A *Pseudomonas putida* species that promotes seed germination in Cymbidium goeringii and its applications

Abstract

This invention discloses a *Pseudomonas putida* strain that promotes seed germination in *Cymbidium goeringii* and its applications. The strain's preservation number is CCTCC No: M 2023259. This *Pseudomonas putida* strain not only promotes seed germination through phosphorus solubilization and IAA production, but also promotes coenzyme A synthesis and enhances alkaloid synthesis in *Cymbidium goeringii* by upregulating the expression of dextropanthenol, nicotinamide, and their mediated coenzyme A and alkaloid synthesis pathways, thereby promoting seed germination and seedling growth. In practical applications, this *Pseudomonas putida* strain can be used as a promoter for *Cymbidium goeringii* seed germination and tissue culture growth, facilitating efficient seed germination and rapid growth of tissue cultures. This is beneficial for promoting the co-germination and propagation of *Cymbidium goeringii*, especially wild species, and has significant practical implications for the in-situ conservation of endangered wild orchid species and even the industrial-scale artificial propagation of these species.

Description

Technical Field

[0001] This invention relates to a strain of *Pseudomonas putida*, specifically to a strain of *Pseudomonas putida* that promotes the germination of *Cymbidium goeringii* seeds and its application in promoting the germination of *Cymbidium goeringii* seeds and the growth of tissue cultures. Background Technology

[0002] Conservation of biodiversity in the wild and endangered Orchidaceae family has been a key focus of ecological and environmental work globally. Despite the established market for rapid propagation of renowned Orchid species through tissue culture, the endangered status of wild Orchids has not improved. To date, Orchids remain a globally protected species, with the entire family listed in China's "National Key Protected Wild Plants List" and also included in the protection scope of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).

[0003] The main reasons for the endangerment of wild orchids are twofold. First, as typical mycorrhizal plants, orchids rely heavily on mycorrhizal fungi for seed germination and seedling growth under natural conditions. In some mature orchids, the mycorrhizal symbiotic system still provides nutrients for normal plant growth and promotes flowering and fruiting. It can be said that from "head to toe," from insect pollination to mycorrhizal symbiosis, a biological symbiotic relationship is evident. Over-reliance on the mycorrhizal symbiotic system has brought great difficulties to species breeding and resource conservation. Second, different genera and species of orchids have significant differences in the maturity time of their capsules and the conditions for seed germination. Different species of orchids also have significant differences in their growth habits and adaptability to growth environment conditions. This makes it difficult to extend the germination-promoting technology for specific orchid varieties to other varieties. In other words, the existing symbiotic germination fungi do not have broad applicability.

[0004] Cymbidium goeringii, also known as Spring Orchid, is highly prized for its elegant flowers and leaves, rich fragrance, and long-standing Chinese orchid culture, earning it the reputation of "the most fragrant orchid in the world." Its high ornamental and economic value has led to rampant poaching of wild Cymbidium goeringii, coupled with habitat destruction and ecological degradation, resulting in the rapid depletion of its germplasm resources. This poses a serious threat to the continuation of wild populations and the protection of genetic diversity. However, effective technologies for the protection and in-situ propagation of Cymbidium goeringii, especially wild varieties, remain scarce. Therefore, to achieve the protection of wild Cymbidium goeringii resources, rapidly and efficiently obtaining more symbiotic fungi that promote seed germination is key to solving the problem of in-situ conservation of this plant.

[0005] Meanwhile, different types of mycorrhizal fungi have different mechanisms for promoting germination and growth in orchids. Current technologies for exploring symbiotic fungi in Cymbidium goeringii mainly focus on orchid mycorrhizal fungi (OMF) and non-mycorrhizal fungi (NMF), with less attention paid to endophytic bacteria that can also form symbiotic relationships with orchid seeds. Even when bacteria are considered, the focus is primarily on the diversity of microbial communities under non-culturable conditions, with few cases of directly utilizing endophytic bacteria under culturable conditions for efficient germination promotion. However, in actual industrial production, bacteria have advantages over fungi, such as easier fermentation, better control, and shorter culture cycles, playing a crucial role in improving host nutrient absorption, growth and development, and disease resistance. Therefore, developing symbiotic bacteria for Cymbidium goeringii seed germination is necessary.

[0006] Furthermore, it is generally accepted in bacterial research that bacteria of different species within the same genus exhibit significant spatial heterogeneity and differences in metabolic activity after colonizing a host (animal or plant), leading to different host-bacterial interaction effects. Therefore, even bacteria of different species within the same genus do not provide a reference for the interaction effects on other bacterial species and plant hosts. Although studies on *Pseudomonas* sp. have found that it can serve as an endophyte in soil or plants, possessing advantages such as strong colonization and reproduction capabilities within plants or soil environments; rapid growth suitable for large-scale fermentation production; rapid utilization of seed and root exudates; synthesis of beneficial secondary metabolites, such as antibiotics, siderophores, and other growth-promoting substances; and strong adaptability to environmental stress, the use of *Pseudomonas putida*, a different species within the same genus, to promote the germination and growth of orchids such as *Cymbidium goeringii* remains unknown. Meanwhile, in the inventors' previous research, it was found that *Pseudomonas putida*, belonging to different species within the same genus, exhibits significant differences in its effects on promoting the germination of *Cymbidium goeringii* seeds and the growth of *Cymbidium goeringii* seedlings. It is not effective in promoting seed germination, nor is it particularly effective in promoting the growth of *Cymbidium goeringii* seedlings. Therefore, obtaining a *Pseudomonas* bacterium capable of promoting efficient germination and rapid growth of *Cymbidium goeringii* is of great significance for the in-situ conservation of endangered wild orchid species and even for the industrial-scale artificial propagation of these species. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a *Pseudomonas putida* strain that promotes the germination of Cymbidium goeringii seeds and its application. This *Pseudomonas putida* strain can not only promote seed germination by phosphate solubilization and secretion of IAA, but also promote the synthesis of coenzyme A and the ability of Cymbidium goeringii to synthesize alkaloids by upregulating the expression of dexpanthenol, nicotinamide and their mediated coenzyme A and alkaloid synthesis pathways, thereby promoting the germination of Cymbidium goeringii seeds and the growth of seedlings.

[0008] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0009] A *Pseudomonas putida* strain that promotes seed germination in Cymbidium goeringii, classified as *Pseudomonas putida* (CH4), has been deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC No: M 2023259.

[0010] The above-mentioned *Pseudomonas putida* that promotes the germination of Cymbidium goeringii seeds is further improved in that the *Pseudomonas putida* is obtained by isolating and purifying the roots of wild Cymbidium goeringii.

[0011] As a general technical concept, the present invention also provides a promoter comprising the aforementioned *Pseudomonas putida*.

[0012] The aforementioned promoter is further improved in that the concentration of *Pseudomonas putida* in the promoter is 1 × 10⁻⁶. 8 CFU / mL ~ 1×10 10 CFU / mL.

[0013] In a further improvement of the aforementioned promoter, the concentration of *Pseudomonas putida* in the promoter is 0.5 × 10⁻⁶. 9 CFU / mL ~1.5×10 9 CFU / mL.

[0014] As a general technical concept, the present invention also provides the application of the above-mentioned promoter in promoting the germination of Cymbidium goeringii seeds.

[0015] The above application, further improved, includes the following steps: mixing the promoter with Cymbidium goeringii seeds, and culturing the Cymbidium goeringii seeds under the action of the promoter; wherein the Cymbidium goeringii seeds are wild Cymbidium goeringii seeds.

[0016] The above application, further improved, includes the following steps: preparing Cymbidium goeringii seeds into a seed suspension, and sowing the seed suspension onto a plate coated with a growth promoter for cultivation; wherein the Cymbidium goeringii seeds are wild Cymbidium goeringii seeds.

[0017] As a general technical concept, the present invention also provides the application of the above-mentioned promoter in promoting the growth of Cymbidium goeringii tissue culture.

[0018] The above application, further improved, includes the following steps: mixing the promoter with Cymbidium goeringii tissue culture, and culturing the Cymbidium goeringii tissue culture under the action of the promoter; wherein the Cymbidium goeringii tissue culture is wild Cymbidium goeringii tissue culture.

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

[0020] To address the shortcomings of existing orchid symbiotic systems, such as long germination cycles and low germination potential and rates, this invention creatively provides a *Pseudomonas putida* strain that promotes seed germination in *Cymbidium goeringii*. This strain, classified as *Pseudomonas putida* (CH4), is deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC No. M 2023259. It is an orchid endophytic bacterium isolated and purified from the roots of wild *Cymbidium goeringii*. Unlike existing conventional orchid endophytic bacteria, this *Pseudomonas putida* strain not only promotes seed germination through phosphate solubilization and IAA secretion, but also promotes coenzyme A synthesis and enhances alkaloid synthesis in *Cymbidium goeringii* by upregulating the expression of dextropanthenol, nicotinamide, and their mediated coenzyme A and alkaloid synthesis pathways, thereby promoting seed germination and seedling growth. In practical applications, the *Pseudomonas putida* strain of the present invention can be used as a promoter for the germination of Cymbidium goeringii seeds and the growth of Cymbidium goeringii tissue culture. It can not only promote the efficient germination of Cymbidium goeringii seeds, but also promote the rapid growth of Cymbidium goeringii tissue culture. Therefore, it can realize the large-scale propagation of Cymbidium goeringii, especially wild Cymbidium goeringii. This is of great significance for the in-situ conservation of endangered wild orchid species, and even the industrial production of artificial breeding. Attached Figure Description

[0021] 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.

[0022] Figure 1 This is a colony morphology diagram of the *Pseudomonas putida* strain in Example 1 of the present invention on vitamin B humic acid agar (HV) medium.

[0023] Figure 2 This is an evolutionary tree diagram of the *Pseudomonas putida* strain in Example 1 of the present invention.

[0024] Figure 3 This is a diagram showing the growth of *Pseudomonas putida* on NBRIP medium in Example 1 of the present invention.

[0025] Figure 4 This is a volcano diagram showing the significantly upregulated metabolites in Cymbidium goeringii (WC) inoculated with CH4 and Cymbidium goeringii (AC) not inoculated with CH4 in Example 1 of the present invention.

[0026] Figure 5 This is a volcano diagram showing the significantly downregulated metabolites in Cymbidium goeringii (WC) inoculated with CH4 and Cymbidium goeringii (AC) not inoculated with CH4 in Example 1 of the present invention.

[0027] Figure 6 This is a volcano diagram showing no significant difference in metabolites between Cymbidium goeringii (WC) inoculated with CH4 and Cymbidium goeringii (AC) not inoculated with CH4 in Example 1 of the present invention.

[0028] Figure 7 This is a principal component analysis diagram of metabolites from Cymbidium goeringii (WC) inoculated with CH4 and Cymbidium goeringii (AC) not inoculated with CH4 in Example 1 of the present invention.

[0029] Figure 8 This is a comparison chart showing the differential expression levels of the metabolites dexpantanol and nicotinamide between Cymbidium goeringii (WC) inoculated with CH4 and Cymbidium goeringii (AC) not inoculated with CH4 in Example 1 of the present invention. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] Example 1:

[0033] A strain of *Pseudomonas putida* that promotes seed germination in Cymbidium goeringii, classified as an endophytic bacterium of orchids, with the accession number CH4, has been 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 CCTCC No: M 2023259.

[0034] The *Pseudomonas putida* that promotes seed germination in the above embodiment is an endophytic bacterium of the orchid family obtained by isolating and purifying the roots of wild *Cymbidium goeringii*.

[0035] The isolation, purification, and screening process of this *Pseudomonas putida* strain was as follows: Fresh wild Cymbidium goeringii was collected from a certain location 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 removal, 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 fresh for use). 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 goeringii 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 vitamin B humic acid agar (HV) 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. Figure 1 As shown.

[0036] As shown in Table 1, a total of 4 dominant strains were selected. Meanwhile, from... Figure 1 It can be seen that the color of strain CH4 on vitamin B humic acid agar (HV) medium is off-white.

[0037] Table 1. Morphological statistics of dominant strains

[0038] strain number Thickness, size Dry / wet, loose / dense color surface edge transparency CH1 thin, small Dry, dense yellow folds irregular opaque CH2 thick, big wet, dense cream smooth tidy opaque CH3 thin, small wet, dense White smooth tidy opaque CH4 thin, small wet, dense off-white smooth tidy opaque

[0039] Molecular identification was performed on strain CH4 selected from Table 1:

[0040] (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 CH4 was as shown in SEQ ID NO.1 of the sequence listing, specifically:

[0041]

[0042] (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. A phylogenetic tree was then constructed using the neighbor-joining method in MEGA 5.1 software, as shown below. Figure 2 As shown. By Figure 2 It can be seen that strain CH4 has a high degree of homology with Pseudomonas putida strain AA7 (KX817279.1). Therefore, it can be preliminarily inferred that strain CH4 belongs to Pseudomonas putida. Thus, strain CH4 is identified as Pseudomonas putida and named Pseudomonas putida CH4.

[0043] Biochemical characteristics of *Pseudomonas putida* (CH4) were identified:

[0044] (1) Activation of bacterial strain: 200 μL of endophytic bacteria (CH4) was inoculated onto LB medium and cultured for 3 days.

[0045] (2) Strain purification: The activated endophytic bacteria were inoculated onto solid LB medium and liquid LB medium respectively and cultured for 3 days.

[0046] (3) IAA production detection: 100 μL of endophytic bacteria were inoculated into LB liquid medium containing 500 mg / L tryptophan and cultured at 30℃ and 170 rpm for 48 h. The supernatant was then centrifuged at 8000 rpm and mixed with IAA colorimetric reagent at a volume ratio of 1:2. The mixture was reacted in the dark for 30 min 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.

[0047] (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.

[0048] (5) Phosphate-solubilizing capacity test: 50 μL of endophytic bacteria was inoculated into the center of NBRIP medium using an Oxford cup and cultured at 30℃ for 7 days. The presence or absence of a clear zone around the endophytic bacteria was observed. The results are shown in Table 2 and... Figure 3 As shown.

[0049] As shown in Table 2, strain CH4 did not show any obvious growth signs, indicating that strain CH4 does not have nitrogen-fixing ability.

[0050] From Table 2 and Figure 3It can be seen that strain CH4 produced a clear zone on NBRIP medium, indicating that strain CH4 has phosphate-solubilizing ability.

[0051] As shown in Table 2, strain CH4 has the ability to produce IAA.

[0052] Table 2. Biochemical characteristics analysis results of *Pseudomonas putida* (CH4).

[0053] strain number Nitrogen fixation Phosphorus dissolution Produced by IAA CH4 - + +

[0054] Effects of *Pseudomonas putida* (CH4) on plant endogenous hormones and metabolites

[0055] (1) Correlation heatmap analysis and principal component analysis (PCA) were performed on the metabolites of Cymbidium goeringii roots inoculated with CH4 (WC) and those not inoculated with CH4 (AC) to identify differentially enriched metabolic components among the samples. Results Figure 4 and Figure 5 As shown.

[0056] Figure 4 This is a volcano diagram showing the significantly upregulated metabolites in Cymbidium goeringii (WC) inoculated with CH4 and Cymbidium goeringii (AC) not inoculated with CH4 in Example 1 of the present invention. Figure 5 This is a volcano diagram showing the significantly downregulated metabolites in Cymbidium goeringii (WC) inoculated with CH4 and Cymbidium goeringii (AC) not inoculated with CH4 in Example 1 of the present invention. Figure 6 This is a volcano diagram showing no significant difference in metabolites between Cymbidium goeringii (WC) inoculated with CH4 and Cymbidium goeringii (AC) not inoculated with CH4 in Example 1 of this invention. Figure 4-6 It can be seen that the fold difference in the expression of metabolites between Cymbidium goeringii (WC) and Cymbidium goeringii (AC) is quite significant, which indicates that the presence of Pseudomonas putida (CH4) promotes the high expression of metabolites in the roots of Cymbidium goeringii.

[0057] Figure 7 This is a principal component analysis diagram of metabolites from Cymbidium goeringii (WC) inoculated with CH4 and Cymbidium goeringii (AC) not inoculated with CH4, as shown in Example 1 of this invention. Figure 7 It can be seen that *Pseudomonas putida* (CH4) produces a significant difference in the composition of metabolites of *Cymbidium goeringii*.

[0058] (2) All metabolites isolated using LC-MS technology were compared and analyzed in the KEGG (Kyoto Encyclopedia of Genes and Genomes, http: / / www.genome.jp / kegg / ) database to obtain information and statistical results of these metabolites in the database. Figure 8This is a comparison chart showing the differential expression levels of the metabolites dexpantanol and nicotinamide between CH4-inoculated Cymbidium goeringii (WC) and uninoculated Cymbidium goeringii (AC) in Example 1 of this invention. Figure 8 It was found that dextropanthenol and nicotinamide were significantly upregulated in Cymbidium goeringii (WC), indicating that *Pseudomonas putida* (CH4) can promote the expression of dextropanthenol and nicotinamide in *Cymbidium goeringii*. Meanwhile, the high expression of the metabolites dextropanthenol and nicotinamide compared to *Cymbidium goeringii* (AC) is related to the growth-promoting mechanism of *Pseudomonas putida* (CH4) on orchids. Specifically, dextropanthenol is a precursor for the synthesis of coenzyme A (CoA) via the pantothenic acid pathway. After entering the body, it can be converted into pantothenic acid, which is then used to synthesize coenzyme A. Coenzyme A promotes the metabolism of proteins, fats, and carbohydrates. Furthermore, coenzyme A is an important compound that carries and transports acyl groups in organisms, and its derivatives, such as acetyl-CoA, are important components in the metabolism of proteins, fats, and carbohydrates. In the synthesis and metabolism of various biomolecules, including the tricarboxylic acid cycle (TCA) and fatty acid oxidation, nicotinamide plays a crucial role in the energy and material cycling of organisms. Nicotinamide, also known as vitamin B3, is a widely distributed vitamin and cofactor in plants. As a cofactor, nicotinamide is known to participate in the synthesis of up to 18 enzymes. In plants, it participates in the nicotinic acid-to-alkaloid pathway, playing an important protective role against oxidative stress. Studies have shown that nicotinic acid compounds have similar functions to plant hormones such as salicylic acid, activating systemic acquired resistance (SAR) and inducing the production of SAR molecular characteristic proteins, resulting in broad-spectrum antibacterial activity against pathogens. Therefore, *Pseudomonas putida* (CH4) can upregulate the expression of dextropanthenol, nicotinamide, and their mediated coenzyme A and alkaloid synthesis pathways, thereby promoting the germination and seedling growth of wild orchids by enhancing plant coenzyme A synthesis and alkaloid synthesis.

[0059] In addition, the plant height and root length of Cymbidium goeringii inoculated with CH4 (WC) and those not inoculated with CH4 (AC) were statistically analyzed, and the results are shown in Table 3. Table 3 shows that the growth of Cymbidium goeringii inoculated with CH4 (WC) was significantly better than that of those not inoculated with CH4 (AC). Furthermore, in terms of plant height, the average plant height of WC was 59 cm, significantly higher than that of AC. Moreover, the average root length of WC was also significantly longer than that of AC. In addition, the root surface of WC was brownish-yellow, while the root surface of AC was whitish.

[0060] Table 3. Statistics on plant height and root length of Cymbidium goeringii (WC, AC)

[0061]

[0062] Example 2

[0063] An accelerator comprising *Pseudomonas putida* (CH4) from Example 1.

[0064] In this embodiment, the concentration of *Pseudomonas putida* in the accelerator 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.

[0065] The above-mentioned method for preparing the accelerator includes the following steps:

[0066] (1) The activated endophytic bacteria (CH4) were inoculated into liquid culture medium and cultured in the dark at 37°C and 180 rpm for 1 day.

[0067] (2) Add LB liquid culture to the liquid culture medium and adjust the concentration of *Pseudomonas putida* 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 fermentation broths of different concentrations of *Pseudomonas putida*, which are the promoters of this invention.

[0068] The application of the above-mentioned promoter in the germination of Cymbidium goeringii seeds specifically involves using Pseudomonas putida at a concentration of 1×10⁻⁶. 9 The fermentation broth at CFU / mL was used as a promoter to cultivate Cymbidium goeringii seeds, including the following steps:

[0069] The concentration of *Pseudomonas putida* was 1 × 10⁻⁶. 9CFU / mL of *Pseudomonas putida* fermentation broth was spread onto culture plates, and four sterile filter paper strips (1*3 cm) were placed on each plate. The control (CK) was not inoculated. The pods were cleaned with detergent, rinsed with water, and diseased areas were removed before being placed in a clean bench. 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 the disinfectant solution was completely removed. The pods were cut open with a sterile scalpel on sterile filter paper, and the 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.

[0070] 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 day 30. Germination potential and germination rate were observed daily, and the average values ​​were taken. The results are shown in Table 4.

[0071] Germination rate (GP) (%) = Number of germinated seeds at the end of the germination test / Total number of seeds tested * 100%.

[0072] Germination potential (GV) (%) = Number of seeds germinated at the beginning of the germination test / Total number of seeds tested * 100%.

[0073] As shown in Table 4, the germination potential and germination rate of Cymbidium goeringii seeds in the experimental group inoculated with Pseudomonas malodorans (CH4) were significantly higher than those in the CK group, increasing by 24% and 34% respectively. This indicates that Pseudomonas malodorans (CH4) of the present invention can effectively promote the germination of Cymbidium goeringii seeds.

[0074] Table 4. Effects of *Pseudomonas putida* (CH4) on the germination potential and germination rate of *Cymbidium goeringii* seeds.

[0075] serial number Germination potential (GV) (%) Germination rate (GP) (%) CH4 59 89 CK 35 55

[0076] In this embodiment, the effects of different *Pseudomonas putida* strains (CH1, CH2, CH3, CH4) on the germination potential and germination rate of *Cymbidium goeringii* seeds were also investigated. Except for the different *Pseudomonas putida* strains, the other conditions were the same as in Example 2, wherein the concentration of *Pseudomonas putida* in the fermentation broth was 1 × 10⁻⁶. 9 CFU / mL, the results are shown in Table 5.

[0077] As shown in Table 5, except for *Pseudomonas putida* (CH4), the other *Pseudomonas putida* (CH1, CH2, CH3) could not effectively promote the germination of *Cymbidium goeringii* seeds. They still had the defect of poor germination promotion effect, and were even worse than the control group (CK).

[0078] Table 5. Effects of different *Pseudomonas putida* species (CH1, CH2, CH3, CH4) on the germination potential and germination rate of *Cymbidium goeringii* seeds.

[0079]

[0080]

[0081] As shown in Tables 4 and 5, the *Pseudomonas putida* (CH4) of this invention can effectively promote the rapid and efficient germination of Cymbidium goeringii seeds.

[0082] Example 3

[0083] The application of a growth promoter in promoting the growth of Cymbidium goeringii tissue culture includes the following steps: using different concentrations of Pseudomonas putida ferment broth from Example 2 as the growth promoter, and culturing Cymbidium goeringii tissue culture under the action of the growth promoter.

[0084] 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. Different concentrations of bacterial suspension were added to the medium as promoters, and the culture was placed in an artificial climate chamber at 25℃, relative humidity 70%–75%, 14 hours of light per day, and a light intensity of 4000 lux. 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.

[0085] After harvesting, the tissue cultures were weighed fresh using an electronic balance, then dried in a 50℃ oven until constant weight, and the dry matter weight was measured. One-way ANOVA was performed on the increases in fresh and dry weight of the tissue cultures in each treatment group, followed by multiple comparisons. The results are shown in Tables 6 and 7.

[0086] As shown in Table 6, the growth of *Cymbidium goeringii* tissue culture increased slowly and steadily during the cultivation process, and the growth of *Cymbidium goeringii* tissue culture in the culture medium under the action of *Pseudomonas putida* (CH4) was significantly higher than that in the control group. Table 6 also shows that different concentrations of *Pseudomonas putida* (CH4) had different growth-promoting effects on *Cymbidium goeringii* tissue culture. With the increase of *Pseudomonas putida* (CH4) fermentation broth concentration, the fresh weight of *Cymbidium goeringii* tissue culture also continuously increased. Compared with the control group, the addition of different doses of endophytic bacteria fermentation broth increased the fresh weight of *Cymbidium goeringii* tissue culture by 189%–256%, with the following intensity: 1.0 (1×10⁻⁶). 9 CFU / mL) > 1.5 (1×10 10 CFU / mL) > 0.5 (1×10 8 CFU / mL) > CK(0).

[0087] Table 6. Effects of different concentrations of *Pseudomonas putida* (CH4) fermentation broth on the fresh weight of *Cymbidium goeringii* tissue culture.

[0088]

[0089] As shown in Table 7, during the cultivation process, adventitious buds continuously differentiated from the rhizomes of each Cymbidium goeringii tissue culture. In the treatment with added Pseudomonas putida (CH4) 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. Table 7 also shows that different concentrations of Pseudomonas putida (CH4) fermentation broth had varying degrees of influence on the differentiation of adventitious buds in Cymbidium goeringii tissue culture. In particular, after the cultivation period stabilized, compared with the control, Pseudomonas putida (CH4) promoted the differentiation of adventitious buds in the Cymbidium goeringii tissue culture, which was beneficial to the proliferation and cultivation of Cymbidium goeringii.

[0090] Table 7. Effects of different concentrations of *Pseudomonas putida* (CH4) fermentation broth on adventitious shoot differentiation of *Cymbidium goeringii* tissue culture.

[0091] concentration Germination potential Germination rate <![CDATA[0.5(1×10 8 CFU / mL)]]> 53.19％ 86.00％ <![CDATA[1.0(1×10 9 CFU / mL)]]> 55.49％ 84.6％ <![CDATA[1.5(1×10 10 CFU / mL)]]> 49.16％ 81.1％ CK(0) 39.00％ 44.74％

[0092] In this embodiment, the effects of different *Pseudomonas putida* strains (CH1, CH2, CH3, CH4) on the growth of *Cymbidium goeringii* tissue culture were also investigated. Except for the different *Pseudomonas putida* strains, the other conditions were the same as in Example 3, wherein the concentration of *Pseudomonas putida* in the fermentation broth was 1 × 10⁻⁶. 9 CFU / mL, the results are shown in Tables 8 and 9.

[0093] As shown in Table 8, except for *Pseudomonas putida* (CH4), the other *Pseudomonas putida* (CH1, CH2, CH3) could not effectively promote the growth of *Cymbidium goeringii* tissue culture. They still had the defect of poor growth promotion effect, and even worse than the control group (CK).

[0094] Table 8. Effects of fermentation broths of different *Pseudomonas putida* (CH1, CH2, CH3, CH4) on the fresh weight of *Cymbidium goeringii* tissue culture.

[0095]

[0096] As shown in Table 9, except for *Pseudomonas putida* (CH4), the other *Pseudomonas putida* (CH1, CH2, CH3) could not effectively promote the growth of adventitious bud differentiation in *Cymbidium goeringii* tissue culture. They still had the defect of poor growth promotion effect, and even worse than the control group (CK).

[0097] Table 9. Effects of fermentation broths of different *Pseudomonas putida* (CH1, CH2, CH3, CH4) on adventitious shoot differentiation of *Cymbidium goeringii* tissue culture.

[0098] serial number Germination potential Germination rate CH4 55.49％ 84.6％ CH3 31％ 35％ CH2 29％ 45％ CH1 24％ 39％ CK 39.00％ 44.74％

[0099] The results in Tables 6 to 9 show that the *Pseudomonas putida* (CH4) of this invention can effectively promote the rapid growth of *Cymbidium goeringii* tissue culture.

[0100] In summary, unlike other existing conventional orchid endophytic bacteria, the *Pseudomonas putida* strain of this invention not only promotes the germination of *Cymbidium goeringii* seeds through phosphorus solubilization and IAA secretion, but also promotes coenzyme A synthesis and enhances the alkaloid synthesis capacity of *Cymbidium goeringii* by upregulating the expression of dextropanthenol, nicotinamide, and their mediated coenzyme A and alkaloid synthesis pathways, thereby promoting seed germination and seedling growth. In practical applications, the *Pseudomonas putida* strain of this invention can serve as a promoter for *Cymbidium goeringii* seed germination and tissue culture growth. It not only promotes efficient seed germination but also accelerates the growth of *Cymbidium goeringii* tissue cultures, facilitating the co-germination and propagation of *Cymbidium goeringii*, especially wild *Cymbidium goeringii*. This has significant practical implications for the in-situ conservation of endangered wild orchid species and even the industrial-scale production of artificial breeding.

[0101] 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. A *Pseudomonas malodorans* strain that promotes seed germination in Cymbidium goeringii (… Pseudomonas putida ), characterized in that, The *Pseudomonas putida* strain, designated CH4, has been deposited at the China Center for Type Culture Collection (CCTCC) on March 6, 2023, with accession number CCTCC No: M 2023259.

2. An accelerator, characterized in that, The promoter comprises the *Pseudomonas putida* as described in claim 1.

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

4. The accelerator according to claim 3, characterized in that, The concentration of *Pseudomonas putida* in the promoter 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 Cymbidium goeringii seeds.

6. The application according to claim 5, characterized in that, Includes the following steps: The growth promoter was mixed with Cymbidium goeringii seeds, and the seeds were cultured under the action of the growth promoter; the Cymbidium goeringii seeds were wild Cymbidium goeringii seeds.

7. The application according to claim 5, characterized in that, Includes the following steps: The seeds of Cymbidium goeringii were made into a seed suspension, and the seed suspension was sown onto a plate coated with a growth promoter for cultivation; the Cymbidium goeringii seeds were wild Cymbidium goeringii seeds.

8. The application of the promoter as described in any one of claims 2 to 4 in promoting the growth of Cymbidium goeringii tissue culture.

9. The application according to claim 8, characterized in that, Includes the following steps: The promoter was mixed with Cymbidium goeringii tissue culture, and the tissue culture was cultured under the action of the promoter; the Cymbidium goeringii tissue culture was wild Cymbidium goeringii tissue culture.

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