 5 ml of a bacterial suspension of *Erwinia carotovora* subsp. *Carotovora* (Jones) Bergey et al. was added, mixed well, poured into petri dishes, and allowed to cool and solidify. Using pre-sterilized toothpicks, bacterial colonies of the isolated kanamycin-degrading strains were collected and inoculated onto agar plates. After incubation at 32°C for 48 hours, the growth of each strain was observed, and strains with a higher ratio of inhibition zone to colony diameter were selected.

[0026] Taking into account the growth ability of the screened strains in the culture medium with kanamycin as the sole carbon source and their ability to antagonize the pathogen of tomato soft rot, a bacterium with strong kanamycin degradation ability and pathogen antagonism ability was obtained, namely PAPM-Y1-2 strain.

[0027] Example 2: Identification of Bacillus compostii PAPM-Y1-2

[0028] (1) Morphological and physiological and biochemical characteristics

[0029] The morphological and physiological-biochemical characteristics of the PAPM-Y1-2 strain are as follows: After culturing on LB medium at 35℃ for 24 hours, the colony diameter is approximately 3-6 mm. The colonies are round, moist, off-white, with irregular edges and a uniform texture. Figure 1 The colony characteristics varied slightly with different incubation times. The bacteria were rod-shaped, Gram-positive, and spore-forming. Figure 2 The fermentation tests for glucose, mannitol, and sorbitol were all positive; the starch hydrolysis test was positive; the nitrate reduction test was positive; the ammonia production test was positive; the catalase test was positive; the VP test was negative; and the indole test was negative.

[0030] (2) 16S rDNA sequence analysis

[0031] The PAPM-Y1-2 strain was inoculated into LB liquid medium and cultured at 35°C and 200 rpm for 24 h. The bacterial cells were collected, and total DNA was extracted. Using this DNA as a template, PCR amplification of the 16S rRNA gene was performed using universal primers for prokaryotic 16S rRNA: F27:5′-AGA GTT TGATCA TGG CTC AG-3′ and F27:5′-AGA GTT TGA TCA TGG CTC AG-3′. The amplification conditions were: 95°C pre-denaturation for 3 min, 94°C denaturation for 1 min, 55°C annealing for 1 min, 72°C extension for 1.5 min, for a total of 30 cycles, followed by a final extension at 72°C for 10 min. The amplified products were separated by 1% agarose gel electrophoresis, recovered using a gel extraction kit, and sequenced. The obtained sequence is shown in SEQ No. 1 of the sequence listing. The measured 16S rDNA sequence was compared with sequences in the GenBank database for multiple sequence homology analysis, and a phylogenetic tree was constructed, such as... Figure 3 As shown. The 16SRNA sequence of strain PAPM-Y1-2 (SEQ No. 1):

[0032]

[0033] Through the above steps, strain PAPM-Y1-2 was identified as Bacillus stercoris. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 21, 2023, with accession number CGMCC No. 27681.

[0034] Example 3: Determination of beneficial characteristics of Bacillus compostii PAPM-Y1-2

[0035] (1) The degradation ability of Bacillus compostii PAPM-Y1-2 on kanamycin

[0036] Bacillus compostii strain PAPM-Y1-2 was inoculated into kanamycin fermentation waste medium and cultured at 35℃ with shaking at 200 rpm for 72 h. The medium without inoculation with Bacillus compostii strain PAPM-Y1-2 served as a blank control. The residual amount of kanamycin in the medium before and after fermentation was determined by liquid chromatography, and the degradation rate was calculated.

[0037] The preparation method of the kanamycin fermentation waste residue culture medium is as follows: after drying, the kanamycin fermentation waste residue is pulverized to 200 mesh, 10g is weighed, 100mL of water is added, the pH is adjusted to 6.5-7.0, and sterilized at 121℃ for 20min. The kanamycin fermentation waste residue is a solid waste generated during the kanamycin fermentation production process, with an organic matter content of 78.54%, nitrogen, phosphorus, and potassium contents of 3.23%, 1.36%, and 0.65%, respectively, and a kanamycin residue of 2.017mg / g.

[0038] The experimental results showed that the residual amount of kanamycin in the blank control treatment was 0.1815 mg / mL, while the residual amount of kanamycin in the experimental group inoculated with Bacillus compostii strain PAPM-Y1-2 was 0.0071 mg / mL. This indicates that Bacillus compostii strain PAPM-Y1-2 has a strong degradation ability for kanamycin.

[0039] (2) The protein-degrading ability of Bacillus compostii PAPM-Y1-2

[0040] Bacillus compostii PAPM-Y1-2 was inoculated onto skim milk powder agar plates using sterilized toothpicks and incubated at 35℃ for 48 hours. The clear zone and colony diameter were then measured, and the clear zone to colony diameter ratio (HC) was calculated. The results showed that Bacillus compostii PAPM-Y1-2 produced a clear zone diameter of 13.2 mm and a colony diameter of 4.1 mm on the skim milk powder agar plate, with an HC value of 3.219. This indicates that the strain has a strong protein-decomposing ability, which is beneficial for its hydrolysis of kanamycin fermentation waste and its growth and reproduction in soil, as well as its decomposition and transformation of soil organic nutrients. The skim milk powder agar plate formulation was: 3.0 g skim milk powder, 1 g glucose, 1.6% agar, 200 mL deionized water, pH 7.0-7.2, sterilized at 108℃ for 15 min.

[0041] (3) Determination of IAA production capacity of Bacillus compostii PAPM-Y1-2

[0042] Bacillus compostii PAPM-Y1-2 was inoculated into LB liquid medium containing L-tryptophan (200 mg / L) and cultured at 35℃ with shaking at 200 rpm for 48 h. 50 μL of the bacterial suspension was dropped onto a white ceramic plate, along with 50 μL of Salkowski colorimetric solution. A positive control was prepared by adding 50 μL of a colorimetric solution containing 50 mg / L IAA. The white ceramic plate was observed after being placed at room temperature in the dark for 30 min; a red color indicated the ability to secrete IAA. The experimental results showed that the Bacillus compostii PAPM-Y1-2 bacterial suspension turned red, indicating that this strain has the ability to secrete IAA. The Salkowski colorimetric solution was formulated as follows: 450 mL of 35% HClO and 1 mL of 0.5 mol / L FeCl3.

[0043] Example 4: Preparation of fermentation broth of Bacillus compostii PAPM-Y1-2

[0044] 1) Strain activation: The Bacillus compostingus PAPM-Y1-2 strain, preserved at low temperature, was transferred to LB solid medium slant tubes and activated by incubation at 35℃ for 24 hours. The LB solid medium formula is as follows: 5g yeast extract, 10g tryptone, 10g sodium chloride, 20g agar powder, 1000mL water, pH 7.5.

[0045] 2) Preparation of seed culture in Erlenmeyer flasks: Activated Bacillus compostii PAPM-Y1-2 bacterial growth was scraped off with an inoculation loop and inoculated into LB liquid medium in Erlenmeyer flasks. The flasks were then incubated at 35°C and 180 rpm with shaking for 24 hours to obtain the seed culture. The LB liquid medium consisted of 5g yeast extract, 10g tryptone, 10g sodium chloride, and 1000mL water, with a pH of 7.5.

[0046] 3) Preparation of inoculum in liquid seed tank: The seed culture from the Erlenmeyer flask was transferred at an inoculation rate of 3% into a seed tank containing LB liquid culture medium and incubated at 35℃ for 24 hours to obtain the inoculum for the seed tank. During incubation, the stirring speed was 200 rpm, the aeration rate was 8 L / min from 0-10 hours, and the aeration rate was 16 L / min from 11-24 hours. The seed tank had a volume of 20 L and contained 16 L of culture medium.

[0047] 4) Transfer the inoculum from the seed tank to a 1000L fermenter at an inoculation rate of 3%. The fermenter contains 700L of fermentation medium and is incubated at 35℃ for 36 hours. During fermentation, the stirring speed is 200 rpm, and the aeration rate is 300-350 L / min for the first 10 hours, then 600-700 L / min. After fermentation, the Bacillus compostii PAPM-Y1-2 fermentation broth is obtained, with a viable cell count of 1.0 × 10⁻⁶. 10 cfu / mL, used for the formulation of biological organic water-soluble fertilizers.

[0048] The fermentation medium is formulated as follows: 120 g / L kanamycin fermentation waste powder, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, and 1.0 g / L neutral protease, with an initial pH of 6.5-7.5. The neutral protease is specified as 100,000 U / g, and its activity unit definition and detection method comply with the national standard GB / T 23527-2009 "Protein Preparations". The liquid fermentation medium is prepared by: preparing the medium according to the formulation, enzymatically hydrolyzing at 50-55℃ for 60-90 min, and then sterilizing at 121℃ for 15-30 min. The kanamycin fermentation waste is solid waste generated during the kanamycin fermentation process, with an organic matter content of 78.54%, nitrogen, phosphorus, and potassium contents of 3.23%, 1.36%, and 0.65%, respectively, and a kanamycin residue of 2.017 mg / g.

[0049] Example 5: Formulation of Bio-organic Water-soluble Fertilizer

[0050] Bio-organic water-soluble fertilizer was prepared using the Bacillus composting spores PAPM-Y1-2 fermentation broth prepared in Example 4. The formula was as follows: 70 parts by weight of fermentation broth, 29.9 parts by weight of low-value fish enzymatic hydrolysate powder, and 0.1 parts by weight of Kathon. The low-value fish enzymatic hydrolysate powder and Kathon were slowly added to the fermentation broth according to the formula, stirring constantly until a uniform consistency without lumps was achieved. This constituted the bio-organic water-soluble fertilizer of the present invention, with an effective component content of ≥2 × 10⁻⁶ viable Bacillus composting spores PAPM-Y1-2. 9 cfu / mL, organic matter content ≥300g / L.

[0051] The preparation method of the low-value fish enzymatic hydrolysate dry powder is as follows: Low-value fish meal is prepared into a substrate concentration of 10% and stirred evenly. The solution is heated at 90°C for 10 minutes to moderately denature the protein. After cooling, a complex protease is added at 3% (percentage of substrate). Enzymatic hydrolysis is carried out at 55°C for 6 hours, followed by enzyme inactivation at 90°C for 10 minutes, and then rapidly cooled to below 50°C. Solid-liquid separation is performed using a plate and frame filter press to obtain a clear filtrate. The filtrate is concentrated using a triple-effect vacuum concentrator to a solid content of over 20%, and then spray-dried. The process conditions are: liquid temperature 50-55°C, inlet temperature 180-200°C, and outlet temperature 70-80°C. The complex protease contains an alkaline protease activity of 100,000 U / g and a neutral protease activity of 50,000 U / g.

[0052] Application Example 1: Application in Tomato Production

[0053] The tomato pot experiment used pots with an inner diameter of 20cm (top), 14cm (bottom), and 16cm (height), each containing 3kg of soil. The experiment was conducted in Dongzhuang Village, Ancheng Town, Pingyin County, Shandong Province. The soil used had a pH of 6.68, an organic matter content of 16.354g / kg, a available nitrogen content of 60.5mg / kg, a available phosphorus content of 72.06mg / g, and a available potassium content of 168.61mg / g. The pot experiment included two treatment groups (T1 and T2) and one control group (CK), with 15 pots in each treatment, and one tomato plant per pot. Treatment group T1 received 15g of the fermentation broth of Bacillus compostii PAPM-Y1-2 prepared in Example 4 of this invention per pot; treatment group T2 received 15g of the bio-organic water-soluble fertilizer prepared in Example 5 of this invention per pot; and the control group CK received 15g of the culture medium used to prepare the fermentation broth of Bacillus compostii PAPM-Y1-2 per pot. Fertilizer was applied via irrigation, 5g each time. The first application was made 7 days after tomato transplanting, followed by applications every 10 days for a total of 3 times. Relevant indicators were measured on day 60 of the pot experiment. Plant height and stem diameter were measured using a measuring tape and vernier calipers, respectively; fresh weight was determined by weighing. Rhizosphere soil enzyme activities were measured: urease was determined using the sodium phenolate colorimetric method, catalase was determined using the potassium permanganate titration method, and sucrase was determined using the DNS colorimetric method.

[0054] The experimental results are shown in Tables 1 and 2. Compared with the control group (CK), the plant height, stem diameter, and fresh weight of treatment groups T1 and T2 were all increased to varying degrees. This indicates that the application of the fermentation broth of Bacillus compostingus PAPM-Y1-2 described in this invention and the bio-organic water-soluble fertilizer prepared with it as the main component both have a promoting effect on tomato growth. Compared with the control group (CK), the activities of urease, catalase, and sucrase in treatment groups T1 and T2 were all increased to varying degrees. This indicates that the fermentation broth of Bacillus compostingus PAPM-Y1-2 described in this invention and the bio-organic water-soluble fertilizer prepared with it as the main component can both increase the enzyme activity in the rhizosphere soil of tomatoes.

[0055] Table 1 Effects on tomato growth

[0056]

[0057] *Different letters in the same column of the table indicate significant differences (P < 0.05).

[0058] Table 2 Effects on enzyme activity in tomato rhizosphere soil

[0059]

[0060] *Different letters in the same column of the table indicate significant differences (P < 0.05).

[0061] Application Example 2: Control of Tomato Soft Rot

[0062] The tomato pot experiment used pots with an inner diameter of 20cm (top) and 14cm (bottom) and a height of 16cm, each containing 3kg of soil. The experiment was conducted in Dongzhuang Village, Ancheng Town, Pingyin County, Shandong Province. The soil used had a pH of 6.68, an organic matter content of 16.354g / kg, available nitrogen content of 60.5mg / kg, available phosphorus content of 72.06mg / g, and available potassium content of 168.61mg / g. The pot experiment included one control group (CK) and one treatment group (T), with 50 tomato seedlings in each group. The control group (CK) was treated with *Erwinia carotene* inoculum solution (1×10⁻⁶) per plant on the first day after transplanting. 6 50 mL of pre-sterilized bio-organic water-soluble fertilizer (CFU / mL) was applied to each tomato plant on days 1, 16, and 31, diluted 100 times. Treatment group T: 1 × 10⁻⁶ cfu / mL Erwinia carotene solution was applied to each plant on day 1 after transplanting. 6 Apply 50 mL of the bio-organic water-soluble fertilizer (cfu / mL) diluted 100 times on days 1, 16, and 31, for a total of 3 applications. Observe the symptoms and calculate the severity and efficacy of the disease on days 20, 40, and 60.

[0063] The disease grading standard uses the whole plant as the indicator, and the specific grades are as follows: Grade 0: No symptoms; Grade 1: Water-soaked green patches first appear on the stem near the ground; Grade 3: The stem tip expands into round or irregular brown spots; Grade 5: The fruit lesions in the mature stage are round, faded white spots; Grade 7: The fruit lesions turn into dirty brown spots; Grade 9: The whole plant and fruit rot.

[0064] Disease index (%) = ∑(Grade value × Number of plants) / (9 × Total number of plants) × 100

[0065] Prevention and control efficacy (%) = (Control disease index - Treatment disease index) / Control disease index × 100

[0066] The experimental results are shown in Table 3. The disease index of tomatoes in the treatment group T was significantly lower than that in the control group CK, and the control efficacy reached 73.91%, 69.33%, and 55.26% on days 20, 40, and 60, respectively.

[0067] Table 3. The effect of bio-organic water-soluble fertilizer on the prevention and control of soft rot in tomatoes.

[0068]

Claims

1. A strain of Bacillus compostii ( Bacillus stercoris PAPM-Y1-2, its accession number is CGMCCNo.27681.

2. The Bacillus composting strain described in claim 1 ( Bacillus stercoris Applications of PAPM-Y1-2 in the production of IAA, protein decomposition, kanamycin degradation, and antagonism of tomato soft rot pathogens.

3. The Bacillus composting strain described in claim 1 ( Bacillus stercoris The fermentation method of PAPM-Y1-2 includes strain activation, seed preparation in Erlenmeyer flasks, strain preparation in liquid seed tanks, and fermentation in liquid fermenters; its characteristic is that... The inoculum was activated using LB medium slant culture in test tubes at a temperature of 35-37℃ for 20-24 hours. Seed preparation in Erlenmeyer flasks and liquid seed tanks was performed using LB liquid medium at a temperature of 32-37℃, with shaking or stirring at 180-220 rpm for 18-30 hours. The inoculum size in the liquid fermenter was 2%-4%, and the culture was carried out at 32-37℃ for 36-48 hours to obtain the fermentation broth of Bacillus compostii PAPM-Y1-2. The liquid fermenter used a composite culture medium, specifically: 100-150 g / L kanamycin fermentation waste powder, 0.3-0.8 g / L potassium dihydrogen phosphate, 0.3-0.8 g / L magnesium sulfate, and 0.8-1.2 g / L neutral protease. Enzymatic hydrolysis was carried out at 50-55℃ for 60-90 minutes followed by sterilization, with an initial pH of 6.5-7.

5.

4. The fermentation broth of Bacillus compostii PAPM-Y1-2 prepared by the fermentation method of claim 3.

5. A bio-organic water-soluble fertilizer with the fermentation broth of Bacillus compostingus PAPM-Y1-2 as described in claim 4 as the main component, characterized in that, Its composition is as follows: by mass, 60-80 parts of Bacillus composting spores PAPM-Y1-2 fermentation broth, 19.85-39.95 parts of low-value fish enzymatic hydrolysate dry powder, and 0.05-0.15 parts of Kathon.

6. The bio-organic water-soluble fertilizer as described in claim 5, characterized in that, By mass fraction, the contents included 70 parts of Bacillus composting spores PAPM-Y1-2 fermentation broth, 29.9 parts of low-value fish enzymatic hydrolysate dry powder, and 0.1 parts of Kathon.

7. The bio-organic water-soluble fertilizer as described in claim 5 or 6, characterized in that, Its effective ingredient content is: Bacillus compostii PAPM-Y1-2 viable bacteria count ≥2×109 cfu / mL, organic matter content ≥300g / L.

8. The method for preparing the bio-organic water-soluble fertilizer as described in claim 5 or 6, characterized in that, Low-value fish enzymatic hydrolysate dry powder and Kathon are slowly added to the fermentation liquid of Bacillus compostii PAPM-Y1-2 while stirring until it is uniform and free of lumps, thus obtaining biological organic water-soluble fertilizer.

9. The application of the bio-organic water-soluble fertilizer according to claim 5 or 6 in improving soil, promoting crop growth and preventing tomato soft rot.

10. The method of using the biological organic water-soluble fertilizer according to claim 5 or 6, characterized in that, When used for crop cultivation, dilute with water 100-1000 times by weight and apply by irrigation. The dosage is 5-10 kg per acre each time, and it should be applied 1-3 times throughout the crop's growth period.

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