Bacillus velezensis for preventing and treating powdery mildew of rosa chinensis and application thereof
By using Bacillus belye TYU1 and its metabolites and volatile gases, the chemical control problem of gray mold in roses was solved, achieving efficient and safe biological control and promoting plant growth.
Patent Information
- Application Number
- CN202611078761.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-20
- Publication Date
- 2026-11-03
AI Technical Summary
Existing chemical control methods for rose gray mold pose risks of increased resistance, pesticide residues, and environmental pollution, and there is a lack of biocontrol strains with growth-promoting properties.
Using Bacillus beleilli strain TYU1 and its metabolites and volatile gases, plant growth was promoted and organophosphates were degraded by inhibiting the growth of Botrytis cinerea, producing ferrophiles and IAA.
It effectively suppresses gray mold in roses, reduces the risk of pesticide resistance, reduces environmental pollution, promotes plant growth, and provides an efficient and safe biological control method.
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Figure CN123027148A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically, to a Bacillus bellis strain for controlling gray mold in roses and its application. Background Technology
[0002] Roses, belonging to the genus Rosa in the family Rosaceae, are one of the world's most important ornamental flowers, possessing high ornamental and economic value. They occupy an important position in cut flower production, landscaping, and the fragrance and cosmetic industries. With the development of protected horticulture and commercial production, the scale of rose cultivation has continued to expand, and its disease problems have become increasingly prominent, seriously restricting the high-quality development of the rose industry.
[0003] Gray mold of roses is a typical fungal disease caused by Botrytis cinerea. It occurs widely around the world and is one of the most serious diseases that threaten rose production, especially during post-harvest preservation.
[0004] Currently, the control of gray mold in roses mainly relies on chemical fungicides, such as dicarboximides, benzimidazoles, and amides. Although chemical control has the advantage of being fast-acting, long-term and large-scale use can easily lead to increased drug resistance in pathogens, as well as pesticide residues, environmental pollution, and ecological security risks, making it difficult to meet the requirements of modern green agriculture and sustainable development.
[0005] With the development of ecological agriculture, biological control has gradually become an important direction for plant disease prevention and control. Utilizing antagonistic microorganisms to inhibit the growth of pathogens has advantages such as environmental friendliness, high safety, and diverse mechanisms of action. Studies have shown that microbial biocontrol bacteria can exert their control effects through multiple pathways, such as competing for nutrients and ecological niches, producing antibacterial substances, and inducing plant systemic resistance. They have shown good application prospects in the control of gray mold. Among many biocontrol microorganisms, Bacillus spp. is considered one of the most promising biological control resources due to its rapid reproduction, strong resistance, broad antibacterial spectrum, and ease of formulation. In particular, Bacillus velezensis has shown good antagonistic effects and application value in the control of various plant diseases.
[0006] However, there are currently few strains that have specific antagonistic effects against gray mold in roses and also have multiple growth-promoting properties. The development and application of related biocontrol agents are still in their early stages. There is an urgent need to develop efficient, safe biocontrol strains and technologies that also have growth-promoting properties to meet the pressing needs of sustainable agricultural development.
[0007] Therefore, there is an urgent need for a Bacillus berberis strain for the prevention and control of gray mold in roses and its application, in order to solve the above problems. Summary of the Invention
[0008] In order to overcome the above-mentioned defects of the prior art, the embodiments of the present invention provide a Bacillus berberis for the prevention and control of gray mold in roses and its application, so as to solve the problems mentioned in the background art.
[0009] To achieve the above objectives, the present invention provides the following technical solution: a Bacillus baileyi for controlling gray mold in roses, comprising Bacillus baileyi strain TYU1 for controlling gray mold in roses. This strain is a Gram-positive bacterium and is deposited at the China General Microbiological Culture Collection Center on August 1, 2025, with the accession number CGMCC No. 35496.
[0010] In a preferred embodiment, the strain has the ability to significantly inhibit the growth and reproduction of Botrytis cinerea.
[0011] In a preferred embodiment, the Bacillus baileyi strain TYU1 or its culture medium, bacterial suspension, fermentation product or volatiles can be used for antibacterial purposes.
[0012] In a preferred embodiment, the Bacillus baileyi strain TYU1 or its culture medium, bacterial suspension, fermentation product or volatiles can be used to prepare plant biocontrol agents.
[0013] In a preferred embodiment, the Bacillus baileyi strain TYU1 or its culture medium, bacterial suspension, fermentation product or volatiles can be used for the prevention and control of plant diseases, especially for the prevention and control of gray mold in plants and / or gray mold in roses.
[0014] In a preferred embodiment, the Bacillus baileyi strain TYU1 or its culture medium, bacterial suspension, fermentation product or volatiles can also be used to promote plant growth, degrade organophosphates, prepare IAA and produce ironophiles.
[0015] The technical effects and advantages of this invention are as follows:
[0016] This invention provides a strain of Bacillus belye TYU1 with antibacterial activity, which can significantly inhibit the growth and reproduction of Botrytis cinerea.
[0017] This strain, along with its metabolites and volatile organic compounds (VOCs), can effectively inhibit the growth of the pathogenic fungus *Gyromyces rubrum* in roses.
[0018] This strain exhibits typical characteristics of plant growth-promoting bacteria, such as dissolving organic phosphorus, producing siderophores, and producing IAA.
[0019] Therefore, Bacillus tyu1 provides a valuable strain resource for the research and development of biocontrol agents for gray mold in roses, and has broad application potential and promotion value. Attached Figure Description
[0020] Figure 1 This is a schematic diagram of the morphological observation of the strain TYU1 of the present invention.
[0021] Figure 2 This is a schematic diagram of the phylogenetic tree constructed based on the 16S rDNA gene sequence of the strain TYU1 of the present invention.
[0022] Figure 3 This is a schematic diagram of the phylogenetic tree constructed based on the gyrB gene sequence of the strain TYU1 of the present invention.
[0023] Figure 4 This is a schematic diagram illustrating the antagonistic effect of the strain TYU1 of the present invention against the causal agent of gray mold on roses.
[0024] Figure 5 This diagram illustrates the inhibitory effect of the volatile gases emitted by strain TYU1 of the present invention on the causal agent of gray mold on roses.
[0025] Figure 6 This is a schematic diagram illustrating the inhibition rate of the sterile filtrate of strain TYU1 of the present invention against the root causal agent of gray mold of rose at gradient concentrations.
[0026] Figure 7 This is a schematic diagram illustrating the detection of growth-promoting capabilities of the TYU1 strain of the present invention. Detailed Implementation
[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0028] As attached Figure 1 Appendix Figure 2 Appendix Figure 3 Appendix Figure 4 Appendix Figure 5 Appendix Figure 6 and attached Figure 7 As shown, the present invention provides a Bacillus velezensis strain for controlling gray mold in roses, including Bacillus velezensis strain TYU1 for controlling gray mold in roses. This strain is a Gram-positive bacterium and is deposited at the China General Microbiological Culture Collection Center on August 1, 2025, with accession number CGMCC No. 35496.
[0029] The strain has the ability to significantly inhibit the growth and reproduction of Botrytis cinerea.
[0030] Bacillus baileyi strain TYU1 or its culture medium, bacterial suspension, fermentation products or volatiles can be used for antibacterial purposes.
[0031] Bacillus baileyi strain TYU1 or its culture medium, bacterial suspension, fermentation products or volatiles can be used to prepare plant biocontrol agents.
[0032] Bacillus baileyi strain TYU1 or its culture medium, bacterial suspension, fermentation products or volatiles can be used for the prevention and control of plant diseases, especially for the prevention and control of gray mold in plants and / or gray mold in roses.
[0033] Bacillus baileyi strain TYU1 or its culture medium, bacterial suspension, fermentation products or volatiles can also be used to promote plant growth, degrade organophosphates, prepare IAA and produce ironophiles.
[0034] Botrytis cinerea was obtained from the Plant Protection Laboratory of the Department of Horticulture and Landscape Architecture, Taiyuan University.
[0035] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.
[0036] Example 1
[0037] Identification of Bacillus belyssus TYU1
[0038] 1. Isolation and morphological observation of strains
[0039] Strain TYU1 was isolated from the rhizosphere soil of roses and is an indigenous microorganism originating from the rhizosphere environment of roses. The collected rhizosphere soil samples were isolated and cultured using the dilution plating method. After multiple rounds of purification, the target strain TYU1 was obtained. This strain was then inoculated onto LB solid medium and incubated overnight at 30℃. The morphological characteristics of single colonies were observed, including color, size, edge morphology, and transparency.
[0040] Morphological observation results as follows Figure 1 As shown, the strain forms irregularly shaped circular colonies on LB solid medium. Figure 1 A), with wavy edges, opaque milky white to light yellow overall, dry surface, rough texture, slightly raised center with irregular wrinkles, no obvious pigment production, Gram staining shows that the strain is rod-shaped ( Figure 1 B), after staining, turns blue-purple, indicating that it is a Gram-positive bacillus.
[0041] 2. Physiological and biochemical characteristics analysis
[0042] According to the "Handbook of Systematic Identification of Common Bacteria," the physiological and biochemical reactions of strain TYU1 were tested (Table 1). Strain TYU1 showed positive reactions in catalase, litmus milk hydrolysis, VP, citrate utilization, D-xylose, L-arabinose, D-mannitol, nitrate reduction, and starch hydrolysis tests, indicating its strong carbon source utilization capacity and certain metabolic activity. It showed negative reactions in oxidase, methyl red, propionate, and gelatin liquefaction tests. Furthermore, strain TYU1 can grow under 7% NaCl conditions and pH 5.7, and exhibits anaerobic growth ability, indicating its adaptability and tolerance to the environment.
[0043] Table 1. Physiological and biochemical identification of biocontrol bacteria
[0044] Physiological and biochemical indicators Test results Physiological and biochemical indicators Test results Oxidase test － L-arabinose test ＋ Catalase test ＋ D-mannitol test ＋ Litmus Milk Hydrolysis Test ＋ Gelatin liquefaction test － Methyl red test － 7% NaCl growth experiment ＋ VP test ＋ Growth test at pH 5.7 ＋ Citrate test ＋ Nitrate reduction test ＋ Propionate test － Starch hydrolysis test ＋ D-xylose test ＋ Anaerobic growth experiment ＋
[0045] Note: + indicates a positive reaction; - indicates a negative reaction.
[0046] 3. Molecular biological identification
[0047] Total DNA was extracted from activated strain TYU1. Using this DNA as a template, PCR amplification of the 16S rDNA gene and gyrB gene of strain TYU1 was performed using the universal bacterial primers 27F / 1492R and the housekeeping gene gyrB primers UP-1S / UP-2Sr, respectively. The primer sequences used are as follows:
[0048] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1);
[0049] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2);
[0050] UP-1S: 5'-GAAGTCATCATGACCGTTCTGCA-3' (SEQ ID NO.3);
[0051] UP-2Sr: 5'-AGCAGGGTACGGATGTGCGAGCC-3' (SEQ ID NO. 4).
[0052] PCR amplification system (20 μL): template DNA 1 μL, 2 × Taq PCR Mix 10 μL, forward / reverse primers 1 μL each, ddH2O 7 μL.
[0053] PCR amplification program: 94℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 57℃ annealing for 30 s, 72℃ extension for 90 s, 40 cycles; 72℃ final extension for 2 min; store at 4℃.
[0054] After purification, the amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequences were compared with the sequences published in the NCBI database for homology, and a phylogenetic tree was constructed using MEGA 11.0 software.
[0055] Molecular identification results such as Figure 2 and Figure 3 As shown: the 16S rDNA gene sequence of strain TYU1 has 99.93% sequence similarity with the DB1S sequence of Bacillus velezensis; the gyrB gene sequence has 100% sequence similarity with the RL-LL12 sequence of Bacillus velezensis. A phylogenetic tree constructed based on these sequences shows that strain TYU1 clusters with several previously reported Bacillus velezensis strains in the same branch, exhibiting high bootstrap support, indicating a close phylogenetic relationship between this strain and Bacillus velezensis. Figure 2 and Figure 3 ).
[0056] Based on the combined morphological characteristics, physiological and biochemical characteristics and molecular biological identification results, strain TYU1 can be finally identified as Bacillus velezensis.
[0057] Example 2
[0058] Antibacterial activity of Bacillus vesiculosus TYU1 against Staphylococcus aureus
[0059] 1. Inhibition detection of Bacillus belyceta TYU1 against Botrytis cinerea, the causal agent of gray mold in roses.
[0060] The inhibitory effect of Bacillus belye TYU1 on Botrytis cinerea was tested using the plate confrontation method. The experimental method was as follows: the strain TYU1 was inoculated on LB solid medium and spread for activation. A single colony was picked and inoculated into LB liquid medium. The culture was shaken at 30℃ and 200 r / min for 12 h to obtain the fermentation broth for use. Sterile filter paper with a diameter of 6 mm was immersed in the fermentation broth for 1 min and then placed on sterile paper to dry.
[0061] Botrytis cinerea cakes (5 mm in diameter) were inoculated in the center of a 90 mm diameter PDA plate. Four sterile filter paper discs containing fermentation broth from different strains were placed 3 cm from the center using a cross-hatching method. A control group inoculated only with Botrytis cinerea cakes was used. Each treatment was repeated five times. All plates were incubated at 26°C and 60% relative humidity for 7 days. Colony growth was observed, and the inhibition rate was determined. The inhibition rate was calculated using the following formula:
[0062] Inhibition rate (%) = (Radius of control pathogen - Radius of treated pathogen) × 100 / (Radius of control pathogen - Radius of fungal cake)
[0063] The antibacterial effect of Bacillus vesiculosus TYU1 against Staphylococcus aureus was detected as follows: Figure 4 As shown, where Figure 4 A represents the colony morphology of the control group. Figure 4 B represents the colony morphology of the experimental group. Figure 4 C represents the measured inhibition rate. The results showed that Bacillus belye TYU1 had an inhibitory effect on Staphylococcus aureus, with an inhibition rate of 69.61%.
[0064] 2. Antibacterial activity of volatile gases from Bacillus belye TYU1 against Staphylococcus aureus
[0065] The inhibitory effect of volatile gases produced by Bacillus belyi TYU1 on Botrytis cinerea was determined using the two-segment petri dish method. 5 μL of TYU1 bacterial suspension was inoculated onto one side of LB medium in a two-segment petri dish, and a Botrytis cinerea cake (5 mm in diameter) was inoculated into the center of the other side of PDA medium. The medium without TYU1 bacterial suspension served as the control group. The dishes were incubated at 26℃ and 60% relative humidity for 7 days. Colony growth was observed and the inhibition rate was measured. Each treatment was repeated 5 times.
[0066] The antibacterial activity of volatile gases from Bacillus belye TYU1 against Staphylococcus aureus is as follows: Figure 5 As shown, where Figure 5 A represents the colony morphology of the control group and the experimental group. Figure 5 B represents the measured inhibition rate. The results show that the volatile gas produced by Bacillus belye TYU1 can also significantly inhibit the growth of Botrytis cinerea, with an inhibition rate of 58.78%.
[0067] 3. Antibacterial activity of Bacillus belye TYU1 sterile filtrate against Botrytis cinerea
[0068] The fermentation broth of strain TYU1 was centrifuged at 8000 rpm for 20 min. The supernatant was filtered six times through a sterile 0.22 μm needle filter and added to sterile PDA medium at proportions of 0%, 1%, 5%, 7%, 10%, and 20% to prepare plates. A 5 mm diameter Botrytis cinerea mycelial cake was inoculated into the center of the PDA medium. Each treatment was repeated five times. The plates were incubated at 26℃ and 60% relative humidity for 7 days. The inhibition rate was measured on the 2nd, 4th, and 7th days of incubation.
[0069] The sterile filtrate of Bacillus baileyi TYU1 showed antibacterial activity against Staphylococcus aureus, such as... Figure 6As shown, the results indicated that the inhibition rate of *Bacillus belye* TYU1 sterile filtrate against *Botrytis cinerea* decreased with increasing culture time, and the 1% sterile filtrate exhibited the strongest antibacterial effect on day 2, with an inhibition rate of 47.98%. Figure 6 A), the 7% sterile filtrate showed the strongest antibacterial effect on day 4, with an inhibition rate of 30.36% (A). Figure 6 B), by day 7, the sterile filtrate treatment had no antibacterial effect ( Figure 6 C).
[0070] Example 3
[0071] Assay of the growth-promoting ability of Bacillus belysin TYU1
[0072] Bacillus belye TYU1 was inoculated onto LB solid medium and cultured for 12 days to obtain single colonies.
[0073] (1) Phosphorus solubility test: A single colony of TYU1 strain was inoculated onto an organic phosphorus bacteria culture medium and cultured for 48 h to observe whether a phosphorus solubility zone appeared;
[0074] (2) IAA production capacity determination: a single colony of TYU1 strain was inoculated into LB liquid medium containing L-tryptophan. After culturing for 3 days, a small amount of Salkowsk colorimetric solution was added and the culture was protected from light for 30 min. The bacterial culture was then observed to see if it turned red.
[0075] (3) Determination of the ability to produce iron: Take a single colony of TYU1 strain and inoculate it on CAS detection medium. After culturing for 3 days, observe whether a yellow or orange transparent ring appears around the colony.
[0076] The results of the growth-promoting function test of Bacillus belyssioides TYU1 are as follows: Figure 7 As shown, strain TYU1 can grow normally on organophosphate bacteria culture medium and produce a distinct clear zone ( Figure 7 A) indicates that this strain has good phosphate-solubilizing ability. The TYU1 strain grows well in a medium containing L-tryptophan, and after treatment with Salkowsk colorimetric solution, the bacterial culture turns red (A). Figure 7 B) indicates that this strain can produce IAA. Furthermore, this strain can also form an orange-yellow transparent zone on the CAS detection medium. Figure 7 C) indicates that it can produce heparin, thereby enhancing the absorption and utilization of iron. The above results show that Bacillus belye TYU1 has the ability to solubilize phosphorus, produce IAA and heparin, and exhibits good growth-promoting ability.
[0077] This invention provides a strain of Bacillus belye TYU1 with antibacterial activity. This bacterium can significantly inhibit the growth and reproduction of Botrytis cinerea. This strain, its metabolites, and volatile organic compounds (VOCs) can effectively inhibit the growth of the pathogen of gray mold in roses. This strain has typical characteristics of plant growth-promoting bacteria, such as dissolving organophosphates, producing siderophores, and producing IAA. Therefore, Bacillus belye TYU1 provides a valuable strain resource for the research and development of biocontrol agents for gray mold in roses, and has broad application potential and promotion value.
[0078] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A Bacillus vesiculosus strain for controlling gray mold in roses, characterized in that: This includes the Bacillus tyU1 strain, which controls gray mold in roses. This strain is a Gram-positive bacterium and is deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 1, 2025, with accession number CGMCC No. 35496.
2. The Bacillus vesiculosus for controlling gray mold in roses according to claim 1, characterized in that: The strain has the ability to significantly inhibit the growth and reproduction of Botrytis cinerea.
3. The application of Bacillus vesiculosus of claim 1 in the prevention and control of gray mold in roses.
4. The application according to claim 3, characterized in that: The application in the prevention and control of gray mold in roses is to inhibit Botrytis cinerea.
5. The application according to claim 3, characterized in that: The aforementioned method for controlling gray mold in roses involves using this strain and its volatile gases and metabolites to control the disease.
6. The application of Bacillus vesiculosus of claim 1 for preventing and controlling gray mold in roses in promoting plant growth.
7. A biocontrol agent for gray mold of roses, characterized in that: Includes the Bacillus bellisi, as described in claim 1, for the prevention and control of gray mold in roses.