Strain, culture product, plant growth promoter and application

Abstract

The invention discloses a strain, a culture product, a plant growth promoter and application. The invention provides a new strain of Sandarinobacter, which is preserved in the China Center for Type Culture Collection on March 18, 2024, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M 2024453. Experimental research shows that the strain has stable indoleacetic acid synthesis capability and can decompose an ethylene precursor ACC to relieve plant stress, so that the strain has the effect of promoting plant growth. In a specific embodiment, the strain has the effect of promoting rice growth, and not only can improve the development degree of rice root systems, but also can improve the rice spike length and the grain number per spike. Therefore, the new strain or the culture product (such as bacterial suspension, culture supernatant and thallus crushing matter) provided by the invention has the prospect of being developed into a medicament for promoting plant growth.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and relates to the discovery and application of new strains, specifically to a strain, culture product, plant growth promoter and its application. Background Technology

[0002] Increased grain production and the development of green agriculture are core demands of agricultural production. The application of highly efficient growth-promoting microorganisms is a key way to improve crop yields and reduce reliance on chemical fertilizers. Plant growth-promoting bacteria (PGPB), as a green agricultural input, have shown great potential in increasing crop yields, enhancing plant stress resistance, and improving soil structure. However, most of the PGPBs reported so far originate from soil, rhizosphere, or single environments, with limited functions and poor stability in complex environments. Traditional isolation methods are inefficient and prone to missing unknown potential microbial resources. In particular, there is a lack of development of dedicated, highly efficient growth-promoting strains for rice. Existing microbial agents generally suffer from unstable growth-promoting effects and limited adaptability.

[0003] Algae-bacteria symbiosis (ABS) has attracted much attention due to its unique nutrient cycle and microecological stability, and is widely used in agricultural wastewater treatment and nutrient recovery. Bacteria in ABS can secrete vitamins, IAA, and other substances to form a stable symbiotic system with microalgae, providing a high-quality resource pool for screening functional strains. However, current research mainly focuses on the purification capacity and energy cycling mechanism of this system in wastewater treatment, and the plant growth-promoting potential of symbiotic bacteria has not been explored in depth. Although my country has abundant microbial resources, research on identifying rice growth-promoting strains from ABS is limited, especially... Sandaracinobacter The growth-promoting properties of this genus of strains have not been systematically studied, and there are no reports of their application in promoting plant growth.

[0004] Based on Sandaracinobacter The discovery of this strain led to the invention of this invention. Summary of the Invention

[0005] The first objective of this invention is to provide a novel bacterial strain, the second objective is to provide a culture product of the strain, the third objective is to provide a plant growth promoter based on the strain or the culture product, and the fourth objective is to provide the application of the strain or the culture product in the preparation of a plant growth promoter.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: A bacterial strain, which is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2024453.

[0007] Application of the above-mentioned strains in the preparation of agents that promote plant growth.

[0008] In one specific embodiment, the plant is rice.

[0009] A culture product obtained by culturing the above-mentioned strain.

[0010] The culture product is selected from one or more of bacterial suspension, culture supernatant and bacterial cell fragments.

[0011] The application of the above-mentioned culture products in the preparation of agents that promote plant growth.

[0012] In one specific embodiment, the plant is rice.

[0013] A plant growth promoter, wherein the active ingredient of the plant growth promoter is the aforementioned strain or culture product.

[0014] Preferably, it also includes a carrier of the active ingredient.

[0015] More preferably, the carrier is a solid, liquid, or semi-solid. Beneficial effects

[0016] This invention provides Sandaracinobacter This is a novel bacterial strain, deposited on March 11, 2024, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024453. Experimental studies have shown that this strain possesses a stable ability to synthesize indoleacetic acid (IAA) and can decompose the ethylene precursor ACC to alleviate plant stress; therefore, this strain has a plant growth-promoting effect. In one specific embodiment, this strain promotes rice growth, not only enhancing root development but also increasing panicle length and the number of grains per panicle. Therefore, this novel bacterial strain or its culture products (such as bacterial suspension, culture supernatant, and bacterial cell fragments) provided by this invention have the potential to be developed into a plant growth-promoting agent. Attached Figure Description

[0017] Figure 1 This is a photograph of the colony morphology of the strain in Example 1 of this application.

[0018] Figure 2 This is a microscopic image of the morphological characteristics of strain T42 in Example 1 of this application under an electron microscope.

[0019] Figure 3 This is a phenotypic comparison of rice treated with strain T42 and the control group under hydroponic conditions for 40 days in Example 1 of this application; where a is the plant phenotype, b is the plant height, and c is the number of tillers per plant.

[0020] Figure 4 This is a comparison diagram of the ear phenotypes of the T42 strain treatment group and the control group under hydroponic conditions in Example 1 of this application; where a is the ear phenotype, b is the ear length, and c is the number of grains per ear.

[0021] Figure 5 This is a comparison of the phenotypes of rice treated with strain T42 and the control group under pot conditions for 80 days in Example 1 of this application; where a is the plant phenotype and b is the plant height.

[0022] Figure 6 This is a comparison of the ear phenotypes of the T42 strain treatment group and the control group under pot cultivation conditions in Example 1 of this application; where a is the ear phenotype, b is the ear length, c is the number of primary branches, and d is the number of grains per ear. Detailed Implementation

[0023] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the specific content of the embodiments. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0024] Example 1: Isolation, Screening and Identification of Strains I. Experimental Materials 1. Culture medium R2A medium: yeast extract 0.5g, tryptone 0.5g, casein hydrolysate 0.5g, glucose 0.5g, soluble starch 0.5g, dipotassium hydrogen phosphate (… Add 0.3g of anhydrous magnesium sulfate, 0.024g of sodium pyruvate, and bring the volume to 1L with tap water.

[0025] Soybean meal culture medium: 200mL of 5% soybean meal supernatant, 0.5g glucose, 0.5g soluble starch, 0.3g sodium pyruvate, 0.024g anhydrous magnesium sulfate, 0.3g dipotassium hydrogen phosphate, and tap water to a final volume of 1L.

[0026] 2. Main reagents Kimura B culture medium was purchased from phytoene.

[0027] Hoagland culture medium was purchased from Xiqingchun Company.

[0028] Salkowski reagent was purchased from phytoene.

[0029] 3. Main Instruments Incubator (202-3AB, Tianjin Test Instruments); AIRTECH Clean Bench Microscope (BX51M, Olympus); Electrophoresis apparatus (DYY-6C, Beijing Liuyi Instruments); PCR instrument (Qiagen); Full-temperature shaking incubator (Shanghai Zhichu Instruments).

[0030] II. Experimental Methods and Results 1. Isolation and screening of strains Water samples were collected from the ABS (microalgae-bacterial symbiotic system) wastewater system in pig farms and transported at 4°C in sterile sampling bottles. The bacterial strains were isolated using the serial dilution plate method, with the following steps: 10 mL of the water sample was placed in 90 mL of sterile water and shaken at 180 rpm for 30 min. Diluent, serially diluted to Take 100 μL of bacterial suspension at each dilution and spread it evenly on R2A agar plates. Incubate at 30°C for 72 h. Pick a single orange-yellow, round, raised colony and streak it three times on R2A agar to obtain a pure culture strain, named T42. Inoculate this strain into R2A liquid medium and incubate at 30°C with shaking at 180 rpm for 48 h. Mix with an equal volume of 50% glycerol (final concentration 25%) and freeze at -80°C for later use.

[0031] 2. Identification of the morphological characteristics of the strain Strain T42 was inoculated onto R2A medium plates and incubated at 30°C for 72 hours. Colony characteristics were observed: colonies were orange-yellow, round, smooth, moist, raised, and had neat edges. Figure 1 Fresh colonies were picked and Gram-stained. Optical microscopy revealed the bacteria to be Gram-negative, while electron microscopy showed them to be short rod-shaped (size: 1.2-2.0 μm long, 0.5-0.8 μm wide). Figure 2 ).

[0032] 3. Determination of the culture characteristics of the strain 3.1 Effects of different soybean meal concentrations on the growth of bacterial strains (1) Preparation of culture medium 1 g / L soybean meal culture medium: Take 20 mL of 5% soybean meal supernatant (containing 1 g of soybean meal), add 80 mL of tap water, and supplement with 0.05 g of glucose, 0.05 g of soluble starch, 0.03 g of sodium pyruvate, 0.0024 g of anhydrous magnesium sulfate, and 0.03 g of disodium hydrogen phosphate. Sterilize and use for later use.

[0033] 2.5 g / L soybean meal culture medium: Take 50 mL of 5% soybean meal supernatant (containing 2.5 g of soybean meal), and use the same components and amounts as "1 g / L soybean meal culture medium". Sterilize and use for later use.

[0034] (2) Test methods The bacterial suspension of T42 was inoculated into 1 g / L soybean meal powder medium and 2.5 g / L soybean meal powder medium at an inoculation rate of 1% (v / v), and cultured on a shaker at 30℃ and 180 rpm. The color change of the medium was observed daily (orange was the growth indicator of the strain), and the time when the orange color appeared was recorded.

[0035] (3) Results In a 1 g / L soybean meal medium, a distinct orange color was observed after 2 days of culture; in a 2.5 g / L soybean meal medium, a distinct orange color was observed after 3 days of culture, indicating that a 1 g / L soybean meal concentration is more suitable for the rapid proliferation of strain T42.

[0036] 3.2 Effects of soluble starch and sodium pyruvate on the growth of the strain (1) Culture medium preparation: Based on 1 g / L soybean meal powder culture medium, 4 treatment groups were set up: Control group (full composition): Contains glucose, soluble starch, and sodium pyruvate; Starch-deficient group: Contains no soluble starch; other components are the same as the control group. Sodium pyruvate deficient group: Contains no sodium pyruvate, other components are the same as the control group; Double deficiency group: It contains neither soluble starch nor sodium pyruvate, and the other components are the same as the control group.

[0037] (2) Test methods Each group was inoculated with T42 bacteria (1%, v / v) and incubated at 37℃ and 180 rpm. The time of orange appearance and the intensity of the color were recorded (using visual scoring: +++ for dark orange, ++ for medium orange, and + for light orange).

[0038] (3) Results The control group showed a deep orange color (+++) after 2 days of culture; both the starch-deficient and sodium pyruvate-deficient groups showed a medium orange color (++) after 3 days; and the double-deficient group showed a light orange color (+) after 3 days. This indicates that soluble starch and sodium pyruvate can synergistically promote the growth of T42 bacteria.

[0039] 3.3 Synergistic effect of glucose and soluble starch and environmental adaptability (1) Effect of soluble starch concentration Prepare a 1 g / L soybean meal culture medium (without glucose), add 0.05 g (final concentration 0.5 g / L) and 0.1 g (final concentration 1 g / L) of soluble starch respectively, inoculate with T42 bacteria and incubate at 37℃ and 180 rpm for 6 days, and observe the orange color change.

[0040] Experimental results: After 2 days of culture, both groups were medium orange (++); after 6 days of culture, the 0.5 g / L starch group was medium orange (++) and the 1 g / L starch group was dark orange (+++), indicating that higher concentrations of soluble starch are beneficial to the long-term growth of the strain.

[0041] (2) Effects of glucose and aseptic conditions Prepare a 1 g / L soybean meal culture medium (containing 0.1 g glucose + 0.1 g soluble starch), and divide it into two groups: ① aseptic inoculation in a laminar flow hood; ② non-aseptic inoculation outside a laminar flow hood. Both groups were incubated at 37°C and 180 rpm for 2 days.

[0042] Experimental results: Both groups showed a deep orange color (+++) after 2 days, with no significant difference in color; and the orange color of the group was deeper than that of the control group without glucose, indicating that glucose can promote the rapid growth of the strain, and that strain T42 has strong tolerance to contamination by other bacteria and good environmental adaptability.

[0043] 4. Molecular biological identification Genomic DNA was extracted from strain T42, and the 16S rRNA gene sequence was amplified using universal primers. Sequencing results were then compared with those from the NCBI database using BLAST. Sandaracinobacter neustonicus The similarity of PAMC28131 was 98.66%; however, further whole-genome sequencing using the Illumina sequencing platform, followed by assembly to obtain the complete genome sequence, and average nucleotide identity (ANI) analysis, showed that it was similar to known... Sandaracinobacter The species' ANI value was below 83%; DNA-DNA hybridization index (DDH) analysis showed that the DDH values ​​of all known species in the same genus were below 35%, meeting the criteria for defining a new species. Genome annotation using the HMMSCA tool revealed several unique metabolic gene clusters; a phylogenetic tree was constructed using MEGA 11 software based on the 16S rRNA gene sequence and the whole genome sequence, showing that strain T42 formed a separate branch, supporting its classification as a new species. Sandaracinobacter It is a new species.

[0044] Those skilled in the art know that the ANI value is the gold standard for defining new species in prokaryotes. The ANI threshold for the same species in prokaryotes is ≥95-96%, while different species within the same genus typically have an ANI <90%. Here, <83% is far below the threshold, and the DDH value supplements it. A value below 70% supports the classification of different species, directly supporting T42 as... Sandaracinobacter This is a new species. Furthermore, a phylogenetic tree was constructed based on dual data sources: 16S rRNA gene (a conserved single gene) and whole genome sequence (whole genome level, higher resolution). T42 formed a separate independent branch. Compared with single gene analysis, the results are more reliable and convincing, which is also the mainstream method for phylogenetic analysis of new species. In addition, the unique metabolic gene clusters annotated by HMMSCA not only supplemented the functional characteristics of the new species, but also provided metabolic evidence for the rationality of its taxonomic status, which meets the research requirements of polymorphic taxonomy.

[0045] The above-mentioned T42 strain ( Sandaracinobacter alcalinohabitumT42 was deposited on March 11, 2024, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024453, located at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The 16S rRNA sequence and genome sequence of T42 have been submitted to GenBank, with access numbers PX695393 and LC901161, respectively.

[0046] Example 2: Detection of functional characteristics of the strain 1. IAA (indoleacetic acid) secretion detection Experimental methods: Strain T42 was inoculated into R2A liquid medium and cultured at 30℃ and 180 rpm for 16 h with shaking. 2 mL of fermentation broth was centrifuged at 8000 rpm for 10 min. The supernatant was mixed with an equal volume of Salkowski reagent and allowed to stand in the dark for 30 min. The concentration was detected by spectrophotometer (wavelength 530 nm) and calculated using the IAA standard curve. Three replicates were performed.

[0047] Experimental results: The fermentation broth of strain T42 was pink in color, and the IAA secretion amount was 108.07±9.04 ng / mL, indicating that it has a stable auxin synthesis ability.

[0048] 2. ACC deaminase activity detection Experimental method: The strain T42 was inoculated into a medium with ACC as the sole nitrogen source and cultured at 30℃ and 180 rpm for 72 h with shaking. The growth was observed. The basal medium without ACC was used as a control.

[0049] Experimental results: Strain T42 grew well on ACC medium, OD 600 The value reached 0.85±0.06, indicating that it possesses ACC deaminase activity and can decompose the ethylene precursor ACC to alleviate plant stress.

[0050] Example 3: Plant growth promoting effect of the strain (hydroponics) I. Experimental Materials and Methods Preparation of T42 bacterial suspension: T42 was cultured in R2A medium for 30 minutes. o C. Shake incubator to OD 600 ≈1.

[0051] The rice variety used was Nipponbare. Plump and uniformly sized seeds were selected, disinfected with 75% ethanol for 15 minutes, rinsed 5 times with sterile water, soaked and germinated until they showed white tips, and then transferred to 1 / 2 Kimura B nutrient solution for seedling cultivation. Treatment was carried out 7 days later.

[0052] The experiment was divided into two groups: the T42 treatment group (30 mL OD was inoculated into 20 L 1 / 2 Hoagland culture medium).600 =1.0 T42 bacterial suspension, control group (20L 1 / 2 Hoagland culture medium inoculated with an equal volume of sterile R2A medium), each group was repeated 3 times, and cultured at 30℃ under 16h light / 8h dark conditions until maturity.

[0053] II. Test Results During the vegetative growth stage (40 days of treatment), Figure 3 The plant height and number of tillers in the T42 treatment group were significantly higher than those in the control group (plant height: 463.27 ± 24.65 mm vs. 424.18 ± 32.92 mm, number of tillers: 4.73 ± 0.65 vs. 3.82 ± 0.98). At maturity ( Figure 4 The T42 treatment plants showed an increase in panicle length of approximately 30% (171.7 ± 24.75 mm vs. 143.2 ± 22.64 mm) and an increase in the number of grains per panicle of approximately 50% (approximately 65.55 grains per panicle) compared to the control group.

[0054] Example 4: Plant growth promoting effect of the strain (potted plants) I. Experimental Materials and Methods Preparation of pre-cultured bacterial solution: T42 was cultured in R2A medium for 30 minutes. o C. Shake incubator to OD 600 ≈1.

[0055] Farmland soil was selected and placed in flowerpots. Three Nipponbare seedlings that had been hydroponically cultured for 7 days were planted in each pot. Two groups were set up: T42 treatment group (30 mL of pre-cultured bacterial solution was mixed in 7 kg of soil) and control group (7 kg of soil was mixed with an equal amount of sterile R2A culture solution). No fertilizer was applied throughout the process. Each group was replicated 3 times and cultured under natural conditions until maturity.

[0056] II. Test Results Post-harvest growth indicators were measured: the plant height in the T42 treatment group was 792.75±46.085 mm, significantly higher than that in the control group (722.818±84.586 mm). Figure 5 The T42 treatment group showed an increase of approximately 30% in ear length and one more primary branch compared to the control group; the T42 treatment group had 58.458±18.209 grains per ear, significantly higher than the control group (38.895±14.282). Figure 6 ).

[0057] In summary: This invention provides SandaracinobacterThis is a novel bacterial strain, deposited on March 11, 2024, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024453. Experimental studies have shown that this strain possesses a stable ability to synthesize indoleacetic acid (IAA) and can decompose the ethylene precursor ACC to alleviate plant stress; therefore, this strain has a plant growth-promoting effect. In one specific embodiment, this strain promotes rice growth, not only enhancing root development but also increasing panicle length and the number of grains per panicle. Therefore, this novel bacterial strain or its culture products (such as bacterial suspension, culture supernatant, and bacterial cell fragments) provided by this invention have the potential to be developed into a plant growth-promoting agent.

[0058] The technical solutions provided in this application have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A strain, characterized in that: This strain is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCCNO: M 2024453.

2. The use of the strain according to claim 1 in the preparation of a plant growth promoter.

3. The application according to claim 2, characterized in that: The plant in question is rice.

4. A culture product, characterized in that: It is obtained by culturing the strain described in claim 1.

5. The culture product according to claim 4, characterized in that: The culture product is selected from one or more of bacterial suspension, culture supernatant and bacterial cell fragments.

6. The use of the culture product according to claim 4 or 5 in the preparation of a plant growth promoter.

7. The application according to claim 6, characterized in that: The plant in question is rice.

8. A plant growth promoter, characterized in that: The active ingredient of this plant growth promoter is the strain described in claim 1 or the culture product described in any one of claims 4 to 5.

9. The plant growth promoter according to claim 8, characterized in that: It also includes a carrier for the active ingredient.

10. The plant growth promoter according to claim 9, characterized in that: The carrier can be solid, liquid, or semi-solid.

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