Application of synergy of ensifer bacteria S281 and bacillus subtilis to promotion of corn growth

Abstract

The invention discloses application of an ensifer bacterium S281 and bacillus subtilis to synergistically promote corn growth, relates to the technical field of microorganisms and application thereof, and discloses a method for promoting corn growth, which comprises the following steps: inoculating S281 into an enrichment culture medium for culture to prepare an ensifer bacterium S281 microbial inoculum; mixing the mixture with a bacillus subtilis inoculant according to a volume ratio of 1: 1, inoculating the mixture into the corn rhizosphere soil, and performing conventional maintenance. According to the microbial composition, rhizosphere soil nitrification is enhanced and corn endogenous cytokinin synthesis is promoted through the ensifer bacteria S281, the microbial composition and the bacillus subtilis generate a synergistic effect in the aspects of root colonization and nutrient activation, overground biomass accumulation of corn can be remarkably promoted, underground root development is promoted, and the yield of corn is increased. And an efficient microbial solution is provided for green agriculture.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms and their applications, and particularly relates to application of Xiphinum bacteria S2_8_1 and Bacillus subtilis in synergistically promoting corn growth. BACKGROUND

[0002] Microorganisms play an important role in promoting sustainable agricultural development, especially in promoting plant growth and improving soil fertility. Among them, plant growth-promoting rhizobacteria can effectively support the healthy growth of crops through various mechanisms such as enhancing nutrient absorption, improving plant stress resistance, and promoting biomass accumulation. Bacillus subtilis, as an important rhizosphere beneficial bacteria, has been widely studied due to its root colonization, nutrient solubilization, and enhanced plant resistance to biotic and abiotic stresses.

[0003] Rhizobium of the Rhizobiaceae family is mainly known for its nitrogen-fixing ability in symbiosis with leguminous plants. Some strains of the Xiphinum genus in this family have been found to have the ability to participate in soil nitrogen cycling and synthesize plant hormones such as cytokinins, thereby playing a potential role in regulating plant growth and chlorophyll metabolism.

[0004] In recent years, the use of various functional microorganisms in combination to achieve synergistic effects has gradually become a research hotspot in the field of agricultural microorganisms. However, current research and practice still focus on the growth-promoting effects of single known strains, and the potential of new strain combinations has not been fully developed. SUMMARY

[0005] The present application provides a microbial combination composed of Xiphinum bacteria S2_8_1 and Bacillus subtilis. Through the functional complementation of the two strains, the combination produces significant synergistic growth-promoting effects, promoting corn root development and biomass accumulation. This provides a more effective microbial solution for improving corn growth performance and provides new microbial resources for the development of composite biological fertilizers.

[0006] To achieve the above-mentioned purposes, the technical solution adopted by the present application is: In a first aspect, the present application provides the application of Xiphinum bacteria S2_8_1 and Bacillus subtilis in synergistically promoting corn growth.

[0007] As a preferred solution, the synergistic promotion of corn growth is at least one of promoting aboveground biomass accumulation, promoting underground root development, or enhancing rhizosphere soil nitrification.

[0008] As a preferred solution, the Xiphinum bacteria S2_8_1 and Bacillus subtilis are applied in the form of bacterial agents, with a volume ratio of 1:1.

[0009] As a preferred solution, the concentration of the Xiphinenna bacteria S2_8_1 agent is 200000-230000 cfu / mL.

[0010] In a second aspect, the present application provides a microbial agent for promoting the growth of corn, which is prepared by mixing the Xiphinenna bacteria S2_8_1 agent and the Bacillus subtilis agent at a volume ratio of 1:1.

[0011] As a preferred solution, the concentration of the Xiphinenna bacteria S2_8_1 agent is 200000-230000 cfu / mL.

[0012] In a third aspect, the present application provides a method for promoting the growth of corn, which comprises the following steps: Step 1: inoculate the Xiphinenna bacteria S2_8_1 into an enrichment medium and cultivate at a constant temperature of 28℃ for 15-25 days to prepare the Xiphinenna bacteria S2_8_1 agent; Step 2: mix the Xiphinenna bacteria S2_8_1 agent prepared in Step 1 and the Bacillus subtilis agent at a volume ratio of 1:1; Step 3: inoculate the mixed agent into the rhizosphere soil of corn, and the inoculation amount is 200 mL per corn plant; Step 4: perform routine maintenance on the corn.

[0013] As a preferred solution, in Step 1, the enrichment medium comprises 3.79 mM (NH4)2SO4, 5.51 mM KH2PO4, 1.81 mM NaH2PO4, 0.059 mM MnSO4 4H2O, 0.12 mM MgSO4 7H2O, 50.0 mM CaCO3, and the pH value of the medium is 7.0-7.2.

[0014] According to the above technical solution, the present application has the following advantages: 1. The present application first uses the Xiphinenna bacteria S2_8_1 in combination with the Bacillus subtilis to show a clear synergistic effect in promoting the growth of corn, which is significantly better than any single strain treatment, specifically, the aboveground biomass of the combined inoculation group is 26% higher than that of the Bacillus subtilis group alone, and the underground biomass is 8% higher than that of the S2_8_1 group alone, achieving the growth promotion effect of "1+1>2". The synergistic effect is due to the functional complementation of the two strains: the Xiphinenna bacteria S2_8_1 can significantly enhance the nitrification of the rhizosphere soil (the rate is increased by more than 2 times compared with the control group) and promote the synthesis of endogenous cytokinin of corn, while the Bacillus subtilis improves the rhizosphere microenvironment, promotes nutrient activation and strengthens root development, providing a solid foundation for the function of S2_8_1, and the two strains synergistically promote the overall growth and development of corn from the root system to the aboveground part.

[0015] 2. The microbial composition and method provided by this invention do not require the use of chemical hormones or complex variety replacement, are environmentally friendly, and pose no risk of residue. The preparation process of this microbial agent is simple, low-cost, and easy to apply, making it highly suitable for widespread application in the cultivation of field crops, cash crops, and flowers, providing a new microbial resource for sustainable agriculture.

[0016] Strain preservation information: The Rhizobiaceae Ensifer bacteria S2_8_1 described in this invention has been deposited at the China Center for Type Culture Collection (CCTCC) on April 14, 2021, in Wuhan, China, with accession number CCTCC NO: M2021374. Attached Figure Description

[0017] Figure 1 Photographs showing the aboveground growth of potted corn plants under different treatments; Figure 2 Photos showing the development of maize roots under different treatments. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0019] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0020] In the following examples, the inoculation amounts were all inoculated according to the corresponding volume ratios.

[0021] Example 1 1. Isolation, screening and identification of Ensifer strain S2_8_1 20g of soil samples were collected from the experimental farm of Henan University of Science and Technology and inoculated into an enrichment medium for cultivation. The medium formulation was: 3.79mM (NH4)2SO4, 5.51 mM KH2PO4, 1.81 mM NaH2PO4, 0.059 mM MnSO4 4H2O, 0.12mM MgSO4 7H2O, 50.0 mM CaCO3, with deionized water added to a final volume of 1000 mL, and the pH adjusted to 7.0–7.2. The medium was sterilized at 121℃ for 20 min and then incubated statically at 28℃ in the dark for 15–25 days. After cultivation, the presence of ammonia-oxidizing bacteria was detected using Griess' reagent. The enriched culture containing the ammonia-oxidizing bacteria was transferred to a new enrichment medium for further cultivation, repeated twice to obtain a purified bacterial enrichment solution.

[0022] Take 0.1 mL of the above enrichment solution and spread it on a solid separation medium containing 2.5 g / L agar (other components are the same as the above enrichment medium), and incubate at 28°C in the dark for 7 days. After single colonies have formed on the plate, pick colonies with clear morphology and appropriate size, and repeat the streak isolation and purification 5 to 10 times until a stable pure strain is obtained.

[0023] Five to ten representative strains with stable nitrification-promoting abilities were selected and inoculated into a liquid enrichment medium. The cultures were incubated at 28°C in the dark for 15 days. The resulting bacterial solution was the *Rhizobiaceae* S2_8_1 bacterial inoculum used in the experiment. The concentration of the *Rhizobiaceae* S2_8_1 bacterial inoculum was 200,000–230,000 cfu / mL. *Bacillus subtilis* was a commonly used commercially available strain. The *Bacillus subtilis* inoculum was prepared using conventional methods in the field and used in subsequent experiments. The *Ensifer* bacteria that promote soil nitrification, named S2_8_1 and taxonomically named *Rhizobiaceae ensifer*, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2021374.

[0024] 2. Effects of combined application of S2_8_1 and Bacillus subtilis on maize growth and rhizosphere nitrification like Figure 1 and Figure 2 As shown, to verify the effects of combined application of *Strombus* S2_8_1 and *Bacillus subtilis* on maize growth and rhizosphere nitrification, the following pot experiment was conducted: 36 pots of maize seedlings with uniform growth and 30 days of emergence were selected, with one seedling in each pot. The organic matter content of the cultivation soil was 16.5 g / kg. The 36 pots of maize were randomly divided into four treatment groups (a, b, c, and d), with 9 pots in each group; each group was further divided into 12 subgroups (a1, a2, a3; b1, b2, b3; c1, c2, c3; d1, d2, d3), with 3 pots in each subgroup, serving as 3 replicates.

[0025] Before inoculation, plant samples were collected from subgroups a1, b1, c1, and d1. The roots, stems, and leaves were dried at 75℃ to constant weight, and the total dry weight was measured and recorded as Ra0, Rb0, Rc0, and Rd0, respectively. At the same time, rhizosphere soil samples were collected from each subgroup, and the nitrification rate was measured using the culture method and recorded as Za0, Zb0, Zc0, and Zd0, respectively, for subsequent comparative analysis.

[0026] Subgroups a2, b2, c2, and d2 were used for inoculation treatment: Subgroup a2 was inoculated with a mixture of 100 mL of *Cymbidium* S2_8_1 inoculum and 100 mL of *Bacillus subtilis* inoculum per pot; Subgroup b2 was inoculated with 200 mL of Bacillus subtilis inoculum per pot; In the c2 subgroup, each pot was inoculated with 200 mL of *Cymbidium* S2_8_1 fungal agent; Each pot in subgroup d2 was inoculated with 200 mL of sterile enrichment medium as a blank control.

[0027] Ten days after inoculation, samples from subgroups a2, b2, c2, and d2 were collected. The dry weights of the roots, stems, and leaves of the plants were measured using the method described above and recorded as Ra1, Rb1, Rc1, and Rd1 for later use. At the same time, the nitrification rate of the rhizosphere soil was measured and recorded as Za1, Zb1, Zc1, and Zd1 for later use.

[0028] Data analysis showed that the dry weight of plants in each treatment group before inoculation was: Ra0 (combined inoculation) = 6.62g / pot, Rb0 (Bacillus subtilis only) = 6.41g / pot, Rc0 (S2_8_1 only) = 6.22g / pot, Rd0 (blank control group) = 6.37g / pot; The nitration rates are Za0 = 2.48 ng / g, Zb0 = 2.33 ng / g, Zc0 = 2.47 ng / g, and Zd0 = 2.54 ng / g.

[0029] There were no significant differences between the different treatments, indicating that the growth of maize and the nitrification rate of the rhizosphere soil were consistent before inoculation.

[0030] Ten days after inoculation, the dry weight of the plants was as follows: Ra1 (5.01g) > Rc1 (4.62g) > Rb1 (4.27g) > Rd1 (3.98g). The dry weight of the combined inoculation group (Ra1) was 17.18% higher than that of the Bacillus subtilis-inoculated group (Rb1) and 9% higher than that of the S2_8_1-inoculated group (Rc1). The rhizosphere soil nitrification rate was as follows: Za1 (7.71ng / g) > Zc1 (4.92ng / g) > Zd1 (2.25ng / g) > Zb1 (1.95ng / g). The nitrification rate of the combined inoculation group (Za1) was 2.96 times and 1.57 times higher than that of the Bacillus subtilis-inoculated group (Zb1) and the S2_8_1-inoculated group (Zc1), respectively. The above results indicate that the combined application of strain S2_8_1 and Bacillus subtilis can significantly promote maize plant growth by enhancing rhizosphere soil nitrification, and the synergistic effect is better than that of single-strain inoculation treatment.

[0031] 3. Effects of combined application of *S. fragilis* S2_8_1 and *Bacillus subtilis* on maize biomass. To clarify the effects of combined application of *Strombus* S2_8_1 and *Bacillus subtilis* on the aboveground and underground biomass of maize, a pot experiment was conducted. Thirty-six uniformly grown maize seedlings (30 days old) were selected, with one seedling per pot. The soil organic matter content was 16.5 g / kg. The 36 pots were randomly divided into four treatment groups (a, b, c, and d), with nine pots in each group. Each group was further divided into 12 subgroups: a1, a2, a3; b1, b2, b3; c1, c2, c3; and d1, d2, d3, with three pots in each subgroup, serving as three biological replicates.

[0032] Before inoculation, plants from subgroups a1, b1, c1, and d1 were taken, and the above-ground parts were cut 5 cm above the stem base. The roots were collected intact, rinsed with clean water to remove soil residue, and the fresh weights of the above-ground and underground parts were weighed and recorded as above-ground biomass Aa0, Ab0, Ac0, Ad0 and underground biomass Ba0, Bb0, Bc0, Bd0 before inoculation for subsequent comparative analysis.

[0033] Subgroups a2, b2, c2, and d2 were used for inoculation treatment: Subgroup a2 was inoculated with a mixture of 100 mL of *Cymbidium* S2_8_1 inoculum and 100 mL of *Bacillus subtilis* inoculum per pot; Subgroup b2 was inoculated with 200 mL of Bacillus subtilis inoculum per pot; In the c2 subgroup, each pot was inoculated with 200 mL of *Cymbidium* S2_8_1 fungal agent; Each pot in subgroup d2 was inoculated with 200 mL of sterile enrichment medium as a blank control.

[0034] Ten days after inoculation, complete plants from subgroups a2, b2, c2, and d2 were collected, and the fresh weights of the aboveground and underground parts were measured using the method described above. These were recorded as aboveground biomass Aa1, Ab1, Ac1, Ad1 and underground biomass Ba1, Bb1, Bc1, Bd1, respectively.

[0035] The test results show that: The aboveground biomass after inoculation was: Aa1 (co-inoculation) = 3.97g, Ab1 (Bacillus subtilis only) = 3.15g, Ac1 (S2_8_1 only) = 3.67g; The underground biomass is: Ba1=5.13g, Bb1=4.61g, Bc1=4.76g.

[0036] The combined inoculation group (a2) showed a 26% increase in aboveground biomass compared to the group inoculated with Bacillus subtilis alone (b2), and an 8% increase in underground biomass compared to the group inoculated with S2_8_1 alone (c2).

[0037] The results above indicate that the combined application of strain S2_8_1 and Bacillus subtilis can synergistically promote the accumulation of biomass in both the aboveground and underground parts of maize, and its effect is better than that of single strain inoculation, showing a significant synergistic effect.

[0038] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.

Claims

1. Application of S2_8_1 bacteria of the genus *Cyclophorus* and *Bacillus subtilis* in synergistic promotion of maize growth.

2. The application according to claim 1, characterized in that: The synergistic promotion of maize growth is manifested in at least one of promoting aboveground biomass accumulation, promoting underground root development, or enhancing rhizosphere soil nitrification.

3. The application according to claim 1 or 2, characterized in that: Both the *S. spp.* S2_8_1 and *Bacillus subtilis* were applied in the form of inoculum at a volume ratio of 1:

1.

4. The application according to claim 3, characterized in that: The concentration of the *Cyclophorus* bacteria S2_8_1 inoculum was 200,000–230,000 cfu / mL.

5. A microbial inoculant for promoting corn growth, characterized in that: The bacterial agent is prepared by mixing S2_8_1 bacterial agent of the genus *Cyclophorus* and Bacillus subtilis bacterial agent at a volume ratio of 1:

1.

6. The microbial agent according to claim 5, characterized in that: The concentration of the *Cyclophorus* bacteria S2_8_1 inoculum was 200,000–230,000 cfu / mL.

7. A method for promoting corn growth, characterized in that: Includes the following steps: Step 1: Inoculate the S2_8_1 bacteria of the genus *Cyclophorus* into the enrichment medium and culture it at a constant temperature of 28°C for 15-25 days to prepare the S2_8_1 bacterial agent of the genus *Cyclophorus*. Step 2: Mix the S2_8_1 bacterial inoculant obtained in Step 1 with the Bacillus subtilis inoculant at a volume ratio of 1:

1. Step 3: Inoculate the mixed microbial agent into the rhizosphere soil of the corn plant at a rate of 200 mL per plant. Step 4: Perform routine maintenance on the corn.

8. The method according to claim 7, characterized in that: In step 1, the enrichment medium contains: 3.79 mM (NH4)2SO4, 5.51 mM KH2PO4, 1.81 mM NaH2PO4, 0.059 mM MnSO4 4H2O, 0.12 mM MgSO4 7H2O, and 50.0 mM CaCO3, and the pH of the medium is 7.0 to 7.2.

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