Bacillus siamensis AQ171 and application thereof
By developing Bacillus siamese AQ171, the problem of the single antibacterial and antifungal effects of existing Bacillus strains has been solved, diversified inhibition of pathogens has been achieved, and a highly effective microbial pesticide candidate strain has been provided.
Patent Information
- Application Number
- CN202510849871.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-24
- Publication Date
- 2025-09-23
AI Technical Summary
Existing Bacillus strains are difficult to achieve dual antibacterial effects of high efficiency against bacteria and fungi, and the types of lipopeptide antibacterial substances secreted are relatively single. There is a lack of microbial agents on the market that can simultaneously achieve strong inhibition of pathogens and have rich and diverse metabolites.
A strain of Bacillus siamensis AQ171 was developed, which can ferment and produce a variety of lipopeptide antimicrobial compounds including bacillus mycin D, peliocin, fengycin and surfactin, which are used to simultaneously inhibit pathogenic fungi and pathogenic bacteria.
Bacillus siamea AQ171 exhibits significant broad-spectrum antibacterial activity and can effectively inhibit red star pathogens and bacterial wilt pathogens. The fermentation crude extract can significantly inhibit fungi at low concentrations and also shows strong antibacterial activity against bacteria at high concentrations. It has a rich variety of metabolites and has important potential for application in agricultural biocontrol.
Smart Images
Figure CN120682991A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological control, and particularly relates to a siam bacillus AQ171 and an application thereof. Background Art
[0002] In the field of biocontrol, Bacillus has long been a key focus of biocontrol research due to its significant advantages, including broad resistance, rapid reproduction, ease of cultivation, rich diversity, and strong stress tolerance. Lipopeptides secreted by Bacillus exhibit diverse biological activities, including antibacterial, antifungal, antiviral, and antitumor properties. Compounds such as iturin, surfactin, and fengycin are the primary lipopeptides produced by Bacillus, playing a vital role in biocontrol.
[0003] However, as research deepens, it has been discovered that there are many different types of Bacillus, and that the metabolite compositions and inhibitory effects against pathogens vary significantly among different Bacillus species. Current research indicates that while Bacillus species show considerable potential in biocontrol, most existing strains have significant limitations. Comparative studies of multiple Bacillus species from the same batch, including Bacillus siamese, Bacillus subtilis, and Bacillus cereus, revealed that these strains struggled to simultaneously achieve highly effective dual antibacterial and antifungal activity, and the types of lipopeptide antimicrobial substances they secreted were relatively limited. Furthermore, lipopeptide compounds such as pelistatin, previously reported to be primarily metabolized by Bacillus subtilis, have been less well-characterized by other Bacillus species. Therefore, there is a lack of microbial agents on the market that can simultaneously achieve potent inhibition of pathogens and produce a rich and diverse array of metabolites. The development of new microbial agents is urgently needed to meet the demand for highly effective biocontrol products in the biocontrol field. Summary of the Invention
[0004] The present invention aims to develop novel microbial agents to meet the demand for highly effective biocontrol products in the field of biological control. To this end, the present invention provides a strain of Bacillus siamensis AQ171. The strain can effectively inhibit both pathogenic fungi and bacteria and ferment to produce lipopeptide antimicrobial compounds, including bacillomycin D, plipastatin, fengycin, and surfactin, to meet the demand for highly effective biocontrol products in the field of biological control.
[0005] The present invention provides a strain of Bacillus siamensis AQ171, with a deposit number of CGMCC No.34134.
[0006] The present invention also provides a microbial agent, wherein the active ingredient of the microbial agent includes the siam Bacillus AQ171 according to claim 1.
[0007] Preferably, the microbial agent further comprises agriculturally acceptable adjuvants.
[0008] Preferably, the agriculturally acceptable excipient comprises one or more of an adsorbent material, a liquid suspension matrix and an embedding material.
[0009] The present invention also provides the use of the siam bacillus AQ171 described in the above technical solution or the microbial agent described in the above technical solution in producing lipopeptide antibacterial compounds and / or inhibiting pathogens.
[0010] Preferably, the lipopeptide antibacterial compound includes one or more of bacillus mycin D, pelistatin, fengycin and surfactin.
[0011] Preferably, the pathogens include pathogenic fungi and / or pathogenic bacteria;
[0012] The pathogenic fungi include red star pathogenic bacteria;
[0013] The pathogenic bacteria include bacterial wilt pathogen.
[0014] The present invention also provides a method for producing lipopeptide antibacterial compounds, comprising the following steps:
[0015] The siam Bacillus AQ171 described in the above technical solution is fermented to obtain a fermentation liquid containing lipopeptide antibacterial compounds.
[0016] Preferably, the lipopeptide antibacterial compound includes bacillus mycin D, pelistatin, fengycin and surfactin at the same time.
[0017] Preferably, the temperature of the fermentation culture is 22° C. to 37° C.; the shaking speed of the fermentation culture is 150 rpm to 200 rpm; and the OD600 value of the fermentation liquid is 0.3.
[0018] Beneficial effects:
[0019] The present invention provides a strain of Bacillus siamensis AQ171, with a deposit number of CGMCC No. 34134. The Bacillus siamensis AQ171 of the present invention has excellent broad-spectrum antibacterial activity, can simultaneously and efficiently inhibit the red star pathogen (Alternaria alternata) and the wilt pathogen (Ralstonia solanacearum), and the antibacterial effect (+++) is significantly better than other Bacillus isolated from the same batch or stored in the laboratory (such as NH4 / 1, Cas02, etc.). HPLC-MS / MS analysis shows that Bacillus siamensis AQ171 can synthesize a variety of lipopeptide antibacterial substances (such as bacillus d, pelistatin, fengycin and surfactin), and the types of metabolites are far more than other strains containing only one or two similar compounds. The crude fermentation extract of Bacillus siamensis AQ171 described in this study significantly inhibits fungi at a low concentration (1 mg / mL) and exhibits strong antibacterial activity against bacteria at 10 mg / mL, highlighting its high bioactivity and stability. Therefore, AQ171 has significant potential for application in agricultural biocontrol and serves as a high-quality candidate for the development of highly effective microbial pesticides.
[0020] Biological deposit information
[0021] Bacillus siamensis AQ171, biologically classified as Bacillus siamensis, is deposited in the General Microbiology Center of the China Culture Collection Administration, abbreviated as CGMCC. The deposit date is April 8, 2025, and the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 34134. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.
[0023] Figure 1 Schematic diagram of the colony morphology of Bacillus siamensis AQ171;
[0024] Figure 2 Schematic diagram of the identification phylogenetic tree of Bacillus siamensis AQ171 strain;
[0025] Figure 3 This is a comparison of the antifungal and antibacterial activities of methanol extracts of different Bacillus sp.
[0026] Figure 4 This is the HPLC-MS / MS test results of methanol phase extracts of different Bacillus. DETAILED DESCRIPTION
[0027]
[0028] The present invention also provides a microbial agent, wherein the active ingredient of the microbial agent includes the siam Bacillus AQ171 according to claim 1.
[0029] As an embodiment, the OD of the microbial agent of the present invention is 600 The value is 0.3. In one embodiment, the microbial agent of the present invention further comprises an agriculturally acceptable excipient. In one embodiment, the agriculturally acceptable excipient of the present invention comprises one or more of an adsorbent material, a liquid suspension matrix, and an embedding material. In one embodiment, the adsorbent material of the present invention may be a solid culture medium. In one embodiment, the suspension matrix of the present invention may be a liquid culture medium.
[0030] As an embodiment, the preparation method of the microbial agent of the present invention comprises the following steps: activating Bacillus siamese AQ171 by NA medium, and fermenting and culturing in NB medium to the logarithmic phase to obtain a fermentation broth. As an embodiment, the fermentation broth of the present invention is adjusted to OD 600 =0.3 working concentration to obtain the microbial agent.
[0031] The present invention also provides the use of the siam Bacillus AQ171 described in the above technical solution or the microbial agent described in the above technical solution for producing lipopeptide antimicrobial compounds and / or inhibiting pathogens. In one embodiment, the lipopeptide antimicrobial compound of the present invention includes one or more of bacillus spp., pelistatin, fengycin, and surfactin. In one embodiment, the siam Bacillus AQ171 of the present invention produces significantly more metabolites than other Bacillus isolated from the same batch.
[0032] In one embodiment, the pathogens described herein include pathogenic fungi and / or pathogenic bacteria; the pathogenic fungi include the pathogen of Alternaria alternata; and the pathogenic bacteria include the pathogen of Ralstonia solanacearum. In one embodiment, the antibacterial effect of the siam Bacillus sp. AQ171 described herein is significantly superior to that of other Bacillus sp. isolated from the same batch. In one embodiment, the crude fermentation extract of the siam Bacillus sp. AQ171 described herein can significantly inhibit fungi at a low concentration (1 mg / mL) and also exhibits strong antibacterial activity against bacteria at 10 mg / mL.
[0033] The present invention also provides a method for producing lipopeptide antibacterial compounds, comprising the following steps:
[0034] The siam Bacillus AQ171 described in the above technical solution is fermented to obtain a fermentation liquid containing lipopeptide antibacterial compounds.
[0035] The present invention ferments and cultures the siam bacillus AQ171 described in the above technical solution to obtain a fermentation broth; the fermentation broth contains lipopeptide antibacterial compounds. As an embodiment, the fermentation culture temperature is 22°C to 37°C; the shaker speed of the fermentation culture is 150rpm to 200rpm; the OD of the fermentation broth of the present invention is 600 The value is 0.3. As another embodiment, the temperature of the fermentation culture is 28°C; the shaker speed of the fermentation culture is 180 rpm. As an embodiment, the fermentation culture of the present invention is carried out by a culture medium. As an embodiment, the culture medium of the present invention can be an oat solid culture medium and / or NB culture medium. As an embodiment, the lipopeptide antibacterial compound of the present invention includes bacillus D, pelistatin, fengyuansu and surfactin at the same time. As an embodiment, the lipopeptide antibacterial compound in the fermentation broth of the present invention can be further extracted with methanol to obtain a methanol extract of the lipopeptide antibacterial compound, and the methanol (GC content ≥99.5%) is purchased from Sinopharm Chemical Reagent Co., Ltd. As an embodiment, after obtaining the methanol extract of the lipopeptide antibacterial compound, the methanol extract can be dried with nitrogen, dissolved in 30% methanol solution, and then sterilized using a filter membrane. As an embodiment, the filter membrane of the present invention can be a 0.22μm filter membrane.
[0036] To further illustrate the present invention, a strain of Bacillus siamensis and its application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.
[0037] Example 1
[0038] Isolation and identification of strains
[0039] (1) Isolation of strains
[0040] Add 10 g of litter and leaf humus to 90 mL of sterilized ultrapure water, shake at 28°C and 180 rpm for 30 min, mix thoroughly and then let stand, take 1 mL of the suspension and dilute it in sequence, take 100 μL of the diluted suspension and spread it on NA solid medium (peptone 10 g / L, sodium chloride 5 g / L, agar 15 g / L, beef extract powder 3 g / L), and culture it in an incubator at 28°C for 5 days. The colony on the NA medium was streaked and purified repeatedly 5 to 8 times. The purified single strain was cultured on the NA medium to obtain the following: Figure 1 The colony shown is numbered AQ171. The colony is a light yellow, round colony with a smooth surface. As the strain grows, the colony gradually becomes wrinkled and matte.
[0041] (2) Identification of strains
[0042] The genomic DNA of AQ171 was extracted, and according to the method of using Norvegian 2×Taq Plus MasterMixⅡ DNA polymerase, the extracted genomic DNA was used as the amplification template, and the 16S rDNA universal primer was used as the amplification primer. The sequence was: upstream primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2), downstream primer (1492R): 5'-TACGGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3). The specific PCR amplification system is shown in Table 1, and the PCR amplification program used is shown in Table 2. PCR amplification was performed on the bacteria respectively. The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results of the 16S rDNA of the strains were Blast aligned in the NCBI database, and the strains with higher sequence similarity were selected. The phylogenetic tree was constructed using MEGA11, as shown in FIG. Figure 2 The results showed that the strain AQ171 was Bacillus siamensis, which was biologically classified as Bacillus siamensis and deposited as a biological deposit.
[0043] Table 1 PCR amplification system
[0044] Reagent name Dosage / μL DNA template 1 Forward primer 1 Reverse primer 1 2×TaqPlusMasterMixⅡ 12.5 <![CDATA[ddH2O]]> 9.5
[0045] Table 2 PCR amplification program
[0046]
[0047] Example 2
[0048] A microbial agent
[0049] 1. Composition of the microbial agent
[0050] Active ingredient: Bacillus siamese AQ171 (deposit number: CGMCC No.34134).
[0051] Culture medium: oatmeal solid medium and NB medium.
[0052] 2. Preparation Method
[0053] Bacillus siamese AQ171 was activated by oatmeal solid medium (OA) and fermented in NB medium to the logarithmic phase to obtain a fermentation broth. The fermentation broth was adjusted to OD 600 =0.3 working concentration. The microbial agent is obtained.
[0054] Example 3
[0055] Antagonistic Effects of Bacillus siamese AQ171 on Pathogenic Fungi and Bacteria
[0056] 1. Bacillus siamensis AQ171 antagonizes pathogenic bacteria
[0057] The purified Siamese Bacillus AQ171 was activated and grown to the logarithmic phase by shaking the flask with NB medium, and inoculated into the center of the NA medium. After dark culture in an incubator at 28 ° C for 24 hours, the bacterial wilt pathogen (Ralstonia solanacearum RS10, see Chu, D., Ilyas, N., Peng, L., Wang, X., Wang, D., Xu, Z., ... & Yuan, Y. (2021). Genomic insights on fighting bacterial wilt by a novel Bacillus amyloliquefaciens strain Cas02. Microbial Biotechnology, 15 (4), 1152-1167.) was cultured to the logarithmic phase. The bacterial suspension was evenly sprayed on the surface of the culture medium to test the antagonistic effect of the strain on bacterial wilt. After 24 hours, the antibacterial effect of the strain on bacterial wilt was judged based on the size of the inhibition zone.
[0058] 2. Antagonism of Bacillus siamensis AQ171 against pathogenic fungi
[0059] The red star pathogen (Alternaria alternate, see Yuan, Yuan, et al. "Production of Antifungal Iturins from Vegetable Straw: A Combined Chemical-Bacterial Process." Bioresource Technology, vol. 378, 2023, p. 129010.) was activated on oatmeal solid medium (OA). A 5mm borer was used to extract a bacterial cake and inoculated into the center of the medium. Purified siam bacillus AQ171 was activated in NB medium shake flasks to the logarithmic phase and inoculated onto both sides of the red star pathogen (or 50 μL was inoculated into the bacterial cake well). The cultures were incubated in the dark at 25°C for 5-7 days, and the antibacterial effects were observed daily. (The above pathogen strains are deposited in the National Agricultural Environmental Microbial Germplasm Resource Bank - Shandong).
[0060] 3. Other comparative strains
[0061] Fifteen other strains isolated and screened from the same batch as Bacillus siamatifolia AQ171 and previously isolated and preserved in the laboratory were selected as control strains. The identification process was the same as that of Bacillus siamatifolia AQ171 in Example 1. The method for antagonizing pathogenic bacteria (Ralstonia solanacearum) was the same as step 1, and the method for antagonizing pathogenic fungi (Alternaria alternate) was the same as step 2. The specific strain identification results and the results of antagonism to pathogenic fungi and pathogenic bacteria are shown in Table 3.
[0062] Table 3 Antagonistic effects of different strains on pathogenic fungi and bacteria and their identification information
[0063]
[0064] In Table 3, "×" indicates that the strain has no inhibitory effect on pathogens; "+" indicates that the strain has an inhibitory effect on pathogens. The number of "+" indicates the strength of the inhibitory effect, with the strongest effect being indicated as "+++." This indicates that among the strains isolated and screened from the same batch and those previously preserved in the laboratory, Bacillus strains possessed the best antibacterial activity. Among them, Bacillus siamensis AQ171 possessed strong antagonism against both pathogenic bacteria and fungi, and exhibited the best antagonism against both Ralstonia solanacearum and Alternaria alternate.
[0065] Example 4
[0066] Isolation and Identification of Antimicrobial Compounds from Bacillus siamese AQ171
[0067] Further, four different strains of Bacillus (AQ171, WL51, HTDT4 and cmc7) with strong antibacterial activity in Example 3 were fermented in large quantities: OD 600 200 μL of a bacterial suspension with a pH value of 0.3 was inoculated into 500 mL of NB medium and fermented at 28°C for 3 days. The resulting fermentation broth was centrifuged, the supernatant was collected, and lyophilized to a powder. The fermentation extract was partially purified using a macroporous adsorption resin. 5 g of the extract was dissolved in 50 mL of distilled water and loaded onto an XAD-16 macroporous resin wet-packed in a glass column (800 mm × 40 mm inner diameter). Elution was performed sequentially with 600 mL of water, 600 mL of a 30% aqueous methanol solution (water:methanol = 7:3, v / v), and 600 mL of a 100% methanol solution (anhydrous methanol, purchased from China National Pharmaceutical Group Co., Ltd.) at a flow rate of 3 mL / min. The 100% methanol phase eluate was collected, rotary evaporated in a 40°C water bath, and weighed to determine the yield. This yielded the purified 100% methanol phase extracts of the different Bacillus species fermentations.
[0068] The methanol extract obtained above was used to test the antibacterial activity against Alternaria alternata and Ralstonia solanacearum at final concentrations of 1 mg / mL and 10 mg / mL, respectively. The specific steps are as follows:
[0069] 100 mg of the purified 100% methanol extract was weighed separately and extracted with 500 μL of anhydrous methanol by vortexing for 5 minutes, followed by centrifugation (8000 rpm for 5 minutes) to collect the supernatant. The precipitate after centrifugation was further extracted with 500 μL of anhydrous methanol. To ensure complete dissolution of the active substance, the extraction operation was repeated three times. After drying with nitrogen, approximately 1500 mL of methanol extract obtained three times was dissolved in 10 mL of 30% methanol solution and sterilized by filtering through a 0.22 μm filter membrane for testing to obtain the purified 100% methanol extract solution.
[0070] The red star pathogen was activated and cultured in oatmeal (OA) medium for about 7 days. 1 mL of the above-mentioned methanol phase extract solution was first dissolved in OA medium and then poured into the plate. After the plate solidified and cooled, a 5mm punch was used to take the red star bacteria cake and place it in the center of the culture medium. The culture was dark-cultured at 25°C for 7 to 10 days, the antibacterial effect was observed, and the antibacterial rate was calculated to obtain the inhibitory effect of different Bacillus metabolites (i.e., methanol phase extract solution) on red star pathogen. Figure 3 As shown in A to D and Table 4, Figure 3 Figure A shows the inhibitory effect of AQ171 metabolites on Alternaria alternata; Figure B shows the inhibitory effect of WL51 metabolites on Alternaria alternata; Figure C shows the inhibitory effect of HTDT4 metabolites on Alternaria alternata; and Figure D shows the inhibitory effect of cmc7 metabolites on Alternaria alternata. Inhibition rate (%) = [(control diameter - treated diameter) / control diameter] * 100.
[0071] After the bacterial wilt is activated in NA medium, it is inoculated into NB medium and shake-cultured. The bacterial suspension cultured to the logarithmic phase is evenly sprayed onto the surface of the NA medium. After the bacterial liquid is air-dried, a hole is punched using a puncher. The center culture medium is removed by an inoculation needle and 50 μL of the above-mentioned methanol extract solution is added to the hole. The culture is dark-cultured in an incubator at 28°C for 24 hours to observe the size of the inhibition zone and obtain the inhibitory effect of different Bacillus metabolites on bacterial wilt. Figure 3 As shown in E to H, Figure 3 E in the figure is the inhibitory effect of AQ171 metabolites on bacterial wilt; F is the inhibitory effect of WL51 metabolites on bacterial wilt; G is the inhibitory effect of HTDT4 metabolites on bacterial wilt; H is the inhibitory effect of cmc7 metabolites on bacterial wilt. Figure 3The results shown in Table 4 showed that the metabolites of the four strains all had strong antifungal activity, but only the metabolites of AQ171 had both strong antifungal and antibacterial activities, especially the inhibition zone radius of AQ171 against bacterial wilt pathogen reached 0.8 cm.
[0072] Table 4 Inhibitory effect of different strains on red star pathogen
[0073] Sample name Antibacterial rate (%) AQ171 82.12±0.12a WL51 84.11±0.10a cmc7 76.85±4.58a HTDT4 41.70±1.70b
[0074] The fermentation product after purification of the above bacteria was further subjected to HPLC-MS / MS detection, and the results were as follows Figure 4 A longitudinal comparison of metabolites revealed that compared with other strains, Bacillus siamensis AQ171 could metabolize a wider range of lipopeptide antimicrobial compounds, including bacillus mycin D, pelistatin, fengycin, and surfactin, while other strains only had one or two of the aforementioned active metabolites.
[0075] Based on the above, it can be seen that the siam bacillus AQ171 provided by the present invention has important application potential in the field of agricultural biocontrol and can be used as a high-quality candidate strain for the development of highly effective microbial pesticides.
[0076] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.
Claims
1. A strain of Bacillus siamensis AQ171, deposited with CGMCC No. 34134.
2. A microbial agent, characterized in that: The active ingredient of the microbial agent includes the siam bacillus AQ171 according to claim 1.
3. The microbial agent according to claim 2, characterized in that The microbial agent further comprises agriculturally acceptable adjuvants.
4. The microbial agent according to claim 3, characterized in that The agriculturally acceptable auxiliary material includes one or more of an adsorption material, a liquid suspension matrix and an embedding material.
5. Use of the Bacillus siamensis AQ171 according to claim 1 or the microbial agent according to any one of claims 2 to 4 in producing lipopeptide antimicrobial compounds and / or inhibiting pathogenic bacteria.
6. The use according to claim 5, characterized in that The lipopeptide antibacterial compound includes one or more of bacillus mycin D, pelistatin, fengycin and surfactin.
7. The use according to claim 5, characterized in that The pathogens include pathogenic fungi and / or pathogenic bacteria; The pathogenic fungi include red star pathogenic bacteria; The pathogenic bacteria include bacterial wilt pathogen.
8. A method for producing lipopeptide antibacterial compounds, characterized in that: The following steps are involved: The siam Bacillus AQ171 according to claim 1 is fermented and cultured to obtain a fermentation liquid containing lipopeptide antibacterial compounds.
9. The method according to claim 8, characterized in that The lipopeptide antibacterial compound includes bacillus mycin D, pelistatin, fengycin and surfactin.
10. The method according to claim 8, characterized in that The fermentation temperature is 22°C to 37°C; the shaker speed is 150 rpm to 200 rpm; The OD of the fermentation broth 600 The value is 0.3.