Application of pseudobacillus strain FD-6 in preparation of product for promoting growth of glossogyne macrophylla and accumulation of soluble protein content

Abstract

The invention relates to application of a pseudobacillus strain FD-6 in preparation of a product for promoting growth of glossogyne macrophylla and accumulation of soluble protein content, and belongs to the technical field of microorganisms. The invention discloses a microbial agent for promoting growth of glossogyne macrophylla and accumulation of soluble protein content of the glossogyne macrophylla. The effective component of the microbial agent is viable bacteria of a single pseudobacillus strain FD-6; the microbial agent can significantly promote the accumulation of root and leaf organ growth amount, biomass and soluble protein content of Gonggutta macrophylla. Wherein the preservation number of the pseudomonas strain FD-6 is CCTCC (China Center for Type Culture Collection) NO: M 20252238, and the pseudomonas strain FD-6 is preserved in the China Center for Type Culture Collection on October 17, 2025; the bacillus sp.FD-6 strain is classified and named as the bacillus sp.FD-6 strain. The bacillus sp.FD-6 strain is bacillus sp.FD-6.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to the application of Pseudomonas strain FD-6 in the preparation of products for promoting the growth of Gynostemma pentaphyllum and the accumulation of soluble protein content. Background Technology

[0002] Large-rooted truffle orchid is a species of orchid in the genus *Truffle* of the family Orchidaceae. Holcoglossum *Cymbidium magna* is a perennial epiphytic herb named for the numerous curved and stout roots at the base of its stem, which is wrapped in leaf sheaths. It primarily grows on tree trunks in evergreen broad-leaved forests at altitudes of 1250-2000 meters, and in my country, it is only distributed in southern to western Yunnan Province. *Cymbidium magna* is listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and is listed as Vulnerable (VU) by the International Union for Conservation of Nature (IUCN), facing a high risk of extinction. my country's *National Key Protected Wild Plants List* explicitly lists the entire genus *Cymbidium* as a Class I protected plant, prohibiting illegal harvesting and trade. This genus is also included in the National Project for the Rescue and Protection of Wild Plants with Extremely Small Populations.

[0003] There are approximately 20 species of *Cymbidium* worldwide, with 18 species discovered and recorded in my country (Checklist of Chinese Plant Species 2025). New distributions are still being reported in their main distribution areas of Myanmar, Thailand, Laos, and Vietnam. Global research on *Cymbidium* species is extremely limited, currently focusing on systematic studies based on nuclear and chloroplast genes, and taxonomic studies based on karyotypes. Furthermore, *Cymbidium macrocarpa* can precisely deliver pollen masses into the stigma cavity at the base of the rostellum by rotating its anther 360° without any pollinating agents or auxiliaries, thus achieving fertilization. This novel reproductive mechanism, evolved by *Cymbidium* to adapt to arid environments lacking pollinating insects, is being studied. Nature Reported in 2006. Plants of the genus *Gnaphalium* have significant scientific research value as experimental materials for studying the spatiotemporal distribution patterns and evolution of epiphytes endemic to East Asia. At the same time, due to their varied flower colors and beautiful corolla shapes, they are also important ornamental flowers with great potential for horticultural development.

[0004] Soluble proteins contain key enzymes at various stages of plant growth, and their levels directly reflect the vigor of plant growth and the flowering potential of horticultural flowers. Furthermore, some soluble proteins participate in the shaping of plant stress resistance characteristics, including heat shock proteins (HSPs), osmotic regulatory proteins (such as dehydrin), antifreeze proteins (AFPs), and metallothioneins (MTs), enhancing the plant's resistance to high temperatures, drought, cold, and heavy metal stress. For *Cymbidium goeringii*, measuring its soluble protein content is of great significance for assessing plant physiological status, cultivation management, and variety selection; especially as an important ornamental flower, soluble protein content is a crucial evaluation indicator for regulating its ornamental quality and stress resistance management. Increasing the soluble protein content of *Cymbidium goeringii* plants can accelerate plant physiological metabolism, promote nitrogen utilization, and regulate earlier flowering; simultaneously, it can also enhance plant resistance and extend the duration of flower morphology. The increase in soluble protein content has an important indicative effect on the screening of superior varieties of *Gymnocalycium mihanovichii*, early warning of stress damage, and optimization of water and fertilizer management.

[0005] Currently, there are no research or technical reports on the propagation of *Cymbidium goeringii*, either domestically or internationally. This invention aims to provide a single microbial agent that promotes root and leaf growth and the accumulation of soluble protein in *Cymbidium goeringii*. This agent achieves effective plant growth and promotes soluble protein synthesis through core microorganisms colonizing the rhizosphere of *Cymbidium goeringii*, breaking through the technical bottleneck in the propagation of this important ornamental horticultural flower and providing technical support for its efficient resource utilization and wild population protection. This microbial agent is readily available, easy to apply, and has no toxic side effects on the soil substrate, making it easy to promote and apply. It is a pioneering achievement both domestically and internationally. Summary of the Invention

[0006] To solve or partially solve the problems existing in related technologies, this invention application is made as follows: On the one hand, a strain of Pseudomonas was provided ( Fictibacillus sp. The strain FD-6, with accession number CCTCC NO:M 20252238, was deposited at the China Center for Type Culture Collection on October 17, 2025; the deposit address is: Wuhan University, Wuhan, China.

[0007] On the other hand, this application also provides the use of Pseudomonas strain FD-6 in the preparation of products for promoting the growth of Gynostemma pentaphyllum.

[0008] On the other hand, this application also provides the application of Pseudomonas strain FD-6 in the preparation of products for promoting the accumulation of soluble protein content in tall rooted charcoal.

[0009] Furthermore, the promotion of *Gymnocalycium mihanovichii* growth includes promoting root growth, promoting leaf growth, and / or increasing overall biomass.

[0010] On the other hand, this application also provides a microbial inoculant for promoting the growth of *Gnaphalium affine*, the active ingredient of which is a single live strain of *Pseudomonas* FD-6. Furthermore, the above-mentioned microbial inoculant is used to promote the accumulation of soluble protein content in *Gnaphalium affine*. Beneficial effects

[0011] 1. The microbial agent provided in this application has a live Pseudomonas strain FD-6 as its active ingredient; the microbial agent effectively promotes the growth of the roots of Oryza sativa, increases the number of roots and the average length of roots; at the same time, it promotes leaf growth, significantly increases the number of leaves and the height of the plant.

[0012] 2. The active ingredient provided in this application is a bacterial agent prepared from live bacteria of Bacillus pseudosporidis strain FD-6, which significantly increases the biomass and accumulation of soluble protein content of the plant by regulating the overall growth of Oryza macrocarpa.

[0013] 3. This application utilizes a microbial agent prepared by applying live bacteria of *Pseudomonas aeruginosa* strain FD-6 as the active ingredient, which significantly increases the soluble protein content of *Gnaphalium affine*. This has the following core value for actual production and cultivation: (1) Promote growth rate and enhance commercial value: Soluble protein is the basis of cell structure and function proteins, and its content is directly related to the cellular biosynthetic activity. A significant increase in content means that the plant grows faster and accumulates more biomass. For *Cymbidium goeringii*, an ornamental plant, this can shorten the seedling and mature plant cultivation cycle, reach marketable size more quickly, thereby reducing time costs and improving production efficiency.

[0014] (2) Enhancing drought resistance and ensuring production stability: Proline and other substances in soluble proteins are important osmotic regulators. Increased content of these substances significantly enhances the plant's water retention capacity and improves drought resistance. In the context of global climate change and frequent extreme weather events, this characteristic is crucial for ensuring the survival rate of *Gymnocalycium mihanovichii*, especially for plants reintroduced into the wild and protected, reducing losses caused by drought and making production and management more resilient.

[0015] (3) Enhancing metabolic vitality and laying the foundation for returning to nature: Soluble protein content is a key indicator of plant vitality. A plant with vigorous metabolism and active protein synthesis has a stronger ability to adapt to environmental changes and resist adversity. Therefore, increasing the soluble protein content directly enhances the overall vitality of the plant, providing a crucial internal guarantee for its successful "return to cultivation" from artificial cultivation environment to natural habitat and for the restoration of wild populations.

[0016] 4. The Pseudomonas strain FD-6 provided by this invention was isolated from wild *Cymbidium goeringii* plants and is naturally occurring. Applying it to *Cymbidium goeringii* plants will not cause additional burden or harm to the environment or other species.

[0017] 5. The microbial agent provided in this application, being a single-strain preparation agent, has a simple preparation process and extremely low application cost, and has broad prospects for development and utilization as well as value for promotion and application. Attached Figure Description

[0018] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings.

[0019] Figure 1 A phylogenetic tree for Pseudomonas strain FD-6 constructed based on the 16S gene sequence.

[0020] Figure 2 This is a colony morphology diagram of Pseudomonas aeruginosa strain FD-6 cultured on agar plates.

[0021] Figure 3 A schematic diagram showing the effect of adding live bacteria of a single strain of Pseudomonas FD-6 as the exogenous active ingredient on the growth of tissue culture seedlings of Oryza macrocarpa (90 days).

[0022] Biological Preservation Instructions: The *Pseudomonas* strain FD-6 disclosed in this application was deposited on October 17, 2025, at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20252238, located at Wuhan University, Wuhan, China; and classified as *Pseudomonas* FD-6. Fictibacillus sp. FD-6. Detailed Implementation

[0023] The alternative embodiments of this application will now be described in more detail. While alternative embodiments of this application have been described, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0024] For clarity, the following examples will be used to provide a detailed description.

[0025] Example 1: Isolation and Identification of Bacterial Strains 1. Isolation and purification method of Pseudomonas strain FD-6 Wild *Dendrobium nobile* individuals were collected from Changning County, Baoshan City, Yunnan Province. The roots, stems, and leaves of *Dendrobium nobile* were rinsed with sterile water to remove surface impurities, and then blotted dry with sterile filter paper. In a clean bench, the individuals were first rinsed with 75% alcohol for 1 minute, then soaked in 0.1% mercuric chloride solution for 2 minutes. The soaked individuals were rinsed three times with sterile water, and the sterile water from the final rinse was used as a control. Using a tissue homogenization method, the roots, stems, and leaves of *Dendrobium nobile* were cut into sample pieces and ground in a sterilized mortar. An appropriate amount of distilled water was added, and the mixture was homogenized. 100 μL of each homogenate was pipetted onto NA, YG, and KB solid media, with each treatment repeated three times. The samples were incubated in the dark at 28°C for 5–7 days to isolate endophytic bacteria from *Dendrobium nobile*.

[0026] After colonies grow on the culture medium, select well-separated and moderately numerous marginal colonies based on differences in color, morphology, and texture. Using an inoculation loop, aseptically pick out all single colonies with different morphological characteristics and inoculate them onto NA-1 solid medium using the streak plate method to obtain a large number of single colonies. Streak each single colony on 2-3 plates. After they regrow, pick out single colonies and inoculate them onto fresh NA-1 solid medium. Repeat this purification process until stably growing bacterial single colonies are obtained, and number the purified single colonies.

[0027] Select a single colony of the purified bacteria strain FD-6 and inoculate it into 1 mL of LB broth. Incubate overnight at 28°C and 180 rpm in a shaker. Then, take 150 μL of the bacterial culture, add 150 μL of 50% sterile glycerol, mix well, and store at -80°C.

[0028] 2. Identification of Pseudomonas strain FD-6 1) Morphological identification: Strain FD-6 was activated and inoculated onto NA-1 solid medium. After incubation in the dark at 28°C for 2-3 days, the color, morphology, and texture of single colonies were photographed and recorded. The isolated endophytic bacteria were preliminarily classified and identified according to the *Manual of Systematic Identification of Common Bacteria* and the *Bergey's Manual of Bacteriological Identification*. Strain FD-6 appeared white on NA-1 medium, with round, wrinkled colonies. Single colonies were round, raised to a diameter of 1-3 mm, with a viscous, opaque texture and intact edges.

[0029] 2) Molecular biological identification: Single colonies of the purified bacterial strain were selected and inoculated into 1 mL of LB broth, then incubated overnight at 28°C and 180 rpm on a shaker. The purified bacterial culture was then sent to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing. The sequence result is (SEQ ID NO.1):

[0030] The 16S rRNA splice sequence obtained from sequencing was aligned for homology using NCBI (https: / / www.ncbi.nlm.nih.gov / ), and a phylogenetic tree was constructed. The phylogenetic results show that strain FD-6 is related to... Fictibacillus enclensis NIO-1003 has the highest phylogenetic relationship and a sequence identity of 98.93%.

[0031] In summary, based on the colony morphology and molecular biological characteristics of strain FD-6, strain FD-6 was identified as... Fictibacillus sp. FD-6, Family Bacillaceae, Genus Pseudomonas ( Fictibacillus It was deposited at the China Center for Type Culture Collection on October 17, 2025, with accession number CCTCC NO:M 20252238, and classified as: *Pseudomonas FD-6*. Fictibacillus sp. FD-6.

[0032] Example 2: Preparation and application of microbial agents 1) Preparation of FD-6 bacterial agent Strain strain FD-6 was inoculated into 100 mL of NA-1 liquid medium supplemented with 500 mg / L tryptophan and cultured at 28 °C with shaking at 200 rpm for 24 h. The bacterial suspension was then adjusted to OD using sterilized liquid medium. 600nm =1.0, FD-6 liquid bacterial agent was obtained, and the bacterial cell density of the agent was 3×10⁻⁶. 6 cfu / ml.

[0033] 2) Co-culture of mycelium and seedlings and its growth-promoting effect on *Gynostemma pentaphyllum* The *Cymbidium goeringii* and *Cymbidium wangii*, provided by the Special Ornamental Trait Formation and Germplasm Innovation Team of the College of Landscape Architecture and Horticulture, Southwest Forestry University, were selected as experimental materials. They were transplanted into a mycotoxin-seedling symbiotic culture medium. The symbiotic culture medium formula consisted of 1 / 2 MS + 30 g / L sucrose + 8 g / L agar + 1 g / L activated carbon, adjusted to pH 6.8. After removing contaminants, each test group consisted of 20 bottles, with 5 seedlings per bottle. 50 μL of FD-6 inoculant was applied near the roots of each tissue-cultured seedling, while the blank control group was treated with an equal volume of sterile liquid culture medium (the same liquid culture medium used in the inoculant preparation). The tissue-cultured seedlings were placed at 26℃ with a light / dark cycle of 12 / 12 h and a light intensity of 1500 Lux for 90 days. After this period, the *Cymbidium goeringii* tissue-cultured seedlings were placed on a clean bench, rinsed with distilled water to remove residual culture medium, blotted dry with sterile filter paper, and their fresh weight was measured and recorded. Plant height was measured using a ruler, from the base of the plant to the highest point of the leaves. If the plant was bent or tilted, it could be gently pulled to straighten, but excessive force should be avoided to prevent damage. The number of leaves was counted leaf by leaf, starting from the base of the stem, using fully unfolded leaves (with clearly visible petioles) as the standard. Yellowed and withered leaves were not counted. The number of roots was counted starting from the base of the stem, counting the visible roots. The three longest main roots were selected, and their lengths were measured and recorded using a ruler. If the roots were bent, they could be gently pulled to straighten, but excessive force should be avoided to prevent damage. After measurement, the grouped plants were placed in an oven at 105℃ for 30 minutes to blanch, then dried at 65℃ until constant weight. After cooling, the dry weight was immediately weighed and recorded.

[0034] The soluble protein content was determined using the Coomassie Brilliant Blue G-250 staining method. Six test tubes were prepared, and 5 mL of bovine serum albumin (BSA) standard solution with concentration gradients of 0-100 μg were added to each tube. Using a pipette, 0.1 mL of each solution was accurately transferred to a 10 mL stoppered test tube. 5 mL of Coomassie Brilliant Blue G-250 reagent was added to each tube, and the tubes were capped and repeatedly inverted to mix. After standing at room temperature for 2 min, the absorbance was measured at 595 nm. A standard curve was plotted with the BSA standard solution concentration as the x-axis and absorbance as the y-axis. Fresh *Gnaphalium affine* seedlings from each treatment group were chopped, mixed, and 0.5 g was weighed and placed in a pre-chilled mortar. 5 mL of pre-chilled 0.1 mol / L (pH 7.0) phosphate buffer was added in portions, along with a small amount of quartz sand to aid grinding. The mixture was ground completely into a homogenate under ice bath conditions and centrifuged at 4000 rpm for 10 min. The resulting supernatant was the extract for each sample. Accurately pipette 0.1 mL of sample extract and react it with Coomassie Brilliant Blue G-250 reagent as described above. Measure the absorbance of each sample at a wavelength of 595 nm and calculate the corresponding protein concentration based on the standard curve.

[0035] 3) Results and Analysis

[0036]

[0037] As shown in Table 1, after adding FD-6 inoculant, compared with the control group (CK), the number of roots, root length, number of leaves, and plant height of *Gymnocalycium mihanovichii* were significantly increased (from...). Figure 3 (This can also be seen visually). However, FD-6 inoculant did not significantly increase the plant growth of *Cymbidium goeringii* (and the accumulation of underground biomass actually decreased), suggesting that FD-6 inoculant is specific to *Cymbidium goeringii* in promoting growth. *Cymbidium goeringii* has long, fleshy leaves that are typically obliquely upright and curved outwards, forming a stepped light-receiving surface. More leaves lead to an increase in the overall photosynthetic efficiency of the plant. As an epiphytic orchid, the number and length of its roots are crucial for physiological adaptation and niche construction. First, root development directly determines the efficiency of *Cymbidium goeringii* in capturing air humidity and rainwater in its native habitat. Dense, deep roots that penetrate deep into bark crevices effectively absorb water during the dry season, keeping the plant alive. Secondly, the thickened cell walls of the root epidermal cells of *Gnaphalium affine*, which may contain mucilage rich in arabinogalactan, slow down the dehydration rate under drought conditions. Simultaneously, during extreme weather events, more roots can maintain overall survival through the sacrificial death of some roots, and the root tips can synthesize flavonoids to resist UV-B radiation. Thirdly, the increased root number enhances the total amount of IAA synthesis, promotes the transport of photosynthetic assimilates to storage organs, and maintains healthy root growth. In terms of ecological adaptation, the increased root number enhances the adhesion to the host trunk, and the dense root system also provides a niche advantage by competing with neighboring plants for basal space. Furthermore, the strigolactones secreted by the roots of *Gnaphalium affine* can attract AM fungi, inhibiting the germination of spores from neighboring mosses and reducing competitors on the epiphytic surface. Therefore, rapid root development is a core strategy for *Gnaphalium affine* to balance resource efficiency and propagation costs in epiphytic habitats, and is of great significance to its growth.

[0038] Table 2 shows that after adding FD-6 inoculant, the plant biomass and soluble protein accumulation of *Cymbidium goeringii* significantly increased compared to the control group (CK), while no significant increase was observed in the *Cymbidium pumilense* group. During colonization and growth, microorganisms, especially the key rhizosphere microorganisms of *Cymbidium goeringii*, can directly stimulate cell division and elongation by secreting auxins, and induce the activation of endogenous hormone synthesis pathways in plants. Some groups can also secrete heptaphilin, which can chelate iron, improve the plant's iron absorption efficiency, and thus promote chlorophyll synthesis and photosynthesis, accelerating the assimilation and fixation of photosynthetic products. The inoculant prepared from *Pseudomonas fusiforme* FD-6 selected in the experiment can also provide more potential intermediate products for the plant's tricarboxylic acid cycle, ultimately promoting protein synthesis. Simultaneously, the upregulation of the proline synthase gene (P5CS), which mediates the increase in soluble protein content, can also enhance the cell's water retention capacity and improve the drought resistance of *Cymbidium goeringii* in the inoculant-seedling co-culture system. In addition, during the colonization of plants in the rhizosphere, Pseudomonas inhibits the growth of pathogens and seizes ecological niches in the rhizosphere complex (SynCom) by secreting antimicrobial peptides (such as cyclic polypeptides). While increasing the expression of resistance proteins (such as chitinase and β-1,3-glucanase), it reduces the energy consumption of plants in the defense response and devotes more resources to growth.

[0039] As shown in Tables 1 and 2, the *Pseudomonas* FD-6 inoculant significantly promotes the growth, biomass, and soluble protein accumulation of the root and leaf organs of *Orchidonia macrocarpa*. As an important epiphytic ornamental orchid, this invention provides an effective safeguard mechanism for optimizing cultivation conditions, reducing propagation costs, and promoting the return to cultivation and protection of wild resources in the context of global climate change.

[0040] In summary, the active ingredient provided is a bacterial agent prepared from live Pseudomonas strain FD-6. The strain has a clear source and is easily obtained. In practical applications, it can effectively improve the growth, biomass, and soluble protein accumulation of the roots and leaves of *Cymbidium ensifolium*. Unlike other inventions that require multiple nutrient solutions or multiple bacterial strains, this application is easy to prepare and inexpensive. Furthermore, the preparation of the bacterial solution using a single strain is simple and easy to promote, effectively filling a technological gap. It provides important technical support for the subsequent development and research of *Cymbidium ensifolium*, an important ornamental orchid, and for the healthy and sustainable development of the industry, achieving a breakthrough in *Cymbidium ensifolium* cultivation technology and realizing green ecological goals. Currently, there are no related technical reports domestically or internationally, making this a pioneering work.

[0041] The various embodiments of this invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. Pseudomonas ( Fictibacillus sp. The application of strain FD-6 in the preparation of products for promoting the growth of *Gynostemma pentaphyllum*; the preservation number of *Pseudomonas* strain FD-6 is: CCTCC NO: M 20252238, and it was deposited at the China Center for Type Culture Collection on October 17, 2025; its classification name is: *Pseudomonas FD-6*. Fictibacillus sp.FD-6.

2. The application according to claim 1, characterized in that, The promotion of *Gymnocalycium mihanovichii* growth includes promoting root growth, promoting leaf growth, and / or increasing overall biomass.

3. Application of *Pseudomonas* strain FD-6 in the preparation of products for promoting the accumulation of soluble protein content in *Gynostemma pentaphyllum*; the preservation number of *Pseudomonas* strain FD-6 is: CCTCC NO: M 20252238, and it was deposited at the China Center for Type Culture Collection on October 17, 2025; its classification name is: *Pseudomonas* FD-6. Fictibacillus sp. FD-6.

4. A microbial inoculant for promoting the growth of *Gymnocalycium mihanovichii*, characterized in that, Its active ingredient is a single live strain of *Pseudomonas* FD-6; the accession number of *Pseudomonas* strain FD-6 is: CCTCC NO: M 20252238, and it was deposited at the China Center for Type Culture Collection on October 17, 2025; its classification name is: *Pseudomonas* FD-6. Fictibacillus sp. FD-6.

5. The microbial agent according to claim 4, characterized in that, The fungal agent is used to promote the accumulation of soluble protein in *Gynostemma pentaphyllum*.

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