Microbial fertilizer and preparation method thereof

Abstract

The present application belongs to the technical field of microbial fertilizer, and more particularly relates to a carbon-based microbial fertilizer and a preparation method thereof and application of the carbon-based microbial fertilizer in promoting growth of tomatoes and improving quality of tomato fruits. The microbial fertilizer comprises the following components in parts by weight: 60-70 parts of biochar, 10-20 parts of functional bacterial agent, 5-10 parts of bentonite, 5-10 parts of bacillus subtilis fragments and 5-10 parts of tea tree essential oil fermentation product.

Description

Technical Field

[0001] This invention belongs to the field of microbial fertilizer technology. More specifically, it relates to carbon-based microbial fertilizers and their preparation methods, as well as their application in promoting tomato growth and improving tomato fruit quality. Background Technology

[0002] Microorganisms can improve the ecological environment of arable soil through their life activities, regulate soil properties, increase the supply of plant nutrients, promote crop growth, and increase crop yield and quality, thereby improving soil and enhancing the quality of agricultural products.

[0003] Biochar possesses physical properties such as a large specific surface area, abundant pore structure, and strong adsorption capacity. [1] It can effectively improve the physical and chemical properties of soil and increase the absorption and accumulation of nutrients and heavy metals by crops. Secondly, the abundant chemical functional groups and surface charge on the surface of biochar also endow it with a higher cation exchange capacity and more significant adsorption performance, firmly fixing microorganisms on it and providing a suitable living environment for them. [2] Combining microorganisms with biochar not only provides a certain degree of sustained-release function. [3] It also makes nutrient utilization more efficient, reduces various environmental problems caused by the unreasonable application of chemical fertilizers, improves fertilizer nutrient utilization, and reduces the use of chemical fertilizers. [4] Based on the unique structure of biochar and the soil-improving effect of microorganisms, it can better repair the soil, regulate the microbial environment and colony structure in the soil, which has good ecological significance and achieves weight reduction, high efficiency and environmental protection.

[0004] However, carbon-based microbial fertilizers still face many technical challenges in actual research and development and application. For example, the high pH of biochar (alkaline carbon can reach pH 9-10) and the porous structure adsorbing signal molecules or nutrients secreted by microorganisms affect the colonization of microorganisms. Secondly, the tar or phenolic substances remaining on the carbon surface may inhibit the activity of microorganisms. Furthermore, the microorganisms are easily deactivated during the subsequent drying process, resulting in a low survival rate of microorganisms in the carbon pores.

[0005] References:

[0006] [1] Ji Changling. Mechanism of biochar-based composite materials synergistically treating chlorinated hydrocarbon-contaminated groundwater by microorganisms [J]. East China University of Science and Technology, 2019.

[0007] [2] Ma, Fengfeng. Adsorption-immobilization of cadmium in loess by biochar and its effect on phytoavailability and its mechanism [D]. Lanzhou: Lanzhou Jiaotong University, 2017

[0008] [3] Ren Hongyang, Wang Bing, Xie Hongli, et al. A method for preparing immobilized bacterial agents based on biochar slow-release nutrient substrate [M].

[0009] [4] Qiu Shaojun, Li Ning, He Ping, et al. Study on changes in nutrient utilization efficiency of chemical fertilizers in spring maize in typical black soil[J]. Chinese Agricultural Science, 2019, 52(16):11. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing carbon-based microbial fertilizers in which it is difficult to maintain the activity of microorganisms, and to provide a carbon-based microbial fertilizer that promotes tomato growth and improves the quality of tomato fruit.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] This invention provides a microbial fertilizer comprising the following components by mass fraction: 60-70 parts biochar, 10-20 parts functional microbial agent, 5-10 parts bentonite, 5-10 parts Bacillus subtilis fragments, and 5-10 parts tea tree essential oil ferment.

[0013] Biochar is a stable, highly aromatic, carbon-rich solid material prepared from biomass feedstock through slow pyrolysis and carbonization under limited oxygen conditions. Commonly used biochar feedstocks in this field are typically plant-based and animal-based; plant-based biochar feedstocks include corn stalks, rice stalks, wheat stalks, corn cobs, sunflower stalks, rice husks, chestnut shells, banana peels, durian shells, and vegetable leaves; animal-based biochar feedstocks include poultry and livestock manure.

[0014] In one embodiment of the present invention, the biochar raw material is selected from rice husks, corn stalks, and rice straw. In a more preferred embodiment, the biochar raw material is selected from corn stalks, which have a higher biochar element content than the other two types, thus providing a more favorable environment for microbial growth.

[0015] In one embodiment of the present invention, the microbial agent is selected from one or more of Bacillus megaterium, Bacillus amyloliquefaciens, and Alcaligenes faecalis; in a more preferred embodiment of the present invention, the microbial agent is composed of Bacillus megaterium, Bacillus amyloliquefaciens, and Alcaligenes faecalis in a weight ratio of 4:3:3.

[0016] The Bacillus megaterium described in this article belongs to the Phosphate Solubilizers. It can secrete organic acids (citric acid, oxalic acid) and dissolve insoluble phosphates (such as Ca(PO4)2). Through multiple mechanisms, it significantly promotes crop growth (nitrogen fixation, phosphorus and potassium solubilization, and plant hormone synthesis), enhances stress resistance (through the secretion of lipopeptide antibiotics, competition with and parasitic control of pathogens; activation of defense pathways and regulation of antioxidant enzyme-induced systemic resistance), and improves soil microecology (degradation of organic matter and regulation of microbial colonies).

[0017] The Bacillus proteolyticus described in this article is a type of probiotic that can secrete proteases and degrade organic nitrogen. It can promote crop growth (organic nitrogen mineralization and nutrient supply, plant hormone regulation), improve soil fertility (organic matter degradation and humification, microbial community regulation), and enhance plant stress resistance (secretion of antimicrobial substances, competitive ecological niche, and induction of systemic resistance) through multiple mechanisms.

[0018] The Alcaligenes faecalis described in this article is a Gram-negative probiotic widely found in soil, water, and plant rhizosphere. It plays a variety of positive roles in regulating tomato growth, including promoting nutrient absorption (nitrogen fixation, phosphorus and potassium solubilization, and regulation of plant hormone synthesis), enhancing disease resistance (secreting antibiotics, inducing systemic resistance (ISR), and inhibiting pathogen colonization), and alleviating abiotic stress (accumulation of osmotic regulators, activation of antioxidant enzymes, and secretion of extracellular polysaccharides).

[0019] The synergistic advantages among the three microorganisms in this paper are reflected in: ① Nitrogen-phosphorus synergy: protein-degrading bacteria release organic nitrogen → Alcaligenes faecalis fixes nitrogen → Bacillus megaterium activates phosphorus, forming a nutrient cycle; ② Stress resistance + growth promotion: ACC deaminase (1-aminocyclopropane-1-carboxylic acid deaminase) secreted by Alcaligenes faecalis alleviates stress, and IAA (Bacillus megaterium) promotes root absorption. Through carrier screening and process optimization, this combination has the potential for nutrient cycling and growth promotion and disease resistance.

[0020] In one embodiment of the present invention, the Bacillus subtilis fragments were prepared by enzymatic hydrolysis. Compared with the ultrasonic method, the enzymatic hydrolysis method can obtain smaller peptidoglycan fragments (1-10 kDa).

[0021] As one embodiment of the present invention, the Bacillus subtilis fragments are prepared by the following steps:

[0022] Step S1: Resuspend the bacterial cells in lysis buffer and adjust the concentration to 1×10⁻⁶. 5 ~1×10 9 CFU / mL;

[0023] Step S2: Add lysozyme and benzyl sulfonyl fluoride, and incubate with gentle shaking at 30-37°C for 30-60 minutes; the addition of benzyl sulfonyl fluoride is to prevent protein degradation.

[0024] Step S3: Sonicate under ice bath for 3-5 minutes, then add Triton X-100; Triton X-100 acts as a detergent and also enhances membrane disruption and assists in disruption.

[0025] Step S4: Inactivate the enzyme, cool, centrifuge, discard the supernatant, and collect the precipitate to obtain cell debris; in this step, enzyme inactivation can be achieved by adding EDTA (final concentration 5mM) to chelate Mg. 2+ This terminates the action of lysozyme.

[0026] In one embodiment of the present invention, in step S1, the lysis buffer is 30-50 mM Tris-HCl + 1 mM EDTA, and the pH is 8.0; in step S2, the final concentrations of lysozyme and benzyl sulfonyl fluoride are 1-2 mg / mL and 1-2 mM, respectively.

[0027] In one embodiment of the present invention, in step S3, the ultrasonic power is 100-200W, and 0.1-0.3% Triton X-100 is added; in step S4, the centrifugation conditions are 4°C, centrifugation at 10,000×g for 10-20 minutes.

[0028] As one embodiment of the present invention, the tea tree essential oil ferment is prepared by the following steps:

[0029] Step A: Dissolve tea tree essential oil in a 30-60% ethanol solution to obtain a tea tree essential oil solution with a concentration of 10-20% by volume;

[0030] Step B: Inoculate the Yeast Extract seed culture into the fermentation medium at an inoculation rate of 1-5% and ferment for 24-48 hours. Then, add tea tree oil solution with a final concentration of 0.1-1% (v / v) and continue fermentation for 48-96 hours.

[0031] Step C: Collect the fermentation broth, centrifuge, take the supernatant, filter, and you have the product.

[0032] In one embodiment of the present invention, the fermentation temperature is 28-30℃, the fermentation pH is 4.0-6.0, the shaking speed is 150-200 rpm, and the aeration rate is 0.5-1.0 vvm.

[0033] In one embodiment of the present invention, the fermentation medium contains 10-30 g / L glucose, 5-10 g / L yeast extract, 1-2 g / L potassium dihydrogen phosphate, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.01-0.1 mg / L biotin, and pH 4.0-6.0.

[0034] During the fermentation process described above, the concentration of tea tree oil (TTO) needs to be controlled within a low range. When it exceeds 1%, it is found to inhibit the growth of the *Yamylostella lipolytica* strain. Secondly, the timing of TTO addition is also important. Adding it at the beginning of fermentation will inhibit mycelial growth, while adding it in the middle of fermentation (when mycelial biomass is most stable at 24–48 h) can avoid the above effects.

[0035] The inventive concept of this invention stems from the unexpected observation that microbial fertilizers containing Bacillus subtilis fragments exhibited superior effects in promoting tomato growth and improving fruit quality. It was speculated that this might be related to the ability to maintain and enhance the activity of the microbial agent. However, in practical applications, it was found that Bacillus subtilis fragments are easily degraded in the soil, making it difficult to maintain their effectiveness. Therefore, the idea was to use biochar to simultaneously adsorb Bacillus subtilis cell fragments and the compound microbial agent, reducing microbial contact and delaying the degradation of Bacillus subtilis fragments. Furthermore, the addition of fermented tea tree oil further enhanced these effects, significantly improving tomato growth, fruit quality, and yield. This may be because certain substances in the fermented tea tree oil, besides exerting antibacterial effects and delaying the degradation of cell fragments by microorganisms, may also induce plant resistance and the secretion of growth hormones.

[0036] The microbial fertilizer provided by this invention incorporates innovative ingredients "Bacillus subtilis fragments" and "tea tree essential oil fermentation products." Both work together in the entire formula to enhance and maintain microbial activity, promote tomato growth, and improve the quality and yield of tomato fruits. This discovery could not be found in the prior art before the application date.

[0037] The present invention also provides a method for preparing the microbial fertilizer, comprising the following steps:

[0038] Step 1) Biochar preparation: Rice husks, corn stalks or rice straw are crushed into 1-2 mm pieces and biochar is prepared by high-temperature slow pyrolysis.

[0039] Step 2) Biochar pretreatment: The biochar is acid-washed to remove alkali and then activated to improve pore size.

[0040] Step 3) Functional microbial agent loading: Mix biochar, functional microbial agent, Bacillus subtilis fragments and tea tree essential oil fermentation material and load into a sealed impregnation tank; impregnate at -0.08 to -0.1 MPa for 30 to 45 minutes; slowly release the vacuum, repeat the impregnation 2 to 3 times, then place on a shaker at 50 rpm and 25°C for 30 to 60 minutes; centrifuge, take the precipitate and pre-freeze at -20 to -40°C for 2 to 5 hours, vacuum dry, and mix with bentonite to obtain the final product.

[0041] In one embodiment of the present invention, the biochar prepared by the high-temperature slow pyrolysis method in step 1) has the characteristics of high porosity, stable chemical properties, and abundant surface functional groups. The specific operating steps of the high-temperature slow pyrolysis method of the present invention are as follows: The pulverized biomass material is sieved, placed in a quartz boat, leveled and compacted, wrapped with tin foil, and then placed in a vacuum tube furnace. 99.999% pure nitrogen is used as the carrier gas and protective gas, the nitrogen flow rate is controlled at 100 mL / min, the heating rate is 20°C per minute, the final pyrolysis temperature is set to 600°C and maintained for 2–3 hours, and after pyrolysis, it is placed in a desiccator and cooled to obtain the final product.

[0042] In one embodiment of the present invention, in step 2), the biochar prepared from rice husks, corn stalks, or rice straw has a pH of approximately 10-11. Acid washing to remove alkali is to lower the pH of the biochar and improve its compatibility with microorganisms. Specific operating steps include: soaking the biochar in 1M HCl for 24 hours, washing with deionized water until neutral, and drying at 60°C; the specific operating steps for pore activation are: placing the biochar in a 105°C oven for 2 hours to remove moisture and volatile substances from the pores.

[0043] As one embodiment of the present invention, step 3) of the vacuum impregnation process includes the following specific steps: Pretreated biochar, functional microbial agents, Bacillus subtilis fragments, and tea tree essential oil fermentation products are mixed in proportion and placed into a sealed impregnation tank; a vacuum is drawn to -0.08 to -0.1 MPa and maintained for 30 to 45 minutes, then the air in the char pores is removed; the vacuum is slowly released, and atmospheric pressure is used to force the bacterial solution, Bacillus subtilis fragments, and tea tree essential oil fermentation products into the biochar pores, repeated 2 to 3 times to improve the loading rate; the impregnated mixture is placed on a shaker at 50 rpm and 25°C for 30 to 60 minutes to promote uniform adsorption; centrifuged at 1000 to 2000 × g for 3 to 5 minutes to remove unadsorbed free bacterial solution; pre-frozen at -20 to -40°C for 2 to 5 hours, vacuum dried for 24 hours, then mixed with bentonite and stored to obtain the final product.

[0044] The present invention also provides the application of the aforementioned microbial fertilizer in promoting tomato growth and improving tomato fruit quality.

[0045] The "promoting tomato growth" mentioned in this article mainly refers to promoting the improvement of plant morphological indicators (plant height, stem diameter, and fresh weight, etc.) and yield; the "improving tomato fruit quality" mentioned in this article mainly refers to improving the soluble solids content, vitamin C content, sugar-acid ratio, and lycopene content of the fruit.

[0046] The present invention has the following beneficial effects:

[0047] (1) The present invention adds “Bacillus subtilis fragments” and “tea tree essential oil fermentation product” to carbon-based microbial fertilizer, which can effectively reduce the loss of functional microbial agents during the production and storage process; the test shows that after being placed at room temperature for 24 months, the number of viable bacteria decreased by 16-21%, while the number of effective viable bacteria in fertilizers without the above ingredients was 30-52%.

[0048] (2) The addition of “Bacillus subtilis fragments” and “tea tree essential oil fermentation product” to carbon-based microbial fertilizer significantly improved the growth-promoting effect of microbial fertilizer on tomatoes, and the difference was statistically significant compared with no addition. The above effect may be due to the positive effect of the two on maintaining the activity of functional microbial agents and inducing plant resistance and growth hormone secretion. Detailed Implementation

[0049] The present invention can be further described through the following embodiments; however, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from its spirit and scope.

[0050] In the following examples, tea tree oil was purchased from Guangzhou Haishi Biotechnology Co., Ltd., catalog number MELAO-W076; Bacillus megaterium was purchased from Shandong Dehe Biotechnology Group Co., Ltd.; Bacillus amyloliquefaciens was purchased from Shanghai Yushao Biotechnology Co., Ltd., catalog number YS-M5717; and Alcaligenes faecalis was purchased from Pusrui (Shanghai) Biomedical Co., Ltd.

[0051] Example 1: Preparation of tea tree oil fermentation product

[0052] Step A, Tea Tree Oil Pretreatment: Dissolve tea tree oil in 50% ethanol solution to obtain a tea tree oil solution with a concentration of 10% by volume.

[0053] Step B, Preparation of *Yersinia lipolytica* seed culture: Pick an appropriate amount of *Yersinia lipolytica* (ATCC90811) from the slant and inoculate it into a test tube containing YPD medium. Activate the strain by shaking and culturing in a shaker at 28–30°C and 200 rpm for 24 h. Inoculate the activated *Yersinia lipolytica* at an inoculation rate of 3% into seed culture medium (containing 20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, pH 4.0–6.0). Cultivate in a shaker at 28–30°C and 200 rpm for 24 h to allow the cells to grow to the logarithmic growth phase, thus obtaining the seed culture.

[0054] Step C, Fermentation: Inoculate the seed culture at a volume fraction of 5% into the fermentation medium (20 g / L glucose, 10 g / L yeast extract, 1 g / L potassium dihydrogen phosphate, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 0.05 mg / L biotin, pH 4.0-6.0) and ferment for 48 h. Then add tea tree oil solution with a final concentration of 0.5% (v / v) and ferment for 96 h. Collect the fermentation broth, centrifuge at 10000×g for 15 minutes at 4℃, take the supernatant and filter through a 0.22 μm ultrafiltration membrane to obtain the final product.

[0055] Example 2: Preparation of tea tree oil fermentation product

[0056] The difference from Example 1 is that in step C, a tea tree essential oil solution with a final concentration of 0.1% (v / v) is added and fermented for 96 hours, while the other parameters are the same as in Example 1.

[0057] Example 3: Preparation of tea tree oil fermentation product

[0058] The difference from Example 1 is that in step C, a tea tree essential oil solution with a final concentration of 1% (v / v) is added and fermented for 48 hours, while the other parameters are the same as in Example 1.

[0059] Example 4: Microbial Fertilizer (parts by weight)

[0060]

[0061]

[0062] Preparation process:

[0063] Step 1) Biochar preparation: Corn stalks are crushed into 1-2 mm pieces. The crushed biomass material is sieved, packed into a quartz boat, flattened and compacted, wrapped with tin foil and placed in a vacuum tube furnace. 99.999% pure nitrogen is used as the carrier gas and protective gas. The nitrogen flow rate is controlled at 100 mL / min, the heating rate is 20℃ per minute, the final pyrolysis temperature is set at 600℃ and maintained for 2 hours. After pyrolysis, it is placed in a desiccator and cooled to obtain corn biochar.

[0064] Step 2) Biochar pretreatment: Soak corn stalk biochar in 1M HCl for 24 hours, wash with deionized water until neutral, and dry at 60℃; place the biochar in a 105℃ oven for 2 hours to remove moisture and volatile substances from the pores;

[0065] Step 3) Preparation of Bacillus subtilis fragments: Resuspend Bacillus subtilis in lysis buffer (30-50 mM Tris-HCl + 1 mM EDTA, pH 8.0) and adjust the concentration to 1 × 10⁻⁶. 9 CFU / mL; add 1 mg / mL lysozyme and 1 mM benzyl sulfonyl fluoride, incubate gently with shaking at 37°C for 30 minutes; sonicate on ice for 5 minutes, add 0.1% Triton X-100 and EDTA (final concentration 5 mM), cool, centrifuge at 10,000×g for 10 minutes at 4°C, discard the supernatant, collect the precipitate, and obtain Bacillus subtilis cell fragments;

[0066] Step 4) Functional microbial agent loading: The bacterial strain was resuspended in deionized water to obtain a bacterial solution; corn straw biochar, bacterial solution, Bacillus subtilis fragments and tea tree essential oil fermentation product were mixed and loaded into a sealed impregnation tank; impregnated at -0.08 MPa for 30 minutes; the vacuum was slowly released, and the impregnation was repeated twice, and then placed on a shaker at 50 rpm and 25℃ for 30 minutes; centrifuged, the precipitate was pre-frozen at -30℃ for 3 hours, vacuum dried for 24 hours, mixed with bentonite, bagged and sealed to obtain the product.

[0067] Example 5: Microbial Fertilizer (parts by weight)

[0068]

[0069]

[0070] The preparation process is described in Example 4.

[0071] Example 6: Microbial Fertilizer (parts by weight)

[0072] Corn biochar 60 copies Bacillus megaterium (500 million / g) 4 copies Bacillus amyloliquefaciens (500 million / g) 3 copies Alcaligenes faecalis (500 million / g) 3 copies Bentonite 10 copies Bacillus subtilis fragments 10 copies Example 3: Tea Tree Oil Fermentation 10 copies

[0073] The preparation process is described in Example 4.

[0074] Comparative Example 1: Microbial Fertilizer

[0075] The difference from Example 4 is that this microbial fertilizer does not contain Bacillus subtilis fragments or tea tree oil fermentation products.

[0076] Comparative Example 2: Microbial Fertilizer

[0077] The difference from Example 4 is that this microbial fertilizer does not contain Bacillus subtilis fragments.

[0078] Comparative Example 3: Microbial Fertilizer

[0079] The difference from Example 4 is that this microbial fertilizer does not contain tea tree essential oil fermentation products.

[0080] Comparative Example 4: Microbial Fertilizer

[0081] The difference from Example 4 is that this microbial fertilizer uses tea tree oil instead of tea tree oil fermentation product.

[0082] Experiment 1: Stability test of functional bacteria

[0083] The microbial fertilizers prepared in Examples 4-6 and Comparative Examples 1-4 were placed in a dry place at room temperature and protected from light for 24 months. The number of viable bacteria was measured after 0 months, 12 months and 24 months. The number of viable bacteria was measured according to the standard method in the "Test Procedure for Microbial Bio-fertilizer Products" (NY / T 2321-2013). The results are shown in Table 1.

[0084] Table 1. Changes in the number of viable bacteria in each product group.

[0085]

[0086] The results showed that the microbial fertilizers prepared in Examples 4-6 suffered less loss during production, were closest to the initial added viable bacteria count, and the decrease in viable bacteria count after 24 months of storage at room temperature was between 16% and 21%. In contrast, the microbial fertilizers prepared in Comparative Examples 1-4 suffered a viable bacteria count loss rate of approximately 16% to 24% during production, and the loss was even greater after 24 months of storage at room temperature, with an effective viable bacteria count loss of 30% to 52%. This indicates that the simultaneous adsorption of Bacillus subtilis fragments and tea tree essential oil fermentation products in biochar can significantly improve the stability of the microbial agent during production and storage, and the two may have a synergistic effect.

[0087] Experiment 2: Effects of microbial fertilizers on tomato growth, yield, and fruit quality

[0088] 1. Materials and Methods

[0089] 1.1 Experimental Materials: The tomato variety used was 'Jingcai 8'. Each treatment consisted of 3 experimental plots, each with an area of ​​60m². 2A total of 18 experimental plots were set up in a randomized block design. Each planting bed was planted with two rows of plants, with double plants per bed, a plant spacing of 35 cm, and a row spacing of 150 cm. All groups received basal fertilizer (800 kg / mu), topdressing (200 kg / mu), and other field management measures were consistent. The drip irrigation topdressing experimental design is shown in Table 2.

[0090] Table 2 Field Trial Design

[0091]

[0092]

[0093] 1.2 Measurement Indicators and Methods

[0094] 1.2.1 Plant growth morphology: During the flowering period, 10 consecutive tomato plants were randomly selected from each experimental area and marked. The plant height, stem diameter, fresh weight and dry weight of the tomato plants were measured. The plant height was measured by measuring the vertical distance from the rootstock to the top of the main stem with a steel tape measure. The stem diameter was measured by measuring the horizontal and vertical diameters 5 cm from the top of the plant with vernier calipers. The average value was taken.

[0095] 1.2.2 Fruit Quality and Yield: After the tomatoes began to set fruit, the number of tomatoes on the first and second trusses was counted. Once each truss of fruit matured, 10 out-of-shape tomatoes from each treatment were randomly harvested, and their weight was measured using an analytical balance. The average weight of a single tomato per truss was calculated. The experiment was conducted in plots of 60m². 2 Statistical output, then converted to per 667m 2 Yield. Five mature tomatoes of uniform size and good appearance were selected from each treatment condition for the determination of tomato quality-related indicators. Vitamin C content in the tomato fruit was determined using the molybdenum blue colorimetric method; soluble solids were detected using a refractometer; the sugar-acid ratio was the ratio of soluble sugar to titratable acid. Lycopene content was determined spectrophotometrically; soluble protein content was determined using the Coomassie Brilliant Blue G250 staining method.

[0096] 1.3 Results Analysis

[0097] 1.3.1 Effects of different treatments on tomato plant morphology: As shown in Table 3 below, compared with the treatment group that applied organic fertilizer (CK), the microbial fertilizer treatment containing Bacillus subtilis fragments and / or tea tree essential oil fermentation significantly increased the average plant height, stem diameter, fresh weight, and dry weight of tomatoes at the flowering period; among them, the tomatoes in the T1 treatment group grew the best, indicating that the combined use of Bacillus subtilis fragments and tea tree essential oil fermentation has a significantly better growth-promoting effect on tomatoes than the use of either alone, and is greater than the sum of the effects of the use of either alone.

[0098] Table 3 Effects of different treatments on tomato plant morphology

[0099]

[0100]

[0101] Compared to CK, # P < 0.05 ## P < 0.01; compared with CK1 group, ^P < 0.05, ^^P < 0.01.

[0102] 1.3.2 Effects of different treatments on tomato fruit quality and yield: As shown in Tables 4 and 5 below, microbial fertilizers containing Bacillus subtilis fragments and tea tree essential oil fermentation can significantly improve the fruit quality and yield of tomatoes, and the effect is significantly better than microbial fertilizers containing Bacillus subtilis fragments or tea tree essential oil fermentation.

[0103] Table 4. Changes in tomato fruit quality under different treatments

[0104] project CK CK1 CK2 CK3 CK4 T1 Lycopene (mg / g) 15.38 16.85 18.21 17.35 16.93 21.82 Soluble solids (%) 5.39 5.68 6.77 6.32 6.05 8.36 Sugar-acid ratio 11.26 12.51 15.64 14.47 13.86 18.52 Vitamin C (mg / 100mL) 11.69 12.25 15.09 13.75 13.45 17.06 Soluble protein (mg / g) 0.76 0.81 0.91 0.87 0.85 1.22

[0105] Table 5 Comparison of tomato yield under different treatments

[0106] Group Average weight of a single fruit (g) Average number of fruits per plant <![CDATA[Yield (kg / 667m 2 )]]> CK 95.4 21.5 4628.75 CK1 97.9 20.6 4485.26 CK2 105.1 22.8 4911.36 CK3 102.6 24.5 5125.49 CK4 101.0 23.5 5002.74 T1 117.6 26.0 5950.82

[0107] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A microbial fertilizer, characterized in that, The product comprises the following components in parts by weight: 60-70 parts biochar, 10-20 parts functional microbial agent, 5-10 parts bentonite, 5-10 parts Bacillus subtilis fragments, and 5-10 parts tea tree essential oil fermentation product; the Bacillus subtilis fragments are prepared by the following steps: Step S1: Resuspend the bacterial cells in lysis buffer and adjust the concentration to 1×10⁻⁶. 5 ~1×10 9 CFU / mL; Step S2: Add lysozyme and benzyl sulfonyl fluoride, and incubate with gentle shaking at 30-37°C for 30-60 minutes; Step S3: Sonicate under ice for 3-5 minutes, then add Triton X-100; Step S4: Inactivate enzymes, cool and centrifuge, discard supernatant, collect precipitate to obtain cell debris; The tea tree essential oil ferment is prepared by the following steps: Step A: Dissolve tea tree essential oil in a 30-60% ethanol solution to obtain a tea tree essential oil solution with a concentration of 10-20% by volume; Step B: Inoculate the Yeast Extract seed culture into the fermentation medium at an inoculation rate of 1-5% and ferment for 24-48 hours. Then, add tea tree oil solution with a final concentration of 0.1-1% (v / v) and continue fermentation for 48-96 hours. Step C: Collect the fermentation broth, centrifuge, take the supernatant, filter, and obtain the final product; The method for preparing the microbial fertilizer includes the following steps: Step 1) Biochar preparation: Rice husks, corn stalks or rice straw are crushed into 1-2 mm pieces and biochar is prepared by high temperature slow pyrolysis. Step 2) Biochar pretreatment: The biochar is acid-washed to remove alkali and then activated to improve pore size. Step 3) Functional microbial agent loading: Mix biochar, functional microbial agent, Bacillus subtilis fragments and tea tree essential oil fermentation material and load into a sealed impregnation tank; impregnate at -0.08~-0.1MPa for 30~45 minutes; slowly release the vacuum, repeat the impregnation 2~3 times, then place on a shaker at 50rpm and 25℃ for 30~60 minutes; centrifuge, take the precipitate and pre-freeze at -20~-40℃ for 2~5 hours, vacuum dry, and mix with bentonite to obtain the final product.

2. The microbial fertilizer according to claim 1, characterized in that, The biochar raw material is selected from one of rice husks, corn stalks, and rice straw.

3. The microbial fertilizer according to claim 1, characterized in that, The functional microbial agent is selected from one or more of Bacillus megaterium, Bacillus amyloliquefaciens, and Alcaligenes faecalis.

4. The microbial fertilizer according to claim 1, characterized in that, In step S1, the lysis buffer is 30-50 mM Tris-HCl + 1 mM EDTA, and the pH is 8.0; in step S2, the final concentrations of lysozyme and benzyl sulfonyl fluoride are 1-2 mg / mL and 1-2 mM, respectively.

5. The microbial fertilizer according to claim 1, characterized in that, In step S3, the ultrasonic power is 100~200W, and 0.1~0.3% Triton X-100 is added; in step S4, the centrifugation conditions are 4℃, centrifugation at 10,000×g for 10~20 minutes.

6. The microbial fertilizer according to claim 1, characterized in that, The fermentation process parameters in step B are as follows: fermentation temperature is 28~30℃, fermentation pH is 4.0~6.0, shaking speed is 150~200rpm, and aeration rate is 0.5~1.0vvm.

7. The application of the microbial fertilizer according to any one of claims 1 to 6 in promoting tomato growth and improving tomato fruit quality.

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