Paenibacillus polymyxa strain for inhibiting root rot of American ginseng and application thereof

Abstract

The invention belongs to the technical field of microorganisms, relates to a biocontrol bacterium Paenibacillus polymyxa (Paenibacillus polymyxa) strain Ba130 for inhibiting root rot of American ginseng, and further relates to a biological preparation containing the strain and application of the strain. The strain Ba130 can effectively inhibit root rot of American ginseng.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and discloses a biocontrol bacterium, namely Bacillus polymyxa, that inhibits root rot in American ginseng and its application. Background Technology

[0002] American ginseng (Panax quinquefolium), also known as Panax quinquefolium or Panax quinquefolium, is a perennial herb belonging to the genus Panax in the family Araliaceae. It was first recorded in my country in the *Illustrated Compendium of Materia Medica*. Like traditional Chinese ginseng, it is a precious medicinal herb. It has a slightly bitter and sweet taste, and is cool in nature. It has the effects of tonifying qi and nourishing yin, clearing heat and promoting body fluids. The word *panax* originally means "panacea" in Greek. It has a history of over 300 years in China. The *Compendium of Materia Medica* states: "Lung qi originates from the kidneys. Most medicines that benefit lung qi are slightly cold, but this one is bitter and cold. It is only beneficial for those suffering from qi deficiency due to excessive fire, cough with phlegm and blood, or fatigue and loss of essence." It has the effects of tonifying qi and nourishing yin, clearing heat and promoting body fluids, and it tonifies without causing dryness and clears without causing heat.

[0003] American ginseng contains a variety of chemical components, mainly including saponins, polysaccharides, organic acids, sterols, flavonoids, polyacetylene, and volatile oils, as well as nutrients such as vitamins, amino acids, carbohydrates, and trace elements. Ginsenosides and polysaccharides are the most abundant and are the main active ingredients of American ginseng. Modern pharmacological studies have shown that American ginseng has anti-tumor, cardiovascular protective, anti-oxidative stress, insulin resistance-improving, lipid-lowering, anti-fatigue, and immune-enhancing effects.

[0004] In 2023, dried roots of American ginseng were included in the list of substances traditionally used as both food and medicinal materials. With the rapid development of my country's health industry, American ginseng, due to its dual nature as both food and medicine, has shown broad application prospects and market potential in the fields of food, health products, and cosmetics. The consumer base for American ginseng is gradually becoming younger, market demand is increasing daily, and the planting area is continuously expanding. Currently, China has become the world's largest consumer and second-largest producer of American ginseng.

[0005] American ginseng has a long growth cycle, typically 4-5 years. With increasing cultivation time, pathogens accumulate in the soil, primarily affecting the roots, threatening both yield and quality. Root rot, often called "plant cancer," is one of the most destructive plant diseases impacting ginseng yield and quality. Normally, the incidence of root rot in medicinal plants ranges from 10% to 30%, but in severe cases, it can reach 70% to 80%, even causing complete crop failure. Research indicates that root rot is related to changes in rhizosphere soil microorganisms. A decrease in beneficial microorganisms allows pathogens such as *Fusarium* and *Ilyonectria* to proliferate, leading to disease primarily affecting the roots and rhizomes of seedlings. The peak incidence occurs in July and August. Initially, the above-ground parts show no obvious symptoms, but later the roots become dark brown and wet-rotted, and the leaves wilt and die. In production, pesticides such as metalaxyl-mancozeb, chlorobromoisocyanuric acid, thiophanate-methyl, methyl thiophanate, tebuconazole, and sennamycin are commonly used for prevention and control. The overuse of pesticides frequently occurs, directly affecting the quality of American ginseng and the safety of its clinical use.

[0006] Therefore, developing green, efficient, and safe biocontrol technologies has become an important research direction. By screening biocontrol strains with antagonistic effects, it is hoped that the use of chemical pesticides can be effectively replaced or reduced, the occurrence of pathogens can be inhibited, and the soil microecological balance can be maintained, thereby significantly improving the yield and quality of American ginseng. Summary of the Invention

[0007] In view of this, the purpose of this application is to provide a biocontrol bacterium, namely Bacillus polymyxa, for inhibiting root rot in American ginseng, the corresponding biological agent, and the method thereof.

[0008] On the one hand, this invention provides a biocontrol bacterium for inhibiting root rot in American ginseng, namely, the Paenibacillus polymyxa strain Ba130. Strain Ba130 was collected in November 2023 from the roots of four-year-old American ginseng collected in Wendeng District, Weihai City, Shandong Province, and was deposited on July 16, 2025, 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, 100101, China; accession number: CGMCCNo. 35250. The taxonomic name of strain Ba130 is Paenibacillus polymyxa.

[0009] In an embodiment of the present invention, the 16S rDNA sequence of strain Ba130 is shown in SEQ ID NO:1.

[0010] In an embodiment of the present invention, the strain Ba130 was inoculated onto TSA medium using the streak plating method and cultured in the dark at 25°C for 2 days. The strain exhibited the following culture characteristics: the cells were rod-shaped, measuring (0.5-1) μm × (2.0-8.0) μm, and were often arranged in pairs or chains; when cultured on soybean casein medium (TSA), the milky white colonies formed were irregularly round, with serrated edges, smooth surfaces, and slight stickiness.

[0011] In a second aspect, the present invention provides a biological agent for inhibiting root rot in American ginseng, comprising the aforementioned Bacillus polymyxa strain Ba130, its culture, or metabolites.

[0012] In the second embodiment, root rot is caused by fungi of the genera *Fusarium* and *Ilyonectria*.

[0013] In a specific implementation, the Fusarium fungus can be, for example, Fusarium oxysporum.

[0014] In a specific implementation plan, the fungus in the genus *Ilyonectria* can be, for example, *Ilyonectria mors-panacis*.

[0015] In a third aspect, the present invention provides a method for inhibiting root rot in American ginseng, the method comprising applying the aforementioned Bacillus polymyxa strain Ba130 or its culture or metabolites to American ginseng.

[0016] In a fourth aspect, the present invention provides the use of the above-mentioned polymyxa strain Ba130 or its culture or metabolites in the following:

[0017] 1) Preparation of biological agents for inhibiting root rot in American ginseng; 2) Method for inhibiting root rot in American ginseng; 3) Nitrogen fixation; 4) Phosphorus solubilization; 5) Protease production; 6) Cellulase production; and 7) Siderophore production.

[0018] The results of the plate confrontation method showed that the polymyxa strain Ba130 of the present invention exhibited a very good inhibitory effect on the growth of the pathogen causing root rot of American ginseng. Attached Figure Description

[0019] Figure 1 The colony morphology characteristics of the pathogenic fungus causing root rot in American ginseng are shown.

[0020] Figure 2 The pathogenicity of the fungus causing root rot in American ginseng was demonstrated. Figure 3 The plate confrontation experiment showed that the biocontrol agent Ba130 inhibited the growth of two pathogenic fungi (C0701 and C0795) of American ginseng.

[0021] Figure 4 The culture characteristics of Ba130 are shown.

[0022] Figure 5 The activity assays of the biocontrol bacterium *Bacillus polymyxa* strain Ba130 are shown: A: nitrogen fixation activity; B: phosphorus solubilization activity; C: cellulose production activity; D: protease production activity; E: siderophore production activity.

[0023] Strain Ba130 was deposited on July 16, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China; accession number: CGMCCNo.35250; and the taxonomic name of strain Ba130 is Paenibacillus polymyxa. Detailed Implementation

[0024] The preferred embodiments of the present invention will now be described in detail with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent, and all such modifications and substitutions fall within the scope of protection claimed in the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] Example 1: Isolation, purification, culture, identification, and pathogenicity testing of pathogens.

[0027] 1.1 Isolation, purification, culture and identification of root rot pathogens

[0028] Four-year-old American ginseng roots were collected from Wendeng District, Weihai City, Shandong Province in November 2023. Pathogens were isolated, purified, and cultured using the tissue block isolation method. Take diseased ginseng roots, rinse them once with sterile water, disinfect them in 75% ethanol for 30 seconds, disinfect them in 5% sodium hypochlorite for 3 minutes, rinse them once with sterile water, soak them in a double antibiotic solution (containing 100 μg / mL streptomycin sulfate and 100 μg / mL potassium penicillin) for 10 minutes, rinse them three times with sterile water, blot them dry with sterile filter paper, and use a sterile scalpel to cut 0.5 cm × 0.5 cm tissue blocks from the junction of diseased and healthy tissue at the junction of diseased and healthy tissue. Inoculate them on PDA circular medium (d = 1.5 cm) containing antibiotics (100 μg / mL streptomycin sulfate and 100 μg / mL potassium penicillin), with 9 blocks per plate. Incubate them at 25°C in the dark until mycelia grow around the tissue blocks. Pick the mycelia from the edge of the colony and transfer them to a new PDA plate. Purify them until the colony morphology is stable and then preserve them.

[0029] Suspected pathogenic fungi were isolated from diseased roots of American ginseng. Two dominant pathogenic fungi, C0701 and C0795, were selected for subsequent pathogenicity verification. (See below) Figure 1 When these two pathogenic fungi were cultured on PDA medium, the colonies of strain C0701 were white with regular edges, a compact texture, and underdeveloped aerial hyphae. The colonies of strain C0795 were purplish-red with well-developed, cotton-like aerial hyphae and regular edges. Based on the colony morphology and DNA sequence characteristics, specifically the intraribosome transcribed spacer (ITS) sequence, strain C0701 was preliminarily identified as *Ilyonectria morspanacis*, and strain C0795 was preliminarily identified as *Fusarium oxysporum*.

[0030] 1.2 Pathogenicity determination of pathogens

[0031] Fresh American ginseng taproots were collected, disinfected with 75% ethanol, and punctured with a sterile inoculation needle. A 4mm diameter pathogenic fungal cake was then inoculated onto the punctured site using a punch. Simultaneously, blank PDA medium was inoculated as a control. Each treatment was repeated six times. The treated American ginseng roots were placed on petri dishes containing sterile filter paper for humidification and incubated at 25℃ for 5-10 days. Results were observed and lesion patterns and disease progression were recorded. The experimental results showed that, compared to the uninoculated control, both tested pathogenic fungi could cause disease in detached American ginseng roots, forming lesions, proving that these two fungi are the causative agents of root rot in American ginseng. Figure 2 ).

[0032] Example 2. Screening of biocontrol bacteria

[0033] 2.1 Isolation and identification of culturable bacteria from the rhizosphere soil of American ginseng

[0034] Referring to the literature (Zhou Yangwei et al., Isolation and Identification of Plant Rhizosphere Soil Bacteria. Chinese Agricultural Science Bulletin, 2015, 31(7):212-217), microorganisms in American ginseng rhizosphere soil samples were isolated using the serial dilution method. 10g of American ginseng rhizosphere soil was weighed and placed in separate Erlenmeyer flasks. 90mL of sterile water was added, and the flasks were shaken thoroughly for 20min on a shaker. After mixing, the mixture was allowed to stand. 1mL of the soil suspension was then serially diluted with sterile water to a final concentration of 10. -7 Multiply by 10, respectively -4 10 -5 10 -6 10 -7 Soil suspensions of various concentrations were used for bacterial isolation. 100 μL of each diluted bacterial suspension was evenly spread onto TSA plates, with 100 μL of sterile water as a blank control. Each concentration gradient was repeated three times, and the plates were incubated at 25°C for 1–2 days. After colonies grew, they were classified according to size, color, surface, and edge characteristics. Colonies of each class were randomly selected proportionally for streak purification until uniform single colonies appeared. These were then inoculated into liquid TSA medium and stored with 25% glycerol at -80°C for later use.

[0035] 2.2 Screening of antagonistic strains of the pathogen causing root rot in American ginseng

[0036] Referring to the literature (Zheng Mengqi et al., Inhibitory effect of 3 biocontrol bacteria against pathogens causing root rot of Polygonatum odoratum. Chinese Journal of Traditional Chinese Medicine, 2023, 48(5)1212-1217), the plate confrontation method was used to detect the antibacterial effect of our isolated soil bacteria against the pathogen causing root rot of American ginseng. Strains with antagonistic effects against the pathogen (i.e., antagonistic bacteria or biocontrol strains) were preliminarily screened for future biological control of root rot in American ginseng. The pathogen obtained in Example 1 was activated on a PDA plate and cultured in the dark at 25℃ for 7 days. Bacterial blocks (d=6mm) were punched around the pathogen colonies and inoculated into the center of a new PDA plate. Simultaneously, 2μL of the rhizosphere bacterial solution obtained above was inoculated as a single bacterium 2mm from the center of the pathogen block. The control was a PDA medium containing only pathogen bacterial cakes, without rhizosphere bacteria for screening. Each group had 3 replicates. Invert the plate and incubate until the control pathogen colonies reach 3 / 4 of the plate. Measure the inhibition radius and calculate the inhibition rate.

[0037] Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%.

[0038] The results showed that strain Ba130, isolated from the rhizosphere of American ginseng, exhibited excellent inhibitory effects on the growth of the pathogen causing root rot in American ginseng. Its inhibition rate against *C. spp.* fungi C0701 reached 81.74%, while its inhibition rate against *Fusarium oxysporum* reached 69.68% (Table 1). Figure 3 ).

[0039] Table 1. Screening results of biocontrol strains that inhibit the pathogen causing root rot in American ginseng.

[0040]

[0041] 2.3 Morphological identification of biocontrol strain Ba130

[0042] Observe the morphology of the colonies. Select a small number of colonies to prepare temporary slides and observe the appearance, size, and other characteristics of the strains under a stereomicroscope. Refer to the reference *Bergey's Manual of Systematic Bacteriology* (2nd edition) for preliminary identification of the strains. Refer to *Manual of Systematic Identification of Common Bacteria*. Inoculate the selected antimicrobial bacteria onto TSA medium using the streak plate method and incubate at 25°C in the dark for 2 days. Observe and record the growth status, size, color, and other morphological characteristics of the colonies.

[0043] The results showed that when strain Ba130 was inoculated onto TSA medium using the streak plating method and incubated in the dark at 25°C for 2 days, it formed milky-white, irregularly round, toothed-edge, smooth-surfaced, and slightly sticky colonies. Figure 4 As shown.

[0044] 2.4 Molecular biological identification of biocontrol strain Ba130

[0045] Species identification of biocontrol bacteria was performed using colony PCR. 16S rDNA PCR amplification of strain Ba130 was conducted using primers 27F-AGAGTTTGATCCTGGCTCAG and 1492RTACGGCTACCTTGTTACGACTT. The PCR reaction system consisted of 8.5 μL ddH2O, 1 μL 27F, 1 μL 1492R, 12.5 μL Taq enzyme, and 2 μL DNA template, with a total reaction volume of 25 μL. PCR cycling was as follows: 95℃ pre-denaturation for 10 min, 1 cycle; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 90 s, 33 cycles; and a final extension at 72℃ for 10 min. The amplified products were sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. BLAST analysis of the sequences in the National Center for Biotechnology Information (NCBI) database was performed, and the taxonomic position of the biocontrol bacteria was determined based on morphological characteristics.

[0046] Based on morphological and DNA sequence characteristics, the biocontrol bacterium Ba130 was preliminarily identified as *Paenibacillus*. spo lymyxa).

[0047] 2.5 Other characteristics of biocontrol bacteria Ba130

[0048] In addition to testing the inhibitory properties of the biocontrol bacterium Ba130 polymyxin Bacillus against the growth of the pathogenic fungus causing root rot in American ginseng, the nitrogen-fixing activity, phosphorus solubilization, siderophore production, protease production, and cellulase production of this strain were also tested. These properties may be related to its ability to inhibit the occurrence of pathogens, and also enrich the potential application range of this strain.

[0049] 2.5.1 Nitrogen fixation: The antagonistic bacterium Ba130 was inoculated onto Assumption solid medium and cultured at 30℃ for 3 days. If the strain grew normally on the solid medium, it indicated that the strain had nitrogen fixation ability. The results showed that strain Ba130 could grow normally on Assumption solid medium, indicating that it had nitrogen fixation potential. Figure 5 A).

[0050] 2.5.2 Phosphorus solubilization: Strain Ba130 was inoculated into an organic phosphorus medium and cultured at 30°C for 5–7 days. The presence of a clear zone around the colonies was observed to determine its phosphorus solubilization ability. Results showed that strain Ba130 produced a clear zone around its colonies after 5 days of culture on organic phosphorus solid medium, suggesting its phosphorus solubilization function. Figure 5 B).

[0051] 2.5.3 Protease Production: Strain Ba130 was inoculated onto the protease detection medium and incubated at 30℃ for 5–7 days. The appearance of a clear halo or a distinct lysis zone around the colony indicates that the strain has the ability to secrete protease; the larger the halo, the stronger the enzyme activity. Results showed that after 5 days of incubation on the protease detection medium, a clear halo appeared around the Ba130 strain, indicating that this strain could produce protease. Figure 5 C).

[0052] 2.5.4 Cellulase Production: Inoculate strain Ba130 in the center of Congo red solid medium and incubate at 30°C for 5–7 days. The appearance of a clear zone around the colony indicates its ability to produce cellulase. After culturing strain Ba130 on cellulase-producing medium for 5 days, a clear zone was produced, indicating that strain Ba130 has the potential to produce cellulase. Figure 5 D).

[0053] 2.5.5 Siderophore Production: Strains Ba130 were inoculated into the center of the heptaphilic assay medium and incubated at 30℃ for 5-7 days. If an orange-yellow halo appeared around the strain, it indicated that it possessed siderophore production ability. The size of the halo was used to determine the siderophore production ability of antagonistic strains. The results showed that strain Ba130 produced an orange-yellow halo after 5 days of incubation on the heptaphilic solid medium, indicating that Ba130 possesses siderophore production function. Figure 5 E).

Claims

1. The Paenibacillus polymyxa strain Ba130, characterized in that, Its preservation number is CGMCCNo.35250.

2. The *Bacillus polymyxa* strain Ba130 according to claim 1, characterized in that, The 16S rDNA sequence of strain Ba130 is shown in SEQ ID NO:

1.

3. A biological agent for inhibiting root rot in American ginseng, comprising the Bacillus polymyxa strain Ba130 as described in claim 1 or 2.

4. The biological agent according to claim 1, characterized in that... The root rot is caused by Fusarium and Ilyonectria.

5. The biological agent according to claim 4, characterized in that... The Fusarium mentioned is Fusarium oxysporum.

6. The biological agent according to claim 4, characterized in that... The red crust mentioned is Ilyonectria mors-panacis.

7. A method for inhibiting root rot in American ginseng, characterized in that, The method includes applying the polymyxa strain Ba130 of claim 1 or 2 or the biological agent of claim 3 to American ginseng.

8. The use of the polymyxa strain Ba130 according to claim 1 or 2 in the following: 1) Prepare biological agents for inhibiting root rot in American ginseng; 2) Methods to suppress root rot in American ginseng; 3) Nitrogen fixation; 4) Phosphorus solubilization; 5) Produces proteases; 6) Produces cellulase; and 7) Iron-producing carrier.

Images