Low-temperature-resistant straw-degrading composite microbial inoculant and application thereof

Abstract

This invention discloses a low-temperature resistant straw-degrading compound microbial agent and its application, belonging to the field of microbial technology. The active ingredients of the compound microbial agent include *Bacillus belye* Y10 and *Streptomyces loucherei* S36. The preservation number of *Bacillus belye* Y10 is CGMCC No. 32927, and the preservation number of *Streptomyces loucherei* S36 is CGMCC No. 32928. The concentration of *Bacillus belye* Y10 in the bacterial solution is 5 × 10⁻⁶. 8 CFU / mL, the concentration of the *Streptomyces louchei* S36 bacterial suspension was 5 × 10⁻⁶ CFU / mL. 6 CFU / mL, and the two are mixed at a volume ratio of 2:1, resulting in a total concentration of 1×10⁻⁶ CFU / mL for the compound bacterial agent. 8 The *Bacillus belyssioides* Y10 and *Streptomyces loucheri* S36 can grow stably at a low temperature of 15℃ and both have cellulose degradation capabilities. The compound microbial agent is used for straw degradation, crop growth promotion, or soil-borne disease control. This invention has the technical effects of low temperature resistance, high straw degradation efficiency, good strain synergy, and combining crop growth promotion with broad-spectrum antibacterial and disease prevention.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a low-temperature resistant straw degradation compound microbial agent and its application. Background Technology

[0002] Straw contains abundant nutrients such as carbon, nitrogen, and potassium, making it an important organic resource. It mainly refers to the stems and leaves remaining after the harvest of mature crops, such as the residues of wheat, corn, soybeans, and cotton. As a major agricultural country, my country produces a huge amount of crop straw, accounting for approximately 20%-30% of the world's total. Returning straw to the field is one of the main methods of straw disposal, effectively replenishing soil organic matter, improving soil structure, and reducing the use of chemical fertilizers. However, straw decomposes slowly in the natural environment and is highly dependent on ambient temperature, resulting in a long degradation cycle and incomplete decomposition. This not only affects subsequent operations, but uncomposted straw can also inhibit the growth of crop seedlings. Especially in cold northern regions, during autumn and winter, and early spring, low temperatures severely restrict the decomposition efficiency of straw.

[0003] Utilizing microorganisms to enhance straw degradation rates is currently an effective technical approach, with existing technologies largely focusing on single cellulose-degrading bacteria. However, single strains struggle to overcome the lignin-cellulose complex barrier during degradation, resulting in limited efficiency and a general lack of ability to inhibit soil-borne pathogens, potentially exacerbating disease risks. Furthermore, current research primarily focuses on mesophilic conditions, while studies on compound microbial agents with highly efficient degradation and disease prevention / growth promotion functions under low-temperature environments are scarce, failing to meet the practical needs of straw return to fields in cold regions and during autumn and winter. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a low-temperature resistant straw degradation compound microbial agent and its application. It has the advantages of low-temperature resistance, high straw degradation efficiency, good strain synergy, and both crop growth promotion and broad-spectrum antibacterial and disease prevention. It solves the technical problems of slow straw decomposition, incomplete degradation, easy inhibition of seedling growth, and aggravation of soil-borne diseases in the cold northern regions and low-temperature seasons.

[0005] This invention is achieved as follows: a low-temperature resistant straw degradation compound microbial agent, wherein the active ingredients of the compound microbial agent include Bacillus belye Y10 and Streptomyces loucheri S36, wherein the preservation number of Bacillus belye Y10 is CGMCC No. 32927 and the preservation number of Streptomyces loucheri S36 is CGMCC No. 32928.

[0006] As a preferred embodiment of the present invention, the concentration of the Bacillus berberis Y10 bacterial solution is 5 × 10⁻⁶. 8 CFU / mL, the concentration of the *Streptomyces louchei* S36 bacterial suspension was 5 × 10⁻⁶ CFU / mL. 6CFU / mL, and the two are mixed at a volume ratio of 2:1, resulting in a total concentration of 1×10⁻⁶ CFU / mL for the compound bacterial agent. 8 CFU / mL.

[0007] As a preferred embodiment of the present invention, the Bacillus belye Y10 and Streptomyces loucheri S36 can grow stably at a low temperature of 15°C and both have the ability to degrade cellulose. After treatment with Congo red staining, the ratio of the diameter of the transparent zone produced by the two to the diameter of the strain is 3.0 and 3.9, respectively.

[0008] As a preferred embodiment of the present invention, it also includes agriculturally acceptable auxiliary materials, which are one or more selected from wheat bran, corn flour, rice bran, diatomaceous earth, and kaolin.

[0009] As a preferred embodiment of the present invention, the compound microbial agent is in the form of powder, wettable powder, or suspension.

[0010] A method for preparing a low-temperature resistant straw degradation compound microbial agent includes the following steps: S1. Strain Culture: Bacillus belye Y10 was inoculated into LB liquid medium and cultured at 28℃ and 180 r / min for 2 days to obtain a concentration of 5×10⁻⁶. 8 Y10 bacterial suspension at CFU / mL; Streptomyces louchei S36 was inoculated into LB liquid medium and cultured at 28℃ and 180 r / min for 5-7 days to obtain a concentration of 5×10⁻⁶ CFU / mL. 6 S36 bacterial suspension at CFU / mL; S2. Combining: Mix the S36 bacterial solution and Y10 bacterial solution obtained in step S1 at a volume ratio of 2:1 to obtain a total concentration of 1×10⁻⁶. 8 CFU / mL compound bacterial agent; S3, Formulation: Add agriculturally acceptable excipients to the compound microbial agent obtained in step S2 to prepare the desired formulation.

[0011] Application of a low-temperature resistant straw degradation compound microbial agent, wherein the compound microbial agent is used for straw degradation, crop growth promotion or soil-borne disease control.

[0012] As a preferred embodiment of the present invention, the straw is the straw of at least one of the following crops: wheat, corn, soybean, and cotton. The crop is at least one of the following: watermelon, American foxtail grass, wheat, corn, soybean, and cotton. The control of soil-borne diseases includes at least one of the following: wheat root rot, corn root rot, cotton verticillium wilt, and watermelon wilt.

[0013] As a preferred embodiment of the present invention, the compound microbial agent is applied by irrigation, seed soaking, or foliar spraying, and the concentration of the compound microbial agent is 1×10⁻⁶ when irrigating or soaking seeds. 8 CFU / mL, the compound microbial agent should be diluted before use when applying it as a foliar spray.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: Excellent low-temperature resistance and high straw degradation efficiency: The Y10 and S36 strains in the compound microbial agent of this invention can grow stably and play a role in a low-temperature environment of 15℃, which can effectively overcome the restriction of low temperature on straw degradation, effectively shorten the straw decomposition cycle, and solve the problem of slow straw degradation when returning straw to the field in cold northern regions and in autumn and winter. The strains have a significant synergistic effect and strong enzyme production capacity: there is no antagonistic effect between strains Y10 and S36, and they can work synergistically. Both can produce high levels of cellulase, which can effectively break through the lignin-cellulose complex barrier and improve the degradation efficiency of straw. Multifunctional and synergistic: This compound microbial agent not only efficiently degrades straw but also significantly promotes crop growth, exhibiting excellent growth-promoting effects on crops such as watermelon and American foxtail grass, increasing plant height, root length, stem diameter, and biomass accumulation. Simultaneously, it demonstrates significant control over various soil-borne diseases, including wheat root rot, corn root rot, cotton verticillium wilt, and watermelon wilt, achieving a synergistic effect of straw degradation, crop growth promotion, and green control of soil-borne diseases. The preparation process is simple and the cost is low: the strain is cultured using conventional LB medium, the raw materials are readily available and low in cost, and the preparation process only requires three core steps: activation, propagation and compounding. No complex equipment or cumbersome operations are required, which can realize large-scale production and greatly reduce the cost of bacterial agent preparation and promotion. The strains are stable and highly adaptable to the environment: After screening and identification, strains Y10 and S36 are stable and highly adaptable to the environment. They can maintain high enzyme activity even under low temperature conditions, making them suitable for widespread application in the field. Green and environmentally friendly, with no residue: The compound microbial agent of this invention uses microorganisms as active ingredients, with no chemical pesticide residues. It is safe for crops and environmentally friendly, and can reduce the use of chemical pesticides and fertilizers. It meets the needs of green and sustainable development in modern agriculture and further enhances the promotion value of this microbial agent. Attached Figure Description

[0015] Figure 1 This is a Congo red staining result diagram of strains Y10 and S36 provided in the embodiments of the present invention.

[0016] Figure 2 This is a graph showing the filter paper strip disintegration test results of strains Y10 and S36 provided in the embodiments of the present invention.

[0017] Figure 3 These are morphological images of solid culture medium plates for strains Y10 and S36 provided in this embodiment of the invention: where A: morphological image of strain Y10 on LB medium plate; B: morphological image of strain S36 on Gao's No. 1 medium plate.

[0018] Figure 4 This is a glucose standard curve provided in an embodiment of the present invention.

[0019] Figure 5 The following is a graph showing the enzyme production capacity of strains Y10 and S36 provided in the embodiments of the present invention: where A: carboxymethyl cellulase (CMCase) activity; B: filter paper enzyme (FPA) activity.

[0020] Figure 6 This describes the straw degradation rate of single and compound inoculants of Actinomycete S36 and Bacillus vesiculosus Y10 provided in the embodiments of the present invention.

[0021] Figure 7 The images shown are scanning electron microscope (SEM) images of straw containing single and compound inoculants of Actinomycete S36 and Bacillus vesiculosus Y10 provided in this embodiment of the invention: Wherein, A: morphology at 50µm and 30µm after water treatment (CK); B: morphology at 50µm and 30µm after inoculation with strain Y10; C: morphology at 50µm and 30µm after inoculation with strain S36; D: morphology at 50µm and 30µm after inoculation with compound inoculant.

[0022] Figure 8 The following is a graph showing the effects of compound microbial agents Y10 and S36 provided in this embodiment of the invention on the physiological indicators of American foxtail grass: where A: chlorophyll and carotenoid content; B: soluble sugar and soluble protein content; C: SOD activity; D: POD activity; CK is the water control group; Y10+S36 is the compound microbial agent treatment group. Detailed Implementation

[0023] To further understand the invention's content, features, and effects, the following embodiments are provided, and detailed descriptions are given in conjunction with the accompanying drawings.

[0024] The structure of the present invention will now be described in detail with reference to the accompanying drawings.

[0025] Example 1: Strain Screening 1.1 Initial screening: The glycerol tubes of the strain stored at -80℃ were activated and propagated using LB and Gao's No. 1 solid medium, respectively. Single colonies were picked and inoculated into selective medium with sodium carboxymethyl cellulose as the sole carbon source. After the strain had grown well, 1 g / L Congo red solution was poured into the plate. After 15-20 min, the staining solution was decanted, and then 1 mol / L NaCl aqueous solution was used to decolorize for 15-20 min. The presence of a clear hydrolysis zone was observed. like Figure 1As shown, the presence of a transparent hydrolysis zone indicates that the strain has cellulose degradation function. The diameter of the transparent hydrolysis zone (D) and the diameter of the colony (d) were measured, and the ratio of the hydrolysis zone diameter to the colony diameter was calculated. The cellulase activity of the isolated strain was initially judged based on the size of the ratio. The larger the ratio, the stronger the enzyme production ability and the stronger the decomposition ability. As shown in Table 1, two strains were obtained through screening.

[0026] Table 1 Comparison of hydrolysis values ​​of strains The formula for LB solid medium is: 10g tryptone, 10g yeast extract, 10g NaCl, and 20g agar; The formula for sodium carboxymethyl cellulose medium is as follows: 10g sodium carboxymethyl cellulose, 2.0g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 1g peptone, 4g ammonium sulfate, and 20g agar; The formula for Gao's No. 1 solid culture medium is as follows: 20g soluble starch, 0.5g sodium chloride, 0.5g magnesium sulfate heptahydrate, 1g potassium nitrate, 0.5g dipotassium hydrogen phosphate, 0.01g ferric sulfate, and 20g agar. All the above culture media were brought to a final volume of 1000 mL with distilled water, the pH was adjusted to 7.0, and then autoclaved at 121°C for 20 minutes before use.

[0027] 1.2 Secondary screening: Filter paper strip disintegration test Two strains with high hydrolysis values ​​were inoculated into their respective liquid culture media to prepare seed culture. These seed cultures were then transferred to filter paper strip culture media at a 5% inoculum size and incubated at 15℃ and 180 rpm for 14 days. The disintegration of the filter paper strips was observed and recorded. Figure 2 As shown, the filter paper strips in Y10 and S36 disintegrated significantly, indicating that both strains still have a strong cellulose degradation ability under low temperature conditions. Filter paper strip culture medium: 3 filter paper strips (1cm x 6cm) per bottle, 0.1g yeast extract, 1g ammonium sulfate, 0.5g magnesium sulfate heptahydrate, 1g potassium dihydrogen phosphate, and distilled water to a final volume of 1000mL.

[0028] Example 2: Strain Identification 2.1 Observation of strain morphology: Strains Y10 were inoculated onto LB medium, and strain S36 was inoculated onto Gao's I medium. Colony morphology was observed after incubation at 28°C. Figure 3 As shown, the results indicate that on LB medium, the colonies of strain Y10 are milky white and round, with a smooth and raised surface, and a viscous texture that is easy to pick up; on Gao's I medium, the colonies of strain S36 are white-brown and round, with a concave center and slightly raised edges, and wavy notched edges.

[0029] 2.2 Molecular biological identification of the strain: Strains Y10 were picked and inoculated onto LB medium. After culturing at 28°C for 48 hours, the bacterial sludge was collected. Strains S36 were picked and inoculated onto Gao's I medium. After culturing at 28°C for 5-7 days, the bacterial sludge was collected. Genomic DNA of strains Y10 and S36 was extracted using the 16S rDNA identification method. Their gene sequences were amplified using universal primers 27F / 1492R. The sequencing results were analyzed using BLAST software in NCBI for similarity searches. The DNA sequence alignment of strain Y10 showed over 99% similarity to *Bacillus velezensis*, therefore it was classified as *Bacillus velezensis*. Its accession number is CGMCC No. 32927. The DNA sequence alignment of strain S36 identified it as *Streptomyces rochei*, with accession number CGMCC No. 32928.

[0030] Example 3: Antagonistic Effect of Strains The two selected strains were subjected to mutual antagonism test, and the results are shown in Table 2. The strains did not antagonize each other (Note: "+" indicates antagonism and "-" indicates no antagonism).

[0031] Table 2 Results of strain antagonism

[0032] Example 4: Determination of enzyme production capacity of the strain 4.1 Glucose standard curve: Prepare a 1 mg / mL glucose standard solution. Take the volumes shown in Table 3, add distilled water to each 10 mL centrifuge tube, and bring the volume to 2 mL. Then add 1.5 mL of DNS solution, shake to mix, and immediately boil in boiling water for 5 minutes. After cooling to room temperature, bring the volume to 10 mL. Measure the absorbance at 540 nm using a microplate reader. Plot a standard curve with the glucose solution concentration in the test tube as the x-axis and the absorbance at 540 nm as the y-axis. The glucose standard curve is shown below. Figure 4 As shown (y = 0.08x + 0.0011, R² = 0.9993).

[0033] Table 3. Preparation of Glucose Standard Solution

[0034] 4.2 Cellulase Activity Assay: Transfer 5% of the bacterial seed culture to the enzyme-producing medium and culture at 15℃ with shaking at 180 rpm. Take samples every 2 days to measure carboxymethyl cellulase activity and filter paper enzyme activity. Take 2 mL of bacterial culture into a centrifuge tube and centrifuge at 8000 rpm at 4℃ for 10 min. The supernatant is the crude enzyme solution.

[0035] CMC enzyme activity assay: Add 0.5 mL of crude enzyme solution to a 10 mL test tube, add 1.5 mL of 1% sodium carboxymethyl cellulose solution, and set up a control group by adding boiled and inactivated crude enzyme solution. Both the experimental group and the control group were set up in triplicate. The reaction was carried out in a 50℃ water bath for 30 min, then 1.5 mL of DNS solution was added, shaken well, and then cooled to room temperature by running water after boiling for 10 min. The absorbance was measured at a wavelength of 540 nm using a spectrophotometer, and the result was converted to glucose concentration according to the glucose standard curve. The CMC enzyme activity was calculated according to (Formula 1-1).

[0036] FPA enzyme activity assay: Add 0.5 mL of crude enzyme solution to a 10 mL test tube. Add an equal amount of boiled and inactivated crude enzyme solution to the control group. Add 1.5 mL of buffer solution and one filter paper (1 cm x 6 cm) to each test tube. Incubate at 50 °C for 60 min. Add 1.5 mL of DNS solution, shake well, incubate at boiling water for 10 min, and then cool to room temperature under running water. Measure the absorbance at 540 nm and calculate the glucose concentration. Calculate the filter paper enzyme activity according to (Formula 1-1).

[0037] (Formula 1-1): Enzyme activity (U / mL) = G × N × 5.56 × 1000 / T Where G is the amount of glucose corresponding to the absorbance value, in mg / mL; N is the dilution factor; T is the reaction time (min); and 5.56 is the amount of substance (μmol) of 1 mg of glucose.

[0038] like Figure 5 As shown, strains Y10 and S36 were found to have good enzyme production capabilities. After 4-6 days of culture, the carboxymethyl cellulase activity reached 14.22 U / mL and 15.28 U / mL, respectively, and the filter paper enzyme activity reached 7.22 U / mL and 8.32 U / mL, respectively.

[0039] Enzyme-producing culture medium: 10g sodium carboxymethyl cellulose, 5g peptone, 2.5g yeast extract, 2g ammonium sulfate, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 5g sodium chloride, and distilled water to a final volume of 1000mL.

[0040] Example 5: Preparation of Compound Microbial Agent Y10 was cultured in LB liquid medium for 2 days at 28℃ and 180 rpm, while S36 was cultured in LB liquid medium for 5-7 days. The concentration of the actinomycete S36 culture was 5 × 10⁻⁶.6 CFU / mL, the concentration of Y10 bacterial culture is 5×10⁻⁶. 8 CFU / mL.

[0041] The compound microbial agent was prepared by mixing S36 and Y10 at a volume ratio of 2:1 to obtain the compound microbial agent, the total concentration of which was 1×10⁻⁶. 8 CFU / mL was used to obtain a straw-degrading compound microbial agent.

[0042] Example 6: Degradation determination of straw by liquid fermentation 6.1 Experimental Design: The strain seed culture was transferred at a 5% inoculum to straw liquid fermentation medium. The culture was carried out at 15℃ with shaking at 180 r / min for 30 days. The straw was then dried and weighed. The degradation rate of the straw by each strain was measured and calculated according to (Formula 2-1). Straw residue samples were plated with gold and the surface morphology was observed using a scanning electron microscope (SEM).

[0043] (Formula 2-1): Degradation rate = (W0 - W1) / W0 × 100% Where W0 is the weight of the straw before fermentation, W 1为 Weight of straw after fermentation.

[0044] 6.2 Experimental Results: like Figure 6 As shown, the degradation rate of the compound bacterial agent inoculation was 36%, significantly higher than that of the control group (CK) and higher than that of the single-strain inoculation group; this was observed by SEM. Figure 7 As shown, the control CK straw surface is smooth and continuous in sheet form with an intact structure; the surface structure of straw inoculated with only a single strain and straw inoculated with a compound strain is significantly damaged, and the surface of straw inoculated with a compound strain is more severely eroded locally, which can significantly damage the lignocellulose barrier on the surface of the straw.

[0045] Straw liquid fermentation medium: 10g sodium carboxymethyl cellulose, 0.5g sodium chloride, 0.5g magnesium sulfate heptahydrate, 1g dipotassium hydrogen phosphate, 2g peptone, 0.5g yeast extract, 2g wheat straw per bottle, and distilled water to a final volume of 1000mL.

[0046] Example 7: The growth-promoting effect of straw-degrading compound microbial agent on watermelon seedlings 7.1 Experimental Design: This experiment used the watermelon variety 'Huaxin' as the material and set up two treatments: a water control (CK) and a compound microbial agent treatment (Y10+S36), with each treatment replicated three times. Seedlings were transplanted when they reached the two-leaf-one-heart stage. The Y10+S36 treatment group was irrigated with the compound microbial agent (concentration 1×10⁻⁶). 8 The control group (CFU / mL) was treated with an equal volume of sterile water; plant height, stem diameter, root length, fresh weight, and dry weight were measured 30 days after treatment.

[0047] 7.2 Experimental Results: Table 4 shows that the plant height, root length, and stem diameter of the treated seedlings increased by 31.93%, 25.19%, and 49.52% respectively compared to the water control (CK), indicating a promoting effect on the overall plant growth. The fresh weight and dry weight of the treated group increased by 48.72% and 58.18% respectively compared to CK, demonstrating that this inoculant significantly promotes the growth and biomass accumulation of watermelon seedlings. (Note: Data in the table are mean ± standard deviation, the same below.) Table 4. Effects of straw-degrading microbial agents on watermelon seedling growth

[0048] Example 8: The growth-promoting effect of straw-degrading compound microbial agent on American foxtail grass 8.1 Experimental Design: This experiment used the American foxtail grass (Pennisetum glaucum) variety 'Jingza 2409' as material: two treatments were set up: water soaking (CK) and compound microbial inoculant soaking (Y10+S36); plump seeds were selected, and the Y10+S36 group was treated with compound microbial inoculant (concentration 1×10). 8 Seeds were soaked in CFU / mL solution, while the control group was treated with sterile water. Each treatment was repeated three times. Plant height, leaf length, leaf width, and fresh weight of aboveground parts and roots were measured. Physiological and biochemical indicators such as chlorophyll content, carotenoid content, soluble protein content, soluble sugar content, superoxide dismutase (SOD) activity, and peroxidase (POD) activity were also measured.

[0049] 8.2 Experimental Results: Table 5 shows that compared with the water control (CK), the compound microbial agent (Y10+S36) treatment significantly promoted the growth of *Pennisetum purpureus*. The plant height with the compound microbial agent was 75.92 cm, an increase of approximately 30.13%, and the aboveground fresh weight reached 21.45 g, an increase of 38.92%. The underground fresh weight increased by 107.79%, leaf width increased, while leaf length showed no significant difference. Figure 8 As shown, by Figure 8 It can be seen that after treatment with the microbial agent, the content of soluble sugar and soluble protein, as well as the activities of SOD and POD, significantly increased, while the content of chlorophyll and carotenoids decreased. This indicates that the compound microbial agent can effectively promote the growth of American foxtail grass and the accumulation of biomass by regulating physiological metabolism and enhancing antioxidant capacity, especially its promoting effect on the growth of underground parts is more prominent.

[0050] Table 5. Effects of straw-degrading microbial agents on the growth of American foxtail grass.

[0051] Example 9: Plate confrontation experiment of straw degradation compound microbial agent To verify the antagonistic ability of the strains (S10 and S36) in the compound microbial agent of the present invention against common plant pathogenic fungi, thereby proving that the microbial agent has the potential effect of inhibiting soil-borne diseases while degrading straw; strains S10 and S36 prepared in Example 1 and activated plant pathogens, including wheat root rot pathogen, corn root rot pathogen, cotton verticillium wilt pathogen, and watermelon wilt pathogen, were placed on PDA solid culture medium plates.

[0052] 9.1 Experimental Design: The plate confrontation method was used. The pathogenic fungal spores were taken with a sterile punch and inoculated into the center of a new PDA plate. S10 and S36 were symmetrically streaked on both sides of the plate about 2 cm away from the pathogenic fungal spores. Plates inoculated only with pathogenic fungal spores were used as a control group. All plates were placed in an incubator at 28℃ and inverted for 5-7 days.

[0053] 9.2 Experimental Results: Measure and record the data, and calculate the antibacterial rate according to (Formula 3-1). (Formula 3-1): Inhibition rate (%) = (Coronary diameter of control group - Pathogen diameter of treatment group) / Colony diameter of control group × 100%; The test results are shown in Table 6. The two biocontrol strains contained in the compound microbial agent of this invention showed different degrees of antagonism against the four tested pathogenic fungi, with inhibition rates ranging from 51.6% to 85.1%, indicating that both are excellent biocontrol strains with broad-spectrum application potential.

[0054] Table 6. Inhibition rate of biocontrol strains against plant pathogenic fungi

[0055] Example 10: Experiment on the control of watermelon wilt disease by straw-degrading compound microbial agent To verify the control effect of the compound microbial agent on watermelon wilt, and to clarify its combined function of inhibiting soil-borne diseases, this study aims to provide a basis for assessing its agricultural application potential.

[0056] 10.1 Experimental Design: The watermelon seedlings were cultured to the two-leaf-one-heart stage before transplanting. This experiment included three treatments: a blank control (CK, watered with only the same amount of water), a pathogen control (FON, inoculated with 1×10⁻⁶ bacteria after transplanting), and a pathogen control (FON, inoculated with 1×10⁻⁶ bacteria after transplanting). 6The treatment included CFU / mL FON bacterial suspension, compound bacterial agent, and pathogen co-treatment (Y10+S36+FON, straw degradation compound bacterial agent prepared in Example 5 was applied 2 days before transplanting, FON bacterial suspension was applied after transplanting, and an equal amount of compound bacterial agent was applied 7 days after transplanting). Each treatment was repeated 3 times. During the treatment, the disease index and control effect of watermelon wilt were calculated according to (Formula 4-1) and (Formula 4-2).

[0057] (Formula 4-1): Disease index = Σ (Disease grade × Number of plants at that disease grade) / (Maximum disease grade × Total number of plants) × 100 (Formula 4-2): Prevention and control effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0058] 10.2 Experimental Results: As shown in Table 7, compared with the FON group inoculated only with pathogens, the disease index of watermelon wilt in the treatment group (Y10+S36+FON) with straw degradation compound microbial agent was significantly reduced, from 96.17 to 33.05, with a control effect of 65.64%. This result indicates that the compound microbial agent can effectively inhibit the occurrence of watermelon wilt, significantly reduce the severity of the disease, and has good potential for biocontrol application. The experimental results fully demonstrate that the straw-degrading compound microbial agent of the present invention not only has the functions of degradation and growth promotion, but also the strains contained therein can effectively inhibit the growth of a variety of plant pathogenic fungi. In field application, it can play a multi-effect role, that is, while degrading straw, it promotes plant growth and prevents and reduces soil-borne diseases caused by pathogens.

[0059] Table 7. Control effect of straw-degrading compound microbial agent on watermelon wilt disease.

[0060] sequence list The 16S rDNA sequence of the Y10 bacterium:

[0061] The 16S rDNA sequence of the S36 bacterium:

[0062] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0063] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A low-temperature resistant straw degradation compound microbial agent, characterized in that: The active ingredients of the compound microbial agent include Bacillus belyssus Y10 and Streptomyces loucheri S36. The preservation number of Bacillus belyssus Y10 is CGMCC No. 32927, and the preservation number of Streptomyces loucheri S36 is CGMCC No. 32928.

2. The low-temperature resistant straw degradation compound microbial agent as described in claim 1, characterized in that: The concentration of the *Bacillus vesiculus* Y10 bacterial culture was 5 × 10⁻⁶. 8 CFU / mL, the concentration of the *Streptomyces louchei* S36 bacterial suspension was 5 × 10⁻⁶ CFU / mL. 6 CFU / mL, and the two are mixed at a volume ratio of 2:1, resulting in a total concentration of 1×10⁻⁶ CFU / mL for the compound bacterial agent. 8 CFU / mL.

3. The low-temperature resistant straw degradation compound microbial agent as described in claim 1, characterized in that: The Bacillus belye Y10 and Streptomyces loucheri S36 can grow stably at a low temperature of 15℃ and both have the ability to degrade cellulose. After Congo red staining, the ratio of the diameter of the transparent zone produced by the two strains to the diameter of the strain is 3.0 and 3.9, respectively.

4. The low-temperature resistant straw degradation compound microbial agent as described in claim 1, characterized in that: It also includes agriculturally accepted auxiliary materials, which are one or more of wheat bran, corn flour, rice bran, diatomaceous earth, and kaolin.

5. The low-temperature resistant straw degradation compound microbial agent as described in claim 1, characterized in that: The compound microbial agent is available in the form of powder, wettable powder, or suspension.

6. The method for preparing the low-temperature resistant straw degradation compound microbial agent according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Strain Culture: Bacillus belye Y10 was inoculated into LB liquid medium and cultured at 28℃ and 180 r / min for 2 days to obtain a concentration of 5×10⁻⁶. 8 Y10 bacterial suspension at CFU / mL; Streptomyces louchei S36 was inoculated into LB liquid medium and cultured at 28℃ and 180 r / min for 5-7 days to obtain a concentration of 5×10⁻⁶ CFU / mL. 6 S36 bacterial suspension at CFU / mL; S2. Combining: Mix the S36 bacterial solution and Y10 bacterial solution obtained in step S1 at a volume ratio of 2:1 to obtain a total concentration of 1×10⁻⁶. 8 CFU / mL compound bacterial agent; S3, Formulation: Add agriculturally acceptable excipients to the compound microbial agent obtained in step S2 to prepare the desired formulation.

7. The application of the low-temperature resistant straw degradation compound microbial agent according to any one of claims 1-5, characterized in that, The compound microbial agent is used for straw degradation, crop growth promotion, or control of soil-borne diseases.

8. The application of the low-temperature resistant straw degradation compound microbial agent as described in claim 7, characterized in that: The straw is the straw of at least one of the following crops: wheat, corn, soybean, and cotton. The crop is at least one of the following crops: watermelon, American foxtail grass, wheat, corn, soybean, and cotton. The soil-borne disease control includes at least one of the following diseases: wheat root rot, corn root rot, cotton verticillium wilt, and watermelon wilt.

9. The application of the low-temperature resistant straw degradation compound microbial agent as described in claim 7, characterized in that: The compound microbial agent is applied by irrigation, seed soaking, or foliar spraying. When irrigating or soaking seeds, the concentration of the compound microbial agent is 1×10⁻⁶. 8 CFU / mL, the compound microbial agent should be diluted before use when applying it as a foliar spray.

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