Bacillus cereus HUB0155 and application thereof

Abstract

The invention discloses a bacillus cereus HUB0155 strain and an application of the bacillus cereus HUB0155 strain. The strain is preserved in Guangdong Microbial Culture Collection Center (GDMCC) on March 16, 2026, and the preservation number is GDMCC No: 67964. The strain has a good antagonistic effect on five kinds of tea-oil tree pathogenic fungi, and the bacteriostasis rate reaches 44.67-70%. As the tea-oil tree endophyte, the HUB0155 is safer to tea-oil tree plants. The bacterial strain is further inoculated on the surfaces of in-vivo camellia oleifera leaves infected by pathogenic bacteria, the number of new leaves of camellia oleifera plants treated by the bacterial liquid is increased, fallen leaves are obviously reduced, and disease spots are inhibited. Therefore, the invention provides a core strain resource for the development of a green prevention and control technology of camellia oleifera diseases, and has obvious development and application prospects in the aspect of large-area biological prevention and control of leaves and fruits of low-yield camellia oleifera forests.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, specifically to a strain of Bacillus cereus HUB0155 and its applications. Background Technology

[0002] Camellia oleifera ( Camellia oleifera Abel . Camellia oleifera is a unique woody oilseed crop in my country, boasting high yields and economic benefits. Beyond its significant economic value, it also plays a crucial role in ecological protection. However, the development of the camellia oleifera industry has consistently been constrained by diseases, with fungal diseases having a particularly prominent impact. Reports indicate that there are 42 types of camellia oleifera diseases, 30 of which are fungal, including anthracnose, soft rot, and root rot. These diseases are prevalent in all camellia oleifera producing areas across the country, severely impacting yield and quality, leading to fruit and leaf drop, yield reductions of 20-30%, and resulting in substantial economic losses.

[0003] Traditional chemical pesticides are commonly used to control fungal diseases in camellia oleifera. However, in recent years, the drawbacks of chemical pesticide control have become increasingly apparent. In contrast, biological control offers a series of advantages, including being environmentally friendly, residue-free, and less prone to developing resistance, making it an ideal alternative to or a reduction in chemical pesticide use. Its core lies in utilizing antagonistic microorganisms or their metabolites to inhibit pathogen infection at its source through mechanisms such as competition, parasitism, antibiosis, and induction of plant resistance, exhibiting significant sustainability. In recent years, the development of biocontrol agents has become a hot topic in the field of green control of camellia oleifera diseases. These agents can address the disease problem at its root while avoiding the negative impacts of chemical pesticides, making them a more sustainable control method. The research and application of biocontrol agents in the field of green control of camellia oleifera diseases has become a key issue.

[0004] Current research on biocontrol agents mainly focuses on beneficial microorganisms such as *Trichoderma* (e.g., *Trichoderma harzianum*, *Trichoderma viride*), *Bacillus* (e.g., *Bacillus amyloliquefaciens*, *Bacillus belyceae*), and *Pseudomonas*, targeting major diseases such as anthracnose, soft rot, and root rot in camellia oleifera. Technological evolution shows a trend towards single-strain agents to compound agents, and from simple formulations to functional formulations such as nanocarrier-based sustained-release agents. Chinese patent application CN117821333A discloses a strain capable of effectively controlling diseases caused by *Neoprothiolane* (…). Neopestalotiopsis clavispora Anthrax bacteria ( Colletotrichum siamense Bacillus cereus is a fungal pathogen that causes strawberry leaf spot, calyx wilt, anthracnose, and wilt, as well as other fungal diseases such as strawberry leaf spot and calyx wilt caused by Fusarium oxysporum. However, the efficacy of Bacillus cereus in controlling leaf and fruit drop pathogens and fungal diseases that directly lead to yield loss in Camellia oleifera has not been disclosed. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a Bacillus cereus ( Bacillus cereus ) HUB0155 strain.

[0006] A second objective of this invention is to provide a biological agent.

[0007] The third objective of this invention is to provide the application of the above-mentioned Bacillus cereus strain HUB0155 or the above-mentioned biological agent in the prevention and control of pathogenic fungi of Camellia oleifera or diseases of Camellia oleifera caused by pathogenic fungi.

[0008] A fourth objective of this invention is to provide the application of the Bacillus cereus strain HUB0155 or the above-mentioned biological agent in promoting the growth of Camellia oleifera.

[0009] The fifth objective of this invention is to provide a method for preventing the drop of fruit and leaves from Camellia oleifera.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a strain of Bacillus cereus ( Bacillus cereus The strain HUB0155 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 16, 2026, with accession number GDMCC No: 67964.

[0011] This invention focuses on the control of fruit and leaf drop diseases in Camellia oleifera through biological control. A strain of Bacillus cereus with stable and highly effective antagonistic activity against pathogenic fungi of Camellia oleifera was isolated and screened from healthy Camellia oleifera tea leaves collected from a Camellia oleifera planting base in Shima Town, Xingning City, Meizhou City, Guangdong Province. Bacillus cereus HUB0155, this strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 16, 2026, with accession number GDMCC No: 67964; classified and named as follows: Bacillus cereusThe depositary address is No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province. Because this strain originates from healthy Camellia oleifera tissue, it possesses natural ecological adaptability and colonization advantages in Camellia oleifera plants. As a member of the Bacillus genus, HUB0155 can form highly resistant spores, tolerating adverse environments such as high temperature, drought, and ultraviolet radiation, facilitating inoculant processing and field storage. As an endophytic bacterium, it can rapidly colonize the roots and leaves of Camellia oleifera and form biofilms, effectively inhibiting the infection of pathogenic microorganisms through competition for nutrients and space. The strain's high adaptability and stress tolerance to the internal environment of Camellia oleifera enable it to stably colonize and form a durable protective barrier in complex rhizosphere and canopy habitats, effectively reducing the incidence of disease in the field and achieving precise control of canopy diseases. Further research revealed that its core mechanism of action not only directly inhibits the growth of pathogens by efficiently scavenging iron, but also induces systemic resistance in camellia and secretes growth-promoting substances. Thus, while protecting leaves and fruits from infection, it enhances tree vigor and reduces fruit drop, achieving a synergistic effect of "disease prevention and control" and "fruit setting protection".

[0012] When this strain was used for pathogen control, the results showed that strain HUB0155 had broad-spectrum and stable antagonistic activity against various pathogenic fungi that cause fruit and leaf drop in Camellia oleifera, such as *Anthracnose* and *Polytrichum platycodon*. It also showed strong antagonistic activity against *Botrytis* species. Neofusicoccum parvum HU1163, Grape seat cavity Botryosphaeria dothidea HU1389, Anthrax genus Colletotrichum camelliae HU1424 and Colletotrichum gloeosporioides genus *Pseudomonas* Neopestalotiopsis saprophytica The inhibition rate of HU1438 reached 44.67-70%. When this strain HUB0155 was inoculated onto the surface of in vivo Camellia oleifera leaves, more new leaves grew, and the disease incidence on these new leaves was significantly reduced, indicating that this strain has high safety for Camellia oleifera. Inoculating the surface of Camellia oleifera leaves infected with the pathogen with strain HUB0155 resulted in more new leaves, significantly reduced leaf drop, and suppressed lesions in the treated plants. As a green control method, the application of strain HUB0155 can significantly reduce the use of chemical pesticides, thereby reducing environmental risks and pathogen resistance, meeting the requirements for high-quality Camellia oleifera production.

[0013] This invention provides a biological agent comprising the aforementioned Bacillus cereus strain HUB0155 or its fermentation broth. HUB0155 not only possesses natural ecological adaptability to its host plant, Camellia oleifera, and can integrate well into the Camellia oleifera growth system, but its fermentation broth can also directly act on Camellia oleifera pathogens, controlling diseases by inhibiting mycelial growth. This strain is easy to cultivate and reproduces rapidly, enabling industrial-scale fermentation production to prepare environmentally friendly biological pesticides, providing a core strain resource for developing green control technologies for Camellia oleifera diseases based on plant-endophytic bacteria interactions.

[0014] This invention provides the application of the above-mentioned Bacillus cereus strain HUB0155 or the above-mentioned biological agent in the prevention and control of pathogenic fungi in Camellia oleifera.

[0015] The present invention also provides the application of the above-mentioned Bacillus cereus strain HUB0155 or the above-mentioned biological agent in the prevention and control of diseases of Camellia oleifera caused by pathogenic fungi.

[0016] Furthermore, the pathogenic fungus mentioned is the pathogenic fungus that causes fruit and leaf drop in Camellia oleifera.

[0017] Furthermore, the pathogenic fungus of Camellia oleifera is selected from the genus Staphylococcus. Neofusicoccum parvum Grape seat cavity belongs to Botryosphaeria dothidea Anthrax genus Colletotrichum camelliae and Colletotrichum gloeosporioides genus *Pseudomonas* Neopestalotiopsis saprophytica One or more of them.

[0018] Preferably, the pathogenic fungus of Camellia oleifera is selected from the genus Staphylococcus. Neofusicoccum parvum HU1163, Grape seat cavity Botryosphaeria dothidea HU1389, Anthrax genus Colletotrichum camelliae HU1424 and Colletotrichum gloeosporioides genus *Pseudomonas* Neopestalotiopsis saprophytica One or more of HU1438.

[0019] Furthermore, the disease mentioned is a fruit and leaf drop disease of Camellia oleifera.

[0020] The present invention also provides the application of the above-mentioned Bacillus cereus strain HUB0155 or the above-mentioned biological agent in promoting the growth of Camellia oleifera.

[0021] Furthermore, promoting the growth of Camellia oleifera refers to promoting the growth of the number of new leaves of Camellia oleifera.

[0022] The present invention also provides a method for preventing fruit and leaf drop in Camellia oleifera, wherein the method involves inoculating Camellia oleifera with the aforementioned Bacillus cereus HUB0155 or the aforementioned biological agent.

[0023] Furthermore, the inoculation method is foliar spraying, root irrigation, or seed spraying or soaking.

[0024] Furthermore, the inoculation method involves spraying, drenching the roots, or spraying or soaking the tea leaves with the aforementioned Bacillus cereus HUB0155 or the aforementioned biological agent together with 0.1% sodium alginate stabilizer.

[0025] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a strain of Bacillus cereus, HUB0155, and its applications. The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 16, 2026, with accession number GDMCC No: 67964. This strain exhibits good antagonistic activity against five pathogenic fungi of Camellia oleifera, with an inhibition rate of 44.67–70%. As an endophytic fungus of Camellia oleifera, HUB0155 is safer for Camellia oleifera plants. Further inoculation of this strain onto the surface of in vivo Camellia oleifera tea leaves infected with pathogens resulted in an increase in the number of new leaves, a significant reduction in leaf drop, and inhibition of lesions in the treated Camellia oleifera plants. Therefore, this invention provides a core strain resource for the development of green control technology for Camellia oleifera diseases, and has significant development and application prospects in the large-scale biological control of leaf and fruit drop in low-yield Camellia oleifera forests. Attached Figure Description

[0026] Figure 1 The results show the morphological identification of strain HUB0155. Among them, Figure 1 In the diagram, A represents the cell morphology of HUB0155; B represents the microscopic morphological characteristics of HUB0155.

[0027] Figure 2 This is a phylogenetic tree constructed based on whole-genome data.

[0028] Figure 3 The study investigated the antagonistic effect of strain HUB0155 against five pathogenic fungi that cause fruit and leaf drop in Camellia oleifera.

[0029] Figure 4 Safety test of strain HUB0155 on Camellia oleifera plants.

[0030] Figure 5 The study investigated the field control efficacy of strain HUB0155 against common camellia oleifera. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0032] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0033] Anthrax Colletotrichum gloeosporioides Purchased from Ningbo Taiste Biotechnology Co., Ltd., product number: TS263664 (product name: Glomerella cingulata ).

[0034] Anthrax Colletotrichum camelliae HU1424 (see Chinese patent application CN 120060000 A) Colletotrichum tomentosaeHU1424), Staphylococcus spp. Neofusicoccum parvum HU1163, Grape seat cavity Botryosphaeria dothidea HU1389, *Plasmodium* genus Neopestalotiopsis saprophytica HU1438 was isolated and identified by the inventor from diseased tea leaves or fruits, and its pathogenicity was verified by Koch's postulates. It is preserved in the Natural Product Chemistry Laboratory of the School of Life Sciences, Huizhou University.

[0035] Example 1: Isolation and Identification of Bacillus cereus HUB0155 1. Isolation of strain HUB0155 This invention involves collecting healthy camellia leaves from a camellia oleifera planting base in Shima Town, Xingning City, Meizhou City, Guangdong Province. After surface disinfection, the leaves are cultured at 28°C in NA medium supplemented with actinomycete ketone using a tissue isolation method, and finally the strain HUB0155 is isolated and purified.

[0036] 2. Identification of strain HUB0155 (1) Morphological identification The strain HUB0155 was cultured on NA medium for 3 days, and the size, shape, color, and edge morphology of the colonies were observed.

[0037] The results are as follows Figure 1 As shown, HUB0155 colonies on NA plates are relatively large, round, short rod-shaped, with smooth edges, milky white color, and a moist texture.

[0038] (2) Molecular identification The strain HUB0155 was sequenced by 16S rRNA and analyzed by phylogenetic tree, and the results are as follows: Figure 2 As shown, it was preliminarily identified as Bacillus cereus (Bacillus cereus). Bacillus cereus Following whole-genome sequencing analysis using both DNBSEQ and Nanopore platforms, a complete circular genome was obtained after data filtering, assembly, and correction. This genome contained one chromosome (5,196,016 bp) and one plasmid (507,038 bp), with a total size of 5,703,054 bp and a GC content of 35.04%. Alignment of the assembled sequence with the NCBI NT database showed that this strain was similar to... Bacillus cereus (TaxID: 1396) has a genome coverage of 98.81%, confirming its species classification as Bacillus cereus. Bacillus cereus ).

[0039] Strain HUB0155 is taxonomically classified under the known Bacillus cereus (Bacillus). Bacillus cereusWhile the strain is not deficient in virulence, its complete genome sequence exhibits unique compositional characteristics, possessing significant scientific research value. Genome annotation analysis revealed that this strain carries 313 virulence factor-related genes (based on VFDB annotation) and 59 antibiotic resistance genes (based on ARDB annotation), providing molecular evidence for its potential antibiotic resistance phenotype. Simultaneously, four CRISPR arrays were predicted on the chromosome, indicating that this strain possesses a CRISPR-Cas adaptive immune system, potentially enhancing its defense against bacteriophage invasion and influencing the horizontal transfer of exogenous genes. Furthermore, prephage sequences were identified in the genome, and annotation using databases such as COG and KEGG revealed numerous gene clusters related to the synthesis and transport of secondary metabolites, suggesting the strain's genetic potential to synthesize novel bioactive compounds. These comprehensive genomic features make HUB0155 an ideal model and important resource for studying the pathogenic mechanisms, environmental adaptations, and evolutionary patterns of Bacillus cereus, as well as developing potential industrial applications. It also lays a solid molecular foundation for subsequent functional genomics and biotechnology applications research.

[0040] Therefore, this invention successfully isolated a strain of endophytic bacteria HUB0155 from healthy tea leaves, which was identified as Bacillus cereus. Bacillus cereus This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 16, 2026, with accession number GDMCC No: 67964; it is classified and named... Bacillus cereus The address of the collection is No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0041] Example 2: Antagonistic effect of strain HUB0155 against five pathogenic fungi causing leaf and fruit drop in Camellia oleifera. I. Experimental Methods Five pathogenic fungi of Camellia oleifera, belonging to the genus *Botrytis*, were extracted from a PDA plate using a sterile punch. Neofusicoccum parvum HU1163, Grape seat cavity Botryosphaeria dothidea HU1389, Anthrax genus Colletotrichum camelliae HU1424 and Colletotrichum gloeosporioides genus *Pseudomonas* Neopestalotiopsis saprophytica HU1438 was used to prepare mycelial blocks by punching holes, and the mycelial blocks were transferred to PDA plates. At a distance of 3 cm from the mycelial blocks, HUB0155 cells were picked up with an inoculation loop for spot inoculation. The plates were incubated at 28℃ for 5 days. The formation of an inhibition zone was observed on the plates, and the diameter of the fungi was measured to calculate the inhibition rate.

[0042] Inhibition rate (%) = [(Coronavirus colony diameter of control pathogen - Coronavirus colony diameter of treatment pathogen) / Coronavirus colony diameter of control pathogen] × 100%.

[0043] II. Experimental Results The outcome of the standoff was as follows Figure 3 As shown in Table 1, strain HUB0155 has a good antagonistic effect on all five pathogenic fungi that cause severe leaf and fruit drop in Camellia oleifera, with an inhibition rate of 44.67-70%.

[0044] Table 1. Antagonistic effects of strain HUB0155 against five pathogenic fungi of Camellia oleifera.

[0045] Example 3: Safety test of strain HUB0155 on Camellia oleifera plants 1. Safety test of tea leaves in vitro HUB0155 was cultured in NB liquid medium at 28℃ and 180 r / min for 24 h to prepare a seed culture. After subculturing at a 5% inoculum, the culture was continued for 2 days to obtain a concentration of approximately 10%. 8 A bacterial suspension of CFU / mL was prepared. Tea leaves were selected, and after surface sterilization, uniform cross-shaped wounds were made. The leaves were then immersed in the bacterial suspension for 30 seconds, repeated three times. Sterile water treatment served as a control. Three leaves were used per group, with three replicates per group. Leaf condition was observed and recorded on days 1, 3, 5, and 7 after treatment.

[0046] The results are as follows Figure 4 As shown, this indicates that the tea leaves (tea leaves) treated with HUB0155 bacterial solution for 5 days... Figure 4 (middle and right images) and control group ( Figure 4 Compared to the left picture, there is no obvious difference. The leaves are not wilted, the color is normal, the appearance is normal, and no obvious lesions are produced. Therefore, HUB0155 is safe for camellia.

[0047] 2. Safety test of living Camellia oleifera plants HUB0155 was cultured in NB medium at 28℃ and 180 rpm to prepare a seed culture. After being transferred for expansion culture for 2 days, it was diluted to 10⁻⁶. 8 CFU / mL bacterial suspension. The following treatments were then performed: (1) Leaf treatment: detached leaves with disinfected surfaces and cross-shaped wounds were soaked in bacterial suspension; (2) Leaf spraying treatment: bacterial suspension was evenly sprayed onto the entire tea plant; (3) Root irrigation treatment: 50 mL of bacterial suspension was applied to the roots of each plant using the five-point method. Each treatment had a sterile water control and was repeated 3 times (3 plants or 3 leaves per repeat). The growth of the plants / leaves was observed and recorded regularly after each treatment.

[0048] The results are as follows Figure 4 As shown, compared with the control group ( Figure 4 Compared to the left image, spraying with HUB0155 bacterial solution is not only safe for camellia oleifera plants, but also results in camellia oleifera plants treated with HUB0155 having more new leaves. Figure 4(The middle and right images show that the disease infection rate of the newly grown leaves is significantly reduced.)

[0049] Example 4: Field control effect of strain HUB0155 on common camellia oleifera I. Experimental Methods To clearly verify the field control efficacy of HUB0155 against the pathogenic fungus causing leaf and fruit drop in Camellia oleifera, the following experiment was conducted under field conditions using Camellia oleifera seedlings. Healthy Camellia oleifera seedlings of uniform growth were selected, and the leaf surface was disinfected by wiping with 75% alcohol, followed by rinsing three times with sterile water. After the leaves were allowed to air dry naturally, a 0.5cm "+" shaped wound was made on both sides of the main vein of the leaf using a sterile scalpel blade, and the tested pathogen (…) was excised. Neofusicoccum parvum HU1163 and Neopestalotiopsis saprophytica The mycelial surface of the HU1438 fungal cake was placed tightly against the wound and secured with breathable tape. Three replicates were used per treatment, with each plantlet inoculated with six leaves. Three days after inoculation, the tape was removed, the initial lesion diameter was measured, and the fungal solution was sprayed. Preparation of the fungal agent: HUB0155 LB fermentation broth was mixed with 0.1% sodium alginate stabilizer at a 1:1 ratio. 20 mL of the mixture was diluted with 180 mL of sterile water to a final concentration of approximately 1×10⁻⁶. 7 Prepare a bacterial suspension at CFU / mL. Spray it evenly onto the surface of the inoculated leaves until the leaves are dripping wet. Measure the diameter of the lesions again 4 days after spraying, and record the amount of leaf drop and the degree of disease infection.

[0050] II. Experimental Results The results are as follows Figure 5 As shown, compared with the control, spraying with HUB0155 bacterial solution can significantly inhibit pathogens. Neofusicoccum parvum HU1163 and Neopestalotiopsis saprophytica HU1438-induced lesion expansion. Neopestalotiopsis saprophytica HU1438 plants treated with HUB0155 bacterial solution showed increased new leaf quantity and significantly reduced disease incidence on new leaves. The control group was inoculated... Neofusicoccum parvum HU1163 tea seedlings experienced 100% leaf drop, while tea seedlings treated with HUB0155 bacterial solution showed significantly reduced leaf drop and suppressed lesions.

[0051] The above specific embodiments are all further illustrations of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, all other modifications and refinements obtained without creative effort are within the scope of protection of the present invention.

Claims

1. A strain of Bacillus cereus ( Bacillus cereus HUB0155 strain, characterized in that, The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 16, 2026, with accession number GDMCC No: 67964.

2. A biological agent, characterized in that, The biological agent comprises the Bacillus cereus strain HUB0155 of claim 1 or the fermentation broth of the Bacillus cereus strain HUB0155 of claim 1.

3. The application of the Bacillus cereus strain HUB0155 of claim 1 or the biological agent of claim 2 in the prevention and control of pathogenic fungi in Camellia oleifera.

4. The application of the Bacillus cereus strain HUB0155 of claim 1 or the biological agent of claim 2 in the prevention and control of diseases of Camellia oleifera caused by pathogenic fungi.

5. The application according to claim 3 or 4, characterized in that, The pathogenic fungus mentioned is the fungus that causes fruit and leaf drop in camellia.

6. The application according to claim 5, characterized in that, The pathogenic fungus of Camellia oleifera is selected from the genus Staphylococcus. Neofusicoccum parvum Grape seat cavity belongs to Botryosphaeria dothidea Anthrax genus Colletotrichum camelliae or Colletotrichum gloeosporioides genus *Pseudomonas* Neopestalotiopsis saprophytica One or more of them.

7. The application according to claim 4, characterized in that, The disease mentioned is the fruit and leaf drop disease of Camellia oleifera.

8. The application of the Bacillus cereus strain HUB0155 of claim 1 or the biological agent of claim 2 in promoting the growth of Camellia oleifera.

9. A method for preventing fruit and leaf drop in camellia oleifera, characterized in that, The method involves inoculating Camellia oleifera with the Bacillus cereus strain HUB0155 of claim 1 or the biological agent of claim 2.

10. The method according to claim 9, characterized in that, The inoculation methods include foliar spraying, root irrigation, or seed spraying and soaking.

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