Bacillus amyloliquefaciens for preventing and treating tea tree anthracnose and application of bacillus amyloliquefaciens

Abstract

The invention belongs to the technical field of microorganisms, and relates to bacillus amyloliquefaciens for preventing and treating tea tree anthracnose and application of the bacillus amyloliquefaciens. The bacillus amyloliquefaciens H-2-3-(1) disclosed by the invention has good broad-spectrum antibacterial activity, can be used for remarkably inhibiting the growth of mycelia of Colletotrichum siamense, and shows an excellent biological prevention and control effect in the aspect of preventing and treating the anthracnose of tea trees. In addition to inhibiting pathogenic bacteria of tea trees, the strain also shows obvious antagonistic activity on various plant pathogenic fungi and has good plant growth promoting ability. The strain has the characteristics of high efficiency, safety and environmental friendliness, and is an ideal candidate resource for developing a tea tree anthracnose biological control fungicide. The application of the method not only helps to improve the green and sustainable level of tea tree disease prevention and control, but also provides important theoretical basis and technical support for research, development and industrialization of tea tree biological prevention and control products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a Bacillus amyloliquefaciens for the prevention and control of anthracnose in tea trees and its application. Background Technology

[0002] Anthracnose is a serious disease threatening tea production, caused by fungi of the genus *Colletotrichum* pp. It is widespread globally, especially prevalent in major tea-producing areas of China. In the early stages, water-soaked, yellowish-brown lesions appear on the leaves, gradually expanding and forming distinct grayish-white necrotic areas. The edges of the lesions are brown or scorched yellow. Later, the leaf tissue becomes necrotic, brittle, and falls off, severely reducing tea yield and quality, causing significant economic losses to the tea industry.

[0003] Currently, the control of anthracnose in tea trees mainly relies on chemical pesticides, such as broad-spectrum fungicides like difenoconazole, azoxystrobin, and prochloraz. While these pesticides are fast-acting, long-term and repeated use can lead to drug resistance in pathogens, reducing their effectiveness and forcing farmers to increase pesticide application, further exacerbating environmental pollution and pesticide residue problems. Furthermore, with increasing consumer awareness of food safety and environmental protection, pesticide residues in tea are receiving growing attention, leading to increasing restrictions on the use of chemical pesticides. Therefore, relying solely on chemical agents to control anthracnose in tea trees does not meet the requirements of sustainable agricultural development and ecological security in modern agriculture.

[0004] In recent years, with the advancement of agricultural technology and the advocacy of ecological agriculture, biological control has gradually become a safe, green, and environmentally friendly method for controlling plant diseases, attracting widespread attention. Bacillus spp., as an emerging biological control resource, has shown great application potential in multiple agricultural disease control fields due to its strong environmental adaptability, high antibacterial activity, and good safety profile. In particular, Bacillus amyloliquefaciens possesses multiple functions, including rapid reproduction, strong resistance, broad-spectrum antibacterial activity, and promotion of plant growth. Existing studies have reported that Bacillus amyloliquefaciens exhibits significant antibacterial activity against various plant diseases such as ginseng rust rot, blueberry crown gall, and peony root rot, demonstrating great application potential in practical production.

[0005] However, there are currently few strains that have specific antagonistic effects against anthracnose in tea trees and also possess 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 promote growth in order to meet the pressing needs of sustainable agricultural development.

[0006] Therefore, to address the aforementioned issues, a strain of Bacillus amyloliquefaciens with significant inhibitory activity against anthracnose pathogens of tea trees and multiple growth-promoting functions was obtained through screening. This provides a safe, efficient, and green new solution for the biological control of anthracnose in tea trees, and has significant economic, social, and ecological implications. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention provides a strain of Bacillus amyloliquefaciens H-2-3-(1) and its application in the prevention and control of anthracnose in tea trees, so as to solve the problems of limited prevention and control effect, high risk of drug resistance and poor ecological safety in the prior art.

[0008] The details are as follows: A strain of Bacillus amyloliquefaciens, H-2-3-(1), is a Gram-positive bacterium used to control anthracnose in tea trees. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, on October 21, 2025, with accession number CGMCCNo. 36236 and classification name Bacillus amyloliquefaciens.

[0009] The colonies of this strain are irregularly round with uneven edges, an opaque central area, and an overall milky white color. It exhibits broad-spectrum inhibitory activity against a variety of common plant pathogenic fungi. It possesses a strong extracellular enzyme secretion capacity, effectively producing cellulase, amylase, and phytase; it also has the ability to dissolve phosphorus, produce siderophores, and fix nitrogen.

[0010] Furthermore, the strain H-2-3-(1) has the ability to significantly inhibit the growth and reproduction of *Anthrax sicca*, *Discosporum aviculare*, *Dystomata chamae*, *Fusarium sclerotium*, *Staphylococcus aureus*, *Anthrax moniliforme*, and *Anthrax spp.*

[0011] The application of Bacillus amyloliquefaciens H-2-3-(1), which is used to prevent anthracnose in tea trees, in the prevention and control of common plant pathogens.

[0012] Furthermore, the common plant pathogens mentioned are *Diaporthe discoidispora*, *Pestalotiopsis theae*, *Fusarium chlamydosporum*, *Botryosphaeria dothidea*, *Colletotrichum musae*, and *Colletotrichum fructicola*.

[0013] The application of Bacillus amyloliquefaciens H-2-3-(1) for the prevention and control of anthracnose in tea trees.

[0014] Furthermore, the application in the prevention and control of anthracnose in tea trees is its application in the preparation of biocontrol agents for anthracnose in tea trees.

[0015] Furthermore, the application in the prevention and control of anthracnose in tea trees is to inhibit the activity of Colletotrichumsiamense, a strain that exhibits a relatively good antagonistic effect against Colletotrichumsiamense.

[0016] Furthermore, the aforementioned prevention and control of tea anthracnose involves using this strain and its metabolites and volatile organic compounds (VOCs) to control tea anthracnose. This strain and its metabolites and VOCs can effectively inhibit the infection of tea anthracnose pathogens and significantly reduce the incidence of tea anthracnose disease.

[0017] Furthermore, the aforementioned prevention and control of anthracnose in tea trees refers to the prevention and control of anthracnose in detached tea leaves.

[0018] The application of Bacillus amyloliquefaciens H-2-3-(1), which is used to prevent and control anthracnose in tea trees, in promoting plant growth.

[0019] A biocontrol agent for anthracnose of tea trees, containing the above-mentioned Bacillus amyloliquefaciens H-2-3-(1) for controlling anthracnose of tea trees.

[0020] The beneficial effects of this invention are: This invention provides a strain of Bacillus amyloliquefaciens H-2-3-(1) with broad-spectrum antibacterial activity, which can significantly inhibit the growth and reproduction of various plant pathogenic fungi, including Anthracnoseus sicca, Discosporum spp., Polychaete chacca, Fusarium tumefaciens, Staphylococcus aureus, Anthracnoseus basilica, and Anthracnoseus spp.

[0021] In addition, this strain and its metabolites and volatile organic compounds (VOCs) can effectively inhibit the infection of anthracnose pathogens in tea trees, significantly reducing the incidence of anthracnose disease in tea trees.

[0022] Therefore, Bacillus amyloliquefaciens H-2-3-(1) provides a valuable strain resource for the research and development of biocontrol agents for anthracnose in tea trees, and has broad application potential and promotion value. Attached Figure Description

[0023] Figure 1 For the morphological observation of H-2-3-(1); where A: colony morphology, B: cell morphology under Gram staining optical microscope (100× oil immersion).

[0024] Figure 2The phylogenetic tree for H-2-3-(1) is constructed based on the 16S rRNA gene sequence.

[0025] Figure 3 The phylogenetic tree for H-2-3-(1) is constructed based on the gyrB gene sequence.

[0026] Figure 4 To investigate the antibacterial activity of Bacillus amyloliquefaciens H-2-3-(1) against different plant pathogenic fungi (left side is CK, right side is antibacterial activity); among them, a: Discospora spp.; b: Discospora chamae; c: Fusarium chrysogenum; d: Staphylococcus aureus; e: Anthracnose basilica; f: Anthracnose basilica.

[0027] Figure 5 To investigate the antagonistic effect of Bacillus amyloliquefaciens H-2-3-(1) on anthracnose fungus of tea tree; where a: control group; b: experimental group.

[0028] Figure 6 A gradient concentration inhibition diagram of Bacillus amyloliquefaciens H-2-3-(1) sterile filtrate against anthracnose pathogens of tea trees.

[0029] Figure 7 The inhibitory effect of volatile gases from Bacillus amyloliquefaciens H-2-3-(1) on anthracnose pathogens of tea trees is shown in the figure. A: LB solid medium control; B: Antibacterial effect of volatile substances from strain H-2-3-(1).

[0030] Figure 8 To investigate the control of Bacillus amyloliquefaciens H-2-3-(1) against anthracnose fungus of tea tree; in which, the left photo (CK): blank LB medium; the right photo: bacterial suspension coated with antagonistic strain.

[0031] Figure 9 To detect the extracellular enzyme production of Bacillus amyloliquefaciens H-2-3-(1); where A: cellulase production capacity, B: amylase production capacity, and C: phytase production capacity.

[0032] Figure 10 To detect the growth-related abilities of Bacillus amyloliquefaciens H-2-3-(1); where A and B: phosphorus solubilization ability; C: iron carrier production ability; D: nitrogen fixation ability. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0034] The experimental materials used in the embodiments of this invention are from the following sources: The Mongkina organic phosphorus medium, PKO inorganic phosphorus medium, and CAS detection medium were all purchased from Qingdao Haibo Biotechnology Co., Ltd. Amylase detection medium (g / L): tryptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, soluble starch 2 g / L, distilled water as solvent; Cellulase detection medium (g / L): tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, sodium carboxymethyl cellulose 10g / L, distilled water as solvent; Phytase medium (%): 1% sodium phytate, 3% glucose, 0.5% (NH4)2SO4, 0.05% KCl, 0.003% MnSO4·4H2O, 0.05% MgSO4·7H2O, 0.003% FeSO4·7H2O, pH 5.5 Nitrogen-fixing medium (g / L): mannitol 10 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, CaSO4 0.1 g / L, CaCO3 5 g / L, agar 18 g / L, pH 7.2, distilled water as solvent; Siamese anthrax ( Colletotrichum siamense ), Discoid spores ( Diaporthe discoidispora ), Tea-like spores ( Pestalotiopsis theae ), Fusarium truncatum ( Fusarium chlamydosporum ), Staphylococcus aureus ( Botryosphaeria dothidea ), banana anthrax bacteria ( Colletotrichum musae ), fruit anthracnose ( Colletotrichum fructicola (From the Key Laboratory of Microbial Resources and Utilization, College of Marine and Biotechnology, Guangxi University for Nationalities)

[0035] Unless otherwise specified, any materials or reagents not described in the embodiments of this invention can be purchased through conventional channels.

[0036] Example 1 Identification of Bacillus amyloliquefaciens H-2-3-(1) 1. Isolation and morphological observation of bacterial strains Strain H-2-3-(1) was isolated from the rhizosphere soil of *Machilus tigrinus* and is an indigenous microorganism originating from the rhizosphere environment of *Machilus tigrinus*. The collected rhizosphere soil samples were isolated and cultured using the dilution plating method, and the target strain H-2-3-(1) was obtained after multiple rounds of purification. The strain was streaked onto LB solid medium and incubated at 30℃ for 24 hours. The morphological characteristics of single colonies were observed, including color, size, edge morphology, and transparency.

[0037] Morphological observation results as follows Figure 1As shown. The strain forms irregularly shaped circular colonies on LB solid medium ( Figure 1 A), with irregular edges, an opaque central area, and an overall milky white color; 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.

[0038] 2. Molecular biological identification Total DNA was extracted from strain H-2-3-(1) after activation. Using this as a template, bacterial universal primers 27F and 1492R and the housekeeping gene were used to extract DNA. gyrB Primers UP-1 and UP-2r were used to target the 16S rRNA gene of strain H-2-3-(1) and gyrB The gene was amplified by PCR. The primer sequences used are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1); 1492R: 5'-TACGGCTACCTTGTTACGAGTT-3' (SEQ ID NO. 2); UP-1: 5'-ATTTGGCGCTGGCGGTTAT-3' (SEQ ID NO. 3); UP-2r: 5'-GGTTTCGGCTGGGCTGGTA-3' (SEQ ID NO. 4).

[0039] The PCR amplification system (50 μL) includes: 1 μL template, 22 μL ddH2O, 2× Taq PCR Mix 25 μL, forward and reverse primers 1 μL each.

[0040] The PCR amplification program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 1 min, 55℃ annealing for 1 min, 72℃ extension for 2 min, 32 cycles; 72℃ extension for 10 min; the amplified products were stored at 4℃.

[0041] After purification, the amplified products were sent to Aoke Biotechnology (Wuhan) Co., Ltd. for bidirectional sequencing. The obtained sequences were compared with the sequences published in the NCBI database for homology, and a phylogenetic tree was constructed using MEGA 7.0 software.

[0042] Molecular identification results such as Figure 2 and Figure 3 As shown: The 16S rRNA gene sequence of strain H-2-3-(1) is similar to that of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Bacillus amyloliquefaciens KC-1 has the highest similarity. Figure 2); gyrB gene sequence and Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens The similarity between T23 and T23 exceeds 99%, and phylogenetic analysis shows that they cluster in the same evolutionary branch. Figure 3 ).

[0043] Based on the combined morphological characteristics and molecular biological identification results, strain H-2-3-(1) can be finally identified as Bacillus amyloliquefaciens (Bacillus). Bacillus amyloliquefaciens ).

[0044] Example 2: Detection of the antibacterial ability of Bacillus amyloliquefaciens H-2-3-(1) The antagonistic effect of *Bacillus amyloliquefaciens* H-2-3-(1) against various plant pathogenic fungi was evaluated using the plate confrontation method. The tested pathogens included *Tetranychus chamaejasminoides* (…). Pestalotiopsis theae ), Discoid spores ( Diaporthe discoidispora ), Tea-like spores ( Pestalotiopsis theae ), Fusarium truncatum ( Fusarium chlamydosporum ), Staphylococcus aureus ( Botryosphaeria dothidea ), banana anthrax bacteria ( Colletotrichum musae ) and fruit anthracnose ( Colletotrichum fructicola Six common plant pathogenic fungi were identified. The experimental method was as follows: H-2-3-(1) was inoculated onto LB solid medium and streaked to activate it. Single colonies were picked and inoculated into LB liquid medium. The culture was shaken at 30℃ and 180 r / min for 24 h to obtain the fermentation broth of the strain for later use. A 5 mm diameter fungal cake of pathogen was inoculated in the center of a PDA plate. 5 μL of H-2-3-(1) fermentation broth was dropped into the plate along a cross shape at a distance of 2.5 cm from the fungal cake. The medium without the fungal broth was used as a control. Each treatment was repeated 3 times. All plates were placed in an incubator at 28℃ for 5 days. The growth of the colonies was observed and the size of the inhibition zone was measured. The inhibition rate (V) was calculated as follows: V = (D CK -D d ) / D CK ×100%. Where D d D represents the diameter (cm) of pathogen colonies in the treatment group. CK The diameter (cm) of pathogen colonies in the control group.

[0045] The antibacterial effect of Bacillus amyloliquefaciens H-2-3-(1) against various pathogens was detected as follows: Figure 4As shown in Table 1, the left side represents the colony morphology of the control group, and the right side represents the colony morphology of the experimental group. The test results showed that Bacillus amyloliquefaciens H-2-3-(1) had varying degrees of inhibitory effects on six common plant pathogens, namely, Discospora spp., Discospora chamaejasminoides, Fusarium oxysporum, Staphylococcus aureus, Anthracnoseus moniliforme, and Anthracnoseus spp. (Table 1), with an inhibition rate of over 50%, indicating that strain H-2-3-(1) has strong broad-spectrum antibacterial activity.

[0046] Table 1. Antibacterial detection of Bacillus amyloliquefaciens H-2-3-(1)

[0047] Example 3: Antibacterial activity of Bacillus amyloliquefaciens H-2-3-(1) against Bacillus anthracis of Siamese spp. 1. Detection of the inhibitory effect of Bacillus amyloliquefaciens H-2-3-(1) on Anthracnose of Tea Tree. The inhibitory effect of Bacillus amyloliquefaciens H-2-3-(1) on Bacillus anthracis was tested using the plate confrontation method. The experimental procedure was the same as in Example 2.

[0048] The inhibitory effect of Bacillus amyloliquefaciens H-2-3-(1) is as follows: Figure 5 As shown in Figure a, the results showed that Bacillus amyloliquefaciens H-2-3-(1) had an inhibitory effect on Bacillus anthracis, with an inhibition rate of 82.12%.

[0049] 2. Antibacterial activity of Bacillus amyloliquefaciens H-2-3-(1) sterile filtrate against Bacillus anthracis of Siamese spp. The antagonistic bacteria fermentation broth was centrifuged at 6000 rpm for 8 min. The supernatant was filtered twice through a 0.22 μm filter membrane and added to sterile PDA medium at proportions of 0%, 1%, 2%, 3%, 5%, 8%, 10%, 12%, 15%, and 20% to prepare culture plates. Anthrax mycelial cakes with a diameter of 5 mm were inoculated into the center of the medium. Each treatment was repeated 3 times and incubated at 30 °C for 7 days.

[0050] The antibacterial activity of sterile filtrate of Bacillus amyloliquefaciens H-2-3-(1) against Bacillus anthracis in Siamese anthrax is as follows: Figure 6 As shown in Table 2, the results indicate that the inhibition rate of the sterile filtrate against Bacillus anthracis increases with increasing concentration. When the concentration reaches 15%, the inhibition rate approaches 100%.

[0051] Table 2. Determination of the inhibitory effects of Bacillus amyloliquefaciens H-2-3-(1) sterile filtrate on Bacillus anthracis of Siamese bacteria using gradient concentrations.

[0052] 3. Antibacterial activity of volatile gases from Bacillus amyloliquefaciens H-2-3-(1) against Bacillus anthracis in Siamese bacteria. The inhibitory effect of volatile gases produced by *Bacillus amyloliquefaciens* H-2-3-(1) on *Anthracis sinensis* was determined using the double-plate inverted plate method. *Bacillus amyloliquefaciens* H-2-3-(1) was inoculated at 2% in 50 mL LB liquid medium and cultured at 30℃ and 180 r / min for 24 h with shaking. 100 μL of the fermentation broth was evenly spread onto LB solid plates, and a 5 mm diameter *Anthracis sinensis* bacterium block was placed in the center of the PDA plate. The two plates were then inverted and sealed, and incubated at 28℃ for 5 days. The control group consisted of plates coated with blank medium instead of plates containing *Bacillus amyloliquefaciens* H-2-3-(1). Each treatment was repeated in triplicate, and the average value was taken.

[0053] The antibacterial activity of volatile gases from *Bacillus amyloliquefaciens* H-2-3-(1) against *Anthrax sicca* is as follows: Figure 7 As shown, the results indicate that the volatile gas produced by Bacillus amyloliquefaciens H-2-3-(1) can also significantly inhibit the growth of Bacillus anthracis, with an inhibition rate as high as 86.25%.

[0054] Example 4: Detection of the control effect of Bacillus amyloliquefaciens H-2-3-(1) on anthracnose of tea trees. Pathogen treatment: Take a 5 mm mycelial block and place it in the center of fresh PDA medium. Incubate for 5 days, then punch a 5 mm hole in the edge of the mycelium for later use.

[0055] Preparation of strain culture medium: strain H-2-3-(1) was inoculated in LB liquid medium and cultured at 30℃ and 180 rpm for 1 day for later use.

[0056] Fresh tea leaf processing: Take tea leaves of the same size and shape, with healthy and spotless surfaces, wash off surface impurities with sterile water and air dry. Apply the bacterial fermentation broth to the surface, then inoculate with activated 5 mm Siamese anthrax bacteria blocks and place them on the tea leaves. Use blank culture medium as a control. Each treatment is repeated three times.

[0057] The control effect of Bacillus amyloliquefaciens H-2-3-(1) on anthracnose of tea trees is as follows: Figure 8 As shown, the results indicate that H-2-3-(1) has a good control effect on anthracnose of tea trees. The leaves of tea trees coated with blank culture medium showed obvious anthracnose spots, and some of them expanded into water-soaked lesions, indicating a more severe disease. The leaves turned yellow and lost their green color. In contrast, the leaves of tea trees coated with culture medium of strain H-2-3-(1) showed only a few minor lesions, indicating a very mild disease. The leaves remained green and healthy, indicating that strain H-2-3-(1) can effectively inhibit *Anthracnose sicca*, slow down the occurrence of anthracnose in tea trees, and has a good control effect on anthracnose in tea trees.

[0058] Example 5: Determination of growth-promoting and enzyme-producing capacity of Bacillus amyloliquefaciens H-2-3-(1) Preparation of single colonies of Bacillus amyloliquefaciens H-2-3-(1): Bacillus amyloliquefaciens H-2-3-(1) was inoculated onto LB solid medium and strewn in three zones for 1 day.

[0059] Single colonies of Bacillus amyloliquefaciens H-2-3-(1) were collected and placed in the center of PKO inorganic phosphorus medium, Monkina organic phosphorus medium, CAS detection medium, cellulase detection medium, amylase detection medium, and phytase detection medium, respectively, and cultured at 30℃ for 5 days. After culturing in cellulase detection medium, the colonies were immersed in 1 mol / L Congo red solution for 10 min, followed by washing and decolorization with 0.9% sodium chloride solution for 10 min. The diameter of the transparent zone was observed and measured. After culturing in amylase detection medium, the colonies were immersed in iodine solution for 1 min, and the diameter of the transparent zone was observed and measured.

[0060] Select a single colony of H-2-3-(1) and streak it in three zones on nitrogen-fixing medium. Incubate at 30℃ for 1-3 days and observe whether it can grow on nitrogen-fixing medium.

[0061] Qualitative detection results of enzyme production characteristics of Bacillus amyloliquefaciens H-2-3-(1) are as follows: Figure 9 As shown, Bacillus amyloliquefaciens H-2-3-(1) was cultured in cellulase detection medium ( Figure 9 A) Amylase detection medium ( Figure 9 B) and phytase detection medium ( Figure 9 In C), a clear transparent zone was produced in all three media. Amylase activity was the highest, with a clear zone to colony diameter ratio of 3.13±0.04; cellulase and phytase activities were relatively low, with clear zone to colony diameter ratios of 1.04±0.06 and 1.69±0.03, respectively. The results indicate that strain H-2-3-(1) produced a clear transparent zone in all three media. Amylase activity was the highest, with a clear zone to colony diameter ratio of 3.13±0.04; cellulase and phytase activities were relatively low, with clear zone to colony diameter ratios of 1.04±0.06 and 1.69±0.03, respectively. These experimental results confirm that strain H-2-3-(1) has a strong extracellular enzyme secretion capacity and can effectively produce cellulase, amylase, and phytase.

[0062] The results of the growth-promoting function test of Bacillus amyloliquefaciens H-2-3-(1) are as follows: Figure 10 As shown, Bacillus amyloliquefaciens H-2-3-(1) was cultured in PKO inorganic phosphorus medium (PKO). Figure 10 A) and Monkina organic phosphorus medium ( Figure 10The strain grew normally in both cultures (B) and produced distinct clear zones, preliminarily indicating that it has good phosphate-solubilizing ability. Furthermore, this strain also formed clear zones on CAS siderophore-producing media. Figure 10 C), indicating that it can secrete siderophores, thereby enhancing the absorption and utilization of iron. Strain H-2-3-(1) can also grow normally on nitrogen-free medium ( Figure 10 D), which preliminarily proves that it has nitrogen-fixing ability. The above results show that strain H-2-3-(1) has the ability to solubilize phosphorus, produce iron carriers and fix nitrogen, showing good growth potential.

[0063] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A Bacillus amyloliquefaciens for controlling anthracnose in tea trees, characterized in that, This strain is a Gram-positive bacterium. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, on October 21, 2025, with accession number CGMCC No. 36236.

2. The Bacillus amyloliquefaciens for controlling anthracnose in tea trees according to claim 1, characterized in that, The strain exhibits a significant ability to inhibit the growth and reproduction of *Anthrax sicca*, *Discosporum*, *Diatomum chamaejasminoides*, *Fusarium chrysogenum*, *Staphylococcus aureus*, *Anthrax moniliforme*, and *Anthrax spp.* 3. The application of Bacillus amyloliquefaciens as described in claim 1 for controlling anthracnose in tea trees in the control of common plant pathogens.

4. The application according to claim 3, characterized in that, The common plant pathogens mentioned are Discoidospora ( Diaporthe discoidispora ), Tea-like spores ( Pestalotiopsis theae ), Fusarium truncatum ( Fusarium chlamydosporum ), Staphylococcus aureus ( Botryosphaeria dothidea ), banana anthrax ( Colletotrichum musae ), fruit anthracnose ( Colletotrichum fructicola ) .

5. The application of Bacillus amyloliquefaciens as described in claim 1 in the prevention and control of anthracnose in tea trees.

6. The application according to claim 5, characterized in that, The application described in the prevention and control of anthracnose in tea trees is to inhibit *Anthracnose sirenus* (Siamese anthracnose fungus). Colletotrichum siamense The role of ).

7. The application according to claim 5, characterized in that, The aforementioned method for controlling anthracnose in tea trees involves using this strain of bacteria, its metabolites, and volatile gases to control the disease.

8. The application according to claim 5, characterized in that, The aforementioned prevention and control of anthracnose in tea trees refers to the prevention and control of anthracnose in detached tea leaves.

9. The application of Bacillus amyloliquefaciens as described in claim 1 for preventing and controlling anthracnose in tea trees in promoting plant growth.

10. A biocontrol agent for anthracnose in tea trees, characterized in that, It contains Bacillus amyloliquefaciens for controlling anthracnose in tea trees as described in claim 1.

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