Aeromonas eel, fungicide containing aeromonas eel and application of aeromonas eel in promoting nitrogen fertilizer absorption of plants

Abstract

The invention relates to the technical field of microorganisms and application thereof, in particular to aeromonas eel, a microbial agent containing the aeromonas eel and application of the aeromonas eel to promotion of nitrogen fertilizer absorption of plants. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.34412. The aeromonas eel provided by the invention is separated and screened by the inventor for the first time, and has the capability of promoting plants to absorb nitrogen fertilizer. In addition, the strain also has the functions of dissolving phosphorus, potassium and silicon, is a multifunctional strain, and can promote plant growth and activate soil nutrients.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms and their applications, and particularly relates to a strain of Aeromonas veronii, a bacterial agent containing the same and a use for promoting plant absorption of nitrogen fertilizer. BACKGROUND

[0002] Nitrogen is a component of proteins and nucleic acids, and is also a basic component of many coenzymes, prosthetic groups, chlorophyll molecules, and some plant hormones (such as auxins and cytokinins) and vitamins (Kindred D R et al., 2008; Sylvester-Bradley R, 2009). Except for some legumes that can fix nitrogen through rhizobia, most plants need to obtain nitrogen sources from the soil through roots, and therefore, increasing nitrogen fertilizer can promote plant growth and development and increase crop yield. However, the intensification of modern agricultural production makes the available nitrogen in the soil very limited (Jiang Zhi-min, 2018). With the continuous development of agricultural production and the increasing demand for increasing crop yield, the amount of nitrogen fertilizer is also increasing year by year. The overuse of chemical fertilizers not only increases the cost of agricultural production, but also has negative effects on the sustainable development of agriculture and the environment: excessive fertilization exceeds the carrying capacity of the land, leading to a decline in soil quality, and in turn causing a series of environmental problems, such as soil acidification and compaction, increased soil-borne diseases, greenhouse gas emissions, excessive fertilizer flowing into rivers, lakes and seas causing water eutrophication, and polluting water resources (Yu Guo, 2022). Improving nitrogen use efficiency meets the needs of the environment and the economy, and can minimize nitrogen loss and optimize the input-output ratio of nitrogen fertilizer (Lin C et al., 2022). Reducing the amount of chemical fertilizer and optimizing fertilization are also the trends of future agricultural development.

[0003] Currently, the research on improving nitrogen utilization efficiency in agriculture mainly focuses on agricultural management, chemistry, and biology. In terms of agricultural management, one is to optimize the fertilization mode, such as optimizing nitrogen management measures, adopting base fertilizer: tillering fertilizer: flower promoting fertilizer: flower preserving fertilizer = 4:2:2:2, which can increase yield by 8.4% compared with the farmers' habit of fertilization (Wang Yuwen et al., 2016); the second is to adjust the planting mode and water management. Some studies have pointed out that straw returning combined with dry-wet alternate irrigation and precise nitrogen reduction is beneficial to dry matter accumulation and distribution, nitrogen absorption and transport, which can improve nitrogen agronomic efficiency and save 20% of nitrogen input (Hu Mingming et al., 2024). In terms of chemistry, the use of chemical inhibitors, such as NBPT (N-butyl thiophosphoryl triamide) and DMPP (3,4-dimethyl pyrazole phosphate), can increase the absorption and utilization of nitrogen by crops, promote crop growth, and increase crop yield (Yan Tingshun et al., 2025). Compared with traditional chemical inhibitors, biological methods are more environmentally friendly. Generally, various biological fertilizer synergists are used, including humic substances, amino acids, oligosaccharides, and microorganisms. Modifying soil with humic substances can improve the stability of aggregates, making them more stable in dry-wet cycles, improving soil aeration, promoting root penetration, improving water use efficiency of plants, reducing soil erosion, and helping to improve nutrient absorption. The application of amino acid synergists can improve plant nutrition by affecting soil processes and directly affecting plant physiology, including improving soil structure, improving the solubility of trace elements in soil, and changing plant root morphology (Li Ruhi et al., 2024). Oligosaccharide synergists such as chitosan can promote plant growth, improve stress resistance, prevent and control crop diseases and pests, regulate soil microbial activity, and reduce soil urease, phosphatase, catalase, and other enzyme activities (Li Jiancheng et al., 2024).

[0004] Microbial fertilizer synergists can use various mechanisms to improve soil fertility, promote plant nutrition and water absorption, increase crop yield, and regulate soil microbial community structure, such as converting insoluble elements in soil into soluble elements by solubilizing nitrogen, phosphorus, and potassium, excreting plant hormones, enhancing iron availability, and dissolving phosphate, inhibiting the production of plant pathogens, protecting plants from abiotic and biotic stress, and underground pollutants (Li Ruhi et al., 2024). Microbial fertilizer synergists can coordinate and supplement chemical fertilizers, minimize the negative impact of chemical inputs, and thus improve the quantity and quality of agricultural products, making it the most promising green development.

[0005] Therefore, it is necessary to develop more microbial synergists with the function of reducing fertilizer and increasing efficiency, and to effectively utilize microbial resources. In view of this, the present application is proposed. SUMMARY

[0006] The purpose of this invention is to provide a strain of Aeromonas hydrophila, a bacterial agent containing the same, and its use in promoting the absorption of nitrogen fertilizer by plants.

[0007] The present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of Aeromonas surae, wherein Aeromonas surae is Aeromonas surae (… Aeromonas encheleia The strain KY1695 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34412.

[0008] In one embodiment, the 16S rDNA sequence of the strain KY1695 is shown in SEQ ID No. 1.

[0009] This application unexpectedly revealed for the first time that when plants were treated with strain KY1695, their growth was significantly enhanced after being drenched with a 0.1% urea solution, with larger leaves and greater plant height. It was speculated that strain KY1695 might promote nitrogen fertilizer absorption in plants. Subsequent in-depth studies confirmed that this strain not only promotes nitrogen fertilizer absorption and utilization but also possesses phosphorus, potassium, and silicon solubilization functions, making it a multifunctional bacterium with both nitrogen-reducing and nitrogen-enhancing effects. 16S rDNA sequencing of strain KY1695 showed that this strain... Aeromonas encheleia With a similarity of 99.19%, strain KY1695 was identified as Aeromonas surimi ( Aeromonas encheleia This is the first report on the weight loss and efficacy enhancement function of this bacterium, and it has great market potential.

[0010] In a second aspect, the present invention provides a microbial agent containing the aforementioned Aeromonas an eel ( Aeromonas encheleia ) strain KY1695 or the aforementioned Aeromonas an eel ( Aeromonas encheleia (A culture of strain KY1695)

[0011] In a third aspect, the present invention provides a fertilizer synergist comprising Aeromonas hydrophila from the first aspect (…). Aeromonas encheleia ) strain KY1695 or the aforementioned second aspect of the bacterial agent.

[0012] In one embodiment, the fertilizer synergist further comprises an oligosaccharide synergist, preferably, the oligosaccharide synergist includes one or more of chitosan oligosaccharide, fucoidan oligosaccharide, chitin oligosaccharide and amino oligosaccharide.

[0013] In a preferred embodiment, the oligosaccharide synergist is chitosan oligosaccharide.

[0014] In a fourth aspect, the present invention provides the aforementioned Aeromonas hydrophila ( Aeromonas encheleiaThe following uses are permitted for strain KY1695, the aforementioned microbial agents, or the aforementioned fertilizer synergists: (1) Promote the absorption and / or utilization of nitrogen fertilizer by plants; and / or (2) Promotes the solubility of phosphorus, potassium and / or silicon.

[0015] In one embodiment, the plant includes crops; more preferably, the crop is one of corn, wheat, rice, peanuts, and soybeans. Most preferably, the plant is corn.

[0016] In a fifth aspect, the present invention provides a method for promoting the absorption and / or utilization of nitrogen fertilizer by plants, the method comprising utilizing the aforementioned Aeromonas hydrophila (…). Aeromonas encheleia Plants are treated with strain KY1695, the aforementioned microbial agent, or the aforementioned fertilizer synergist.

[0017] In one embodiment, the treatment includes root irrigation and / or foliar spraying of plants with a liquid containing the aforementioned Aeromonas anguillarum strain KY1695, the aforementioned microbial agent, or the aforementioned fertilizer synergist.

[0018] Preservation information: Aeromonas hydrophila provided in this application ( Aeromonas encheleia The strain, named KY1695, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is April 30, 2025, and the accession number is CGMCC No. 34412. It was confirmed as a viable strain by the collection center on April 30, 2025.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) Aeromonas hydrophila of the present invention ( Aeromonas encheleia The strain KY1695 was first discovered and screened by the inventors. This strain can promote the absorption and utilization of nitrogen fertilizer by plants, and has the functions of phosphorus solubilization, potassium solubilization and silicon solubilization. It is a multifunctional strain with good comprehensive performance and has broad application prospects in soil improvement and crop growth promotion.

[0021] (2) The strain KY1695 of the present invention or the bacterial agent containing it can be compounded with oligosaccharide synergists to form a compound fertilizer synergist. It can work synergistically with oligosaccharide synergists such as chitosan oligosaccharide to jointly achieve the effect of reducing fertilizer and increasing efficiency for crops such as corn. It has the potential to be developed into a microbial fertilizer and has a wide market application prospect.

[0022] (3) The strain KY1695 of the present invention and the compound fertilizer synergist formulated with oligosaccharide synergists can reduce the application of chemical nitrogen fertilizers, reduce the cost of agricultural production, and reduce environmental pollution, which is of great significance in agricultural pollution prevention and control. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Aeromonas encheleia The diagram shows the plant growth of the KY1695 strain treatment group, the Bacillus subtilis treatment group, and the CK (control) group after being sprayed with 0.1% urea solution in the embodiments of the present invention. Figure 1 This is a comparison between the KY1695 bacterial solution treatment group and the CK treatment group under different concentrations of urea in this embodiment of the invention; Figure 2 This is a comparison of the KY1695 bacterial solution treatment group and the 92068 bacterial solution treatment group under different concentrations of urea in this embodiment of the invention; Figure 3 This is a comparison of the corn growth in each treatment group under 0.1% urea conditions in this embodiment of the invention; Figure 4 The results show the detection results of the phosphorus-solubilizing, potassium-solubilizing, and silicon-solubilizing abilities of strain KY1695 and control Bacillus subtilis 92068 in the embodiments of the present invention. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] It should be noted that, unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied.

[0027] This invention provides a strain of Aeromonas elegans, wherein Aeromonas elegans is Aeromonas elegans (Figure 5 Aeromonas Strain KY1695, deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 34412, has the 16S rDNA sequence shown in SEQ ID No. 1. The results of 16S DNA sequencing of strain KY1695 indicate that its base sequence is similar to that of strain KY1695. encheleia Aeromonas With a high homology of over 99.86%, it was identified as *Aeromonas surae*. This strain was collected by the inventor and deposited at the China General Microbiological Culture Collection Center on April 30, 2025.

[0028] The present invention also provides a bacterial agent, characterized in that the bacterial agent contains the aforementioned Aeromonas an eel ( encheleia ) strain KY1695 or the aforementioned Aeromonas an eel ( Aeromonas encheleia The bacterial agent contains a culture of strain KY1695. Aeromonas encheleia The strain KY1695 is used as the main active ingredient.

[0029] In a specific implementation plan, the *Aeromonas elegans* Aeromonas encheleia The culture type of strain KY1695 can be any one of the following: culture extract, lyophilized powder, fermentation broth, fermentation broth precipitate, fermentation broth supernatant, or fermentation broth extract.

[0030] In a specific implementation scheme, the microbial agent is a solid or liquid microbial agent; the *Aeromonas eelus* contained in the microbial agent... (Aeromonas encheleia The total viable count of strain KY1695 is at least 1 × 10⁻⁶. 7 cfu·g -1 Or 1×10 7 cfu·mL -1 In some specific embodiments, the total viable count of Aeromonas eelusae in the microbial agent is 10. 8 cfu·mL -1 10 9 cfu·mL -1 10 10 cfu·mL -1 , or 10 8 cfu·g -1 10 9 cfu·g -1 10 10 cfu·mL -1 .

[0031] In a specific implementation scheme, the bacterial agent can be obtained by using the aforementioned Aeromonas hydrophila (eel aeromonas). (Aeromonas encheleia Aeromonas The microbial agent is obtained by liquid fermentation culture of strain KY1695. The microbial agent also contains commonly used carriers, such as solid or liquid carriers, specifically, bentonite, calcium carbonate, zeolite, starch; or vegetable oil, mineral oil, and water. In specific embodiments, the microbial agent is applied by spraying, watering, or as a base fertilizer.

[0032] The present invention also provides a fertilizer synergist, wherein the fertilizer synergist comprises the aforementioned Aeromonas hydrophila ( encheleia (Strain KY1695 or the aforementioned bacterial agent.) The fertilizer enhancer of the present invention can be used as a component of bio-organic fertilizer, containing the aforementioned Aeromonas hydrophila ( Aeromonas encheleia The KY1695 strain is inoculated into organic fertilizer and stirred evenly to obtain a bio-organic fertilizer containing fertilizer synergists. The fertilizer synergists of this invention can promote plant growth and improve soil fertility. Additionally, they can be used as plant growth promoters or nitrogen nutrient improvers for promoting the growth of crops and vegetables.

[0033] In one embodiment, the fertilizer synergist further comprises an oligosaccharide synergist, preferably chitosan oligosaccharide. Chitosan oligosaccharide, also known as chitosan oligosaccharide or chitosan oligomer, is a low-polymerization, water-soluble sugar composed of 2-10 glucosamine molecules linked by β-(1,4)-glycosidic bonds. It appears as a white or off-white powder and has good water solubility. In a specific embodiment, commercially available chitosan oligosaccharide powder can be diluted 5000-7000 times and mixed with Aeromonas hydrophila KY1695 bacterial solution to prepare the fertilizer synergist. In a preferred embodiment, commercially available chitosan oligosaccharide powder is diluted 6000 times and mixed with Aeromonas hydrophila KY1695 bacterial solution to prepare a compound fertilizer synergist. Chitosan oligosaccharide and Aeromonas hydrophila KY1695 bacterial solution have a synergistic effect in promoting nitrogen absorption and utilization by plants.

[0034] The present invention also provides the aforementioned Aeromonas hydrophila ( Aeromonas encheleia The following uses are permitted for strain KY1695, the aforementioned microbial agents, or the aforementioned fertilizer synergists: (1) Promote the absorption and / or utilization of nitrogen fertilizer by plants; and / or (2) Promotes the solubility of phosphorus, potassium and / or silicon.

[0035] Aeromonas hydrophila of the present invention ( Aeromonas encheleiaThe strain KY1695, and inoculants or fertilizer synergists containing it, can promote the absorption and utilization of nitrogen fertilizer by plants and have the ability to promote plant growth. By applying the Aeromonas hydrophila inoculant provided by this invention, the amount of chemical nitrogen fertilizer applied to target crops can be reduced, agricultural production costs can be reduced, and environmental pollution can be reduced.

[0036] The Aeromonas hydrophila strain KY1695 of the present invention or an inoculum containing it can also be used for phosphorus solubilization, potassium solubilization and silicon solubilization, which can improve soil fertility and promote plant growth.

[0037] This invention also provides a method for promoting the absorption and / or utilization of nitrogen fertilizer by plants, the method comprising treating plants with the aforementioned Aeromonas encheleia strain KY1695, the aforementioned microbial agent, or the aforementioned fertilizer synergist. Applying Aeromonas encheleia strain KY1695, the aforementioned microbial agent, or the aforementioned fertilizer synergist to the roots and / or leaves of plants can promote the conversion of nitrogen fertilizer into nitrogen nutrients that can be effectively absorbed by the plants; the plants are crops such as corn, peanuts, or soybeans, preferably corn, such as the Huangnuo 589 corn variety.

[0038] Aeromonas eel (using Aeromonas esculenta) Aeromonas encheleia Using strain KY1695, the aforementioned microbial agent, or the aforementioned fertilizer synergist for root irrigation and / or foliar spraying can promote the absorption of nitrogen from the soil and improve nitrogen utilization efficiency. This invention experimentally demonstrates that, compared to the CK control group, the nitrogen fertilizer utilization rate of Aeromonas hydrophila (*Aeromonas hydrophila*) is significantly higher. Aeromonas encheleia The nitrogen fertilizer utilization rate of the experimental group treated with strain KY1695 increased by 6.8%, while the nitrogen fertilizer utilization rate of the mixed treatment group of KY1695 bacterial culture and chitosan increased by 19%.

[0039] In a specific implementation plan, the nitrogen fertilizer contains 0.04% to 0.1% urea.

[0040] Aeromonas hydrophila of the present invention ( Aeromonas encheleia Strain KY1695 or compound fertilizer synergists containing KY1695 bacterial solution and chitosan oligosaccharides can improve crop nitrogen absorption efficiency and increase the content of nutrients such as phosphorus, potassium, and silicon while reducing the application of chemical fertilizers, thereby improving crop yield and quality. In particular, strain KY1695 and compound fertilizer synergists containing chitosan oligosaccharides have a synergistic effect on promoting plant nitrogen fertilizer absorption.

[0041] The Aeromonas encheleia strain KY1695 or a bacterial solution containing KY1695 and a compound fertilizer synergist containing chitosan oligosaccharide of the present invention can be used to develop new microbial fertilizers to achieve multiple goals such as promoting crop growth, increasing yield, increasing planting benefits and reducing environmental pollution.

[0042] Example: 1. Materials 1.1 Strains: Aeromonas an eel ( Aeromonas encheleia KY1695 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34412.

[0043] Commercial growth promoter strain Bacillus subtilis ( Aeromonas encheleia )92068, originating from the Institute of Agricultural Resources Zoning, Chinese Academy of Agricultural Sciences.

[0044] 1.2 Culture medium R2A liquid medium: 0.5 g yeast extract, 0.5 g peptone, 0.5 g tryptone, 0.5 g glucose, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.3 g sodium pyruvate, 0.05 g magnesium sulfate heptahydrate, 1 L water.

[0045] Potassium-solubilizing solid culture medium: 10 g glucose, 0.5 g yeast extract, 1 g ammonium sulfate, 2 g disodium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 1 g calcium carbonate, 1 g potassium feldspar, 15 g agar, 1 L water.

[0046] Inorganic phosphorus solid culture medium: 0.5 g yeast extract, 10 g glucose, 0.5 g ammonium sulfate, 0.02 g potassium chloride, 0.1 g magnesium sulfate heptahydrate, 0.03 g ferrous sulfate, 0.03 g manganese sulfate, 5 g tricalcium phosphate, 15 g agar, 1 L water.

[0047] Silicon-free solid culture medium: 0.5 g yeast extract, 0.5 g peptone, 0.5 g tryptone, 0.5 g glucose, 0.5 g soluble starch, 0.3 g dipotassium hydrogen phosphate, 0.3 g sodium pyruvate, 0.05 g magnesium sulfate heptahydrate, 5 g magnesium silicate, 15 g agar, 1 L water.

[0048] 1.3 Main Reagents and Seeds 1.3.1 Reagents Chitosan oligosaccharide was prepared and provided by Kang Shengyuan (Zhaoqing) Biotechnology Co., Ltd., and can also be purchased through commercial channels; 10% urea-15N was purchased from Maclean's, and all other reagents were domestically produced analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0049] 1.3.2 Seeds The corn seeds were Huangnuo 589, purchased from Qingdao Jiaoyan Seedling Co., Ltd.

[0050] 2. The discovery that strain KY1695 promotes nitrogen fertilizer absorption in plants. In experiments using microorganisms from the strain library of Kang Shengyuan (Zhaoqing) Biotechnology Co., Ltd. to promote plant growth, it was found that the KY1695 strain treatment group showed significant growth after being drenched with a 0.1% urea solution during the growth process, with larger leaves and taller plants. Bacillus subtilis As shown in the figure. It is speculated that it may promote the absorption of nitrogen fertilizer by plants, so it was used as a new research object to carry out a series of experiments on improving the absorption of nitrogen fertilizer by plants.

[0051] 3. 16S rDNA sequencing of strain KY1695 3.1 Extraction of bacterial DNA using the CTAB method (1) Inoculate a single colony into 5 mL of R2A and incubate overnight at 30°C; (2) Take 1 mL of seed culture medium and inoculate it into 100 mL of R2A liquid, and incubate at 37℃ and 220 r / min for 16 hours. (3) Centrifuge at 5000 r / min for 10 minutes and discard the supernatant; (4) After centrifugation and washing with 10 mL TE, dissolve the bacterial cells with 10 mL TE, mix well, and store at -20℃ for later use. (5) Take 3.5 mL of bacterial suspension, add 184 μL of 10% SDS, mix well, add 37 μL of 10 mg / mL proteinase K, mix well, and incubate at 37°C for 1 hour; (6) Add 740 μL of 5 mol / L NaCl, then add 512 μL of CTAB / NaCl, mix well, and incubate at 65℃ for 10 minutes; (7) Add an equal volume of chloroform / isoamyl alcohol, mix well, centrifuge at 10000 r / min for 5 minutes, and retain the supernatant; (8) Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) to the supernatant, mix well, centrifuge at 10000r / min for 5 minutes, and retain the supernatant; (9) Add 0.6 times the amount of isopropanol, mix well, centrifuge at 10000 r / min for 5 minutes, collect the DNA precipitate, and wash the DNA precipitate with 70% ethanol by centrifugation. (10) Dissolve DNA in 1 mL TE, add RNase A to a final concentration of 20 μg / mL, and store at 4℃.

[0052] 3.2 Amplification and Sequencing PCR amplification of 16S rDNA was performed using universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID No. 2) and 1492r (5'-GGTTACCTTGTTACGACTT-3', SEQ ID No. 3). PCR reaction conditions were: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 60 s, for 30 cycles. PCR products were subjected to 1.5% agarose gel electrophoresis, and the PCR products were recovered, purified, and sequenced after agarose gel electrophoresis.

[0053] 3.3 16S rDNA sequencing results of strain KY1695 The 16S rDNA sequence of strain KY1695 is as follows, Sequence 1:

[0054] Based on the obtained 16S rDNA sequence (SEQ ID No. 1) of KY1695, homologous sequences were searched in GenBank and compared with each other. Simultaneously, sequence alignment analysis was performed with the 16S RNA database (Chun's lab) recognized by the International Committee for Bacteriology. The results showed that the 1455-base sequence of this strain was homologous with that of strain [unclear - possibly a specific strain name]. Figure 1 With a similarity of 99.19%, KY1695 has been confirmed as [the correct name / product]. Aeromonas encheleia .

[0055] 4. Verification of the weight-loss and synergistic effects of strain KY1695 on corn. 4.1 Growth of maize treated with KY1695 bacterial solution at different urea concentrations 4.1.1. Processing Group (1) Control group: Commercial multifunctional strain Bacillus subtilis 92068 treatment group, CK (blank) treatment group; (2) Experimental group: Treatment group with strain KY1695; (3) Urea concentration: Based on a 0.1% urea solution, groups were set up with a gradient reduction of 20% to 0.08% urea solution, 0.06% urea solution, and 0.04% urea solution. For each of the above urea concentrations, three replicates were set up for the control group and the experimental group. Note: The concentration of urea solution refers to the mass-volume concentration (w / v, mass / volume percentage concentration).

[0056] 4.1.2. Methods (1) Select corn seeds of uniform size and plump kernels and plant them in vermiculite pots. After they sprout, leave 3 corn seedlings of similar size and growth in each pot and pull out the excess seedlings. This can reduce the differences caused by the seeds themselves. (2) OD 600 The 92068 and KY1695 strains with a concentration of 0.05 were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of R2A liquid medium and cultured at 30 °C and 200 r / min for 2 days to obtain bacterial suspensions. (3) Dilute each of the above bacterial solutions to 10. 8 CFU / mL, each group was watered 3-4 times until the corn seedlings absorbed the solution, and then urea solution of the corresponding concentration was applied. The changes in the corn growth process of different treatment groups were observed, the growth of corn in each treatment group was compared, and their chlorophyll, plant height and biomass data were recorded.

[0057] 4.1.3. Results and Analysis Data were analyzed using SPSS Statistics 27. One-way ANOVA and Duncan's new multiple range test were used to test for significance of differences. Aeromonas encheleia and Figure 2 It can be seen that, under different concentrations of urea, the growth of the KY1695 bacterial solution treatment group was the best compared with the CK treatment group and the 92068 bacterial solution treatment group. This is further confirmed by the data in Tables 1 and 2 below.

[0058] Table 1. Comparison of SPAD values ​​and plant height of maize in different treatment groups under different urea concentrations.

[0059] Note: Different lowercase letters in the same column indicate significant differences at the 5% level (P<0.05).

[0060] Table 2. Comparison of fresh and dry weight of corn in different treatment groups under different urea concentrations.

[0061] As shown in Table 1, the SPAD value and plant height of the KY1695 bacterial solution group were higher than those of the other two groups at the same urea concentration under all urea concentration conditions. Table 2 shows that in addition to the good above-ground growth of the corn, the root system of the KY1695 bacterial solution group was also very well developed. Even under low concentrations of 0.04% and 0.06% urea, the growth of the KY1695 bacterial solution group was excellent, indicating that even with reduced urea nitrogen fertilizer application, the growth of corn was not affected after irrigating with KY1695 bacterial solution, and its growth was better than that of the CK group or the growth-promoting bacteria 92068 group. This further confirms that KY1695 bacteria has the effect of reducing fertilizer use and enhancing plant efficiency.

[0062] 5. Synergistic effect of strain KY1695 and fertilizer synergist on promoting nitrogen fertilizer absorption by plants. 5.1 Growth of maize treated with KY1695 bacterial solution and chitosan oligosaccharide followed by 0.1% urea solution 5.1.1. Processing Group (1) Control group: CK blank group, 92068 bacterial solution group, chitosan oligosaccharide group, KY1695 bacterial solution group.

[0063] (2) Experimental group: KY1695 bacteria and chitosan oligosaccharide mixture group.

[0064] 5.1.2. Methods (1) Select corn seeds of uniform size and plump kernels and plant them in vermiculite pots. After they sprout, leave 3 corn seedlings of similar size and growth in each pot and pull out the excess seedlings. This can reduce the differences caused by the seeds themselves. (2) OD60 KY1695 strain with a concentration of 0.05 was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of R2A liquid medium. After incubation at 30°C and 200 rpm for 2 days, the KY1695 bacterial suspension was obtained and diluted to 10⁻⁵. 8 CFU / mL; (3) Place 10 8 CFU / mL of 92068 bacterial culture, chitosan oligosaccharide solution diluted 6000 times, 10 8 CFU / mL KY1695 bacterial culture, chitosan oligosaccharide diluted 6000 times, and 10 8 The corresponding groups were irrigated with a mixture of CFU / mL KY1695 bacteria (hereinafter referred to as the mixture) and water. Each group was irrigated 3-4 times, and then 0.1% urea solution was applied multiple times. The changes in maize growth in different treatment groups were observed, the growth of maize in each treatment group was compared, and their chlorophyll SPAD value, plant height and biomass data were recorded.

[0065] 5.1.3. Results and Analysis as follows Figure 3 As shown, the corn growth in the chitosan oligosaccharide group, the KY1695 bacterial solution group, and the mixed solution group was excellent. Based on the data in Table 3, the mixed solution group showed the best performance in terms of SPAD value, plant height, and corn biomass, followed by the chitosan oligosaccharide group and the KY1695 bacterial solution group. Therefore, it can be concluded that the KY1695 bacterial solution and chitosan oligosaccharide have a synergistic effect in promoting nitrogen fertilizer absorption in corn.

[0066] Table 3. Comparison of chlorophyll content, plant height, and fresh and dry weight of maize in different treatment groups under 0.1% urea conditions.

[0067] Note: Different lowercase letters in the same column indicate significant differences at the 5% level (P<0.05).

[0068] 5.2 The nitrogen fertilizer utilization rate of maize in each treatment group was detected using the 15N tracer method. 5.1.1. Processing Group (1) Control group: CK blank group, 92068 bacterial solution group, chitosan oligosaccharide group, KY1695 bacterial solution group; (2) Experimental group: KY1695 bacteria and chitosan oligosaccharide mixture group.

[0069] 5.1.2. Methods (1) 15N-labeled corn: As before, after germination, leave 3 corn seedlings of similar size and growth in each pot, and mix them with a 6000-fold diluted chitosan oligosaccharide solution. 8The corresponding treatment groups were irrigated with CFU / mL KY1695 bacterial solution, mixed solution, and water. Each group was irrigated with the treatment solution 3-4 times. Then, 15N-labeled urea was used as nitrogen fertilizer and applied in small amounts multiple times. 325 mg of nitrogen fertilizer was applied to each pot of corn throughout the entire growth process.

[0070] (2) Sample collection: After collecting the above-ground plant samples, the surface soil and impurities were washed off. The washed corn samples were placed in an oven and dried at 100℃ for 30 minutes to kill the green. The killed corn samples were then dried at 80℃ to constant weight, weighed, pulverized using a grinding device, and passed through a 100-mesh sieve. The nitrogen fertilizer utilization rate of each group of corn was calculated using a stable isotope mass spectrometer.

[0071] (3) The main calculation method is as follows: Nitrogen fertilizer utilization rate (%) = absolute abundance of 15N in plants × biomass / [application amount of 15N urea × nitrogen content of urea × abundance of 15N urea] × 100 (325 mg of 15N urea was applied to each group, the nitrogen content of urea was 46.7%, and the abundance of 15N urea reagent used was 10%).

[0072] 5.1.3. Results and Analysis Table 4 shows the nitrogen fertilizer utilization rates of maize in each group. It can be seen that the nitrogen fertilizer utilization rates of the KY1695 bacterial solution treatment group, the chitosan oligosaccharide treatment group, and the KY1695 bacterial solution-chitosan oligosaccharide mixed treatment group were all higher than those of the CK control group and the 92068 bacterial solution group, indicating that these treatment groups can improve the absorption of nitrogen fertilizer by maize. Among them, the mixed solution group showed the most significant improvement, with a 19% increase in nitrogen fertilizer utilization rate compared to the CK control group. This result is consistent with the previous experimental conclusion and further demonstrates that KY1695 bacterial solution can improve the absorption of nitrogen fertilizer by maize, and that its combination with chitosan oligosaccharide has a synergistic effect.

[0073] Table 4. Results of nitrogen fertilizer utilization rate of maize in each group detected by 15N tracer method

[0074] 6. Strain KY1695 has the functions of solubilizing silicon, potassium, and phosphorus. 6.1 Detection of the silicon-solubilizing, potassium-solubilizing, and phosphorus-solubilizing abilities of strain KY1695 6.1.1. Method (1) Detection of silicon solubility: KY1695 bacteria and 92068 control bacteria were inoculated on the same silicon solubility solid medium with an inoculation needle and incubated at 30℃ for 3 days. The presence of transparent zones was observed.

[0075] (2) Potassium solubilizing ability test: KY1695 bacteria and 92068 control bacteria were inoculated on the same potassium solubilizing solid medium with an inoculation needle and incubated at 30℃ for 3 days. The presence of a clear zone was observed.

[0076] (3) Phosphate solubilization capacity test: KY1695 bacteria and 92068 control bacteria were inoculated on the same inorganic phosphorus solid culture medium with an inoculation needle and incubated at 30℃ for 3 days. The presence of a transparent zone was observed.

[0077] 6.1.2. Test Results from Figure 4 As can be seen from A in the data, both control bacterium 92068 and strain KY1695 have potassium-solubilizing capabilities, but strain KY1695 exhibits significantly better potassium-solubilizing ability; from Figure 5 As can be seen from B, the control strain 92068 has no silicon-dissolving ability, while strain KY1695 has a certain silicon-dissolving ability; from Figure 5 As shown in Figure C, both control bacterium 92068 and strain KY1695 possess phosphorus-solubilizing capabilities, but strain KY1695 exhibits a more pronounced degradation zone, indicating that its phosphorus-solubilizing ability is slightly better than that of bacterium 92068. Therefore, whether it's potassium, silicon, or phosphorus solubilization, strain KY1695 demonstrates better degradation effects than control bacterium 92068, suggesting it is a more comprehensive multifunctional bacterium with superior soil nutrient activation capabilities.

[0078] 7. Conclusion This invention discovered a strain KY1695 that promotes nitrogen fertilizer absorption in plants. Sequence analysis of the 16SDNA of this strain revealed that the 1455 base sequence of this strain is consistent with that of the strain... Figure 5 With a similarity of 99.19%, strain KY1695 was identified as... Aeromonas encheleia Aeromonas encheleia The experimental results show that strain KY1695 significantly promotes nitrogen fertilizer absorption in plants, possesses the function of reducing fertilizer use and enhancing efficiency, and also has certain phosphorus, potassium, and silicon solubilizing functions, making it a more comprehensive multifunctional bacterium. When used in combination with chitosan oligosaccharide, it has a synergistic effect on promoting nitrogen fertilizer absorption in corn, and can be developed as a compound microbial fertilizer enhancer with broad development prospects and environmentally friendly characteristics.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Aeromonas hydrophila, characterized in that, The Aeromonas chencheleia strain mentioned is Aeromonas chencheleia strain KY1695, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 34412.

2. The Aeromonas hydrophila of claim 1, wherein the 16S rDNA sequence of strain KY1695 is shown in SEQ ID No.

1.

3. A microbial agent, characterized in that, The bacterial agent contains Aeromonas encheleia strain KY1695 as described in claim 1 or claim 2, or a culture of Aeromonas encheleia strain KY1695.

4. A fertilizer synergist, characterized in that, The fertilizer enhancer comprises the microbial agent as described in claim 3.

5. The fertilizer synergist according to claim 4, wherein the fertilizer synergist further comprises an oligosaccharide synergist, preferably, the oligosaccharide synergist comprises one or more of chitosan oligosaccharide, fucoidan oligosaccharide, chitin oligosaccharide and amino oligosaccharide.

6. The fertilizer synergist according to claim 5, wherein the oligosaccharide synergist is chitosan oligosaccharide.

7. Aeromonas hydrophila according to claim 1 or claim 2 ( Aeromonas encheleia Uses of strain KY1695, the microbial agent according to claim 3, or the fertilizer synergist according to any one of claims 4 to 6 in the following: (1) Promote the absorption and / or utilization of nitrogen fertilizer by plants; and / or (2) Promotes the solubility of phosphorus, potassium and / or silicon.

8. The use according to claim 7, characterized in that, The plants include crops; more preferably, the crops are one of corn, wheat, rice, peanuts and soybeans.

9. A method for promoting the absorption and / or utilization of nitrogen fertilizer by plants, characterized in that, The method includes using Aeromonas hydrophila as described in claim 1 or 2 (… Aeromonas encheleia The plant is treated with strain KY1695, the microbial agent according to claim 3, or the fertilizer synergist according to any one of claims 4 to 6.

10. The method according to claim 9, characterized in that, The treatment includes root irrigation and / or foliar spraying of plants with a liquid containing the Aeromonas hydrophila strain KY1695 of claim 1 or 2, the bacterial agent of claim 3, or the fertilizer synergist of any one of claims 4 to 6.

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