Synthetic microbial flora for preventing and treating panax notoginseng root rot as well as preparation method and application of synthetic microbial flora

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a synthetic microbial flora for preventing and treating panax notoginseng root rot as well as a preparation method and application of the synthetic microbial flora. The invention relates to a synthetic microbial community. The synthetic microbial community is prepared from neosphingomonas sp. BW11, sphingomonas sp. BW12, sphingomyza sp. BW15, Borsella sp. BG233, variovorax sp. BJ219, pseudomonas sp. BG5 and stenotrophomonas sp. BJ8. The synthetic microbial flora provided by the invention can effectively inhibit the growth of panax notoginseng root rot mycelia, reduce the morbidity of panax notoginseng root rot, and realize effective prevention and treatment of panax notoginseng root rot; and the synthesized microbial flora is a natural microbial combination, does not contain chemical pesticide components, is green and safe, is environment-friendly, and meets the development requirements of green agriculture.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a synthetic microbial flora for preventing and controlling root rot in Panax notoginseng, its preparation method, and its application. Background Technology

[0002] Panax notoginseng, a traditional and precious Chinese medicinal herb, is a perennial plant belonging to the Araliaceae family and the Panax genus. It possesses significant medicinal and health-promoting value and is a core ingredient in many traditional Chinese medicines (such as Yunnan Baiyao and Compound Danshen Dripping Pills) and health products. However, its cultivation is often plagued by root rot, a disease primarily caused by Fusarium oxysporum. This disease is characterized by rapid spread, significant damage, and difficulty in control. The incidence rate in medicinal plants is typically 10%-30%, but can reach over 70% in severe cases, even causing complete crop failure, earning it the industry moniker of "plant cancer." Current control methods mainly employ two approaches: agronomical measures, such as field hygiene management and crop rotation; and chemical methods, including soil disinfection. While these methods have some effectiveness, the use of chemical agents disrupts the rhizosphere microecological balance and leads to pesticide residues and heavy metal contamination in the medicinal materials, seriously affecting the quality of the medicine and its therapeutic effects.

[0003] In recent years, rhizosphere microbial regulation technology has become a research focus for the control of soil-borne diseases due to its environmentally friendly characteristics. However, existing research has mostly focused on the development of single strains, and practical applications have revealed problems such as low colonization efficiency and weak environmental adaptability, leading to unstable control effects. Therefore, researchers have begun to turn to the study of compound microbial agents and synthetic microbial communities, hoping to improve the stability and sustainability of disease control through the synergistic effects between microorganisms.

[0004] The information disclosed in this background section is only intended to enhance understanding of the overall background of the invention and should not be considered as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a synthetic microbial community for preventing and treating root rot of Panax notoginseng, as well as its preparation method and application, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A synthetic microbial community, wherein the synthetic microbial community comprises *Sphingosine monocytogenes* BW11, *Sphingosine monocytogenes* BW12, *Sphingobacterium* BW14, *Sphingosine monocytogenes* BW15, *Gnaphalium glutamicum* BJ219, *Pseudomonas* BG5, and *Oligotrophomonas* BJ8; wherein, The neosphingosine bacterium BW11 was purchased from the China General Microbiological Culture Collection Center. Novosphingobium kunmingenseAccession number: CGMCC NO. 1.12274; The *Sphingomonas* BW12 strain was *Sphingomonas yunnanensis* purchased from the China General Microbiological Culture Collection Center. Sphingomonas yunnanensis Accession number: CGMCC NO. 1.15275; The *Sphingomonas* BW14 was *Sphingomonas siyangensis* purchased from the China General Microbiological Culture Collection Center. Sphingobacterium siyangense Accession number: CGMCC NO. 1.6855; The sphingosine box bacteria BW15 was a ginseng-derived sphingosine box bacteria purchased from the China General Microbiological Culture Collection Center. Sphingopyxis ginsengisoli Accession number: CGMCC NO. 1.9098; The glutathione BJ219 is *Polyborophione*. Variovorax boronicumulans BRS2-19 was deposited at the China General Microbiological Culture Collection Center on May 28, 2025, with accession number CGMCC NO.34701; The Pseudomonas BG5 is a type of Pseudomonas marginalis. Pseudomonas marginalis BRS125 was deposited on May 28, 2025 at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC NO.34702; The oligotrophomonas BJ8 is a strain Stenotrophomonas humi BRS348 was deposited on May 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC NO. 34703.

[0007] Furthermore, the viable counts of the aforementioned *Sphingosine monocytogenes* BW11, *Sphingosine monocytogenes* BW12, *Sphingobacterium* BW14, *Sphingosine box bacteria* BW15, *Gnaphalium glutamicum* BJ219, *Pseudomonas* BG5, and *Oligotrophomonas* BJ8 were all 1.0 × 10⁻⁶. 8 cfu / mL.

[0008] The present invention also provides the application of the synthetic microbial flora in the prevention and control of root rot in Panax notoginseng.

[0009] Furthermore, the pathogen causing root rot in Panax notoginseng includes Fusarium oxysporum.

[0010] Furthermore, the specific method for controlling root rot of Panax notoginseng using the synthetic microbial flora is as follows: the synthetic microbial flora solution is applied to the substrate around the roots of Panax notoginseng at a rate of 5 mL / plant, and then applied again after an interval of 5 days.

[0011] The present invention also provides the application of the synthetic microbial community in the prevention and control of plant diseases caused by Fusarium oxysporum infection.

[0012] Furthermore, the plant disease mentioned includes root rot of Panax notoginseng.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The synthetic microbial flora of this invention can effectively inhibit the mycelial growth of Panax notoginseng root rot fungus, reduce the incidence of Panax notoginseng root rot, and achieve effective prevention and control of Panax notoginseng root rot; moreover, the synthetic microbial flora of this invention is a natural microbial combination, does not contain chemical pesticide components, is green and safe, environmentally friendly, and meets the requirements of green agriculture development.

[0014] Preservation Information Boron glutathione Variovorax boronicumulans BRS2-19 was deposited on May 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 34701. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Marginal Pseudomonas Pseudomonas marginalis BRS125 was deposited on May 28, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 34702. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. strain Stenotrophomonas humi BRS348 was deposited on May 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 34703. The depository address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Attached Figure Description

[0015] Figure 1 This is the result of the potted plant experiment of the present invention. Detailed Implementation

[0016] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0017] 1. Materials and Methods 1.1 Test strains 1.1.1 Purchasing strains The strains BW11, BW12, BW14, and BW15 of *Sphingosinophila* in this application were purchased from the China General Microbiological Culture Collection Center. Their relevant information is as follows: Neosphingosine mononitrate BW11 was purchased from the China General Microbiological Culture Collection Center in Kunming. Novosphingobium kunmingense Accession number: CGMCC NO. 1.12274; Sphingosomalidone BW12 is *Sphingosomalidone yunnanensis* purchased from the China General Microbiological Culture Collection Center. Sphingomonas yunnanensis Accession number: CGMCC NO. 1.15275; The *Sphingomonas* BW14 was *Sphingomonas siyangensis* purchased from the China General Microbiological Culture Collection Center. Sphingobacterium siyangense Accession number: CGMCC NO. 1.6855; Sphingosine Box BW15 is a type of ginseng sphingosine box purchased from the China General Microbiological Culture Collection Center. Sphingopyxis ginsengisoli Collection number: CGMCC NO. 1.9098.

[0018] 1.1.2 Isolation of bacterial strains This application isolated *Pseudomonas hummi* BJ219, *Pseudomonas* BG5, and *Oligotrophozoa* BJ8 from the roots of *Panax notoginseng*. Molecular identification methods were used, employing universal primers (Table 1) for molecular amplification and sequencing. BLAST comparisons with data from the BNCI website were then performed to determine their phylogenetic relationships. A phylogenetic tree was constructed using MEGA software to identify *Pseudomonas hummi* BJ219, *Pseudomonas* BG5, and *Oligotrophozoa* BJ8.

[0019] Table 1 Primers and Sequences

[0020] The identification result is: BJ219 is *Polyborophanthus*. Variovorax boronicumulans ; Pseudomonas BG5 is a marginal Pseudomonas. Pseudomonas marginalis ; Oligotrophomonas BJ8 is a strain Stenotrophomonas humi .

[0021] 1.2 Test pathogens Fusarium oxysporum isolated from roots of Panax notoginseng diseased root rot samples ( Fusarium oxysporum ).

[0022] 1.3 Test Culture Medium LB liquid medium: 10.0 g tryptone, 5.0 g yeast extract, 5.0 g sodium chloride, 1000 mL sterile water; PDA medium: 250g potato, 20g glucose, 10g agar powder, 1000mL sterile water.

[0023] 1.3 Construction of synthetic microbial communities The tested strains *Sphingosine monocytogenes* BW11, *Sphingosine monocytogenes* BW12, *Sphingosine box bacteria* BW15, *Boswellia* BG233, *Gnaphalium affine* BJ219, *Pseudomonas* BG5, and *Oligotrophomonas* BJ8 were inoculated into LB liquid medium and cultured at 28°C and 200 rpm for 12 h. The viable cell count was then adjusted to 1.0 × 10⁻⁶. 8 cfu / mL; mix the bacterial solutions of 7 strains in equal volume ratio to obtain the synthetic microbial community SynCom.

[0024] 1.4 Flat Plate Standoff Test The inhibitory activity of bacterial suspensions from seven bacterial strains and the synthetic microbial community SynCom against the mycelial growth of the tested pathogens was determined using the plate confrontation culture method. Specifically, the tested pathogens were inoculated onto PDA medium and activated at 30°C. Mycelial discs with a diameter of 8 mm were then punched along the edge of the colony. These discs were transferred to the center of a PDA plate (9 cm diameter), and 5 μL of bacterial suspension was inoculated at four points 2.5 cm from the center of the plate. Each treatment was repeated four times. A plate inoculated only with mycelial discs served as a blank control. All plates were incubated at 30°C. When the colony diameter in the blank control reached two-thirds of the plate diameter, the colony diameter was measured, and the inhibition rate was calculated. The results are shown in Table 2.

[0025]

[0026] Table 2 Results of the plate standoff test

[0027] Note: Different lowercase letters in the table represent significant differences between treatments. P <0.05, ANOVA, Duncan's method) Table 2 shows that the inhibition rates of single-strain bacterial solutions against the mycelial growth of the tested pathogen *Fusarium oxysporum* varied considerably, and were significantly lower than the inhibition rate of the synthetic microbial community SynCom. This indicates that the synthetic microbial community SynCom of the present invention can improve the inhibitory effect on *Fusarium oxysporum*, the pathogen causing root rot in Panax notoginseng.

[0028] 1.5 Pot Experiment Each pot contains 4 kg of sterilized soil, and 5-6 three-year-old Panax notoginseng seedlings are transplanted into each pot. After the transplanted Panax notoginseng has recovered from transplanting, SynCom bacterial culture solution is inoculated into the soil around the Panax notoginseng plants, 5 mL per plant. This treatment is repeated once after 5 days with the same inoculation amount. A control group treated with LB liquid medium is used, and each treatment is replicated 5 times. Five days after the second SynCom inoculation, the phloem of the Panax notoginseng root is punctured with a 20 mL syringe needle, and 1×10⁻⁶ cells are injected. 7 cfu / mL F.oxysporum Inoculate the rhizosphere of Panax notoginseng with a conidial suspension at a rate of 5 mL per plant. Samples were taken 50 days after inoculation to observe disease occurrence and the proportion of diseased plants was recorded. Results are shown below. Figure 1 .

[0029] Depend on Figure 1 It can be seen that, compared with the control group that was only inoculated with pathogens, inoculation with the SynCom bacterial community solution can effectively reduce the incidence of root rot in Panax notoginseng. This indicates that the SynCom synthetic microbial community of the present invention effectively reduces the incidence of root rot in Panax notoginseng, achieving effective prevention and control of root rot in Panax notoginseng.

[0030] In summary, the synthetic microbial flora of the present invention can effectively inhibit the mycelial growth of Panax notoginseng root rot fungus, reduce the incidence of Panax notoginseng root rot, and achieve effective prevention and control of Panax notoginseng root rot; moreover, the synthetic microbial flora of the present invention is a natural microbial combination, does not contain chemical pesticide components, is green and safe, environmentally friendly, and meets the requirements of green agriculture development.

[0031] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A synthetic microbial community, characterized in that, The synthetic microbial community consists of *Neosphingomonas* BW11, *Sphingomonas* BW12, *Sphingobacterium* BW14, *Sphingomonas-boxella* BW15, *Gnaphalium* BJ219, *Pseudomonas* BG5, and *Oligotrophomonas* BJ8; among which... The neosphingosine bacterium BW11 was purchased from the China General Microbiological Culture Collection Center. Novosphingobium kunmingense Accession number: CGMCC NO. 1.12274; The *Sphingomonas* BW12 strain was *Sphingomonas yunnanensis* purchased from the China General Microbiological Culture Collection Center. Sphingomonas yunnanensis Accession number: CGMCC NO. 1.15275; The *Sphingomonas* BW14 strain was *Sphingomonas siyangensis* purchased from the China General Microbiological Culture Collection Center. Sphingobacterium siyangense Accession number: CGMCC NO. 1.6855; The sphingosine box bacteria BW15 was a ginseng-derived sphingosine box bacteria purchased from the China General Microbiological Culture Collection Center. Sphingopyxis ginsengisoli Accession number: CGMCC NO. 1.9098; The glutathione BJ219 is *Polyborophione*. Variovorax boronicumulans BRS2-19 was deposited at the China General Microbiological Culture Collection Center on May 28, 2025, with accession number CGMCC NO.34701; The Pseudomonas BG5 is a type of Pseudomonas marginalis. Pseudomonas marginalis BRS125 was deposited on May 28, 2025 at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC NO.34702; The oligotrophomonas BJ8 is a strain Stenotrophomonas humi BRS348 was deposited on May 28, 2025, at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC NO. 34703.

2. The synthetic microbial community according to claim 1, characterized in that, The viable counts of the following bacteria were all 1.0 × 10⁸: *Sphingosine monocytogenes* BW11, *Sphingosine monocytogenes* BW12, *Sphingobacterium* BW14, *Sphingosine cascade* BW15, *Gnaphalium affine* BJ219, *Pseudomonas* BG5, and *Oligotrophomonas* BJ8. 8 cfu / mL.

3. The application of the synthetic microbial community described in claim 1 in the prevention and control of root rot in Panax notoginseng.

4. The application according to claim 3, characterized in that, The pathogen causing root rot in Panax notoginseng includes Fusarium oxysporum.

5. The application according to claim 3, characterized in that, Specifically, the synthetic microbial flora solution is applied to the substrate around the roots of Panax notoginseng at a rate of 5 mL per plant, and then applied again after an interval of 5 days.

6. The application of the synthetic microbial community according to claim 1 in the prevention and control of plant diseases caused by Fusarium oxysporum infection.

7. The application according to claim 6, characterized in that, The plant diseases mentioned include root rot of Panax notoginseng.

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