Synthetic microbial agent for promoting rice growth, preparation method and application thereof

Abstract

This invention discloses a synthetic microbial agent for promoting rice growth, its preparation method, and its application, belonging to the fields of agricultural microbial technology and crop growth promotion technology. The synthetic microbial agent is composed of *Masses aurea* (…). Massilia aurea ) MA and Serratia oryzae Made from SO. This invention screened rice to obtain two strains: *Massezia aurea* MA and... Serratia oryzae So, the two strains are complementary in hormone regulation, nitrogen-related functions, and protease activity. Verification has shown that the two strains can be combined into a synthetic inoculum, which, when applied to the rhizosphere of rice plants, significantly increases plant height, stem diameter, number of tillers, number of leaves, root length, root fresh weight, aboveground fresh weight, and aboveground dry weight, as well as chlorophyll content; it also regulates the supply of inorganic nitrogen in the rhizosphere. This invention provides a new method for promoting rice growth.

Description

Technical Field

[0001] This invention relates to the fields of agricultural microbial technology and crop growth promotion technology, and in particular to a synthetic microbial agent for promoting rice growth, its preparation method and application. Background Technology

[0002] Rice is a major food crop in my country, making an irreplaceable contribution to ensuring national food security. During its growth and development, rice faces challenges such as the need for efficient root development and nutrient utilization, optimized nitrogen-density interactions, and stress resistance and stable yield. Plant growth-promoting rhizobacteria (PGPRs) are beneficial microorganisms that can colonize the rhizosphere and promote plant growth directly or indirectly. The mechanisms by which PGPRs promote plant growth include: biological nitrogen fixation, phosphorus and potassium solubilization, production of plant hormones (such as IAA, gibberellins, and cytokinins), synthesis of siderophores, secretion of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase to reduce stress-induced ethylene levels, production of antibiotics and extracellular enzymes to control plant pathogens, and induction of plant systemic resistance (ISR).

[0003] Significant progress has been made in the application of PGPR (Polymerase-Glycerol Propagated Reproductive Factors) in rice production. Studies have shown that Bacillus subtilis can synthesize IAA (Indigenous Acid) through the tryptophan pathway and work in conjunction with the IAA synthesized by the plant itself to stimulate root growth and directly promote plant development. Inoculation of rice with Bacillus subtilis under salt stress can increase endogenous IAA content, thereby significantly increasing seedling height, root length, fresh weight, and dry weight. PGPRs such as Pseudomonas aeruginosa, nitrogen-fixing bacteria, and Enterobacteriaceae can improve soil nutrient availability through mechanisms such as biological nitrogen fixation, phosphorus and potassium solubilization, and the production of siderophores, promoting rice growth and yield formation. Currently, there is no evidence to suggest that *Morse roseum* can be combined with... Serratia oryzae Reports on the use of compound inoculants in rice. Summary of the Invention

[0004] The purpose of this invention is to provide a synthetic microbial agent for promoting rice growth, its preparation method, and its application, thereby solving the problems existing in the prior art. The synthetic microbial agent provided by this invention, when applied to the rhizosphere of rice plants, can significantly increase plant height, stem diameter, number of tillers, number of leaves, root length, root fresh weight, aboveground fresh weight, and aboveground dry weight, as well as chlorophyll content; it can also regulate the supply of inorganic nitrogen in the rhizosphere. This invention provides a new method for promoting rice growth.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a synthetic microbial agent for promoting rice growth, wherein the synthetic microbial agent is composed of *Mosaicium aureum* (…). Massilia aurea ) MA and Serratia oryzae Made of SO; The accession number of the golden muscarinic bacteria MA is GDMCC No: 68034, the accession date is April 2, 2026, the depositary institution is Guangdong Provincial Center for Microbial Culture Collection, the depositary address is Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The Serratia oryzae The accession number of SO is GDMCC No: 68035, the accession date is April 2, 2026, the depositary institution is Guangdong Provincial Center for Microbial Culture Collection, the depositary address is Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] Optionally, the number of viable bacteria in the synthetic microbial agent is 10. 8 ~10 9 CFU / mL.

[0007] The present invention also provides a method for preparing the aforementioned synthetic bacterial agent, comprising mixing the fermentation broth of *Mosaicium aureum* MA with... Serratia oryzae The steps for mixing SO fermentation broth.

[0008] Optionally, the golden bouillon MA fermentation broth and the Serratia oryzae The volume ratio of SO fermentation broth is 1:1.

[0009] The present invention also provides the application of the synthetic microbial agent in promoting rice growth.

[0010] Optionally, the promotion of rice growth is manifested in increasing one or more of the following indicators: plant height, stem diameter, number of tillers, number of leaves, root length, root fresh weight, aboveground fresh weight, and aboveground dry weight.

[0011] Optionally, the promotion of rice growth manifests as an increase in the chlorophyll content of the plant.

[0012] The present invention also provides a method for promoting rice growth, including the step of treating rice with the aforementioned synthetic microbial agent.

[0013] Optionally, the method of treating rice includes applying the synthetic microbial agent to the rhizosphere of rice plants.

[0014] The present invention discloses the following technical effects: This invention screened two strains of bacteria in rice: *Mosaicella aurea* (…). Massilia aurea ) MA and Serratia oryzaeSO and the two strains are complementary in hormone regulation, nitrogen-related functions, and protease activity. It has been verified that strains MA and SO can be combined into a synthetic inoculant, which, when applied to the rhizosphere of rice plants, significantly improves plant height, stem diameter, tiller number, leaf number, root length, root fresh weight, aboveground fresh weight, and aboveground dry weight, as well as chlorophyll content. It also regulates the supply of inorganic nitrogen in the rhizosphere and, while promoting growth, does not significantly disturb soil active carbon and microbial biomass carbon levels. Furthermore, this invention reveals that the combined inoculant is not a simple replication of the effects of a single strain, but rather induces cross-pathway molecular remodeling in the root system. In summary, this invention provides a novel method for promoting rice growth. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Potentially beneficial microbial groups that are positively correlated with rice quality; Figure 2 Potentially beneficial microbial groups that are positively correlated with rice yield; Figure 3 Morphological characteristics of two isolated microbial strains that promote rice growth. Figure 4 A phylogenetic tree of strain MA constructed based on the 16S rRNA gene sequence; Figure 5 A phylogenetic tree of strain SO constructed based on the 16S rRNA gene sequence; Figure 6 The results of experiments on the antagonism and synergistic effects between strain MA and strain SO; Figure 7 The results of experiments on the effects of different microbial inoculation treatments on the aboveground phenotype of rice plants; Figure 8 Experimental results showing the effects of different microbial inoculation treatments on rice growth indicators; (a): plant height; (b): stem diameter; (c): chlorophyll content; (d): number of tillers; (e): number of leaves; (f): root length; (g): root fresh weight; (h): aboveground fresh weight; (i): aboveground dry weight. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Example 1: Screening of bacterial strains significantly associated with rice yield and quality Rhizosphere soil samples were collected from different rice plants at the rice maturity stage, and yield and quality indicators were measured simultaneously. High-throughput sequencing was used to analyze the rhizosphere bacterial community composition, and candidate microorganisms significantly associated with yield and quality were screened at the amplicon sequence variant (ASV) level. Random forest models were constructed with yield and quality indicators as response variables to screen biomarkers with high explanatory contributions to yield and quality. Results showed that ASV1466 had a high contribution to the quality-related models (…). Figure 1 ASV10820 makes a high contribution to output-related models. Figure 2 ).

[0023] The above candidate ASVs were classified and annotated, with ASV1466 belonging to... Massilia genus, ASV10820 belongs to SerratiaThe above results indicate that ASV1466 and ASV10820 can serve as candidate functional strains associated with rice growth promotion for the subsequent construction of growth-promoting agents.

[0024] Furthermore, the correlation between mature rhizosphere microbial communities and yield and overall quality index was most pronounced. Through a combined screening using correlation analysis, random forest, variance decomposition, co-occurrence network, and differential analysis, 221 microbial taxa significantly associated with yield and quality were identified. Massilia genus and Serratia The corresponding ASVs can be back-matched to ASV1466 and ASV10820 respectively.

[0025] Example 2 Isolation and Identification of Potential Growth-Promoting Microorganisms Targeting the differentially expressed ASVs screened in Example 1, potential growth-promoting microbial strains were isolated from rice rhizosphere soil samples. 10 g of rhizosphere soil sample was weighed and placed in an Erlenmeyer flask containing 90 mL of sterile water. The sample was incubated at 25°C and 180 r / min with constant shaking for 30 min. After standing for 1 min, the supernatant was collected, and the soil microbial suspension was serially diluted to 10⁻¹⁰ using a 10-fold serial dilution method. -5 10 -6 and 10 -7 Take 0.2 mL of bacterial suspension at each dilution and spread it evenly on a TSB medium plate. Incubate at 28°C for about 3 days.

[0026] Single colonies were selected based on differences in colony morphology and purified multiple times on fresh TSB agar plates using the streak plate method to obtain morphologically stable pure cultures. The purified single colonies were then transferred to slant agar and stored at 4°C for later use.

[0027] The isolated strains were then subjected to molecular biological identification. DNA was extracted from the strains using an alkaline lysis method, and the full-length 16S rRNA gene was amplified and sequenced using universal primers 27F and 1492R. The obtained sequences were submitted to the NCBI database for BLAST alignment analysis, and the taxonomic position of the strains was determined based on the phylogenetic tree construction results. The results showed that representative strains corresponding to the key ASVs screened in Example 1 were successfully obtained, among which the strain corresponding to ASV1466 was identified as… Massilia aurea It was named MA, and the strain corresponding to ASV10820 was identified as Serratia oryzae Let's name it SO.

[0028] Colony morphology observation of the above strains showed that both strains exhibited stable colony morphological characteristics. The colony morphologies of strains MA and SO are shown in the figures below. Figure 3The 16S rDNA of the above strains is shown in SEQ ID NO.1-2. Further phylogenetic analysis results indicate that strain MA is related to... Massilia aurea The most closely related strain SO is... Serratia oryzae The closest related species are shown in their phylogenetic trees. Figure 4 and Figure 5 .

[0029] Therefore, strain MA was identified as *Masséella aurea* (…). Massilia aurea The sample, named MA, was deposited on April 2, 2026, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 68034), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0030] The strain SO was identified as Serratia oryzae Named SO, it was deposited on April 2, 2026 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 68035, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0031] The indoleacetic acid (IAA) production capacity, siderophore production capacity, cellulase activity, protease activity, phosphate solubility, and growth on nitrogen-fixing and combined nitrogen-fixing media were measured for strains MA and SO. The results showed that MA possessed the ability to produce IAA, SO grew stably on Ashby's nitrogen-fixing medium, and both strains could grow on combined nitrogen-fixing medium and exhibited protease activity. Although both strains could grow on organic and inorganic phosphorus media, their solubility zones were not obvious, and they did not show significant siderophore production or cellulose degradation phenotypes. These results indicate that MA and SO are complementary in terms of hormone regulation, nitrogen-related functions, and protease activity.

[0032] SEQ ID NO.1 ( Massilia aurea 16S rDNA): SEQ ID NO.2( Serratia oryzae 16S rDNA):

[0033] Example 3: Verification of antagonistic / synergistic effects among strains of synthetic bacterial agents To verify whether there is an antagonistic effect between strain MA and strain SO obtained in Example 2, the interaction between strain MA and strain SO was detected using a plate confrontation culture method. Simultaneously, fermentation supernatants were obtained through fermentation culture, and cross-interaction experiments were conducted to evaluate the effect of fermentation products on the growth of the other strain.

[0034] Specifically, strains MA and SO were inoculated into sterile LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride) and cultured at 30℃ and 180 r / min for 24 h with shaking to obtain fermentation broth. The supernatant was collected after centrifugation. For plate confrontation culture, one strain was inoculated onto the surface of a medium plate, and the other strain was inoculated at the center or corresponding position. The plates were cultured at 28℃ for 2 days, and the presence of a clear inhibition zone or growth inhibition was observed. In the cross-effect experiment of fermentation supernatant, the fermentation supernatant of strain MA was applied to strain SO, and the fermentation supernatant of strain SO was applied to strain MA, and the effects on the growth of the other strain were observed.

[0035] The results showed that no obvious antagonism was observed between strain MA and strain SO during plate confrontation culture; cross-treatment of fermentation supernatant also did not significantly inhibit the growth of the other strain. Figure 6 The above results indicate that strain MA and strain SO have good compatibility and can be used together to construct a synthetic growth-promoting agent, which can then be used to verify the growth-promoting effect on rice.

[0036] Example 4: Verification of Synthetic Growth-Promoting Bacterial Agents in Promoting Rice Growth The rice used in this embodiment is Liangliangyou 1484 (National Approval Rice No. 20233102), provided by Hunan Agricultural University.

[0037] Fermentation broth preparation: Strains MA and SO were inoculated into sterile LB liquid medium and cultured at 30℃ and 180 r / min for 24 h with shaking to obtain fermentation broth. The viable cell counts of MA and SO fermentation broths were 8.2 × 10⁻⁶, respectively. 8 and 6.8×10 8 CFU / mL.

[0038] Preparation of mixed inoculum (synthetic inoculum): Mix equal volumes of MA fermentation broth and SO fermentation broth, with a viable cell count of 7.4 × 10⁻⁶. 8 CFU / mL.

[0039] To verify the growth-promoting effects of strains MA and SO on rice, a pot experiment was conducted using rice as the experimental material. An uninoculated treatment was set up as the control group (CK), and groups with strains MA (M), SO (S), and a mixture of both strains (MS) were established. The fermentation broths of each treatment group were serially diluted 100-fold with sterile water to prepare the corresponding working bacterial solutions. Using the root irrigation method, 100 mL of the working bacterial solution was evenly applied to the rhizosphere of each plant. All other cultivation and management conditions remained consistent.

[0040] The growth status of rice plants under different treatments was investigated during the growing season, and indicators such as plant height, stem diameter, chlorophyll content, number of tillers, number of leaves, root length, root fresh weight, aboveground fresh weight, and aboveground dry weight were measured.

[0041] The effects of different microbial inoculation treatments on the aboveground phenotype of rice plants are shown in the results. Figure 7 The effects of different microbial inoculation treatments on various growth indicators of rice are shown in the following figures. Figure 8 The results showed that, compared with the uninoculated control, strains MA, SO, and their mixtures all promoted rice growth to varying degrees. The MS treatment was superior to MA and SO treatments alone in several key growth indicators. Specifically, in terms of plant height, the M, S, and MS treatments increased by 7.9%, 8.5%, and 17.2% compared with the control (CK), respectively. The MS treatment further increased the height by 8.7% compared with the optimal single-strain treatment and was significantly higher than the M and S treatments. In terms of root length, the M, S, and MS treatments increased the root length by 15.9%, 11.8%, and 25.8% compared with the control (CK), respectively. The MS treatment further increased the root length by 9.9% compared with the optimal single-strain treatment and was significantly higher than the M and S treatments. In terms of leaf number, the M, S, and MS treatments increased the leaf number by 16.2%, 25.8%, and 51.5% compared with the control (CK), respectively. The MS treatment further increased the leaf number by 25.7% compared with the optimal single-strain treatment and was significantly higher than the other treatments.

[0042] In terms of biomass accumulation, the MS treatment was significantly superior to single-strain treatments. Aboveground fresh weight increased by 58.2%, 67.2%, and 137.5% compared to the control (CK) under M, S, and MS treatments, respectively. The MS treatment further increased above the M and S treatments by 50.1% and 42.0%, respectively, showing significant differences. Aboveground dry weight only saw a significant increase under the MS treatment, increasing by 114.5% compared to the CK, and further increasing by 51.0% and 47.5% compared to the M and S treatments, respectively. These results indicate that while MA and SO inoculation alone can promote rice growth in some indicators, the MS treatment, combining both, showed superior effects to any single-strain treatment in key indicators such as plant height, root length, leaf number, aboveground fresh weight, and aboveground dry weight. This demonstrates that the MA and SO combination is significantly superior to single-strain treatments in promoting the growth of small-grain hybrid rice.

[0043] Example 5: Effects of Synthetic Growth-Promoting Bacterial Agents on Rhizosphere Nitrogen Supply and Root Molecular Response Based on the pot experiment in Example 4, rhizosphere soil samples were collected from different treatments, and the contents of ammonium nitrogen, nitrate nitrogen, soluble organic carbon, and microbial biomass carbon were measured. The results showed that, compared with the uninoculated control, the M, S, and MS treatments significantly increased the soil ammonium nitrogen content, by 675.9%, 507.2%, and 537.6%, respectively; the MS treatment increased the soil nitrate nitrogen content by 24.5%, significantly higher than the control and single-strain treatments; there were no significant differences in soluble organic carbon and microbial biomass carbon content among the treatments. These results indicate that the synthetic growth-promoting bacterial agent of this invention can regulate the supply of inorganic nitrogen in the rhizosphere and, while exerting its growth-promoting effect, does not significantly disturb the levels of active carbon and microbial biomass carbon in the soil.

[0044] Further analysis of rice roots from each treatment was conducted using a combined transcriptome and metabolome. The results showed that single-strain MA treatment primarily affected basal carbon and nitrogen metabolism, amino acid metabolism, and some redox processes in the roots. MS combination treatment, in addition to these effects, further involved pathways related to aromatic amino acid metabolism, phenylpropanoid metabolism, glutathione metabolism, flavonoid metabolism, and plant hormone regulation. This indicates that the combined microbial agents did not simply replicate the effects of single strains, but rather induced cross-pathway molecular remodeling in the roots.

[0045] Joint transcriptomic and metabolomic analyses revealed that MS combined treatment, on the one hand, reorganized the conversion of phenylalanine to downstream branches of phenylpropane and reduced the accumulation of key metabolites in the phenylpropane pathway, such as caffeic acid, sinapic acid, sinaponic acid, and coniferaldehyde; on the other hand, it regulated the glutathione-ascorbic acid cycle and related redox networks, rebalancing glutathione turnover, peroxide scavenging, detoxification processes, and reducing power supply. These changes collectively promoted the synergistic adjustment of root carbon and nitrogen metabolism, secondary metabolism, and antioxidant regulation, providing a molecular metabolic basis for increased plant height, root length, tiller formation, and biomass accumulation in rice.

[0046] The embodiments described above 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 synthetic microbial agent for promoting rice growth, characterized in that, The synthetic microbial agent is composed of *Masses aureus* (… Massilia aurea ) MA and Serratia oryzae Made of SO; The preservation number of the golden bouillabata MA is GDMCC No: 68034; The Serratia oryzae The accession number of SO is GDMCC No: 68035.

2. The synthetic microbial agent as described in claim 1, characterized in that, The number of viable bacteria in the synthetic microbial agent is 10. 8 ~10 9 CFU / mL.

3. The method for preparing the synthetic microbial agent as described in claim 1 or 2, characterized in that, Including the fermentation broth of *Mosaicium aureum* (MA) with Serratia oryzae The steps for mixing SO fermentation broth.

4. The preparation method according to claim 3, characterized in that, The golden bouillon MA fermentation broth and the Serratia oryzae The volume ratio of SO fermentation broth is 1:

1.

5. The application of the synthetic microbial agent as described in claim 1 or 2 in promoting rice growth.

6. The application as described in claim 5, characterized in that, The promotion of rice growth is manifested in increasing one or more of the following indicators: plant height, stem diameter, number of tillers, number of leaves, root length, root fresh weight, aboveground fresh weight, and aboveground dry weight.

7. The application as described in claim 5, characterized in that, The promotion of rice growth is manifested in increasing the chlorophyll content of the plant.

8. A method for promoting rice growth, characterized in that, The step includes treating rice with the synthetic microbial agent as described in claim 1 or 2.

9. The method as described in claim 8, characterized in that, The method for treating rice includes applying the synthetic microbial agent to the rhizosphere of rice plants.

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