A strain of *Pseudomonas gingerae* YND5, its inoculum and its application

Abstract

This invention provides a strain of *Pseudomonas gingerae* YND5, an inoculant, and its applications, belonging to the field of bio-agricultural technology. This invention provides a strain of *Pseudomonas gingerae* (… Pseudomonas ‘gingeri’ YND5, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37512, demonstrates a significant antagonistic effect of *Pseudomonas gingerica* YND5 on *Fusarium aromaticum*, a fungus that causes diseases in *Gastrodia elata*. This provides an important basis for its development as a potential biocontrol agent and is of great significance for the implementation of biocontrol of diseases in *Gastrodia elata*.

Description

Technical Field

[0001] This invention belongs to the field of bio-agricultural technology, and in particular relates to a strain of Pseudomonas gingerii YND5, its inoculant, and its application. Background Technology

[0002] Gastrodia elata ( Gastrodia elata Bl. Gastrodia elata is a traditional and precious Chinese medicinal herb. Its dried tuber is used medicinally; it is neutral in nature, sweet in taste, and enters the liver meridian. It has the effects of calming the liver and extinguishing wind, and relieving convulsions. The growth and development of Gastrodia elata is mainly based on its tuber and is closely related to the rhizosphere microbial community. However, currently, there is a lack of key rhizosphere soil microorganisms that can effectively control diseases of Gastrodia elata. Summary of the Invention

[0003] The purpose of this invention is to provide a strain of *Pseudomonas gingerae* YND5, a fungal agent, and its application. The *Pseudomonas gingerae* strain of this invention can antagonize fungi that cause diseases of *Gastrodia elata*.

[0004] This invention provides a strain of *Pseudomonas gingerae* (… Pseudomonas 'gingeri' YND5 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37512.

[0005] The present invention also provides a bacterial agent comprising *Pseudomonas gingerae* YND5 as described in the above-described scheme.

[0006] Preferably, the bacterial agent comprises a bacterial solution; the concentration of *Pseudomonas gingerologica* YND5 in the bacterial solution is OD0.05. 600 =0.6.

[0007] The present invention also provides a method for preparing the microbial agent described in the above-mentioned scheme, comprising the following steps: The bacterial agent was obtained by inoculating *Pseudomonas gingerica* YND5 onto LB liquid medium and culturing it.

[0008] This invention also provides the application of *Pseudomonas gingerae* YND5 as described in the above-mentioned scheme, the fungal agent described above, or the fungal agent prepared by the above preparation method in antagonizing pathogenic fungi of *Gastrodia elata*, wherein the pathogenic fungus of *Gastrodia elata* is *Fusarium aromaticum* (…). Fusarium redolens ).

[0009] Preferably, the antagonistic fungi that cause Gastrodia elata disease include those that inhibit the growth of Fusarium aromaticum mycelium.

[0010] This invention also provides a method for antagonizing pathogenic fungi of Gastrodia elata, by applying the *Pseudomonas gingera* YND5 described in the above scheme, the fungal agent described above, or the fungal agent prepared by the above preparation method; wherein the pathogenic fungus of Gastrodia elata is *Fusarium aromaticum*.

[0011] This invention provides a strain of *Pseudomonas gingerae* YND5, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37512. *Pseudomonas gingerae* YND5 of this invention exhibits significant antagonistic activity against *Fusarium aromaticum*, a fungus that causes diseases in *Gastrodia elata*, providing important evidence for its development as a potential biocontrol agent and holding significant importance for the implementation of biocontrol of diseases in *Gastrodia elata*.

[0012] Biological Preservation Instructions *Pseudomonas gingerica* YND5, suggested classification name Pseudomonas 'gingeri' It was deposited on January 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 37512. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 Phylogenetic tree of YND5; Figure 2 YND5 was cultured at 30℃ for 24 h using LB culture. Figure 3 Gram staining results for YND5; Figure 4 Morphological diagram of YND5 antagonistic Fusarium; Figure 5 The growth curve of YND5 after antagonizing Fusarium; Figure 6 The inhibition rate of YND5 against Fusarium; Figure 7 Morphological diagram of the antagonistic Fusarium moniliforme on Gastrodia elata by YND5. Detailed Implementation

[0015] This invention provides a strain of *Pseudomonas gingerae* YND5, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37512.

[0016] The *Pseudomonas gingerica* YND5 of this invention is a rhizosphere soil bacterium of *Gastrodia elata*, isolated from surface soil of *Gastrodia elata* and soil surrounding mycelial cords of *Armillaria mellea* collected in Yunnan.

[0017] The morphological characteristics of *Pseudomonas gingerae* YND5 of this invention include: Gram-negative staining and rod-shaped cells. After culturing on LB medium for 24 h, the colonies are round, milky white, and smooth. These morphological and Gram-stained characteristics are consistent with... Pseudomonas The morphological characteristics of the genera are similar.

[0018] The physiological and biochemical characteristics of *Pseudomonas gingerae* YND5 of the present invention include: conforming to... Pseudomonas Major cellular fatty acid characteristics of the genus; API 50CH data of strain YND5 are consistent with... Pseudomonas Biochemical metabolic characteristics of the genus The nucleotide sequence of the 16S rRNA gene of *Pseudomonas gingera* YND5 of the present invention is shown in SEQ ID NO.1.

[0019] The present invention also provides a bacterial agent comprising *Pseudomonas gingerae* YND5 as described in the above-described scheme.

[0020] In one embodiment, the bacterial agent comprises a bacterial solution; the concentration of *Pseudomonas gingerologica* YND5 in the bacterial solution is OD0.05. 600 =0.6; the concentration of the bacterial solution is (1~9)×10 8 CFU / mL.

[0021] The present invention also provides a method for preparing the microbial agent described in the above-mentioned scheme, comprising the following steps: The bacterial agent was obtained by inoculating *Pseudomonas gingerica* YND5 onto LB liquid medium and culturing it.

[0022] In one embodiment, the culture includes oscillation culture; the culture temperature is 25°C; the oscillation culture frequency is 180~200 rpm; and the culture time is 24~36 h.

[0023] This invention also provides the application of *Pseudomonas gingerae* YND5 described in the above-described scheme, the described inoculum agent, or the inoculum agent prepared by the described preparation method in antagonizing fungi that cause diseases of *Gastrodia elata*; wherein the fungus causing diseases of *Gastrodia elata* is *Fusarium aromaticum*. As one embodiment, the antagonism against fungi causing diseases of *Gastrodia elata* includes inhibiting the mycelial growth of *Fusarium aromaticum*.

[0024] This invention also provides a method for antagonizing pathogenic fungi of Gastrodia elata, by applying the *Pseudomonas gingera* YND5 described in the above scheme, the fungal agent described above, or the fungal agent prepared by the above preparation method; wherein the pathogenic fungus of Gastrodia elata is *Fusarium aromaticum*.

[0025] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a strain of *Pseudomonas gingerae* YND5, its inoculum, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1 I. Strain Isolation and Screening 1. Culture medium: 1) LB medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, 15 g agar; 2) Modified PDA medium: 20 g glucose, 33 g wheat bran, 4 g potato extract powder, 15 g agar; The liquid culture medium is prepared in the same way as above, but without adding agar.

[0027] The above culture medium was diluted to 1 L with ultrapure water and autoclaved at 121°C for 15 min. The solid culture medium was dispensed into 90 mm Petri dishes, with 20 mL of culture medium added to each dish. After cooling, it was ready for use.

[0028] 2. Sampling Location: Soil samples were collected from the surface of Gastrodia elata and from the soil surrounding the mycelial cords of Armillaria mellea in Yunnan Province. The samples were identified as fresh tubers of Gastrodia elata, an orchid species.

[0029] 3. Handling method: 1) Isolation of rhizosphere bacteria from Gastrodia elata The collected *Gastrodia elata* and *Armillaria mellea* mycelia were rinsed with sterile water in a clean bench to remove surface dirt and sand, and the rinsing solution was collected. The surface moisture of the rinsed *Gastrodia elata* and *Armillaria mellea* mycelia was absorbed using sterile filter paper, and the samples were placed in 50 mL sterile centrifuge tubes. Sterile water was added, and the tubes were placed in an ultrasonic cleaner and ultrasonicated for 5 min. The ultrasonicated *Gastrodia elata* and *Armillaria mellea* mycelia were removed, and the surface of the *Gastrodia elata* was rinsed again with sterile water. The rinsed *Gastrodia elata* was then dried with sterile filter paper and ground into a homogenate in a sterile mortar. The rinsing solutions from both rinses were combined with the sterile water used for ultrasonic treatment of the *Gastrodia elata* and *Armillaria mellea* mycelia to create an aqueous extract of the rhizosphere soil bacteria from the *Gastrodia elata* mycorrhizae. Each material was treated three times.

[0030] 2) Bacterial suspension dilution and bacterial isolation (1) Dilution of bacterial culture Accurately pipette 1 mL of each of the *Gastrodia elata* homogenate and soil bacteria aqueous extract into sterile centrifuge tubes, and dilute with 9 mL of sterile water to obtain diluted stock solutions of the *Gastrodia elata* homogenate and soil bacteria aqueous extract. Each treatment was repeated three times. Then, the diluted stock solutions of the *Gastrodia elata* homogenate and soil bacteria aqueous extract were diluted 10 mL each. 2 10 3 10 4 10 5 10 6 The different concentrations of diluted solutions were obtained by multiplying the concentrations and set aside for later use.

[0031] (2) Bacterial isolation Prepare LB solid and liquid culture media. Take a dose diluted to 10...6 Two 200 μL solutions of *Gastrodia elata* homogenate and soil bacterial aqueous extract were spread onto LB agar plates, three times in total. The plates were incubated at 25 °C for 3–5 days, with daily observation of bacterial colony growth. The grown colonies were then transferred to 1 mL of the corresponding liquid culture medium and incubated overnight at 25 °C with a shaker at 200 rpm. After multiple streak-line separation and purification processes, a pure bacterial culture was obtained. Based on the morphology and characteristics of the bacteria on the culture medium, preliminary classification was performed, and duplicates were removed. The purified bacteria were named YND5.

[0032] II. Strain Identification 1. Molecular identification: The 16S rRNA gene sequence was determined using universal primers 27F and 1492R for the bacterial 16S rRNA gene, yielding a 1443 bp gene fragment. Specific steps: Ten replicates of YND5 bacterial culture cultured overnight on a shaker were amplified by PCR using primers 27F (5'-agagtttgatcctggctcag-3', SEQ ID NO.2) and 1492R (5'-tacggttaccttgttacgactt-3', SEQ ID NO.3) and then sent for identification. The PCR reaction system (25 μL) consisted of: 0.4 μL of each primer, 2 μL of YND5 bacterial culture, 12.5 μL of 2×Taq PCR Mix, and ddH2O to a final volume of 25 μL. PCR reaction conditions: Denaturation was performed at 94℃ for 3 min, followed by 35 cycles of denaturation at 94℃ for 25 s, annealing at 55℃ for 25 s, extension at 72℃ for 90 s, and a final extension at 72℃ for 5 min. 5 μL of the extracted PCR product was electrophoresed on a 1% agarose gel. The PCR product with a bright band at 1500 bp was sent for sequencing, and the sequencing results were compared with those from NCBI. 800 μL of YND5 bacterial culture was added to 800 μL of 50% sterile glycerol and mixed thoroughly. The mixture was then stored at -80℃. The comparison results showed that YND5 was… Pseudomonas ' gingeri The sequencing results are shown in SEQ ID NO.1, specifically:

[0033] 2. 16S rRNA gene sequence (1443 bp) and phylogenetic analysis of strain YND5 16S rRNA gene sequence alignment analysis based on the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) showed that strain YND5 is similar to known strains. Pseudomonas 'gingeri' NCPPB 3146 showed the highest similarity (99.92%), and the similarities of other model bacteria are shown in Table 1. A phylogenetic tree of strain YND5 and related taxa based on 16S rRNA gene sequences was constructed, as follows: Figure 1 .

[0034] Table 1. Comparative analysis of 16S rRNA gene sequences of strain YND5

[0035] 3. Morphological identification 1) See the colony morphology photograph of strain YND5. Figure 2 The bacteria were Gram-negative under YND5 staining and the cells were rod-shaped. After culturing on TSA medium for 24 h, the colonies were round, milky white, and smooth. These morphological and Gram-staining characteristics are consistent with... Pseudomonas The morphological characteristics of the genera are similar. See the Gram staining photograph of strain YND5. Figure 3 .

[0036] 4. Physiological and biochemical identification 1) Fatty acid data of strain YND5 The fatty acid composition of strain YND5 was determined by the Microbial Fatty Acid Rapid Identification System (MIDI). The main fatty acid of the tested strain was found to be C. 16:0 C 17:0 cyclo, C 12:0 2OH and C 12:0 3OH, with contents of 32.53%, 20.10%, 7.45%, and 6.15%, respectively. The specific fatty acid results are shown in Table 2, which are consistent with... Pseudomonas The main cellular fatty acid characteristics of the genus.

[0037] Table 2 Fatty acid data for strain YND5

[0038] 2) API 50CH data for strain YND5 API 50CH test results: Positive reactions included L-arabinose, D-xylose, and D-fucose; weakly positive reactions included D-ribose, D-galactose, D-fructose, D-mannose, D-mannitol, D-trehalose, and D-arabinol; negative reactions included control, glycerol, erythrose, D-arabinose, L-xylose, D-ribitol, methyl-β-D-xylopyranoside, D-glucose, L-sorbose, L-rhamnose, galactitol, inositol, D-sorbitol, and methyl-α-D-pyranoside. Mannosides, methyl-α-D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, aescin, salicin, D-cellobiose, D-maltose, D-lactose (bovine), D-merinobiose, D-sucrose, inulin, D-melinobiose, D-raffinose, starch, glycogen, xylitol, gentiobiose, D-melinobiose, D-lythose, D-tagatose, L-fucose, L-arabinol, potassium gluconate, 2-ketogluconate, and 5-ketogluconate are detailed in Table 3. (Compliant) Pseudomonas Biochemical metabolic characteristics of the genus.

[0039] Table 3. API 50CH data for strain YND5

[0040] Note: + indicates positive; - indicates negative; w indicates weak positive.

[0041] Example 2: Functional Identification of Strains 1. Culture medium: 1) PDA medium: 6 g potato extract powder, 20 g glucose, 20 g agar powder (pH 5.6); 2) LB medium: 10 g peptone, 5 g yeast extract, 10 g NaCl, 15 g agar.

[0042] Add ultrapure water to the culture medium to a final volume of 1 L, and autoclave at 121℃ for 15 min. Dispense the solid culture medium into 90 mm Petri dishes, adding 20 mL of culture medium to each dish, and let cool before use.

[0043] 2. Preparation of bacterial strains and pathogens 1) Activation and fermentation of YND5 Activation: Streak the bacterial strain preserved in glycerol tubes on LB plates, incubate at 25°C for 16-24 hours, and pick single colonies.

[0044] Fermentation: Select a single colony and inoculate it into LB liquid medium. Incubate at 25°C with shaking at 180-200 rpm for 24-36 hours until the bacterial concentration reaches approximately 1×10⁻⁶. 8 CFU / mL.

[0045] 2) Preparation of pathogen (Fusarium) spore solution Procedure: Save the target Fusarium (Aromatic Fusarium, Fusarium redolens (LY) Inoculate onto PDA plates and activate at 25°C for 5-7 days. After sporulation, add sterile water to the plate and gently scrape the surface of the colonies with a spreader. Filter through double-layered sterile gauze to remove hyphae, and collect the filtrate as the spore suspension. Count the spores using a hemocytometer and adjust the spore concentration to 1×10⁻⁶ with sterile water. 6 ~1×10 7 per mL.

[0046] 3. In vitro antibacterial verification Procedure: Inoculate a 5 mm diameter Fusarium mycelium disc at the center of a PDA plate. Simultaneously inoculate 10 μL of *Pseudomonas gingerica* suspension at symmetrical positions 2 cm from the center. Figure 4 A plate containing only Fusarium was set up as a control. These plates were incubated at 25°C. The colony radius of Fusarium was observed and measured every 3 days, and the growth rate and inhibition rate of the pathogen were calculated. Growth rate (mm / d) = (final colony diameter - initial colony diameter) / (incubation days - initial days) Inhibition rate = [(control radius - treatment radius) / control radius] × 100%.

[0047] 4. In vivo efficacy experiment of Gastrodia elata Inoculation was performed using the tubers of Gastrodia elata.

[0048] Material preparation: Select healthy, disease-free, and uniformly sized Gastrodia elata tubers. Wash off the surface soil with clean water, then disinfect the surface with 75% alcohol and air dry. Inoculation and treatment protocols: Blank control group (CK): Inoculate tubers with sterile water. Pathogen group (LY): Inoculate tubers with Fusarium spores. Antagonistic bacteria group (YND5): Inoculate tubers with Pseudomonas aeruginosa spores. Combined treatment group (YND5+LY): Inoculate tubers with Pseudomonas aeruginosa spores first, then inoculate with Fusarium spores 24 hours later.

[0049] Specific inoculation and culture methods: (1) Inoculation: Use a sterile toothpick to prick 4-6 small holes about 2-3 mm deep in the middle of the Gastrodia elata tuber. (2) Inoculation: Use a pipette to inject 10 μL of the corresponding bacterial solution or spore solution into the hole. (3) Moisturizing: After inoculation, cover the inoculation site with a wet cotton ball or plastic wrap and place it in a culture dish lined with moist sterile filter paper. (4) Culture: Place in an incubator at 25 ℃ and maintain a high humidity environment (relative humidity > 90%) for 10-15 days, during which time the disease incidence is observed.

[0050] Antagonistic screening of over 100 isolated soil bacteria from *Gastrodia elata* against *Fusarium* fungal diseases was conducted, and the bacterium YND5 was found to have a relatively good antagonistic effect against these fungal diseases. Phenotypic and data observation revealed that YND5 significantly antagonized the growth rate of *Fusarium* compared to untreated *Fusarium*. (See [link to relevant documentation]). Figures 4-7 .

[0051] To explore the biocontrol potential of bacteria from microbial resources, this embodiment systematically screened a bacterial strain YND5 isolated and purified from the rhizosphere soil of *Gastrodia elata*. The key pathogenic fungus causing diseases in *Gastrodia elata*—*Fusarium* (… Fusarium spp. Using YND5 as the target, the antagonistic activity was evaluated using the plate confrontation method. The results showed that a bacterium designated YND5 exhibited a particularly prominent antagonistic effect. Figure 4 As shown, under the same culture conditions, compared with the uninoculated blank control, the mycelial growth of *Fusarium* treated with YND5 was significantly inhibited, as evidenced by clearly visible inhibition zones, sparse and deformed colony edges, and severely limited expansion ability. Further quantitative analysis showed that YND5 inhibited *Fusarium* mycelial growth by as much as 61% (data expressed as mean 61.08871 ± 0.09 standard deviation, analyzed by t-test). P <0.01 (the difference is statistically significant), see [link / reference]. Figure 6 Based on the combined phenotype and data, this example reports for the first time that strain YND5 has a significant antagonistic effect on *Fusarium oxysporum* LY, a fungus that causes disease in *Gastrodia elata* isolated from the field, providing important experimental evidence for its development as a potential biocontrol agent.

[0052] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of *Pseudomonas gingerae* ( Pseudomonas 'gingeri' YND5, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 37512.

2. A microbial agent, characterized in that, It includes the *Pseudomonas gingera* YND5 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent comprises a bacterial solution; the concentration of *Pseudomonas gingerae* YND5 in the bacterial solution is OD. 600 =0.

6.

4. The method for preparing the microbial agent according to claim 2 or 3, characterized in that, Includes the following steps: The bacterial agent was obtained by inoculating *Pseudomonas gingerica* YND5 onto LB liquid medium and culturing it.

5. The application of the *Pseudomonas gingerae* YND5 according to claim 1, the fungal agent according to claim 2 or 3, or the fungal agent prepared by the preparation method according to claim 4 in antagonizing pathogenic fungi of *Gastrodia elata*, wherein the pathogenic fungus of *Gastrodia elata* is *Fusarium aromaticum* (…). Fusarium redolens ).

6. The application according to claim 5, characterized in that, The antagonistic fungi that cause diseases of Gastrodia elata include those that inhibit the growth of Fusarium aromaticum mycelium.

7. A method for antagonizing fungi that cause diseases in Gastrodia elata, characterized in that, The fungus used is *Pseudomonas gingerica* YND5 as described in claim 1, the fungal agent as described in claim 2 or 3, or the fungal agent prepared by the preparation method described in claim 4; the pathogenic fungus of *Gastrodia elata* is *Fusarium aromaticum*.

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