Plant disease biocontrol bacillus cereus strain LCBC246 and development and application thereof

Abstract

The invention belongs to the technical field of microbial prevention and control of plant diseases and insect pests, and particularly discloses a plant disease biocontrol bacillus cereus strain LCBC246 which is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the strain is CGMCC No.33323. The invention further discloses a preparation method of the plant disease biocontrol bacillus cereus strain LCBC246. The LCBC246 strain provided by the invention is a gram-positive bacterium, has a very strong bacteriostatic effect on colletotrichum fucicola, the bacteriostatic rate is 92.1%, the disease control rate on the colletotrichum fucicola is 90.8%, and the LCBC246 strain has a very good inhibitory effect on other important plateau characteristic crop pathogenic bacteria and also has a remarkable crop growth promoting property. The bacterial inhibition rates of fermentation liquor and volatile substances of the strain LCBC246 on colletotrichum gloeosporioides of macadamia nuts are 62.70% and 76.58% respectively; the bacillus amyloliquefaciens can generate sterilization metabolites such as cellulase, amylase and siderophore, and has a very strong broad-spectrum bacteriostatic effect on various plant pathogenic fungi. Therefore, the strain LCBC246 and the metabolite thereof have the application value of developing bacterial biopesticides.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for plant diseases and pests, specifically relating to the biocontrol strain LCBC246 of Bacillus cereus, which has a highly effective inhibitory effect on plant pathogens such as Macadamia anthracnose fungus, and its prepared biocontrol agents. Background Technology

[0002] Macadamia nuts (Macadamia integrifolia Maiden & Betche), named for their origin in Australia, are now widely cultivated in many countries and regions, including the United States, Australia, Indonesia, and Thailand. Introduced to my country in the 1980s, macadamia nuts are now widely grown in various southern provinces. The nuts are rich in nutrients, primarily containing fats, carbohydrates, and proteins, as well as abundant calcium, phosphorus, iron, and vitamins B1 and B2. The kernels are fragrant, crisp, and smooth, with a unique creamy aroma, making them among the world's highest quality edible nuts, often referred to as the "Queen of Nuts" and the "King of Nuts," with a flavor and texture far superior to other nuts such as cashews. In recent years, Lincang and other areas in Yunnan Province have prioritized macadamia nuts as a key industry in their highland specialty agriculture development. By 2024, the planting area had reached nearly 300,000 hectares, accounting for 90% of China's total macadamia nut planting area and more than half of the global total, making it a core production area and the largest production base globally. Annual output value has exceeded 7 billion yuan, making it an important economic crop that the region is vigorously developing.

[0003] Anthracnose is a common and important disease of macadamia nuts. Recent research by the author has shown that its pathogens are Colletotrichum fucticola and C. gloeosporiodes. These two fungi also cause important foliar diseases on many legumes, fruit trees, vegetables, medicinal herbs and flowering plants, causing significant economic losses due to their severe reduction in the yield and quality of these crops.

[0004] Currently, chemical control is the most widely used method for controlling crop diseases, and anthracnose is no exception. However, long-term and excessive use of chemical pesticides can lead to problems such as crop residues, environmental pollution, and the development of pesticide resistance in pathogens. Biological control, on the other hand, can avoid these problems associated with chemical pesticides. Therefore, the discovery of biocontrol microorganisms for plant diseases and the development of microbial pesticides have become an important and hot research area in biology in recent years. To date, important biocontrol microorganisms for plant diseases discovered mainly include several types of bacteria such as Bacillus spp., Pseudomonas spp., Acinetobacter spp., and Streptomyces spp. Among them, biological fungicides developed from Bacillus subtilis and Bacillus licheniformis have been commercialized and widely used in the control of various crop diseases.

[0005] Macadamia integrifolia, tea tree (Camellia sinensis), coffee (Coffeaarabica), tobacco ( Nicotiana cordifolia, N. benthamiana Crops and medicinal herbs (e.g., Leucosceptrum canum, Polygala crotalarioides) are characteristic crops of the Yunnan-Guizhou Plateau in my country, widely cultivated and possessing significant economic value. However, research on biocontrol agents and biological agents targeting diseases of these plateau-specific crops is rarely reported in the literature.

[0006] In recent years, many national invention patents related to Bacillus cereus have been granted, such as: a Bacillus cereus strain CZBC1 that dissolves pond Oscillatoria and its application (CN103352010 B), an alkali- and chromium-resistant Bacillus cereus strain (strain FZUY01) and its application (CN 114164119 B), a disease-suppressing and growth-promoting Bacillus cereus strain YT2-1C and its application (CN 115044502 B), and a Bacillus cereus (strain GW-01) agent and its application in eliminating cypermethrin in animals (CN115141764 B), etc. The strains involved in these patents come from different provinces and ecological environments (soil or ponds, etc.), and each has its own important application value. The Bacillus cereus strain LCBC246 in this application was isolated from anthracnose tissue of macadamia nuts in Yunnan. It not only has an inhibitory effect of more than 90% on anthracnose of macadamia nuts and the pathogen, but also has a strong broad-spectrum antibacterial effect on anthracnose pathogen of tea trees, root rot pathogen of Polygala tenuifolia, and wilt pathogen of tobacco, and also has a significant plant growth-promoting effect. Summary of the Invention

[0007] This invention yielded a biocontrol bacterium strain, LCBC246, exhibiting strong inhibitory effects against important crop pathogens such as anthracnose in macadamia nuts and tea trees, and also demonstrating excellent control over a variety of diseases. It was identified as *Bacillus cereus* using morphological and molecular biological methods. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 33323. Studies revealed that it interferes with and inhibits pathogenic growth through the production of cellulase, amylase, and ferrophosphate. This biocontrol bacterium is non-pathogenic to plants; on the contrary, it significantly promotes plant growth. Its main biological characteristics were clarified through experimental analysis, leading to the optimization of its optimal culture conditions. These findings provide a valuable strain for further research and development of biological fungicides for controlling plant diseases and lay the technical foundation for the creation of biological agents.

[0008] Discovery and identification of strain LCBC246: Twenty-eight pure culture bacterial strains were isolated and purified from anthracnose lesions on macadamia leaves using the nutrient medium plate isolation method. After repeated antagonistic tests against *Colletotrichum fructicola*, the strain with the strongest inhibitory effect against this pathogen, LCBC246, was selected (see attached). Figure 1 It was identified as Bacillus cereus through morphological and molecular biological methods.

[0009] Morphological characteristics of LCBC246 (with appendix) Figure 2 Preliminary identification: After streaking on nutrient agar plates and incubating at 37°C in the dark for 48 h, the resulting colonies were round or nearly round, milky white, opaque, with a waxy surface and irregular edges, and a diameter of 3.2–12.6 mm (n=30). The bacteria were Gram-positive after staining. Under a 1000× microscope, the bacteria appeared as short rods, straight or slightly curved, occurring singly or in chains of two or more cells, measuring 2.24–3.19 × 0.65–0.91 μm (n=30). Based on these characteristics and referring to Bergey's Handbook of Bacteriological Identification, 8th Edition, and other literature, LCBC246 was preliminarily identified as a Bacillus sp.

[0010] Molecular identification of LCBC246: The ITS and 16S rDNA sequences of strain LCBC246 were obtained by PCR amplification and were 539 bp and 1410 bp, respectively (see attached image). Figure 3The results of online BLAST-N alignment analysis of the two sequences uploaded to the NCBI GenBank database showed that strain LCBC246 had 100% similarity to both Bucillus cereus strains ATCC 14579 and LT15-MRL in the NCBI database (536 / 536 bp; 1410 / 1410 bp). A neighbor-joining phylogenetic tree based on the 16S rDNA sequences of each bacterium was constructed using MAGA11 (see attached). Figure 4 The results showed that LCBC246 and Bacillus cereus strain JCM1465 were located on the same terminal branch, with Bootstrap support of 100%.

[0011] Based on the above morphological and molecular biological identification results, LCBC246 was finally identified as Bacillus cereus.

[0012] Inhibitory effects of LCBC246 on important crop pathogens: LCBC246 showed inhibition rates of 72.4%–92.1% against 10 plant pathogens, including Colletotrichum gloeosporiodes and C. fructicola (macadamia nut anthracnose), Rhizoctonia solani (wheat sheath blight), Epicoccus latusicollum (corn white spot), Botrytis cinerea (fruit tree mold), Fusarium oxysporium (tobacco wilt), C. camelliae (tea thorn spores), Pseudopestalotiopsos camelliae (tea spores), Curvularia eragrostidis (leaf spot of Polygala tenuifolia), and C. fioriniae (rice flower anthracnose). These results indicate that LCBC246 has a strong inhibitory effect on a variety of crop pathogens.

[0013] The antibacterial mechanism of LCBC246: The volatile substances of LCBC246 showed inhibition rates of 73.1% and 57.6% against *Colletotrichum spp.* and *Fusarium oxysporum*, respectively. The antagonistic bacterial filtrates diluted 5 and 10 times showed inhibition rates of 77.6% and 58.2% against *Colletotrichum spp.* and 71.3% and 54.7% against *Fusarium oxysporum*, respectively. It can produce amylase, cellulase, protease, and ferrophosphate, which interfere with the normal physiological and biochemical processes of *Colletotrichum spp.*, thereby invading and destroying the cell structure and growth of the pathogen, preventing infection and disease in host plants.

[0014] Optimized culture conditions for LCBC246: The suitable temperature range for LCBC246 growth and sporulation is 15℃~50℃, with an optimum temperature of 37℃~40℃. Growth ceases at 4℃ and above 50℃. Colonies can form at pH 4~13, with an optimum pH of pH 5~7. Light has no significant effect on colony formation. LCBC246 forms colonies fastest on NA and Czapek's agar, and can also grow normally on PDA and PSA. Growth is slower on other tested carbon source media. Peptone and yeast extract are suitable for LCBC246 growth, while the other two nitrogen sources are less suitable. LCBC246 can grow on all nine tested culture media plates, but grows fastest and sporulates most vigorously on nutrient medium (NA).

[0015] The Bacillus cereus strain LCBC246 provided by this invention is identified as Bacillus cereus. The strain is deposited in vivo at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.33323.

[0016] The application of Bacillus cereus LCBC246 in inhibiting the growth of plant pathogens and in the preparation of agents to inhibit the growth of plant pathogens, including Colletotrichum fructicola, Colletotrichum gloeosporiodes, Rhizoctonia solani, Epicoccus latusicollum, Botrytis cinerea, Fusarium oxysporium, Colletotrichum camelliae, Pseudopestalotiopsos camelliae, Curvularia eragrostidis, or Colletotrichum fioriniae.

[0017] Application of Bacillus cereus LCBC246 in the control of crop anthracnose and in the preparation of crop disease control agents. These crop diseases mainly include anthracnose of macadamia nuts (Colletotrichum fructicola), tea (Colletotrichum camelliae), and rice flower (Colletotrichum fioriniae). Because LCBC246 preparations have strong inhibitory effects on pathogens causing root rot, wilt, leaf spot, and gray mold in crops, they can also be used for the prevention and control of these crop diseases.

[0018] The biocontrol agent of LCBC246 comprises the aforementioned Bacillus cereus, its fermentation broth, and volatile preparations.

[0019] Biological control methods for crop diseases. The method involves applying the aforementioned Bacillus cereus LCBC246 bacterial suspension preparation, which can effectively control anthracnose of African nut (Colletotrichum fructicola), tea (Colletotrichum camelliae), and rice flower (Colletotrichum fioriniae). Attached Figure Description

[0020] The application materials for this invention include the following figures:

[0021] Figure 1 Image of LCBC246, the antagonistic bacterium with the strongest inhibitory effect against macadamia anthracnose.

[0022] Figure 2 Morphological characteristics of Bacillus cereus LCBC246;

[0023] Note: A. Colonies on NA plate; B. Colonies observed under a stereomicroscope (5×); C. Bacterial cells observed under a microscope (1000×); The box in the upper right corner shows magnified bacterial cells, and the arrow points to bacterial spores.

[0024] Figure 3 The sequence diagram of ITS and 16S rDNA of strain LCBC246 obtained by PCR amplification;

[0025] Figure 4 . Neighbor-joining phylogenetic tree diagram of antagonistic bacteria LCBC246 strain and related species;

[0026] Note: The neighbor-joining phylogenetic tree based on ITS and 16S rDNA splicing sequences was constructed using MEGA11 software. The scale bar shows the degree of difference between splicing sequences of paired species, and the number at each node represents the autonomous support rate that the paired species are the same species.

[0027] Figure 5 A diagram illustrating the growth-promoting effect of Bacillus cereus LCBC246 on wheat seedlings.

[0028] Figure 6 Safety evaluation of Bacillus cereus LCBC246 on different plants.

[0029] Note: A. Negative control (macadamia leaves soaked in sterilized water); B. Positive control (macadamia leaves inoculated with anthracnose fungus 7 days later); leaves of the other 10 plant species were treated with 10... 9 The sample was photographed after being soaked in / mL Bacillus cereus solution and cultured for 21 days. Detailed Implementation

[0030] Note: Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased. The invention will be further described in detail below with reference to examples.

[0031] Note: The antagonistic bacterium LCBC246 provided by this invention, whose taxonomic name is Bacillus cereus, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33323. The address of the collection center is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is January 7, 2025. This biological material was accepted and registered by the collection center on January 7, 2025. The microbial material was tested by the collection center on January 7, 2025, and the result was "viable".

[0032] The LCBC246 strain provided by this invention is derived from anthracnose-infected tissue of macadamia nuts grown in Yunnan. It is a Gram-positive bacterium and has a strong inhibitory effect on Colletotrichum fructicola, the causal agent of macadamia anthracnose, with an inhibition rate of 92.1% and a disease control rate of 90.8% for this anthracnose disease. The fermentation broth and volatile substances of strain LCBC246 showed inhibition rates of 62.70% and 76.58% against *Colletotrichum gloeosporiodes*, the causal agent of macadamia anthracnose, respectively. It can produce cellulase, amylase, and ferrophile, and is effective against various plant pathogenic fungi, including *Colletotrichum fructicola*, *Colletotrichum gloeosporiodes*, *Rhizoctonia solani*, *Epicoccus latusicollum*, *Botrytis cinerea*, *Fusarium oxysporium*, *Colletotrichum camelliae*, *Pseudopestalotiopsos camelliae*, *Curvularia eragrostidis*, and *Colletotrichum ferniensis*. All of the LCBC246 strains (fioriniae) exhibit strong antibacterial activity, with inhibition rates ranging from 72.4% to 92.1%. Treatment with LCBC246 bacterial suspension significantly promotes wheat seed germination and seedling growth. Therefore, strain LCBC246 and its metabolites have significant economic value for the development of bacterial biopesticides.

[0033] The following experimental research cases were mainly implemented in this invention project:

[0034] Implementation Case 1: Obtaining the antagonistic bacterium LCBC246, Bacillus cereus

[0035] Samples of root zone soil, branches and leaves, and anthracnose-infected tissue were collected from macadamia nut orchards in different locations in Lincang, Yunnan Province. These samples were then isolated and purified using NA agar plates, yielding 28 pure bacterial cultures. These strains were then subjected to antagonistic tests against *C. fruiticola*, the causal agent of macadamia anthracnose, to screen for antagonistic bacteria with strong antagonistic activity. The screening results showed that strain LCBC246, isolated from macadamia anthracnose tissue, exhibited strong antagonistic activity against *C. fruiticola*. Figure 1 The highest positivity rate was 92.36 ± 2.68% (n=5), which was significantly higher than other antagonistic bacterial strains. Therefore, further in-depth research should be conducted on this bacterium.

[0036] Implementation Case 2: Morphological Identification of LCBC246

[0037] The strain was inoculated onto 90 mm diameter PDA plates and incubated at 37°C in the dark for 48 h. Colony morphology was observed, and the diameter of 30 colonies was measured. The shape, color, size, and other characteristics of the colonies were described. Gram staining was performed to determine whether the bacteria were Gram-negative or Gram-positive based on the final color of the cells. The shape of the bacterial cells was observed and photographed under a microscope, and the size of 30 bacterial cells was measured. Based on the observation and measurement results, and with reference to authoritative bacterial classification literature such as Bergey's Manual of Bacteriology, 8th Edition, the taxonomic genus of LCBC246 was preliminarily determined.

[0038] Research observation results: The colonies formed were round or nearly round, milky white, opaque, with a waxy surface and irregular edges, and a diameter of 3.2–8.6 mm (n=30); the bacterial cells were Gram-positive (purple) after staining; under a 1000× microscope, the bacterial cells appeared as short rods, straight or slightly curved, occurring singly or in chains of two to more cells, with a size of 2.24–3.19 × 0.65–0.91 μm (n=30) (see...). Figure 2 Based on these characteristics and with reference to authoritative literature such as Bergey's Handbook of Bacteriological Identification, LCBC246 was preliminarily identified as a Bacillus bacterium.

[0039] Implementation Case 3: Molecular Biological Identification of LCBC246

[0040] A suitable amount of bacterial cells were scraped from LCBC246 colonies cultured on nutrient medium (NA) for 48 h. Genomic DNA was extracted from the mycelium using the cetyltrimethylammonium bromide (CTAB) method. The ITS and TEF1-1α gene sequence fragments were amplified from the DNA by PCR using primer pairs (ITS1 / ITS4 5'-TCCGTAGGTGAACCTGCGG-3' / 5'-TCCTCCGCTTATTGATATGC-3') and (5'-ATGGGTAAGGAGGACAAGAC-3' / 5'-GGAGGTACCAGTGATCATGTT-3). The amplified products were recovered, purified, and sent to Kunming Shuoqing Biotechnology Co., Ltd. for sequencing.

[0041] Identification results: The gene sequences of ITS and TEF1-1α are 539 bp and 1410 bp, respectively. Figure 3 ). Connect the two sequences online ( https: / / blast.ncbi.nlm.nih.gov / Blast.cgiBLAST-n comparative analysis was performed separately; ITS and 16S rRNA sequences of related species of Bacillus were downloaded from the NCBIGenBank database, and a phylogenetic tree was constructed using the spliced ​​sequences of these two sequences (see attached). Figure 4 The results showed that LCBC246 clustered with Bacillus sereus strain DC3 on the same terminal branch, with a bootstrap support of 100%. Therefore, LCBC246 was ultimately identified as Bacillus sereus. In the recent "three-domain classification system of cellular biology," it belongs to the domain Bacteria, kingdom Bacteria, phylum Firmicutes, and class Bacillales.

[0042] Implementation Case 4: Antimicrobial Effect Test of LCBC246 Strain on Important Crop Pathogens

[0043] Ten pathogenic fungi of the tested plants (see Table 1) were inoculated onto PDA agar plates and incubated in the dark at 25°C for 5 days. Mycelial cakes with a diameter of 5 mm were then collected using a punch. Each fungal cake was placed in the center of a 90 mm diameter agar plate. Bacillus cereus LCBC246 was spotted at 25 mm intervals around the fungal cakes. For the control, sterile water was spotted around the fungal cakes. Each treatment was repeated five times. After inoculation, the plates were incubated at 28°C in the dark for 7 days. The area of ​​the fungal colonies was then measured, and the inhibition rate of LCBC246 against each pathogen was calculated as [=(control colony area - treated colony area) / control colony area × 100%].

[0044] The experimental results (Table 1) show that Bacillus cereus LCBC246 has a strong inhibitory effect on the growth of 10 plant pathogens, with an inhibition rate of 72.4%–92.1%. Among them, the inhibition rate against four crop anthracnose pathogens is higher than 80%, which indicates that LCBC246 has a strong inhibitory or bactericidal effect on these crop pathogens.

[0045] Table 1. Antibacterial effects of Bacillus cereus LCBC246 against 10 important crop pathogens

[0046]

[0047] Note: Colony diameter and area are the mean ± standard error of three replicates. The inhibitory effect of LCBC246 treatment on 10 pathogens was highly significant (P<0.001).

[0048] Implementation Case 5: Disease Control Efficacy Test of Bacillus cereus LCBC246 against Several Anthrax Diseases

[0049] Healthy macadamia seedlings, potted tea seedlings, and rice flower seedlings were prepared in the greenhouse; LCBC246 cultured on PDA plates for 14 days was prepared to a concentration of approximately 10. 8-9 A Trichoderma solution with spores / mL was used to uniformly spray the leaves of three plant seedlings. 24 h after foliar spraying with LCBC246 antagonistic bacteria, a spore suspension of *Colletotrichum gloeosporioides*, *Colletotrichum chrysogenum*, and *Colletotrichum ferniensis* (approximately 10 mL) was then applied. 6 Foliar spraying was performed on macadamia seedlings, tea seedlings, and rice flower seedlings using spores / mL. The negative control was sprayed only with pathogen spore solution, while the positive control was applied 24 hours after applying difenoconazole solution, followed by pathogen spore solution. Each treatment was replicated with 10 pots of plants. After treatment, seedlings were placed in black plastic bags in the dark and kept moist for 24 hours, then placed in a growth chamber (25℃, RH>80%, 10h / d light) for cultivation. Disease severity (%) was observed and recorded after 21 days, and the disease control rate (%) was calculated.

[0050] The experimental results (Table 2) show that the control efficacy of Bacillus cereus LCBC246 against anthracnose in macadamia nuts, tea trees and rice flowers is 86.9%~90.8%, which is slightly lower than the control efficacy of 40% difenoconazole suspension (91.2%~94.89%).

[0051] Table 2. Control effects of Bacillus cereus LCBC246 on several crop diseases

[0052]

[0053] Note: Disease severity % is the average of 10 replicates ± standard error; Disease control rate = (treatment - control) / control × 100%.

[0054] Implementation Case 6: Test on the growth-promoting effect of Bacillus cereus LCBC246 on plants

[0055] Taking the effects of LCBC246 on wheat seed germination and seedling growth as an example, wheat seeds were treated separately with sterilized water (control) and LCBC246 bacterial suspension (approximately 10 μL). 9After soaking for 24 hours, the seeds were removed and evenly sown on moistened straw paper or filter paper in petri dishes (50 seeds / dish, 3 replicates per dish). Germination was observed and recorded after 2 days at room temperature and under natural light. Seedling height, root length, and fresh weight were measured after 14 days to analyze the effects of LCBC246 on wheat seed germination and seedling growth. [Note: Using wheat and other cereal seeds for the experiment is convenient and time-efficient. Therefore, wheat seeds were used as a representative sample in this study to measure the effects of LCBC246 on plant seed germination and seedling growth.]

[0056] From the appendix Figure 6 It can be seen that LCBC246 bacterial suspension treatment has a significant promoting effect on wheat seed germination and seedling growth. Detailed measurement data (Table 3) show that, compared with the control, LCBC246 bacterial suspension treatment significantly (P<0.01) increased seed germination rate, plant height, and root length, with the four growth indicators increasing by 17.5% to 33.9%, indicating that LCBC246 has a very significant (P<0.01) growth-promoting effect on crops.

[0057] Table 3. Effects of LCBC246 bacterial suspension treatment on wheat seed germination and seedling growth

[0058]

[0059] Note: Seed germination rate is the mean ± standard error of three replicates; observations were recorded 2 days after germination; seedling height, root length, and single plant weight are the mean ± standard error of 20 seedlings, measured 14 days after germination; growth promotion rate = (control - treatment) / control; statistical comparative analysis showed that the differences in all indicators between the LCBC246 treatment and the control reached a highly significant level (P<0.01).

[0060] Case Study 7: Determination of the Pathogenicity (Safety) of Bacillus cereus LCBC246 in Different Crops

[0061] The 10 plants tested ( Figure 6These are important crops characteristic of the Yunnan Plateau, including macadamia (Macadamia integrifolia), tea (Camellia sinensis), small-bean coffee (Coffea arabica), wax apple (Syzygium samarangense), grape (Vitis vinifera), rice flower (Leucosceptrum canum), cowpea (Vignasinensis), corn (Zea mays), gardenia (Fructus gardeniae), and camphor tree (Camphora officinarum). Healthy leaves of these plants were collected from natural fields and analyzed using a Bacillus cereus LCBC246 bacterial suspension (10... 9 The leaves of macadamia nuts were soaked in sterile water for 10 min ( / mL); the negative control was soaked in sterile water for 10 min, and the positive control was inoculated with Colletotrichum fructicola mycelium. Each treatment was repeated with 3 leaves. Leaves of different treatments were placed on moistened filter paper in steel trays and the trays were sealed tightly with plastic wrap. After treatment, the leaves were placed in saturated humidity and darkness for 24 h, and then transferred to a growth chamber at 25℃, RH>90% and 10 h / d light. After 9 days, the lesions on the positive control were observed and photographed. After 21 days, the leaves of the negative control and the 10 types of leaves treated with Bacillus cereus LCBC246 were observed and photographed.

[0062] Results: Macadamia leaves inoculated with *Colletotrichum gloeosporioides* (positive control) developed typical anthracnose lesions 9 days later. The negative control macadamia leaves and leaves of 10 plant species treated with *Bacillus cereus* LCBC246 suspension remained healthy after 21 days, showing no disease symptoms. (See attached image) Figure 6 The results showed that LCBC246 is not pathogenic and its application to plants will not cause "phytotoxicity".

[0063] Implementation Case 8: Antibacterial Activity Test of Volatile Products from Bacillus cereus LCBC246

[0064] Colletotrichum fructicola, the anthracnose pathogen of macadamia nuts, was selected as the representative pathogen. The inhibitory effect of volatile metabolites of LCBC246 on the pathogen was determined using the "double-plate inversion method". Colonies cultured at 28℃ in the dark for 5 days were used. LCBC246 bacterial suspension was dropwise inoculated into the center of a PDA plate, and a pathogenic bacterial cake (5 mm in diameter) was inoculated into the center of another PDA plate. The two plates were then inverted and sealed with sealing film. A PDA plate inoculated with the pathogen and then inverted with a PDA plate containing sterile water was used as a control. Each treatment was repeated three times. After 7 days of incubation at 28℃ in the dark, the diameter of the pathogenic colonies was observed and measured. The colony area was calculated, and the inhibition rate of the antagonistic fungal volatile substances against the pathogen was calculated [=(control colony area - treatment colony area) / control colony area × 100%].

[0065] The test results showed that the volatile substances of LCBC246 had an inhibition rate of 65.6% against *Colletotrichum cereus*, which preliminarily indicates that the production of volatile substances with antibacterial effects is one of the important mechanisms by which *Bacillus cereus* LCBC246 inhibits the growth of pathogens.

[0066] Implementation Case 9: Antibacterial Activity Test of Bacillus cereus LCBC246 Fermentation Broth

[0067] 0.5 mL of antagonistic bacterial suspension was inoculated into 50 mL of NB liquid medium and cultured at 30 °C with shaking at 200 times / min for 36 h to obtain bacterial suspension. The bacterial suspension was centrifuged at 6000 rpm for 15 min at room temperature, and the supernatant was collected and filtered through a 0.22 µm microporous membrane to remove bacterial cells, obtaining fermentation filtrate. The fermentation filtrate was mixed with PDA medium at ratios of 1:4 and 1:9 (v / v) to obtain 5× and 10× dilution media, respectively, with ordinary PDA medium as a control. *C. fructicola*, the anthracnose causal agent of macadamia nuts, was activated on PDA plates. A 5 mm diameter mycelial cake was collected from the edge of the colony and inoculated into the center of the plate, with each treatment repeated three times. After incubation at 28 °C in the dark for 5 days, the colony size was measured and recorded, and the inhibition rate (%) was calculated.

[0068] The test results showed that the antagonistic bacterial filtrate diluted 5× and 10× times had inhibition rates of 77.4% and 58.2% against the tested *Colletotrichum gloeosporioides*, respectively. This indicates that the fermentation broth of LCBC246 antagonistic bacteria has a strong inhibitory effect on plant pathogens.

[0069] Implementation Case 10: Analysis of the Physiological and Biochemical Mechanisms of LCBC246's Antibacterial Effect

[0070] Following the methods described in Bergey's Manual of Bacterial Identification (8th Edition) and other literature, the reactions of LCBC246 to glucose fermentation, lactose fermentation, D-mannose fermentation, starch hydrolysis, citric acid decomposition, cellulose hydrolysis, protein hydrolysis, hydrogen sulfide production, indoleacetic acid, gelatin liquefaction test, and VP test were determined; referencing Huang et al. (2024). [1] He Zhang et al. (2015) [2] The methods were used to determine the production of catalase, β-1,3-glucanase, protease, chitinase and cellulase by the strain. [Note: References [1]. Huang Xun, Feng Jiawen, Jin Chunlin, et al. Identification of antagonistic bacteria JYC1217 for potato scab and its biocontrol and growth-promoting characteristics [J]. Journal of Plant Protection, 2024, 51(3): 684-697; [2]. Zhang Bin, Qiao Junqing, Liang Xuejie, et al. Evaluation of antagonistic bacteria between Fusarium wilt and Ralstonia solanacearum [J]. Journal of Plant Protection, 2015, 42(03): 353-361.]

[0071] The test results (Table 4) show that strain LCBC246 can decompose and utilize glucose and liquefy gelatin, but it cannot decompose lactose, D-mannose, citric acid, or indoleacetic acid, nor does it produce hydrogen sulfide, which is consistent with the description of Bacillus cereus in the literature. In addition, LCBC246 can produce hematoxylin, catalase, β-1,3-glucanase, and chitinase, with a particularly strong ability to produce cellulase and amylase.

[0072] Table 4. Physiological and biochemical test results of LCBC246 antagonistic bacterial strains

[0073]

[0074] Note: - Negative; + Positive; ++ Strongly Positive

[0075] Implementation Case 12: Biological Characteristics and Culture Condition Optimization Experiment of Bacillus cereus LCBC246

[0076] The effects of different temperatures, pH levels, light exposure times, culture media, and C and N sources on the growth of LCBC246 antagonistic bacteria were tested. For each biological test, the required culture media were prepared in advance according to the formula, sterilized at 121℃ (0.10~0.12 MPa) for 30 min, and then poured into Φ90 mm Petri dishes to prepare agar plates. Simultaneously, the LCBC246 antagonistic bacteria strain stored at 4℃ was taken out, placed at room temperature for 24 h, and then inoculated onto nutrient medium (NA) plates. After incubation at 37℃ for 3 days, an appropriate amount of bacterial cells was used to prepare approximately 10... 8CFU / mL bacterial suspensions were spread onto different treatments and placed on culture plates. After incubation at 37°C for 3 days (except for temperature tests), colonies were counted. Each treatment was repeated three times.

[0077] ① Culture medium test. The growth of pathogens on eight culture media, including NA, PDA, CDA, LB, CMA, PCA, WA and MEA, was tested.

[0078] ② Carbon source experiment. Using CDA as the basal medium (control), appropriate amounts of potato starch, corn starch, soluble starch, maltose, glucose, sucrose, lactose, or galactose were added to prepare eight different carbon source media with a carbon source content of 30 g / L.

[0079] ③ Nitrogen source test. Using CDA as the basal medium, yeast powder, peptone, soy protein, ammonium sulfate, and ammonium chloride were added to prepare a medium with a nitrogen source content of 2.0 g / L.

[0080] ④ Temperature test. Using PDA agar plates, 15 temperature treatments were set up: -20℃, 0, 4, 10, 15, 20, 25, 30, 34, 37, 40, 45, 50, 55, and 60℃. After 48 hours of treatment, the number of colonies per plate was counted, and the average colony count for each treatment was calculated.

[0081] ⑤ pH test. Using PDA medium plates, set 11 pH values ​​(pH 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and pH 13) for treatment. Adjust the pH of the medium to the required pH value using 1.0 mol / mL HCl solution and 1.0 mol / mL NaOH solution.

[0082] ⑥ Light test. Using PDA culture medium plates, light durations of 0, 4, 8, 12, 18, and 24 h / d were set during the culture period.

[0083] ⑦ Aerobic test. Inoculate LCBC246 bacterial suspension onto beef extract peptone agar (NA) plates and incubate in a conventional incubator (aerobic) and in a carbon dioxide incubator (anaerobic) after sealing the plates with sealing film.

[0084] The results showed that Bacillus cereus LCBC246 could grow or maintain activity within the temperature range of 10℃ to 50℃, with the optimal temperature being 37℃. Growth ceased at 0℃ and above 37℃. LCBC246 could grow within the pH range of 4 to 13, with the optimal pH being pH 6.5. LCBC246 grew fastest under 0–4 (hrs) of light per day, and could grow under 6–24 (h / d) of light per day, but growth gradually slowed down with increasing light exposure (Table 5).

[0085] Table 5. Effects of temperature, pH and light duration on the growth of LCBC246

[0086]

[0087] Note: Each observation in each experiment is the average of three replicates; different superscript letters indicate significant differences between different carbon or nitrogen source treatments (P<0.01).

[0088] LCBC246 was able to form colonies normally on all eight tested culture media plates, with nutrient medium (NA) being its optimal medium. Among the eight tested carbon sources, glucose and maltose were the optimal carbon sources for LCBC246, and it could also form colonies normally on the other carbon source media. Peptone and beef extract were the most suitable for the growth of LCBC246, and it could also produce colonies on the other six tested nitrogen source media (Table 6).

[0089] In addition, LCBC246 can grow normally and form colonies in an anaerobic environment, but it grows faster under aerobic conditions and forms significantly more colonies than under anaerobic conditions (P<0.01).

[0090] Table 6. Effects of different carbon and nitrogen sources on the growth of LCBC246

[0091]

[0092] Note: Each value is the mean ± standard deviation of three replicates; different superscript letters indicate significant differences between different carbon or nitrogen source treatments (P<0.01).

Claims

1. A Bacillus cereus ( Bacillus cereus strain LCBC246, characterized in that, Category Naming: Bacillus cereus; It is deposited at the China General Microbiological Culture Collection Center, with the strain accession number CGMCC No. 33323.

2. The Bacillus cereus in claim 1 ( Bacillus cereus strain LCBC246 and its prepared biocontrol agent, which are effective against macadamia anthracnose ( Colletotrichum gloeosporiodes and Colletotrichum. fructicola ), Tea Thorn Colloides ( Colletotrichum. camelliae ), Anthracnose fungus ( Colletotrichum. fioriniae ), wheat sheath blight fungus ( Rhizoctonia solani ), corn white spot disease fungus ( Epicoccus latusicollum Fruit tree mold () Botrytis cinerea ), Tobacco wilt pathogen ( Fusarium oxysporium ), Tea false disc polychaete ( Pseudopestalotiopsos camelliae ) and leaf spot disease of Polygala tenuifolia ( Curvularia eragrostidis It has a very strong antibacterial effect on plant pathogens such as macadamia nuts, tea trees and rice plants (inhibition rate 72.4%-92.1%); under inoculation conditions, it has a good control effect on anthracnose of macadamia nuts, tea trees and rice plants (disease control rate 86.9%-90.8%).

3. The volatile substances and filtrate produced by the Bacillus cereus strain LCBC246 in claim 1 have significant inhibitory effects on both Colletotrichum spp. and Fusarium oxysporum; it can produce proteases, amylases, cellulases, and ferrophiles to interfere with the normal physiological and biochemical processes of pathogens, thereby invading and destroying the cell tissue structure and growth and development of pathogens, and preventing pathogens from infecting and causing disease in host plants.

4. The optimal culture conditions for Bacillus cereus strain LCBC246 in claim 1 are as follows: the optimal growth temperature is 37℃~40℃, the optimal pH value is pH5~7; colonies form fastest on NA and Czapek's medium; yeast extract and peptone are suitable for the growth of LCBC246.

5. The use of Bacillus cereus LCBC246 as described in claim 1 in inhibiting the growth of plant pathogens or in preparing agents to inhibit the growth of plant pathogens, characterized in that, The pathogens include *Colletotrichum caryophyllum*. (Colletotrichum fructicola ), Discus spicula ( C. gloeosporiodes ), Tea Thorn Colloides ( C. camelliae ), Feline spirochete ( C. fioriniae Rhizoctonia solani ( ) Rhizoctonia solani ), broad-necked coccidioidomyces ( Epicoccus latusicollum ), Botrytis cinerea ( Botrytis cinerea Fusarium oxysporum ( Fusarium oxysporium ), Tea false disc polychaete ( Pseudopestalotiopsos camelliae ) and Microsporum simonii (Curvularia) eragrostidis ).

6. The application of the Bacillus cereus strain LCBC246 according to claim 1 in the control of crop anthracnose or in the preparation of agents for controlling crop diseases, characterized in that, Specifically, this includes macadamia anthrax ( Colletotrichum. fructicola Anthracnose of tea trees Colletotrichum. camelliae ) and rice flower anthracnose ( Colletotrichum. fioriniae Plant diseases.

7. A biocontrol agent for LCBC246, characterized in that, Fermentation broth and / or volatile preparations containing Bacillus cereus LCBC246 as described in claim 1.

8. A biological control method for crop diseases, using the Bacillus cereus LCBC246 biocontrol agent as described in claim 7 to control diseases of crops with highland characteristics, including macadamia anthracnose, tea anthracnose, rice flower anthracnose, wheat sheath blight, corn white spot disease, strawberry gray mold, tobacco wilt, tea brown spot disease, and Southwest China Polygala leaf spot disease.

9. The use of the Bacillus cereus strain LCBC246 of claim 1 in the production of amylase, cellulase, protease and ferrophile.

10. The effect of the Bacillus cereus strain LCBC246 suspension according to claim 1 in promoting wheat seed germination and seedling growth.

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