A Bacillus strain X21 with disease-preventing and growth-promoting effects on Camellia oleifera and its applications.

Abstract

This invention discloses a Bacillus X21 strain with disease-preventing and growth-promoting effects on Camellia oleifera and its applications. The Bacillus X21 strain, with accession number CCTCC M 20253060, was deposited at the China Center for Type Culture Collection on December 31, 2025. The Bacillus X21 strain of this invention has a round, waxy, pale yellow colony morphology and can produce heparin, IAA, and cellulose, exhibiting phosphorus-solubilizing and nitrogen-fixing abilities. It can significantly promote the growth and development of Camellia oleifera. This strain has antagonistic effects against Camellia oleifera anthracnose and shows sensitivity to multiple antibiotics, making it suitable for the biological control of Camellia oleifera anthracnose. Microbial agents prepared using this strain have disease-resistant and growth-promoting functions against Camellia oleifera, effectively reducing soil microecological damage caused by the application of chemical agents.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically, it relates to a Bacillus strain that has disease prevention and growth promotion effects on Camellia oleifera and its application. Background Technology

[0002] Anthracnose was first identified by Penzig in 1882. This fungus can cause infection in a variety of woody and herbaceous plants. Anthracnose is a major and common disease in camellia oleifera cultivation areas in my country. It spreads rapidly, is difficult to control, and primarily affects the fruit, leaves, and branches of camellia oleifera, especially older leaves, causing widespread leaf drop and weakening the tree. Several species of the *Anthracnose* genus can cause diseases in camellia oleifera, but the pathogenicity varies among species. Currently reported pathogens in camellia oleifera include *Colletotrichum gloeosporioides*, *Colletotrichum siamense*, *C. horri*, *Colletotrichum foetida*, *Colletotrichum camelliaense*, *Colletotrichum cava*, and *Colletotrichum cryptosporum*. At present, chemical pesticides remain the main control method, primarily using carbendazim, thiophanate-methyl, and prochloraz. However, long-term application leads to large amounts of these pesticides entering the soil and water environment, causing pesticide residues and other problems, which are detrimental to the healthy development of the camellia oleifera industry.

[0003] In recent years, previous researchers have discovered some anthracnose-resistant strains that can effectively control anthracnose in Camellia oleifera, providing new control strategies for the development of biopesticides. Studies have isolated antagonistic bacteria against anthracnose from Camellia oleifera leaves, bark, and fruit, finding that Bacillus amyloliquefaciens P-14 has a good inhibitory effect on the pathogen of Camellia oleifera anthracnose. Its main component is C15 bacillomycin D, and its antibacterial effect originates from the benzene ring derivatives of organic acid esters produced by its metabolism, which can promote the growth of plant buds and roots.

[0004] Camellia oil (Camellia oleifera Abel .) Camellia oleifera is the most widely cultivated woody oilseed tree species in southern my country, covering an area of ​​over 75 million mu (approximately 5 million hectares). It boasts high economic value and a promising market prospect. It also possesses health, ecological, and edible value. However, during its growth period, Camellia oleifera is frequently affected by anthracnose, which negatively impacts the plant's growth and development, as well as the yield of tea seed oil. In severe cases, yield reductions can reach 30% to 50%, or even result in total crop failure. Utilizing biological control methods to manage anthracnose in Camellia oleifera can overcome the problems of pesticide residues and resistance associated with chemical control. Summary of the Invention

[0005] This application provides a Bacillus X21 strain that has disease prevention and growth-promoting effects on Camellia oleifera, which can effectively promote the growth and development of Camellia oleifera and improve the prevention and control of Camellia oleifera anthracnose.

[0006] This invention provides a Bacillus strain X21 that has disease-preventing and growth-promoting effects on Camellia oleifera ( ). Bacillussp. X21), with accession number CCTCC M 20253060, was deposited on December 31, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China, 430072).

[0007] This invention isolates and screens a Bacillus strain X21 from the rhizosphere of Camellia oleifera in Liuyang City, Hunan Province. It can produce ironophiles, IAA and cellulose, and has the ability to solubilize phosphorus and fix nitrogen. It can effectively promote the growth and development of Camellia oleifera. It also has an antagonistic effect on anthracnose and is sensitive to antibiotics.

[0008] The present invention also provides a biological agent that has disease prevention and growth promotion effects on Camellia oleifera, comprising the above-mentioned Bacillus X21 culture as an active ingredient.

[0009] Preferably, the above-mentioned Bacillus X21 culture is prepared by the following method: Bacillus X21 is inoculated into LB liquid medium and cultured at 28°C. After enrichment and centrifugation, a bacterial suspension with OD600=0.8 is prepared using sterile water.

[0010] Preferably, the application of the above-mentioned Bacillus X21 and biological agents in promoting the growth and development of Camellia oleifera involves promoting the activity of biological enzymes in Camellia oleifera, increasing the activity of antioxidant enzymes and defense enzymes in the leaves, promoting the growth of Camellia oleifera plant height, increasing the diameter at ground level, increasing the fresh and dry weight of the above-ground and underground parts, and promoting the elongation and growth of Camellia oleifera roots.

[0011] The present invention also provides a method for preventing and controlling anthrax in camellia oleifera, comprising the step of applying the above-mentioned Bacillus X21 or the above-mentioned biological agent to camellia oleifera.

[0012] Experiments of this invention show that the Bacillus X21 of this invention has the ability to dissolve inorganic phosphorus, fix nitrogen, and produce IAA, among other growth-promoting abilities.

[0013] Experiments of this invention show that the Bacillus X21 of this invention has the ability to secrete protease, cellulase, amylase, β-1,3-glucanase, and ferrophile.

[0014] Experiments of this invention show that the Bacillus X21 of this invention has an antagonistic effect on anthrax in Camellia oleifera, is sensitive to antibiotics, and can be used for the prevention and treatment of anthrax in Camellia oleifera.

[0015] Experiments of this invention show that Bacillus X21 of this invention can effectively inhibit the growth of anthrax pathogens in Camellia oleifera, thereby reducing the area of ​​lesions.

[0016] Experiments of this invention show that Bacillus X21 of this invention can effectively promote the growth of Camellia oleifera, change the activity of rhizosphere soil enzymes in Camellia oleifera, and enrich the rhizosphere colony structure.

[0017] Experiments of this invention show that Bacillus X21 of this invention can change the root morphology and structure of Camellia oleifera and promote root growth.

[0018] Experiments of this invention show that Bacillus X21 of this invention can enhance the activity of antioxidant enzymes and defensive enzymes in tea leaves.

[0019] The present invention has the following beneficial effects: 1. The Bacillus X21 strain obtained by screening in this invention has disease prevention and growth promotion effects on Camellia oleifera. It can produce ironophiles, IAA and cellulose, and has the ability to solubilize phosphorus and fix nitrogen. It can promote the growth and development of Camellia oleifera and can be widely used as a bio-fertilizer with good market prospects.

[0020] 2. This invention demonstrates through in vitro experiments, pot experiments, and field trials that Bacillus X21 has an antagonistic effect on anthracnose in Camellia oleifera and is sensitive to antibiotics, thus it can be used for the prevention and control of anthracnose in Camellia oleifera. Pot experiments show that Bacillus X21 can promote the elongation and growth of Camellia oleifera roots, thereby improving the root system's ability to absorb soil nutrients and increasing the activity of antioxidant and defensive enzymes in Camellia oleifera tea leaves.

[0021] 3. This invention demonstrates through pot experiments and field trials that Bacillus X21 can alter the rhizosphere soil microbial community structure and increase rhizosphere soil enzyme activity in Camellia oleifera. Therefore, Bacillus X21 of this invention is a strain with highly efficient biocontrol and growth-promoting effects, and can be used to prepare disease-resistant and growth-promoting microbial agents. Attached Figure Description

[0022] Figure 1 The results of the initial screening of rhizosphere biocontrol bacteria in Experiment Example 1 of this invention are shown below. The bacteria are numbered from left to right as follows: C1, C4, C5, C6, C8, C10, C14, C15, C22, X12, X21, X23, X29, X33, X85, Q6, Q8, Q9, Q11, Q12, Q13, Q18, Q19, D22, D23, D24, D55, D57, D58, and anthracnose. Figure 2 The phylogenetic tree of Bacillus X21 based on the 16S rDNA sequence in Experimental Example 2 of this invention; Figure 3 The figure shows the results of the growth-promoting and antibacterial properties of Bacillus X21 in different culture media in Experimental Example 3 of the present invention, wherein: (A) inorganic phosphorus, (B) nitrogen fixation, (C) ironophile, (D) IAA, (E) protease, (F) cellulase, (G) β-1,3-glucanase, and (H) amylase. Figure 4 This is a growth curve of Bacillus X21 in Experimental Example 3 of the present invention; Figure 5 This shows the growth of Bacillus X21 strains under different sodium chloride concentrations in Experimental Example 3 of the present invention; Figure 6 This shows the growth of Bacillus X21 strains under different pH conditions in Experimental Example 3 of the present invention; Figure 7 Experimental Example 4 of this invention demonstrates the control effect of Bacillus X21 bacterial suspension on anthracnose of Camellia oleifera detached leaves; Figure 8 To illustrate the control effect of Bacillus X21 bacterial suspension on anthracnose in Camellia oleifera field during Experiment Example 6 of this invention, A and B: Controls before and after spraying without bacterial suspension; C and D: Controls before and after spraying with bacterial suspension. Figure 9 This is a statistical analysis of the changes in rhizosphere enzyme activity of Camellia oleifera under different treatments in Experimental Example 8 of the present invention. Figure 10 This is a bar chart showing the percentage abundance of rhizosphere bacteria in Camellia oleifera under different treatments in Experimental Example 8 of this invention. Detailed Implementation

[0023] The specific embodiments of this disclosure are described in detail below with reference to the accompanying drawings. Specific details of the implementation are set forth in the following description to facilitate understanding of the invention. Unless otherwise specified, all materials used in this embodiment are commercially available products.

[0024] The culture media used in the various experimental examples of this invention were: Ashby medium (nitrogen fixation), Alexandrov medium (potassium solubilization), NBRIP inorganic phosphorus solubilization medium, Monkina organic phosphorus solubilization medium, CAS detection medium, fungal growth medium (PDA), protease medium, amylase medium, β-1,3-glucanase medium, cellulase medium, LB medium, and CAS solid medium. For the specific formulations and preparation methods of the above culture media, please refer to the relevant records in the literature: "The Influence of Siderogenic Bacteria on Peanuts in Calcareous Purple Soils," "Screening and Identification of Antagonistic Bacteria for Potato Dry Rot and Determination of Their Growth-Promoting Characteristics," "Isolation and Identification of Growth-Promoting Bacteria in the Root Soil of Oxyphenidyl and Their Effect on Seedling Growth," and "Isolation and Identification of Endophytic Bacteria in Banana Fusarium Wilt-Resistant Strains and Their Antibacterial and Growth-Promoting Effects."

[0025] Experimental Example 1: Screening and Identification of Rhizosphere Biocontrols in Camellia oleifera This invention employs a 5-point sampling method. Rhizosphere soil from 12-year-old 'Changlin 40' camellia trees in the camellia oleifera cultivation area of ​​Changchong Town, Liuyang City, Hunan Province was selected. The litter layer and the top 5 cm of soil were removed. The 5-20 cm layer of rhizosphere soil was collected using the "root-shaking method" and transported back to the laboratory in a portable bag with an ice pack for the isolation of biocontrol strains. 10g of soil sample was weighed and placed in a 90mL Erlenmeyer flask containing sterile water. The flask was shaken thoroughly for 15-30 minutes to prepare a soil suspension. Then, under sterile conditions, 1mL of the soil suspension was injected into a test tube containing 9mL of sterile water and mixed thoroughly to prepare 10... -3 10 -4 10 -5 Prepare dilution solutions. Spread 0.1 mL of each dilution solution onto the corresponding LB agar plate, incubate at 28°C for 48 h, and record colony morphology, color, transparency, and other characteristics. Select colonies with significant differences in characteristics, purify the cultured bacteria, incubate on LB slant agar for 48 h, and then store at 4°C for later use.

[0026] The routine tissue isolation method for the anthracnose pathogen of Camellia oleifera (Fructus anthracnoseae): First, treat the tissue at the junction of diseased and healthy tissue with 75% alcohol, disinfect with 0.1% mercuric chloride solution, rinse thoroughly with sterile water, purify and culture in PDA medium, and store at 4℃ for later use.

[0027] Anthrax pathogen mycelium with a diameter of 6 mm was inoculated in the center of each PDA plate, and isolated bacterial strains were inoculated 2.5 cm away from the periphery. One bacterial strain was inoculated per plate, with three points symmetrically inoculated. The plates were incubated at 25℃. Meanwhile, plates inoculated only with anthrax pathogen mycelium served as a control. The inhibition rate was measured when the control mycelium reached the edge of the plate.

[0028] Inhibition rate (%) = [(Coronavirus diameter of control group - Coronavirus diameter of treatment group) / (Coronavirus diameter of control group - Diameter of original mycelium)] × 100% (Formula 1).

[0029] See appendix Figure 1 Preliminary screening results of rhizosphere biocontrol bacteria in Camellia oleifera: A total of 29 strains with inhibitory effects on anthracnose pathogens were isolated and purified. The inhibition range of the strains was found to be 8.89% to 76%, among which two strains had an inhibition rate of over 60%, numbered X21 and X23 respectively.

[0030] Table 1. Preliminary screening statistics of biocontrol strains in the rhizosphere of Camellia oleifera.

[0031] Secondary screening of biocontrol bacteria in the rhizosphere of Camellia oleifera: Three mycelial cakes of biocontrol bacteria X21 and X23, which showed an inhibition rate ≥60% in the initial screening, were inoculated into 250mL Erlenmeyer flasks containing 50mL of beef extract peptone broth. The flasks were incubated at 28℃ and 140r / min with shaking for 3 days. A 6mm diameter sterile punch was used to make a well in the center of a PDA plate. The culture medium in the well was removed, and 20µL of the initially screened biocontrol bacteria X21 and X23 were injected into the well. Mycelial cakes of the tested pathogen (6mm in diameter) were inoculated around the well at a distance of 3.0cm from the well. This process was repeated for 3 groups. The plates were incubated at a constant temperature of 28℃, with a control group inoculated only with the tested fruit anthracnose pathogen. The inhibition rate was measured when the fast-growing pathogen control reached the edge of the petri dish, and calculated using Formula 1.

[0032] Based on the initial selection results, two strains, X21 and X23, with an inhibition rate of over 60%, were selected for secondary screening. The experimental results showed that the inhibition rate of the two strains against anthracnose of Camellia oleifera was 35.4% to 47.0%, with the inhibition rate of X21 being higher than that of X23 at 47.0%. Therefore, this biocontrol strain numbered X21 was used for subsequent identification and experiments.

[0033] Table 2. Statistical Table of Rescreening of Rhizosphere Biocontrol Strains in Camellia oleifera

[0034] Example 2: Identification of biocontrol strain X21 1. Morphological identification: The colonies of biocontrol bacteria strain X21 on LB solid medium are pale yellow, opaque, round, and waxy in texture; the colonies of strain X21 on NA solid medium are white, opaque, round, and waxy in texture.

[0035] 2. Physiological and Biochemical Identification: Partial physiological and biochemical identification of biocontrol bacteria strain X21 is shown in Table 3. Rhizosphere bacteria showed positive results for Gram staining, catalase test, oxidase test, methyl red test, and VP, but negative results for citrate reaction. This strain can utilize glucose, nitrate, and hydrolyzed gelatin, and interacts with Bacillus spp. (…). Bacillus The characteristics of the bacterial strain are basically consistent. Based on morphological characteristics, the biocontrol strain X21 was preliminarily identified as a Bacillus genus.

[0036] Table 3. Physiological and biochemical experimental results of biocontrol bacteria strain X21

[0037] 3. Molecular identification: The genome of biocontrol bacteria strain X21 was extracted and amplified by PCR using universal primers for bacterial 16S rRNA gene sequence amplification. The primer sequences were: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The PCR amplification procedure was the same as in the reference "Influence of Different Nutrients on the Diversity of Cultivable Soil Bacteria". The PCR amplification products were sequenced, and the sequencing results of the 16S rRNA are shown in SEQ ID NO.1.

[0038] The sequence was homology-aligned with that of strain X21 in the NCBI gene bank. Bacillus mycoides The similarity of strain X21 (NR024697.1:651-1477) reached 92.55%, and strain X21 was identified as belonging to the genus Bacillus of the phylum Firmicutes, and named as such. Bacillus Sp. X21. The 16S rRNA gene sequence of Bacillus X21 was imported into NCBI's BLAST program for alignment, and known sequences with high homology were selected for phylogenetic tree construction. The results are shown in the appendix. Figure 2 .

[0039] Bacillus X21 was deposited on December 31, 2025, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China 430072, China), with accession number CCTCC M 20253060.

[0040] Experimental Example 3: Bacillus X21 ( Bacillus sp. X21) Active substances, drug resistance and acid-base adaptability 1. Antibacterial active substances: The ability of Bacillus X21 to produce heptaphilin was detected using CAS solid medium. Various enzymes were measured using different culture media, including β-1,3-glucanase, protease, and cellulase. The halo area around the colony is also observed. This indicates enzyme activity.

[0041] 2. Production of life-promoting substances: Qualitative and quantitative analysis of indoleacetic acid (IAA) production was performed. The potassium-solubilizing ability of the strains was determined using potassium feldspar medium, and the phosphorus-solubilizing ability was studied using the PVK method. The formation of clear zones around colonies in each culture medium was observed, and standard curves were used to calculate phosphorus-solubilizing and potassium-solubilizing abilities.

[0042] 3. Antibiotic Resistance: The sensitivity of Bacillus anthelminticus X21 to various antibiotics was detected using the disk agar diffusion method. Bacillus anthelminticus X21 bacterial suspension, activated and stored at -80℃, was diluted appropriately and spread onto the surface of LB solid medium. The medium was then air-dried in a clean bench, and antibiotic tablets were placed in the center. The medium was incubated overnight at 28℃, and colony growth was observed, with the diameter of the resulting clear zone measured.

[0043] 4. Growth curve determination: A single colony of purified Bacillus X21 was inoculated into LB liquid medium and incubated at 28℃ and 180 r·min. -1 The cells were cultured in a shaker with three replicates. The absorbance (OD600) at 600 nm was measured every 2 hours, and growth curves were plotted based on the OD600 values ​​at different times.

[0044] 5. Acid-base adaptability: Bacillus X21 strain was inoculated into LB liquid medium and incubated overnight at 28 ℃ and 180 r / min for 12 h. 10 μL of the bacterial culture was then inoculated into LB liquid medium at different pH values, with three replicates per pH gradient. The culture was incubated at 28 ℃ for 24 h, and the absorbance at OD600 was measured.

[0045] See appendix Figure 3 According to Table 4 below, the results show that Bacillus X21 has the ability to dissolve inorganic phosphorus, fix nitrogen, and produce IAA, and also has the ability to secrete protease, cellulase, amylase, β-1,3-glucanase, and ferrophile.

[0046] Table 4. Statistical Table of Rhizosphere Growth Promotion and Antibacterial Properties

[0047] Referring to the results in Table 5, Bacillus X21 showed sensitivity to macrolides such as erythromycin, tetracycline, minocycline, and gentamicin; aminoglycosides such as neomycin, kanamycin, amikacin, penicillin, and ampicillin; penicillins such as oxacillin, piperacillin, cefazolin, cefadroxil, and cefazolin; and cephalosporins such as cefoperazone, ceftriaxone, and ceftazidime. This indicates that Bacillus X21 can be controlled by antibiotics, which meets the standards for the use of biocontrol microorganisms.

[0048] Table 5. Statistical table of Bacillus X21 drug susceptibility test results.

[0049] See appendix Figure 4The results showed that the adaptation period was 0-1h: when Bacillus X21 was first inoculated onto the culture medium, the number of cells did not increase, but the metabolic activity gradually increased. The logarithmic growth phase was 1-2h: the number of cells increased exponentially, and the growth rate reached its maximum. The stationary phase was 2-48h: the number of cells tended to stabilize, and the growth rate was equal to the death rate.

[0050] Depend on Figure 5 , Figure 6 It can be seen that Bacillus X21 can grow normally in LB liquid medium with NaCl concentration between 1% and 7%, and Bacillus X21 can also grow normally at pH values ​​between 5.0 and 9.0.

[0051] Experiment Example 4: Bacillus X21 Inhibits Anthracnose on Isolated Tea Leaf Leaves The specific methods used in this experiment were referenced in the literature "Screening, Identification and Antagonistic Effect Analysis of Biocontrol Bacteria for Camellia anthrax".

[0052] Preparation of Bacillus X21 bacterial suspension: A Bacillus X21 bacterial suspension was prepared using LB medium and cultured on a shaker at 28℃ and 120 r / min for 2 days. The concentration of the bacterial suspension was adjusted to OD600 = 0.8 for later use.

[0053] Healthy, uniformly sized two-year-old tea leaves were selected as inoculation subjects. The leaves were disinfected with 75% alcohol for 30 seconds, then washed three times with sterile water and dried. The epidermis of the leaves was punctured but not pierced with a disposable syringe needle. Two punctures were made on each leaf, and the leaves were placed in a petri dish lined with sterile absorbent paper.

[0054] Preventive treatment: Leaves were first sprayed with Bacillus X21 bacterial suspension. Seven days after inoculation, pathogenic blocks were then inoculated onto the punctured areas of the leaves. Ten treatments were applied in total. Control treatment: Pathogenic blocks were first inoculated onto the punctured areas of Camellia oleifera leaves. Seven days after inoculation, leaves were sprayed with Bacillus X21 bacterial suspension. Ten leaves were inoculated for each treatment and placed in a 28℃ constant temperature incubator for humidification. The lesion area was measured after 15 days.

[0055] Rdisease = [(Ndisease - Ntotal) / Ntotal] × 100%. Where: Rdisease is the incidence rate, Ndisease is the number of diseased leaves, and Ntotal is the total number of leaves. Rcontrol = [(R0 - R1) / R0] × 100%. Where: Rcontrol represents the control effect, R0 is the control incidence rate, and R1 is the treatment incidence rate.

[0056] After culturing in petri dishes for 15 days, see [link / reference]. Figure 7As shown in Table 6, treatment T1 involved initial inoculation with the pathogen, followed by inoculation with Bacillus X21 suspension after disease onset. The disease incidence rates of Camellia oleifera varieties 'Huajin', 'Huashuo', and 'Huaxin' were 35.5%, 37.15%, and 54.56%, respectively. After inoculation with Bacillus X21 suspension, the control efficiencies for 'Huajin', 'Huashuo', and 'Huaxin' were 64.5%, 62.85%, and 45.44%, respectively. Among these, 'Huaxin' had the highest disease incidence, while 'Huajin' had the highest control efficiency. The T2 treatment involved inoculating the plants with a suspension of Bacillus X21, followed by inoculation with anthracnose pathogen blocks. The incidence rates of the 'Huajin', 'Huashuo', and 'Huaxin' varieties were 1.49%, 0.56%, and 0.76%, respectively, with control efficiencies of 98.51%, 99.44%, and 99.24%. This indicates that Bacillus X21 also has an inhibitory effect on the growth of the anthracnose pathogen in Camellia oleifera, reducing the area of ​​pathogenic lesions and demonstrating a significant antibacterial effect.

[0057] Table 6. Control efficacy of Bacillus X21 bacterial suspension against anthracnose in detached Camellia oleifera leaves.

[0058] Experiment Example 5: Control of Anthracnose in Potted Camellia oleifera Seedlings with Bacillus X21 Potted control experiment: A suspension of Bacillus X21 was prepared on LB medium and cultured for 2 days in a shaker at 28℃ and 120 r / min. The concentration of the Bacillus X21 suspension was adjusted to OD600=0.8. One-year-old Camellia oleifera potted seedlings with uniform growth were selected, and each seedling was inoculated with 3 leaves. The control group (CK) was inoculated only with anthracnose pathogen blocks and sprayed with water.

[0059] As shown in Table 7, the indoor pot experiment results indicate that Bacillus X21 significantly inhibits the anthracnose pathogen of Camellia oleifera. The control efficiency of 'Huajin' (A1) was significantly higher than CK (98.97%), and the control efficiency of 'B2' was significantly higher than C3 (86.26%). The control efficiency of 'Huashuo' (A1) was significantly higher than CK (92.94%), and the control efficiency of 'B2' was significantly higher than C3 (83.43%). The control efficiency of 'Huaxin' (A1) was significantly higher than CK (92.83%), and the control efficiency of 'B2' was significantly higher than C3 (79.05%).

[0060] Table 7. Control efficacy of Bacillus X21 against potted Camellia oleifera seedlings.

[0061] Experimental Example 6: Control of anthracnose in Camellia oleifera fields using Bacillus X21 Camellia oleifera 'Changlin 40' infected with the disease was selected as the research object, and an experimental area and a blank control area were set up. A Bacillus X21 bacterial suspension with an OD600 of 0.8 was sprayed, with each spray applied enough to completely wet the leaves of the entire Camellia oleifera tree. Two treatments were implemented: the control group was sprayed with water, and the experimental group was sprayed with Bacillus X21 bacterial suspension, with 10 Camellia oleifera trees in each treatment. Day 0 was defined as the time after spraying with Bacillus X21. Leaves from different treatments were collected 30 days later to analyze the activity of defensive enzymes in the leaves. Ten leaves were collected from each tree, and 100 leaves were collected from each treatment. Disease analysis was then performed.

[0062] As shown in Table 8, field trials indicate that Bacillus X21 can effectively control the anthracnose pathogen of Camellia oleifera. The control efficiency (which includes the control rate, and in in vivo trials, control efficiency and control rate have the same meaning) of Camellia oleifera was significantly higher than that of the control group, at 70.57%.

[0063] Table 8. Statistical table of the field control efficacy of Bacillus X21 against anthracnose in Camellia oleifera.

[0064] From Table 9, Figure 8 It was found that spraying with Bacillus X21 bacterial suspension significantly increased the activity of defense enzymes in the leaves of Camellia oleifera plants. Compared with the control group, the D1 treatment significantly increased the activities of CAT (112.51%), POD (220.03%), SOD (113.72%), PAL (68.93%), and PPO (64.37%). This indicates that Bacillus X21 can put plants in a positive defense response state, thereby enhancing plant stress resistance; moreover, increasing PAL and PPO activities can strengthen plant secondary metabolism and improve internal homeostasis. CAT refers to catalase, POD to peroxidase, SOD to superoxide dismutase, PAL to phenylalanine ammonia-lyase, and PPO to polyphenol oxidase.

[0065] Table 9. Defensive enzyme activity in plant leaves

[0066] Experiment Example 7: Bacillus X21 promotes the growth of potted Camellia oleifera seedlings Seed stratification: Collect seeds of superior Camellia oleifera varieties 'Asus,' 'Huajin,' and 'Huaxin,' stratify them in sand, maintaining a constant moisture content during the stratification process, and sow the seeds after they have rooted.

[0067] Seedling substrate: Acidic red soil from Central South University of Forestry and Technology was used. It was disinfected with wettable carbendazim, covered with film for 5 days, and air-dried. The soil was then passed through a 10-mesh sieve and mixed with vermiculite perlite in a ratio of 4:1:1. The mixture was then divided into seedling pots with a height × diameter (25cm × 16cm).

[0068] Preparation of bacterial agent: Bacillus X21 was inoculated into LB liquid medium and enriched in a shaker at 28℃ and 180r / min for 12h. After centrifugation at 8000r / min for 5min, a bacterial suspension with OD600=0.8 was prepared using sterile water and stored in a refrigerator at 4℃ for later use.

[0069] Experimental Treatments: Three Camellia oleifera varieties, 'Huashuo', 'Huajin', and 'Huaxin', were used. The treatments included inoculation with Bacillus X21 and a control (CK). Bacillus X21 suspension was inoculated into the roots of Camellia oleifera seedlings using a root drenching method, with 250 ml of the inoculum per seedling. CK was treated with 250 ml of sterile water. The treatment period was 3 months, with root drenching applied twice, once every 30 days. CK: Inoculated only with pathogen blocks and sprayed with water; Treatment groups: A1: drenched only with Bacillus X21 suspension; B2: drenched with Bacillus X21 suspension 7 days after pathogen inoculation; C3: inoculated only with pathogen blocks. Each treatment consisted of 25 potted plants.

[0070] Indicator measurements: Three months after inoculation, the height and diameter of the Camellia oleifera seedlings were measured using a ruler and vernier calipers; at harvest, plants were collected to measure biomass; and root morphology was measured using a root scanner.

[0071] The statistical results in Table 10 show that the seedling height of 'Huajin', 'Huaxin', and 'Huashuo' seedlings after inoculation with the fungus A1 and B2 treatments was significantly higher than that of the CK and C3 treatments. The plant height of 'Huajin' seedlings in the A1 treatment was 14% higher than that in the CK treatment, and in the B2 treatment, it was 10% higher than that in the C3 treatment. The ground diameter of 'Huajin' seedlings in the A1 treatment was 7.4% higher than that in the CK treatment, and in the B2 treatment, it was 35.6% higher than that in the C3 treatment. The fresh weight of the aboveground parts of 'Huajin' seedlings in the A1 treatment was 50.5% higher than that in the CK treatment, and in the B2 treatment, it was 54.3% higher than that in the C3 treatment. The fresh weight of the underground parts of 'Huajin' seedlings in the A1 treatment was 36.9% higher than that in the CK treatment, and in the B2 treatment, it was 14.2% higher than that in the C3 treatment. The dry weight of the aboveground parts of 'Huajin' seedlings in the A1 treatment was 62.1% higher than that in the CK treatment, and in the B2 treatment, it was 73.8% higher than that in the C3 treatment. The diameter at rootstock of the 'ASUS' A1 treatment increased by 147.8% compared to the control (CK), and the diameter of the B2 treatment increased by 7.7% compared to the C3 treatment. The fresh weight of the above-ground parts of the 'ASUS' A1 treatment increased by 104.2% compared to the control (CK), and the fresh weight of the underground parts of the 'ASUS' A1 treatment increased by 21.3% compared to the control (CK). The plant height of the 'Huaxin' A1 treatment increased by 28.4% compared to the control (CK); the fresh weight of the above-ground parts of the B2 treatment increased by 293.7% compared to the C3 treatment; and the fresh weight of the underground parts of the B2 treatment increased by 220.7% compared to the C3 treatment.

[0072] Table 10 Biomass of Camellia oleifera seedlings under different treatments

[0073] As shown in Table 11, compared with the CK treatment, the treatment inoculated with Bacillus X21 generally increased the total root length, root surface area, root volume, and number of root tips in Camellia oleifera. The total root length of A1 in the 'Huaxin' treatment increased by 8% compared to CK; the total root length of B2 in the 'Huajin', 'Huashuo', and 'Huaxin' treatments increased by 36.1%, 57.2%, and 107.5% compared to C3, respectively. The root surface area of ​​B2 in the 'Huajin', 'Huashuo', and 'Huaxin' treatments increased by 40%, 131.4%, and 58% compared to C3, respectively. Compared to the control (CK), the root diameter of Camellia oleifera treated with 'Huashuo' and 'Huaxin' increased by 2.7% and 53% in A1, respectively; the root diameter of Camellia oleifera treated with 'Huajin' and 'Huashuo' increased by 2.7% and 13.4% in B2, respectively, compared to C3; the root volume of Camellia oleifera treated with 'Huashuo' increased by 13.6% in A1, and by 46.2% and 22.6% in B2, respectively, compared to C3; the number of root tips in A1 treated with 'Huashuo' increased by 60.7% compared to CK; and the number of root tips in B2, respectively, increased by 26.8% and 43.9% compared to C3, compared to B2, respectively. These results indicate that inoculation with Bacillus X21 can promote the elongation and growth of Camellia oleifera roots, thereby increasing the range of soil nutrient absorption by the Camellia oleifera roots.

[0074] Table 11 Root morphology and structure of Camellia oleifera seedlings under different treatments

[0075] Experimental Example 8: Bacillus X21 promotes the bio-enzyme activity and enriches the rhizosphere microbial community of Camellia oleifera seedlings. The bioenzyme activity and rhizosphere microbial community of each treatment group in Experiment Example 7 were measured. For example... Figure 9As shown, the activity of catalase B2 in soils of 'Huajin' and 'Huashuo' increased by 21.49% and 0.7% respectively compared to treatment C3; the activity of alkaline phosphatase A1 in soils of 'Huashuo' and 'Huaxin' increased by 175.1% and 86.9% respectively compared to treatment CK, and the activity of alkaline phosphatase B2 in soils of 'Huajin' and 'Huashuo' increased by 110% and 43% respectively compared to treatment C3; the activity of urease A1 in soils of 'Huajin', 'Huashuo', and 'Huaxin' increased by 6.3%, 26.2%, and 20% respectively compared to treatment CK, and the activity of urease B2 in soils of 'Huajin', 'Huashuo', and 'Huaxin' increased by 7.8%, 22.5%, and 56% respectively compared to treatment C3. The acid phosphatase activity in the soil of 'Huajin', 'Huashuo', and 'Huaxin' plants increased by 64.5%, 82%, and 92.5% respectively compared to the control (CK) treatment; the acid phosphatase activity in the soil of 'Huajin', 'Huashuo', and 'Huaxin' plants increased by 13.5% and 58.2% respectively compared to the control (C3) treatment; the sucrase activity in the soil of 'Huajin', 'Huashuo', and 'Huaxin' plants increased by 23.4%, 35.9%, and 7.3% respectively compared to the control (CK) treatment; the sucrase activity in the soil of 'Huajin', 'Huashuo', and 'Huaxin' plants increased by 5.71%, 16.7%, and 32% respectively compared to the control (C3) treatment; indicating that Bacillus X21 can promote the bioenzyme activity of Camellia oleifera.

[0076] See Figure 10 As shown, after inoculation with Bacillus X21, the top 10 dominant phyla in the rhizosphere microbiota of Camellia oleifera were Proteobacteria (…). Proteobacteria Unclassified bacteria, Acidobacteria Acidobacteriota ), Green Curvature ( Chloroflexi ), Actinobacteria ( Actinobacteriota Bacteroidetes ( Bacteroidota ), Myxococcus (Myxococcota) Firmicutes (Firmicutes) ), Verrucous microbes ( Verrucomicrobiota ), Bacillus phylum ( Gemmatimonadota The top 10 dominant bacterial genera are: Paucibacter Unclassified bacteria, unclassified anaerobic bacteria Anaerolineae Uncultured forest soil bacteria, unclassified acid bacteria Acidobacteriales ), unclassified Elsterales , Candidatus Koribacter genus *Acidophilus* Acidothermus ), Pseudomonas spp. Pseudomonas ), Candidatus Solibacter .

Claims

1. A Bacillus strain X21 that has disease-preventing and growth-promoting effects on Camellia oleifera ( Bacillus sp. X21), characterized in that, The accession number is CCTCC M 20253060, and it was deposited at the China Center for Type Culture Collection on December 31, 2025.

2. A biological agent with disease-preventing and growth-promoting effects on camellia oleifera, characterized in that, A culture containing Bacillus X21 as described in claim 1 is used as the active ingredient.

3. The biological agent according to claim 2, characterized in that, The culture was prepared by inoculating Bacillus X21 in LB liquid medium and culturing at 28°C. After enrichment and centrifugation, a bacterial suspension with OD600=0.8 was prepared using sterile water.

4. The application of Bacillus X21 as described in claim 1 or the biological agent as described in claim 2 in promoting the growth and development of Camellia oleifera, wherein the promotion of Camellia oleifera growth is achieved by promoting the activity of biological enzymes in Camellia oleifera, increasing the activity of antioxidant enzymes and defense enzymes in leaves, promoting the growth of Camellia oleifera plant height, increasing the diameter of Camellia oleifera and the fresh and dry weight of the above-ground and underground parts, and promoting the elongation and growth of Camellia oleifera roots.

5. A method for preventing and controlling anthracnose in Camellia oleifera, characterized in that, The procedure includes applying Bacillus X21 as described in claim 1 or the biological agent as described in claim 2 to Camellia oleifera.

Images