Bacillus velezensis mutant strain XG5-1 for preventing and treating root rot of angelica sinensis as well as cultivation method and application of bacillus velezensis mutant strain XG5-1

Abstract

The invention relates to a bacillus velezensis mutant strain for preventing and treating root rot of traditional Chinese medicinal material angelica sinensis as well as a cultivation method and application of the bacillus velezensis mutant strain. The strain is a mutant strain which is obtained by carrying out ultraviolet mutation breeding and screening on a wild strain separated from healthy angelica sinensis rhizosphere soil and is high in stability and excellent in antagonism. Through 16S rDNA (ribosomal deoxyribonucleic acid) sequence determination and phylogenetic analysis, the strain is preliminarily identified to belong to bacillus spp. The strain provided by the invention is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M 2025959, and the preservation date is May 6, 2025. The strain has a good antagonistic effect on common pathogenic bacteria, namely fusarium tricinctum and Mucor racemosus, of the root rot of the angelica sinensis, the growth speed of the pathogenic bacteria can be remarkably reduced, and the incidence rate of the root rot of the angelica sinensis can be effectively reduced under greenhouse and field conditions. The invention provides a novel biological resource for green prevention and control of diseases of Chinese herbal medicines such as angelica sinensis, and has a good industrial transformation prospect.

Description

Technical Field

[0001] The invention relates to a Bacillus velezensis strain for preventing and treating root rot of Chinese medicinal material Angelica sinensis. The strain is obtained from angelica rhizosphere soil bacteria Bacillus velezensis XG5 through ultraviolet mutagenesis. The strain exhibits good antagonism against common pathogens of angelica sinensis root rot, Fusarium tricinctum and Mucor racemosus, can significantly reduce the growth rate of the pathogens, and effectively reduce the incidence of angelica sinensis root rot under greenhouse and field conditions. The strain belongs to the category of microbial pesticides, biocontrol agents and green prevention and control technologies for Chinese medicinal materials. Background Art

[0002] Angelica sinensis (Oliv.) Diels is a traditional Chinese medicinal herb, primarily grown in Gansu, Shaanxi, and Sichuan. It boasts numerous pharmacological benefits, including nourishing and activating blood circulation, regulating menstruation, and alleviating pain. It is a commonly used herb in clinical gynecological medicine. With the rapid development of Traditional Chinese Medicine (TCM) and the growing demand for health products, the area cultivated for Angelica sinensis has expanded annually. However, this has led to increasingly prominent diseases, particularly root rot, which has become a major constraint on its development.

[0003] Angelica root rot is a typical soil-borne disease that typically breaks out in the seedling stage or mid-to-late growth stages, causing root rot and browning of tissues, severely impacting the yield and commercial value of the medicinal herb. Research has shown that pathogenic fungi such as Fusarium tricinctum and Mucor racemosus are the primary cause of this disease. These fungi often inhabit the soil or rhizosphere, can persist in the field for long periods, and can be spread by irrigation water and agricultural tools, making prevention and control challenging.

[0004] Currently, the prevention and control of angelica root rot primarily relies on soil treatment and chemical pesticides. However, long-term use of chemical pesticides not only leads to the development of resistance in pathogens but also may damage the rhizosphere microecological system, thereby affecting the accumulation of active ingredients and the ecological safety of angelica. Furthermore, the accumulation of chemical pesticide residues poses a threat to angelica exports and drug safety, severely restricting the green development of the Chinese herbal medicine industry. Therefore, the search for safe, green, and effective alternative prevention and control methods is urgent.

[0005] In recent years, plant-based and microbial-based biocontrol technologies have developed rapidly in the agricultural field. Among them, Bacillus velezensis, as a biocontrol strain with disease resistance, growth promotion and environmental adaptability, can produce a broad-spectrum inhibitory effect on a variety of pathogenic fungi by secreting a variety of antimicrobial peptides, organic acids and volatile compounds. At the same time, this type of strain can colonize in the rhizosphere of plants, stimulate the systemic resistance of the host plant, and enhance the plant's own defense ability against diseases. However, there are still relatively few studies on Bacillus velezensis in the field of traditional Chinese medicine, especially in the biological control of angelica root rot. Functional strain resources with strong targeting, clear antagonistic ability and good adaptability have not yet been fully developed. In order to meet the needs of the green development of the angelica industry, it is urgent to screen and create efficient and stable biocontrol strains from the authentic medicinal material planting environment, and develop its formulation application technology to achieve ecological precision prevention and control of angelica root rot. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to overcome the problems of heavy reliance on chemical pesticides, easy development of resistance by pathogens, pesticide residue pollution and high environmental risks in the current prevention and control of angelica root rot, and to provide a Bacillus velezensis XG5-1 mutant strain with good antagonistic activity and genetic stability and its use in the prevention and control of angelica root rot.

[0007] Another object of the present invention is to provide a method for isolating the Bacillus velez mutant strain XG5-1 from the rhizosphere soil of Angelica sinensis and obtaining it through ultraviolet mutagenesis breeding.

[0008] The strain has good antagonistic effects on common pathogens of angelica root rot, Fusarium tricinctum and Mucor racemosus, and can be used to prepare a microbial agent for preventing and controlling angelica root rot.

[0009] Technical solution: The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a Bacillus velezensis strain XG5-1 for preventing and treating angelica root rot. The strain is derived from angelica rhizosphere soil and obtained through ultraviolet mutagenesis breeding. It has excellent genetic stability and significant antagonistic activity. Microbial 16SrDNA sequencing identified it as Bacillus spp. It is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 2025959, classified as Bacillus velezensis XG5-1, and the deposit date is May 6, 2025.

[0011] The Bacillus velezensis XG5-1 strain provided by the present invention has the following colony morphological characteristics after being cultured on LB solid medium at 37° C. for 16 hours:

[0012] The colonies are round or nearly round, with slightly wavy edges. The surface is moist, smooth, and slightly glossy. They are milky white or off-white, opaque to slightly transparent. They are moist and slightly sticky to the touch. They are medium-sized, approximately 1–3 mm in diameter, moderately raised or slightly flattened, and evenly distributed. They do not produce pigment, with no noticeable color diffusion. The underside is pale yellow or nearly white. No obvious pigment deposition or diffusion zones were observed during incubation.

[0013] The present invention provides a method for isolating and fermenting the Bacillus velezensis XG5-1 strain, comprising shaking, inoculation, isolation, mutagenesis, culture, and preservation, and the specific steps are as follows:

[0014] a) Oscillation: Add the rhizosphere soil of Angelica sinensis to sterile water, incubate with shaking, and allow to stand to prepare a series of soil dilution solutions;

[0015] b) Isolation: The soil dilution was inoculated into LB solid medium and purified repeatedly to obtain a single colony;

[0016] c) Mutagenesis: Different mutant strains were obtained by ultraviolet irradiation mutagenesis;

[0017] d) Antibacterial screening: Screen different mutants for their antibacterial properties to obtain mutant strains with strong stability and excellent antagonism;

[0018] e) Cultivation and storage: The mutant strain was cultured in LB liquid medium with shaking, mixed with glycerol and stored in a freezer.

[0019] As a preferred embodiment, the separation, fermentation and mutagenesis culture method of the mutagenized strain of Bacillus velezinsis described above comprises the following steps:

[0020] a) Oscillation: Add the rhizosphere soil of Angelica sinensis to sterile water, oscillate at 37°C and 160 rpm for 1 to 2 hours, and let it stand for 30 to 60 minutes to prepare 10 -1 to 10 -6 of soil dilution;

[0021] b) Separation: Take 0.1mL 10 -4 to 10 -6 The soil dilution was inoculated into LB solid medium and purified repeatedly to obtain a single colony;

[0022] c) Mutagenesis: The strain was placed 36 cm away from a UV lamp and irradiated with UV light for 5, 10, 15, 20, 30, 40, 60, and 90 seconds, respectively, to obtain different mutant strains;

[0023] d) Antibacterial screening: Different mutants were screened for their antibacterial properties against Fusarium thunbergii and Mucor circinelloides, and the mutant strain XG5-1 of Bacillus velezensis was obtained;

[0024] e) Cultivation and Storage: The mutagenized strain XG5-1 was cultured in LB liquid medium at 37°C with shaking at 160 rpm for 12-24 hours, mixed with glycerol, and stored in a freezer.

[0025] As a more preferred embodiment, the separation, fermentation and mutagenesis culture method of the mutagenized strain of Bacillus velezinoff is as follows:

[0026] a) Oscillation: 5 g of Angelica rhizosphere soil was added to 100 mL of sterile water, shaken at 37°C and 160 rpm for 2 hours, and allowed to stand for 30 minutes to prepare 10 -1 to 10 -6 of soil dilution;

[0027] b) Separation: Take 0.1mL 10 -5 to 10 -6 The soil dilution was inoculated into LB solid medium and purified repeatedly to obtain a single colony;

[0028] c) Mutagenesis: The strain was placed 36 cm away from a UV lamp and irradiated with UV light for 5, 10, 15, 20, 30, 40, 60, and 90 seconds, respectively, to obtain different mutant strains;

[0029] d) Antibacterial screening: Different mutants were screened for their antibacterial properties against Fusarium thunbergii and Mucor circinelloides, and the mutant strain XG5-1 of Bacillus velezensis was obtained;

[0030] e) Cultivation and Storage: The mutagenized strain XG5-1 was cultured in LB liquid medium at 37°C with shaking at 160 rpm for 24 hours, mixed with 50% glycerol at a ratio of 1:1, and stored at -80°C.

[0031] The Bacillus Velez strain XG5-1 for preventing and treating angelica root rot provided by the present invention was tested for in vitro antibacterial activity by a plate standoff method, and was found to be capable of inhibiting the growth of Fusarium tricinctum and Mucor racemosus, common pathogenic fungi of angelica root rot, showing a certain antagonistic effect.

[0032] The mutant strain XG5-1 of Bacillus velezensis provided by the present invention has an improved ability to antagonize Fusarium thunbergii and Mucor circinelloides in vitro compared with the wild-type strain, which is manifested by an increased inhibition rate.

[0033] The isolated Bacillus Velez XG5-1 strain of the present invention can be used to prevent and control root rot of angelica sinensis and promote the healthy growth of angelica sinensis plants. The fermentation broth of Bacillus Velez XG5-1 obtained by the fermentation method can be directly contacted with the root system of angelica sinensis plants through dilution treatment at different concentrations and used as a biocontrol agent during angelica sinensis cultivation. By interacting with the rhizosphere microbial community of angelica sinensis, Bacillus Velez XG5-1 can inhibit the occurrence of root rot of angelica sinensis, reduce the incidence of root rot, enhance the disease resistance and growth performance of angelica sinensis plants, and improve the yield and efficacy of angelica sinensis.

[0034] Beneficial effects: Compared with existing research, the present invention has the following advantages:

[0035] The Bacillus velezensis XG5-1 strain provided by the present invention has significant effects on preventing and controlling Fusarium tricinctum and Mucor racemosus, and performs well in preventing and controlling angelica root rot, effectively reducing the impact of the disease on angelica yield. The microbial agent of the present invention is a green, low-cost, and efficient biocontrol product that can replace the use of chemical pesticides and meet the needs of green and healthy angelica cultivation. This technology can be widely used in the angelica industry, providing strong technical support for achieving sustainable green development. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a front view of a colony of the mutagenic strain Bacillus velezensis XG5-1;

[0037] Figure 2 This is a back view of a colony of the mutagenic strain Bacillus velezensis XG5-1;

[0038] Figure 3 The effects of the mutant strain Bacillus velezensis XG5-1 on Mucor circinelloides and Fusarium oxysporum (left: effect on Mucor circinelloides; right: effect on Fusarium oxysporum);

[0039] Figure 4 The relationship between UV mutagenesis time and lethality of Bacillus velez XG5;

[0040] Figure 5 This is a comparison of the antibacterial rates of Bacillus velez XG5 and XG5-1 against Mucor circinelloides and Fusarium oxysporum. Specific implementation plan

[0041] The present invention can be better understood based on the following examples. However, it is readily understood by those skilled in the art that the specific fermentation culture processes and functional evaluations described in the examples are only for illustrating the present invention and should not and will not limit the present invention described in detail in the claims.

[0042] The chemical reagents used in the following examples can be obtained from conventional commercial sources.

[0043] Example 1

[0044] A new Bacillus velez strain XG5-1 was isolated and induced by the following method:

[0045] a) Oscillation: peel off the soil around the roots of Angelica sinensis, shake off and collect the soil on the rhizosphere surface, take 5g of soil and place it in a 100mL triangular flask with sterile water, shake it in a constant temperature shaker at 37℃ at 160r / min for 2h, let it stand for 30min, and get 10 -1 The soil stock solution was then pipetted with a liquid transfer gun to draw 1 mL of the supernatant and add it to 9 mL of sterile water. The mixture was shaken thoroughly to obtain 10 -2 Soil dilution, and so on, to prepare 10 -1 -10 -6 of soil dilution;

[0046] b) Separation: draw 0.1mL10 -4 , 10 -5 , 10 -6 Inoculate the soil dilution into prepared LB solid medium, with three replicates for each dilution. Invert the plate and incubate at 37°C, observing the colony growth over time. Use an inoculation loop to inoculate bacteria of different morphologies onto the LB solid medium, and repeatedly purify until a single colony is obtained.

[0047] c) Mutagenesis:

[0048] 1. Concentration screening

[0049] Take 5 centrifuge tubes, each containing 9 mL of sterile water, and number them 10-1, 10-2, 10-3, 10-4, and 10-5. Prepare another 9 sets of LB sterile culture dishes and number them 10-4, 10-5, and 10-6, with each number corresponding to 3 culture dishes. Pick a single colony on the LB solid culture medium and inoculate it into a shaking tube containing 5 mL of liquid culture medium. Place it on a 37°C constant temperature shaker and shake at 200 rpm for 20 hours. Pipette 1 mL of the culture supernatant and add it to the centrifuge tube numbered 10-1 and shake evenly. Then, take 1 mL of the bacterial suspension in the 10-1 tube and add it to the tube numbered 10-2. And so on, dilute step by step to 10-5. Accurately aspirate 10-3, 10-4, and 10-5 bacterial suspensions (0.1 mL each) and inoculate them into the corresponding numbered culture dishes respectively, spreading them evenly. After the bacterial solution has dried slightly, invert the plate and incubate in a 30°C incubator for 12 hours. Count and record the number of colonies after incubation, and adjust the bacterial solution to a concentration that produces 300-500 individual colonies on the plate. Inoculate a single XG5 colony into a shaker tube containing 5 mL of liquid culture medium and incubate at 37°C on a shaker at 200 rpm for 20 hours. After a 10,000-fold dilution, the number of colonies on the plate will be 357 ± 22.34.

[0050] 2. Screening of mutagenesis conditions

[0051] Take 0.1 mL of the prepared bacterial suspension and spread it evenly on LB solid culture medium. Expose the culture medium to a 20W UV lamp at a distance of 36 cm from the UV lamp for 5 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 40 seconds, 60 seconds and 90 seconds of UV irradiation respectively. After treatment, wrap the culture dish with tin foil and place it in a dark incubator at 30°C for culture. Estimate the number of single colonies by plate counting method, and calculate the lethality based on the unirradiated control group. Select the UV irradiation time with a lethality between 85% and 90% as the optimal irradiation condition. Figure 4 The optimal mutagenesis condition finally screened out was exposing the culture medium to a 20W ultraviolet lamp at a distance of 36 cm for 15 seconds.

[0052]

[0053] 3. Screening of mutagenic strains

[0054] Ten single colonies with the largest colony diameter and fastest growth after UV mutagenesis were selected and inoculated onto slanted LB plates and stored at 4°C until further use. The mutagenized strains were screened for antimicrobial activity using the plate standoff method. First, an appropriate amount of culture medium of the mutagenized strain was spread onto LB solid medium. A 6mm borer was used to punch holes in the plate. Inoculate the culture medium with a colony of Mucor circinelloides at four points on the upper, lower, left, and right sides of the medium. Subsequently, an appropriate amount of spores of Mucor circinelloides was spread onto polydimethylsiloxane (PDA) medium. A 6mm borer was used to punch holes in the plate. The colony was then inoculated in the center of the medium containing the biocontrol bacteria colony. The culture was then incubated at 30°C. Subsequently, an appropriate amount of spores of Fusarium trifasciatum was spread onto PDA medium. The colony was then inoculated onto the plate containing the Fusarium trifasciatum colony at four points on the upper, lower, left, and right sides of the plate. The culture was then incubated at 30°C. A control group consisted of the original strain, which had not been mutagenized, and received the same treatment conditions. After culturing for 24 hours, the diameter of the inhibition zone was measured and recorded, and the changes in the antibacterial activity of the strains before and after mutagenesis were compared. The specific results are shown in Table 1.

[0055]

[0056] Table 1 Inhibitory rate of wild-type XG5 and its variants against Mucor circinelloides and Fusarium oxysporum

[0057]

[0058]

[0059] c) Cultivation and preservation:

[0060] The bacterium XG5-1 with the best antibacterial activity screened by mutagenesis according to Table 1 was picked into a conical flask containing LB liquid culture medium, shaken at 160 r / min at 37°C for 24 h, and placed into a sterilized cryopreservation tube with 50% glycerol at a volume ratio of 1:1. The tube was mixed evenly, sealed with a sealing film, and stored in a -80°C ultra-low temperature refrigerator.

[0061] Example 2

[0062] The new Bacillus Velezii XG5-1 strain isolated by the present invention was molecularly identified by the following method:

[0063] The Bacillus velezensis XG5-1 strain, fermented and cultured according to the method of Example 1, was sent to Shanghai Paisonno Biotechnology Co., Ltd. for 16S rDNA sequencing. The upstream primer 27F (5'-AGAG TTTGATCCTGGCTCAG-3') and the downstream primer 1492R (5'-GGTTACCTTGTTACGACTT-3') were used. The 16S rDNA sequence was compared against the ribosomal database (ncbi.nlm.nih.gov). Blast sequence analysis showed, as shown in Table 1, that the XG5-1 strain shared 99.65% homology with Bacillus velezensis. The strain was identified as a Bacillus species and presumably Bacillus velezensis.

[0064] The sequence of the bacteria is:

[0065]

[0066] Table 2 Sequence alignment results of XG5-1 strain

[0067]

[0068]

[0069] The above results demonstrate that the present invention screened and obtained a new rhizosphere bacterium, Bacillus velez XG5-1, and established methods for its isolation, cultivation, mutagenesis, preservation, and application in preventing and treating root rot in angelica. Bacillus velez XG5-1 exhibits significant efficacy in preventing and treating root rot in angelica, effectively inhibiting the growth of pathogenic bacteria, enhancing the disease resistance of angelica plants, and promoting healthy growth. The microbial agent prepared according to the methods described herein can effectively prevent and treat root rot in angelica, improve root health in angelica plants, and increase yield, possessing significant practical application value.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mutagenized strain of Bacillus velez for preventing and treating angelica root rot, characterized in that: The strain was obtained by ultraviolet mutagenesis breeding of Bacillus velezensis and was deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 2025959. The strain was classified as Bacillus velezensis XG5-1.

2. The separation, fermentation and mutagenesis culture method of the induced strain of Bacillus velez according to claim 1, characterized in that: The following steps are involved: a) Oscillation: Add the rhizosphere soil of Angelica sinensis to sterile water, incubate with shaking, and allow to stand to prepare a series of soil dilution solutions; b) Isolation: The soil dilution was inoculated into LB solid medium and purified repeatedly to obtain a single colony; c) Mutagenesis: Different mutant strains were obtained by ultraviolet irradiation mutagenesis; d) Antibacterial screening: Screen different mutants for their antibacterial properties to obtain mutant strains with strong stability and excellent antagonism; e) Cultivation and storage: The mutant strain was cultured in LB liquid medium with shaking, mixed with glycerol and stored in a freezer.

3. The separation, fermentation and mutagenesis culture method of the induced strain of Bacillus Velezii according to claim 2, wherein: The following steps are involved: a) Oscillation: Add the rhizosphere soil of Angelica sinensis to sterile water, oscillate at 37°C and 160 rpm for 1 to 2 hours, and let it stand for 30 to 60 minutes to prepare 10 -1 to 10 -6 of soil dilution; b) Separation: Take 0.1mL 10 -4 to 10 -6 The soil dilution was inoculated into LB solid medium and purified repeatedly to obtain a single colony; c) Mutagenesis: The strain was placed 36 cm away from a UV lamp and irradiated with UV light for 5, 10, 15, 20, 30, 40, 60, and 90 seconds, respectively, to obtain different mutant strains; d) Antibacterial screening: Different mutants were screened for their antibacterial properties against Fusarium thunbergii and Mucor circinelloides, and the mutant strain XG5-1 of Bacillus velezensis was obtained; e) Cultivation and Storage: The mutagenized strain XG5-1 was cultured in LB liquid medium at 37°C with shaking at 160 rpm for 12-24 hours, mixed with glycerol, and stored in a freezer.

4. The separation, fermentation and mutagenesis culture method of the induced strain of Bacillus Velezii according to claim 3, wherein: The following steps are involved: a) Oscillation: 5 g of Angelica rhizosphere soil was added to 100 mL of sterile water, shaken at 37°C and 160 rpm for 2 hours, and allowed to stand for 30 minutes to prepare 10 -5 of soil dilution; b) Separation: Take 0.1mL 10 -5 The soil dilution was inoculated into LB solid medium and purified repeatedly to obtain a single colony; c) Mutagenesis: The strain obtained in step b) was placed at a distance of 36 cm from a UV lamp and subjected to UV irradiation for 15 seconds to induce mutagenesis, thereby obtaining a mutant strain; d) Antibacterial screening: The mutants were screened for antibacterial activity against Fusarium thunbergii and Mucor circinelloides, and the mutant strain XG5-1 with good antibacterial activity of Bacillus velezensis was obtained; e) Cultivation and Storage: The mutagenized strain XG5-1 was cultured in LB liquid medium at 37°C with shaking at 160 rpm for 24 hours, mixed with 50% glycerol at a ratio of 1:1, and stored at -80°C.

5. Use of the mutated strain of Bacillus velezensis according to claim 1 in the preparation of a microbial agent for preventing and treating angelica root rot.

6. Use of the mutated strain of Bacillus velezensis according to claim 1 in the preparation of a microbial agent for controlling Fusarium tricinctum and Mucor racemosus.

7. A microbial agent for preventing and treating angelica root rot, characterized in that: It includes Bacillus velezensis XG5-1.

8. A multi-effect composite biological fertilizer for preventing and treating angelica root rot, characterized in that: It includes Bacillus velezensis XG5-1.

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