Multifunctional composite microbial inoculant and application thereof in rice straw returning to field

Abstract

This invention belongs to the field of microbial agents, and specifically discloses a multifunctional compound microbial agent and its application in rice straw return to the field, which is composed of Bacillus velezensis BD3, Bacillus halotolerans BN15, Bacillus velezensis CB13 and Bacillus amyloliquefaciens CB156. The multifunctional compound microbial agent of this invention can improve the efficiency of straw decomposition in the field, promote rice yield increase, and inhibit the growth of crop pathogens. When the multifunctional compound microbial agent is sprayed directly after straw is returned to the field, the degradation rate of rice straw increases by 14.87% after 70 days, and it can significantly inhibit the growth of rice blast fungus, achieving a control efficacy of 67.14% against rice blast. The multifunctional compound microbial agent alone can increase rice yield by 8.55%, and spraying the multifunctional compound microbial agent under the condition of direct straw return to the field can improve the yield reduction caused by straw return to the field, achieving a rice yield increase of 3.83%. The multifunctional compound microbial agent effectively solves the problems of slow straw decomposition, crop yield reduction, and large accumulation of pathogens and high disease incidence when straw is directly returned to the field.

Description

Technical Field

[0001] This invention relates to the field of microbial agents, and in particular to a multifunctional compound microbial agent and its application in rice straw return to the field. Background Technology

[0002] Returning straw to the field can effectively reduce soil bulk density and improve soil structure; increase soil organic carbon and nutrient content, and improve the composition of microbial communities. At the same time, returning straw to the field promotes the cycling and efficient utilization of farmland nutrients, replaces some chemical fertilizers, and plays an important role in protecting and utilizing black soil resources, ensuring food security, and promoting sustainable agricultural development.

[0003] my country has approximately 737 million tons of collectable straw resources, with a direct straw return rate of 52.33% and a return volume of 400 million tons. However, with the widespread adoption and increasing frequency of straw return, several pressing issues have emerged: First, straw decomposition is slow. Straw decomposition in the field relies heavily on soil microorganisms, but low soil microbial activity leads to slow decomposition, impacting subsequent operations and crop growth. Second, crop yields decrease after straw return. Incomplete straw crushing, excessively long straw, or excessive application result in loose soil with high air content, affecting seed germination and root growth. Furthermore, the high nitrogen content in crop straw can cause an imbalance in the soil's carbon-nitrogen ratio after return, hindering crop growth and leading to reduced yields. Third, the straw-return planting model leads to pathogen accumulation and increased disease incidence. Pathogens carried by straw enter the soil, increasing the number of pathogens as primary sources of infection and raising the risk of disease in subsequent crops. Returning straw to the field significantly increases the incidence and severity of rice sheath blight and rice blast, as well as soil-borne diseases in corn and wheat. This directly leads to huge yield losses in subsequent crops.

[0004] To address crop yield reduction caused by straw returning to the field, the type and amount of fertilizer should be adjusted according to soil conditions and crop needs to ensure sufficient nutrients to meet crop growth requirements. Additionally, applying microbial fertilizers during straw returning can promote crop growth and improve fertilizer efficiency. Regarding the high incidence of crop diseases caused by straw returning, the core solution is to effectively control the pathogen population. Some straw decomposition-promoting microbial agents have antibacterial effects, but their control efficacy is not significant. Applying microbial agents simultaneously with straw returning can control diseases, but their effects are singular and cannot promote straw decomposition; simultaneous application of both can easily lead to antagonistic reactions, causing the agents to become ineffective. Developing a multifunctional compound microbial agent for straw returning can effectively control the pathogen population, reduce crop disease incidence, promote crop growth, and ultimately increase crop yield while promoting straw decomposition. This is an effective solution to the above problems and is of great significance for the efficient and safe return of straw to the field and the efficient utilization of straw resources.

[0005] Therefore, those skilled in the art have provided a multifunctional compound microbial agent to solve the problems mentioned in the background art. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a multifunctional compound microbial agent and its application in rice straw return to the field.

[0007] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0008] One objective of this invention is to provide a multifunctional compound microbial agent, composed of Bacillus velezensis BD3, Bacillus halotolerans BN15, Bacillus velezensis CB13, and Bacillus amyloliquefaciens CB156; wherein the accession number of Bacillus velezensis BD3 is GDMCC.No. 64386, with an accession date of March 4, 2024; the accession number of Bacillus halotolerans BN15 is GDMCC.No. 64999, with an accession date of August 8, 2024; and the accession number of Bacillus velezensis CB13 is GDMCC.No. Accession number 65000, deposited on August 8, 2024; and accession number GDMCC.No 65001, deposited on August 8, 2024, are both deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The second objective of this invention is to provide an application of a multifunctional compound microbial agent in straw return to the field.

[0010] Specifically, the amount of rice straw returned to the field is 9000 kg / hm². 2 Rice straw is crushed into 3-5cm pieces and evenly spread on the surface of the paddy field. A multifunctional compound microbial agent and urea are then sprayed on top. The concentration of the compound microbial agent is 1×10⁻⁶. 8 CFU / mL, application rate 45 L / hm 2 The urea application rate is 30 kg / hm². 2 Rotary tillage returns the straw to the soil, followed by watering, and frequent submerged irrigation, alternating between dry and wet conditions.

[0011] Compared with existing technologies, the multifunctional compound microbial agent of this invention can improve the efficiency of straw decomposition in the field, promote rice yield increase, and inhibit the growth of crop pathogens. After 70 days of full straw return to the field, the degradation rate of rice straw by the compound microbial agent was 54.87%, which was 14.87% higher than the control. After 100 days, the degradation rate of rice straw by the compound microbial agent was 62.30%, which was 13.11% higher than the control. By maturity, the degradation rate of straw by the compound microbial agent reached 62.77%, compared with 59.8% in the control group. The compound microbial agent alone increased rice yield by 8.55%. Under the condition of full straw return to the field, spraying the microbial agent can improve the yield reduction caused by straw return to the field, achieving a rice yield increase of 3.83%. The compound microbial agent significantly inhibited the growth of rice blast fungus, with a control efficacy of 67.14% against rice blast. It effectively solves the problems of slow straw decomposition, crop yield reduction, large accumulation of pathogens, and high disease incidence. Attached Figure Description

[0012] Figure 1 Clear zones produced by strains BD3, BN15, CB156, and CB13 on selection medium.

[0013] Figure 2 The inhibitory effects of candidate strains on rice and maize pathogenic fungi are as follows: A. Ustilaginoidea virens; B. Magnaporthe oryza; C. Rhizoctonia solani; D. Fusarium fujikuroi; E. Exserohilumturcicum; F. Bipolari maydis; G. Curularia lunata.

[0014] Figure 3 The manganese peroxidase, laccase, cellulase, and filter paper enzyme activities of fermentation broths from different bacterial strain combinations were measured as follows: 1: BN15; 2: CB156; 3: BD3; 4: BN15+CB156; 5: BN15+BD3; 6: CB156+BD3; 7: BN15+CB156+BD3; 8: BN15+CB156+BD3+CB13; 9: BN15+CB156+BD3+CB118.

[0015] Figure 4 The degradation rates of rice straw by fermentation broths of different bacterial strains were as follows: 1: BN15; 2: CB156; 3: BD3; 4: BN15+CB156; 5: BN15+BD3; 6: CB156+BD3; 7: BN15+CB156+BD3; 8: BN15+CB156+BD3+CB13; 9: BN15+CB156+BD3+CB118.

[0016] Figure 5This is a phylogenetic tree of four bacteria based on their 16S rRNA sequences.

[0017] Figure 6 The degradation rate (a) and degradation efficiency (b) of rice straw by the multifunctional compound microbial agent.

[0018] Figure 7 The effects of a multifunctional compound microbial agent on rice plant height (a) and tillering (b).

[0019] Figure 8 The effect of multifunctional compound microbial agent on inhibiting the growth of rice blast fungus. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0021] Example 1: Screening of Four Strains

[0022] I. Isolation of bacteria from paddy field soil

[0023] Five soil samples, each weighing 200-300g, were obtained from winter paddy fields using a five-point sampling method. The samples were thoroughly mixed, and 20g of each sample was placed in an Erlenmeyer flask containing 50mL of sterile water. The flasks were then incubated at 15℃ and 180r / min with shaking for 1 hour, followed by static sedimentation. The supernatant was collected. Using the dilution plating method, 10μL of the diluted sample was diluted 10... 3 10 4 10 5 10 6 and 10 7 The supernatant was evenly spread onto LB medium and incubated at 15°C for 24 hours. Single colonies were picked for purification, and the purified bacteria were stored in a -76°C refrigerator with 30% glycerol.

[0024] II. Bacterial Isolation from Pig Biogas Slurry

[0025] Take 100 mL of pig biogas slurry in a sterile container, centrifuge at 4℃ and 5000 r / min for 10 min, and collect the supernatant; use the dilution plating method to dilute 1 mL of the supernatant 10 times. 3 10 4 10 5 10 6 and 10 7 Take 100 μL of the solution and spread it evenly on LB medium. Incubate at 20°C for 24 h. Pick a single colony for purification. After purification, store the purified product in a -76°C freezer with 30% glycerol.

[0026] III. Isolation of Insect Gut Bacteria

[0027] Insect intestinal tissue was collected under aseptic conditions, ground with sterile water, and centrifuged at 5000 r / min for 10 min at 4℃. The supernatant was collected. Using the dilution plating method, 1 mL of the supernatant was diluted 10 times. 3 10 4 10 5 10 6 and 10 7 Take 100 μL of the solution and spread it evenly on LB medium. Incubate at 20°C for 24 h. Pick a single colony for purification. After purification, store the purified product in a -76°C freezer with 30% glycerol.

[0028] IV. Initial Screening of High-Yield Lignocellulase Strains

[0029] Initial screening was performed using cellulose-Congo red medium and aniline blue medium. Strains were inoculated onto the center of plates and cultured at 20℃ for 7 days. The diameter of the clear zone and the diameter of the strain were then investigated. The results of the clear zone diameters of the four bacteria used to construct the inoculum on the screening medium are shown in Table 1. Figure 1 The diameters of the clear zone of strains BD3, BN15, CB156 and CB13 on cellulose-Congo red medium were 53.82 mm, 48.64 mm, 47.01 mm and 44.12 mm, respectively, and the clear zone ratios on aniline blue medium were 6.51, 5.18, 4.83 and 3.44, respectively.

[0030] Table 1. Diameter of the transparent zone produced by the strains on the selection medium.

[0031]

[0032] Assay of lignocellulase activity of candidate strains

[0033] The lignocellulase activity of the strains obtained in the initial screening was determined using a kit method. The cellulase activities of the four bacteria used to construct the inoculum are shown in Table 2. Strain BN15 had the highest manganese peroxidase and laccase activities, at 4405.33 U / L and 438.31 U / L, respectively, followed by CB156. Strain BD3 had the highest filter paper enzyme activity, at 306.80 U / L, followed by CB156 and BN15. Strain CB13 had the highest carboxymethyl cellulase activity, at 139.81 U / mL.

[0034] Table 2 shows the lignocellulase activities of some strains.

[0035]

[0036]

[0037] V. Degradation rate of rice straw by the strain

[0038] After cleaning the rice straw, cut it into small pieces of about 3cm and dry it at 80℃ until constant weight. Weigh 10g of dried straw into a sterile Erlenmeyer flask (250ml), add 100mL of sterile water and 10mL of candidate bacterial suspension (1×10⁻⁶). 7 (CFU / mL), with 110 ml of sterile water added to the blank control. After culturing at 20℃ for 15 days, the straw was rinsed with water and dried at 80℃ to constant weight. The remaining straw was weighed, and the straw degradation rate was calculated using the weight loss method. The degradation rates of rice straw by the four bacterial strains used to construct the inoculum are shown in Table 2. The degradation rates of rice straw by BD3, CB156, CB13, and BN15 were 24.70%, 23.6%, 23%, and 22.9%, respectively.

[0039] VI. Screening of Antagonistic Bacteria for Crop Pathogenic Fungi

[0040] The plate confrontation culture method was used. Rice and maize pathogenic fungal discs were inoculated into PDA medium, and candidate strains were inoculated 3 cm from the center of the discs. The culture was carried out at 20℃. The diameter of the control strains was measured when the control strains had fully grown on the plate, and the growth inhibition rate was calculated. The four bacterial strains CB13, CB156, BD3, and BN15 that were used to construct the inoculum agent all showed significant inhibitory effects against the main pathogenic fungi of rice and maize (Table 3). Figure 2 ).

[0041] Table 3 shows the inhibition rates of some strains against fungal pathogens.

[0042]

[0043]

[0044] VII. Antagonism assay among candidate strains

[0045] Based on the diameter of the clear zone on the screening medium, the activity of degrading enzymes, the degradation rate of rice straw, and the inhibition rate of crop pathogens, strains CB13, CB118, CB156, BD3, BN15, and PY1 were selected for antagonism testing. The results showed that some bacteria exhibited antagonistic effects (Table 4). Finally, strains CB156, BD3, and BN15, which did not antagonize each other, were selected to construct the basic bacterial community, while strains CB13 and CB118 inhibited each other and were used as candidate strains to construct the composite bacterial community.

[0046] Table 4 Results of interbacterial antagonism test

[0047] strain CB118 BD3 CB156 PY1 CB13 BN15 CB118 √ √ × × √ BD3 √ √ × √ √ CB156 √ √ × √ √ PY1 × × × √ √ CB13 × √ √ √ √ BN15 √ √ √ √ √

[0048] VIII. Construction of Microbial Agents

[0049] Basic bacterial communities were constructed using strains CB156, BD3, and BN15, and the results are as follows: Figure 3The results showed that the CB156+BD3+BN15 combination exhibited high degrading enzyme activity and rice straw degradation rate. Using CB156+BD3+BN15 as the base microbial community, and combining it with CB13 and CB118 strains, the results also showed high degrading enzyme activity and rice straw degradation rate. The CB156+BD3+BN15+CB13 combination produced manganese peroxidase, laccase, cellulase, and filter paper enzyme activities of 7709.33 U / L, 450.65 U / L, 154.67 U / mL, and 309.18 U / L, respectively. Figure 3 ); The degradation rate of rice straw after 15 days was 24.9% ( Figure 4 The sterile filtrate of the compound microbial agent showed inhibition rates of 54.12%, 60.57%, 63.02%, and 40.81% against rice false smut, rice blast, rice sheath blight, and rice seedling blight, respectively (Table 5).

[0050] Table 5. Inhibition rate of aseptic fermentation broth of compound microbial agent against rice pathogens.

[0051] Test pathogenic fungi Antibacterial rate (%) rice false smut fungus Ustilaginoideavirens 54.12±0.88 Magnaportheoryzae, the rice blast fungus 60.57±0.69 Rhizoctonia solani, the pathogen of rice sheath blight 63.02±0.32 rice seedling blight pathogen Fusarium fujikuroi 40.81±1.34

[0052] IX. Molecular identification of strains:

[0053] Genomic DNA was extracted from four bacterial strains using a rapid bacterial extraction kit. PCR amplification and sequencing of the 16S rDNA sequence were performed using universal bacterial primers 27F and 1492R. The PCR amplification conditions were: 95℃ for 5 min, 95℃ for 1 min, 56℃ for 2 min, 72℃ for 2 min, 30 cycles, followed by 72℃ for 10 min. The sequences were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The nucleotide sequences of strains CB13, CB156, BD3, and BN15 were uploaded to GenBank, with accession numbers OP430814, PP809059, PP033752, and PP809060, respectively. The 16S rDNA sequences of the four strains are shown in Table 6.

[0054] Table 6. 16S rDNA sequences of four bacterial strains

[0055]

[0056]

[0057] The obtained 16S rDNA sequence was compared with the nucleotide sequences in GenBank. Phylogenetic analysis was performed on Bacillus strains with high similarity, and *Escherichia coli* was selected as the external reference sequence. Figure 5The results showed that strains CB13 and BD3 were identified as Bacillus velezensis, CB156 as Bacillus amyloliquefaciens, and BN15 as Bacillus halotolerans.

[0058] Four strains have been deposited at the Guangdong Provincial Center for Microbial Culture Collection. The accession numbers for strains BD3, BN15, CB13, and CB156 are GDMCC.No 64386, GDMCC.No 64999, GDMCC.No 65000, and GDMCC.No 65001, respectively.

[0059] Example 2: Study on the effects of multifunctional compound microbial agent on promoting decomposition and increasing yield of rice straw

[0060] To verify that the multifunctional microbial agent constructed in the above embodiments can promote straw decomposition and increase yield, the following experiments were conducted:

[0061] The development of the multifunctional compound microbial agent involved field trials conducted from April 22, 2024 to October 25, 2024. Four treatments were established: ① CK; ② compound microbial agent; ③ full rice straw return to the field; ④ full rice straw return to the field + compound microbial agent. On April 22, straw return to the field was initiated. Rice straw was pulverized into 3-5 cm pieces, and 40 g of straw was placed in a mesh bag and buried at a depth of 20 cm in the paddy fields of treatments 3 and 4. The trials were repeated 21 times. At 10, 20, and 30 days after straw return, and at the seedling, tillering, heading, and maturity stages, the mesh bags were removed, the rice straw was cleaned, and dried in an 80℃ oven to constant weight. The degradation rate of the rice straw was determined using the loss-in-weight method. The straw return rate in the field trials was 9000 kg / hm². 2 The field plots are 5m × 6m in size. Rice straw is shredded into 3-5cm pieces, and 25kg of straw is returned to the field per plot. The straw is evenly spread on the surface of the paddy field, and a compound microbial agent is sprayed at a concentration of 1×10⁻⁶. 8 The application rate was 1.25L CFU / mL. 90g of urea was evenly spread and the straw was returned to the soil by rotary tillage. Then, water was added and the soil was irrigated frequently by submerging the soil, alternating between dry and wet conditions. The seedlings were transplanted on May 28. The changes in rice plant height and tillering were investigated during the tillering stage, and the yield was investigated after maturity.

[0062] Results of the compound microbial agent's degradation efficiency on rice straw: The degradation rate and efficiency of straw were measured at 10, 20, and 30 days after straw return to the field, at the seedling stage, tillering stage, heading stage, and maturity stage. The results showed that... Figure 6The compound microbial agent showed a higher degradation rate of rice straw than the control. On day 30 after straw return to the field, the degradation rate reached 36.53%, significantly higher than the control. The average degradation efficiency over 30 days was also significantly higher than the control. From the tillering to the heading stage, the degradation rate of rice straw by the compound microbial agent was consistently significantly higher than the control. On day 70, the degradation rate was 54.87%, 14.87% higher than the control. On day 100, the degradation rate was 62.30%, 13.11% higher than the control. By maturity, the degradation rate reached 62.77%, compared to 59.8% in the control group. The degradation efficiency of the compound microbial agent on rice straw was consistently higher than the control group. However, by maturity, there was no significant difference in degradation efficiency between the two groups. The results indicate that the compound microbial agent significantly improved the degradation efficiency of rice straw.

[0063] Results of the compound microbial agent on rice yield increase experiment: Rice plant height and tillering were investigated four times during the tillering stage. Figure 7 There were no significant differences in plant height among the four treatments. Compared with the control, the inoculant treatment significantly increased the number of rice tillers, with an increase of 17.73%. The treatments of full straw return to the field and full straw return to the field plus inoculant treatment increased the number of rice tillers, but not to a significant degree. Rice yield was investigated after maturity (Table 7). The results showed that the rice yield of the control group was 9604.92 kg / hm². 2 The yield of the single application of compound microbial agent was 10426 kg / hm. 2 The yield increased by 8.55% compared to the control; the yield of the treatment with full straw return to the field without the application of multifunctional microbial agents was 9009.93 kg / hm². 2 Compared to the control, the yield decreased by 6.19%, and the yield of the treatment with microbial agent spraying under the condition of full straw return to the field was 9803.25 / hm². 2 Compared with the control, the yield increased by 3.83%. The results show that the application of compound microbial agent alone significantly improved rice tillering and yield, with a yield increase of 8.55%. The application of compound microbial agent in combination with straw return to the field can mitigate the yield reduction caused by straw return to the field, resulting in a rice yield increase of 3.83%.

[0064] Table 7. Effects of multifunctional compound microbial agents and straw return to the field on rice yield.

[0065] deal with <![CDATA[Yield (kg / hm 2 )]]> Production growth rate % CK 9604.92±213.91ab —— Compound microbial agent 10426.59±196.30b 8.55％ Rice straw returned to the field 9009.93±517.03a -6.19％ Rice straw returning to the field + compound microbial agent 9803.25±524.74ab 3.83％

[0066] To verify that the multifunctional compound microbial agent constructed in the above embodiments can inhibit the growth of rice blast in rice straw and reduce the incidence of disease in seedlings, the following experiments were conducted:

[0067] Experimental Methods: A pot experiment on disease control was conducted in early April. The experimental treatments were: ① rice straw + rice blast fungus; ② rice straw + rice blast fungus + inoculant. The pot dimensions were 40cm × 60cm × 28cm (length × width × height). The straw return rate was 9000 kg / hm². 2 Based on the area of ​​each potted plant, 0.21 kg of straw was returned to the field per pot. After being crushed, the straw was inoculated with rice blast fungus at a dosage of 100 mL and a concentration of 1×10⁻⁶. 5 CFU / mL, followed by inoculation with a compound bacterial fermentation broth, at a volume of 10 mL and a concentration of 1×10⁻⁶. 8 CFU / mL was mixed with 40 kg of soil and placed in pots. Rice seedlings were transplanted in mid-May, with 2 rows per pot, 4 holes per row, and 3 seedlings per hole. Soil samples were collected at 10, 20, 30, 50, 70, 100, and 170 days. The content of rice blast fungus in the soil and straw mixture samples was detected by qRT-PCR. Rice blast fungus specific primers: 28SMF / 28SMR: 28SMF(5'-ACCCTACTGATGACCTCG-3') and 28SMR.

[0068] (5'-GTGTCAAAATTACAATACGC-3'); During the yellow ripening stage of rice, survey the entire panicle and grade each panicle according to the rate of loss due to disease.

[0069] The grading method is as follows:

[0070] Level 0: No disease;

[0071] Grade 1: Less than 5% loss per ear (individual branches affected);

[0072] Level 3: 6%–20% loss per ear (about one-third of the branches and stalks are affected);

[0073] Grade 5: 21%–50% loss per ear (disease on the neck or main axis, grains half-empty);

[0074] Level 7: 51%–70% loss per ear (neck disease, most ears are empty);

[0075] Grade 9: 71-100% loss per ear (caused by disease at the neck of the ear, resulting in white ears).

[0076] Method for calculating the disease index:

[0077]

[0078] In the formula: CK1—disease index of the control area;

[0079] PT1—Treatment area disease index.

[0080] Experimental results: Soil samples were collected on days 10, 20, 30, 50, 70, 100, and 170, and the content of rice blast fungus was detected by qRT-PCR. The results are as follows: Figure 8 As shown in the figure. The results showed that from day 10 to day 30, the compound microbial agent could inhibit rice blast fungus, but not significantly (P>0.05); from day 50 to day 170, the compound microbial agent significantly inhibited the growth of rice blast fungus in straw (P<0.05), with an inhibition rate of 74.12% against rice blast fungus;

[0081] On September 12, 2024, a survey of disease incidence and disease index was conducted, and the results are shown in Table 8. The incidence rate of rice blast fungus in the control group was 70.90%, and the disease index was 25.20. The incidence rate of rice blast fungus in the treatment group was 28.80%, and the disease index was 8.28. The multifunctional compound microbial agent achieved a control efficacy of 67.14% against rice blast.

[0082] Table 8. Control efficacy of multifunctional compound microbial agent against rice blast fungus.

[0083] deal with Incidence rate (%) Disease index Preventive efficacy (%) Rice straw + rice blast fungus 70.90 25.20 —— Rice straw + rice blast fungus + inoculant 28.80 8.28 67.14

[0084] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A multifunctional compound microbial agent, characterized in that, It is composed of *Bacillus velezensis* BD3, *Bacillus halotolerans* BN15, *Bacillus velezensis* CB13, and *Bacillus amyloliquefaciens* CB156; the accession number of *Bacillus velezensis* BD3 is GDMCC.No. 64386, deposited on March 4, 2024; the accession number of *Bacillus halotolerans* BN15 is GDMCC.No. 64999, deposited on August 8, 2024; and the accession number of *Bacillus velezensis* CB13 is GDMCC.No. Accession number 65000, deposited on August 8, 2024; and accession number GDMCC.No 65001, deposited on August 8, 2024, for Bacillus amyloliquefaciens CB156, are both deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of the multifunctional compound microbial agent as described in claim 1 in rice straw return to the field.

3. The application of the multifunctional compound microbial agent according to claim 2 in rice straw return to the field, characterized in that: The amount of rice straw returned to the field is 9000 kg / hm². 2 Rice straw is crushed into 3-5cm pieces and evenly spread on the surface of the paddy field. A multifunctional compound microbial agent and urea are then sprayed on top. The concentration of the compound microbial agent is 1×10⁻⁶. 8 CFU / mL, application rate 45 L / hm 2 The urea application rate is 30 kg / hm². 2 Rotary tillage returns the straw to the soil, followed by watering with shallow, frequent irrigation, alternating between dry and wet conditions.

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