Enterobacter sp. for promoting germination of orchid seeds and application thereof

Abstract

The application discloses multiple strains of enterobacter for promoting seed germination of orchidaceae and application of the enterobacter, and the strain preservation numbers of the enterobacter are CCTCC No: M 2023255, CCTCC No: M 2023256 and CCTCC No: M 2023258 in sequence.The enterobacter of the application can not only promote seed germination through nitrogen fixation, phosphorus dissolution and IAA secretion, but also can comprehensively enhance the immune ability of a plant, improve the antibacterial and bacteriostatic ability of a wild orchid plant, promote absorption and utilization of useful substances by the plant, and further promote seed germination and differentiation of seedling adventitious buds, and can be used as a promoting agent for promoting efficient seed germination of orchidaceae and rapid growth of orchid tissue culture, so that large-scale propagation of orchidaceae, especially wild orchidaceae, can be realized, and the application has important practical significance for effectively protecting endangered wild orchid species.

Description

Technical Field

[0001] This invention relates to Enterobacteriaceae and their applications, specifically to multiple Enterobacteriaceae strains that promote the germination of orchid seeds and their applications in promoting orchid seed germination and tissue culture growth. Background Technology

[0002] Currently, the artificial tissue culture propagation of renowned orchid species has reached a significant market scale, but the endangered status of wild orchids has not improved. To protect the biodiversity of endangered orchid species, it is necessary to promote the propagation and conservation of species, including wild orchids. For wild orchids, in-situ propagation and conservation in the wild is typically required. This method cannot be achieved through artificial division techniques, or it is too costly and impractical, making it difficult to meet practical needs. Therefore, obtaining a method for in-situ propagation of orchids is crucial for effectively protecting the biodiversity of endangered orchid species.

[0003] Orchids, as typical mycorrhizal plants, are absolutely dependent on mycorrhizal fungi for seed germination and seedling growth under natural wild conditions. Therefore, the in-situ conservation of wild orchids and the effective protection of wild Cymbidium goeringii resources can be achieved by rapidly and efficiently obtaining more symbiotic fungi that promote orchid seed germination and seedling growth. However, different types of mycorrhizal fungi have varying effects on promoting germination and growth in orchids, and their mechanisms of action are not yet clear. Furthermore, the symbiotic germination mechanisms between different orchid species and their symbiotic bacteria vary greatly. Therefore, it is unknown whether these symbiotic bacteria promote germination of other orchid species, or whether they protect and promote the growth of critical seedlings after germination. This necessitates determining a suitable symbiotic system for each orchid species under study in practical applications, thus increasing the difficulty of orchid diversity conservation. Therefore, finding a suitable symbiotic system between orchid plants and symbiotic bacteria is of great significance for promoting the effective protection, in-situ propagation and conservation of orchid plants, especially wild orchid plants.

[0004] Currently, techniques for utilizing multiple symbiotic bacteria to effectively protect and propagate orchids, especially wild orchids, are still quite scarce, and there are no reports of using Enterobacteriaceae to promote orchid seed germination or seedling growth. Furthermore, the inventors' previous research revealed significant differences in the germination-promoting effects and seedling growth-promoting effects of Enterobacteriaceae from different species within the same genus, indicating that they are not effective in promoting seed germination or seedling growth. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide an Enterobacter bacillus that promotes the germination of orchid seeds and its application.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: An Enterobacterium that promotes seed germination in the Orchidaceae family; the Enterobacterium is classified as follows: Enterobacter ludwigii The specimen, designated HW6, has been deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC No: M 2023255.

[0007] The aforementioned Enterobacterium that promotes the germination of orchid seeds is further improved in that the Enterobacterium is obtained by isolation and purification from the roots of wild Cymbidium faberi.

[0008] An Enterobacterium that promotes seed germination in the Orchidaceae family; the Enterobacterium is classified as follows: Enterobacter asburiae The specimen, designated HT1, has been deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC No: M 2023256.

[0009] The aforementioned Enterobacterium that promotes the germination of orchid seeds is further improved in that the Enterobacterium is obtained by isolation and purification from the roots of wild Cymbidium faberi.

[0010] An Enterobacterium that promotes seed germination in the Orchidaceae family; the Enterobacterium is classified as follows: Enterobacter asburiae The specimen, designated CW2, has been deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC No: M 2023258.

[0011] The aforementioned Enterobacterium that promotes the germination of orchid seeds is further improved in that the Enterobacterium is obtained by isolation and purification from the roots of wild Cymbidium goeringii.

[0012] As a general technical concept, the present invention also provides a promoter comprising at least one of the above-described Enterobacteriaceae.

[0013] The aforementioned promoter is further improved by comprising a suspension of Enterobacter bacilli; wherein the concentration of Enterobacter bacilli in the suspension is 1 × 10⁻⁶. 8 CFU / mL ~ 1×10 10 CFU / mL.

[0014] The aforementioned promoter is further improved in that it is obtained by mixing any two types of *Enterobacter* bacterial suspensions in a volume ratio of 1:1; or by mixing HW6, HT1, and CW2 bacterial suspensions in a volume ratio of 1:1:1 to 2:1:1; and the concentration of *Enterobacter* in the *Enterobacter* bacterial suspension is 0.5 × 10⁻⁶. 9 CFU / mL ~1.5×10 9 CFU / mL.

[0015] As a general technical concept, the present invention also provides the application of the above-mentioned promoter in promoting the germination of orchid seeds or promoting the growth of orchid tissue cultures.

[0016] A further improvement to the above application, when using a promoter to promote the germination of orchid seeds, includes the following steps: mixing the promoter with orchid seeds and culturing the orchid seeds under the action of the promoter; wherein the orchid seeds are wild orchid seeds; and wherein the wild orchid seeds include wild Cymbidium goeringii seeds or wild Cymbidium faberi seeds.

[0017] The above-mentioned application, in a further improvement, involves using a promoter to accelerate the germination of orchid seeds, including the following steps: preparing orchid seeds into a seed suspension, and sowing the seed suspension onto a plate coated with a promoter for cultivation; the orchid seeds are wild orchid seeds; the wild orchid seeds include wild Cymbidium goeringii seeds or wild Cymbidium faberi seeds.

[0018] The above-mentioned application, in a further improvement, when using a promoter to promote the growth of orchid tissue culture, includes the following steps: mixing the promoter with the orchid tissue culture, and culturing the orchid tissue culture under the action of the promoter; wherein the orchid tissue culture is a wild orchid tissue culture; wherein the wild orchid tissue culture includes wild Cymbidium goeringii tissue culture or wild Cymbidium faberi tissue culture.

[0019] Compared with the prior art, the advantages of the present invention are as follows: To address the shortcomings of existing orchid symbiotic systems, such as long germination cycles and low germination potential and rates, this invention creatively provides multiple Enterobacteriaceae strains that promote orchid seed germination. The first Enterobacteriaceae strain is classified and named... Enterobacter ludwigii The first strain, code-named HW6, is deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC No: M 2023255. It is an endophytic bacterium of the orchid family, isolated and purified from the roots of wild Cymbidium faberi. The second strain of Enterobacter is classified as... Enterobacter asburiaeThe sample, code-named HT1, is deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC No.: M2023256. It is an endophytic bacterium of the orchid family, isolated and purified from the roots of wild Cymbidium faberi. The third Enterobacter strain is classified as follows: Enterobacter asburiae The invention, code-named CW2, is deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC No: M 2023258. It is an endophytic bacterium of the orchid family, isolated and purified from the roots of wild Cymbidium goeringii. Unlike existing conventional orchid endophytic bacteria, the Enterobacteriaceae (HW6, HT1, CW2) of this invention not only promote seed germination through nitrogen fixation, phosphorus solubilization, and IAA secretion, but also comprehensively enhance the plant's immunity by upregulating the expression of melibiose, L-asparagine, and syringic acid. This improves the antibacterial and bacteriostatic abilities of wild orchids, promotes the absorption and utilization of useful substances, and thus promotes seed germination and the differentiation of adventitious buds in seedlings. In practical applications, the Enterobacter strains of this invention can be used as promoters for orchid seed germination and orchid tissue culture growth. They not only promote efficient seed germination but also accelerate the growth of orchid tissue cultures. More importantly, when different types of Enterobacter strains are combined, interspecific signaling can induce adaptive regulation of colony structure, composition, and abundance, thereby altering the topological characteristics of the molecular ecological network of orchid rhizosphere microbiota. Specifically, this manifests as enhanced network connectivity and a stronger core population. Changes in the taxa (a type of bacteria) ultimately lead to co-evolutionary relationships among species, maintaining biodiversity while enhancing ecosystem stability. This not only encourages various Enterobacter strains to secrete more beneficial components for promoting germination and growth, significantly improving the germination effect on orchid seeds and the growth effect on orchid seedlings, but also significantly increases the nitrogen-fixing bacteria community in plants, which is beneficial for improving the fertility of the rhizosphere soil of orchids and further promoting the growth of orchids. Therefore, it is more conducive to the large-scale propagation of orchids, especially wild orchids, and has important practical significance for the in-situ conservation of endangered wild orchid species and even the industrial production of artificial breeding. Attached Figure Description

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0021] Figure 1 This is an evolutionary tree diagram of the Enterobacter strain in Example 1 of the present invention.

[0022] Figure 2 This is a comparison chart showing the differential expression levels of the metabolite melibiose in Cymbidium faberi (WH) inoculated with HW6 and Cymbidium faberi (AH) not inoculated with HW6 in Example 1 of the present invention.

[0023] Figure 3 This is an evolutionary tree diagram of the Enterobacter strain in Example 2 of the present invention.

[0024] Figure 4 This is a comparison chart showing the differential expression levels of the metabolite L-Asparagine in Cymbidium faberi (WH) inoculated with HT1 and Cymbidium faberi (AH) not inoculated with HT1 in Example 2 of the present invention.

[0025] Figure 5 This is an evolutionary tree diagram of the Enterobacter strain in Example 3 of the present invention.

[0026] Figure 6 This is a comparison chart showing the differential expression levels of the metabolite syringic acid in Cymbidium goeringii (WC) inoculated with CW2 and Cymbidium goeringii (AC) not inoculated with CW2 in Example 3 of the present invention.

[0027] Figure 7 This is a bar chart showing the LDA values ​​of microorganisms in the rhizosphere soil of Cymbidium goeringii inoculated with HW6, HT1, and CW2 in Example 7 of the present invention.

[0028] Figure 8 This is a bar chart showing the LDA values ​​of microorganisms in the rhizosphere soil of Cymbidium faberi inoculated with HW6 in Example 7 of the present invention.

[0029] Figure 9 This is a bar chart showing the LDA values ​​of microorganisms in the rhizosphere soil of Cymbidium faberi inoculated with HT1 in Example 7 of the present invention.

[0030] Figure 10 This is a bar chart showing the LDA values ​​of microorganisms in the rhizosphere soil of Cymbidium faberi inoculated with CW2 in Example 7 of the present invention. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0032] In the following embodiments, unless otherwise specified, the raw materials and instruments used are commercially available, the processes used are conventional processes, and the equipment used is conventional equipment.

[0033] Example 1: An Enterobacterium that promotes seed germination in orchids; this Enterobacterium is an endophytic bacterium of orchid plants, and is classified as... Enterobacter ludwigiiThe accession number, HW6, is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on March 6, 2023. The accession number is CCTCC No. M2023255.

[0034] The Enterobacterium that promotes orchid seed germination in the above embodiment is an endophytic bacterium of orchid plants obtained by isolating and purifying the roots of wild Cymbidium faberi.

[0035] The isolation, purification, and screening process of this Enterobacter strain was as follows: Fresh wild Cymbidium orchids were collected, and the roots were carefully washed with clean water and left to dry overnight at room temperature. Healthy, undamaged orchid root segments were cut. Random and uniform samples were taken, and the root segments were surface disinfected. Specifically, the root segments were first immersed in 124 mM disodium hydrogen phosphate buffer and sonicated for 1 min. After removal, they were placed in anhydrous ethanol for 1 min on a sterile operating table, then immersed in 6% sodium hypochlorite solution for 6 min, soaked in anhydrous ethanol for 30 s, then rinsed with sterile water for 30 s, then soaked in 2.5% sodium thiosulfate solution for 10 min, and then soaked in 10% NaHCO3 solution for 10 min to inhibit endophytic fungi. Finally, the surface was rinsed 3 times with sterile water to remove residual solution (the 6% sodium hypochlorite solution and the 10% NaHCO3 solution were prepared and used immediately). Take 200 μL of the final sterile water rinse solution and spread it on LB medium. Incubate at 30℃ for 27 h to verify surface disinfection. If no colonies grow, it proves that the surface disinfection is thorough. Air dry the cleaned orchid root segments in a sterile operating table. Cut the dried wild Cymbidium root segments into small segments about 1 cm long using scissors (heated to red-hot) in a sterile operating table. Place these segments evenly on water agar (WA) medium, seal with film, and label. Set up 5 replicates and incubate in a 30℃ constant temperature incubator. Observe the precipitation of bacteria in the isolation medium daily, select the more prominent endophytic bacterial colonies, and observe their morphological characteristics, including color, surface dryness / wetness, colony size, density, and edge morphology. Immediately transfer the precipitated bacteria to LB medium for streaking. After colony growth, transfer single colonies to new LB medium for further incubation. Repeat streaking 3-4 times until pure bacteria are obtained, completing the purification process. The results are shown in Table 1.

[0036] As shown in Table 1, a total of 3 dominant strains were selected, among which strain HW6 was milky white on water agar (WA) medium.

[0037] Table 1. Morphological statistics of dominant strains

[0038] Molecular identification was performed on strain HW6 selected from Table 1: (1) The PCR amplification products were sequenced using the sequencing platform of Shanghai Meiji Biopharmaceutical Technology Co., Ltd. 16S rDNA amplicon sequencing results showed that the 16S rRNA gene sequence of strain HW6 is as shown in SEQ ID NO.1 of the sequence listing, specifically: 5'--3'.

[0039] (2) The obtained sequences were compared on the NCBI website. Twenty pseudo-homologous bacterial species and one exogenous bacterial species (Escherichia coli was used in this study) were selected to construct an NJ phylogenetic tree in MEGA software. The phylogenetic tree was constructed using the neighbor-joining method in MEGA 5.1 software, as shown below. Figure 1 As shown. By Figure 1 It can be seen that strain HW6 and Enterobacter ludwigii strain EN119 (NR042349.1) It exhibits high homology. Therefore, it can be preliminarily inferred that strain HW6 belongs to... Enterobacter ludwigii (Enterobacterium). Therefore, strain HW6 was identified as Enterobacterium and named... Enterobacter ludwigii HW6 (NCBI comparison showed the highest similarity to 99%, which should be an unknown new species).

[0040] Biochemical characteristics of Enterobacter (HW6) were identified: (1) Activation of bacterial strain: 200µL of endophytic bacteria (HW6) was inoculated onto LB medium and cultured for 3 days.

[0041] (2) Strain purification: The activated endophytic bacteria were inoculated onto solid LB medium and liquid LB medium respectively and cultured for 3 days.

[0042] (3) IAA production detection: 100µL of endophytic bacteria were inoculated into LB liquid medium containing 500mg / L tryptophan and cultured at 30℃ and 170rpm for 48h. The supernatant was then centrifuged at 8000rpm and mixed with IAA colorimetric reagent at a volume ratio of 1:2. The mixture was reacted in the dark for 30min and the color change was observed. If the reaction solution turned pink, it proved that IAA was produced. The results are shown in Table 2.

[0043] (4) Nitrogen fixation capacity test: 50 µL of endophytic bacteria were inoculated in the center of Ashby medium using Oxford cups and cultured in a constant temperature incubator at 30℃ for 7 days. The growth status of endophytic bacteria was observed, and the results are shown in Table 2.

[0044] (5) Phosphate solubility test: 50 µL of endophytic bacteria were inoculated in the center of NBRIP medium using Oxford cups and cultured in a constant temperature incubator at 30℃ for 7 days. The results are shown in Table 2.

[0045] The results showed that strain HW6 grew well, with colonies exhibiting a clear tendency to spread outwards, indicating its nitrogen-fixing ability. Strain HW6 produced a distinct clear zone on NBRIP medium, demonstrating its phosphate-solubilizing ability. Strain HW6 also demonstrated the ability to produce IAA (isocyanate-associated amino acids).

[0046] Table 2. Biochemical characteristics analysis results of Enterobacter (HW6)

[0047] Effects of Enterobacter 6 (HW6) on plant endogenous hormones and metabolites (1) Correlation heatmap analysis and PCA principal component analysis were performed on the metabolites of Cymbidium faberi (WH) roots inoculated with HW6 and Cymbidium faberi (AH) roots not inoculated with HW6 to identify differentially enriched metabolic components among the samples.

[0048] The results showed a significant fold difference in metabolite expression between Cymbidium faberi (WH) inoculated with HW6 and Cymbidium faberi (AH) not inoculated with HH6, indicating that the presence of Enterobacter 6 (HW6) promotes efficient metabolite expression in the roots of wild Cymbidium faberi. Furthermore, HW6 inoculation resulted in substantial differences in the composition of the metabolites.

[0049] (2) All metabolites isolated by LC-MS technology were compared and analyzed in the KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ) database to obtain the data information and statistical results of these metabolites in the database. Figure 2 This is a comparison chart showing the differential expression levels of the metabolite melibiose between Cymbidium faberi (WH) inoculated with HW6 and Cymbidium faberi (AH) not inoculated with HW6 in Example 1 of this invention. Figure 2 It was found that melibiose was significantly upregulated in the white blood cell (WH) metabolite, indicating that Enterobacter 6 (HW6) can promote melibiose production in Cymbidium faberi. Furthermore, the high expression of melibiose, compared to the active white blood cell (AH) metabolite, may be a key mechanism by which endophytes promote the growth and development of orchid plants. Specifically, melibiose molecules can be directly absorbed into the plant's circulatory system and exert their effects, enhancing the plant's antibacterial and antimicrobial capabilities. Therefore, Enterobacter 6 (HW6) enhances the antibacterial and antimicrobial capabilities of wild orchids by promoting the synthesis of the melibiose metabolite, and can promote seed germination and adventitious bud differentiation in orchids.

[0050] Example 2: An Enterobacterium that promotes seed germination in orchids; this Enterobacterium is an endophytic bacterium of orchid plants, and is classified as... Enterobacter asburiae The specimen, designated HT1, is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, on March 6, 2023, with accession number CCTCCNo: M 2023256.

[0051] The Enterobacterium that promotes orchid seed germination in the above embodiment is an endophytic bacterium of orchid plants obtained by isolating and purifying the roots of wild Cymbidium faberi.

[0052] The isolation, purification, and screening process of this Enterobacter strain was as follows: Fresh wild Cymbidium orchids were collected, and the roots were carefully washed with clean water and left to dry overnight at room temperature. Healthy, undamaged orchid root segments were cut. Random and uniform samples were taken, and the root segments were surface disinfected. Specifically, the root segments were first immersed in 124 mM disodium hydrogen phosphate buffer and sonicated for 1 min. After removal, they were placed in anhydrous ethanol for 1 min on a sterile operating table, then immersed in 6% sodium hypochlorite solution for 6 min, soaked in anhydrous ethanol for 30 s, then rinsed with sterile water for 30 s, then soaked in 2.5% sodium thiosulfate solution for 10 min, and then soaked in 10% NaHCO3 solution for 10 min to inhibit endophytic fungi. Finally, the surface was rinsed 3 times with sterile water to remove residual solution (the 6% sodium hypochlorite solution and the 10% NaHCO3 solution were prepared and used immediately). Take 200 μL of the final sterile water rinse solution and spread it on LB medium. Incubate at 30℃ for 27 h to verify surface disinfection. If no colonies grow, it proves that the surface disinfection is thorough. Air dry the cleaned orchid root segments in a sterile operating table. Cut the dried wild Cymbidium root segments into small segments about 1 cm long using scissors (heated to red-hot) in a sterile operating table. Place these segments evenly on yeast extract agar (TWYE) medium, seal with film, and label. Set up 5 replicates and incubate in a 30℃ constant temperature incubator. Observe the precipitation of bacteria in the isolation medium daily, select the more prominent endophytic bacterial colonies, and observe their morphological characteristics, including color, surface dryness / wetness, colony size, density, and edge morphology. Immediately transfer the precipitated bacteria to LB medium for streaking. After colony growth, transfer single colonies to new LB medium for further incubation. Repeat streaking 3-4 times until pure bacteria are obtained, completing the purification process. The results are shown in Table 3.

[0053] As shown in Table 3, two dominant strains were selected, among which strain HT1 was milky white on yeast extract agar (TWYE) medium.

[0054] Table 3. Morphological statistics of dominant strains

[0055] Molecular identification was performed on strain HT1 selected from Table 3: (1) The PCR amplification products were sequenced using the sequencing platform of Shanghai Meiji Biopharmaceutical Technology Co., Ltd. The results showed that the 16S rRNA gene sequence of strain HT1 is as shown in SEQ ID NO.2 of the sequence listing, specifically: 5'--3'.

[0056] (2) The obtained sequences were compared on the NCBI website. Twenty pseudo-homologous bacterial species and one exogenous bacterial species (Escherichia coli was used in this study) were selected to construct an NJ phylogenetic tree in MEGA software. The phylogenetic tree was constructed using the neighbor-joining method in MEGA 5.1 software, as shown below. Figure 3 As shown. By Figure 3 It can be seen that strain HT1 and Enterobacter asburiae strain JCM6051 (NR024640.1)They exhibit high homology. Therefore, it can be preliminarily inferred that strain HT1 belongs to... Enterobacter asburiae (Enterobacterium). Therefore, strain HT1 was identified as Enterobacterium and named... Enterobacter asburiae HT1 (NCBI comparison showed the highest similarity at 99%, suggesting it is an unknown new species).

[0057] Biochemical characteristics of Enterobacter (HT1) were identified: (1) Activation of bacterial strain: 200µL of endophytic bacteria (HT1) was inoculated onto LB medium and cultured for 3 days.

[0058] (2) Strain purification: The activated endophytic bacteria were inoculated onto solid LB medium and liquid LB medium respectively and cultured for 3 days.

[0059] (3) IAA production detection: 100µL of endophytic bacteria were inoculated into LB liquid medium containing 500mg / L tryptophan and cultured at 30℃ and 170rpm for 48h. The supernatant was then centrifuged at 8000rpm and mixed with IAA colorimetric reagent at a volume ratio of 1:2. The mixture was reacted in the dark for 30min and the color change was observed. If the reaction solution turned pink, it proved that IAA was produced. The results are shown in Table 4.

[0060] (4) Nitrogen fixation capacity test: 50 µL of endophytic bacteria were inoculated in the center of Ashby medium using Oxford cups and cultured in a constant temperature incubator at 30℃ for 7 days. The growth status of endophytic bacteria was observed, and the results are shown in Table 4.

[0061] (5) Phosphate solubility test: 50 µL of endophytic bacteria were inoculated in the center of NBRIP medium using Oxford cups and cultured in a constant temperature incubator at 30℃ for 7 days. The results are shown in Table 4.

[0062] The results showed that strain HT1 grew well, with colonies exhibiting a clear tendency to spread outwards, indicating its nitrogen-fixing ability. Strain HT1 produced a distinct clear zone on NBRIP medium, demonstrating its phosphate-solubilizing ability. Strain HT1 also demonstrated the ability to produce IAA (isocyanate-associated amino acids).

[0063] Table 4. Biochemical characteristics analysis results of Enterobacteriaceae (HT1)

[0064] Effects of Enterobacteriaceae (HT1) on plant endogenous hormones and metabolites (1) Correlation heatmap analysis and PCA principal component analysis were performed on the metabolites of Cymbidium faberi (WH) roots inoculated with HT1 and Cymbidium faberi (AH) roots not inoculated with HT1 to discover the differentially enriched metabolic components among the samples.

[0065] The results showed a significant fold change in metabolite expression between AH and WH, indicating that the presence of Enterobacter 1 (HT1) promotes efficient metabolite expression in wild Cymbidium faberi roots. Furthermore, HT1 inoculation resulted in substantial differences in metabolite composition.

[0066] (2) All metabolites isolated by LC-MS technology were compared and analyzed in the KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ) database to obtain the data information and statistical results of these metabolites in the database. Figure 4 This is a comparison graph showing the differential expression levels of the metabolite L-asparagine in Cymbidium faberi (WH) inoculated with HT1 and Cymbidium faberi (AH) not inoculated with HT1 in Example 2 of this invention. Figure 4 It was found that L-Asparagine was significantly upregulated in the white blood cell (WH) assay, indicating that Enterobacter 1 (HT1) can promote L-Asparagine production in Cymbidium faberi. Furthermore, the high expression of the metabolite L-Asparagine, relative to the white blood cell (AH) assay, may be a key mechanism by which endophytes promote the growth and development of orchid plants. Specifically, L-Asparagine may inhibit the growth and development of Cymbidium faberi, the pathogen of bacterial wilt (Ralstonia solanaceae). Ralstoniasolanacearum QL-Rs1115 (Ralstonia solanacearum) has an inhibitory effect on beneficial bacteria Bacillus amyloliquefaciens T-5 (… Bacillus amylolique The growth of *Faciens T-5* is promoted, and L-asparagine also has a growth-promoting effect. Therefore, *Enterobacterium* (HT1) can promote the germination of wild orchid seeds and the germination and growth of seedlings by promoting the synthesis of the metabolite L-asparagine.

[0067] Example 3: An Enterobacterium that promotes the germination of Cymbidium goeringii seeds; this Enterobacterium is an endophytic bacterium of orchids and is classified as follows: Enterobacter asburiae The accession number, CW2, is deposited at the China Center for Type Culture Collection, located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on March 6, 2023, with accession number CCTCCNo: M 2023258.

[0068] The Enterobacterium that promotes the germination of Cymbidium goeringii seeds in the above embodiment is an endophytic bacterium of orchid plants obtained by isolating and purifying the roots of wild Cymbidium goeringii.

[0069] The isolation, purification, and screening process of this Enterobacter strain was as follows: Fresh wild Cymbidium goeringii was collected from a certain area in Hubei Province. The roots were carefully washed with clean water and left to dry overnight at room temperature. Healthy, undamaged orchid root segments were cut. Random and uniform samples were taken, and the root segments were surface disinfected. Specifically, the root segments were first immersed in 124 mM disodium hydrogen phosphate buffer and sonicated for 1 min. After being removed, they were placed in anhydrous ethanol for 1 min on a sterile operating table, then immersed in a 6% sodium hypochlorite solution for 6 min, soaked in anhydrous ethanol for 30 s, then rinsed with sterile water for 30 s, then soaked in a 2.5% sodium thiosulfate solution for 10 min, and then soaked in a 10% NaHCO3 solution for 10 min to inhibit endophytic fungi. Finally, the surface was rinsed three times with sterile water to remove any residual solution (the 6% sodium hypochlorite solution and the 10% NaHCO3 solution were prepared and used immediately). Take 200 μL of the final sterile water rinse solution and spread it on LB medium. Incubate at 30℃ for 27 h to verify surface disinfection. If no colonies grow, it proves that the surface disinfection is thorough. Air dry the cleaned orchid root segments in a sterile operating table. Cut the dried wild Cymbidium root segments into small segments about 1 cm long using scissors (heated to red-hot) in a sterile operating table. Place these segments evenly on water agar (WA) medium, seal with film, and label. Set up 5 replicates and incubate in a 30℃ constant temperature incubator. Observe the precipitation of bacteria in the isolation medium daily, select the more prominent endophytic bacterial colonies, and observe their morphological characteristics, including color, surface dryness / wetness, colony size, density, and edge morphology. Immediately transfer the precipitated bacteria to LB medium for streaking. After colony growth, transfer single colonies to new LB medium for further incubation. Repeat 3-4 times until pure bacteria are obtained, completing the purification process. The results are shown in Table 5.

[0070] As shown in Table 5, two dominant strains were selected, and strain CW2 was white on water agar (WA).

[0071] Table 5. Morphological statistics of dominant strains

[0072] Molecular identification was performed on strain CW2 selected from Table 5: (1) The PCR amplification products were sequenced using the sequencing platform of Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for 16S rDNA amplicon sequencing. The results showed that the 16S rRNA gene sequence of strain CW2 is as shown in SEQ ID NO.3 of the sequence listing, specifically: 5'--3'.

[0073] (2) The obtained sequences were compared on the NCBI website. Twenty pseudo-homologous bacterial species and one exogenous bacterial species (Escherichia coli was used in this study) were selected to construct an NJ phylogenetic tree in MEGA software. The phylogenetic tree was constructed using the neighbor-joining method in MEGA 5.1 software, as shown below. Figure 5 As shown. By Figure 5 It can be seen that strain CW2 and Enterobacter asburiae strainJCM6051(NR024640.1) and Enterobacter sp strain WL4 (MN540923.1) showed high homology. Therefore, it can be preliminarily inferred that strain CW2 belongs to... Enterobacter asburiae (Enterobacterium). Therefore, strain CW2 was identified as Enterobacterium and named... Enterobacter asburiae CW2 (NCBI comparison showed the highest similarity to 99%, suggesting it is an unknown new species).

[0074] Biochemical characteristics of Enterobacter (CW2) were identified: (1) Activation of bacterial strain: 200µL of endophytic bacteria (CW2) was inoculated onto LB medium and cultured for 3 days.

[0075] (2) Strain purification: The activated endophytic bacteria were inoculated onto solid LB medium and liquid LB medium, respectively, and cultured for 3 days. In this invention, the LB medium consisted of 10g peptone, 5g yeast extract, 10g glucose, 15g agar, 1000mL ultrapure water, pH 7.0±0.2, sterilized at 121℃ and 1×10⁵ Pa for 25min. Liquid culture was performed without agar.

[0076] (3) IAA production detection: 100µL of endophytic bacteria were inoculated into LB liquid medium containing 500mg / L tryptophan and cultured at 30℃ and 170rpm for 48h. The supernatant was then centrifuged at 8000rpm and mixed with IAA colorimetric reagent at a volume ratio of 1:2. The mixture was reacted in the dark for 30min and the color change was observed. If the reaction solution turned pink, it proved that IAA was produced. The results are shown in Table 6.

[0077] (4) Nitrogen fixation capacity test: 50 µL of endophytic bacteria were inoculated in the center of Ashby medium using Oxford cups and cultured in a constant temperature incubator at 30℃ for 7 days. The growth status of endophytic bacteria was observed, and the results are shown in Table 6.

[0078] (5) Phosphate solubility test: 50 µL of endophytic bacteria were inoculated in the center of NBRIP medium using Oxford cups and cultured in a constant temperature incubator at 30℃ for 7 days. The results are shown in Table 6.

[0079] The results showed that strain CW2 grew well, with colonies exhibiting a clear tendency to spread outwards, indicating its nitrogen-fixing ability. Strain CW2 produced a distinct clear zone on NBRIP medium, demonstrating its phosphate-solubilizing ability. Strain CW2 also demonstrated the ability to produce IAA (isocyanate-associated amino acids).

[0080] Table 6. Biochemical characteristics analysis results of Enterobacter (CW2)

[0081] Effects of Enterobacteriaceae (CW2) on plant endogenous hormones and metabolites (1) Correlation heatmap analysis and PCA principal component analysis were performed on the metabolites of Cymbidium goeringii (WC) roots inoculated with CW2 and Cymbidium goeringii (AC) roots not inoculated with CW2 to discover the differentially enriched metabolic components among the samples.

[0082] The results showed a significant fold change in metabolite expression between WC and AC, indicating that the presence of Enterobacter 2 (CW2) promotes efficient metabolite expression in the roots of wild Cymbidium faberi. Furthermore, inoculation with CW2 resulted in substantial differences in metabolite composition.

[0083] (2) All metabolites isolated by LC-MS technology were compared and analyzed in the KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ) database to obtain the data information and statistical results of these metabolites in the database. Figure 6 This is a comparison chart showing the differential expression levels of the metabolite syringic acid in Cymbidium goeringii (WC) inoculated with CW2 and Cymbidium goeringii (AC) not inoculated with CW2 in Example 3 of this invention. Figure 6 It was found that syringic acid was significantly upregulated in WC, indicating that Enterobacter cylindrica (CW2) can promote the production of syringic acid in Cymbidium faberi. Furthermore, compared to AC, the highly expressed metabolite syringic acid can inhibit bacterial biofilm formation. For example, syringic acid can significantly reduce the total number of bacteria in the soil around the plant roots, and it can significantly inhibit the potential for bacterial biofilm formation, which may be related to its antibacterial properties. Syringic acid can also enrich short-chain organic acid degrading bacteria. Therefore, Enterobacter cylindrica (CW2) can enhance the antibacterial ability of plants by promoting the synthesis of the metabolite syringic acid.

[0084] Example 4 An accelerator comprising Enterobacter (HW6) from Example 1.

[0085] In this embodiment, the concentration of Enterobacteriaceae in the promoter is 1×10⁻⁶. 8 CFU / mL ~ 1×10 10 CFU / mL, specifically 1×10 8 CFU / mL, 1×10 9 CFU / mL, 1×10 10 CFU / mL.

[0086] The above-mentioned method for preparing the accelerator includes the following steps: (1) The activated endophytic bacteria (HW6) were inoculated into liquid culture medium and cultured in the dark at 37°C and 180 rpm for 1 day.

[0087] (2) Add LB liquid culture medium to the liquid culture medium and adjust the concentration of Enterobacteriaceae in the bacterial suspension to 1×10⁻⁶. 8 CFU / mL, 1×10 9 CFU / mL, 1×10 10 CFU / mL was used to obtain different concentrations of Enterobacter fermentation broth, which is the promoter of this invention.

[0088] An accelerator comprising Enterobacter (HT1) from Example 2.

[0089] In this embodiment, the concentration of Enterobacter (HT1) in the promoter is 1×10⁻⁶. 8 CFU / mL ~ 1×10 10 CFU / mL, specifically 1×10 8 CFU / mL, 1×10 9 CFU / mL, 1×10 10 CFU / mL.

[0090] The above-mentioned method for preparing the accelerator includes the following steps: (1) The activated endophytic bacteria (HT1) were inoculated into liquid culture medium and cultured in the dark at 37°C and 180 rpm for 1 day.

[0091] (2) Add LB liquid culture medium to the liquid culture medium and adjust the concentration of Enterobacteriaceae in the bacterial suspension to 1×10⁻⁶. 8 CFU / mL, 1×10 9 CFU / mL, 1×10 10 CFU / mL was used to obtain different concentrations of Enterobacter fermentation broth, which is the promoter of this invention.

[0092] An accelerator comprising Enterobacter (CW2) from Example 3.

[0093] In this embodiment, the concentration of Enterobacter (CW2) in the promoter is 1×10⁻⁶. 8 CFU / mL ~ 1×10 10 CFU / mL, specifically 1×10 8 CFU / mL, 1×10 9 CFU / mL, 1×10 10 CFU / mL.

[0094] The above-mentioned method for preparing the accelerator includes the following steps: (1) The activated endophytic bacteria (CW2) were inoculated into liquid culture medium and cultured in the dark at 37°C and 180 rpm for 1 day.

[0095] (2) Add LB liquid culture medium to the liquid culture medium and adjust the concentration of Enterobacteriaceae in the bacterial suspension to 1×10⁻⁶. 8 CFU / mL, 1×10 9 CFU / mL, 1×10 10 CFU / mL was used to obtain different concentrations of Enterobacter fermentation broth, which is the promoter of this invention.

[0096] Example 5 The application of a promoter in orchid seed germination specifically involves using Enterobacteriaceae (HW6, HT1, CW2) prepared in Example 4 at a concentration of 1×10⁻⁶. 9 The fermentation broth at CFU / mL was used as a promoter to cultivate Cymbidium faberi or Cymbidium goeringii seeds, including the following steps: The concentration of Enterobacteriaceae was 1×10⁻⁶. 9 Fermentation broths of Enterobacteriaceae (HW1, HW2, HW6, HT1, HT2) at CFU / mL were spread onto culture plates, with four sterile filter paper strips (1*3cm) placed on each plate. The control (CK) was left uninoculated. The pods were cleaned with detergent, rinsed with water, and diseased areas were removed before being placed in a clean bench. After sterilization with 75% ethanol solution for 30 seconds, the plates were transferred to 5% sodium hypochlorite solution for 30 minutes, followed by rinsing 3-4 times with sterile water to ensure complete removal of the disinfectant solution. The pods were cut open with a sterile scalpel on sterile filter paper, and the Cymbidium seeds were scattered into 1 / 2 MS medium supplemented with exogenous hormones to prepare a seed suspension. The seed suspension was then evenly sown onto the plates containing bacteria and sterile filter paper strips, as well as the control plate. Each treatment was repeated three times. The plates were incubated in the dark in a sterile room for 120 days, and the dynamics of seed germination were observed.

[0097] The concentration of Enterobacteriaceae was 1×10⁻⁶. 9CFU / mL Enterobacter (CW1, CW2) fermentation broth was spread onto culture plates, with four sterile filter paper strips (1*3cm) placed on each plate. The control (CK) was left uninoculated. The pods were cleaned with detergent, rinsed with water, and diseased areas were removed before being placed in a laminar flow hood. The plates were disinfected with 75% ethanol solution for 30 seconds, then transferred to 5% sodium hypochlorite solution for 30 minutes. The plates were rinsed 3-4 times with sterile water to ensure complete removal of the disinfectant solution. The pods were cut open with a sterile scalpel on sterile filter paper, and the Cymbidium goeringii seeds were scattered into 1 / 2 MS medium supplemented with exogenous hormones to prepare a seed suspension. The seed suspension was then evenly sown onto the plates containing bacteria and sterile filter paper strips, as well as the control plate. Each treatment was repeated three times. The plates were incubated in the dark in a sterile room for 120 days, and the germination dynamics were observed.

[0098] For each treatment of orchid plants, seeds of equal mass were collected and replicated three times. The appearance of an embryo was used as a germination signal, and germination breaking through the seed coat was observed in the treatment groups on days 30-40. Germination potential and germination rate were observed daily, and average values ​​were taken. The results are shown in Tables 7 and 8.

[0099] Germination rate GP (%) = Number of germinated seeds at the end of the germination test / Total number of seeds tested * 100%.

[0100] Germination potential (GV) (%) = Number of seeds germinated at the beginning of the germination test / Total number of seeds tested * 100%.

[0101] As shown in Table 7, the germination potential and germination rate of Cymbidium faberi seeds in the experimental groups inoculated with Enterobacteriaceae (HW6, HT1) were significantly higher than those in the control group (CK), increasing by 54% and 61%, and 38% and 59%, respectively. This indicates that the Enterobacteriaceae (HW6, HT1) of this invention can effectively promote the germination of Cymbidium faberi seeds. Meanwhile, as shown in Table 7, apart from Enterobacteriaceae (HW6, HT1), other Enterobacteriaceae (HW1, HW2, HT2) could not effectively promote the germination of Cymbidium faberi seeds, still exhibiting poor germination-promoting effects, even worse than the control group (CK). Therefore, it is evident that the Enterobacteriaceae (HW6, HT1) of this invention can effectively promote the rapid and efficient germination of Cymbidium faberi seeds.

[0102] Table 7. Effects of different Enterobacteriaceae (HW1, HW2, HW6, HT1, HT2) on the germination potential and germination rate of Cymbidium faberi seeds.

[0103] Table 8 shows that the germination potential and germination rate of Cymbidium goeringii seeds in the experimental group inoculated with Enterobacter (CW2) were significantly higher than those in the control group, increasing by 13% and 36%, respectively. This indicates that the Enterobacter (CW2) of this invention can effectively promote the germination of Cymbidium goeringii seeds. Meanwhile, as shown in Table 8, apart from Enterobacter (CW2), other Enterobacter species (CW1) did not effectively promote the germination of Cymbidium goeringii seeds, still exhibiting poor germination-promoting effects, even worse than the control group (CK). Therefore, it is evident that the Enterobacter (CW2) of this invention can effectively promote the rapid and efficient germination of Cymbidium goeringii seeds.

[0104] Table 8. Effects of different Enterobacteriaceae (CW1, CW2) on the germination potential and germination rate of Cymbidium goeringii seeds.

[0105] Example 6 The application of a growth promoter in promoting the growth of orchid tissue cultures specifically involves using the Enterobacter fermentation broth from Example 4 as a growth promoter to cultivate Cymbidium faberi or Cymbidium goeringii tissue cultures under the action of the growth promoter, including the following steps: The subcultured Cymbidium faberi tissue culture (a mixture of aseptically sown seeds that germinate into rhizomes and adventitious buds, referred to as Cymbidium faberi tissue culture) was weighed fresh using an electronic balance (balance accuracy 0.0001 g) and transferred to a conventional rhizome differentiation induction medium with an Enterobacter concentration of 1×10⁻⁶. 9 CFU / mL of Enterobacter fermentation broth (HW1, HW2, HW6, HT1, HT2) was used as a promoter and added to the culture medium. The culture was then placed in an artificial climate chamber at 25°C, 70%–75% relative humidity, 14 hours of light per day, and a light intensity of 4000 lux. The tissue cultures were harvested after 35 days. During the culture period, the degree of bud differentiation was assessed every 7 days.

[0106] The subcultured Cymbidium goeringii tissue culture (a mixture of aseptically sown seeds that germinate into rhizomes and adventitious buds, referred to as Cymbidium goeringii tissue culture) was weighed fresh using an electronic balance (balance accuracy 0.0001g) and transferred to a conventional rhizome differentiation induction medium with an Enterobacter concentration of 1×10⁻⁶. 9 CFU / mL of Enterobacter (CW1, CW2) fermentation broth was used as a promoter and added to the culture medium. The culture was then placed in an artificial climate chamber at 25°C, 70%–75% relative humidity, 14 hours of light per day, and 4000 lux light intensity. The tissue culture was harvested after 35 days. During the culture period, the degree of bud differentiation of the tissue culture was statistically analyzed every 7 days.

[0107] After harvesting, the fresh weight of the tissue culture was measured using an electronic balance. One-way ANOVA was then performed on the increase in fresh weight of the tissue culture in each treatment group, and multiple comparisons were conducted. The results are shown in Tables 9 to 12.

[0108] As shown in Table 9, the growth of *Cymbidium faberi* tissue culture increased slowly and steadily during the culture process, and the growth of *Cymbidium faberi* tissue culture in the culture medium treated with *Enterobacterium* (HW6, HT1) was significantly higher than that in the control group. Table 9 also shows that, apart from *Enterobacterium* (HW6, HT1), other *Enterobacterium* strains (HW1, HW2, HT2) could not effectively promote the growth of *Cymbidium faberi* tissue culture, exhibiting poor growth-promoting effects, even worse than the control group (CK). Therefore, the *Enterobacterium* strains (HW6, HT1) of this invention can effectively promote the rapid growth of *Cymbidium faberi* tissue culture.

[0109] Table 9. Effects of different Enterobacteriaceae (HW1, HW2, HW6, HT1, HT2) on the fresh weight of Cymbidium faberi tissue culture.

[0110] As shown in Table 10, during the cultivation process, adventitious buds continuously differentiated from the rhizomes of each Cymbidium faberi tissue culture. In the treatment with the addition of Enterobacterium fermentation broth (HW6, HT1), the Cymbidium faberi tissue culture was bright green, with a large number of robust adventitious buds and rapid growth; the control Cymbidium faberi tissue culture was pale green, with fewer adventitious buds and slower growth. Furthermore, Table 10 shows that, apart from Enterobacterium faberi (HW6, HT1), other Enterobacterium species (HW1, HW2, HT2) could not effectively promote the differentiation of adventitious buds in Cymbidium faberi, and were even worse than the control group.

[0111] Table 10 Effects of different Enterobacteriaceae (HW1, HW2, HW6, HT1, HT2) on the germination potential and germination rate of adventitious buds in Cymbidium faberi.

[0112] As shown in Table 11, the growth of *Cymbidium goeringii* tissue culture increased slowly but steadily during the cultivation process, and the growth of *Cymbidium goeringii* tissue culture in the culture medium under the action of *Enterobacterium* (CW2) was significantly higher than that of the control group. Table 11 also shows that, apart from *Enterobacterium* (CW2), other *Enterobacterium* strains (CW1) did not effectively promote the growth of *Cymbidium goeringii* tissue culture, still exhibiting poor growth-promoting effects, even worse than the control group (CK). Therefore, it is evident that the *Enterobacterium* (CW2) of this invention can effectively promote the rapid growth of *Cymbidium goeringii* tissue culture.

[0113] Table 11 Effects of different Enterobacteriaceae (CW1, CW2) on the fresh weight of Cymbidium goeringii tissue culture

[0114] As shown in Table 12, during the cultivation process, adventitious buds continuously differentiated from the rhizomes of each Cymbidium goeringii tissue culture. In the treatment with the addition of Enterobacterium (CW2) fermentation broth, the Cymbidium goeringii tissue culture was bright green, with a large number of robust adventitious buds and rapid growth; the control Cymbidium goeringii tissue culture was pale green, with fewer adventitious buds and slower growth. Furthermore, Table 12 shows that, apart from Enterobacterium (CW2), other Enterobacteriaceae (CW1) did not effectively promote the differentiation of adventitious buds in Cymbidium goeringii, and were even worse than the control group.

[0115] Table 12 Effects of different Enterobacteriaceae (CW1, CW2) on the differentiation, germination potential, and germination rate of adventitious buds in Cymbidium goeringii.

[0116] In this embodiment, the growth-promoting effect of a growth promoter obtained by mixing fermentation broths of three Enterobacteriaceae on Cymbidium faberi tissue culture was also investigated. Except for the different growth promoters, the other conditions were the same as in Example 6, and the structures are shown in Table 13.

[0117] In Table 13, the promoter was prepared by mixing equal volumes of the fermentation broths of various Enterobacteriaceae, and the concentration of Enterobacteriaceae in each fermentation broth was 1×10⁻⁶. 9 CFU / mL. As shown in Table 13, the CFU / mL of Cymbidium faberi tissue culture was significantly increased after treatment with the promoter obtained by combining HW6+HT1+CW2, indicating that the promoter obtained by combining HW6+HT1+CW2 is more effective in promoting the growth of orchid tissue culture.

[0118] Table 13 shows the effect of a growth promoter obtained by mixing fermentation broths of three Enterobacteriaceae on the fresh weight of Cymbidium faberi tissue culture.

[0119] Example 7 This study investigated the effects of single-strain growth promoters (HW6), (HT1), and (CW2) prepared in Example 4, as well as a growth promoter obtained by mixing equal volumes of fermentation broths from three Enterobacteriaceae (HW6, HT1, and CW2), on the rhizosphere microbiota of Cymbidium faberi. Specifically, the tissue culture obtained after the tissue culture experiment in Example 6 was used as potted plants. The soil consisted of red soil, substrate soil, and vermiculite, mixed in a 3:5:2 ratio, and sterilized under high pressure and heat before use. A conventional temperature-controlled, humidity-controlled pot experiment was conducted. After 60 days, rhizosphere soil was collected for 16S rDNA high-throughput sequencing. The resulting microbiota underwent OTU cluster analysis and species annotation using the NR database. Subsequently, LEfSe multi-level species difference discriminant analysis was performed: the non-parametric factorial Kruskal-Wallis (KW) sum-rank test was used to detect microbiota with significant abundance differences; the taxonomic level was selected from Phylum to Genus; the multi-group comparison strategy was one against all; and the abundance was normalized. The significantly enriched microbial species in each inoculant treatment group were screened using the discriminant value (LDA>3), and the results are as follows: Figure 7-10 As shown.

[0120] Figure 7 This is a bar chart showing the LDA values ​​of microorganisms in the rhizosphere soil of Cymbidium goeringii inoculated with HW6, HT1, and CW2 in Example 7 of the present invention.

[0121] Figure 8 This is a bar chart showing the LDA values ​​of microorganisms in the rhizosphere soil of Cymbidium faberi inoculated with HW6 in Example 7 of the present invention.

[0122] Figure 9 This is a bar chart showing the LDA values ​​of microorganisms in the rhizosphere soil of Cymbidium faberi inoculated with HT1 in Example 7 of the present invention.

[0123] Figure 10 This is a bar chart showing the LDA values ​​of microorganisms in the rhizosphere soil of Cymbidium faberi inoculated with CW2 in Example 7 of the present invention.

[0124] contrast Figure 7-10 The results show that, compared with inoculation with a single strain, the inoculation of three Enterobacteriaceae (HW6, HT1 and CW2) in this invention significantly enriches the nitrogen-fixing bacterial community, while effectively regulating the community structure, composition and abundance, and changing the topological characteristics of the molecular ecological network of orchid rhizosphere microbiota. Specifically, this is manifested in enhanced network connectivity, changes in the keystone taxa, and ultimately the formation of species co-evolutionary relationships, which can maintain biodiversity while also improving ecosystem stability.

[0125] In summary, unlike existing conventional orchid endophytic bacteria, the *Enterobacterium* strain of this invention not only promotes seed germination through nitrogen fixation, phosphorus solubilization, and IAA secretion, but also comprehensively enhances the plant's immunity by upregulating the expression of melibiose, L-asparagine, and syringic acid. This improves the antibacterial and bacteriostatic abilities of wild orchids, promotes the absorption and utilization of useful substances, and thus promotes seed germination and the differentiation of adventitious buds in seedlings. In practical applications, the *Enterobacterium* strain of this invention can be used as a promoter for orchid seed germination and orchid tissue culture growth. It not only promotes efficient seed germination but also accelerates the growth of orchid tissue cultures. More importantly, when different *Enterobacterium* strains are combined, interspecific signal transduction can induce adaptive regulation of colony structure, composition, and abundance, thereby altering the topological characteristics of the molecular ecological network of orchid rhizosphere microbiota. Specifically, this manifests as enhanced network connectivity and the emergence of keystone species. Changes in the taxa (a type of bacteria) ultimately lead to co-evolutionary relationships among species, maintaining biodiversity while enhancing ecosystem stability. This not only encourages various Enterobacter strains to secrete more beneficial components for promoting germination and growth, significantly improving the germination effect on orchid seeds and the growth effect on orchid seedlings, but also significantly increases the nitrogen-fixing bacteria community in plants, which is beneficial for improving the fertility of the rhizosphere soil of orchids and further promoting the growth of orchids. Therefore, it is more conducive to the large-scale propagation of orchids, especially wild orchids, and has important practical significance for the in-situ conservation of endangered wild orchid species and even the industrial production of artificial breeding.

[0126] The above embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. An Enterobacterium that promotes the germination of orchid seeds, characterized by, The Enterobacteriaceae are Enterobacteriaceae ( Enterobacter ludwigii HW6, Enterobacteriaceae ( Enterobacter asburiae HT1 or Enterobacteriaceae ( Enterobacter asburiae CW2; the Enterobacteriaceae ( Enterobacter ludwigii HW6, has been deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC No: M 2023255; the Enterobacter ( Enterobacter asburiae HT1, has been deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC No: M 2023256; the Enterobacter (HT1) Enterobacter asburiae CW2 has been deposited at the China Center for Type Culture Collection on March 6, 2023, with accession number CCTCCNo: M 2023258.

2. An accelerator, characterized in that, The promoter comprises at least one Enterobacter as described in claim 1.

3. The accelerator according to claim 2, characterized in that, The promoter is the Enterobacter suspension according to claim 1; the concentration of Enterobacter in the Enterobacter suspension is 1×10⁻⁶. 8 CFU / mL ~ 1×10 10 CFU / mL.

4. The accelerator according to claim 3, characterized in that, The promoter is obtained by mixing any two strains of *Enterobacter*, with a volume ratio of 1:1; or, the promoter is obtained by mixing strains of HW6, HT1, and CW2, with a volume ratio of 1:1:1; the concentration of *Enterobacter* in the *Enterobacter* suspension is 0.5 × 10⁻⁶. 9 CFU / mL ~1.5×10 9 CFU / mL.

5. The application of the promoter as described in any one of claims 2 to 4 in promoting the germination of orchid seeds or promoting the growth of orchid tissue cultures; wherein the orchid seeds are wild orchid seeds; the wild orchid seeds include wild Cymbidium goeringii seeds or wild Cymbidium faberi seeds; the orchid tissue cultures are wild orchid tissue cultures; the wild orchid tissue cultures include wild Cymbidium goeringii tissue cultures or wild Cymbidium faberi tissue cultures.

6. The application according to claim 5, characterized in that, When using a germination promoter to promote the germination of orchid seeds, the following steps are included: mixing the germination promoter with the orchid seeds and culturing the orchid seeds under the action of the germination promoter.

7. The application according to claim 5, characterized in that, When using a germination promoter to promote the germination of orchid seeds, the following steps are included: preparing orchid seeds into a seed suspension, and sowing the seed suspension onto a plate coated with the germination promoter for cultivation.

8. The application according to claim 5, characterized in that, When using a growth promoter to promote the growth of orchid tissue culture, the following steps are included: mixing the growth promoter with the orchid tissue culture, and culturing the orchid tissue culture under the action of the growth promoter.

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