Microbial compound fermentation inoculant, straw fermentation product and application of straw fermentation product

Abstract

The invention discloses a microbial compound fermentation inoculant, a straw fermentation product and application of the straw fermentation product, and belongs to the technical field of agricultural microorganisms. The preparation method comprises the following steps: compounding bacillus subtilis, trichoderma harzianum and trichoderma longibrachiatum to form a microbial compound fermentation inoculant; when the microbial compound fermentation inoculant is used for fermenting ginger straws, the content of pyrogallic acid in a prepared straw fermentation product can be synergistically improved. The pyrogallic acid has an obvious antibacterial effect on fusarium moniliforme and fusarium laminatum which cause the apple continuous cropping obstacle; the growth of apple seedlings planted in continuous cropping soil can be effectively promoted. Therefore, the straw fermentation product disclosed by the invention can be used for preventing and treating apple continuous cropping obstacles, improving the biomass and root activity of apple seedlings under continuous cropping conditions and reducing the quantity of harmful fusarium in continuous cropping soil.

Description

A microbial compound fermentation agent, straw fermentation products and their applications Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a microbial compound fermentation agent, straw fermentation products, and their applications. Background Technology

[0002] In recent years, many apple orchards have shown serious signs of aging and urgently need renewal and renovation. However, due to limited land resources, replanting apple trees on the same plot of land is unavoidable, leading to the widespread occurrence of apple replanting disease. Apple replanting disease slows the growth of young trees, inhibits root growth, reduces yield, and can even cause tree death. This disease can cause up to 50% economic loss throughout the entire life cycle of an orchard, seriously hindering the sustainable development of the apple industry.

[0003] Currently, harmful fungi, especially Fusarium, are the main cause of continuous cropping obstacles in apple orchards in the Bohai Rim region of China. Studies have shown that Fusarium species, including *Fusarium moniliforme* and *Fusarium moniliforme*, are frequently found in the soil of replanted apple orchards in the Bohai Rim region of China, and they exhibit high pathogenicity in seedlings in Pingyi. Existing technologies for mitigating continuous cropping obstacles in apples include crop rotation and the use of chemical fumigants. However, crop rotation is time-consuming and not easily implemented on a large scale (Fan et al., 2022); chemical fumigants pose serious potential threats to the environment and human health and are being phased out of agriculture (Raymaekers et al., 2020). Therefore, it is crucial to find a green, pollution-free, simple, and easy-to-implement method to alleviate continuous cropping obstacles in apples.

[0004] The straw from some crops can alter the diversity of soil fungi and microorganisms in continuously cropped soils, reducing pathogen damage. However, direct use of straw presents challenges such as slow natural decomposition and susceptibility to pests and diseases, which are key limitations on the resource utilization of straw. Therefore, this study explores fermenting straw and applying the fermentation products to continuously cropped apple orchard soils to both mitigate pathogen damage and address the problems associated with direct straw utilization. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a microbial compound fermentation agent, straw fermentation products and their applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In the first aspect of the present invention, a microbial compound fermentation agent is provided, which is compounded from Bacillus subtilis, Trichoderma harzianum and Trichoderma longifolia in a ratio of (1-3):(1-3):(1-3) of viable bacteria.

[0007] The Bacillus subtilis has the accession number CGMCC NO.31990 and is described in patent CN119020234A.

[0008] Trichoderma harzianum has the accession number ACCC32526; Trichoderma longifolia has the accession number ACCC32060.

[0009] Preferably, the microbial compound fermentation agent is composed of Bacillus subtilis, Trichoderma harzianum, and Trichoderma longicornis in a live cell ratio of 1:1:1. Verification has shown that the synergistic effect of the above strains during mixed fermentation at this ratio is the most significant, resulting in the highest content of the target product.

[0010] Preferably, the total viable count of the microbial compound fermentation agent is greater than or equal to 10. 8 CFU / ml. This viable cell count has been verified to ensure the synergistic fermentation ability of the mixed strains, guaranteeing stable fermentation results.

[0011] In a second aspect, the present invention provides the application of the above-mentioned microbial compound fermentation agent in the production of pyrogallic acid from fermented straw.

[0012] Pyrogallol (1,2,3-trihydroxybenzene, CAS 87-66-1) is an important polyphenolic organic compound. The inventors discovered that certain concentrations of pyrogallol have a significant inhibitory effect on *Fusarium moniliforme* and *Fusarium proliferatum*, which cause continuous cropping obstacles in apples; moreover, it can effectively promote the growth of apple seedlings, which is of positive significance for alleviating continuous cropping obstacles in apples.

[0013] The microbial compound fermentation agent of the present invention can be used to ferment straw (especially ginger straw), which can significantly increase the content of pyrogallol in the straw fermentation product. The prepared straw fermentation product can be used to prevent and control apple continuous cropping obstacles.

[0014] In a third aspect, the present invention provides a straw fermentation product prepared by the following method: straw is crushed and sterilized, and then inoculated with the above-mentioned microbial compound fermentation agent, the pH is adjusted to 7.0-8.0, water is added to adjust the moisture content to 50-70%, and fermentation is carried out at 32°C for 30-40 days to obtain the straw fermentation product.

[0015] Preferably, the straw is ginger straw; ginger straw is rich in lignin, cellulose and other components, and has the strongest synergistic effect with the microbial compound fermentation agent of the present invention, and the fermentation product has the highest content of pyrogallol.

[0016] Preferably, the amount of the microbial compound fermentation agent introduced is 0.5-1.0% of the straw weight; selecting the above-mentioned amount can comprehensively balance fermentation efficiency and economic benefits, and achieve better fermentation results.

[0017] In a fourth aspect, the present invention provides the use of the above-mentioned straw fermentation product in the following (1) or (2): (1) alleviating apple continuous cropping obstacles; (2) preparing a formulation to alleviate apple continuous cropping obstacles.

[0018] In the above applications, the amount of straw fermentation products added is 1% of the mass of the apple rhizosphere soil.

[0019] The beneficial effects of the present invention are as follows: (1) The present invention combines Bacillus subtilis with preservation number CGMCC NO.31990, Trichoderma harzianum with preservation number ACCC32526 and Trichoderma longicornis with preservation number ACCC32060 to form a microbial compound fermentation agent; the microbial compound fermentation agent of the present invention can be used to ferment ginger straw, which can synergistically increase the content of pyrogallic acid in the prepared straw fermentation product.

[0020] (2) Pyrogallic acid has a significant inhibitory effect on Fusarium moniliforme and Fusarium proliferatum, which cause continuous cropping obstacles in apples; it can also effectively promote the growth of apple seedlings planted in continuously cropped soil. Therefore, the straw fermentation product of the present invention can be used to prevent and control continuous cropping obstacles in apples, increase the biomass and root activity of apple seedlings under continuous cropping conditions, and reduce the number of harmful Fusarium fungi in continuously cropped soil. Attached Figure Description

[0021] Figure 1: Results of antibacterial test of different concentrations of pyrogallol.

[0022] Figure 2: Effects of different amounts of pyrogallol on the growth of apple seedlings. Detailed Implementation

[0023] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If specific experimental conditions are not specified in the embodiments, they are generally performed under conventional conditions or according to the conditions recommended by the reagent company; the reagents and consumables used in the following embodiments, unless otherwise specified, can be obtained commercially. Specifically: the preservation number of *Bacillus subtilis* is CGMCC NO.31990, recorded in patent CN 119020234A. The preservation number of *Trichoderma harzianum* is ACCC32526, and the preservation number of *Trichoderma longifolia* is ACCC32060, both from the China Agricultural Microbiological Culture Collection Center. Pyrogallic acid, CAS number: 87-66-1. Ginger straw was taken from fields in Laiwu; the ginger straw used in the embodiments was planted in the same plot at the same time, and the composition of the ginger straw showed no significant difference.

[0025] Example 1: Preparation of Microbial Compound Fermentation Agent Activated Bacillus subtilis was inoculated into LB liquid medium and incubated at 37°C and 180 rpm. -1 After culturing on a shaker for 24 hours, Bacillus subtilis bacterial suspension was obtained; the activated Trichoderma harzianum was inoculated into PDA liquid medium and incubated at 28℃ and 180 rpm. -1 After 3 days of shaking culture, *Trichoderma harzianum* culture was obtained; the activated *Trichoderma longicornis* was inoculated into PDA liquid medium and incubated at 28°C and 180 rpm. -1 After culturing in a shaker for 3 days, a long-branched Trichoderma solution was obtained.

[0026] The viable cell counts in Bacillus subtilis, Trichoderma harzianum, and Trichoderma longifolia cultures were all adjusted to 1×10⁻⁶. 8 CFU / ml; then, by mixing at a volume ratio of 1:1:1, a microbial composite fermentation agent was prepared, resulting in a total viable count of 1×10⁻⁶ CFU / ml. 8 CFU / ml.

[0027] Example 2: Preparation of Microbial Compound Fermentation Agent The preparation methods for Bacillus subtilis, Trichoderma harzianum, and Trichoderma longicornis inoculum were the same as in Example 1, except that the viable cell count in each inoculum was adjusted to 1×10⁻⁶. 8 CFU / ml; then compounded at a volume ratio of 3:1:1 to prepare a microbial compound fermentation agent.

[0028] Example 3: Preparation of Microbial Compound Fermentation Agent The preparation methods for Bacillus subtilis, Trichoderma harzianum, and Trichoderma longicornis inoculum were the same as in Example 1, except that the viable cell count in each inoculum was adjusted to 1×10⁻⁶. 8CFU / ml; then compounded at a volume ratio of 1:3:3 to prepare a microbial compound fermentation agent.

[0029] Example 4: Preparation of ginger straw fermentation product. Ginger straw was dried, pulverized through an 80-mesh sieve, and sterilized at 121℃ for 20 min. Then, under aseptic conditions, the microbial compound fermentation agent prepared in Example 1 was inoculated at an amount of 0.7% of the ginger straw mass. The pH was adjusted to 7.0, and sterile water was added to adjust the moisture content to 60%. Fermentation was carried out at 32℃ for 30 days to obtain the ginger straw fermentation product.

[0030] Example 5: Preparation of ginger straw fermentation product. Ginger straw was dried, pulverized through an 80-mesh sieve, and sterilized at 121℃ for 20 min. Then, under aseptic conditions, the microbial compound fermentation agent prepared in Example 1 was inoculated at an amount of 0.5% of the ginger straw mass. The pH was adjusted to 8.0, and sterile water was added to adjust the moisture content to 50%. Fermentation was carried out at 32℃ for 40 days to obtain the ginger straw fermentation product.

[0031] Example 6: Preparation of ginger straw fermentation product. Ginger straw was dried, pulverized through an 80-mesh sieve, and sterilized at 121℃ for 20 min. Then, under aseptic conditions, the microbial compound fermentation agent prepared in Example 1 was inoculated at an amount of 1.0% of the ginger straw mass. The pH was adjusted to 7.0, and sterile water was added to adjust the moisture content to 55%. Fermentation was carried out at 32℃ for 35 days to obtain the ginger straw fermentation product.

[0032] Comparative Example 1: The preparation method of Bacillus subtilis bacterial suspension was the same as in Example 1, except that the viable cell count in the Bacillus subtilis bacterial suspension was adjusted to 1×10⁻⁶. 8 CFU / ml; used as fermentation agent A.

[0033] After drying, the ginger straw was crushed and passed through an 80-mesh sieve, and then sterilized at 121℃ for 20 min. Then, under aseptic conditions, fermentation inoculant A was inoculated at an amount of 0.7% of the ginger straw mass. The pH was adjusted to 7.0, and sterile water was added to adjust the moisture content to 60%. Fermentation was carried out at 32℃ for 30 days to obtain ginger straw fermentation product A.

[0034] Comparative Example 2: The preparation methods of Bacillus subtilis and Trichoderma harzianum cultures were the same as in Example 1, except that the viable cell count in both cultures was adjusted to 1×10⁻⁶. 8 CFU / ml; then compounded at a volume ratio of 1:1 to prepare fermentation agent B, so that the total viable count in fermentation agent B is 1×10⁻⁶. 8 CFU / ml.

[0035] After drying, the ginger straw was crushed and passed through an 80-mesh sieve, and then sterilized at 121℃ for 20 min. Then, under aseptic conditions, fermentation inoculant B was inoculated at an amount of 0.7% of the ginger straw mass. The pH was adjusted to 7.0, and sterile water was added to adjust the moisture content to 60%. Fermentation was carried out at 32℃ for 30 days to prepare ginger straw fermentation product B.

[0036] Comparative Example 3: The preparation methods of Bacillus subtilis and Trichoderma longicornis bacterial suspensions were the same as in Example 1, except that the viable cell count in both Bacillus subtilis and Trichoderma longicornis bacterial suspensions was adjusted to 1×10⁻⁶. 8 CFU / ml; then compounded at a volume ratio of 1:1 to prepare fermentation inoculum C, so that the total viable count in fermentation inoculum C is 1×10⁻⁶. 8 CFU / ml.

[0037] After drying, ginger straw was crushed and passed through an 80-mesh sieve, and then sterilized at 121℃ for 20 min. Then, under aseptic conditions, fermentation inoculant C was inoculated at an amount of 0.7% of the ginger straw mass. The pH was adjusted to 7.0, and sterile water was added to adjust the moisture content to 60%. Fermentation was carried out at 32℃ for 30 days to obtain ginger straw fermentation product C.

[0038] Example 1: Determination of Pyrogallic Acid Content in Straw Fermentation Products 1. Experimental Method: The pyrogallic acid content in the straw fermentation products prepared in Examples 4-6 and Comparative Examples 1-3 was determined. The specific method is as follows: The straw fermentation products were dried in an oven. 1.000 g of uniformly ground straw fermentation products was accurately weighed into a 50 mL centrifuge tube. 20.00 mL of 70% methanol aqueous solution was added. The mixture was vortexed for 1 minute and ultrasonically extracted for 30 minutes. The mixture was centrifuged at 8000 rpm for 15 minutes. The supernatant was transferred to a 50 mL volumetric flask. The residue was extracted once more with 10 mL of 70% methanol. The supernatants were combined and diluted to 50 mL with 70% methanol. An appropriate amount of the crude extract was filtered through a 0.22 µm filter membrane. The filtrate was stored in a brown sample bottle at 4℃ in the dark for later analysis. All extraction operations were performed under dark conditions.

[0039] Accurately weigh 10.00 mg of pyrogallol standard into a small beaker and dissolve it in a small amount of methanol. Quantitatively transfer the solution to a 100 mL amber volumetric flask and dilute to the mark with pH 5.5 buffer. Shake well to prepare a standard stock solution, preparing fresh each time. Add 0, 0.2, 0.5, 1, 2, 3, and 4 mL of the standard stock solution to stoppered colorimetric tubes S0-S6, respectively. Add 2.0 mL of pH 5.5 buffer to each volumetric flask. Add 1.0 mL of 0.1 M FeCl3 colorimetric reagent. Next, dilute to the 10 mL mark with distilled water and immediately tighten the cap. Vortex the volumetric flasks for about 10 seconds to ensure thorough mixing. Place all the colorimetrically developed volumetric flasks in a 25°C water bath, protected from light, and let stand for 15 minutes to ensure the reaction is complete and stabilized. During the measurement, tube S0 (reagent blank) was used as a reference, and cuvettes with a 1 cm optical path were used to measure the absorbance values ​​of tubes S1 to S6 sequentially at a wavelength of 540 nm. Finally, a standard curve was plotted with absorbance (A) as the ordinate (Y-axis) and the corresponding concentration (C, unit µg / mL) as the abscissa (X-axis), and a linear regression equation was obtained by fitting the curve.

[0040] Take an appropriate amount of the prepared sample filtrate and serially dilute it with pH 5.50 acetate buffer at appropriate ratios (usually 10-200 times) until the absorbance value of the diluted solution falls within the linear range of the standard curve (0.2-0.8). Use a two-tube method to eliminate matrix interference. For each diluted sample solution, prepare two 10 mL colorimetric tubes for parallel operation: one is the sample colorimetric tube (T tube), and the other is the corresponding sample blank tube (B tube).

[0041] The specific operation is as follows: T tube: accurately add 1 mL of sample diluent, then add 2.0 mL of buffer and 1.0 mL of Fe³⁺ colorimetric reagent in sequence, and finally dilute to the mark with ultrapure water.

[0042] Tube B: Accurately add the same volume of sample diluent, then add 2.0 mL of buffer solution and 1.0 mL of 0.10 mol / L HCl solution (to replace the colorimetric reagent), and then dilute to the mark with ultrapure water.

[0043] Immediately tighten the caps on both tubes and vortex for about 10 seconds to ensure thorough mixing. Then place all the volumetric flasks with the developed colorimetric ...

[0044] Next, using the solution in tube B as a reference, the absorbance of the solution in tube T was measured at a wavelength of 540 nm, and recorded as the net absorbance A_net. Three replicates were performed for each sample.

[0045] Pyrogallic acid content (mg / g) = (C 测 10 D 总 V 总 ) / (Vs m 1000)C 测 : Concentration (μg / mL) calculated from the standard curve 10: Final volume of the colorimetric reaction (mL) D 总 V: Total dilution factor from crude extract to colorimetric solution (product of all dilution steps) 总 : Total volume of sample extract (50.00 mL in this experiment) Vs : Volume of sample diluent added during color development (mL) m : Mass of sample weighed (g) 1000 : Unit conversion factor (μg to mg).

[0046] 2. Experimental results: The results of the determination of pyrogallol content in the straw fermentation products prepared in Examples 4-6 and Comparative Examples 1-3 (average of 3 parallels) are shown in Table 1.

[0047] Table 1: Results of pyrogallic acid content determination The results showed that the combination of three strains of Bacillus subtilis, Trichoderma harzianum, and Trichoderma longicornis as a microbial compound agent had a significant synergistic effect on increasing the content of pyrogallic acid in straw fermentation products compared with fermentation using Bacillus subtilis alone (Comparative Example 1), fermentation using Bacillus subtilis + Trichoderma harzianum (Comparative Example 2), and fermentation using Bacillus subtilis + Trichoderma longicornis (Comparative Example 3).

[0048] Experimental Example 2: Performance Evaluation of Pyrogallic Acid 1. Antibacterial Performance Evaluation: Fusarium moniliforme and Fusarium proliferatum were used as test pathogens. Pyrogallic acid was prepared into solutions with concentrations of 10 mg / L, 100 mg / L, 500 mg / L and 1000 mg / L, respectively, and antibacterial tests were conducted using a plate confrontation test.

[0049] As shown in Figure 1, compared with the control, different concentrations of pyrogallol had significant antibacterial effects against Fusarium moniliforme and Fusarium proliferatum, and the antibacterial effect increased accordingly with increasing concentration.

[0050] 2. Growth-promoting performance evaluation: The experiment was conducted in the solar greenhouse (36.20°N, 117.12°E) of Daizong Campus of Shandong Agricultural University from October to November 2025.

[0051] Using 3 kg of soil from a continuously cropped apple orchard per pot as a baseline, five treatments were established: no pyrogallic acid (CK), 8.4 mg pyrogallic acid (J1), 16.8 mg pyrogallic acid (J2), 33.6 mg pyrogallic acid (J3), and 50.4 mg pyrogallic acid (J4). Uniformly growing, disease- and pest-free apple M26 seedlings were selected and transplanted into corresponding plastic pots for each treatment, with one seedling per pot. Each treatment was replicated six times.

[0052] After transplanting, all treatments underwent uniform pruning, irrigation, and pest and disease management. One month after transplanting, three pots from each treatment were randomly selected for sampling to determine plant biomass.

[0053] The results are shown in Figure 2 and Table 2.

[0054] Table 2: Effects of different concentrations of pyrogallic acid on the growth of M26 seedlings The results showed that different concentrations of pyrogallic acid (PGA) significantly affected the growth of M26 seedlings. All treatment groups (J1-J4) were significantly superior to the control group (CK) in terms of plant height, fresh weight, and dry weight, exhibiting an overall trend of initial increase followed by decrease. Specifically, the J3 treatment group (with 33.6 mg of PGA) showed the most outstanding performance: plant height reached 14.37 cm, approximately 55.7% higher than CK; fresh weight was 2.93 g, an increase of 41.5%; and dry weight was 0.97 g, an increase of 29.3%. Although the J4 treatment group (with 50.4 mg of PGA) had slightly lower values ​​than J3, it was still significantly superior to J1, J2, and CK.

[0055] In summary, the appropriate addition of pyrogallol can effectively promote the growth of M26 seedlings and has a positive effect on alleviating apple continuous cropping obstacles.

[0056] Experiment Example 3: Pot Experiment 1. Experimental Method: The pot experiment was conducted at the experimental base of the National Apple Engineering Technology Research Center of Shandong Agricultural University. The soil used in the pot experiment was taken from a 34-year-old apple orchard in Manzhuang Town, Tai'an City, Shandong Province. Multiple random samples were taken from an area 80 cm away from the trunk and 10-40 cm deep, and mixed thoroughly.

[0057] The experiment was set up with the following 5 treatments: CK: Only continuous cropping soil was used as a control.

[0058] T1: Add the ginger straw fermentation product prepared in Example 4 to the continuously cropped soil. The amount of ginger straw fermentation product added is 1% of the mass of the continuously cropped soil.

[0059] T2: Add ginger straw fermentation product A prepared in Comparative Example 1 to the continuously cropped soil. The amount of ginger straw fermentation product A added is 1% of the mass of the continuously cropped soil.

[0060] T3: Add ginger straw fermentation product B prepared in Comparative Example 2 to the continuously cropped soil. The amount of ginger straw fermentation product B added is 1% of the mass of the continuously cropped soil.

[0061] T4: Add ginger straw fermentation product C prepared in Comparative Example 3 to the continuously cropped soil. The amount of ginger straw fermentation product C added is 1% of the mass of the continuously cropped soil.

[0062] Seedlings of Pingyi sweet tea with uniform growth and free from pests and diseases were selected and transplanted into clay pots, one seedling per pot, with eight replicates per treatment. After transplanting, all plants underwent uniform pruning, watering, and pest and disease management. Samples were taken and various indicators were measured three months after transplanting.

[0063] (1) Plant biomass measurement: Three pots were randomly selected from each treatment. The plant height was measured with a ruler and the stem diameter (ground diameter) near the ground was measured with a vernier caliper.

[0064] (2) Root activity determination: Root activity is the core indicator for measuring the absorption, synthesis and metabolism capacity of plant roots, and directly reflects the plant growth status. Root activity is usually measured in units of "the amount of TTC reduced per gram of fresh root per hour (μg·g⁻¹·h⁻¹)"; it is determined by the triphenyltetrazolium chloride reduction method (TTC method), as follows: When collecting plant samples, after rinsing the seedling roots clean, take 0.5 g of white root, cut it into 2 cm long segments, put it in a test tube, add 0.4% TTC and phosphate buffer (1 / 15 mol / L, pH=7.0) in equal volumes and mix 10 mL. For the control, first add 2 mL of 1 mol / L sulfuric acid to terminate the reaction in advance, then add 0.4% TTC and phosphate buffer (1 / 15 mol / L, pH=7.0) in equal volumes and mix 10 mL. Seal the tube and place it in a 37℃ constant temperature incubator for 4 h before taking it out. Except for the control, add 2 mL of 1 mol / L sulfuric acid to terminate the reaction. After standing for 15 min, take out the root, absorb the solution dry, and put it back into the test tube. Add 10 mL of 95% ethanol to each test tube, seal the tube, and extract for 24 h until the root turns white. Dilute 3-5 times according to the color and measure the color at 485 nm.

[0065] (3) Real-time fluorescence quantitative analysis of harmful soil fungi: 0.2 g of soil samples stored at -20℃ or -80℃ were weighed and DNA was extracted using the Soil Genomic DNA Kit. Subsequently, the gene copy number of Fusarium in soil DNA from different treatments was analyzed in real-time using the SYBR Premix Ex Taq™ Kit (TaKaRa) and the CFX96™ ThermalCycler (Bio-Rad).

[0066] The primer sequences for pPCR of Fusarium proliferatum are as follows: qPCR-F. proliferatum-F: 5′-GATCGGCGAGCCCTTGCGGCAAG-3′; (SEQ ID NO.1) qPCR-F. proliferatum-R: 5′-CGCCGCGTACCAGTTGCGAGGGT-3′. (SEQ ID NO.2) The primer sequences for pPCR of Fusarium moniliforme are as follows: qPCR-F. moniliforme-F: 5′-GACTCGCGAGTCAAATCGCGT-3′; (SEQ ID NO.3) qPCR-F. moniliforme-R: 5′-GGGGTTTAACGGCGTGGCC-3′. (SEQ ID NO.4) 2. Experimental results: (1) Results of plant biomass determination: The results of biomass determination for different treatments are shown in Table 3.

[0067] Table 3: Results of plant height and diameter at breast height under different treatments The results showed that the straw fermentation product prepared in Example 4 could significantly increase the plant height and ground diameter of apple seedlings in continuously cropped soil.

[0068] (2) Root vigor test results for different treatments are shown in Table 4.

[0069] Table 4: Root vigor measurement results under different treatments The results showed that, compared with treatment groups T2-T4, the straw fermentation product prepared by Example 4 could synergistically improve the root vigor of apple seedlings in continuously cropped soil.

[0070] (3) Results of soil harmful fungi detection: The results of real-time fluorescence quantitative detection of harmful fungi in soils under different treatments are shown in Table 5.

[0071] Table 5: Results of the determination of harmful fungi in soil under different treatments Data showed that the gene copy numbers of both Fusarium species in the soil of each treatment were lower than those of the continuous cropping control (CK), with the treatment using ginger straw fermentation products prepared in Example 4 (T1) showing the most significant effect. This result indicates that adding an appropriate amount of ginger straw fermentation products can effectively reduce the number of harmful Fusarium species in the soil.

[0072] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A microbial compound fermentation agent, characterized in that, It is a compound of Bacillus subtilis, Trichoderma harzianum and Trichoderma longiflorum in a ratio of (1-3):(1-3):(1-3) of viable bacteria; the preservation number of Bacillus subtilis is CGMCC NO.31990; the preservation number of Trichoderma harzianum is ACCC32526; the preservation number of Trichoderma longiflorum is ACCC32060.

2. The microbial compound fermentation agent according to claim 1, characterized in that, The microbial compound fermentation agent is composed of Bacillus subtilis, Trichoderma harzianum, and Trichoderma longifolia in a live cell ratio of 1:1:

1.

3. The microbial compound fermentation agent according to claim 1 or 2, characterized in that, The total viable count of the microbial compound fermentation agent is greater than or equal to 10. 8 CFU / ml.

4. The application of the microbial compound fermentation agent according to claim 1 in the production of pyrogallic acid from fermented straw.

5. A straw fermentation product, characterized in that, The product is prepared by the following method: after crushing and sterilizing the straw, inoculating it with the microbial compound fermentation agent according to any one of claims 1-3, adjusting the pH to 7.0-8.0, adding water to adjust the moisture content to 50-70%, and fermenting at 32℃ for 30-40 days to obtain the straw fermentation product.

6. The straw fermentation product according to claim 5, characterized in that, The straw in question is ginger straw.

7. The straw fermentation product according to claim 5, characterized in that, The amount of the microbial compound fermentation agent applied is 0.5-1.0% of the straw weight.

8. The use of the straw fermentation product of claim 5 in the following (1) or (2): (1) alleviating apple continuous cropping obstacles; (2) preparing a formulation to alleviate apple continuous cropping obstacles.

9. The application according to claim 8, characterized in that, The amount of straw fermentation products added is 1% of the mass of the apple rhizosphere soil.

Images