A bacterial composition for promoting the growth and saponin accumulation of panax notoginseng

Abstract

The application discloses a bacterial composition for promoting the growth and saponin accumulation of Panax notoginseng, which is composed of three strains, including Paenarthrobacter nicotinovorans SQ 15, Bacillus velezenis SQ 24 and Pseudomonas nitroreducens SQ 63, which are preserved in the China General Microbiological Culture Collection Center with the preservation numbers of CGMCC No.31366, CGMCC No.31369 and CGMCC No.31367 respectively. The three strains of beneficial bacteria have the functions of resisting diseases and promoting growth, can promote the growth of the tuberous roots of Panax notoginseng, increase the saponin accumulation in the roots of Panax notoginseng, prevent the root rot of Panax notoginseng and improve the quality of Panax notoginseng.

Description

Technical Field

[0001] This invention relates to the fields of bacterial strain technology and cultivation of Chinese medicinal materials, specifically to a bacterial composition that promotes the growth of Panax notoginseng and the accumulation of saponins. Background Technology

[0002] Panax notoginseng, a perennial herb belonging to the genus Panax in the family Araliaceae, is used medicinally for its dried roots and rhizomes. It is believed to have blood-activating, stasis-removing, swelling-reducing, and pain-relieving effects, and is a unique and precious traditional Chinese medicine. The size of the main root is often used as an indicator of quality. In the market, the number of roots per 500 grams of Panax notoginseng represents its grade and medicinal value; larger roots generally command higher prices. Saponins are the main active ingredients responsible for the medicinal effects of Panax notoginseng. Modern pharmacological studies have shown that saponins have protective effects on the cardiovascular system, anti-thrombotic effects, blood sugar and lipid regulation, anti-tumor effects, immune enhancement, and anti-aging effects, making them widely applicable in clinical practice. The root is the primary extraction site for saponins; therefore, increasing the accumulation of saponins in the root is crucial for cultivating high-quality Panax notoginseng. However, Panax notoginseng has strict environmental requirements, and its yield and quality are easily limited by factors such as soil and climate. The decline in quality caused by the expansion of planting areas has already begun to emerge. Furthermore, Panax notoginseng has a long growth cycle, requiring three years in the ground before harvest. Continuous cropping poses significant obstacles, and soil-borne diseases are highly prevalent during its growth cycle, severely impacting yield and quality. Therefore, improving yield and quality is one of the key challenges in Panax notoginseng cultivation.

[0003] Rhizosphere soil microorganisms are crucial for crop growth and health. Studies have shown that rhizosphere soil microorganisms can promote the growth of medicinal plants, enhance their resistance to stress and disease, and promote the synthesis and accumulation of active ingredients. In recent years, the relationship between the rhizosphere microecology and the quality of Chinese medicinal herbs has received increasing attention. Many probiotics have been applied to the cultivation of medicinal plants and have been proven to improve yield or quality. The application of microbial agents has become an important means to alleviate the overuse of pesticides and chemical fertilizers in the cultivation of medicinal plants and to improve the effectiveness and safety of Chinese medicinal herbs. IAA stands for indoleacetic acid. Currently, IAA-producing strains are used on plants, as recorded in existing patents (CN202111311796.9), but their use in Panax notoginseng is not observed. Furthermore, microbial agents specifically developed for Panax notoginseng are still very limited, and single agents are unlikely to comprehensively solve the problems of diseases, poor growth, and quality decline that occur in Panax notoginseng production. Summary of the Invention

[0004] The purpose of this invention is to provide a bacterial composition that promotes the growth and saponin accumulation of Panax notoginseng. Addressing the issues of slow growth, susceptibility to disease, and poor quality of Panax notoginseng, this invention screens beneficial bacterial strains from the rhizosphere soil of Panax notoginseng that can antagonize Panax notoginseng pathogens and produce IAA (inotropic acid), and combines them. The effects on major Panax notoginseng pathogens, biomass, root growth, and saponin accumulation are verified under laboratory and field conditions. This aims to lay the foundation for developing beneficial microbial agents that promote the growth and saponin accumulation of Panax notoginseng, thereby promoting tuberous root growth, increasing saponin accumulation in Panax notoginseng roots, preventing root rot, and improving the quality of Panax notoginseng.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A type of *Paenarthrobacter nicotinovorans* SQ 15, used to produce IAA and inhibit *Fusarium oxysporum*;

[0007] The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 31366; the deposit date of accession number CGMCC No. 31366 is July 19, 2024, and it is located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] A type of Bacillus velezenis, specifically Bacillus velezenis SQ 24, is used to inhibit Fusarium oxysporum, Fusarium solani, and Cyclospora demise.

[0009] The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31369. The deposit date for accession number CGMCC No. 31369 is July 19, 2024, and the location is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0010] A nitroreducing Pseudomonas bacterium, Pseudomonas nitroreducens SQ 63, is used to inhibit Fusarium oxysporum, Fusarium solani, and Cyclospora nitroreducens.

[0011] The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31367. The deposit date for accession number CGMCC No. 31367 is July 19, 2024, and it is located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. A bacterial composition promoting the growth and saponin accumulation of Panax notoginseng, the bacterial composition comprising: *Paenarthrobacter nicotinovorans* SQ 15, *Bacillus velezenis* SQ 24, and *Pseudomonas nitroreducens* SQ 63 as described in claim 1.

[0012] Application of a bacterial composition that promotes the growth and saponin accumulation of Panax notoginseng in promoting the growth of Panax notoginseng.

[0013] Application of a bacterial composition that promotes the growth and saponin accumulation of Panax notoginseng in promoting saponin accumulation in Panax notoginseng roots.

[0014] Furthermore, the method applied is as follows:

[0015] Single-strain seed cultures of SQ 15, SQ 24, and SQ 63 were inoculated onto culture medium and cultured on a shaker. After incubation, the culture medium was removed by centrifugation, and the cultures were resuspended in sterile water. The OD values ​​were then calculated. 600 The value determines the dilution factor, and the solution is prepared with concentrations of 10. 8 A mixed bacterial culture of SQ 15, SQ 24, and SQ 63 at cfu / mL;

[0016] After Panax notoginseng seedlings emerge, the plants are irrigated with a mixed bacterial solution.

[0017] Furthermore, the culture medium is selected from LB liquid medium, comprising: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder.

[0018] Furthermore: 200 mL of mixed bacterial solution was added to each Panax notoginseng plant, and it was added again after a one-month interval, with the mixed bacterial solution being added three times consecutively.

[0019] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0020] (1) Based on the rhizosphere soil microorganisms of Panax notoginseng, this invention uses a variety of culture media to isolate as many soil bacteria as possible, screens the antibacterial activity and IAA production activity of the pathogens of Panax notoginseng root rot, and finally identifies SQ 15, SQ24 and SQ63. At the same time, the suspensions of SQ 15, SQ 24 and SQ63 are prepared by propagation and pot and field experiments to obtain a bacterial combination that can promote the growth of Panax notoginseng tubers, increase the content of Panax notoginseng saponins and prevent Panax notoginseng root rot.

[0021] (2) This invention can effectively promote the growth of Panax notoginseng tubers and increase the accumulation of Panax notoginseng saponins, avoiding the problem of excessive growth of Panax notoginseng tubers and reduced active ingredients caused by the abuse of growth regulators in Panax notoginseng cultivation; it can improve the beneficial microbial community in the soil and enhance the protection of the ecological environment; it can antagonize the pathogens of Panax notoginseng root rot and has a preventive and control effect on Panax notoginseng root rot, reducing the input cost of Panax notoginseng disease management, and at the same time providing new materials for the development of medicinal microbial agents.

[0022] (3) The method of this invention is scientific, reliable, and stable; the SQ 15, SQ 24, and SQ 63 strains are derived from the rhizosphere soil of Panax notoginseng, have a strong ability to adapt to the soil environment of Panax notoginseng, and can improve the soil microbial ecological environment. After using the mixed bacterial solution, it can effectively promote the fresh weight growth of Panax notoginseng seedlings by 23.45%, promote the dry weight growth of 3-year-old Panax notoginseng tubers by 9.57% to 23.87%, increase the accumulation of saponins by 8.17% to 26.52%, and reduce the risk of quality decline caused by blindly increasing production and abusing growth regulators; the SQ 24 and SQ 63 strains have an inhibition rate of 12.38% to 65.95% against the growth of various root rot pathogens of Panax notoginseng in the culture medium. This bacterial composition can also prevent the occurrence of root rot of Panax notoginseng, further ensuring the yield increase of Panax notoginseng. Attached Figure Description

[0023] Figure 1 This is a plate contrast diagram of the strains SQ 15, SQ 24, and SQ63 of this invention with Fusarium oxysporum, Fusarium solani, and Cyclospora destructosa.

[0024] Figure 2 This is a verification diagram showing the production of IAA by the strains SQ 15, SQ 24, and SQ63 of this invention.

[0025] Figure 3 The images show the morphological characteristics of strains SQ 15, SQ 24, and SQ63 of this invention.

[0026] Figure 4 This is a phylogenetic diagram of the 16S sequence homology of strains SQ15, SQ24, and SQ63 of this invention.

[0027] Figure 5 Images showing the growth of Panax notoginseng seedlings after application of the bacterial composition of the present invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] Example 1:

[0030] I. Isolation of rhizosphere soil bacteria from Panax notoginseng

[0031] Soil bacteria were isolated using a variety of culture media, including the following five:

[0032] (1) LB medium: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agar powder 15g / L;

[0033] (2) NA medium: peptone 10g / L, beef extract powder 3g / L, sodium chloride 5g / L, agar powder 15g / L;

[0034] (3) TSA medium: tryptone 17.0 g / L, soybean papain hydrolysate 3.0 g / L, sodium chloride 5.0 g / L, agar powder 15 g / L;

[0035] (4) R2A medium: yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, potassium dihydrogen phosphate 0.3 g / L, anhydrous magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15.0 g / L;

[0036] (5) Modified Gao's No. 1 culture medium: soluble starch 20g / L, sodium chloride 0.5g / L, potassium nitrate 1g / L, dimethyl hydrogen phosphate 0.5g / L, magnesium sulfate 0.5g / L, ferrous sulfate 0.01g / L, agar powder 15g / L.

[0037] Weigh 5g of rhizosphere soil from Panax notoginseng and mix it with 45mL of sterile water. Place the mixture in a shaker at 25℃ and 180r / min for 30min to prepare a soil suspension. Let it stand for 1h, and after separation, take 1mL of the upper soil suspension and dilute it 1000 times, 10000 times, and 100000 times with sterile water. Take 100μL of the 1000-fold diluted soil suspension and spread it evenly on the above LB, NA, TSB, R2A, and modified Gao's No. 1 medium. Take 100μL of the 10000-fold diluted soil suspension and spread it evenly on the above LB, NA, TSB, R2A, and modified Gao's No. 1 medium. Take 100μL of the 100000-fold diluted soil suspension and spread it evenly on the above LB, NA, TSB, R2A, and modified Gao's No. 1 medium. The culture medium diluted with sterile water was inverted and incubated at 37°C for 24 hours to obtain single colonies. Diluting with sterile water at concentrations of 10,000 and 100,000 times was found to be too low, resulting in only a few to a dozen colonies growing on the plates. For single-cell purification, a 1,000-fold dilution was used, resulting in dozens to over 100 colonies growing on the plates. Single colonies of NA, TSB, R2A, and modified Gao's No. 1 could all grow on LB medium; therefore, LB medium was used for purification, as the same medium facilitates observation of colony morphology. Single colonies with different morphologies were selected and streaked onto new LB medium for purification. After multiple streaking purifications, purified single colonies were obtained for later use.

[0038] Purified single colonies were picked from the purified culture medium and inoculated into LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride). The culture was incubated at 37°C and 220 rpm for 1 day. 1 mL of the bacterial culture was mixed with 1 mL of 50% sterile glycerol and stored at -80°C as a single-cell seed culture. This seed culture was used for screening antagonistic bacteria and IAAs of pathogens, and for inoculation when preparing mixed bacterial cultures.

[0039] II. Screening and Identification

[0040] 2.1 Screening of antagonistic bacteria against pathogens

[0041] 2.11 Screening of antagonistic bacteria against pathogens

[0042] Using PDA medium (12 g / L potato extract, 20 g / L glucose, 15 g / L agar), *Fusarium oxysporum*, *Fusarium solani*, and *Strombocytosporum destructum*, the pathogens causing root rot of Panax notoginseng, were used as target bacteria. The target bacteria were inoculated in the center of the PDA medium, and the isolated and purified single-bacterial seed solution was spot-inoculated 2 cm from the center of the plate. Plates without bacterial inoculation served as controls. Each treatment was repeated 5 times. All plates of *Fusarium oxysporum* and *Fusarium solani* were inverted in a 30°C incubator, and all plates of *Strombocytosporum destructum* were inverted in a 25°C incubator. After 7 days of growth, the diameter of the target pathogens was measured.

[0043] 2.12 Screening results of antagonistic bacteria against pathogens

[0044] Based on visual morphological observation, the antagonistic bacteria of purified single-colony pathogens were selected. Only SQ 15, SQ 24 and SQ 63 showed significant antagonistic effects against Panax notoginseng pathogens. The results are shown in Table 1.

[0045] Table 1. Antagonistic effects of strains against the pathogenic fungus causing root rot in Panax notoginseng.

[0046]

[0047]

[0048] Note: Lowercase letters in the same column of the table represent significant differences in the antagonistic effects of the strains on the pathogen, P<0.05.

[0049] Depend on Figure 1 As shown in Table 1, after 7 days of growth, the colony diameters of the three pathogens on plates inoculated with SQ 24 and SQ 63 were significantly smaller (P<0.05) than those on the CK plate, indicating that SQ 24 and SQ 63 had significant inhibitory effects on all three pathogens. Furthermore, the colony diameters of the three pathogens on plates inoculated with SQ 24 were also significantly smaller (P<0.05) than those on plates inoculated with SQ 63. The inhibition rate of the antagonistic bacteria against the pathogens was calculated using [(pathogen diameter on CK plate - pathogen diameter on plate inoculated with antagonistic bacteria) / pathogen diameter on CK plate]. The average inhibition rates of SQ 24 against *Fusarium oxysporum*, *Fusarium solani*, and *Cyclocarya spp.* reached 47.52%, 65.95%, and 49.52%, respectively, while SQ 24 showed significantly lower inhibition rates. The average inhibition rates of SQ 24 against the three pathogens were 20.21%, 31.45%, and 12.38%, respectively, indicating that the inhibitory effect of SQ 24 was significantly better than that of SQ 63. The colony diameter of Fusarium oxysporum on the SQ15 inoculated plate was 2.52 cm, which was significantly lower (P<0.05) than that on the CK plate (2.82 cm), indicating that SQ 15 had a slight inhibitory effect on Fusarium oxysporum.

[0050] 2.2 Screening of IAA-producing strains

[0051] 2.21 Screening of IAA-producing strains

[0052] The isolated and purified single-strain seed culture was inoculated into LB liquid medium containing L-tryptophan (100 mg / L) and incubated at 37°C and 220 rpm for 1 day. 50 μL of the bacterial culture was dropped onto a white ceramic plate, and 50 μL of pre-prepared Salkowski colorimetric solution (a mixture of 50 mL of 35% perchloric acid and 1 mL of 0.5 mol / L FeCl3 solution) was added simultaneously. LB liquid medium containing L-tryptophan (100 mg / L) without bacterial inoculation served as a negative control. The white ceramic plate was placed at room temperature in the dark for 30 minutes and observed; a red color indicated that the strain could produce IAA.

[0053] 2.22 Screening results of IAA-producing strains

[0054] When L-tryptophan, as a precursor for IAA synthesis, is added to the culture medium, IAA-producing strains can convert it to IAA via the tryptophan pathway; while indole compounds can react with Fe in the presence of perchloric acid. 3+ A color reaction occurs, known as the Salkowski color reaction, in which indoleacetic acid (IAA) produces a pink to red color, while tryptophan produces a yellow color. For example... Figure 2 As shown, SQ 15 bacterial culture turned pink after being added to Salkowski colorimetric solution, indicating that SQ 15 can produce IAA using tryptophan; while SQ 24 and SQ 63 bacterial cultures and the negative control turned pale yellow, indicating that SQ 24 and SQ 63 cannot produce IAA via the tryptophan pathway.

[0055] 2.3 Identification of antagonistic and growth-promoting bacteria

[0056] 2.31 Identification of antagonistic and growth-promoting bacteria

[0057] Strains SQ 15, SQ 24 and SQ 63 were inoculated into LB solid medium and cultured at 37℃ and 220 r / min for 7 days. Single colony morphology was then observed.

[0058] Strains SQ 15, SQ 24 and SQ 63 were inoculated into LB liquid medium (10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride) and cultured at 37°C and 220 rpm for 1 day. 1 mL of the bacterial culture was then used to extract bacterial genomic DNA using a bacterial extraction kit (TIANBampBacteria DNA Kit, Tiangen Biotech Co., Ltd., Beijing). Bacterial DNA was amplified using 16S universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-TACGGCTACCTTGTACGACTT-3'). The PCR reaction system included: 25 μL of 2×Master Mix, 2 μL each of forward and reverse primers, 19 μL of dd water, and 2 μL of DNA template. The PCR conditions were: 95℃ pre-denaturation for 5 min; 95℃ melting for 40 s, 52℃ annealing for 1 min, 72℃ extension for 90 s, 35 cycles; and 72℃ holding for 10 min. The obtained PCR products were sent to Sangon Biotech Co., Ltd. for sequencing. The obtained 16S sequences of the strain were analyzed for homology using NCBI-BLAST, and sequences with high similarity were obtained. A phylogenetic tree was constructed using MEGA 6.0 software.

[0059] 2.32 Identification results of antagonistic and growth-promoting bacteria

[0060] On LB solid medium, SQ 15 colonies were yellow, opaque, with a raised and moist surface and regular edges. Their 16S nucleic acid sequence was identified as *Paenarthrobacter nicotinovorans* by NCBI-BLAST. SQ24 colonies had relatively regular edges, a raised and moist surface, a viscous texture, and were milky white and opaque. Their 16S nucleic acid sequence was identified as *Bacillus velezensis* by NCBI-BLAST. SQ 63 colonies were pale yellow and translucent, with a smooth surface and regular edges. Their 16S nucleic acid sequence was identified as *Pseudomonas nitroreducens* by NCBI-BLAST. Figure 3 and Figure 4 ).

[0061] In summary, colonies were selected and purified based on visual morphological observation. The purified single strains underwent antagonistic screening against the pathogen causing root rot in Panax notoginseng and IAA functional screening, resulting in single strains SQ 15, SQ 24, and SQ 63, which are effective in preventing root rot or promoting the growth of Panax notoginseng. The PCR products of these three single strains were sequenced, and the 16S sequences were analyzed for homology using NCBI-BLAST. A phylogenetic tree was constructed using MEGA 6.0 software to identify the species of these three single strains. Finally, the three single strains were identified as: *Paenarthrobacter nicotinovorans* SQ 15, *Bacillus velezenis* SQ 24, and *Pseudomonas nitroreducens* SQ 63.

[0062] III. Application

[0063] Application method of SQ 15, SQ 24 and SQ 63 strains in promoting the growth and saponin accumulation of Panax notoginseng: Single-strain seed cultures of SQ 15, SQ 24 and SQ 63 were inoculated into LB liquid medium and cultured at 37℃ and 220 rpm for 2 days on a shaker. The bacterial culture was then centrifuged at 5000 rpm to remove the medium. The bacterial cells were resuspended in an appropriate amount of sterile water. The growth rate was determined based on OD... 600 The dilution factor was determined, and the final solution was prepared with three strains of bacteria at concentrations of 10... 8 A mixed bacterial solution of CFU / mL was used. After the Panax notoginseng seedlings emerged, each plant was irrigated with 200 mL of a 10 CFU / mL solution. 8 A mixed bacterial culture of SQ 15, SQ 24, and SQ 63 (cfu / mL) (approximately 4200-4500L per acre) is added again after a one-month interval, and this process is repeated three times.

[0064] 3.1 Pot experiment on the promotion of Panax notoginseng seedling growth by SQ 15, SQ 24 and SQ 63

[0065] 3.11 Experimental Location: Panax notoginseng cultivation greenhouse, School of Geographical Sciences, Nanjing Normal University

[0066] 3.12 Pre-experiment treatment: Vermiculite and perlite were mixed in a 4:1 ratio, sterilized, and then filled into small flower pots with a diameter of 8 cm and a height of 12 cm. Plump and uniformly sized Panax notoginseng seeds were selected and planted in the substrate, 5 seeds per pot, at a planting depth of 1-1.5 cm. After planting, the substrate was covered. A total of 40 pots were planted, and then each pot was watered with 150 mL of 1 / 4 Hoagland nutrient solution.

[0067] 3.13 Preparation of mixed bacterial suspension of SQ 15, SQ 24, and SQ63: Single-strain seed cultures of the isolated SQ 15, SQ 24, and SQ63 strains were inoculated into LB liquid medium and cultured on a shaker at 37°C and 220 rpm for 2 days. The bacterial suspension was then centrifuged at 5000 rpm to remove the medium, and the bacterial cells were resuspended in an appropriate amount of sterile water. The bacterial culture was then analyzed based on OD... 600 The dilution factor was determined, and the final solution was prepared with three strains of bacteria at concentrations of 10... 8 A mixed bacterial culture with cfu / mL.

[0068] 3.14 Experimental Treatment: One week after planting the Panax notoginseng seeds, 40 pots were randomly divided into a control group (20 pots) and an experimental group (20 pots). Each pot in the experimental group was watered with the three strains of bacteria from 3.12 at concentrations of 10... 8 50 mL of a mixed bacterial solution with a concentration of CFU / mL was added, and the control group was watered with an equal volume of 50 mL of sterilized water. This process was repeated once more after the Panax notoginseng seedlings emerged. During the planting period, an equal volume of 1 / 4 Hoagland's nutrient solution was added as needed based on the moisture level of the substrate. Two months after planting, the fresh weight of the Panax notoginseng seedlings and root growth were recorded.

[0069] 3.15 Test Results: As shown in Table 2 and Figure 5 As shown.

[0070] Table 2. Effects of bacterial composition on the growth of Panax notoginseng seedlings.

[0071]

[0072] Note: Lowercase letters in the same row of the table indicate significant differences between the experimental group and the control group (P < 0.05).

[0073] The addition of bacterial combination significantly increased the fresh weight of Panax notoginseng seedlings by 23.45%, increased the maximum leaf width by 11.86%, and increased the leaf length by 9.79%. Root analysis showed that the addition of bacterial combination had no significant effect on the root growth of Panax notoginseng seedlings. The total root length, root surface area, average root diameter, and root volume of the experimental group of Panax notoginseng seedlings were slightly increased, indicating that the addition of bacterial combination can make the roots of Panax notoginseng robust to a certain extent, while the number of root tips and branches was slightly reduced.

[0074] 3.2 SQ 15, SQ 24 and SQ 63 promote the growth of Panax notoginseng. Application in Qiubei field.

[0075] 3.21 Experimental Location: Panax notoginseng planting base in Xiaoxinzai Village, Shupi Township, Qiubei County, Wenshan Prefecture (104°09′E, 23°51′N)

[0076] 3.22 Preparation of mixed bacterial suspensions of SQ 15, SQ 24, and SQ 63: Single-strain seed cultures of SQ 15, SQ 24, and SQ 63 were inoculated into LB liquid medium and incubated at 37°C and 220 rpm for 2 days. Then, the bacterial suspensions were centrifuged at 5000 rpm to remove the medium, and the bacterial cells were resuspended in an appropriate amount of sterile water. The OD values ​​of the bacterial suspensions were calculated beforehand. 600 A standard curve was plotted for the three strains using the values ​​and bacterial concentrations obtained from the plate count method. Then, the standard curves were compared with the measured OD values. 600 The dilution factor was determined, and the final solution was prepared with three strains of bacteria at concentrations of 10... 8 Prepare a mixed bacterial culture of CFU / mL for later use.

[0077] 3.23 Experimental Methods: The experiment was conducted in Qiubei County, using three-year-old Panax notoginseng as the test subject. After the three-year-old Panax notoginseng seedlings emerged uniformly, plots with good growth and similar size were selected. A control group and experimental groups supplemented with bacterial agents (SQ 15, SQ 24, SQ 63) were set up. Each group had three replicates, with 300 Panax notoginseng plants per replicate. The mixed bacterial solution was added by root irrigation, with 200 mL of the solution added per plant. The control group received an equal amount of water. The solution was added again after a one-month interval, for a total of three consecutive applications. At harvest time, the Panax notoginseng plants were collected, and the biomass (plant height, aboveground fresh weight, underground dry weight) and saponin (notoginsenoside R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd) content were measured.

[0078] 3.24 Test results: as shown in Table 3.

[0079] Table 3. Effects of bacterial composition on the growth and saponin accumulation of Panax notoginseng from Qiubei.

[0080]

[0081] Note: Lowercase letters in the same row of the table indicate significant differences between the experimental group and the control group, P<0.05.

[0082] As shown in Table 3, the selected bacterial combinations (SQ 15, SQ 24, SQ 63) significantly (P<0.05) increased the biomass of both the aboveground and underground parts of Panax notoginseng. The experimental group showed a 16.69% increase in plant height, a 31.23% increase in aboveground fresh weight, and a 23.15% and 23.87% increase in underground fresh and dry weight, respectively, compared to the control group. This indicates that the bacterial combination effectively promotes the growth of Panax notoginseng. Although the bacterial combination relatively reduced the total saponin content of Panax notoginseng, its significant increase in underground biomass resulted in a 5.02% increase, a 28.72% increase, a 22.80% increase, and an 8.71% increase in the accumulation of notoginseng saponins R1, Rb1, Rd, and total saponins per plant compared to the control group, respectively. This suggests that the bacterial combination can increase the accumulation of notoginseng saponins to a certain extent.

[0083] 3.3 SQ 15, SQ 24 and SQ 63 promote the growth of Panax notoginseng in stone forest field application

[0084] 3.31 Experimental Location: Panax notoginseng planting base in Shitouzhai Village, Nuohei Village Committee, Guishan Town, Shilin County, Kunming City (103°32′E, 24°41′N)

[0085] 3.32 Preparation of mixed bacterial suspensions of SQ 15, SQ 24, and SQ 63: Single-strain seed cultures of SQ 15, SQ 24, and SQ 63 were inoculated into LB liquid medium and incubated at 37°C and 220 rpm for 2 days. Then, the bacterial suspensions were centrifuged at 5000 rpm to remove the medium, and the bacterial cells were resuspended in an appropriate amount of sterile water. The OD values ​​of the bacterial suspensions were determined beforehand based on the OD values ​​of the bacterial suspensions. 600 A standard curve was plotted for the three strains using the values ​​and bacterial concentrations obtained from the plate count method. Then, the standard curves were compared with the measured OD values. 600 The dilution factor was determined, and the final solution was prepared with three strains of bacteria at concentrations of 10... 8 Prepare a mixed bacterial culture of CFU / mL for later use.

[0086] 3.33 Test Method:

[0087] The experiment was conducted in Shilin County, using three-year-old Panax notoginseng as the test subject. After the three-year-old Panax notoginseng seedlings emerged uniformly, plots with good growth and similar size were selected. A control group and experimental groups supplemented with bacterial compositions (SQ 15, SQ 24, SQ 63) were set up, with three replicates per group and 300 Panax notoginseng plants per replicate. The mixed bacterial solution was added by root irrigation, with 200 mL of the solution added per Panax notoginseng plant. The control group received an equal amount of water. The same method was repeated after one month, for a total of three consecutive applications. At harvest time, the three-year-old Panax notoginseng plants were collected, and the biomass (plant height, aboveground fresh weight, underground dry weight) and saponin (ginsenoside R1, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd) content were measured.

[0088] 3.34 Test results: as shown in Table 4.

[0089] Table 4. Effects of bacterial composition on the growth and saponin accumulation of Panax notoginseng.

[0090]

[0091]

[0092] Note: Lowercase letters in the same row of the table indicate significant differences between the experimental group and the control group, P<0.05.

[0093] The bacterial combinations (SQ 15, SQ 24, and SQ 63) significantly (P<0.05) increased the biomass of both the aboveground and underground parts of *Panax notoginseng*, increasing the fresh weight of the aboveground parts, fresh weight of the underground parts, and dry weight of the underground parts by 12.7%, 9.57%, and 11.05%, respectively. Furthermore, these bacterial combinations also increased the content and accumulation of ginsenosides in *Panax notoginseng*. Specifically, the experimental group showed increases in the content of ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, and total ginsenosides compared to the control group by 20.91%, 14.22%, 21.34%, and 14.04%, respectively, while their accumulation increased by 33.66%, 26.86%, 34.60%, and 26.52%, respectively. These results indicate that the bacterial combinations of SQ 15, SQ 24, and SQ 63 can promote the growth of *Panax notoginseng* and the synthesis and accumulation of its active ingredient, saponins.

[0094] Laboratory pot experiments showed that applying the bacterial composition of SQ 15, SQ 24 and SQ 63 described in this invention (mixed bacterial solution 10) yielded positive results. 8 The bacterial composition (CFU / mL) effectively promoted the fresh weight growth of Panax notoginseng seedlings by 23.45%, increased the maximum leaf width by 11.86%, and the leaf length by 9.79%. Field experiments further demonstrated that the application of the bacterial composition described in this invention (4200-4500L per acre at a concentration of 10% after Panax notoginseng seedling emergence, every one month) effectively promoted the fresh weight growth of Panax notoginseng seedlings by 23.45%, increased the maximum leaf width by 11.86%, and the leaf length by 9.79%). 8 A single application of a mixed bacterial solution (cfu / mL, repeated three times) can promote the dry weight increase of three-year-old Panax notoginseng tubers by 9.57%–23.87% and increase saponin accumulation by 8.17%–26.52%. Furthermore, the strains SQ 24 and SQ 63 described in this invention exhibited inhibition rates of 12.38%–65.95% against various Panax notoginseng root rot pathogens in culture medium. In summary, the bacterial composition described in this invention has the potential to promote Panax notoginseng growth, increase saponin accumulation, and ensure high-quality and high-yield production of Panax notoginseng. It may also prevent the occurrence of root rot in Panax notoginseng, further guaranteeing improved quality and yield.

[0095] Although the invention has been described herein with reference to several illustrative embodiments, it should be understood that many other modifications and implementations can be devised by those skilled in the art, which will fall within the scope and spirit of the principles disclosed herein. More specifically, various variations and modifications can be made to the components and / or layout of the subject matter arrangement within the scope of the disclosure, drawings, and claims. Besides variations and modifications to the components and / or layout, other uses will be apparent to those skilled in the art.

Claims

1. A bacterial composition that promotes the growth of Panax notoginseng and the accumulation of saponins, characterized in that: The bacterial composition consists of *Nodobacterium nicotinate* (… Paenarthrobacter nicotinovorans SQ 15, Bacillus velezenis; SQ 24, Nitroreducing Pseudomonas ( Pseudomonas nitroreducens Composed of SQ 63; SQ 15 of *Bacillus stomatitis* is used to produce IAA and inhibit *Fusarium oxysporum*. Its preservation number is CGMCC No. 31366. Bacillus belye SQ 24 is used to inhibit Fusarium oxysporum, Fusarium solani, and Fusarium demise; its preservation number is CGMCC No. 31369. Nitro-reducing Pseudomonas SQ 63 is used to inhibit Fusarium oxysporum, Fusarium solani, and Cyclospora demise; its preservation number is CGMCC No. 31367.

2. The use of the bacterial composition of claim 1, which promotes the growth and saponin accumulation of Panax notoginseng, in promoting the growth of Panax notoginseng.

3. The application of the bacterial composition for promoting the growth and saponin accumulation of Panax notoginseng as described in claim 1 in promoting saponin accumulation in Panax notoginseng roots.

4. The application according to claim 3, characterized in that: The application method is as follows: Single-strain seed solutions of SQ 15, SQ 24 and SQ 63 were inoculated into the culture medium and cultured on a shaker. After culture, the culture medium was removed by centrifugation and the culture was resuspended in sterile water. The dilution factor was determined according to the OD600 value to prepare mixed bacterial solutions of SQ 15, SQ 24 and SQ 63 with concentrations of 108 cfu / mL. After Panax notoginseng seedlings emerge, the plants are irrigated with a mixed bacterial solution.

5. The application according to claim 4, characterized in that: The culture medium selected is LB liquid medium, which includes: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride and 15 g / L agar powder.

6. The application according to claim 4, characterized in that: Each Panax notoginseng plant was given 200 mL of mixed bacterial solution, and the solution was added again after one month. The mixed bacterial solution was added three times in total.

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