Pseudomonas bacterial combination, complex microbial inoculant and application thereof

Abstract

This invention provides a Pseudomonas spp. bacterial composition, a compound bacterial agent, and their applications, belonging to the field of microbial technology. This invention provides a Pseudomonas spp. bacterial composition, comprising Pseudomonas spp. bacteria (… Pseudomonas arcuscaelestis JF1 and ( Pseudomonas hutmensis JF2 and JF2 are growth-promoting bacteria screened from the rhizosphere soil of Corydalis yanhusuo. Pseudomonas bacteria JF1 and JF2 have the ability to solubilize phosphorus and produce iron carriers. As growth promoters, they can be used synergistically to promote the growth of Corydalis yanhusuo seedlings. In particular, they have a significant effect on promoting the accumulation of total alkaloids in Corydalis yanhusuo tubers. They can be used in the artificial cultivation of Corydalis yanhusuo, providing a theoretical basis for improving the yield and quality of Corydalis yanhusuo. They have important guiding significance for the rational and effective use of microorganisms to improve the quality and yield of "Qin medicine"—Corydalis yanhusuo.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a combination of Pseudomonas bacteria, a compound bacterial agent, and their applications. Background Technology

[0002] Corydalis ( Corydalis yanhusuo Corydalis (WTWang) is a highly effective herb for promoting blood circulation, removing blood stasis, regulating qi, and relieving pain, especially renowned for its analgesic properties. Corydalis thrives in warm, humid, and cool climates, and is best suited for cultivation in humus-rich, loose, and well-drained sandy loam. The size of the Corydalis tuber is a crucial indicator of its quality; larger tubers indicate superior quality and higher market prices. Corydalis tubers contain alkaloids with strong analgesic, sedative, and hypnotic effects; higher alkaloid content in the tuber indicates superior quality. Rhizosphere microorganisms are the plant's second genome, playing a vital role in plant growth and development. How to utilize rhizosphere growth-promoting bacteria to enhance the growth of Corydalis and increase the content of key active ingredients (total alkaloids) is a technical problem that needs to be solved. Summary of the Invention

[0003] The purpose of this invention is to provide a combination of Pseudomonas bacteria, a compound bacterial agent, and their applications. The combination of Pseudomonas bacteria and the compound bacterial agent of this invention can both promote the growth of Corydalis rhizome and increase the content of total alkaloids, the key active ingredient of Corydalis rhizome.

[0004] This invention provides an assemblage of Pseudomonas bacteria, including Pseudomonas bacteria deposited at CGMCC on June 11, 2025. Pseudomonas arcuscaelestis JF1 and Pseudomonas bacteria deposited at CGMCC on June 11, 2025 ( Pseudomonas hutmensis The Pseudomonas bacteria JF1 has the accession number CGMCC NO.34846; the Pseudomonas bacteria JF2 has the accession number CGMCC NO.34847.

[0005] Preferably, the ratio of the effective viable counts of Pseudomonas bacteria JF1 to Pseudomonas bacteria JF2 is 1:1.

[0006] The present invention also provides a compound bacterial agent comprising the Pseudomonas spp. bacterial combination described in the above-described scheme.

[0007] Preferably, the compound bacterial agent comprises a bacterial suspension of Pseudomonas spp. JF1 and a bacterial suspension of Pseudomonas spp. JF2; the volume ratio of the bacterial suspensions of Pseudomonas spp. JF1 and JF2 is 1:1; the OD values ​​of the bacterial suspensions of Pseudomonas spp. JF1 and JF2 are... 600 They are 0.8 respectively.

[0008] The present invention also provides the application of the above-described combination of Pseudomonas bacteria or the described compound bacterial agent in promoting the growth of Corydalis rhizome and / or increasing the total alkaloid content in Corydalis rhizome tubers.

[0009] Preferably, promoting the growth of Corydalis includes promoting the growth of Corydalis seedlings and / or promoting the growth of Corydalis tubers; the plant includes Corydalis.

[0010] Preferably, the promotion of Corydalis seedling growth includes at least one of 1) and 2):

[0011] 1) Promotes the growth of the above-ground and / or underground parts of Corydalis seedlings;

[0012] 2) Increase the fresh weight and / or dry weight of the whole Corydalis seedlings;

[0013] The promotion of Corydalis tuber growth includes at least one of (1) and (2):

[0014] (1) Increase the transverse diameter and / or longitudinal diameter of Corydalis tubers;

[0015] (2) Increase the fresh weight and / or dry weight of Corydalis tubers.

[0016] Preferably, the promotion of Corydalis growth is achieved by a combination of Pseudomonas bacteria dissolving inorganic phosphorus and / or secreting siderophores.

[0017] The present invention also provides a method for promoting the growth of Corydalis and / or increasing the total alkaloid content in Corydalis tubers, comprising the following steps: applying the combination of Pseudomonas bacteria described in the above scheme or the compound bacterial agent to Corydalis seedlings.

[0018] Preferably, the method of application includes root irrigation.

[0019] This invention provides an assemblage of Pseudomonas bacteria, comprising Pseudomonas bacteria ( Pseudomonas arcuscaelestis JF1 and ( Pseudomonas hutmensisThe *Pseudomonas* bacteria JF1 and JF2 are CGMCC NO.34846 and CGMCC NO.34847, respectively. In this invention, *Pseudomonas* bacteria JF1 and JF2 are growth-promoting bacteria screened from the rhizosphere soil of *Corydalis yanhusuo*. *Pseudomonas* bacteria JF1 and JF2 possess phosphorus-solubilizing and siderophore-producing abilities. As growth promoters, they can be used synergistically to promote the growth of *Corydalis yanhusuo* seedlings, especially showing a significant effect on the accumulation of total alkaloids in *Corydalis yanhusuo* tubers. They can be used in the artificial cultivation of *Corydalis yanhusuo*, providing a theoretical basis for improving the yield and quality of *Corydalis yanhusuo*. This has important guiding significance for the rational and effective use of microorganisms to improve the quality and yield of *Corydalis yanhusuo*, a traditional Chinese medicine from Qin.

[0020] Biological Preservation Instructions

[0021] Pseudomonas bacteria ( Pseudomonas arcuscaelestis JF1 was deposited on June 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34846.

[0022] Pseudomonas bacteria ( Pseudomonas hutmensis JF2 was deposited on June 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34847. Attached Figure Description

[0023] 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.

[0024] Figure 1 The colony characteristics of JF1 and JF2 bacteria are shown; where A represents JF1 ( Pseudomonas arcuscaelestis B is JF2 ( Pseudomonas hutmensis );

[0025] Figure 2 Gram staining results for JF1 and JF2 bacteria; where A represents JF1 ( Pseudomonas arcuscaelestis B is JF2 ( Pseudomonas hutmensis );

[0026] Figure 3The phosphorus solubility characteristics of JF1 and JF2 are given; where A represents JF1 and B represents JF2.

[0027] Figure 4 The characteristics of the iron carrier produced by JF1 and JF2 are given; where A represents JF1 and B represents JF2.

[0028] Figure 5 IAA characteristics are generated for JF1 and JF2; where A represents JF1 and B represents JF2.

[0029] Figure 6 To verify the growth-promoting effects of JF1 and JF2; where A shows the seedling growth status before root irrigation; B shows a comparison of growth in multiple pots, with the 6 pots on the left representing the seedling growth status of the JF1+JF2 treatment group and the 6 pots on the right representing the seedling growth status of the control group (TSA treatment group); C shows a comparison of growth in individual pots, with the left side showing the seedling growth status of the JF1+JF2 treatment group 30 days after root irrigation and the right side showing the seedling growth status of the control group (TSA treatment group) 30 days after root irrigation.

[0030] Figure 7 The image shows a comparison of whole-plant corydalis seedlings from the JF1+JF2 treatment group and the TSA control group after 30 days of root irrigation treatment; where A is the TSA control group and B is the JF1+JF2 treatment group.

[0031] Figure 8 Phenotypic comparison of Corydalis seedlings and tubers in the JF1+JF2 bacterial treatment group and the TSA control group after 30 days of root irrigation treatment;

[0032] Figure 9 A comparison of the total alkaloid content in Corydalis tubers between the JF1+JF2 bacterial treatment group and the TSA control group after 30 days of root irrigation treatment. Detailed Implementation

[0033] This invention provides an assemblage of Pseudomonas bacteria, comprising Pseudomonas bacteria ( Pseudomonas arcuscaelestis JF1 and ( Pseudomonas hutmensis The Pseudomonas bacteria JF1 has the accession number CGMCC NO.34846; the Pseudomonas bacteria JF2 has the accession number CGMCC NO.34847.

[0034] In this invention, the Pseudomonas bacteria JF1 and JF2 were screened from the rhizosphere soil of Corydalis rhizome in Shangyuanguan Town, Chenggu County, Hanzhong City, Shaanxi Province.

[0035] In this invention, the Pseudomonas bacteria JF1 and JF2 have strong phosphorus-solubilizing and siderophore-producing abilities; the combination of Pseudomonas bacteria has a significant promoting effect on the growth of the aboveground parts and tubers of Corydalis seedlings, effectively promoting the growth of Corydalis seedlings, and significantly promoting the accumulation of total alkaloids in Corydalis tubers.

[0036] In this invention, the nucleotide sequence of the 16S rDNA of the Pseudomonas bacterium JF1 is shown in SEQ ID NO.1.

[0037] In this invention, the nucleotide sequence of the 16S rDNA of the Pseudomonas bacterium JF2 is shown in SEQ ID NO.2.

[0038] In this invention, the colonies of the Pseudomonas spp. JF1 and JF2 are bright yellow, smooth, and round with a slightly raised center. After Gram staining, they are Gram-negative bacteria. Under an oil immersion microscope (100x magnification), strains JF1 and JF2 are rod-shaped bacteria.

[0039] In this invention, the Pseudomonas bacteria JF1 and JF2 do not fix nitrogen or secrete auxin.

[0040] In one embodiment, the ratio of the effective viable counts of Pseudomonas bacteria JF1 to Pseudomonas bacteria JF2 is 1:1.

[0041] The present invention also provides a compound bacterial agent comprising the Pseudomonas spp. bacterial combination described in the above-described scheme.

[0042] In one embodiment, the compound bacterial agent comprises a bacterial suspension of Pseudomonas spp. JF1 and a bacterial suspension of Pseudomonas spp. JF2; the volume ratio of the bacterial suspensions of Pseudomonas spp. JF1 and JF2 is 1:1; the OD values ​​of the bacterial suspensions of Pseudomonas spp. JF1 and JF2 are... 600 They are 0.8 respectively.

[0043] As one embodiment, the preparation method of the bacterial suspensions of Pseudomonas spp. JF1 and JF2 includes the following steps:

[0044] The *Pseudomonas* bacteria JF1 and JF2 were inoculated separately into TSA liquid medium and cultured with shaking to obtain bacterial suspensions. The OD of the bacterial suspensions was then adjusted. 600 The value is 0.8; the temperature of the shaking culture is 25℃; the rotation speed of the shaking culture is 200 rpm; and the shaking culture time is 1 day.

[0045] The present invention also provides the application of the above-described combination of Pseudomonas bacteria or the described compound bacterial agent in promoting the growth of Corydalis rhizome and / or increasing the total alkaloid content in Corydalis rhizome tubers.

[0046] In one implementation, promoting Corydalis growth includes promoting the growth of Corydalis seedlings and / or promoting the growth of Corydalis tubers; the plant includes Corydalis.

[0047] As one implementation, the promotion of Corydalis seedling growth includes at least one of 1) and 2):

[0048] 1) Promotes the growth of the above-ground and / or underground parts of Corydalis seedlings;

[0049] 2) Increase the fresh weight and / or dry weight of the whole Corydalis seedlings;

[0050] The promotion of Corydalis tuber growth includes at least one of (1) and (2):

[0051] (1) Increase the transverse diameter and / or longitudinal diameter of Corydalis tubers;

[0052] (2) Increase the fresh weight and / or dry weight of Corydalis tubers.

[0053] In one implementation, the promotion of Corydalis growth is achieved by a combination of Pseudomonas bacteria that dissolve inorganic phosphorus and / or secrete siderophores.

[0054] This invention also provides a method for promoting the growth of Corydalis yanhusuo and / or increasing the total alkaloid content in Corydalis yanhusuo tubers, comprising the following steps:

[0055] Apply the above-described combination of Pseudomonas bacteria or the aforementioned compound bacterial agent to Corydalis seedlings.

[0056] As one implementation method, the application includes root irrigation.

[0057] As one implementation method, the application rate for root irrigation is 30 mL per plant, calculated based on a bacterial suspension of Pseudomonas spp. JF1 and Pseudomonas spp. JF2; the OD values ​​of the bacterial suspensions of Pseudomonas spp. JF1 and Pseudomonas spp. JF2 are... 600 They are 0.8 respectively.

[0058] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a combination of Pseudomonas bacteria, a compound bacterial agent, and their applications provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0059] Example 1

[0060] I. Strain Isolation and Screening

[0061] Preparation of TSA solid medium: Weigh 15 g tryptone, 5 g soybean peptone, 5 g sodium chloride, and 15 g agar, add water to a final volume of 1 L, adjust the pH to 7.3 ± 0.2, and autoclave at 121℃ for 15 min. The liquid medium used in the experiment was the solid medium formula without agar.

[0062] At the Corydalis yanhusuo experimental planting base in Shangyuanguan Town, Chenggu County, Hanzhong City, Shaanxi Province, a five-point sampling method was used, selecting 20 Corydalis yanhusuo plants at each point. The plants were dug up along with the soil, and large clumps of soil around the tubers were shaken off, leaving about 1 mm of soil adhering to the surface of the tubers. Then, an appropriate amount of undamaged tubers were selected and placed in a sterile plastic bottle. The soil adsorbed on the surface of the tubers was washed off with about 100 mL of PBS buffer. The washing was repeated twice. The soil suspension was placed in a 250 mL Erlenmeyer flask and cultured on a shaker (25℃, 120 r / min) for 1 h, followed by sonication at 140 kHz for 40 s.

[0063] Take 1 mL of soil suspension and dilute it stepwise to 10. -4 Dilute 200 μL to 10 times the volume. -4 A soil suspension of a certain concentration was spread onto TSA medium. After the surface of the medium dried, the culture was inverted and incubated at 25°C for 16–18 h. Newly grown single colonies were picked and cultured until no new colonies grew. The grown bacterial colonies were then transferred to 1 mL of the corresponding liquid medium and incubated overnight on a shaker (200 rpm, 25°C). After multiple streak isolation and purification in three zones, pure bacterial cultures were obtained. Based on the morphology and characteristics of the bacteria on the medium, preliminary classification and removal of duplicates were performed. The purified bacteria were named JF1 and JF2.

[0064] II. Strain Identification

[0065] 1. Identification and preservation of JF1 and JF2 strains

[0066] Ten replicates of JF1 and JF2 bacterial cultures cultured overnight on a shaker were amplified by PCR using primers 27F (5'-agagtttgatcctggctcag-3', SEQ ID NO.3) and 1492R (5'-tacggttaccttgttacgactt-3', SEQ ID NO.4) and then sent for identification. The PCR reaction system (25 μL) consisted of: 1 μL of each primer, 1 μL of JF1 and JF2 bacterial cultures respectively, 12.5 μL of 2×Taq PCR Mix, and ddH2O to a final volume of 25 μL. The PCR reaction conditions were as follows: denaturation at 94℃ for 3 min, followed by denaturation at 94℃ for 25 s, annealing at 55℃ for 25 s, extension at 72℃ for 1.5 min, for 35 cycles, and a final extension at 72℃ for 5 min. Five 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 the EzBioCloud database (https: / / www.ezbiocloud.net / ). Eighty-hundred μL of JF1 and JF2 bacterial cultures were added to eighty-hundred μL of 50% sterile glycerol and mixed thoroughly. The mixture was then stored at -80℃. The comparison results showed that JF1 was... Pseudomonas arcuscaelestis pseudomonas hutmensis

[0067] 2. Morphological identification of strains

[0068] Based on the colony characteristics of the isolated strain after purification and culture, such as... Figure 1 Table 1 shows that the colonies of JF1 and JF2 bacteria are bright yellow, smooth, and round with a slightly raised center. After Gram staining, they are Gram-negative bacteria. Under an oil immersion microscope (100x magnification), JF1 and JF2 strains are rod-shaped bacteria. Figure 2 (As shown).

[0069] Table 1. Colony morphology of JF1 and JF2

[0070]

[0071] 3. Physiological and biochemical identification

[0072] Physiological and biochemical analyses of JF1 and JF2 bacteria were performed in accordance with the "Handbook for Systematic Identification of Common Bacteria": Gram staining, carbohydrate decomposition, VP determination, methyl red, citrate utilization, starch hydrolysis, indole, catalase, oxidase, hydrogen sulfide, and gelatin liquefaction experiments. The results are shown in Table 2.

[0073] Table 2 Physiological and biochemical characteristics of JF1 and JF2

[0074]

[0075] III. Functional Identification of Strains

[0076] 1. Nitrogen fixation

[0077] Nitrogen fixation of JF1 and JF2 bacteria was analyzed using Ashby nitrogen-free medium. Based on the characteristics of Ashby medium, only nitrogen-fixing strains can grow normally on this medium. The activated bacterial suspensions (OD200) of JF1 and JF2 bacteria were then analyzed. 600 = 0.5) was added to Ashby liquid medium (1:50 / v:v) and cultured at 25℃ and 200 rpm for 1 day. The bacterial culture was then streaked onto Ashby solid medium and cultured three times consecutively. If the strain grew normally, it was considered to have nitrogen-fixing activity. (5 replicates) The results showed that JF1 and JF2 strains could not grow normally, indicating that these two strains did not have nitrogen-fixing activity.

[0078] 2. Phosphorus dissolution

[0079] Phosphate solubilization of JF1 and JF2 bacteria was analyzed using PVK phosphate-solubilizing medium (10 g glucose, 0.5 g (NH4)2SO4, 0.03 g manganese sulfate monohydrate, 0.03 g FeSO4·7H2O, 0.3 g sodium chloride, 0.3 g magnesium sulfate heptahydrate, 0.3 g KCl, 2.5 g Ca3(PO4)2, 15 g agar, diluted to 1 L with water (pH=7.4±0.2), autoclaved at 121℃ for 15 min). Two μL of activated JF1 and JF2 bacterial suspensions (OD200) were collected using the spot collection method. 600 =0.5), and inoculated onto PVK phosphate-solubilizing medium. After incubation at 25℃ in the dark for 7 days, the formation of a transparent phosphate-solubilizing zone around the colony was observed to determine whether the strain had phosphate-solubilizing activity. The ratio of the diameter of the transparent zone (D) to the diameter of the colony (d) indicated the strength of the strain's activity. A blank LB solid medium (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar, diluted to 1 L with water, autoclaved at 121℃ for 15 min) was used as a control group, with 5 replicates. The results showed that JF1 and JF2 strains formed transparent zones around their colonies, and the average phosphate-solubilizing zone ratios (D / d) were 2.6 and 3.2, respectively, indicating that these two strains had strong phosphate-solubilizing activity. Figure 3 As shown in Table 3.

[0080] Table 3. Determination of phosphate solubility diameter in JF1 and JF2 strains

[0081]

[0082] 3. Role as a carrier for iron production

[0083] The siderophoretic activity of JF1 and JF2 bacteria was analyzed using CAS (Croatian Blue) medium (purchased from Qingdao Haibo Biotechnology Co., Ltd., catalog number HB9132; 10.87 g was weighed, added to 1000 mL of sterile water, boiled until completely dissolved, then autoclaved at 121℃ for 15 min, mixed well, and poured into plates). 2 μL of activated JF1 and JF2 bacterial suspensions (OD) were collected separately using the spot-drop method. 600=0.5), and inoculated onto CAS solid medium. After incubation at 25℃ in the dark for 7 days, the formation of a yellow halo around the colony was observed. The ratio of the halo diameter (D) to the colony diameter (d) indicates the strength of the strain's ability and determines whether the strain has siderogenic activity. A blank LB solid medium (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar, diluted to 1 L with water, autoclaved at 121℃ for 15 min) was used as the control group, with 5 replicates. The results showed that yellow halos formed around the JF1 and JF2 bacteria, and the average halo ratios (D / d) were 4.04 and 4.38, respectively, indicating that JF1 and JF2 bacteria have strong siderogenic activity. Figure 4 As shown in Table 4.

[0084] Table 4. Determination of the diameter of the yellow halo surrounding siderophores produced by JF1 and JF2 strains.

[0085]

[0086] 4. Detection of indole-3-acetic acid (IAA) production

[0087] Qualitative analysis of IAA production in strain J-2 was performed using the Salkowski colorimetric method. Single colonies of activated JF1 and JF2 strains were inoculated into LB liquid medium without L-tryptophan (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S20082) and LB liquid medium containing L-tryptophan (1.0 g / L), respectively, and cultured at 25℃ and 200 rpm for 1 day. Using the L-tryptophan-free LB liquid fermentation medium as a control, the fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant was mixed with 2 mL of Salkowski colorimetric solution (purchased from Feijing Biotechnology Co., Ltd., catalog number PH1941) and incubated in the dark for 30 min. The color of the solution was observed to determine whether the strain had IAA production capacity. The results showed that the solution color did not turn red before and after treatment with JF1 and JF2 strains, indicating that JF1 and JF2 strains did not produce IAA, therefore no further quantitative analysis was performed. Figure 5 As shown in Table 5.

[0088] Table 5. IAA Characteristic Verification of JF1 and JF2 Products

[0089]

[0090] Note: + indicates a positive reaction; - indicates a negative reaction.

[0091] Example 2

[0092] Evaluation of the growth-promoting effect of Corydalis yanhusuo strains in pot experiments on seedlings

[0093] 1. Root irrigation with bacterial solution

[0094] The identified JF1 ( Pseudomonas arcuscaelestis ) and JF2 ( Pseudomonas hutmensis The bacterial suspension was inoculated into TSA liquid medium and cultured at 25°C with shaking at 200 rpm for 1 day. The OD of the obtained bacterial suspension was adjusted. 600 =Approximately 0.8, used for subsequent root irrigation with bacterial solution, while the control treatment was TSA liquid culture medium treated in the same way.

[0095] 2. Sample selection and processing

[0096] About 30 Corydalis seeds of suitable size and fullness were selected and subjected to low-temperature germination treatment. After germination, Corydalis seeds were planted in pots (all Corydalis seeds were disinfected with 75% ethanol before planting, and all soil was sterilized at 121℃ for 15 minutes. One Corydalis seedling was planted in each pot and the same weight of sterilized soil was added). After all the seeds had germinated, they were divided into groups, and 15 Corydalis seedlings of the same growth and health status were selected from each group and labeled as JF1, JF2, JF1+JF2, and TSA control.

[0097] 3. Verification of the effect of promoting fertility

[0098] On the 8th day after the Corydalis seedlings sprouted in pots (growth status as follows) Figure 6 As shown in Figure A), *Corydalis yanhusuo* seedlings were treated with 30 mL / plant of root-drenching bacterial solution, while an equal volume of sterile TSA liquid culture medium was used as a control group (TSA control group). After root-drenching treatment, the *Corydalis yanhusuo* seedlings were placed in a sterile light incubator for natural growth. During the growth period, they were watered with sterile water every 3 days, with day and night temperatures of 22℃ and 18℃, a light duration of 14 h, and a darkness duration of 10 h. After 30 days of root-drenching treatment, the above-ground and underground lengths, whole plant fresh weight, dry weight, tuber transverse and longitudinal diameters, fresh weight, dry weight, and total alkaloid content of the *Corydalis yanhusuo* tubers in each treatment group were measured (growth status after treatment is shown in Figure A). Figure 6 (As shown in B and C). (Above-ground part: The height from the top of each Corydalis plant to the surface of the soil in the flowerpot was measured using a ruler; Below-ground part: After the above-ground part of the Corydalis plant was measured, the above-ground part was cut off with scissors, and the length of the below-ground part was measured with a ruler after the plant was dug out; Dry weight determination: After washing the soil, the plant was freeze-dried, and the dry weight was measured after the freeze-drying was completed; Total alkaloid content (3 replicates): The total alkaloid content determination kit was purchased from Baisha Biotechnology Co., Ltd., catalog number BL1762B).

[0099] 4. Results Analysis

[0100] Figure 7 The image shows a comparison of whole-plant growth of Corydalis seedlings in the JF1+JF2 treatment group and the TSA control group after 30 days of root drenching treatment. The image shows that the overall growth of Corydalis seedlings treated with JF1+JF2 bacterial solution was better than that treated with TSA medium alone. Furthermore, the aboveground parts of the seedlings treated with the bacterial solution were longer than those in the TSA medium treatment group. Figure 8 Tables 6 and 7 show the comparison of aboveground and underground part length, whole plant fresh weight, dry weight, tuber transverse diameter, longitudinal diameter, fresh weight, and dry weight of Corydalis seedlings in the JF1+JF2 experimental group and the TSA control group after 30 days of root drenching treatment. The tables show that after root drenching with JF1+JF2 bacterial solution, the overall Corydalis seedlings treated with this solution showed better aboveground and underground part length, whole plant fresh weight, dry weight, tuber transverse diameter, longitudinal diameter, fresh weight, and dry weight compared to those treated with only TSA medium. In particular, the aboveground growth was significantly promoted, increasing by 31.95% compared to the TSA control group (p<0.05). Figure 9 Table 8 shows a comparison of the total alkaloid content in Corydalis tubers treated with JF1+JF2 bacteria and the TSA control group after 30 days of root irrigation treatment. The results indicate that the total alkaloid content in Corydalis tubers treated with JF1+JF2 bacteria was significantly increased (p<0.05), by 163.73% compared to the TSA control group. Tables 6, 7, and 8 further demonstrate that the combined application of JF1+JF2 bacteria significantly improved the fresh and dry weight of Corydalis seedlings, the transverse and longitudinal diameters of Corydalis tubers, and the total alkaloid content compared to applying JF1 or JF2 alone. These results indicate that the synergistic treatment of JF1+JF2 bacteria can effectively promote the growth of Corydalis seedlings.

[0101] Table 6. Phenotypic comparison of Corydalis seedlings in the JF1+JF2 bacterial treatment group and the TSA control group 30 days after root drenching treatment.

[0102]

[0103] Table 7. Phenotypic comparison of Corydalis tubers between the JF1+JF2 bacterial treatment group and the TSA control group 30 days after root drenching treatment.

[0104]

[0105] Table 8 Comparison of total alkaloid content after JF1+JF2 treatment

[0106]

[0107] 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. An assemblage of Pseudomonas bacteria, characterized in that, Including Pseudomonas bacteria deposited at CGMCC on June 11, 2025. Pseudomonas arcuscaelestis JF1 and Pseudomonas bacteria deposited at CGMCC on June 11, 2025. Pseudomonas hutmensis JF2; The Pseudomonas spp. bacteria Pseudomonas arcuscaelestis JF1 has the accession number CGMCCNO.34846; the described Pseudomonas bacteria Pseudomonas hutmensis JF2 has the accession number CGMCCNO.34847.

2. The Pseudomonas spp. bacterial assemblage according to claim 1, characterized in that, The Pseudomonas spp. bacteria Pseudomonas arcuscaelestis JF1 and Pseudomonas bacteria Pseudomonas hutmensis The effective viable bacteria ratio of JF2 is 1:

1.

3. A compound microbial agent, characterized in that, Includes the Pseudomonas spp. bacterial assemblage as described in claim 1 or 2.

4. The compound microbial agent according to claim 3, characterized in that, The compound microbial agent includes Pseudomonas bacteria. Pseudomonas arcuscaelestis JF1 bacterial suspension and Pseudomonas spp. Pseudomonas hutmensis JF2 bacterial suspension; the aforementioned Pseudomonas spp. bacteria Pseudomonas arcuscaelestis JF1 bacterial suspension and Pseudomonas spp. Pseudomonas hutmensis The volume ratio of the bacterial suspension of JF2 was 1:1; the Pseudomonas bacteria... Pseudomonas arcuscaelestis JF1 bacterial suspension and Pseudomonas spp. Pseudomonas hutmensis OD of JF2 bacterial suspension 600 They are 0.8 respectively.

5. The application of the Pseudomonas spp. bacterial combination as described in claim 1 or 2, or the compound bacterial agent as described in claim 3 or 4, in promoting the growth of Corydalis yanhusuo and / or increasing the total alkaloid content in Corydalis yanhusuo tubers.

6. The application according to claim 5, characterized in that, The promotion of Corydalis growth includes promoting the growth of Corydalis seedlings and / or promoting the growth of Corydalis tubers.

7. The application according to claim 6, characterized in that, The promotion of Corydalis seedling growth includes at least one of 1) and 2): 1) Promotes the growth of the above-ground and / or underground parts of Corydalis seedlings; 2) Increase the fresh weight and / or dry weight of the whole Corydalis seedlings; The promotion of Corydalis tuber growth includes at least one of (1) and (2): (1) Increase the transverse diameter and / or longitudinal diameter of Corydalis tubers; (2) Increase the fresh weight and / or dry weight of Corydalis tubers.

8. The application according to claim 5, characterized in that, The promotion of Corydalis growth is achieved by a combination of Pseudomonas bacteria that dissolve inorganic phosphorus and / or secrete siderophores.

9. A method for promoting the growth of Corydalis yanhusuo and / or increasing the total alkaloid content in Corydalis yanhusuo tubers, characterized in that, Includes the following steps: Apply the combination of Pseudomonas bacteria as described in claim 1 or 2, or the compound bacterial agent as described in claim 3 or 4, to Corydalis seedlings.

10. The method according to claim 9, characterized in that, The application method includes root irrigation.

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