Bacillus velezensis HUB0159 capable of effectively preventing and controlling pathogenic fungi causing tea-oil tree fallen leaves and application of bacillus velezensis HUB0159

Abstract

The invention discloses bacillus velezensis HUB0159 capable of effectively preventing and controlling pathogenic fungi causing tea-oil tree fallen leaves and application of the bacillus velezensis HUB0159. The strain is preserved in Guangdong Microbial Culture Collection Center (GDMCC) on March 16, 2026, and the preservation number is GDMCC No: 67963. The strain HUB0159 has an efficient antagonistic effect on five main pathogenic fungi causing tea-oil tree fallen leaves, and the bacteriostasis rate can reach 32.33-67.78%. When the strain is inoculated to the surface of an in-vivo camellia oleifera leaf infected by pathogenic bacteria, the result shows that the number of new leaves of a camellia oleifera plant is increased, fallen leaves are obviously reduced, and scab is inhibited. Therefore, the bacillus velezensis HUB0159 with the effects of promoting growth and preventing diseases, provided by the invention, provides a core strain resource for developing a green biological pesticide aiming at the tea-oil tree leaf falling diseases, and has an important application value for constructing a tea-oil tree disease ecological prevention and control technical system.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically, it relates to a strain of Bacillus vesicles HUB0159 that can effectively control the pathogenic fungus that causes leaf drop in Camellia oleifera and its application. Background Technology

[0002] Camellia oleifera ( Camellia oleifera Camellia oleifera (Abel.) is an important woody oilseed tree species in my country, and its tea oil products have high nutritional value and broad market prospects. However, with the expansion of Camellia oleifera planting area and the increase in continuous cropping years, fungal diseases have become increasingly serious, becoming a key bottleneck restricting the improvement of the industry's quality and efficiency. Leaf and fruit drop caused by pathogenic fungal infections are particularly prominent, causing significant economic losses to growers. The pathogens causing these diseases are complex, often exhibiting characteristics of multiple pathogens causing complex infections, among which the genus *Metasequoia* (*Abel.*) is particularly prevalent. Diaporthe sp.), genus Diplostomum (sp.), genus Diplostomum Lasiodiplodia sp.) and Trichoderma ( Trichoderma sp.) fungi are several dominant pathogens that have been reported frequently and caused serious damage in recent years, and have a significant impact on the leaf drop of Camellia oleifera.

[0003] Currently, biological control, with its outstanding advantages such as good environmental compatibility, low likelihood of developing resistance, and strong sustainability, is gradually becoming the mainstream direction of disease control technology research and development. However, most biocontrol strains are isolated from soil or other crops, not from the camellia oleifera plant itself. This often results in poor compatibility and unsatisfactory colonization ability after entering the camellia oleifera's internal environment. In field application, this can easily lead to fluctuating efficacy and short-lasting effects.

[0004] Bacillus belesiensis ( Bacillus velezensis Bacillus (B) is a Gram-positive aerobic bacterium belonging to the genus Bacillus, and is currently a widely used broad-spectrum biocontrol bacterium and probiotic in the fields of agriculture and biological control. Chinese patent CN116121104A discloses a strain of Bacillus belyeidae DY-7, which is effective against Aspergillus niger (…). Aspergillus niger ), cordycetes ( Talaromyces funiculosus ), Yunnan Trichoderma ( Trichodermayunnanense ), Penicillium chrysogenum ( Penicillium chrysogenum All of them showed significant antagonistic effects. However, there is currently no publicly available information on the application of Bacillus belye in the simultaneous control of multiple pathogenic fungi that cause leaf drop in Camellia oleifera. Summary of the Invention

[0005] The purpose of this invention is to overcome the aforementioned defects and deficiencies in the prior art and to provide a *Bacillus belyssae* (B) Bacillus velezensis ) HUB0159 strain.

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

[0007] A third objective of this invention is to provide the application of the above-mentioned Bacillus vesicularis HUB0159 or the above-mentioned bacterial agent / biological agent in the prevention and control of pathogenic fungi in Camellia oleifera.

[0008] The fourth object of the present invention is to provide the application of the above-mentioned Bacillus vesiculosus HUB0159 or the above-mentioned bacterial agent / biological agent in the prevention and control of diseases of Camellia oleifera caused by pathogenic fungi.

[0009] The fifth objective of this invention is to provide the application of the aforementioned Bacillus vesiculosus HUB0159 or the above-mentioned bacterial agent / biological agent in promoting the growth of Camellia oleifera.

[0010] The sixth objective of this invention is to provide a method for preventing leaf drop in camellia oleifera.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a strain of Bacillus belye ( Bacillus velezensis The strain HUB0159 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 16, 2026, with accession number GDMCC No: 67963.

[0012] This invention addresses the shortcomings of existing biological control methods for camellia diseases, such as the lack of specific strains targeting deciduous pathogenic fungi and the weak colonization ability of existing exogenous biocontrol bacteria. It provides an endophytic bacterium, HUB0159, isolated from healthy camellia plants in Shima Town, Xingning City, Meizhou City, Guangdong Province. Morphological observation and molecular biological identification confirmed it to be *Bacillus belyesense*. Bacillus velezensis The strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 16, 2026, with accession number GDMCC No: 67963; it was classified and named... Bacillus velezensis The address of the collection is No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0013] Plate confrontation experiments showed that the HUB0159 strain was effective against five major pathogenic fungi that cause leaf drop in Camellia oleifera, including *Cryptococcus pyrenoidosa*. Diaporthe inconspicua ), false cocoa brown disporum ( Lasiodiplodia pseudotheobromae ) and Neodiscrystosporum ( Neopestalotiopsis eucalypticola Fusarium oxysporum ( Fusarium oxysporum ) and Trichoderma ( Trichoderma simileAll strains exhibited stable and highly efficient antagonistic activity, with inhibition rates ranging from 32.33% to 67.78%, demonstrating broad-spectrum antibacterial properties. Further inoculation of strain HUB0159 onto the surface of in vivo Camellia oleifera leaves infected with pathogens (*Diplosporum pseudococcoccus* and *Polytrichum neomycetes*) resulted in increased new leaf quantity, significantly reduced leaf drop, and suppressed lesions in the treated Camellia oleifera plants. As an endophytic fungus of Camellia oleifera, HUB0159 possesses natural affinity and colonization advantages with the host, enabling it to establish a stable population within the plant and achieve sustained suppression of pathogens. This strain combines growth-promoting potential with broad-spectrum antibacterial properties, effectively inhibiting pathogen infection and reducing leaf drop while promoting healthy plant growth, providing core strain resources and technical support for the green control of Camellia oleifera diseases. This invention discovers an endophytic bacterium derived from Camellia oleifera itself that can effectively colonize and has broad-spectrum antagonistic activity against deciduous pathogenic fungi. This is of practical significance for improving the green prevention and control system for Camellia oleifera diseases. Based on this, the development of biological pesticides is in line with the disease management strategy driven by plant-endophytic bacteria interaction and has practical application prospects in building a sustainable Camellia oleifera disease prevention and control system.

[0014] The present invention provides a microbial agent / biological preparation containing one or more of the above-mentioned Bacillus belye HUB0159 and its culture medium, fermentation broth, bacterial suspension and bacterial cells.

[0015] Furthermore, the preparation method of the culture medium, fermentation broth, bacterial suspension, and bacterial cells involves inoculating and culturing Bacillus belye HUB0159 to obtain a culture medium or fermentation broth; centrifuging the culture medium and resuspending it to obtain a bacterial suspension; and centrifuging the culture medium to obtain bacterial cells. This culture condition is simple, has a short growth cycle, and stable genetic traits, making it suitable for large-scale fermentation.

[0016] The present invention also provides the application of the above-mentioned Bacillus vesicularis HUB0159 or the above-mentioned bacterial agent / biological agent in the prevention and control of pathogenic fungi in Camellia oleifera.

[0017] The present invention also provides the application of the above-mentioned Bacillus vesicularis HUB0159 or the above-mentioned bacterial agent / biological agent in the prevention and control of diseases of Camellia oleifera caused by pathogenic fungi.

[0018] Furthermore, the pathogenic fungus of Camellia oleifera is the pathogenic fungus that causes Camellia oleifera leaves to fall off.

[0019] Furthermore, the pathogenic fungus of Camellia oleifera is selected from *Cryptocoryne* (…). Diaporthe inconspicua ), false cocoa brown disporum ( Lasiodiplodia pseudotheobromae ), Neodiscorydalis polychaete ( Neopestalotiopsis eucalypticola Fusarium oxysporum ( Fusarium oxysporum ) and Trichoderma ( Trichoderma simile One or more of the following.

[0020] Furthermore, the disease mentioned is a leaf drop disease of Camellia oleifera.

[0021] The present invention also provides the application of the above-mentioned Bacillus vesiculosus HUB0159 or the above-mentioned bacterial agent / biological agent in promoting the growth of Camellia oleifera.

[0022] Furthermore, promoting the growth of Camellia oleifera refers to promoting the growth of the number of new leaves of Camellia oleifera.

[0023] The present invention also provides a method for preventing leaf drop in Camellia oleifera, wherein the method comprises inoculating Camellia oleifera with the above-mentioned Bacillus cereus HUB0159 or the above-mentioned bacterial agent / biological agent.

[0024] Furthermore, the inoculation method is foliar spraying, root irrigation, or seed spraying or soaking.

[0025] Furthermore, the inoculation method involves spraying, drenching the roots, or spraying or soaking the tea leaves with the aforementioned Bacillus cereus HUB0159 or the aforementioned biological agent together with 0.1% sodium alginate stabilizer.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a strain of *Bacillus vesiculosus* HUB0159 that can effectively control the pathogenic fungi causing leaf drop in *Camellia oleifera* 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: 67963. This strain HUB0159 is effective against five major pathogenic fungi causing leaf drop in *Camellia oleifera*, including *Cryptococcus pyrenoidosa* (…). Diaporthe inconspicua ), false cocoa brown disporum ( Lasiodiplodia pseudotheobromae ) and Neodiscrystosporum ( Neopestalotiopsis eucalypticola Fusarium oxysporum ( Fusarium oxysporum ) and Trichoderma ( Trichoderma simile All of these bacteria exhibit highly effective antagonistic effects, with inhibition rates reaching 32.33% to 67.78% or higher under plate confrontation culture. Further, when this strain HUB0159 was inoculated onto the surface of in vivo Camellia oleifera leaves infected with pathogens (*Diplosporum pseudococcoccus* and *Polytrichum neomycetes*), the number of new leaves in the treated Camellia oleifera plants increased, leaf drop was significantly reduced, and lesions were inhibited. As an endophytic bacterium of Camellia oleifera, strain HUB0159 has strong affinity with the plant and high safety for use. The strain HUB0159 provided by this invention, as an endophytic Bacillus with both growth-promoting and disease-preventing effects, provides a core strain resource for developing green biological pesticides targeting leaf drop diseases in Camellia oleifera, and has significant application value for constructing an ecological control technology system for Camellia oleifera diseases. Attached Figure Description

[0027] Figure 1The results show the morphological identification of strain HUB0159. Among them, Figure 1 In the diagram, A represents the cell morphology of HUB0159; B represents the microscopic morphological characteristics of HUB0159.

[0028] Figure 2 Phylogenetic tree constructed based on 16S rRNA of strain HUB0159.

[0029] Figure 3 The study investigated the antagonistic effect of strain HUB0159 against five pathogenic fungi that cause leaf drop in Camellia oleifera.

[0030] Figure 4 The study investigated the field control efficacy of strain HUB0159 against 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] Fusarium oxysporum Fusarium oxysporum Purchased from Ningbo Taiste Biotechnology Co., Ltd., product number: TS333268.

[0034] Hidden metasomatitis Diaporthe inconspicua False cocoa bromide Lasiodiplodia pseudotheobromae Neodiscrystal Neopestalotiopsis eucalypticola Trichoderma Trichoderma simile The inventor isolated and identified the sample from Camellia oleifera and preserved it in the Natural Product Chemistry Laboratory of the School of Life Sciences, Huizhou University.

[0035] Example 1: Isolation and Identification of Bacillus belye HUB0159 1. Isolation of strain HUB0159 This invention involves collecting healthy camellia leaves from a camellia oleifera planting base in Shima Town, Xingning City, Meizhou City, Guangdong Province. After washing and surface disinfection, small pieces are cut and inoculated into NA medium with the tissue section. The medium is then placed at a constant temperature of 28°C for incubation. Colony changes are observed regularly, and newly grown endophytic bacteria are promptly picked and purified in a new medium. The isolated single colonies are purified and cultured for 2-3 generations to finally isolate and purify strain HUB0159.

[0036] 2. Identification of strain HUB0159 (1) Morphological identification The strain HUB0159 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, HUB0159 colonies on NA plates are relatively large and round, with a diameter of 2-4 mm. They are viscous and easily picked up with an inoculation loop. The surface of the colonies is slightly smooth and slightly raised with wrinkles, and the edges are slightly irregular. They are translucent to opaque and milky white in color.

[0038] (2) Molecular identification Endophytic bacteria HUB0159 were activated with NA medium and then transferred to LB medium. The culture was incubated at 37°C with shaking for 24 h until the culture became turbid. The culture was then transferred to two 2 mL centrifuge tubes, and DNA was extracted using a column-based method with a bacterial and fungal genome extraction kit from Wuhan Nanomagnetic Biotechnology Co., Ltd. The extracted DNA was sent to a sequencing company for 16S rRNA sequencing. The sequences were uploaded to GenBank for BLAST sequence alignment, and a phylogenetic tree was constructed using mega11 software.

[0039] The strain HUB0159 was sequenced by 16S rRNA and analyzed by phylogenetic tree, and the results are as follows: Figure 2 As shown, the strain was identified as Bacillus belye (B. belye). Bacillus velezensis This invention successfully isolated an endophytic bacterium from healthy tea leaves of the tea plantation. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 16, 2026, with accession number GDMCC No: 67963; and classified as follows: Bacillus velezensis The address of the collection is No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0040] Example 2: Antagonistic effect of strain HUB0159 against five pathogenic fungi causing leaf drop in Camellia oleifera. Five strains of Camellia oleifera pathogenic fungi on PDA plates were extracted using a sterile punch. Diaporthe inconspicua ), false cocoa brown disporum ( Lasiodiplodia pseudotheobromae ) and Neodiscrystosporum ( Neopestalotiopsis eucalypticola Fusarium succinate ( ) Fusarium succisae ) and Trichoderma ( Trichoderma simile The purified HUB0159 was subjected to a confrontation screening with pathogenic fungi of Camellia oleifera: A certain amount of endophytic bacteria HUB0159 was picked up with a toothpick and inoculated into PDA medium. Antagonistic bacteria were inoculated at one symmetrical point at the same distance from the center of each medium. The medium was incubated at 28℃ for 48 h. On the third day, fungal mycelial cakes were inoculated at another symmetrical point and incubated at 28℃ for another 72 h. The confrontation was observed, and it was observed whether an inhibition zone was formed on the plate. The diameter of the test fungus was measured and the inhibition rate was calculated.

[0041] Inhibition rate (%) = [(Coronavirus colony diameter of control pathogen - Coronavirus colony diameter of treatment pathogen) / Coronavirus colony diameter of control pathogen] × 100%.

[0042] The results are as follows Figure 3 As shown in Table 1, the plate confrontation results showed that strain HUB0159 had a good antagonistic effect on all five pathogenic fungi that cause diseases of Camellia oleifera, with an inhibition rate of 32.33% to 67.78%.

[0043] Table 1. Antagonistic effect of HUB0159 against five pathogenic fungi of Camellia oleifera.

[0044] Example 3: Control effect of strain HUB0159 on Camellia oleifera field HUB0159 was used in a field control experiment on Camellia oleifera seedlings. After disinfecting the tea leaves with alcohol and sterile water, a 0.5cm "+" shaped wound was created, and the fungus (Diplosporium pseudococovenenans) was introduced. Lasiodiplodia pseudotheobromae ), Neodiscorydalis polychaete ( Neopestalotiopsis eucalypticola The mycelial surface of the fungal cake was applied to the wound and secured with tape. After 3 days, the tape was removed, and the initial lesion was measured. A mixed bacterial solution (20 mL of 0.1% sodium alginate stabilizer + 20 mL of HUB0159 bacterial solution + 180 mL of sterile water, concentration approximately 1×10⁻⁶) was then applied. 7 The fungicide (CFU / mL) was sprayed onto the inoculated leaves, with blank and positive controls included. The lesions were observed and measured again after 4 days to compare the antibacterial effect.

[0045] The results are as follows Figure 4 As shown, compared with the control, after spraying with HUB0159 bacterial solution, the leaf drop of Camellia oleifera seedlings inoculated with Diplostomum simonii and Polytrichum nematodes were significantly improved and leaf lesions were significantly inhibited. The Camellia oleifera seedlings inoculated with Polytrichum nematodes and treated with HUB0159 bacterial solution had more new leaves and the disease infection of the new leaves was significantly reduced.

[0046] 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 belye ( Bacillus velezensis HUB0159 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: 67963.

2. A microbial agent / biological preparation, characterized in that, The bacterial agent / biological agent contains one or more of the following: Bacillus berberis HUB0159 as described in claim 1, its culture medium, fermentation broth, bacterial suspension, and bacterial cells.

3. The microbial agent / biological preparation according to claim 2, characterized in that, The preparation methods for the culture medium, fermentation broth, bacterial suspension, and bacterial cells are as follows: inoculating and culturing Bacillus belye HUB0159 to obtain a culture medium or fermentation broth; centrifuging the culture medium and resuspending it to obtain a bacterial suspension; and centrifuging the culture medium to obtain bacterial cells.

4. The application of Bacillus vesiculosus HUB0159 as described in claim 1 or the bacterial agent / biological agent as described in claim 2 in the control of pathogenic fungi in Camellia oleifera.

5. The application of Bacillus vesiculosus HUB0159 as described in claim 1 or the inoculant / biological agent as described in claim 2 in the prevention and control of diseases of Camellia oleifera caused by pathogenic fungi.

6. The application according to claim 4 or 5, characterized in that, The pathogenic fungus mentioned is the pathogenic fungus that causes leaf drop in Camellia oleifera.

7. The application according to claim 6, characterized in that, The pathogenic fungus of Camellia oleifera was selected from *Cryptospira* (…). Diaporthe inconspicua ), false cocoa brown disporum ( Lasiodiplodia pseudotheobromae ), Neodiscorydalis polychaete ( Neopestalotiopsis eucalypticola Fusarium oxysporum ( Fusarium oxysporum ) and Trichoderma ( Trichoderma simile One or more of the following.

8. The application according to claim 5, characterized in that, The disease mentioned is the leaf drop disease of Camellia oleifera.

9. The application of Bacillus vesiculosus HUB0159 of claim 1 or the bacterial agent / biological agent of claim 2 in promoting the growth of Camellia oleifera.

10. A method for preventing leaf drop in Camellia oleifera, characterized in that, The method involves inoculating Camellia oleifera with the Bacillus berberis HUB0159 of claim 1 or the bacterial agent / biological agent of claim 2.

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