A composite biological preparation and application thereof

Abstract

This invention belongs to the field of agricultural biotechnology, specifically relating to the application of a compound biological agent in agriculture. The compound biological agent comprises live engineered Bacillus subtilis bacteria and a 3% glucose solution; wherein the engineered Bacillus subtilis bacteria are obtained through genetic modification using Bacillus subtilis as the substrate bacteria, and the live bacteria concentration is ≥1×10¹. 0 The 3% glucose solution (CFU / mL) is a 3% (w / v) aqueous solution of glucose, and the two are mixed at a volume ratio of 1:(800-1200). This invention's compound biological agent combines the soil microecological regulation and root growth promotion capabilities of Bacillus subtilis engineered live bacteria with the sugar metabolism regulation effects of 3% glucose. The synergistic effect of these two agents significantly promotes beet root growth, increases the activity of beet sugar metabolism enzymes, and increases the accumulation of sucrose and soluble sugars in the tubers. Simultaneously, it improves the rhizosphere soil microecological environment of beet and enhances soil phosphorus activation capacity. This provides an environmentally friendly and efficient biological agent for high-quality and high-yield beet cultivation, and has promising agricultural application prospects.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a compound biological agent and its application in promoting sugar beet growth and sugar accumulation, particularly to a compound biological agent composed of engineered live Bacillus subtilis bacteria and 3% glucose, and its application in high-quality and high-yield sugar beet cultivation. Background Technology

[0002] Sugar beets are a globally important source of sugar and an important economic crop, with their root sugar content directly determining the production efficiency and economic benefits of the sugar industry. Currently, traditional methods for increasing the sugar content of sugar beets in agricultural production mainly rely on the application of chemical fertilizers and plant growth regulators. While these methods can promote growth to some extent, they have many drawbacks: excessive application of chemical fertilizers can damage soil structure, reduce soil fertility, and lead to eutrophication of water bodies; improper use of chemical regulators can easily lead to a decline in sugar beet quality and pesticide residues, and long-term use can cause sugar beets to develop resistance, gradually reducing the regulatory effect. Therefore, the development of environmentally friendly, safe, and efficient biological agents has become a research hotspot in the field of sugar beet cultivation.

[0003] Glucose, as an important carbon source and signaling molecule in plants, can be effectively regulated by applying appropriate concentrations of exogenous glucose. Studies have shown that root application of a 3% glucose solution can significantly increase the activity of sugar metabolism enzymes such as sucrose phosphate synthase (SPS) and sucrose synthase (SS) in sugar beets. By regulating the expression of key genes such as MYB44 and MYB15, it promotes sucrose synthesis and transport, increasing the accumulation of soluble sugars and sucrose in the roots. However, a single 3% glucose solution can only regulate sugar accumulation at the plant's own metabolic level and cannot improve the microecological environment of the rhizosphere soil in sugar beets. Problems such as available phosphorus deficiency, imbalanced microbial community structure, and weak colonization capacity of beneficial microorganisms in the soil will still limit nutrient absorption and growth of sugar beets, thus affecting the sugar accumulation effect.

[0004] Bacillus subtilis is a widely used beneficial microorganism in agriculture. Genetically engineered Bacillus subtilis can efficiently colonize the rhizosphere of sugar beets, continuously secreting "microbe-crop" interaction signaling substances such as acetoin and 2,3-butanediol. When applied to the soil, live Bacillus subtilis can effectively regulate the rhizosphere microbial community structure, increase the abundance of phosphate-solubilizing bacteria and alkaline phosphatase activity, promote soil phosphorus activation, and directly promote crop root growth, enhancing the crop's ability to absorb nutrients. However, live Bacillus subtilis alone lacks direct regulatory effects on the plant's own sugar metabolism pathways and cannot promote the synthesis and accumulation of sucrose in sugar beets at the molecular level. Furthermore, the colonization and proliferation of live bacteria in the soil require carbon source support, and the activity of live bacteria is easily affected by the soil environment when applied alone, limiting the regulatory effect.

[0005] In summary, neither a single 3% glucose solution nor Bacillus subtilis engineered live bacteria is sufficient to promote sugar beet growth and sugar accumulation. Currently, there are no reports on compound biological agents that combine the two, utilizing glucose to provide a carbon source for the live bacteria and synergistically regulating the rhizosphere soil microecology and sugar metabolism of sugar beets. This invention prepares a compound biological agent by combining Bacillus subtilis engineered live bacteria with a 3% glucose solution in a specific ratio. The 3% glucose provides signaling molecules and a carbon source for sugar metabolism in sugar beets, and also provides nutrients for the colonization and proliferation of the engineered live bacteria in the rhizosphere. The engineered live bacteria improve the soil microecology and promote root growth. The two work synergistically to achieve a triple effect of soil microecological regulation, root growth promotion, and plant sugar metabolism regulation, significantly improving sugar beet growth indicators and sugar accumulation, providing a new technical solution for high-quality and high-yield sugar beet cultivation. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a compound biological agent comprising engineered live Bacillus subtilis bacteria and a 3% glucose solution. The synergistic effect of the two can significantly promote the growth and sugar accumulation of sugar beets, while improving the rhizosphere soil microecology.

[0007] Another objective of this invention is to provide a method for preparing the above-mentioned compound biological agent, which is simple to operate, low in cost, can maintain the activity of live bacteria to the greatest extent, and is suitable for large-scale production.

[0008] Another objective of this invention is to provide the application of the above-mentioned compound biological agent in promoting the growth and sugar accumulation of sugar beets. The application method is simple, the regulatory effect is significant, and it is suitable for large-scale agricultural promotion.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A compound biological agent comprises live engineered Bacillus subtilis bacteria and a 3% glucose solution; the engineered Bacillus subtilis bacteria are genetically engineered bacteria obtained by knocking out the α-subunit gene of acetoin dehydrogenase and the phosphoacetyltransferase gene in the genome of Bacillus subtilis as the substrate bacteria, while expressing the acetyllactone synthase gene and the α-acetyllactone decarboxylase gene.

[0010] The viable bacterial concentration of this engineered bacteria is ≥1×10⁻⁶. 10 CFU / mL; the 3% glucose solution is a glucose aqueous solution with a mass-volume fraction of 3%; the volume ratio of the engineered Bacillus subtilis live bacteria to the 3% glucose solution is 1:(800-1200).

[0011] The engineered Bacillus subtilis strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 11, 2026, with accession number CGMCC No. 37899; the Bacillus subtilis strain was deposited at the same center on March 3, 2026, with accession number CGMCC No. 37834.

[0012] Preferably, the amino acid sequence of the α subunit of the acetoin dehydrogenase is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.2; the amino acid sequence of the phosphoacetyltransferase is shown in SEQ ID NO.3, and the nucleic acid sequence is shown in SEQ ID NO.4; the amino acid sequence of the acetyllactate synthase is shown in SEQ ID NO.5, and the nucleic acid sequence is shown in SEQ ID NO.6; the amino acid sequence of the α-acetyllactate decarboxylase is shown in SEQ ID NO.7, and the nucleic acid sequence is shown in SEQ ID NO.8.

[0013] Preferably, the method for preparing the engineered live Bacillus subtilis includes the following steps: (1) Seed culture preparation: Bacillus subtilis engineered bacteria were inoculated into LB solid activation medium and cultured at 30-37℃ for 12-24h. Single colonies were picked and inoculated into liquid seed culture medium and cultured at 30-37℃ and 100-200rpm for 12-16h until the OD600 was 1.0-2.0 to obtain the first-grade seed culture. (2) Expansion culture: Inoculate the primary seed culture into the liquid expansion culture medium at an inoculation rate of 1%-5% v / v, and culture at 30-37℃, stirring speed of 100-300 rpm, and aeration rate of 0.5-2.0 vvm for 18-48 h. During the culture, maintain the pH at 6.5-7.5 using an automatic control system until the viable cell concentration is ≥1×10⁻⁶. 10 CFU / mL was used to obtain a live bacterial solution of engineered Bacillus subtilis; Both the liquid seed culture medium and the liquid expansion culture medium contain 10-50 g / L of carbon source, 5-30 g / L of nitrogen source, 1-5 g / L of potassium dihydrogen phosphate, 0.5-2 g / L of magnesium sulfate heptahydrate, 0.5-2 g / L of potassium chloride, 0.1-1 mg / L of vitamin B1, and 1-5 mL / L of trace element solution.

[0014] More preferably, the carbon source is at least one of glucose, sucrose, and starch, with glucose being preferred; the nitrogen source is at least one of peptone, yeast extract, and ammonium sulfate, with peptone and yeast extract being preferred; the trace element solution contains iron, manganese, and zinc ions and is prepared by mixing FeSO4, MnSO4, and ZnSO4 in an equimolar ratio.

[0015] Preferably, the volume ratio of the engineered Bacillus subtilis live bacteria to the 3% glucose solution is 1:1000. At this ratio, the synergistic effect of the two is most significant, which can not only ensure the effective colonization of the engineered bacteria and the soil microecological regulation effect, but also maximize the promotion of sugar metabolism in sugar beets, while avoiding excessive glucose concentration causing osmotic stress to sugar beet roots.

[0016] The present invention also provides a method for preparing the above-mentioned compound biological agent, comprising the following steps: slowly adding the engineered live bacterial solution of Bacillus subtilis to a 3% glucose aqueous solution at a volume ratio of 1:(800-1200), and gently stirring at 20-30℃ and 50-100rpm for 5-10 minutes until the mixture is uniform, thereby obtaining the compound biological agent.

[0017] This invention also provides the application of the above-mentioned compound biological agent in promoting the growth and / or sugar accumulation of sugar beets.

[0018] Preferably, the application involves applying the compound biological agent to the soil in which the sugar beet is cultivated. The application time is when the third pair of true leaves of the sugar beet seedlings have fully unfolded (about 35 days after emergence). At this time, the sugar beet grows vigorously, the root system begins to develop rapidly, and the response to external signals is significant. This is a key period for regulating growth and sugar accumulation. The application is carried out 3 times, with an interval of 5 days between each application, and the application amount is 15-20 L / mu each time.

[0019] More preferably, the compound biological agent does not require additional dilution before application and can be directly applied to the roots via drip irrigation or fertigation to ensure full contact between the agent and the beet roots. Application should be avoided during the midday high-temperature period and should be carried out before 10:00 AM or after 4:00 PM to prevent the inactivation of live bacteria and the decomposition of glucose due to high temperatures. After application, a small amount of clean water should be irrigated promptly to reduce the osmotic pressure of the soil solution, promote the colonization of the engineered live bacteria in the rhizosphere and the absorption of glucose by the beet.

[0020] In the compound biological agent of this invention, the engineered live Bacillus subtilis bacteria and the 3% glucose solution produce a significant synergistic effect, specifically manifested in: 1. 3% glucose provides nutritional support for the engineered bacteria: As an easily available carbon source, glucose can provide energy for the colonization and proliferation of Bacillus subtilis engineered bacteria in the rhizosphere of sugar beets, improve the survival rate and colonization efficiency of the bacteria in the soil, and ensure that the engineered bacteria continuously secrete signaling substances such as acetoin and 2,3-butanediol, so as to play a role in soil microecological regulation and root growth promotion. 2. Engineered live bacteria lay the physiological foundation for the regulation of glucose metabolism: The signaling substances secreted by engineered live bacteria can promote the growth of beet roots, increase the root absorption area, and enhance the beet's ability to absorb glucose and nutrients such as nitrogen, phosphorus, and potassium in the soil. At the same time, engineered bacteria can optimize the rhizosphere microbial community, improve the soil phosphorus activation capacity, and provide sufficient mineral nutrition for beet sugar metabolism. 3. The two work synergistically to regulate sugar metabolism in beets: 3% glucose directly acts as a signaling molecule to regulate the expression of key genes such as MYB44 and MYB15 in beets, increase the activity of sugar metabolism enzymes such as SPS and SS, and promote sucrose synthesis and transport; the engineered live bacteria improve soil microecology and root growth, providing sufficient raw materials and energy for sugar metabolism. The two work synergistically at multiple levels, including molecular, cellular, plant, and soil, to significantly increase the sugar accumulation in beets. 4. No synergistic antagonism and good environmental compatibility: Glucose is a natural carbon source and engineered bacteria are safe and beneficial microorganisms. When the two are combined, there is no chemical antagonism, no toxic or harmful substances are produced, and the soil environment can be improved, realizing the green and sustainable development of sugar beet cultivation.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The compound biological agent of this invention combines live engineered Bacillus subtilis bacteria with 3% glucose solution in a specific ratio, achieving for the first time the combination of live bacteria colonization nutrient supply, soil microecological regulation, and plant sugar metabolism regulation. The 3% glucose and the live engineered bacteria form an interactive support system, and the synergistic effect is significantly better than that of a single agent, which can more effectively promote the growth and sugar accumulation of sugar beets. 2. The effective components of the compound biological agent of this invention are engineered live Bacillus subtilis bacteria and glucose. It is environmentally friendly, safe, and residue-free. It will not damage the soil structure or cause pollution to agricultural products, and meets the development requirements of modern green agriculture. 3. The preparation method of the compound biological agent of the present invention is simple to operate, with low temperature and gentle stirring throughout the process, which can maintain the activity of engineered bacteria to the greatest extent. The raw materials are readily available and inexpensive, and no complicated equipment is required, making it suitable for large-scale production. 4. The application method of the compound biological agent of the present invention is simple, requiring no special application equipment. It can be applied by drip irrigation or fertigation. The application amount is small and the regulatory effect is significant. It can significantly increase the root biomass, plant height and above-ground fresh weight of sugar beets, while increasing the activity of SPS and SS enzymes in tubers and increasing the content of sucrose and soluble sugars. It is suitable for large-scale agricultural promotion. 5. The engineered live bacteria in the compound biological agent of this invention can colonize the rhizosphere of sugar beets for a long time and continuously play a role in regulating the soil microecology. This not only improves the yield and quality of sugar beets in the current season, but also improves soil fertility, laying the foundation for the growth of subsequent crops, and has long-term agricultural application value. Attached Figure Description

[0022] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is the phylogenetic tree of SPB1. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0024] All reagents used in the embodiments of this invention are commercially available analytical grade reagents, all culture media are conventional culture media, and all operations without special instructions are routine microbiological experimental operations; the viable cell concentration is determined by plate counting method.

[0025] The contents disclosed in the patent filed by the applicant on the same day (invention title: an engineered strain of Bacillus subtilis, its fermentation products, construction method and application) are included in this application as the contents disclosed in this application.

[0026] The Bacillus subtilis engineered strain used in this embodiment of the invention is a genetically engineered strain obtained by knocking out the α-subunit gene of acetoin dehydrogenase (amino acid SEQ ID NO. 1, nucleic acid SEQ ID NO. 2) and the phosphoacetyltransferase gene (amino acid SEQ ID NO. 3, nucleic acid SEQ ID NO. 4) and expressing the acetolactate synthase gene (amino acid SEQ ID NO. 5, nucleic acid SEQ ID NO. 6) and the α-acetolactate decarboxylase gene (amino acid SEQ ID NO. 7, nucleic acid SEQ ID NO. 8) in Bacillus subtilis as the substrate bacteria (this Bacillus subtilis was deposited at the China General Microbiological Culture Collection Center on March 3, 2026, with accession number CGMCC No. 37899).

[0027] Among them, SEQ ID NO.1: amino acid sequence of acetoin dehydrogenase α subunit. MELLKREGLSLTEEKALWMYQKMLEIRGFEDKVHELFAQGVLPGFVHLYAGEEAVAVGVCAHLHDGDSITSTHRGHGHCIAKGCDLDGMMAEIFGKATGLCKGKGGSMHIADLDKGMLGANGIVGGGFTLACGSALTAKYKQTKYVSVCFFGDGANNQGTFHEGLN LAAVWNLPVVFVAENNGYGEATPFEYASACDSIADRAAAYNMPGVTVDGKDILAVYQAAEEAIERARNGGGPSLIECMTYRNYGHFEGDAQTYKTKDERVEHLEEKDAIQGFKNYLLKETDANKLSDIEQRVSESIEKAVSFSEDSPYPKDSELLTDVYVSYEKGGM The DNA sequence of the α subunit of acetoin dehydrogenase includes the sequence shown in SEQ ID NO.2.

[0028] SEQ ID NO.2: The amino acid sequence of the phosphoacetyltransferase includes the sequence shown in SEQ ID NO.3.

[0029] SEQ ID NO.3; VADLFSTVQEKVAGKDVKIVFPEGLDERILEAVSKLAGNKVLNPIVIGNENEIQAKAKELNLTLDGVKIYDPHTYEDMEDLVQAFVERRKGKATEEQARKALLDENYFGTMLVYKGLADGLVSGAAHSTADTVRPALQIIKTKEGVKKTSGVFIMARGEEQ YVFADCAINIAPDSQDLAEIAIESANTAKMFDIEPRVAMLSFSTKGSAKSDETEKVADAVKIAKEKAPELTLDGEFQFDAAFVPSVAEKKAPDSEIKGDANVFVFPSLEAGNIGYKIAQRLGNFEAVGPILQGLNMPVNDLSRGCNAEDVYNLALITAAQAL The DNA sequence of the phosphorylated acetyltransferase includes the sequence shown in SEQ ID NO.4.

[0030] SEQ ID NO.4: GTGGCAGATTTATTTTCAACAGTGCAAGAAAAAGTAGCTGGAAAAGACGTTAAAATTGTATTTCCTGAAGGCTTAGACGAGCGTATTTTAGAAGCGGTCAGCAAGCTTGCGGGAAACAAAGTGCTGAATCCGATTGTGATCGGCAATGAAAATGAGATCCAAGCAAAAGCAAAAGAATTGAACCTTACGCTGGACGGCGTTAAGATTTATGATCCTCATACATATGAAGACATGGAAGACCTTGTACAAGCATTCGTAGAACGCCGCAAAGGCAAAGCGACAGAAGAACAGGCTCGCAAAGCGTTATTAGACGAGAACTACTTCGGTACAATGCTGGTGTATAAAGGCCTTGCAGACGGACTCGTAAGCGGAGCTGCTCACTCGACAGCTGACACTGTCCGCCCGGCTCTTCAAATCATCAAAACAAAAGAAGGCGTGAAAAAGACTTCAGGCGTGTTCATCATGGCTCGCGGAGAAGAGCAATACGTATTCGCAGATTGCGCGATCAACATTGCGCCTGACAGCCAAGATCTTGCCGAGATTGCGATCGAAAGTGCCAATACGGCAAAAATGTTTGACATTGAGCCTCGCGTGGCAATGCTCAGCTTCTCTACAAAAGGCTCAGCAAAATCTGATGAAACAGAAAAAGTAGCGGATGCAGTGAAAATCGCGAAAGAAAAAGCGCCTGAACTGACACTTGACGGCGAATTCCAATTTGATGCTGCATTTGTTCCATCTGTAGCTGAGAAAAAAGCGCCGGATTCCGAGATCAAAGGGGACGCTAACGTATTCGTATTCCCAAGCCTTGAAGCAGGAAACATCGGCTATAAAATTGCTCAGCGTTTGGGCAACTTTGAAGCGGTAGGACCAATCCTGCAAGGTTTAAATATGCCTGTAAACGACCTTTCAAGAGGATGTAACGCTGAAGATGTTTACAATCTCGCATTAATTACAGCGGCGCAAGCACTGTAA SEQ ID NO.5: Amino acid sequence of acetolactate synthase VLTKATKEQKSLVKNRGAELVVDCLVEQGVTHVFGIPGAKIDAVFDALQDKGPEIIVARHEQNAAFMAQAVGRLTGKPGVVLVTSGPGASNLATGLLTANTEGDPVVALAGNVIRADRLKRTHQSLDNAALFQPITKYSVEVQDVKNIPEAVTNAFRIASAGQAGAAFVSFPQDVVNEVTNTKNVRAVAAPKLGPAADDAISAAIAKIQTAKLPVILVGMKGGRPEAIKAVRKLLKKVQLPFVETYQAAGTLSRDLEDQYFGRIGLFRNQPGDLLLEQADVVLTIGYDPIEYDPKFWNVNGDRTIIHLDEIIADIDHAYQPDLELIGDIPSTINHIEHDAVKVEFAEREQKILSDLKQYMHKDEQVPADWKSDRAHPLEIVKELRNAVDDHVTVTCDIGSHAIWMSRYFRSYEPLTLMISNGMQTLGVALPWAIGASLVKPGEKVVSVSGDGGFLFSAMELETAVRLKAPIVHIVWNDSTYDMVAFQQLKKYNRTSAVDFGNIDIVKYAESFGATGLRVESPDQLADVLRQGMNAEGPVIIDVPVDYSDNINLASDKLPKEFGELMKTKAL SEQ ID NO.6: Nucleic acid sequence of acetolactate synthase SEQ ID NO.7: α-acetolactate decarboxylase amino acid sequence: MKRESNIQVLSRGQKDQPVSQIYQVSTMTSLLDGVYDGDFELSEIPKYGDFGIGTFNKLDGELIGFDGEFYRLRSDGTATPVQKGDRSPFCSFTFFTPDMTHKIDVKMTREDFEKEINSMLPSRNLF YAIRIDGLFKKVQTRTVELQEKPYVPMVEAVKTQPIFNFDNVRGTIVGFLTPAYANGIAVSGYHLHFIDEGRNSGGHVFDYVLEDCTVTISQKMNMNLRLPNTADFFNANLDNPDFAKDIETTEGSPE SEQ ID NO.8: α-acetolactate decarboxylase nucleic acid sequence: ATGAAACGAGAAAGCAACATTCAAGTGCTCAGCCGTGGTCAAAAAGATCAGCCTGTGAGCCAGATTTATCAAGTATCAACAATGACTTCTCTATTAGACGGAGTATATGACGGAGATTTTGAACTGTCAGAGATTCCGAAATATGGAGACTTCGGTATCGGAACCTTTAACAAGCTTGACGGAGAGCTGATTGGGTTTGACGGCGAATTTTACCGTCTTCGCTCAGACGGAACCGCGACACCGGTCCAAAAAGGAGACCGTTCACCGTTCTGTTCATTTACGTTCTTTACACCGGACATGACGCACAAAATTGATGTGAAAATGACACGCGAAGACTTTGAAAAAGAGATCAACAGCATGCTGCCAAGCAGAAACTTATTTTATGCAATTCGTATTGACGGATTGTTTAAAAAGGTGCAGACAAGAACGGTAGAACTTCAAGAAAAACCTTACGTGCCAATGGTTGAAGCGGTCAAAACACAGCCGATTTTCAACTTCGACAACGTGAGAGGAACAATCGTAGGTTTCTTGACACCAGCTTATGCAAACGGAATCGCCGTTTCAGGCTATCACCTGCACTTCATTGACGAAGGACGCAATTCAGGCGGACACGTTTTTGACTATGTGCTTGAGGATTGCACGGTTACGATTTCTCAAAAAATGAACATGAATCTCAGACTTCCGAACACAGCGGATTTCTTTAATGCGAATCTGGATAACCCTGATTTTGCGAAAGATATCGAAACAACTGAAGGAAGCCCTGAATAA Among them, the construction method of the engineered Bacillus subtilis: Using the genomic DNA of *Bacillus subtilis* CGMCC No. 37834 as a template, the upstream and downstream homologous arms of the acetoin dehydrogenase α-subunit gene and the phosphoacetyltransferase gene were amplified by PCR. The upstream and downstream genes were ligated to an antibiotic resistance selection gene using overlap PCR to obtain linear DNA of the acetoin dehydrogenase α-subunit and phosphoacetyltransferase fusion gene. This DNA was then transformed into competent *Bacillus subtilis* cells, and the transformants were identified by genomic PCR amplification and sequencing. Using the *Bacillus subtilis* CGMCC No. 37834 genome as a template, the acetolactate synthase gene and α-acetolactate decarboxylase gene were amplified by PCR and ligated to plasmid pMA5 using overlap PCR with the HpaII promoter. Through these steps, engineered *Bacillus subtilis* strain CGMCC No. 37899 was obtained.

[0031] The basal cell strain *Bacillus subtilis* SPB1 was deposited on March 3, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 37834. The 16S rRNA gene sequence (1472 bp) of this strain SPB1 is shown below, and the phylogenetic tree is as follows. Figure 1 As shown.

[0032] The above results indicate that the strain is Bacillus subtilis.

[0033] The Bacillus subtilis engineered strain and its fermentation products have significant effects on promoting growth and increasing crop yield. The following is an experimental verification of its effects.

[0034] 1) Preparation of fermentation products from engineered Bacillus subtilis strains: Seed culture preparation: Inoculate the engineered Bacillus subtilis strain with preservation number CGMCC No.37899 into a solid activation medium (such as LB agar medium) and culture at 30-37℃ for 12-24 hours; pick a single colony and inoculate it into a liquid seed medium (composition similar to the main fermentation medium, but concentration halved), and culture under the same conditions with shaking (100-200 rpm) for 12-16 hours until the OD600 reaches 1.0-2.0 to obtain the first-grade seed culture.

[0035] Main fermentation process: The primary seed culture is transferred to the fermenter at an inoculum rate of 1%-5%, and the fermenter is filled with culture medium. A fed-batch fermentation strategy is employed: During the initial fermentation stage (0-12 hours), the temperature is controlled at 30-37℃, pH at 6.5-7.5, the stirring speed at 100-200 rpm, and the aeration rate at 1.0-1.5 vvm to promote cell growth; after entering the product synthesis phase (12-72 hours), a residual sugar concentration of 5-10 g / L is maintained by feeding a carbon source (such as glucose solution), and the dissolved oxygen level is optimized (20%-40%) to induce the accumulation of acetoin and 2,3-butanediol. The fermentation cycle typically lasts 48-72 hours, yielding the fermentation product. The culture medium is a liquid fermentation medium containing a carbon source, a nitrogen source, inorganic salts, and growth factors. The carbon source is selected from at least one of glucose, sucrose, and starch, with a concentration of 10-50 g / L. The nitrogen source is selected from at least one of peptone, yeast extract, and ammonium sulfate, with a concentration of 5-30 g / L. The inorganic salts include phosphates, magnesium salts, and potassium salts, with potassium dihydrogen phosphate at a concentration of 1-5 g / L, magnesium sulfate heptahydrate at a concentration of 0.5-2 g / L, and potassium chloride at a concentration of 0.5-2 g / L. The growth factors include vitamins and trace elements, with vitamin B1 at a concentration of 0.1-1 mg / L. The pH of the culture medium is adjusted to 6.5-7.5.

[0036] Post-processing: Step 1: Inactivate the bacterial strain, maintaining the fermenter temperature at 121℃ (approximately 0.12-0.15 MPa pressure) for 20-30 minutes; Step 2: Centrifuge the fermentation broth, maintaining a centrifugation speed ≥5000 rpm / min for ≥30 minutes, and collect the supernatant. The supernatant contains small molecule "microorganism-crop" interaction signaling substances acetoin and 2,3-butanediol.

[0037] The above fermentation products contain the small-molecule "microbe-crop" interaction signaling substances acetoin and 2,3-butanediol. In crop cultivation, such as wheat, corn, tomatoes, and peppers, applying fermentation products containing these small-molecule "microbe-crop" interaction signaling substances can promote root growth and increase crop yield.

[0038] Specifically, the preparation example of fermentation products from engineered Bacillus subtilis bacteria (Sample 2 below): This embodiment details the specific process of preparing fermentation products using engineered Bacillus subtilis strain (preservation number CGMCC No. 37899). The final product contains 100 g / L of acetoin and 10 g / L of 2,3-butanediol.

[0039] Strain activation: The engineered bacteria were taken from the -80℃ glycerol storage tube and streaked onto LB solid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, pH 7.0) and incubated in a 37°C incubator for 24 hours to obtain single colonies.

[0040] Seed culture: Pick a single colony and inoculate it into 100 mL of liquid seed culture medium (composition: glucose 15 g / L, peptone 7.5 g / L, yeast extract 5 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 0.75 g / L, potassium chloride 0.5 g / L, microbial B1 0.25 mg / L, pH 7.0) in a 500 mL shake flask. Incubate at 37°C and 200 rpm for 12 hours until the OD600 is about 1.5. This is the first-stage seed culture.

[0041] Primary fermentation: The primary seed culture was inoculated into a 5 L fermenter (working volume 3 L) at a 2% inoculation rate. The culture medium composition in the tank was as follows: glucose 30 g / L, peptone 15 g / L, yeast extract 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 1.5 g / L, potassium chloride 1 g / L, microbial B1 0.5 mg / L, and trace element solution (containing FeSO4, MnSO4, ZnSO4) 2 mL / L. The pH was adjusted to 7.0. Fermentation conditions were controlled as follows: temperature 37°C, stirring speed 150 rpm, aeration rate 1.2 vvm, and pH maintained at 7.0 ± 0.2 by automatic addition of acid (1 M HCl) or alkali (1 M NaOH). During fermentation, samples were taken every 12 hours to detect OD600, residual sugar concentration (using DNS method), and metabolites (analyzed by HPLC). After 24 hours of fermentation, glucose solution (500 g / L) was added to maintain the residual sugar concentration at 5-8 g / L.

[0042] Fermentation endpoint and product harvest: When fermentation reaches 60 hours, the concentration of acetoin reaches 100 g / L, the concentration of 2,3-butanediol reaches 10 g / L, and the cell OD600 is stable at 15-20, which is judged as the fermentation endpoint.

[0043] The fermentation broth underwent post-treatment: First, the temperature in the fermenter was raised to 121℃ (tank pressure 0.12-0.15 MPa) and maintained for 20 minutes to inactivate the bacterial strain. Then, the fermentation broth was centrifuged at 5000 rpm for 30 minutes at 4℃, and the supernatant was collected, which is the fermentation product containing the signal substance. This product is colorless and transparent, with a pH of approximately 6.5, and can be used directly in agricultural applications or further concentrated.

[0044] Validation analysis: The concentrations of acetoin and 2,3-butanediol in the product were verified by high-performance liquid chromatography (HPLC), and the purity of the substances was confirmed by gas chromatography-mass spectrometry (GC-MS). This example demonstrates that the fermentation technology can stably produce high yields of the target signaling substance, providing a foundation for subsequent applications.

[0045] 2) The following are the effects of the fermentation products of the engineered Bacillus subtilis strain in this application (sample 1 refers to the same thing in each of the following tests, and sample 2 refers to the same thing): Rapeseed field experiment: This experiment was conducted at the open-field plot experimental base of Sinochem Agriculture (Linyi) R&D Center Co., Ltd. The tested vegetable was Shanghai Bok Choy. The experiment consisted of three treatment groups, arranged in a randomized block design with three replicates. The plot area was 15m². 2 (3m × 5m). The specific processing is as follows: CK: Control group, rapeseed was treated with an equal amount of water at the three-leaf stage.

[0046] T1: Sample 1, dosage 1L / mu, diluted 1000 times (i.e., diluted with 1000L / mu of water), applied by fertigation at the three-leaf stage of rapeseed (approximately 15-20 days after sowing). Sample 1 contains 100g / L of acetoin and 10g / L of 2,3-butanediol. Preparation method: Dissolve 100g of pure acetoin and 10g of pure 2,3-butanediol in 1L of ultrapure water. The pure acetoin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number A109410, with a purity of 99%; the pure 2,3-butanediol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number B424414, with a purity of 99%.

[0047] T2: Sample 2, with the same dosage and application method as T1. Sample 2 is the fermentation product of the Bacillus subtilis engineered bacteria fermentation product preparation example, wherein the acetoin content is 100 g / L and the 2,3-butanediol content is 10 g / L.

[0048] The previous crop at the experimental site was melons, and the soil was fertile loam. Before sowing, the soil was deeply tilled, and 30 kg of compound fertilizer per mu (approximately 0.067 hectares) was applied as basal fertilizer. The fertilizer was purchased from Sinochem Shandong Fertilizer Co., Ltd., with a specification of N-P2O5-K2O=15-15-15. Sowing was carried out on March 10, 2024, using a row sowing method with a row spacing of 15 cm. After sowing, a thin layer of soil was applied and the soil was thoroughly watered. After emergence, seedlings were thinned to three leaves, and the plant spacing was adjusted to 8-10 cm to ensure uniform growth. Unified water, fertilizer, and pest and disease management was implemented throughout the entire growth period.

[0049] During the rapeseed harvest period (approximately 40 days after sowing), 20 representative plants were collected from each plot for measurement. Root biomass: The complete root system was carefully dug up, washed, blanched at 105℃ for 30 minutes, and dried at 80℃ to constant weight. The dry weight (g / plant) was then measured.

[0050] Yield measurement: 2m from the center of the harvest plot 2 Weigh all plants within the area and calculate the fresh weight and yield per acre (kg / acre).

[0051] The results are as follows:

[0052] The results show that both T1 and T2 can increase rapeseed root biomass, with T1 and T2 increasing by 28.0% and 64.0% respectively compared to CK, and T2 increasing by 28.1% compared to T1. Both T1 and T2 can also increase rapeseed yield, with T1 and T2 increasing by 10.4% and 21.6% respectively compared to CK, and T2 increasing by 10.1% compared to T1. This example confirms that applying either Sample 1 or Sample 2 (1 L / acre, diluted 1000 times) during rapeseed cultivation can effectively promote rapeseed root growth and increase yield.

[0053] Wheat field trial: This experiment was conducted at the open-field plot experimental base of Sinochem Agriculture (Linyi) R&D Center Co., Ltd. The tested wheat variety was Jimai 22 (a semi-winter variety). The experiment consisted of three treatment groups, arranged in a randomized block design with three replicates. The plot area was 20 m². 2 (4m × 5m). The specific processing is as follows: CK: Control group, which received the same amount of water during the wheat's greening stage.

[0054] T1: Sample 1, dosage 2L / mu, diluted 1000 times (2000L / mu of water), applied by irrigation during the wheat greening stage (before jointing).

[0055] T2: Sample 2, dosage and application method are the same as T1.

[0056] The previous crop in the experimental field was summer maize, and the soil type was brown soil with medium fertility. Deep plowing was carried out before sowing, and 50 kg of compound fertilizer (N-P2O5-K2O=15-15-15) was applied per mu as basal fertilizer. Wheat was sown mechanically in rows on October 12, 2023, with a row spacing of 25 cm, and the basic seedling density was controlled at 1.8 million plants per mu. Uniform water and fertilizer management and pest and disease control were implemented throughout the entire growth period to ensure consistent environmental conditions across all treatments.

[0057] Representative plant samples were collected from each plot at the wheat maturity date (June 5, 2024) for testing: Root biomass: Dig up roots from the 0-20 cm soil layer, wash them, blanch them at 105℃ for 30 minutes, dry them at 80℃ to constant weight, and weigh the dry weight (g / plant).

[0058] Yield determination: The dry weight of the grains was measured after threshing the middle three rows of plants in the actual harvest plot, and the yield per mu (kg / mu) was calculated. As shown in the table below, treatments T1 and T2 were significantly better than CK in terms of root biomass, yield, and soil enzyme activity, with T2 showing the best results.

[0059]

[0060] The root biomass of treatments T1 and T2 increased by 27.1% and 55.3% respectively compared to the control (CK), and the yield increased by 6.9% and 15.3% respectively. Compared with T1, T2 showed an increase of 22.2% in root biomass and 7.8% in yield per acre.

[0061] This embodiment confirms that applying either Sample 1 or Sample 2 (2 L / acre, diluted 1000 times) during the wheat greening stage can effectively promote wheat root growth and increase yield. Sample 2 (T2) showed the most significant effect, providing a reliable basis for expanding the application of this technology in grain crops.

[0062] Maize field trial: This experiment was conducted at the open-field plot experimental base of Sinochem Agriculture (Linyi) R&D Center Co., Ltd. The tested maize variety was Zhengdan 958 (a compact, high-density tolerant variety). The experiment consisted of three treatment groups, arranged in a randomized block design with three replicates, and each plot was 30 m². 2 (5m × 6m). The specific processing is as follows: CK: Control group, which received the same amount of water during the corn jointing stage.

[0063] T1: Sample 1, dosage 2L / mu, diluted 1000 times (2000L / mu of water), applied by irrigation during the corn jointing stage.

[0064] T2: Sample 2, dosage and application method are the same as T1.

[0065] The previous crop at the experimental site was winter wheat, and the soil type was alluvial brown soil with moderate fertility. Deep plowing (25 cm depth) was carried out before sowing, and 50 kg of compound fertilizer (N-P2O5-K2O=15-15-15) was applied. Corn was precision-planted mechanically on June 15, 2024, with a row spacing of 60 cm, a plant spacing of 28 cm, and a planting density of 4000 plants / mu. Uniform water and fertilizer management and pest and disease control were implemented throughout the entire growth period to ensure consistent environmental conditions across all treatments.

[0066] At the corn maturity date (October 10, 2024), representative plant samples were collected from each plot for testing. Root biomass: Dig up roots from the 0-30 cm soil layer, wash them, blanch them at 105℃ for 30 minutes, dry them at 80℃ to constant weight, and weigh the dry weight (g / plant).

[0067] Yield determination: The dry weight of the grains was measured after threshing the middle four rows of plants in the actual plot, and the yield per mu (kg / mu) was calculated.

[0068] As shown in the table below, treatments T1 and T2 were significantly better than the control (CK) in terms of root biomass, yield, and soil enzyme activity, with T2 showing the best results.

[0069] The root biomass of treatments T1 and T2 increased by 25.4% and 54.6% respectively compared to the control (CK), and the yield increased by 11.8% and 22.1% respectively. The root biomass and yield of treatment T2 increased by 23.3% and 9.2% compared to T1.

[0070] This embodiment confirms that applying either Sample 1 or Sample 2 (2 L / acre, diluted 1000 times) during the jointing stage of maize can effectively promote maize root growth, increase yield, and enhance the activation capacity of rhizosphere soil phosphorus, with Sample 2 (T2) showing the most significant effect. These results further validate the application potential of the tested samples on major food crops and provide a reliable basis for expanding the application of this technology in high-yield maize cultivation.

[0071] Tomato field trial: This experiment was conducted at the open-field plot experimental base of Sinochem Agriculture (Linyi) R&D Center Co., Ltd. The tested tomato variety was Provence (a large-fruited pink variety). The experiment consisted of three treatment groups, arranged in a randomized block design with three replicates. The plot area was 25 m². 2 (5m×5m). The specific processing is as follows: CK: Control group, which was irrigated with the same amount of water during the tomato flowering period.

[0072] T1: Sample 1, dosage 2L / mu, diluted 1000 times (1000L / mu of water), applied by drip irrigation during the tomato flowering period (when the first cluster of flowers opens).

[0073] T2: Sample 2, dosage and application method are the same as T1.

[0074] The previous crop in the experimental field was leafy vegetables, and the soil type was light loam. Deep tillage was carried out before sowing, and 3000 kg of well-rotted cow manure and 40 kg of compound fertilizer (N-P2O5-K2O=15-15-15) were applied per acre as basal fertilizer. Tomatoes were raised in plug trays on February 20, 2024, and transplanted on March 25, with a row spacing of 60 cm and a plant spacing of 35 cm, resulting in a planting density of approximately 3200 plants per acre. Uniform water and fertilizer management and pest and disease control were implemented throughout the entire growth period to ensure consistent environmental conditions across all treatments.

[0075] During the peak tomato fruiting period (June 15, 2024), representative plant samples were collected from each plot for testing. Root biomass: Dig up roots from the 0-25 cm soil layer, wash them, blanch them at 105℃ for 30 minutes, dry them at 80℃ to constant weight, and weigh them (g / plant).

[0076] Yield determination: All fruits in the plot were harvested in three batches, and the total fresh weight was measured and converted into yield per mu (kg / mu).

[0077] As shown in the table below, treatments T1 and T2 were significantly better than CK in terms of root biomass, yield, soil enzyme activity and quality indicators, with T2 showing the best results.

[0078]

[0079] The root biomass of treatments T1 and T2 increased by 26.4% and 55.2% respectively compared to the control (CK), and the yield increased by 15.5% and 26.7% respectively. Compared with T1, T2 showed a 22.8% increase in root biomass and a 9.7% increase in yield per acre.

[0080] This embodiment confirms that applying either Sample 1 or Sample 2 (2 L / acre, diluted 1000 times) during the tomato flowering period can effectively promote tomato root growth, increase yield, improve fruit quality, and enhance rhizosphere soil phosphorus activation capacity, with Sample 2 (T2) showing the most significant effect. These results further validate the application potential of the tested samples in fruit and vegetable crops, providing a reliable basis for expanding the application of this technology in high-quality, high-yield cultivation of greenhouse tomatoes.

[0081] The above test results show that the Bacillus subtilis engineered strain of this application has significant technical effects. The fermentation products prepared by it have significant effects on promoting crop root growth, increasing crop yield, and activating rhizosphere soil phosphorus.

[0082] This application utilizes the superior technical effects of the engineered Bacillus subtilis strain modified as described above. The live Bacillus subtilis strain is combined with a 3% glucose solution to form a biological agent. The synergistic effect of the two can significantly promote the growth and sugar accumulation of sugar beets, while improving the rhizosphere soil microecology.

[0083] Example 1: Preparation of engineered live Bacillus subtilis culture 1. Seed culture preparation: The engineered Bacillus subtilis bacteria were removed from the -80℃ glycerol tube and streaked onto LB solid activation medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar, pH 7.0) and incubated at 37℃ for 20 h. Uniform single colonies were picked and inoculated into 100 mL of liquid seed culture medium (20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 0.5 mg / L vitamin B1, 2 mL / L trace element solution, pH 7.0) and placed in a 500 mL shake flask. The culture was carried out at 37℃ and 200 rpm for 14 h until the OD600 reached 1.8, thus obtaining the primary seed culture. 2. Expansion Culture: The primary seed culture was inoculated into a 5L fermenter (working volume 3L) at an inoculation rate of 2% v / v. The liquid expansion culture medium in the tank consisted of: glucose 30g / L, peptone 15g / L, yeast extract 10g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate heptahydrate 1.5g / L, potassium chloride 1g / L, vitamin B1 0.5mg / L, and trace element solution (FeSO4:MnSO4:ZnSO4=1:1:1, concentration 0.1mol / L) 2mL / L, with the pH adjusted to 7.0. The culture conditions were: 37℃, stirring speed 150rpm, aeration rate 1.2vvm, and pH maintained at 7.0±0.2 by automatically adding 1M HCl or 1M NaOH. Samples were taken for analysis after 36 hours of culture. 3. Viable Bacteria Detection: The viable bacteria concentration in the culture was determined using the plate count method, and the results showed a viable bacteria concentration of 3.5 × 10⁻⁶. 10 CFU / mL was used to obtain a live bacterial suspension of engineered Bacillus subtilis, which was then sealed and stored at 4°C for later use.

[0084] Example 2: Preparation of Compound Biological Agent 1. Prepare a 3% glucose solution: Weigh 30g of anhydrous glucose, add it to 1000mL of deionized water, stir until completely dissolved, and obtain a 3% glucose aqueous solution by mass and volume. Keep at room temperature for later use. 2. Compounding: The Bacillus subtilis engineered live bacterial solution prepared in Example 1 was slowly added to the above 3% glucose aqueous solution at a volume ratio of 1:1000. The mixture was gently stirred for 8 minutes at 25°C and 80 rpm until homogeneous, thus obtaining the compound biological agent of this invention; the live bacterial concentration of this agent was approximately 3.5 × 10⁻⁶. 7 CFU / mL, after preparation, seal and store at 25℃ and use within 72 hours.

[0085] Example 3: Preparation of Compound Biological Agent The Bacillus subtilis engineered live bacterial suspension prepared in Example 1 was added to a 3% glucose aqueous solution at a volume ratio of 1:800. The mixture was gently stirred at 20°C and 50 rpm for 10 minutes until homogeneous, yielding a compound biological agent with a live bacterial concentration of approximately 4.4 × 10⁻⁶. 7 CFU / mL.

[0086] Example 4: Preparation of Compound Biological Agent The engineered live Bacillus subtilis culture prepared in Example 1 was added to a 3% glucose aqueous solution at a volume ratio of 1:1200. The mixture was gently stirred at 30°C and 100 rpm for 5 minutes until homogeneous, yielding a compound biological agent with a live bacteria concentration of approximately 2.9 × 10⁻⁶. 7 CFU / mL.

[0087] Example 5: Verification of the Application Effect of Compound Biological Agents in Sugar Beet Cultivation 1. Experimental materials: The beet variety tested was Marunouchi crossbred red beet; the experimental soil was cultivated soil with available nitrogen of 64.7 mg / kg, available phosphorus of 53.3 mg / kg, available potassium of 157 mg / kg, organic matter of 6.17 g / kg, and pH of 7.27; the experimental preparation was the compound biological preparation prepared in Example 2, and a water control group (CK1), a 3% glucose solution group (CK2, single component), and a Bacillus subtilis engineered live bacteria liquid group (CK3, diluted at 1:1000, single component) were set up.

[0088] 2. Experimental Design: The experiment was conducted in a greenhouse using flowerpots with an outer diameter of 31 cm, an inner diameter of 26.3 cm, and a height of 19.4 cm. The soil consisted of a mixture of 90% topsoil and 10% substrate. After seedling cultivation, sugar beets were transplanted into flowerpots 35 days after emergence (when the third pair of true leaves had fully unfolded), with one seedling per pot. Four treatment groups were set up, with three replicates per group and ten pots per replicate. Root application was initiated on the day of transplanting for each treatment group, with three applications at 5-day intervals. The application rate was 15 L / mu (approximately 0.067 hectares), applied via irrigation, avoiding the midday high-temperature period. A small amount of water was applied after each application. Water and fertilizer management was consistent throughout the entire growth period, with the temperature maintained at 15-20℃ and daily light exposure ≥6 hours.

[0089] 3. Measurement Indicators and Methods: (1) Growth indicators: 30 days after the third application (leaf cluster stage), the plant height, petiole length, aboveground fresh weight and root biomass of each beet were measured; the root biomass was measured by digging up the whole root system, washing it, blanching it at 105℃ for 30 minutes, drying it at 80℃ to constant weight, and weighing the dry weight. (2) Sugar metabolism enzyme activity: During the sugar accumulation period of beet tubers (53 days after emergence), beet tuber samples were collected, and the activities of sucrose synthase (SS) and sucrose phosphate synthase (SPS) were measured using the BOXBIO kit. (3) Sugar content: During the sugar beet harvest period (90 days after emergence), sugar beet root samples were collected, and the soluble sugar and sucrose content were determined using the BOXBIO kit; (4) Soil and viable bacteria indicators: Soil samples were collected from the rhizosphere of beets during the harvest period to measure soil alkaline phosphatase activity (Ultra-Kelco micro-method kit) and soil phosphate-solubilizing bacteria abundance (16S rRNA high-throughput sequencing). At the same time, the colonization of Bacillus subtilis engineered bacteria in the rhizosphere soil was determined by plate counting method.

[0090] 4. Test Results (1) Effects on sugar beet growth indicators, the results are shown in Table 1: Table 1 Effects of different treatments on sugar beet growth indicators

[0091] Table 1 shows that, compared with the water control group, both the 3% glucose solution group and the engineered live bacteria group improved the growth indicators of sugar beets to varying degrees. The compound biological agent group of this invention showed the most significant promoting effect: plant height increased by 27.5% compared to CK1, 23.8% compared to CK2, and 13.9% compared to CK3; petiole length increased by 39.8% compared to CK1, 27.4% compared to CK2, and 16.2% compared to CK3; aboveground fresh weight increased by 44.0% compared to CK1, 30.5% compared to CK2, and 21.2% compared to CK3; root biomass increased by 80.0% compared to CK1, 56.1% compared to CK2, and 26.4% compared to CK3. This indicates that the synergistic effect of the two components in the compound biological agent can significantly promote the vegetative growth and root development of sugar beets, and the substantial increase in root biomass lays a solid foundation for nutrient absorption and sugar accumulation.

[0092] (2) Effects on the activity of beet sugar-metabolizing enzymes, the results are shown in Table 2: Table 2. Effects of different treatments on the activity of sugar-metabolizing enzymes in beet roots (U / g)

[0093] As shown in Table 2, the activities of SS and SPS enzymes in the compound biological agent group of this invention were significantly higher than those in other groups: SS activity increased by 93.4% compared to CK1, 43.4% compared to CK2, and 60.2% compared to CK3; SPS activity increased by 115.4% compared to CK1, 44.8% compared to CK2, and 82.6% compared to CK3. This indicates that the compound biological agent can significantly improve the activity of key enzymes in beet sugar metabolism and greatly enhance sucrose synthesis efficiency.

[0094] (3) Effects on sugar content of beet roots, results are shown in Table 3: Table 3. Effects of different treatments on sugar content in beet roots (%)

[0095] Table 3 shows that the compound biological agent group of this invention had the highest contents of both soluble sugar and sucrose: the soluble sugar content increased by 81.0% compared with CK1, 38.2% compared with CK2, and 51.0% compared with CK3; the sucrose content increased by 85.7% compared with CK1, 41.1% compared with CK2, and 61.1% compared with CK3. This indicates that the compound biological agent can significantly promote the accumulation of sugar in beet tubers, greatly improving the quality and sugar production value of beets.

[0096] (4) Effects on beet rhizosphere soil and colonization of live bacteria, the results are shown in Table 4: Table 4. Effects of different treatments on rhizosphere soil and colonization of live bacteria in sugar beets

[0097] Table 4 shows that the soil alkaline phosphatase activity, phosphate-solubilizing bacteria abundance, and engineered bacteria colonization in the compound biological agent group of this invention were significantly higher than those in other groups: alkaline phosphatase activity increased by 78.4% compared to CK1, 63.1% compared to CK2, and 26.9% compared to CK3; phosphate-solubilizing bacteria abundance increased by 145.0% compared to CK1, 118.8% compared to CK2, and 27.6% compared to CK3; and engineered bacteria colonization increased by 210.3% compared to CK3. This indicates that 3% glucose provided sufficient carbon source for the viable engineered bacteria, significantly improving their colonization efficiency in the rhizosphere. The efficient colonization of engineered bacteria further optimized the rhizosphere microbial community and enhanced soil phosphorus activation capacity.

[0098] Example 6: Comparison of the application effects of compound biological agents with different compounding ratios Three groups of compound biological agents with volume ratios of 1:800 (Example 3), 1:1000 (Example 2), and 1:1200 (Example 4) were prepared. A sugar beet cultivation experiment was conducted according to the experimental method of Example 5. The sucrose content of the sugar beets at harvest and the colonization of engineered bacteria in the rhizosphere soil were measured. The results are shown in Table 5. Table 5. Application effects of compound biological agents with different compounding ratios

[0099] Table 5 shows that when the volume ratio of Bacillus subtilis engineered live bacteria to 3% glucose solution is 1:1000, the compound biological agent has the best effect in increasing the sucrose content of sugar beets. At the same time, the colonization of engineered bacteria is maintained at a high level, taking into account both the colonization efficiency of live bacteria and the sugar accumulation effect, which is the optimal compound ratio. When the ratio is 1:800, although the colonization of live bacteria is slightly higher, the relative proportion of glucose is lower, and the sugar metabolism regulation effect is reduced. When the ratio is 1:1200, the concentration of live bacteria is too low, the soil microecological regulation effect is insufficient, and the sugar accumulation effect is poor.

[0100] Comparative Design and Verification To further verify the rationality of the compound ratio of the compound biological agent of the present invention, the specificity of glucose concentration, and the effectiveness of the engineered bacteria, the following 6 comparative groups were set up. All of them were carried out in beet cultivation experiments according to the experimental method of Example 5. The two core indicators of sucrose content in beet at harvest and rhizosphere soil alkaline phosphatase activity were measured. The results are summarized in Table 6.

[0101] Comparative Example 1: Mixture ratio 1:500 (too high proportion of live bacteria) The Bacillus subtilis engineered live bacterial suspension from Example 1 was mixed with 3% glucose solution at a volume ratio of 1:500 to prepare a compound preparation (live bacterial concentration 7.0 × 10⁻⁶). 6 (CFU / mL), the remaining preparation and application conditions are the same as in Example 2.

[0102] Comparative Example 2: A compound formulation was prepared by mixing the Bacillus subtilis engineered live bacteria solution from Example 1 with a 3% glucose solution at a volume ratio of 1:2000 (live bacteria concentration 1.75 × 10⁻⁶). 7 (CFU / mL), the remaining preparation and application conditions are the same as in Example 2.

[0103] Comparative Example 3: Glucose concentration 1% (not 3%) The Bacillus subtilis engineered live bacterial solution from Example 1 was mixed with 1% glucose solution at a volume ratio of 1:1000 to prepare a compound preparation. The remaining preparation and application conditions were the same as in Example 2.

[0104] Comparative Example 4: Glucose concentration 5% (not 3%) The Bacillus subtilis engineered live bacterial solution from Example 1 was mixed with 5% glucose solution at a volume ratio of 1:1000 to prepare a compound preparation. The remaining preparation and application conditions were the same as in Example 2.

[0105] Comparative Example 5: Wild-type Bacillus subtilis live bacteria as replacement for engineered bacteria Wild-type Bacillus subtilis live bacterial solution (live bacterial concentration 3.5 × 10⁻⁶) 10 A compound preparation was prepared by mixing CFU / mL with 3% glucose solution at a volume ratio of 1:1000. The remaining preparation and application conditions were the same as in Example 2.

[0106] Comparative Example 6: Commercially available live Bacillus subtilis bacteria as a substitute for engineered bacteria Commercially available Bacillus subtilis live bacterial solution (purchased from Genlido Biotechnology Co., Ltd., product: Root Crown, live bacterial concentration 3.5×10⁻⁶) was used. 10 A compound preparation was prepared by mixing CFU / mL (non-genetically modified) with 3% glucose solution at a volume ratio of 1:1000. The remaining preparation and application conditions were the same as in Example 2.

[0107] Summary of comparative test results Table 6 Comparison of core indicators between each comparative example and the experimental group of this invention

[0108] Comparative analysis of results Reasonableness of compound ratio: In Comparative Example 1 (1:500), the proportion of live bacteria was too high and glucose was relatively insufficient, resulting in a significant decrease in the effect of sugar metabolism regulation; in Comparative Example 2 (1:2000), the proportion of live bacteria was too low and the soil micro-ecological regulation capacity was insufficient. Both of these results in a significant decrease in the core indicators, proving that 1:800-1200 is the optimal compound ratio range, and 1:1000 has the best effect. Glucose concentration specificity: Comparative Example 3 (1% glucose) had insufficient carbon source and signaling molecules, which could not meet the needs of viable bacterial colonization and beet sugar metabolism; Comparative Example 4 (5% glucose) had too high a concentration, which caused slight osmotic stress to beet roots and inhibited the activity of some rhizosphere microorganisms, proving that 3% is the optimal effective concentration of glucose. Effectiveness of the engineered bacteria: Comparative examples 5 (wild type) and 6 (commercially available Bacillus subtilis) were not genetically modified and could not efficiently secrete signaling substances such as acetoin and 2,3-butanediol. Their effects on soil microecological regulation and root growth promotion were extremely poor, with core indicators decreasing by more than 36%. This proves that the genetically engineered Bacillus subtilis of this invention is the key to achieving synergistic effects.

[0109] Industrial applicability The compound biological agent of this invention achieves the synergistic effect of Bacillus subtilis engineered live bacteria and 3% glucose, and has four functions: live bacteria colonization and nutrient supply, soil microecological regulation, root growth promotion and plant sugar metabolism regulation. It can significantly promote sugar beet growth, increase sugar metabolism enzyme activity, increase root sugar accumulation, improve the rhizosphere soil environment and enhance soil fertility. Its preparation method is simple to operate, the raw materials are readily available, the live bacteria activity is maintained throughout the process, the application method is simple and does not require special equipment, and it is suitable for large-scale production and agricultural promotion.

[0110] Furthermore, the compound biological agent of this invention can also be applied to other food crops and cash crops such as wheat, corn, tomato, pepper, and rapeseed, and has a broad-spectrum crop regulation effect, which can further expand its agricultural application scope. At the same time, the compounding concept of this agent can provide a reference for the compound application of other beneficial microorganisms and plant signaling molecules, and has important technical promotion value.

[0111] The above embodiments and comparative examples are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound biological agent, characterized in that, The mixture includes live engineered Bacillus subtilis bacteria and a 3% glucose solution. The engineered Bacillus subtilis bacteria are genetically engineered bacteria obtained by knocking out the α-subunit gene of acetoin dehydrogenase and the phosphoacetyltransferase gene in the genome of Bacillus subtilis as the substrate bacteria, while expressing the acetyllactone synthase gene and the α-acetyllactone decarboxylase gene.

2. The compound biological agent according to claim 1, characterized in that, The viable bacterial concentration of the engineered bacteria is ≥1×10¹ 0 CFU / mL; the 3% glucose solution is a glucose aqueous solution with a mass-volume fraction of 3%; the volume ratio of the engineered Bacillus subtilis live bacteria to the 3% glucose solution is 1:(800-1200).

3. The compound biological agent according to claim 1, characterized in that, The amino acid sequence of the α subunit of the acetoin dehydrogenase is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.2; the amino acid sequence of the phosphoacetyltransferase is shown in SEQ ID NO.3, and the nucleic acid sequence is shown in SEQ ID NO.4; the amino acid sequence of the acetolactate synthase is shown in SEQ ID NO.5, and the nucleic acid sequence is shown in SEQ ID NO.6; the amino acid sequence of the α-acetolactate decarboxylase is shown in SEQ ID NO.7, and the nucleic acid sequence is shown in SEQ ID NO.

8.

4. The compound biological agent according to claim 1, characterized in that, The method for preparing the engineered live Bacillus subtilis includes the following steps: 1) Seed culture preparation: Bacillus subtilis engineered bacteria were inoculated into LB solid activation medium and cultured at 30-37℃ for 12-24h. Single colonies were picked and inoculated into liquid seed medium and cultured at 30-37℃ with shaking at 100-200rpm for 12-16h until the OD600 was 1.0-2.0 to obtain primary seed culture; 2) Expansion culture: Inoculate the primary seed culture into liquid expansion culture medium at an inoculation rate of 1%-5% v / v, and culture at 30-37℃, stirring speed of 100-300 rpm, and aeration rate of 0.5-2.0 vvm for 18-48 h. Maintain the pH at 6.5-7.5 during the culture process until the viable cell concentration is ≥1×10¹. 0 CFU / mL was used to obtain a live bacterial solution of engineered Bacillus subtilis; Both the liquid seed culture medium and the liquid expansion culture medium contain 10-50 g / L of carbon source, 5-30 g / L of nitrogen source, 1-5 g / L of potassium dihydrogen phosphate, 0.5-2 g / L of magnesium sulfate heptahydrate, 0.5-2 g / L of potassium chloride, 0.1-1 mg / L of vitamin B1, and 1-5 mL / L of trace element solution.

5. The compound biological agent according to claim 4, characterized in that, The carbon source is at least one of glucose, sucrose, and starch; the nitrogen source is at least one of peptone, yeast extract, and ammonium sulfate; and the trace element solution contains iron, manganese, and zinc ions.

6. The compound biological agent according to claim 2, characterized in that, The volume ratio of the engineered Bacillus subtilis live bacteria to the 3% glucose solution is 1:1000.

7. A method for preparing the compound biological agent according to any one of claims 1-6, characterized in that, Includes the following steps: Add the engineered live bacterial solution of Bacillus subtilis to a 3% glucose aqueous solution at a volume ratio of 1:(800-1200), and gently stir for 5-10 minutes at 20-30℃ and 50-100rpm until the mixture is homogeneous to obtain the compound biological agent.

8. The use of the compound biological agent according to any one of claims 1-6 in promoting the growth and / or sugar accumulation of sugar beets.

9. The application according to claim 8, characterized in that, The application involves applying the compound biological agent to the roots of the sugar beet cultivation soil. The application time is when the third pair of true leaves of the sugar beet seedlings have fully unfolded. The application is carried out 3 times, with an interval of 5 days between each application, and the application amount is 15-20 L / mu each time. Preferably, the compound biological agent is applied to the roots by drip irrigation or fertigation to ensure that the agent fully contacts the sugar beet roots. The application should be avoided during the midday high temperature period and should be carried out before 10 am or after 4 pm. After application, a small amount of clean water should be irrigated in time to promote the colonization of live bacteria and glucose absorption.

10. The application according to claim 8, characterized in that, The compound biological agent can significantly increase the plant height, petiole length, aboveground fresh weight and root biomass of sugar beets, increase the activity of sucrose synthase and sucrose phosphate synthase in sugar beet tubers, increase the sucrose and soluble sugar content in tubers, and at the same time increase the activity of alkaline phosphatase in the rhizosphere soil of sugar beets, increase the abundance of phosphate-solubilizing bacteria in the soil, and optimize the structure of the rhizosphere microbial community.

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