Microbial composition with flower promoting effect and application of microbial composition in regulation and control of flowering time of corn

Abstract

The invention discloses a microbial composition with a flowering promoting effect and application of the microbial composition in regulation and control of the flowering time of corn, and belongs to the technical field of agricultural microorganisms. The microbial composition comprises Ensifer strain S281 and bacillus subtilis; a method for promoting precocious flowering of corn comprises the following steps: culturing S281 in an enrichment culture medium to prepare a microbial inoculum; and mixing with a bacillus subtilis inoculant according to a volume ratio of 1: 1, inoculating the mixture into the rhizosphere soil of the corn, and maintaining the corn until the corn blooms. The microbial composition disclosed by the invention can enhance rhizosphere nitrification and regulate endogenous hormone level through the Ensifer strain S281, and can generate a synergistic effect with bacillus subtilis, so that crops such as corn and the like can be obviously promoted to head in advance, and the flowering time can be 2-7 days earlier than that of an untreated control group; and a new microbial solution is provided for realizing green and accurate regulation and control of the growth period of crops.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural microorganisms, and particularly relates to a microbial composition with a flower promoting effect and application thereof in regulation of corn flowering time. BACKGROUND

[0002] The flowering time of plants is a key link for regulating the growth cycle, enhancing environmental adaptability and optimizing agricultural production management. Taking corn as an example, the early or late of the heading stage not only directly affects irrigation and fertilization strategies, but also determines whether the crops can complete the pollination and seed setting process in the best climate window. At present, the flowering time is mainly regulated by means of variety replacement, exogenous hormone treatment or nutrient regulation in production, but there are problems such as high cost, complex operation or strong environmental sensitivity.

[0003] In recent years, plant growth-promoting rhizobacteria have attracted increasing attention in regulating crop growth and development. Studies have shown that some beneficial microorganisms can indirectly regulate the flowering time of plants by regulating the level of endogenous hormones (such as cytokinins, ethylene, etc.) and affecting the nutrient absorption and transport process. Bacillus subtilis is a common plant growth-promoting bacteria, which has significant effects on improving plant stress resistance and promoting biomass accumulation, but its role in specific regulation of the flowering stage is limited. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a microbial composition composed of Ensifer strain S2_8_1 and Bacillus subtilis and its application in promoting early flowering of corn. The composition utilizes the synergistic effect of S2_8_1 strain combined with Bacillus subtilis to effectively regulate the flowering time of corn, and has the comprehensive advantages of green safety, simple operation, stable effect and easy popularization.

[0005] To achieve the above purpose, the technical solution adopted by the present application is: In a first aspect, the present application provides a microbial composition for promoting early flowering of corn, which comprises Ensifer strain S2_8_1 and Bacillus subtilis.

[0006] As a preferred solution, the Ensifer strain S2_8_1 and Bacillus subtilis are both applied in the form of bacterial agents, and the volume ratio is 1:1.

[0007] As a preferred solution, the concentration of the Ensifer strain S2_8_1 bacterial agent is 200000-230000 cfu / mL.

[0008] In a second aspect, the present application provides the application of the combined application of Ensifer strain S2_8_1 and Bacillus subtilis in promoting early flowering of corn.

[0009] As a preferred solution, the Ensifer strain S2_8_1 and the Bacillus subtilis are both applied in the form of a microbial inoculant, in a volume ratio of 1:1.

[0010] As a preferred solution, the concentration of the Ensifer strain S2_8_1 microbial inoculant is 200000-230000 cfu / mL.

[0011] In a third aspect, the present application provides a method for promoting early flowering of corn, comprising the following steps: Step 1: inoculate the Ensifer strain S2_8_1 into an enriched culture medium and cultivate under constant temperature conditions to prepare an Ensifer strain S2_8_1 microbial inoculant; Step 2: prepare a Bacillus subtilis microbial inoculant; Step 3: mix the Ensifer strain S2_8_1 microbial inoculant prepared in Step 1 and the Bacillus subtilis microbial inoculant prepared in Step 2 in a volume ratio of 1:1 to obtain a mixed microbial inoculant; Step 4: inoculate the mixed microbial inoculant into the rhizosphere soil of corn, with an inoculation amount of 200 mL per corn plant; Step 5: maintain the corn until flowering.

[0012] As a preferred solution, in Step 1, the formula of the enriched culture medium is: 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, add deionized water to 1000 mL, adjust the pH to 7.0-7.2, and cultivate under constant temperature conditions at 28℃ for 15 days.

[0013] As a preferred solution, in Step 1, the concentration of the Ensifer strain S2_8_1 microbial inoculant is 200000-230000 cfu / mL.

[0014] According to the above technical solution, the present application has the following beneficial effects: The microbial composition and the application method thereof provided by the application have a significant synergistic effect of promoting flowering by inoculating the Ensifer strain S2_8_1 and Bacillus subtilis in the rhizosphere of corn. The synergistic effect is caused by the functional complementation of the two strains: the Ensifer strain S2_8_1 significantly enhances the nitrification rate of the rhizosphere soil (which can be increased by more than 2 times of the blank control group), and converts nitrogen into nitrate nitrogen which is more easily absorbed, and promotes the synthesis of endogenous plant cytokinin, driving the conversion of corn from vegetative growth to reproductive growth; at the same time, the Bacillus subtilis strengthens the colonization of the root system, activates soil nutrients and promotes the overall healthy growth of the plant, not only provides a favorable rhizosphere microenvironment for the function of the Ensifer strain S2_8_1, but also accumulates the necessary biomass basis for reproductive growth, and the two synergistically promote the significant effect of advancing the flowering period (heading stage) of corn by 2-7 days.

[0015] 2. The application method of the application is simple to operate and easy to popularize. Compared with the traditional regulation means relying on variety replacement or foliar spraying of exogenous hormones, the culture medium for preparing the composition inoculant of the application has clear components and mild culture conditions, and field application only needs to be inoculated in the rhizosphere once at the seedling stage, without the need for complex equipment or frequent operation, and can be used as an important technical means for regulating the growth period of crops in green agricultural production.

[0016] Strain preservation information: the Ensifer strain S2_8_1 described in the application, which is classified and named as Rhizobiaceae Ensifer, has been preserved in the China Center for Type Culture Collection on April 14, 2021, and the preservation address is Wuhan University, China, and the preservation number is CCTCC NO: M2021374. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Flowering period (heading) photos of corn plants under different treatments; Figure 2 Columnar chart of average flowering time of corn in each treatment group. DETAILED DESCRIPTION

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

[0019] The experimental methods described in the following examples are all conventional methods in the art unless otherwise specified; and the reagents and materials can be obtained through commercial channels unless otherwise specified.

[0020] The volume or concentration of the inoculant is implemented according to the experimental proportion, and the relevant inoculation amount is calculated by volume.

[0021] Example 1 1. Isolation, screening and identification of Ensifer strain S2_8_1 A soil sample of 20 g was collected from the experimental farm of Henan University of Science and Technology and inoculated into an enrichment culture medium. The medium formula was: 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 deionized water was added to 1000 mL, and the pH was adjusted to 7.0-7.2. The medium was sterilized at 121°C for 20 min, and then incubated at 28°C in the dark for 15-25 days. After incubation, the presence of ammonia-oxidizing bacterial strains was detected using Griess reagent, and the enrichment culture solution showing the growth of ammonia-oxidizing bacterial strains was transferred to a new enrichment culture medium for continuous culture, and the culture was repeated twice to obtain a purified bacterial enrichment solution.

[0022] 0.1 mL of the above enrichment solution was taken and spread on a solid separation medium containing 2.5 g / L agar (other components were the same as the above enrichment medium), and incubated at 28°C in the dark for 7 days. After single colonies formed on the plate, colonies with clear morphology and appropriate size were picked and repeated streak separation and purification for 5-10 times until a stable growing pure strain was obtained.

[0023] 5-10 representative strains with stable nitrification-promoting ability were selected and inoculated into a liquid enrichment culture medium, and incubated at 28°C in the dark for 15 days. The obtained bacterial solution was the S2_8_1 bacterial inoculant of the Ensifer bacteria used in the experiment, and the concentration of the S2_8_1 bacterial inoculant of the Ensifer bacteria was 200000-230000 cfu / mL. Bacillus subtilis was a commonly used commercially available strain, and the Bacillus subtilis bacterial inoculant was prepared by a conventional method in the art and used in subsequent experiments. The above Ensifer bacteria with soil nitrification-promoting effect were named S2_8_1, and the classification name was Rhizobiaceae ensifer, which had been preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M2021374.

[0024] 2. Effect of combined application of Ensifer S2_8_1 and Bacillus subtilis on corn flowering time To verify the effect of combined application of the Ensifer strain S2_8_1 and Bacillus subtilis in promoting early flowering of corn, the following pot culture test was carried out: 36 pots of corn seedlings with uniform emergence and growth were selected, one plant per pot, and the planting substrate was a field soil with an organic matter content of 16.5 g / kg. The corn plants were randomly divided into four treatment groups, nine pots per group, and the specific treatments were as follows: Group a is the joint inoculation group (S2_8_1 + Bacillus subtilis): inoculate Ensifer strain S2_8_1 with Bacillus subtilis inoculant mixed solution, 100 mL each inoculant; Group b is the single inoculation of Bacillus subtilis group (Bacillus): inoculate 200 mL of Bacillus subtilis inoculant per pot; Group c is the single inoculation of S2_8_1 group (S2_8_1): inoculate 200 mL of S2_8_1 inoculant per pot; Group d is the blank control group (CK): no inoculation of any inoculant.

[0025] All pots were planted on April 1, 2025.

[0026] After inoculation, all pots were placed in a unified greenhouse environment for daily maintenance, keeping the irrigation and light conditions consistent. The growth state of the plants was continuously observed, and the flowering (heading) time was recorded with the exposure of corn pollen as the flowering marker.

[0027] As shown in Figure 1 and Figure 2 , there are significant differences in flowering time among the different treatment groups: the joint inoculation group (group a) flowers earliest, with an average heading time of July 1, 2025; the single inoculation of S2_8_1 group (group c) flowers on July 3, 2025; while the single inoculation of Bacillus subtilis group (group b) and the blank control group (group d) have the same flowering time, both on July 8, 2025.

[0028] The results show that single inoculation of S2_8_1 strain can promote corn to flower earlier, but the joint inoculation group S2_8_1 and Bacillus subtilis has a more prominent effect, with flowering time 7 days earlier than the blank control group and 2 days earlier than the single inoculation of S2_8_1 strain. There is no significant difference in heading time between the single inoculation of Bacillus subtilis group and the blank control group, indicating that its flower-promoting effect is limited. Bacillus subtilis alone does not show obvious flower-promoting effect. This experiment confirms the functional potential of Ensifer strain S2_8_1 in regulating the flowering time of corn and the feasibility of its synergistic application with Bacillus subtilis, providing an effective solution for microbial regulation of crop growth period.

[0029] 3. Synergistic effect of Ensifer S2_8_1 and Bacillus subtilis on corn growth and flowering To comprehensively evaluate the regulation of Ensifer strain S2_8_1 and its combined application with Bacillus subtilis on the growth and flowering time of maize, the following pot experiments were carried out. Thirty-six pots of maize with consistent growth and 30-day-old seedlings were selected, with one plant per pot, and the cultivation substrate was a field soil with an organic matter content of 16.5 g / kg. The plants were randomly divided into four treatment groups (a, b, c, and d), with nine pots per group. Each group was further divided into three subgroups (a1 / a2 / a3, b1 / b2 / b3, c1 / c2 / c3, d1 / d2 / d3), with three pots per subgroup, for destructive sampling and continuous observation at different growth stages. The treatment settings were as follows: Group a was the combined inoculation group (S2_8_1 + Bacillus): inoculated with a mixture of Ensifer strain S2_8_1 and Bacillus subtilis inoculant, 100 mL of each inoculant; Group b was the single Bacillus subtilis inoculation group (Bacillus): inoculated with 200 mL of Bacillus subtilis inoculant per pot; Group c was the single S2_8_1 inoculation group (S2_8_1): inoculated with 200 mL of S2_8_1 inoculant per pot; Group d was the blank control group (CK): no inoculant was applied.

[0030] Before inoculation, the initial aboveground and underground biomass of each group was recorded, and rhizosphere soil was collected for nitrification rate determination to ensure that there were no significant differences in the initial state of each group. On the 10th day after inoculation, the second subgroup of each group (a2, b2, c2) was taken for destructive sampling to determine the fresh weight of the aboveground and underground parts, respectively, and recorded as Aa1, Ab1, Ac1, and Ba1, Bb1, Bc1. The results were as follows: Aboveground biomass (g / plant): Aa1 = 3.97, Ab1 = 3.15, Ac1 = 3.67; Underground biomass (g / plant): Ba1 = 5.13, Bb1 = 4.61, Bc1 = 4.76.

[0031] The aboveground biomass of the combined inoculation group (group a) was 26% higher than that of the single Bacillus subtilis treatment group (group b), and the underground biomass was 8% higher than that of the single S2_8_1 treatment group (group c).

[0032] The nitrification rate of the rhizosphere soil of the above-mentioned subgroups was determined at the same time, and recorded as Za1, Zb1, Zc1, Zd1. The results showed: Nitrification rate (ng / g): Za1 = 7.71, Zb1 = 1.95, Zc1 = 4.92, Zd1 = 2.25.

[0033] The S2_8_1 strain, especially when combined with Bacillus subtilis, can significantly improve the nitrogen conversion efficiency of rhizosphere soil, thereby promoting plant growth.

[0034] During the subsequent growth process, the heading time of the remaining plants in each group was continuously observed. The results are as follows: the combined inoculation group (a1, a3 subgroup) headed on July 1, 2025; the S2_8_1 alone group (c1, c3 subgroup) headed on July 3; the Bacillus subtilis group (b1, b3 subgroup) and the control group (d1, d3 subgroup) all headed on July 8.

[0035] The above results show that Ensifer S2_8_1 can lay a material and hormone foundation for the advance of the reproductive growth stage (heading) of corn by enhancing rhizosphere nitrification, promoting nutrient absorption and plant growth; its combined application with Bacillus subtilis further shows a synergistic effect, which can more significantly shorten the flowering time.

[0036] This experiment confirms that the combined application of Ensifer strain S2_8_1 and Bacillus subtilis can significantly enhance the soil nitrogen conversion capacity, improve plant biomass, and systematically accelerate the transition of corn from vegetative growth to reproductive growth by regulating the nutritional and hormonal state of plants, ultimately achieving a significant flowering effect.

[0037] It should be noted that the above-mentioned embodiments should be understood as illustrative rather than limiting the scope of protection of the present application, and the scope of protection of the present application is subject to the claims. For those skilled in the art, some non-essential improvements and adjustments to the present application without departing from the spirit and scope of the present application still fall within the scope of protection of the present application.

Claims

1. A microbial composition for promoting early flowering of maize, characterized in that, The composition comprises Ensifer strain S2_8_1 and Bacillus subtilis.

2. The microbial composition according to claim 1, characterized in that: The Ensifer strain S2_8_1 and Bacillus subtilis were both applied in the form of inoculum, with a volume ratio of 1:

1.

3. The microbial composition according to claim 2, characterized in that: The concentration of the Ensifer strain S2_8_1 inoculum was 200,000–230,000 cfu / mL.

4. Application of Ensifer strain S2_8_1 in combination with Bacillus subtilis in promoting early flowering of maize.

5. The application according to claim 4, characterized in that: The Ensifer strain S2_8_1 and Bacillus subtilis were both applied in the form of inoculum, with a volume ratio of 1:

1.

6. The application according to claim 4, characterized in that: The concentration of the Ensifer strain S2_8_1 inoculum was 200,000–230,000 cfu / mL.

7. A method for promoting early flowering of corn, characterized in that: Includes the following steps: Step 1: Inoculate Ensifer strain S2_8_1 into enrichment medium and culture it under constant temperature conditions to prepare Ensifer strain S2_8_1 bacterial agent; Step 2, prepare Bacillus subtilis inoculant; Step 3: Mix the Ensifer strain S2_8_1 bacterial agent obtained in Step 1 and the Bacillus subtilis bacterial agent obtained in Step 2 at a volume ratio of 1:1 to obtain a mixed bacterial agent; Step 4: Inoculate the mixed microbial agent into the rhizosphere soil of the corn plant at a rate of 200 mL per plant. Step 5: Care for the corn until it flowers.

8. The method according to claim 7, characterized in that: In step 1, the enrichment culture medium is formulated as follows: 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, with deionized water added to bring the volume to 1000 mL, the pH adjusted to 7.0–7.2, and the culture is kept at a constant temperature of 28°C for 15 days.

9. The method according to claim 7, characterized in that: In step 1, the concentration of the Ensifer strain S2_8_1 inoculum is 200,000 to 230,000 cfu / mL.

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