Preparation method and application of biological bacterial fertilizer for preventing and treating continuous cropping obstacles of apple trees

Abstract

The invention belongs to the field of application of microorganisms, and discloses a preparation method and application of a biological bacterial fertilizer for preventing and treating continuous cropping obstacles of apple trees. The biological bacterial fertilizer for preventing and treating continuous cropping obstacles of apple trees comprises a compound bacterial liquid and a fermentation carrier, the composite bacterial liquid is a common fermentation bacterial liquid of bacillus velezensis FF-6 and LchtB-7, and a fermentation carrier is composed of thoroughly decomposed chicken manure and straw dry powder. The compounding ratio of FF-6 to LchtB-7 in the compound bacterial liquid is 1: 1, the compounding ratio of composted chicken manure to dry straw powder in the fermentation carrier is 3: 1, and the ratio of the compound bacterial liquid to the fermentation carrier is 1: 10 (mL: g). The required raw materials are simple and easy to obtain, the preparation method is simple, and compared with common chemical fertilizers in the market, the chemical fertilizer is green, environmentally friendly and safe to the environment and has good development and utilization prospects.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of microbial application technology, and in particular relates to a preparation method and application of a biological fertilizer for preventing and controlling continuous cropping obstacles of apple trees. Background Art

[0002] Apple continuous cropping disorder is a common problem in the apple industry. It manifests itself as poor growth, reduced yields, and increased pest and disease infestations after consecutive apple trees are planted on the same plot. The main causes include imbalances in soil microbial communities, accumulation of harmful substances, depletion of soil nutrients, and autotoxicity of root secretions. While current preventative measures for this problem (such as soil disinfection, crop rotation, and the use of disease-resistant rootstocks) have shown some effectiveness, they still pose challenges such as long cycles, high costs, and environmental risks.

[0003] As an emerging measure to prevent and control continuous cropping problems, bio-fertilizer has many advantages, such as being green and safe, having a long lasting effect, and having comprehensive functions. However, there are currently few high-efficiency bio-fertilizer products on the market specifically for preventing and controlling continuous cropping problems for apple trees, which leads to fruit farmers facing a dual dilemma of technical support and product selection when preventing and controlling continuous cropping problems.

[0004] In view of the above analysis, the technical problems that need to be solved urgently in the existing technology are:

[0005] The development of a biological fertilizer that can effectively improve soil microbial communities, degrade self-toxic substances, and increase soil fertility has important practical significance and market value for solving the problem of continuous cropping of apple trees. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a preparation method and application of a biological fertilizer for preventing and controlling continuous cropping obstacles of apple trees.

[0007] The present invention is achieved by providing a biological fertilizer for preventing and treating continuous cropping problems in apple trees, characterized in that the biological fertilizer for preventing and treating continuous cropping problems in apple trees comprises a composite bacterial solution and a fermentation carrier; the composite bacterial solution is a co-fermentation bacterial solution of Bacillus velezensis FF-6 and LchtB-7, and the fermentation carrier is decomposed chicken manure fertilizer and straw powder. The composite bacterial solution has a composite ratio of FF-6 to LchtB-7 of 1:1, the fermentation carrier has a composite ratio of decomposed chicken manure to straw powder of 3:1, and the composite bacterial solution is inoculated into the fermentation carrier at a ratio of 1:10 (mL:g).

[0008] Furthermore, the composite bacterial liquid is a bacterial liquid fermented by the biological strains FF-6 and LchtB-7, and its preparation method specifically includes: culturing and activating FF-6 and LchtB-7; inoculating the activated strains into a modified LB liquid culture medium for fermentation to obtain seed liquid, mixing the two seed liquids in a 1:1 ratio to prepare a composite seed liquid, and inoculating the composite seed liquid at a ratio of 1% into a modified LB liquid culture medium for fermentation to obtain a fermentation liquid.

[0009] Furthermore, the improved LB liquid culture medium is an LB liquid culture medium optimized for composite bacterial liquid fermentation, and the specific proportions of its components are: 10 g of tryptone, 7.5 g of yeast extract, 7.5 g of sodium chloride, and 1000 mL of deionized water.

[0010] Another object of the present invention is to provide a method for preparing a biological fertilizer for preventing and controlling continuous cropping obstacles of apple trees, the method specifically comprising:

[0011] S1: First, the composite bacterial solution was fermented, and the activated FF-6 and LchtB-7 strains were inoculated into the modified LB liquid medium and cultured at 28°C and 180 rpm until the OD 600 =0.6 (absorbance at 600 nm wavelength, the same below) as the seed solution, the two seed solutions were mixed in a 1:1 ratio to prepare a composite seed solution, and the composite seed solution was inoculated into a modified LB liquid medium at a ratio of 1% and cultured at 28°C and 180 rpm for 30 hours to obtain a composite bacterial solution;

[0012] S2: sterilizing the decomposed chicken manure and straw powder under high pressure and high temperature, drying at a constant temperature until the moisture content is less than 30%, and mixing them evenly in a ratio of 3:1 (decomposed chicken manure: straw powder) to prepare a fermentation carrier;

[0013] S3: Inoculate the composite bacterial solution into the fermentation carrier at an inoculum rate of 10%, control the moisture content to 30% by adding sterile water or constant temperature drying, seal it, and place it in a dark environment at 25°C for 9 days to obtain the primary product of biological fertilizer;

[0014] S4: The primary biofertilizer product obtained in the previous step is dried at a constant temperature to control the moisture content to be less than 20%, and then subjected to a powdering treatment to obtain powdered biofertilizer BF-1.

[0015] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0016] First, the Bacillus FF-6 isolated from the rhizosphere soil of healthy apple trees was deposited at the General Microbiological Center of the China National Culture Collection Center on March 20, 2025, with the deposit number CGMCC No. 33876 and classified as Bacillus velezensis; the endophytic Bacillus LchtB-7 isolated from the bark of healthy Chinese flowering crabapple was deposited at the General Microbiological Center of the China National Culture Collection Center on March 7, 2025, with the deposit number CGMCC No. 33739 and classified as Bacillus velezensis.

[0017] The Bacillus velezensis FF-6 and LchtB-7 used in the present invention can effectively inhibit the growth of main pathogenic microorganisms such as Fusarium oxysporum in apple continuous cropping obstacles, can degrade the autotoxic substance phloridzin in the apple rhizosphere, can improve the microbial community structure of continuous cropping soil, and at the same time have a certain growth-promoting effect on apple seedlings in the continuous cropping soil environment.

[0018] (3) The component ratio and fermentation conditions of the fermentation carrier described in the present invention have been optimized through single-factor and orthogonal experiments, resulting in a higher fermentation efficiency of the composite bacterial liquid described in the present invention and a larger number of viable bacteria in the product.

[0019] (4) The raw materials required for the present invention are simple and easily available, the preparation method is simple, and compared with common chemical fertilizers on the market, it is green and environmentally friendly, safe for the environment, and has good development and utilization prospects.

[0020] Second, the present invention combines two strains of Bacillus velezensis in a 1:1 ratio and co-ferments them with well-rotted chicken manure to form a four-in-one mode of "bacteriostasis-detoxification-repair-growth promotion". At the same time, all materials used in the experiment are special materials in the northwest apple production area, including the special rootstock "Malus prunifolia" in the northwest production area and the common "yellow cotton soil" in continuous cropping orchards in northern Shaanxi, which improves the pertinence of the product of the present invention and breaks the blank of special biological bacterial fertilizers for continuous cropping obstacle orchards of apple trees in the northwest production area.

[0021] The present invention overcomes the technical prejudice that "chicken manure is acidic and not suitable for solving continuous cropping obstacles". Uncompletely decomposed chicken manure is acidic and is also likely to carry a large number of pathogenic bacteria, resulting in poor repair effect on continuous cropping soil that is already severely acidic and has microbial imbalance; by using well-rotted chicken manure for fermentation in the present invention, its pH is close to neutral and the number of pathogenic bacteria is also greatly reduced. At the same time, the two strains of Bacillus velezensis described in the present invention have high antibacterial activity and broad-spectrum properties, and can play a good inhibitory role on the remaining pathogenic bacteria therein. After testing, the pH value of the fermented bacterial fertilizer product is 6.2, close to neutral, and the content of mold and miscellaneous bacteria is 2.3×10 4cfu / g, which is far lower than the national standard, eliminates the impact of chicken manure acidity and pathogens on fertilizer performance and breaks the technical prejudice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of a method for preparing a biological fertilizer for preventing and controlling continuous cropping obstacles of apple trees provided by an embodiment of the present invention;

[0023] Figure 2 The inhibitory effects of FF-6 and LchtB-7 provided in the examples of the present invention on Fusarium oxysporum and Fusarium solani are as follows: A: Fusarium solani control; B: FF-6 × Fusarium solani; C: LchtB-7 × Fusarium solani; D: Fusarium oxysporum control; E: FF-6 × Fusarium oxysporum; F: LchtB-7 × Fusarium oxysporum;

[0024] Figure 3 This is the degradation effect of FF-6 and LchtB-7 on phlorizin provided in the embodiments of the present invention;

[0025] Figure 4 This is the compatibility test result of FF-6 and LchtB-7 provided in the embodiment of the present invention, with LchtB-7 in the horizontal direction and FF-6 in the vertical direction;

[0026] Figure 5 is the culture medium OD provided in the embodiment of the present invention 600 (absorbance at 600 nm, the same below) and OD of each component content in LB liquid culture medium 600 ;

[0027] Figure 6 This is the solid fermentation optimization test result provided by the embodiment of the present invention;

[0028] Figure 7 This is the effect of the BF-1 biological fertilizer provided by the embodiment of the present invention on the physiological indicators of apple seedlings in continuous cropping soil. The data without significance mark in the figure are all extremely significant (P Value < 0.001);

[0029] Figure 8 This is the restoration effect of the BF-1 biological fertilizer provided by the embodiment of the present invention on the enzyme activity of the rhizosphere soil of apple seedlings in the continuous cropping soil. The data without significance mark in the figure are all extremely significant (P Value < 0.001);

[0030] Figure 9 This is the effect of the BF-1 biological fertilizer provided by the embodiment of the present invention on the repair of the microbial community structure in the continuous cropping soil. The data without significance marks in the figure are all extremely significant (P Value < 0.001). DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] The present invention provides a biofertilizer for preventing and treating continuous cropping problems in apple trees, comprising a composite bacterial solution and a fermentation carrier. The composite bacterial solution is a fermentation solution of Bacillus velezensis FF-6 and LchtB-7, and the fermentation carrier is composed of decomposed chicken manure and dry straw powder. The composite bacterial solution contains FF-6 and LchtB-7 in a 1:1 ratio, the fermentation carrier contains decomposed chicken manure and dry straw powder in a 3:1 ratio, and the composite bacterial solution is inoculated into the fermentation carrier at a ratio of 1:10 (mL:g).

[0033] The composite bacterial liquid is a bacterial liquid fermented by the biological strains FF-6 and LchtB-7. The preparation method thereof specifically comprises: culturing and activating FF-6 and LchtB-7; inoculating the activated strains into a modified LB liquid culture medium for fermentation to obtain seed liquid; mixing the two seed liquids in a 1:1 ratio to prepare a composite seed liquid; and inoculating the composite seed liquid into a modified LB liquid culture medium at a ratio of 1% for fermentation to obtain a fermentation liquid.

[0034] The improved LB liquid culture medium is an LB liquid culture medium optimized for composite bacterial liquid fermentation, and the specific proportions of its components are: 10 g of tryptone, 7.5 g of yeast extract, 7.5 g of sodium chloride, and 1000 mL of deionized water.

[0035] like Figure 1 As shown, the embodiment of the present invention provides a method for preparing a biological fertilizer for preventing and controlling continuous cropping obstacles of apple trees, comprising the following steps:

[0036] S1: First, the composite bacterial solution was fermented, and the activated FF-6 and LchtB-7 strains were inoculated into the modified LB liquid medium at 28°C and shaken at 180 rpm (180 revolutions per minute, the same below) until the OD 600 =0.6 (absorbance at 600 nm wavelength, the same below) as the seed solution, the two seed solutions were mixed in a 1:1 ratio to prepare a composite seed solution, and the composite seed solution was inoculated into a modified LB liquid medium at a ratio of 1% and cultured at 28°C and 180 rpm for 30 hours to obtain a composite bacterial solution;

[0037] S2: sterilizing the decomposed chicken manure and straw powder under high pressure and high temperature, drying at a constant temperature until the moisture content is less than 30%, and mixing them evenly in a ratio of 3:1 (decomposed chicken manure: straw powder) to prepare a fermentation carrier;

[0038] S3: Inoculate the composite bacterial solution into the fermentation carrier at an inoculum rate of 10%, control the moisture content to 30% by adding sterile water or constant temperature drying, seal it and place it in a dark environment at 25°C for 9 days (natural day, i.e. 24 hours, the same below) to obtain the initial product of biological fertilizer.

[0039] S4: The primary biofertilizer product obtained in the previous step is dried at a constant temperature to control the moisture content to be less than 20%, and then subjected to a powdering treatment to obtain powdered biofertilizer BF-1.

[0040] Example 1 Antibacterial Effects of Biocontrol Bacteria FF-6 and LchtB-7 on Fusarium oxysporum and Fusarium solani

[0041] (1) Flat panel confrontation:

[0042] The antibacterial effects of the biocontrol bacteria FF-6 and LchtB-7 against Fusarium oxysporum and Fusarium solani were tested using the plate standoff method. Sterile filter paper discs (5 mm in diameter) were completely soaked in FF-6 or LchtB-7 seed solution. The surface bacterial solution was scraped off and the discs were then affixed to solid PDA (potato dextrose agar) plates. Two discs were affixed to each plate, maintaining a constant distance between them. A sterile hole punch was used to punch holes along the edges of 3-day-old activated Fusarium oxysporum and Fusarium solani. A bacterial cake was then affixed between two filter paper discs, hyphae facing downward, and incubated inverted at 25°C for 5 days. A control treatment containing only the fungus cake was used. Six replicates were performed for each treatment. When the control group had grown to cover the entire plate, the plates were removed and photographed to observe whether antibacterial activity was observed.

[0043] (2) Experimental results:

[0044] like Figure 2 As shown in the figure, the biocontrol bacteria FF-6 and LchtB-7 had significant inhibitory effects on Fusarium oxysporum and Fusarium solani.

[0045] Example 2 Evaluation of the degradation effect of biocontrol bacteria FF-6 and LchtB-7 on phlorizin

[0046] (1) Solid culture test:

[0047] Use a sterile toothpick to dip the activated bacterial liquid of biocontrol bacteria FF-6 and LchtB-7, and streak them on solid phlorizin culture medium and phloroglucinol culture medium plates respectively. Incubate them upside down at 28℃ for 7 days and observe and record. If colonies grow, it means that the biocontrol bacteria can degrade phlorizin and phloroglucinol, otherwise the biocontrol bacteria do not have the degradation ability.

[0048] Formula of solid phlorizin culture medium: 0.5 g potassium dihydrogen phosphate, 0.5 g dipotassium hydrogen phosphate, 1 g ammonium sulfate, 0.2 g magnesium sulfate, 0.1 g sodium chloride, 0.02 g calcium chloride, 0.872 g phlorizin, 15 g agar powder, and 1000 mL deionized water.

[0049] (2) Liquid culture test:

[0050] Single colonies of activated biocontrol bacteria FF-6 and LchtB-7 were picked and placed in liquid phlorizin culture medium and phloroglucinol culture medium test tubes, cultured at 28°C and 180 r / min for 3 days, and the color of the liquid in the test tubes was observed. If the color changed, it means that the two phenolic acids can be converted into other substances by the biocontrol bacteria.

[0051] Liquid phlorizin culture medium formula: 0.5 g potassium dihydrogen phosphate, 0.5 g dipotassium hydrogen phosphate, 1 g ammonium sulfate, 0.2 g magnesium sulfate, 0.1 g sodium chloride, 0.02 g calcium chloride, 0.872 g phlorizin, 1000 mL deionized water.

[0052] (3) Test results:

[0053] like Figure 3 As shown, the biocontrol bacteria FF-6 and LchtB-7 can grow on solid phlorizin culture medium and turn the liquid phlorizin culture medium into orange-yellow, indicating that the biocontrol bacteria FF-6 and LchtB-7 can effectively degrade phlorizin, an autotoxin of apple roots.

[0054] Example 3 Compatibility Determination of FF-6 and LchtB-7

[0055] (1) Compatibility determination:

[0056] Pipette 10 μL of FF-6 seed solution onto one side of a PDA plate. Repeat three times on the same side. Place the plate upright, allowing the seed solution to flow down and air-dry until the flow mark is clear. Add 10 μL of LchtB-7 seed solution onto the other, perpendicular side of the plate. Repeat this process until the two seed solution flow marks intersect perpendicularly. Then, incubate the plate at 28°C for 16 hours and observe for bacterial growth at the intersection.

[0057] (2) Test results:

[0058] like Figure 4 As shown in the figure, bacteria grew normally at each intersection of FF-6 and LchtB-7 bacterial solutions, and no obvious inhibition zone appeared, indicating that FF-6 and LchtB-7 have good compatibility and have the potential to form an efficient composite microbial flora.

[0059] Example 4 Optimization of fermentation conditions of composite bacterial liquid

[0060] (1) Screening of the optimal culture medium:

[0061] LB liquid medium, NB liquid medium, TY liquid medium, SOB liquid medium, CM liquid medium, YG liquid medium, and YSP liquid medium were prepared according to the formula. A 250 mL conical flask with a liquid volume of 100 mL was used to inoculate 1 mL of the composite seed solution. The culture was carried out at 28 ° C and 180 r / min for 30 h. The OD of the bacterial solution of each culture medium was measured. 600 , each treatment was repeated 5 times.

[0062] The LB medium formula is: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, and 1000 mL deionized water.

[0063] The NB culture medium formula is: 5 g peptone, 3 g beef extract, 5 g sodium chloride, and 1000 mL deionized water.

[0064] The TY culture medium formula is: 10 g tryptone, 5 g yeast extract, and 1000 mL deionized water.

[0065] The SOB culture medium formula is: 20 g tryptone, 5 g yeast extract, 0.5 g sodium chloride, 0.186 g potassium chloride, 0.95 g magnesium chloride, and 1000 mL deionized water.

[0066] The CM culture medium formula is: 10 g peptone, 5 g beef extract, 5 g sodium chloride, and 1000 mL deionized water.

[0067] The YG culture medium formula is: 10 g yeast extract, 20 g glucose, and 1000 mL deionized water.

[0068] The YSP culture medium formula is: 10 g yeast extract, 20 g glucose, 10 g peptone, and 1000 mL deionized water.

[0069] (2) Optimization of culture medium composition:

[0070] The optimal culture medium components screened out were further optimized. Except for deionized water, six gradients of 0.25%, 0.5%, 0.75%, 1%, 1.25%, and 1.5% were set for each major component. The single-factor experimental method was used to optimize each major component separately.

[0071] (3) Test results:

[0072] like Figure 5 As shown, when LB liquid culture medium is selected, the OD of the composite bacterial solution is 600The highest concentration indicates that the composite culture medium has the highest biomass. Further optimization of the LB liquid medium composition revealed that the composite culture medium with 1% tryptone, 0.75% yeast extract, and 0.75% sodium chloride (g:mL) had the highest biomass. This formulation was named modified LB liquid medium and subsequently used as the liquid fermentation medium for the strain.

[0073] Example 5 Optimization of solid fermentation conditions for biological fertilizer

[0074] (1) Fermentation carrier screening:

[0075] Decomposed cow dung, decomposed sheep dung and decomposed chicken dung were selected as bacterial fertilizer fermentation carriers respectively. After sterilization, the moisture content was controlled at 50%, and the composite seed liquid was inoculated at a ratio of 50 mL / kg. The fermentation temperature was adjusted to 28°C. Samples were taken after fermentation for 7 days. 5 g of each treatment was added to 50 mL of deionized water and shaken at 28°C for 30 minutes. The supernatant was diluted step by step and evenly spread on LB plates. The bacterial count was recorded after incubation at 28°C for 12 hours. Three replicates were set for each treatment.

[0076] (2) Optimization of fermentation conditions:

[0077] Different fermentation temperatures, straw addition amounts, and initial moisture contents were set, and the above three fermentation conditions were optimized using a single-factor experiment. The inoculation and counting methods were the same as (1).

[0078] (3) Response surface optimization:

[0079] According to the results of the single-factor experiment, an orthogonal experimental plan was designed using Design-Expert software. A solid fermentation optimization experiment was carried out according to the plan. A three-dimensional visualization model of the fermentation conditions was drawn based on the results, and the optimal fermentation conditions were obtained.

[0080] (4) Test results:

[0081] like Figure 6 As shown in the figure, when fully decomposed chicken manure is selected as the base, the number of viable bacteria in the bacterial fertilizer is the largest. At the same time, the solid fermentation temperature, straw addition amount, and initial moisture content were preliminarily optimized, and the optimal temperature was determined to be 24-28°C, the optimal straw addition amount was 20-40%, and the optimal initial moisture content was 20-40%. Based on the above ranges, response surface optimization was continued to obtain the relationship between the number of viable bacteria and fermentation conditions: Y = 27.60 + 1.13A + 1.23B - 0.88C + 0.45AB - 0.64AC - 0.43BC - 2.39A 2 -4.55B 2 -2.05C 2(Y: viable bacteria count, A: initial moisture content, B: fermentation temperature, C: straw addition amount), and the optimal fermentation conditions were obtained: initial moisture content 36.4%, fermentation temperature 28.7°C, and straw addition amount 24%.

[0082] Example 6 Preparation of biological fertilizer

[0083] (1) Liquid fermentation:

[0084] The FF-6 and LchtB-7 strains stored at 4°C were inoculated onto LB solid medium and activated at 28°C for 1 day. The activated FF-6 and LchtB-7 strains were respectively inoculated into modified LB liquid medium and cultured at 28°C with shaking at 180 r / min until the OD 600 =0.6 as the seed solution, the two seed solutions were mixed in a ratio of 1:1 to prepare a composite seed solution, and the composite seed solution was inoculated into a modified LB liquid culture medium at a ratio of 1% and cultured at 28°C and 180 r / min for 30 hours to obtain a composite bacterial solution.

[0085] The LB solid culture medium formula is as follows: 10 g tryptone, 5 g yeast extract, 5 g sodium chloride, 15 g agar, and 1000 mL deionized water.

[0086] The modified LB liquid culture medium formula: 10 g tryptone, 7.5 g yeast extract, 7.5 g sodium chloride, and 1000 mL deionized water.

[0087] (2) Solid fermentation:

[0088] Decomposed chicken manure and straw powder were sterilized under high pressure and high temperature, dried at a constant temperature until the moisture content was less than 30%, and then mixed uniformly in a ratio of 3:1 (decomposed chicken manure: straw powder) to prepare a fermentation carrier. A composite bacterial solution was inoculated into the fermentation carrier at a 10% inoculum rate, and the moisture content was controlled to 30% by adding sterile water or drying at a constant temperature. After sealing, the carrier was placed in a dark environment at 25°C and incubated for 9 days to obtain the primary biofertilizer product. The product was then dried at a constant temperature to control the moisture content to less than 20%, and then subjected to a powdering process to obtain powdered biofertilizer BF-1.

[0089] Example 7: Growth-promoting effect of biofertilizer BF-1 on apple seedlings in continuous cropping soil

[0090] (1) Soil treatment:

[0091] Rhizosphere soil from a 20-year-old apple orchard (hereafter referred to as continuously cropped soil) served as a control (CK). This continuously cropped soil was fumigated 15 days in advance as treatment 1 (T1). 5% BF-1 bacterial fertilizer was added to the continuously cropped soil as treatment 2 (T2). 5% decomposed chicken manure was added to the continuously cropped soil as treatment 3 (T3). Each treatment was mixed and placed in pots, with 5 kg of soil per pot. A single apple seedling was transplanted into the center of each pot and maintained as normal for 60 days.

[0092] (2) Growth measurement:

[0093] The growth indicators of plants in pots were measured every month. The specific indicators and measurement methods are shown in the table below:

[0094]

[0095] (3) Test results:

[0096] The results are shown in Table 1. The plants treated with BF-1 microbial fertilizer were significantly better than those treated with continuous cropping soil (CK) and decomposed chicken manure (T3) in terms of plant height, ground diameter, aboveground fresh weight, aboveground dry weight, underground fresh weight and underground dry weight, indicating that BF-1 microbial fertilizer can promote the growth of apple seedlings in a continuous cropping soil environment.

[0097] Table 1 Effect of BF-1 biological fertilizer on the growth promotion of apple seedlings in continuous cropping soil

[0098]

[0099] Example 8 Effects of biological fertilizer BF-1 on some physiological indicators of apple seedlings in continuous cropping soil

[0100] (1) Soil treatment:

[0101] The soil treatment was the same as in Example 4.

[0102] (2) Chlorophyll content determination:

[0103] Grind 0.1 g of leaves with 10 mL of 80% acetone to extract chlorophyll. Centrifuge and collect the supernatant to record A. 663 (absorbance at a wavelength of 663 nm, the same below), A 645 (Absorbance at a wavelength of 645 nm, the same below), calculate the chlorophyll content according to the formula: total chlorophyll (mg / g) = (8.02×A 663 +20.21×A 645 )×0.1.

[0104] (3) Determination of relative photosynthetic rate:

[0105] Use a photosynthetic analyzer to set parameters according to the program and record the instrument readings.

[0106] (4) Relative conductivity measurement:

[0107] Wash the leaves and chop them, soak them in deionized water for 2 hours, measure the initial conductivity C1, boil them for 10 minutes, measure the total conductivity C2 after cooling, and calculate the relative conductivity according to the formula: relative conductivity = C1 / C2×100%.

[0108] (5) Determination of relative water content:

[0109] The leaves were weighed fresh weight (FW), then soaked in distilled water for 8 h, weighed saturated fresh weight (TW), and then dried with hot air at 60°C to constant weight, weighed dry weight (DW), and the relative water content was calculated according to the formula: relative water content = (TW-DW) / (FW-DW) × 100%.

[0110] (6) Test results:

[0111] like Figure 7 As shown in the data, the chlorophyll content, relative photosynthetic rate and relative water content of the plants treated with BF-1 bacterial fertilizer were significantly higher than those in the continuously cropped soil (CK) and the decomposed chicken manure treatment (T3), and the relative electrical conductivity was significantly lower than those in the continuously cropped soil (CK) and the decomposed chicken manure treatment (T3), indicating that BF-1 bacterial fertilizer can promote the photosynthesis of apple seedlings, increase the metabolic rate of apple seedlings and enhance the cold resistance of apple seedlings in the continuously cropped soil environment.

[0112] Example 9 Effect of biological fertilizer BF-1 on enzyme activities in the rhizosphere soil of apple seedlings in continuous cropping soil

[0113] (1) Soil treatment:

[0114] The soil treatment was the same as in Example 4.

[0115] (2) Catalase assay:

[0116] The method was based on the instructions of the Solarbio soil catalase (S-CAT) kit;

[0117] (3) Sucrase assay:

[0118] The method was based on the instructions of the Solarbio soil sucrase (S-SC) kit;

[0119] (4) Urease assay:

[0120] The method refers to the instructions of the Solarbio soil urease (S-UE) kit;

[0121] (5) Neutral phosphatase assay:

[0122] The method was referred to the instructions of the Solarbio soil neutral phosphatase (S-NP) kit.

[0123] (6) Test results:

[0124] like Figure 8 As shown in the figure, the activities of catalase, sucrase, urease and neutral phosphatase in the rhizosphere of plants treated with BF-1 bacterial fertilizer were significantly higher than those in the continuously cropped soil (CK) and the decomposed chicken manure treatment (T3), indicating that BF-1 bacterial fertilizer can repair the enzyme activity of continuously cropped soil.

[0125] Example 10 Effect of biological fertilizer BF-1 on microbial community structure in continuous cropping soil

[0126] (1) Soil treatment:

[0127] The soil treatment was the same as in Example 4.

[0128] (2) Microbial count:

[0129] Take 10 g of each treated soil and add it to 100 mL of deionized water to make a soil suspension. Add 2 small steel balls and shake at 28°C and 180 r / min for 30 min. Dilute it step by step into a sterile environment to different multiples. Pipette 100 μL of the diluted suspension and evenly spread it on solid LB, solid PDA and solid Gao's No. 1 medium plates. The LB medium was placed at 28°C for 16 h, the PDA medium was placed at 25°C for 3 d, and the Gao's No. 1 medium was placed at 28°C for 7 d. After the culture was completed, the lines were counted.

[0130] Gao's medium No. 1 formula: 20 g soluble starch, 1 g potassium nitrate, 0.5 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.5 g sodium chloride, 15 g agar powder, 1000 mL deionized water. Dissolve the starch with a small amount of cold water before preparation.

[0131] (3) Test results:

[0132] like Figure 9 As shown in the data, the number of bacteria in the soil treated with BF-1 bacterial fertilizer increased significantly, the number of fungi decreased significantly, and the ratio of bacteria to fungi was significantly higher than that in the continuously cropped soil (CK) and the decomposed chicken manure treatment (T3), indicating that BF-1 bacterial fertilizer can repair the microbial community structure of continuously cropped soil.

[0133] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A biological fertilizer for preventing and treating continuous cropping of apple trees, characterized in that: The biological fertilizer includes a composite bacterial liquid and a fermentation carrier; the composite bacterial liquid is prepared by co-fermenting Bacillus velezensis FF-6 and LchtB-7 in a ratio of 1:1, the fermentation carrier is prepared by mixing decomposed chicken manure and straw dry powder in a ratio of 3:1, and the inoculation ratio of the composite bacterial liquid to the fermentation carrier is 1:10 (mL:g).

2. The biological fertilizer according to claim 1, characterized in that The preparation method of the composite bacterial liquid comprises: The activated FF-6 and LchtB-7 strains were inoculated into modified LB liquid medium for fermentation to obtain seed liquid; The two seed solutions are mixed in a ratio of 1:1 to obtain a composite seed solution; The composite seed liquid was inoculated into a modified LB liquid culture medium at an inoculation rate of 1% for fermentation culture to obtain the composite bacterial liquid.

3. The biological fertilizer according to claim 2, characterized in that The modified LB liquid culture medium includes the following components: 10 g of tryptone, 7.5 g of yeast extract, 7.5 g of sodium chloride, and 1000 mL of deionized water.

4. The biological fertilizer according to claim 1, characterized in that The preparation method of the fermentation carrier comprises: The decomposed chicken manure and straw powder are sterilized under high pressure and high temperature respectively, and dried at a constant temperature until the moisture content is less than 30%; The fermentation carrier is obtained by mixing decomposed chicken manure and straw dry powder in a ratio of 3:

1.

5. The biological fertilizer according to claim 1, characterized in that After the fermentation is completed, the biological fertilizer is dried at a constant temperature until the water content is less than 20%, and then subjected to a powdering process to obtain a powdered biological fertilizer.

6. A method for preparing a biological fertilizer for preventing and controlling continuous cropping obstacles of apple trees, characterized in that: The following steps are involved: S1: The activated FF-6 and LchtB-7 strains were inoculated into modified LB liquid medium respectively, and cultured at 28°C and 180 rpm with shaking until OD600 = 0.6 to obtain seed liquid, and the seed liquids were mixed at a ratio of 1:1 to form a composite seed liquid, and then the composite seed liquid was inoculated into modified LB liquid medium at a ratio of 1% and cultured at 28°C and 180 rpm with shaking for 30 hours to obtain a composite bacterial liquid; S2: sterilizing the decomposed chicken manure and straw powder under high pressure and high temperature, drying them at a constant temperature until the moisture content is less than 30%, and then mixing them in a ratio of 3:1 to obtain a fermentation carrier; S3: inoculating the composite bacterial solution into a fermentation carrier at an inoculum size of 10%, controlling the moisture content to 30% by adding sterile water or drying at a constant temperature, sealing the carrier, and culturing the carrier in a dark environment at 25° C. for 9 days to obtain a primary biofertilizer product; S4: drying the primary bio-fertilizer product at a constant temperature until the moisture content is less than 20%, and then subjecting it to a powdering treatment to obtain powdered bio-fertilizer BF-1.

7. The preparation method according to claim 6, characterized in that The formula of the modified LB liquid culture medium is: 10 g of tryptone, 7.5 g of yeast extract, 7.5 g of sodium chloride, and 1000 mL of deionized water.

8. The preparation method according to claim 6, characterized in that The endpoint of the shaking culture in step S1 is based on OD600=0.6 as the optimal seed solution concentration to ensure the activity of the composite bacterial solution.

9. The preparation method according to claim 6, characterized in that During the fermentation process in step S3, the water content is adjusted by adding sterile water or constant temperature drying to maintain it within the range of 30%, thereby ensuring a suitable environment for microbial fermentation.

10. The preparation method according to claim 6, characterized in that In step S4, the constant temperature drying temperature of the primary biofertilizer product is controlled within the range of 40-60° C. to maintain the microbial activity to the greatest extent and obtain powdered biofertilizer.

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