Application of soybean meal fermented by Bacillus natto and Aspergillus oryzae in alleviating the obstacle of continuous apple cropping

Abstract

The invention discloses an application of a soybean meal product obtained by composite fermentation of Bacillus natto and Aspergillus oryzae in alleviating apple continuous cropping obstacles, and belongs to the technical field of agricultural microorganisms. The invention adopts Bacillus natto and Aspergillus oryzae to compositely ferment soybean meal to prepare a soybean meal fermentation product rich in antimicrobial peptides. Applying the soybean meal fermentation product to continuous cropping soil can promote the growth of replanted apple saplings, increase soil enzyme activity, reduce soil phenolic acid content, inhibit the growth of harmful fungi in the soil, optimize the continuous cropping soil microbial environment, and alleviate the occurrence of apple continuous cropping obstacles.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural cultivation, and in particular to application of a soybean meal product produced by composite fermentation of Bacillus natto and Aspergillus oryzae in alleviating the obstacle of continuous apple cropping. Background Art

[0002] Currently, harmful fungi, particularly Fusarium, are the primary cause of continuous cropping problems in apples. Studies have shown that when isolating and identifying harmful fungi in the soil of replanted apple orchards in the Bohai Rim region of China, Fusarium species were frequently found. Specifically, these species include Fusarium oxysporum, Fusarium laminarum, Fusarium solani, and Fusarium moniliforme, and exhibited high pathogenicity to seedlings in Pingyi County. Existing measures to mitigate continuous cropping problems in apples include crop rotation and the use of chemical fumigants. However, crop rotation is time-consuming and difficult to implement on a large scale (Fan et al., 2022). Chemical fumigants pose a serious potential threat to the environment and human health and are being phased out of agriculture (Raymaekers et al., 2020). Therefore, it is crucial to seek a green, pollution-free, and simple method to mitigate the occurrence of continuous cropping problems in apples.

[0003] Research has found that microbial fermentation of precursor proteins can produce bioactive peptides with antimicrobial activity, referred to as antimicrobial peptides (AMPs) (Chai et al., 2019; Thyab et al., 2020). AMPs possess broad-spectrum antimicrobial activity and can effectively combat bacterial and fungal infections in agricultural plants (Plavsin et al., 2017). Soybean meal, a byproduct of soybean oil extraction, is an important and readily available plant protein source. Compared to traditional organic materials, soybean meal is rich in protein and a variety of amino acids. However, in typical production processes, soybean meal often results in significant waste of protein resources due to its low economic value and low utilization rate.

[0004] Therefore, the use of inexpensive soybean meal as a precursor protein for microbial solid-state fermentation could improve soybean meal utilization and alleviate the occurrence of apple continuous cropping problems through the production of antimicrobial peptides. However, there are currently no reports on the application of soybean meal fermented with Bacillus natto and Aspergillus oryzae to alleviate apple continuous cropping problems. Summary of the Invention

[0005] In response to the above-mentioned prior art, the present invention aims to provide a method for using a soybean meal product fermented with Bacillus natto and Aspergillus oryzae to alleviate apple continuous cropping problems. By adding a fermentation solution composed of Bacillus natto and Aspergillus oryzae fermentation solutions to soybean meal and subjecting it to solid-state fermentation, a soybean meal fermentation product rich in antimicrobial peptides is obtained. This product is then applied to continuously cropped soil to promote the growth of replanted apple saplings, increase soil enzyme activity, reduce soil phenolic acid content, inhibit the growth of harmful fungi in the soil, optimize the soil microbial environment, and alleviate the occurrence of apple continuous cropping problems.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A first aspect of the present invention provides a method for preparing a composite fermented soybean meal product, comprising the following steps:

[0008] The soybean meal is disinfected and then water is added to obtain a soybean meal mixture, a fermentation liquid is introduced into the soybean meal mixture, and the mixture is fermented at 32-33° C. and a pH of 6.5-7.5 for 25-35 days to obtain a composite fermented soybean meal product.

[0009] Preferably, the sterilization temperature is 105-121°C and the sterilization time is 15-20 minutes.

[0010] More preferably, the sterilization temperature is 121° C. and the sterilization time is 20 minutes.

[0011] Preferably, the mass ratio of water to sterilized soybean meal is 1:(4-6).

[0012] Further preferably, the mass ratio of water to sterilized soybean meal is 1:5.

[0013] Preferably, the fermentation broth is prepared by compounding Nanobacillus fermentation broth and Aspergillus oryzae fermentation broth in a mass ratio of 1: (0.5-1.5).

[0014] Further preferably, the strain deposit number of the Bacillus natto is CCTCC KB 20081433, and the strain deposit number of the Aspergillus oryzae is ACCC 30789;

[0015] More preferably, the effective viable bacteria count in the natto Bacillus subtilis fermentation broth is 1.3×10 8 -1.8×10 8 CFU / mL, the spore concentration in Aspergillus oryzae fermentation broth was 3.5×10 8 -4.0×10 8 CFU / mL.

[0016] More preferably, the natto Bacillus subtilis fermentation broth is prepared by the following method: inoculating the activated natto Bacillus subtilis into LB liquid culture medium, incubating at 37°C, 180 r·min -1 Cultivate on a shaking platform for 18-24 hours to obtain the natto Bacillus subtilis fermentation liquid;

[0017] The Aspergillus oryzae fermentation broth is prepared by the following method: inoculating the activated Aspergillus oryzae into a PDA liquid culture medium, and stirring at 28°C and 180 r·min -1 After culturing on a shaking table for 2-3 days, Aspergillus oryzae fermentation liquid was obtained.

[0018] Preferably, the mass ratio of fermentation broth to soybean meal is 1:(8-12).

[0019] More preferably, the mass ratio of fermentation broth to soybean meal is 1:10.

[0020] Preferably, in step (2), the fermentation is shallow tray aerobic solid fermentation, the fermentation temperature is 32-33° C., the fermentation time is 25-35 days, and the fermentation pH is 7.

[0021] More preferably, the fermentation temperature is 32.5° C. and the fermentation time is 30 days.

[0022] The second aspect of the present invention provides a composite fermented soybean meal product.

[0023] The third aspect of the present invention provides the use of the composite fermented soybean meal product in the following (1) or (2):

[0024] (1) Alleviate the obstacles of continuous apple cropping;

[0025] (2) Prepare microbial preparations to alleviate the problems of continuous cropping of apples.

[0026] A fourth aspect of the present invention provides a method for alleviating apple continuous cropping obstacles, comprising the following steps:

[0027] The composite fermented soybean meal product was applied to the apple rhizosphere soil.

[0028] Preferably, the added amount of the composite fermented soybean meal product is 0.5% of the mass of the rhizosphere soil.

[0029] Beneficial effects of the present invention:

[0030] The present invention uses Bacillus natto and Aspergillus oryzae as fermentation strains, performs solid-state fermentation on soybean meal, and obtains a soybean meal fermentation product rich in antimicrobial peptides. The obtained soybean meal fermentation product is applied to the soil of apple continuous cropping, which can promote the growth of replanted apple saplings, increase soil enzyme activity, reduce soil phenolic acid content, inhibit the growth of harmful fungi in the soil, optimize the soil microbial environment of continuous cropping, and reduce the occurrence of apple continuous cropping problems. At the same time, the selection of soybean meal as the fermentation raw material not only improves the utilization rate of soybean meal, but also provides a green, pollution-free, simple and easy method to reduce the occurrence of apple continuous cropping problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : bovine serum albumin standard curve;

[0032] Figure 2 :The effects of soybean meal fermentation products from different strains on the gene copy number of Fusarium spp.

[0033] Figure 3:Effects of different treatments on the growth of Pingyi sweet tea seedlings;

[0034] Figure 4 :Effects of different treatments on soil enzyme activities of Camellia sinensis seedlings;

[0035] Figure 5 : Effects of different treatments on soil microorganisms of Camellia sinensis seedlings. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0037] As described in the background art, the continuous cropping problem of apples leads to the widespread occurrence of apple replant disease, which slows down the growth of young fruit trees, inhibits the growth of the root system, reduces the yield and even causes the death of trees, resulting in economic losses. The measures used in the existing technology to solve the continuous cropping problem of apples are time-consuming, difficult to promote or pose a potential threat to the environment.

[0038] Based on this, the present invention utilizes precursor proteins through microbial fermentation to produce antimicrobial peptides with antimicrobial activity. Because antimicrobial peptides have broad-spectrum antimicrobial activity, they can effectively combat bacterial and fungal infections in agricultural plants. Furthermore, soybean meal is rich in protein and amino acids. Thus, by selecting a suitable bacterial strain to ferment soybean meal, the resulting soybean meal fermentation product contains antimicrobial peptides. This can be applied to soils with continuous apple cropping problems to alleviate the problem, promote the growth of fruit tree seedlings and roots, and increase yield.

[0039] The present invention adopts bacillus natto and aspergillus oryzae to ferment, wherein, bacillus natto is a kind of green and environmentally friendly probiotic, with heat-resistant and acid-alkali-resistant, anti-extrusion characteristic, bacillus natto has broad-spectrum antimicrobial action simultaneously, its metabolism produced enzyme system is rich, can produce nattokinase, gamma-polyglutamic acid, polymyxin, bacitracin and 2, the physiologically active substances such as 6-pyridinedicarboxylic acid, can effectively suppress the growth of pathogenic bacteria. The hyphae of aspergillus oryzae is composed of various cells, is a kind of aerobic fungus, adapts to acid-base ability strongly, all can play good enzyme fermentation activity within most of pH value ranges, and aspergillus oryzae can produce various enzymes, including protease, amylase, cellulase, phytase etc.

[0040] Therefore, the composite fermentation of soybean meal with Bacillus natto and Aspergillus oryzae was used to form a soybean meal fermentation product rich in antimicrobial peptides. Applying the soybean meal fermentation product to the continuously cropped soil can promote the growth of replanted apple saplings, increase soil enzyme activity, reduce soil phenolic acid content, inhibit the growth of harmful fungi in the soil, optimize the microbial environment of the continuously cropped soil, and reduce the occurrence of apple continuous cropping obstacles.

[0041] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments. The test materials used in the embodiments of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0042] Bacillus natto was purchased from China Center for Type Culture Collection with the strain collection number of CCTCC KB 20081433, and Aspergillus oryzae was purchased from China Agricultural Microbiological Culture Collection Center with the strain collection number of ACCC 30789.

[0043] PDA medium: 200.0 g peeled potatoes, 20.0 g glucose, 20.0 g agar, 1000 mL distilled water;

[0044] LB medium: peptone 10.0 g, yeast extract powder 5.0 g, NaCl 10.0 g, distilled water 1000 mL;

[0045] Gao's medium No. 1: soluble starch 20.0 g, NaCl 0.5 g, KNO3 1.0 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, FeSO4·7H2O 0.01 g, agar 20 g, distilled water 1000 mL.

[0046] The pathogen of Fusarium proliferatum used in the present invention is the ARD specialized type Fusarium proliferatum MR5, and its classification name is also called "Fusarium proliferatum". The MR5 strain is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee with a deposit number of CGMCC NO.22426 and a deposit date of May 17, 2021, which is recorded in patent CN113817613A. Strain MR5 is pathogenic only to apple rootstocks and belongs to the apple specialized type. The used Fusarium moniliforme (F. moniliforme), Fusarium solani (F. solani), and Fusarium oxysporum (F. oxysporum) are all existing standard strains derived from Shandong Agricultural University. The public can obtain them from the applicant for repeating this experiment.

[0047] Example 1: Composite fermentation of soybean meal with Bacillus natto and Aspergillus oryzae

[0048] (1) The activated Bacillus subtilis natto was inoculated into LB liquid medium and incubated at 37°C and 180 r·min -1The shaker culture was carried out for 18-24 hours to obtain the fermentation liquid of Bacillus natto. The activated Aspergillus oryzae was inoculated into the PDA liquid medium and the mixture was stirred at 28℃ and 180r·min. -1 The mixture was shaken and cultured for 2-3 days to obtain Aspergillus oryzae fermentation liquid, and the Bacillus natto fermentation liquid and Aspergillus oryzae fermentation liquid were mixed in a mass ratio of 1:1 to obtain fermentation liquid;

[0049] Among them, the effective viable bacteria count in the natto Bacillus subtilis fermentation broth was 1.6×10 8 CFU / mL, the spore concentration in Aspergillus oryzae fermentation broth was 3.9×10 8 CFU / mL;

[0050] (2) sterilizing the soybean meal at 121° C. for 20 minutes, and then adding sterilized water to obtain a soybean meal mixture, wherein the mass ratio of sterilized water to sterilized soybean meal is 1:5;

[0051] The pH of the soybean meal mixture was adjusted to 7, and the fermentation liquid was inoculated into the soybean meal mixture, wherein the mass ratio of the fermentation liquid to the soybean meal was 1:10. The mixture was stirred evenly and spread flat on a sterilized tray. After covering with sterile gauze and a lid, a shallow tray aerobic solid fermentation was carried out at 32.5°C. After fermentation for 30 days, a soybean meal fermentation product was obtained.

[0052] Comparative Example 1:

[0053] The difference between this comparative example and Example 1 is that no fermentation liquid was added.

[0054] Comparative Example 2:

[0055] The difference between this comparative example and Example 1 is that the fermentation broth is only Bacillus natto fermentation broth. The number of viable bacteria in the fermentation broth in this comparative example is consistent with that in Example 1.

[0056] Comparative Example 3:

[0057] The difference between this comparative example and Example 1 is that the fermentation broth is only Aspergillus oryzae fermentation broth. The number of viable bacteria in the fermentation broth in this comparative example is consistent with that in Example 1.

[0058] Test Example 1: Extraction of antibacterial substances from soybean meal fermentation products

[0059] (1) Sterile water was added to the soybean meal fermentation product obtained in Example 1, wherein the mass ratio of the soybean meal fermentation product to the sterile water was 1:20, and the mixture was extracted at 180 rpm for 3 h to obtain an extract. The extract was centrifuged at 6000 rpm for 20 min to remove large solid particles, thereby obtaining a supernatant.

[0060] (2) Take 50 mL of the supernatant in a round-bottom flask and place it on a rotary evaporator to concentrate it about 10 times to obtain the supernatant concentrate, which is then frozen in a -20°C refrigerator for later use;

[0061] (3) Add 3 mol / L HCl dropwise to the remaining supernatant to adjust the pH to 2.0. Let it stand at 4°C overnight and then centrifuge at 4°C and 4000 rpm for 30 min. Discard the supernatant to obtain the precipitate.

[0062] (4) The precipitate obtained in step (3) was washed twice with HCl at pH = 2.0, and then 90% ethanol was added and stirred evenly. The pH was adjusted to 7.0 with 3 mol / L NaOH. After standing at room temperature for 5 hours, the polypeptide substances were extracted, and then centrifuged at 4°C and 4000 r / min for 30 minutes to obtain an extract. The extract was concentrated by rotary evaporation and freeze-dried to obtain a crude polypeptide extract, which was frozen in a -20°C refrigerator for use.

[0063] The fermented soybean meal product obtained in Example 1 was centrifuged and filtered, and crude polypeptide extracts were extracted for subsequent experiments.

[0064] Test Example 2: Antibacterial Test

[0065] The soybean meal mixture prepared in Comparative Example 1 and the soybean meal fermentation products prepared in Example 1 and Comparative Examples 2-3 were subjected to antibacterial tests. The test strains were Fusarium oxysporum, Fusarium solani, Fusarium solani, and Fusarium moniliforme. The antibacterial tests were conducted using a plate standoff test. The specific test method was as follows:

[0066] (1) Extracting crude polypeptide extracts from the soybean meal mixture prepared in Comparative Example 1 and the soybean meal fermentation products prepared in Comparative Examples 2 and 3, using the same preparation method as in Experimental Example 1;

[0067] (2) 2 mL of sterilized water was added to the soybean meal mixture prepared in Comparative Example 1 and the crude polypeptide extracts extracted from the soybean meal fermentation products prepared in Example 1 and Comparative Examples 2-3, respectively, to dissolve the crude polypeptide extracts to obtain crude polypeptide extract solutions;

[0068] Equal amounts of the soybean meal mixture prepared in Comparative Example 1, the crude polypeptide extract solution extracted from the soybean meal fermentation products prepared in Example 1 and Comparative Examples 2-3, and sterile water (control group) were added to sterilized unsolidified PDA medium, mixed well, poured into a plate medium, and allowed to stand for solidification to obtain a culture medium.

[0069] (3) The cultured Fusarium oxysporum, Fusarium solani, Fusarium solani, and Fusarium moniliforme, four pathogenic Fusarium strains of apple continuous cropping obstacles, were punched with a 0.5 cm diameter punch to obtain a 0.5 cm diameter bacterial cake, and the bacterial cake was inoculated into each culture medium prepared in step (2) and cultured at a constant temperature of 28°C;

[0070] (4) When the pathogen colonies in the control group with sterile water covered the entire culture dish, the diameters of the pathogen colonies in the three treatment groups of soybean meal fermentation product polypeptide crude extract were measured and recorded, and the inhibition rates were calculated respectively. The results are shown in Table 1.

[0071] Wherein, the antibacterial rate (%) = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0072] Table 1 Inhibition rate of soybean meal fermentation products on harmful fungi in apple continuous cropping problems

[0073]

[0074] As can be seen from Table 1, the crude polypeptide extracts extracted from the soybean meal fermentation products obtained in Comparative Examples 1-3 and Example 1 can inhibit the growth of four harmful fungi in apple continuous cropping obstacles to varying degrees. Among them, the crude polypeptide extract in the soybean meal product of the composite fermentation of Bacillus natto and Aspergillus oryzae (Example 1) has the most obvious inhibitory effect on the four harmful fungi, with inhibition rates of 75.55%, 71.92%, 68.25% and 70.59% on Fusarium oxysporum, Fusarium glomeruloneum, and Fusarium solani, respectively; the effect of Comparative Example 3 is second best, followed by Comparative Example 2, and the effect of Comparative Example 1 is the worst. Therefore, selecting Bacillus natto and Aspergillus oryzae composite fermentation soybean meal can more effectively prevent and control the growth of harmful Fusarium in apple continuous cropping obstacles.

[0075] Test Example 3: Indoor test

[0076] 1. Experimental Design

[0077] This study was conducted in the Apple Continuation and Microbiology Laboratory of Shandong Agricultural University from October to December 2021.

[0078] A total of four treatments were set up in the experiment: continuous cropping control (CK1), unfermented soybean meal treatment (SM1), soybean meal product fermented by Bacillus subtilis natto treatment (SM2), soybean meal product fermented by Aspergillus oryzae treatment (SM3), and soybean meal product fermented by Bacillus subtilis natto and Aspergillus oryzae combined treatment (SF).

[0079] Among them, after the soybean meal fermentation product was fermented for 30 days, the crude polypeptide extract in the soybean meal fermentation product was extracted and its polypeptide content was determined. The fermented soybean meal fermentation product was applied to the continuously cropped soil for 30 days and then the gene copy number of Fusarium solani and Fusarium solani in the soil was determined.

[0080] The experimental soil was selected from a 34-year-old apple orchard in Manzhuang Town, Tai'an City, Shandong Province. The addition rate of soybean meal fermented by Bacillus natto, soybean meal fermented by Aspergillus oryzae, and soybean meal fermented by Bacillus natto and Aspergillus oryzae was 0.5% of the continuous cropping soil mass.

[0081] 2. Measurement indicators:

[0082] (1) Determination of polypeptide content:

[0083] The polypeptide content of fermented soybean meal was determined by Coomassie Brilliant Blue method. The specific steps are as follows:

[0084] Take 50 μL of the polypeptide crude extract solution sample from each treatment group, dilute it 20 times with 0.9% NaCl solution to obtain the polypeptide extract dilution solution, take 1.0 mL of the polypeptide crude extract dilution solution from each group and 4.0 mL of Coomassie brilliant blue dye solution, mix them, let them stand at room temperature for 3 minutes, and read the absorbance value A at a wavelength of 595 nm. 595nm , substitute the absorbance value of the peptide crude extract dilution into the standard curve, and calculate the peptide concentration corresponding to each peptide crude extract dilution. Among them, the absorbance value of bovine serum albumin solution with a concentration gradient of 0-100μg / mL is measured to develop a bovine serum albumin standard curve, such as Figure 1 As shown, the standard curve equation is: y = 0.0061x + 0.0453, R 2 =0.9904.

[0085] The peptide concentration corresponding to each dilution of the crude polypeptide extract sample is multiplied by the dilution multiple to obtain the corresponding peptide concentration (μg / mL) of the original crude polypeptide extract solution and the polypeptide content in the sample (sample volume 50 μL).

[0086] Wherein, polypeptide content (μg) = original polypeptide crude extract solution concentration (μg / mL) × (50 / 1000) (mL).

[0087] (2) Determination of Fusarium gene copy number:

[0088] Total soil DNA was extracted using the EZNATM soil DNA extraction kit (OMEGA), and the gene copy numbers of Fusarium oxysporum and Fusarium solani in the soil were quantitatively analyzed using a CFX96TM Thermal (Cycler Bio-Rad). The results are shown in Table 3.

[0089] 3. Test results:

[0090] (1) Determination of polypeptide content in soybean meal fermented by different strains

[0091] Table 2 Determination of polypeptide content in soybean meal products fermented by different strains

[0092]

[0093] Table 3 Correlation analysis between polypeptide content and inhibition rate of Fusarium oxysporum

[0094]

[0095] As can be seen from Table 2, there are significant differences in the peptide concentrations in the crude polypeptide extract solutions obtained by acid precipitation from unfermented soybean meal (SM1), soybean meal fermented with Bacillus natto (SM2), soybean meal fermented with Aspergillus oryzae (SM3), and soybean meal fermented with Bacillus natto and Aspergillus oryzae (SF).

[0096] As can be seen from Table 3, the correlation coefficient between the polypeptide content in the crude polypeptide extract corresponding to the fermented soybean meal product and the antibacterial rate of Fusarium oxysporum is greater than 0.99, which indicates that the antibacterial effect is highly correlated with their polypeptide content. The higher the polypeptide content, the stronger the antibacterial activity. This also shows that the more polypeptides produced, the higher the content of antibacterial polypeptides with antibacterial activity contained in the crude polypeptide extract. The polypeptide content in the crude polypeptide extract corresponding to the soybean meal (SF) fermented by Bacillus natto and Aspergillus oryzae is higher than that of the single bacteria fermentation, and the corresponding antibacterial rate of Fusarium oxysporum is greater than that of the single bacteria fermentation. This also proves that the composite fermentation of soybean meal by Bacillus natto and Aspergillus oryzae can produce more antibacterial active substances.

[0097] (2) Effects of soybean meal fermented by different strains on Fusarium in continuous cropping soil

[0098] Through the quantitative analysis of oxysporum and Fusarium solani in the continuous cropping soil after the soybean meal products were treated with different strains of fermentation, Figure 2 It can be seen that the results showed that the application of fermentation products significantly reduced the number of Fusarium solani and Oxysporium solani. Among them, the treatment of soybean meal fermented by Bacillus natto and Aspergillus oryzae (SF) had the best effect, with the number of Oxysporium solani and Fusarium solani reduced by 59.75% and 63.00% respectively compared with the continuous cropping control (CK); although the unfermented soybean meal (SM1), the soybean meal product fermented by Bacillus natto (SM2), and the soybean meal product fermented by Aspergillus oryzae (SM3) also reduced the number of harmful Fusarium solani, the effect was slightly worse than the soybean meal fermented by Bacillus natto and Aspergillus oryzae (SF).

[0099] Test Example 4: Potted Plant Experiment

[0100] 1. Experimental Design:

[0101] The potted experiment was conducted from May to October 2022 at the National Apple Engineering Technology Research Center's experimental base and the Apple Replanting and Microbiology Laboratory at Shandong Agricultural University. Soybean meal was purchased commercially; the soil used was selected from a 35-year-old apple orchard in Manzhuang Town, Tai'an City, Shandong Province. Soil was randomly sampled from multiple locations at a depth of 10-40 cm, 80 cm from the tree trunk, and mixed evenly. The soil was a brown loam.

[0102] Four treatments were set up in the experiment: continuous cropping soil control (CK1), continuous cropping soil fumigated with methyl bromide (CK2), continuous cropping soil treated with unfermented soybean meal (SM), and continuous cropping soil treated with the product of soybean meal fermented by Bacillus natto and Aspergillus oryzae (SF).

[0103] The continuously cropped soil is soil from a 35-year-old apple orchard, and the added amounts of unfermented soybean meal, Bacillus natto and Aspergillus oryzae composite fermented soybean meal products are all 0.5% of the mass of the continuously cropped soil.

[0104] The experimental plants were seedlings of Pingyi sweet tea, with 8 replicates per treatment. Normal water and fertilizer management was carried out uniformly after planting. Samples were taken from each treatment in mid-August and relevant indicators were measured.

[0105] 2. Measurement indicators:

[0106] Biomass determination: Use a tape measure and vernier caliper to measure the plant height and ground diameter. When measuring the fresh mass of the plant, first wash the plant with tap water, dry it and measure it with an electronic balance, then dry it at 80℃ to constant weight and measure the dry mass.

[0107] Determination of photosynthetic rate: The intercellular CO2 concentration (C i ), net photosynthetic rate (P n ), stomatal conductance (G s ) and transpiration rate (T r ).

[0108] Determination of soil enzyme activities: urease activity was determined by indigo blue colorimetry; catalase activity was determined by potassium permanganate titration; neutral phosphatase activity was determined by disodium phenyl phosphate colorimetry; and sucrase activity was determined by 3,5-dinitrosalicylic acid colorimetry.

[0109] Determination of soil microbial community structure: The plate count method was used. The soil samples were diluted and spread on LB medium, PDA medium, and Gao's No. 1 medium. The number of culturable bacteria, fungi, and actinomycetes in the soil was counted, and the ratio of soil bacteria to fungi was calculated. Total soil DNA was extracted using the EZNATM soil DNA extraction kit (OMEGA), and the CFX96TM Thermal (Cycler Bio-Rad) was used to quantify the gene copy numbers of four harmful Fusarium species in the soil. Soil alpha diversity was determined by high-throughput sequencing, which was performed by Shanghai Meiji Biotechnology Co., Ltd.

[0110] Determination of soil phenolic acids: ASE350 rapid solvent extractor (Dionex, USA) was used to extract soil phenolic acids, and the extracted soil phenolic acids were analyzed by Waters e2695 high performance liquid chromatography (Waters, USA) to determine the types and contents of phenolic acids.

[0111] Data processing: The experimental data were calculated and plotted using Microsoft Excel 2010 and Origin 2021. SPSS26.0 software was used to compare means and one-way ANOVA, and Duncan's new multiple range method was used for significant difference analysis.

[0112] 3. Test results:

[0113] (1) Growth-promoting effect of soybean meal products fermented by Bacillus natto and Aspergillus oryzae on Pingyi sweet tea seedlings

[0114] Table 4 Effects of soybean meal products fermented by Bacillus natto and Aspergillus oryzae on the biomass of potted Pinyi sweet tea seedlings

[0115] Treatment group Plant height (cm) Ground diameter (mm) Fresh weight (g) Dry weight (g) CK1 59.33±0.88d 6.33±0.23d 36.90±0.49d 18.35±0.17d CK2 75.33±0.88a 10.66±0.03a 85.52±1.79a 42.61±1.31a SM 64.00±1.15c 8.23±0.05c 58.61±0.90c 27.53±1.41c SF 69.33±0.67b 9.40±0.20b 65.13±1.93b 32.96±2.19b

[0116] Depend on Figure 3 As shown in Table 4, all three treatments promoted the growth of Pingyi sweet tea seedlings to varying degrees under potted conditions. Plant height, ground diameter, fresh weight, and dry weight in the continuous cropping soil (SF) treated with a combined fermentation of soybean meal product from Bacillus natto and Aspergillus oryzae increased by 16.8%, 48.58%, 76.48%, and 79.62%, respectively, compared to the continuous cropping soil control (CK1). All parameters in the continuous cropping soil (SM) treated with unfermented soybean meal were lower than those in the continuous cropping soil (SF) treated with a combined fermentation of soybean meal product from Bacillus natto and Aspergillus oryzae.

[0117] Although the growth of Pingyi sweet tea seedlings treated with the continuous cropping soil (SF) treated with the combined fermentation soybean meal product of Bacillus natto and Aspergillus oryzae failed to exceed that of the continuous cropping soil (CK2) treated with methyl bromide fumigation, the continuous cropping soil (SF) treated with the combined fermentation soybean meal product of Bacillus natto and Aspergillus oryzae significantly promoted the growth of Pingyi sweet tea seedlings and had obvious differences compared with the continuous cropping soil control (CK1) and the continuous cropping soil (SM) treated with unfermented soybean meal.

[0118] (2) Effects of soybean meal fermentation products of Bacillus natto and Aspergillus oryzae on the photosynthetic characteristics of Pingyi sweet tea seedlings

[0119] Table 5 Effects of soybean meal products fermented by Bacillus natto and Aspergillus oryzae on photosynthetic characteristics of potted Pinyi sweet tea seedlings

[0120]

[0121] The photosynthetic capacity of plants is closely related to plant growth, and the level of photosynthesis directly affects the quality of crop production. As shown in Table 5, compared with the continuous cropping soil control (CK1), the net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) of the continuous cropping soil (SF) treated with a soybean meal product fermented by Bacillus natto and Aspergillus oryzae increased by 65.53%, 84.14%, 18.14%, and 67.82%, respectively. Compared with the continuous cropping soil (SM) treated with unfermented soybean meal, the net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) of the continuous cropping soil (SF) treated with a soybean meal product fermented by Bacillus natto and Aspergillus oryzae increased by 20.07%, 34.58%, 8.45%, and 19.67%, respectively.

[0122] It can be seen that although the growth of Pingyi sweet tea seedlings treated with the continuous cropping soil (SF) treated with the composite fermentation soybean meal product of Bacillus natto and Aspergillus oryzae failed to exceed that of the continuous cropping soil (CK2) treated with methyl bromide fumigation, the continuous cropping soil (SF) treated with the composite fermentation soybean meal product of Bacillus natto and Aspergillus oryzae significantly promoted the photosynthetic parameters of Pingyi sweet tea seedlings compared with the continuous cropping soil control (CK1) and the continuous cropping soil (SM) treated with unfermented soybean meal.

[0123] (3) Effects of soybean meal fermentation products of Bacillus natto and Aspergillus oryzae on soil enzyme activities of Pingyi sweet tea seedlings

[0124] from Figure 4 Significant differences in soil enzyme activity were observed between treatments. Specifically, treatment with a soybean meal product fermented with Bacillus natto and Aspergillus oryzae in the continuous cropping soil (SF) significantly promoted soil enzyme activity in Pingyi sweet tea seedlings, with sucrase, urease, phosphatase, and catalase activities increasing by 92.72%, 30.59%, 115.81%, and 19.55%, respectively, compared to the continuous cropping soil control (CK1). Soil enzyme activity decreased after methyl bromide fumigation, demonstrating a close relationship between soil enzyme activity and the number and activity of soil microorganisms.

[0125] (4) Effects of soybean meal fermentation products of Bacillus natto and Aspergillus oryzae on soil microorganisms of Pingyi sweet tea seedlings

[0126] Depend on Figure 5 b. Figure 5 It can be seen from eh that the number of culturable fungi and the gene copy number of harmful Fusarium in the soil treated with the soybean meal product of Bacillus natto and Aspergillus oryzae were significantly reduced, but Figure 5 a and Figure 5c It can be seen that the number of culturable bacteria and actinomycetes in the soil increased significantly, and Figure 5 d It can be seen that the ratio of bacteria to fungi in the soil increased significantly.

[0127] Soil Alpha diversity is used to reflect the species abundance and diversity of samples, including the richness index Ace and Chao, and the diversity index Shannon and Simpson. High-throughput sequencing revealed that the fungal Alpha diversity indexes of different treatments were significantly different. Figure 5 i and Figure 5 It can be seen that the Ace index and Chao index of SF are significantly higher than those of CK1, CK2 and SM. Figure 5 k shows that the Shannon index has the same trend as the Ace index and Chao index, indicating that the species richness of fungi in the continuous cropping soil (SF) treated with soybean meal product fermented by Bacillus natto and Aspergillus oryzae is higher. The Simpson index is used to estimate the diversity of microorganisms in soil samples. The higher the value, the lower the community diversity. Figure 5 lIt can be seen that the fungal diversity of SF treatment was significantly higher than that of CK1, CK2 and SM treatments, and showed significant differences.

[0128] (5) Effects of soybean meal fermentation products of Bacillus natto and Aspergillus oryzae on phenolic acid content in soil of Pingyi sweet tea seedlings

[0129] Table 6 Effects of soybean meal fermentation products of Bacillus natto and Aspergillus oryzae on soil phenolic acid content under continuous cropping conditions

[0130]

[0131]

[0132] As shown in Table 6, the soybean meal product fermented with Bacillus natto and Aspergillus oryzae effectively reduced the content of phenolic acids in continuously cropped soil. Overall, the degradation capacity for soil phenolic acids ranked CK2 > SF > SM. The contents of phlorizin, ferulic acid, benzoic acid, and vanillin in continuously cropped soil treated with the soybean meal product fermented with Bacillus natto and Aspergillus oryzae were reduced by 48.62%, 48.2%, 66.10%, and 47.15%, respectively, compared to the continuously cropped soil control (CK1). The contents of phlorizin, ferulic acid, benzoic acid, and vanillin in continuously cropped soil treated with the soybean meal product fermented with Bacillus natto and Aspergillus oryzae were reduced by 33.79%, 16.18%, 57.22%, and 18.10%, respectively, compared to the continuously cropped soil treated with unfermented soybean meal (SM).

[0133] Test Example 5: Field Test

[0134] 1. Experimental Design

[0135] In April 2022, field verification was carried out in continuous cropping orchards in Shilibao Village, Xicheng Town, Qixia City, Yantai City, Shandong Province (35°02′N, 118°36′E) and Liujiagou Town, Penglai District, Yantai City, Shandong Province (37°77′N, 120°89′E).

[0136] The experimental materials were two-year-old grafted apple seedlings, Fujixin 2001 in Qixia orchard and Yanfu 10 in Penglai orchard, with M9T337 as the rootstock. When establishing the orchard, a planting trench was dug in the soil of the continuous apple cropping area, and a 40 cm square planting hole was dug in the trench.

[0137] Three treatment groups were set up in each experimental site, namely: continuous cropping soil control (CK), continuous cropping soil treated with unfermented soybean meal (SM), and continuous cropping soil treated with the product of soybean meal fermented by Bacillus natto and Aspergillus oryzae (SF).

[0138] The continuous cropping soil is the soil of the planting holes in the continuous cropping apple orchard, the soybean meal product obtained by the composite fermentation of Bacillus natto and Aspergillus oryzae is the soybean meal fermentation product obtained in Example 1, and the unfermented soybean meal is the soybean meal obtained in Comparative Example 1 without inoculation of the fermentation solution.

[0139] The addition amount of the unfermented soybean meal, the soybean meal fermented by Bacillus natto and Aspergillus oryzae composite was 0.5% of the mass of the continuous cropping soil.

[0140] After planting the saplings, unified water and fertilizer management will be implemented, and the plant height, ground diameter, trunk circumference and branch length of the saplings will be measured in August 2022.

[0141] 3. Test results:

[0142] Table 7 Effects of different treatments on the growth of two-year-old grafted apple seedlings

[0143]

[0144]

[0145] Table 7 shows that in the experimental orchards in Qixia City, the SF treatment increased plant height, ground diameter, trunk circumference, and branch length by 33.33%, 47.52%, 59.09%, and 101.25% compared to the CK treatment, respectively. Furthermore, the growth vigor of the SF-treated plants was better than that of the SM-treated plants. In the experimental orchards in Penglai District, the SF treatment increased plant height, ground diameter, trunk circumference, and branch length by 32.75%, 26.10%, 41.51%, and 24.00%, respectively, compared to the CK treatment. Furthermore, the growth vigor of the SF-treated plants was significantly better than that of the SM-treated plants. Therefore, the application of a fermented soybean meal product derived from Bacillus natto and Aspergillus oryzae significantly improved the growth of two-year-old grafted apple seedlings.

[0146] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for preparing a composite fermented soybean meal product, characterized in that: The following steps are involved: After the soybean meal is disinfected, water is added to obtain a soybean meal mixture, and fermentation liquid is added to the soybean meal mixture, and fermentation is carried out at 32-33° C. and pH 6.5-7.5 for 25-35 days to obtain a composite fermented soybean meal product; The fermentation bacterial liquid is prepared by compounding the fermentation liquid of Bacillus natto and the fermentation liquid of Aspergillus oryzae in a mass ratio of 1:(0.5-1.5); the strain preservation number of the Bacillus natto is CCTCC KB 20081433, and the strain preservation number of the Aspergillus oryzae is ACCC 30789.

2. The method for preparing the composite fermented soybean meal product according to claim 1, characterized in that: The disinfection temperature is 105-121℃, and the disinfection time is 15-20 min; the mass ratio of water to disinfected soybean meal is 1: (4-6).

3. The method for preparing the composite fermented soybean meal product according to claim 1, characterized in that: The natto Bacillus subtilis fermentation liquid is prepared by the following method: inoculating the activated natto Bacillus subtilis into LB liquid culture medium, and incubating at 37°C, 180 r•min -1 The mixture was cultured in a shaking table for 18-24 hours to obtain the natto Bacillus subtilis fermentation liquid; The Aspergillus oryzae fermentation liquid is prepared by the following method: inoculating the activated Aspergillus oryzae into a PDA liquid culture medium, and heating the culture medium at 28°C and 180 r•min -1 After culturing in a shaking table for 2-3 days, Aspergillus oryzae fermentation liquid was obtained.

4. The method for preparing the composite fermented soybean meal product according to claim 1, characterized in that: The mass ratio of fermentation liquid to soybean meal is 1:(8-12).

5. The method for preparing the composite fermented soybean meal product according to claim 1, characterized in that: The number of effective live bacteria in the fermentation liquid of Bacillus natto is 1.3×10 8 -1.8×10 8 CFU / mL, the spore concentration in the fermentation liquid of Aspergillus oryzae was 3.5×10 8 -4.0×10 8 CFU / mL.

6. The composite fermented soybean meal product obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the composite fermented soybean meal product according to claim 6 in the following (1) or (2): (1) Alleviate obstacles to continuous apple cropping; (2) Prepare preparations to alleviate the obstacles caused by continuous cropping of apples.

8. A method for alleviating the obstacle of continuous cropping of apples, characterized in that: The following steps are involved: The composite fermented soybean meal product according to claim 6 is applied to the rhizosphere soil of apples.

9. The method for alleviating the obstacle of continuous cropping of apples according to claim 8, characterized in that: The addition amount of composite fermented soybean meal product was 0.5% of the rhizosphere soil mass.

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