Application of bacillus velezensis lh-bls012 in plant disease biological control and growth promotion

Abstract

The application discloses Bacillus velezensis LH-BLS012 (CGMCC No. 34146) or an engineering bacterium modified through natural, artificial mutagenesis or gene editing, and application of the engineering bacterium in plant disease biological control and growth promotion. The LH-BLS012 or the engineering bacterium thereof can be colonized in plant roots for a long time, and fermentation culture solution, bacterial powder and / or secondary metabolites of the LH-BLS012 or the engineering bacterium thereof can effectively control fungal diseases of forest trees, greening plants and crops, and can promote yield increase of the crops by 19.3%-24.7%.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial technology, specifically relating to the application of Bacillus belye LH-BLS012 strain, its engineered strain and its metabolites in the biological control and growth promotion of fungal diseases in forest trees, landscaping plants and crops. Background Technology

[0002] Bacillus velezensis, a Gram-positive bacterium with multiple biological activities, has sparked a surge in research and application worldwide in recent years. With its strong antagonistic abilities, plant growth-promoting properties, environmental adaptability, and high safety, this bacterium has become a shining star in the fields of biological control, green agriculture, and environmental protection, demonstrating enormous potential and becoming a research focus in multiple fields such as agriculture, animal husbandry, and aquaculture.

[0003] According to reports, the number of Bacillus velezensis strains currently registered and preserved in global microbial culture centers exceeds 3,000, of which approximately 65% ​​originate from soil, 18% from plant endophytes, and 9% from marine environments. There are approximately 1,870 patents related to "Bacillus velezensis," with China (CNIPA) accounting for 46%, the United States (USPTO) for 15%, and South Korea (KIPO) for 12%. Related research in my country mainly focuses on biocontrol agents, lipopeptide preparation, strain modification, and formulation processes; while international research emphasizes genetic engineering and the use of metabolites in new drugs. Notably, Europe launched a "low-risk active substance green channel" in 2024, granting eight patents for Bacillus velezensis formulations, significantly accelerating industrialization.

[0004] In agriculture and forestry, *Bacillus belyssiensis* has become a core alternative resource for reducing chemical pesticide use. In agriculture, registered crops include over 30 species such as rice, wheat, corn, tomato, pepper, and peanut, with its functional positioning expanding from simple fungicide application to multiple services including growth promotion, stress resistance, and phosphorus and potassium solubilization. While its application in forestry started later, *Bacillus belyssiensis* has seen rapid growth: in Northeast China's red pine forests, Southern China's banana plantations, and Southwest China's walnut forests, it has been used to control damping-off, root rot, and canker, significantly reducing the use of chemical fungicides. Formulations have also expanded from wettable powders and suspensions to storage-resistant microcapsules and dispersible oil suspensions, adapting to various scenarios such as aerial spraying, drip irrigation, and seed coating.

[0005] However, the large-scale promotion of Bacillus belyss in agriculture and forestry still faces three main bottlenecks. First, severe strain homogenization: over 70% of global patents and registered products are concentrated on a few type strains such as FZB42 and LSSC-5, leading to genetic uniformity in the field and an accumulation of resistance risks. Second, insufficient ecological adaptability: significant differences in soil organic matter and indigenous microbial communities across different climates cause existing commercial strains to experience an average colonization rate decrease of over 25% when transferred across regions, affecting the stability of their efficacy. Third, outdated large-scale production processes: high-density fermentation easily leads to problems such as low spore synchronization rate and decreased lipopeptide activity; simultaneously, the survival rate of formulations rapidly declines under storage and transportation conditions above 35℃. Furthermore, the lack of unified field trial standards and risk assessment systems prolongs the registration cycle and keeps promotion costs high. To overcome these challenges, it is urgent to expand and supplement with high-quality new strains, establish a precise strain screening platform based on whole-genome information, develop regionalized compound formulation technologies, and improve quality control standards throughout the entire process from fermentation to application.

[0006] The *Bacillus belyssus* LH-BLS012 of this invention was isolated from the roots of tropical plants in Bawangling, Hainan. The research team analyzed its entire genome, functional genes, and active metabolites. Compared with most strains isolated from soil, it exhibits better plant symbiosis, stronger heat and salt tolerance, and better antibacterial activity and life-promoting properties. Summary of the Invention

[0007] One of the objectives of this invention is to provide the natural strain of Bacillus belyss LH-BLS012 and its engineered strains through natural or artificial mutagenesis or gene editing.

[0008] The Bacillus velezensis of this invention was deposited on April 9, 2025, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 34146.

[0009] The taxonomic position of Bacillus belyssus LH-BLS012 was established in this invention. Purified single-clone cells were selected and cultured overnight in LB broth at 30°C and 180 rpm with shaking. The entire genome of LH-BLS012 was extracted from the fermentation broth. 16SRNA was amplified, with a size of 1486 bp (see attached image). Figure 1 Sequencing and sequence alignment confirmed that LH-BLS012 belongs to the taxonomic group *Bacillus velezensis*. (See appendix) Figure 2 ).

[0010] Primer sequences:

[0011] 27F: AGAGTTTGATCMTGGCTCAG

[0012] 1492R: GGTTACCTTGTTACGACTT

[0013] The present invention relates to the engineered strain of Bacillus belyssus LH-BLS012. Through natural or artificial means such as ultraviolet radiation, random mutations can be induced in the genome of the natural LH-BLS012 strain. Superior mutant strains with high lipopeptide metabolism content, faster reproduction, and stable traits are selected. Using technologies such as CRISPR, Base Editing, and Prime Editing, specific gene fragments or bases of LH-BLS012 are precisely edited to obtain engineered strains with greater phytochemical production, colonization ability, or life-promoting activity than the natural strain.

[0014] The second objective of this invention is to provide a novel antibacterial active ingredient from the metabolites of Bacillus belyssus LH-BLS012. The genome of a single colony of LH-BLS012 was extracted and subjected to third-generation whole-genome sequencing. The results showed that its genome consists of a circular chromosome and a large plasmid, with a total size of approximately 4,036,528 bp, containing nearly 3,900 coding genes (see appendix). Figure 3 AntiSMASH comparison analysis predicted that its antibacterial substances contain at least 13 active ingredients. Transatpks-Nrps, Bacteriocin-Nrps, and Thiopeptide showed high similarity to previously reported sequences; while the similarity of Thiopeptide and PKS-like gene sequences to other reported similar sequences was only 4% and 7%, respectively, and the similarity to surfactantind was also significantly different, indicating the presence of unique new active components. Proteins, peptides, lipopeptides, and other metabolites were isolated and collected using protein chromatography, ammonium sulfate precipitation, and methanol extraction. Antibacterial activity plate experiments identified lipopeptides as the main active substance. Further mass spectrometry analysis confirmed that the main antibacterial active substances were iturobrine, surfactantind with a novel structure, and Butirosin. These active ingredients can be obtained by directly obtaining crude extracts from fermentation broth, by separation with methanol or ammonium sulfate to obtain purer isolates, or by biosynthesis to obtain large quantities of highly pure active substances.

[0015] The third objective of this invention is to provide *Bacillus belyssiensis* LH-BLS012 microbial inoculant. A. Liquid inoculant: Purified single-clone cells are selected and cultured in LB broth at 30°C and 180 rpm with shaking. During the culture process, glucose and antifoaming agents are added, and the pH is adjusted until the spore concentration in the *Bacillus belyssiensis* fermentation broth reaches more than 10 billion CFU / mL. This original fermentation broth can be used directly as an inoculant, or the cell concentration can be adjusted according to the registered content and other auxiliary components can be added for use as a liquid bactericide or microbial fertilizer; B. Powdered inoculant: The original fermentation broth is initially concentrated and then dried using a spray drying tower or freeze-dried. Dry and process into powder. This is the original powder of the microbial agent. The concentration of the microbial cells can be adjusted according to the registered content, and other auxiliary ingredients such as humic acid and fulvic acid can be added to use it as a powdered fungicide or microbial fertilizer. C. Granular microbial fertilizer: Mix the original powder of Bacillus vesiculosus with humic acid, fulvic acid and other auxiliary ingredients in a ratio of 1:100-1:10, and granulate it through processes such as disc or spraying. The resulting granular product, ranging in size from rapeseed to mung bean, is microbial fertilizer (the specific concentration of active ingredients in the final product should not be lower than the microbial cell concentration content specified in the registration).

[0016] Another objective of this invention is to provide a method for applying Bacillus vesiculosus LH-BLS012 microbial inoculant in the biocontrol of plant diseases and the promotion of plant growth. For the control of foliar diseases or the promotion of foliar growth, LH-BLS012 fermentation broth or compound preparations, or LH-LS012 powder, can be diluted with water and applied as a spray. For the control of root diseases, the promotion of root growth, or the conditioning and improvement of soil microbial flora, fermentation broth, compound solutions, or powder can be diluted with water for root irrigation. Granular inoculants are typically applied as base fertilizer or top dressing. However, regardless of whether it is used for aboveground spraying or root irrigation, the final concentration of LH-BLS012 inoculant should be maintained at 100 million CFU / mL or higher. Attached Figure Description Figure 1 Electrophoresis diagram of 16S rRNA gene amplification of Bacillus belyssus LH-BLS012. The left image shows MK; the right image shows the 1486bp amplified fragment of Bacillus belyssus LH-BLS012. Figure 2 The 16S rRNA gene sequence of Bacillus belyssus LH-BLS012. Figure 3 The comparison results with known Bacillus velezensis strains confirmed its taxonomic status as Bacillus velezensis. Figure 4 The complete genome map of Bacillus belyssus LH-BLS012. Figure 5A diagram of active metabolites of Bacillus vesiculosus LH-BLS012g, with the locations of the biosynthetic gene clusters of the main antibacterial active substances marked. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to Examples 1-7.

[0018] Examples are given to illustrate the present invention, but the present invention is not limited to the following embodiments.

[0019] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0020] Example 1: Inhibitory effect of Bacillus belye LH-BLS012 on plant pathogenic fungi

[0021] I. Experimental Materials and Methods

[0022] 1. Experimental Materials

[0023] The tested pathogens were: Fusarium solani (ginger-leaf rot fungus), Calonectriailicicola (lychee-leaf rot fungus), and Coniella quercicola (eucalyptus-leaf rot fungus).

[0024] Test reagents: difenoconazole, 95% purity, commercially available; Bacillus vesiculosus CGMCC No. 14384, commercially available; Bacillus vesiculosus LH-BLS012, proprietary strain.

[0025] 2. Experimental Methods

[0026] Reagent preparation: Prepare a 10ppm solution for later use; CGMCC No.14384, LH-BLS012, LB culture medium, 30℃, 180rpm shaking culture for 72h, and filter out all bacterial cells from the fermentation broth for later use.

[0027] Treatment setup: Difenoconazole was added to PDA medium to prepare plates with a final concentration of 1000 ppm (diluted 100,000 times). Fermentation filtrate of CGMCC No. 14384 and LH-BLS012 was diluted to prepare plates with a final concentration diluted 100 times. A blank control was also set up. Each treatment was repeated in 4 replicates, with 4 plates per replicate. After inoculation with the pathogenic fungus, the plates were cultured until the colony diameter of the blank control reached approximately 80% of the plate's surface area before investigation.

[0028] Data collection: The diameter of each plate was measured, the average value was taken, and the inhibition rate was calculated. Inhibition rate (%) = (CK colony diameter - treated colony diameter) / CK colony diameter × 100%.

[0029] II. Experimental Results

[0030] The survey results (as shown in Table 1) indicate that the positive control chemical agent difenoconazole (1000 ppm) had inhibition rates of 93.01%, 93.56%, and 92.49% against Fusarium solani, Lychee erythroderma, and Eucalyptus necrophorum, respectively; the positive control biological agent CGMCC No. 14384 had inhibition rates of 73.03%, 47.83%, and 38.01% against Fusarium solani, Lychee erythroderma, and Eucalyptus necrophorum, respectively; and the inhibition rates of HL-BLS012 were 78.72%, 59.86%, and 58.97%, respectively. Although the inhibition rate of Bacillus belyceta HL-BLS012 was significantly lower than that of the control chemical agent, it still showed better inhibition rates against Fusarium solani, Lychee erythroderma, and Eucalyptus globulus under the same conditions compared to the commercially available CGMCC No.14384 agent, with increases of 5.69, 12.03, and 20.96 percentage points respectively. This demonstrates its superior potential for antibacterial application.

[0031] Table 1. Survey on the inhibitory effect of LH-BLS012 on plant pathogenic fungi.

[0032]

[0033] Example 2: Colonization of Bacillus belye LH-BLS012 in plant roots

[0034] I. Experimental Materials and Methods

[0035] 1. Experimental materials

[0036] The tested strain was Bacillus belyssus HL-BLS012, labeled with GFP (fluorescent green protein); the tested crop was pepper; the tested culture media were LB solid medium and LB liquid medium; the tested reagents included Tween 80; the tested instruments included pipettes, filter paper, electronic balance, autoclave, laminar flow hood, ultrasonic cleaner, and mortar.

[0037] 2. Experimental Methods

[0038] After being disinfected with sodium hypochlorite, chili seeds are sown in seedling trays using sterile sandy loam soil that has been sterilized at high temperature. When the chili seedlings have 3-4 true leaves, they are transplanted into flowerpots with a diameter of 13.5cm filled with sterile soil.

[0039] GFP-HL-BLS012 fermentation broth was prepared by shaking LB culture medium at 30℃ and 180rpm for 72h. After the pepper seedlings were transplanted and survived, the bacterial solution was used for root irrigation at a rate of 10mL per pot.

[0040] Starting from the first day after root irrigation treatment, samples were taken every 7 days, and tests were conducted 14 times over a period of 3 months at days 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, and 91 to clarify the colonization of GFP-HL-BLS012 in the rhizosphere soil and roots of chili peppers.

[0041] Sampling and testing methods: Pour out all the soil from the flowerpot and mix thoroughly. Measure 100 mL of the mixture and add it to a final volume of 200 mL of sterile water containing 1% Tween 80. Sonicate for 5 minutes, mix thoroughly, dilute, and spread on plates. Analyze the colonization of bacteria in the root zone. Remove the entire chili pepper root, shake off and wash the soil from the root surface, weigh, and grind thoroughly in a sterile mortar. Dilute with sterile water containing 1% Tween 80 and spread on plates. Analyze the colonization of bacteria in the roots.

[0042] II. Results and Analysis

[0043] The colonization dynamics of LH-BLS012 in soil and roots are shown in Table 2 below.

[0044] Table 2. Survey of the colonization dynamics of LH-BLS012 in soil and roots.

[0045]

[0046] The results showed that Bacillus belye LH-BLS012 could successfully colonize both the rhizosphere soil and roots of chili peppers, and the colonization dynamics showed obvious temporal variation patterns, with significant differences in inoculum at different stages (P < 0.05).

[0047] In the rhizosphere soil, the colonization of microorganisms reached its highest value at the initial stage of treatment (1 day), at 398.0 ± 7.8 × 10⁻⁶. 6 CFU / g soil was significantly higher than at other times (marked as days); then it dropped sharply to 38.0 ± 1.7 × 10⁻⁶ at 7 days. 6 The CFU / g soil (labeled as ab) concentration fluctuated over 14–91 days. A rebound occurred at day 35, reaching 25.7 ± 2.6 × 10⁻⁶. 6 CFU / g soil (labeled as a) was significantly higher than at 21 days (95.7 ± 3.8 × 10⁻⁶). 4 CFU / g soil (labeled as c), 63d (81.0 ± 1.5 × 10⁻⁶) 4 CFU / g soil, marked as c), etc.; up to 91 days, 53.3 ± 4.3 × 10⁻⁶ CFU / g soil was still detectable in the rhizosphere soil. 4 The presence of live bacteria at CFU / g in the soil indicates that this strain can achieve long-term colonization in the rhizosphere soil.

[0048] In the root tissue, the colonizing microbial population generally showed a trend of "increasing - peaking - decreasing". One day after treatment, the microbial population was 228.0 ± 5.1 × 10⁻⁶. 5 CFU / g root, then gradually increased, reaching 82.7 ± 6.8 × 10 at 21 days. 6 CFU / g root (labeled as b), further increased to 100.7 ± 0.9 × 10⁻⁶ after 28 days. 6 CFU / g root (labeled c), this stage is the peak period of root colonization; although the inoculum decreased after 35 days, it still remained at a high level (81.7 ± 1.8 × 10⁻⁶). 6 CFU / g root (labeled b); up to 91 days, 75.7 ± 4.7 × 10 CFU / g root was still detectable in the root. 4 The CFU / g of viable bacteria in the roots (labeled as b) was significantly higher than that at 63 days (34.3 ± 2.7 × 10⁻⁶). 5 CFU / g root, labeled as a), 70d (40.0 ± 3.2 × 10) 5 CFU / g root, marked as a) etc.

[0049] In summary, Bacillus belye LH-BLS012 can not only colonize the rhizosphere soil of chili peppers, but also effectively invade and colonize the root tissues. Moreover, its colonization in the roots is more persistent and the peak inoculum is higher. This provides an important microbiological basis for its biocontrol or growth-promoting effects by occupying an ecological niche for a long time.

[0050] Example 3: Biocontrol effect of Bacillus belyceta var. var. LH-BLS012 against boxwood powdery mildew.

[0051] I. Experimental Materials and Methods

[0052] 1. Test materials

[0053] The control agent was triadimefon, 20% concentration, commercially available; the strain was Bacillus berberis HL-BLS012, a proprietary strain; the test crop was Buxus macrocarpa; and the target disease was powdery mildew.

[0054] 2. Test methods

[0055] The experiment was conducted on large-leaved boxwoods with relatively uniform powdery mildew incidence, cultivated by workers at the Beijing Zoo. A blank control (no treatment) was set up, while a positive control was established using triadimefon at the recommended concentration. Treatments were performed with HL-BLS012 bacterial suspension at final concentrations of 0.1, 1, and 1 billion CFU / mL. Each treatment was replicated four times, with three boxwood plants per replicate. The solution was sprayed evenly on both sides of the leaves until it just dripped. Starting with the first application, the solution was applied every seven days for a total of three applications.

[0056] The investigation began 7 days after the last application of the pesticide. Grading standards: Grade 0: 0 diseased leaves, no disease in the plant; Grade 1: less than 10% of the total number of leaves, mild disease; Grade 2: 11%-30% of the total number of leaves, moderate disease; Grade 3: 31%-50% of the total number of leaves, severe disease; Grade 4: more than 51% of the total number of leaves, severe disease. Disease index (%) = ∑(Grade number × Number of plants in the same grade) / (Total number of plants × 4) × 100%. Control effect (%) = (Disease index of blank control group – Disease index of experimental group) / Disease index of blank control group × 100%.

[0057] II. Results and Analysis

[0058] The results of the experiment are shown in Table 3 below.

[0059] Table 3. Efficacy of LH-BLS012 against Boxwood Powdery Mildew

[0060]

[0061] The experimental results show that:

[0062] Differences in Disease Index Among Different Treatments: Significant differences were found in the disease index of boxwood powdery mildew across the treatment groups. The control group (CK) had the highest average disease index, reaching 69.89±2.10%, indicating severe boxwood powdery mildew incidence under natural conditions. Among the drug and bacterial suspension treatment groups, treatment with different concentrations of Bacillus belycei LH-BLS012 bacterial suspension and triadimefon treatment effectively reduced the disease index. Specifically, the 1 billion CFU / mL LH-BLS012 bacterial suspension treatment group had the lowest average disease index at 14.89±2.91%; followed by the triadimefon treatment group at 20.28±2.91%; the 100 million CFU / mL bacterial suspension treatment group had an average disease index of 32.81±3.71%; and the 100 million CFU / mL bacterial suspension treatment group had a relatively high average disease index of 54.78±3.06%. From the statistical differences of the data, the average disease index of the 1 billion CFU / mL bacterial suspension treatment group and the triadimefon treatment group was at the same statistical level (both c), which was significantly lower than that of the 0.1 billion CFU / mL and 1 billion CFU / mL bacterial suspension treatment groups (both b). The 0.1 billion CFU / mL and 1 billion CFU / mL bacterial suspension treatment groups were significantly lower than the blank control group (a).

[0063] Comparison of control effects of different treatments: The control effects of different treatments on boxwood powdery mildew also showed significant differences. The 1 billion CFU / mL LH-BLS012 bacterial suspension treatment group had the best control efficacy, reaching 78.70%; the triadimefon treatment group had a control efficacy of 70.97%; the 100 million CFU / mL bacterial suspension treatment group had a control efficacy of 53.06%; and the 10 million CFU / mL bacterial suspension treatment group had a relatively low control efficacy of 21.62%. It is worth noting that the 1 billion CFU / mL bacterial suspension treatment group had a slightly higher control efficacy than the traditional chemical agent triadimefon treatment group, demonstrating excellent biocontrol potential. Although the 100 million CFU / mL bacterial suspension treatment group had a lower control efficacy than the triadimefon treatment group, it still reached a moderately high level of control efficacy. The 10 million CFU / mL bacterial suspension treatment group had a relatively low control efficacy, but it could still inhibit the development of the disease to a certain extent.

[0064] Relationship between bacterial concentration and control efficacy: Treatment results with different concentrations of *Bacillus vesiculosus* LH-BLS012 bacterial suspensions showed that within the experimental concentration range (0.1-1 billion CFU / mL), the control efficacy exhibited a significant upward trend with increasing suspension concentration. At a concentration of 0.1 billion CFU / mL, the control efficacy was only 21.62%; when the concentration was increased to 100 million CFU / mL, the control efficacy increased significantly to 53.06%; and when the concentration was further increased to 1 billion CFU / mL, the control efficacy reached 78.70%, achieving a significant leap in efficacy. This result indicates that *Bacillus vesiculosus* LH-BLS012 has a significant concentration effect on the control of boxwood powdery mildew, with higher concentrations of bacterial suspensions more effectively controlling the disease.

[0065] In summary, Bacillus berreatus LH-BLS012 exhibits good biocontrol efficacy against boxwood powdery mildew, especially at a concentration of 1 billion CFU / mL, where its efficacy is superior to or comparable to that of the traditional chemical agent triadimefon, demonstrating its potential as a substitute for chemical agents in the control of boxwood powdery mildew. Furthermore, in practical applications, spraying with a bacterial solution at a concentration of 100 million CFU / mL or higher can be considered to achieve better control results.

[0066] Example 4: Biocontrol effect of Bacillus belyceta var. berberis LH-BLS012 against eucalyptus scorch disease

[0067] I. Experimental Materials and Methods

[0068] 1. Test materials

[0069] The control agent was benzoyl pyrazole ester, 40% content, commercially available; Bacillus berberis HL-BLS012 strain was a proprietary strain; the test plant was eucalyptus; the target disease was leaf blight.

[0070] 2. Test methods

[0071] A pot experiment was conducted in a greenhouse in Beijing. Eucalyptus seedlings of uniform growth were selected and uniformly inoculated with conidia of *Calonectria ilicicola*, the pathogen causing eucalyptus scorch disease. The seedlings were cultured under moist conditions until leaf symptoms appeared. A blank control (no treatment) was set up, and benzoyl pyrazolite was used as a positive control at the recommended concentration. Three treatments were applied: HL-BLS012 bacterial suspension at final concentrations of 0.1, 1, and 1 billion CFU / mL. Each treatment was replicated four times, with three eucalyptus plants per replicate. The solution was sprayed evenly on both sides of the leaves until just dripping. Starting with the first application, applications were made every seven days, for a total of three applications.

[0072] The investigation begins 7 days after the last application of the pesticide. Grading standards: Grade 0 (No disease): No symptoms of disease are observed on the leaves or young shoots; leaves are normal in color (dark green or emerald green), without yellowing, curling, or necrosis; young shoots are vigorous, without wilting or browning. Grade 1 (Mild disease): Disease is mainly concentrated on the lower, older leaves or a small number of young shoots; ≤10% of the total number of leaves are affected, manifesting as yellowish-brown necrotic spots on the leaf edges or in localized areas, without obvious curling or shedding; ≤5% of young shoots are affected, with only a few shoot tips showing slight browning or wilting, not affecting the overall growth of the plant. Level 2 (Moderate Disease): The disease spreads to the lower and middle leaves and some tender shoots, affecting 11%-30% of the total number of leaves. Large areas of yellowish-brown to brown necrotic spots appear on the diseased leaves, some leaves curl and scorch, and a small number of leaves fall off. The disease incidence rate on tender shoots is 6%-20%, with the browning length not exceeding 1 / 3 of the shoot length. Some tender shoots stop growing, but healthy new shoots still appear on the upper part of the plant. Level 3 (Severe Disease): The disease spreads to the upper and middle leaves and most tender shoots, significantly worsening the condition. 31%-50% of the total number of leaves are affected, with a large number of leaves scorched, curled, and falling off. "Bare" branches appear on the lower and middle parts of the plant. The disease incidence rate on tender shoots is 21%-50%, with most tender shoots showing browning exceeding 1 / 3 of their length. Some tender shoots die, and plant growth is significantly inhibited, with weakened apical dominance. Level 4 (severe disease): The entire plant is severely affected, with almost no healthy leaves or effective tender shoots. Diseased leaves account for ≥51% of the total number of leaves on the plant. Leaves wither and fall off over a large area, leaving only a few top leaves or no leaves at all. The proportion of tender shoots affected is ≥51%, with most tender shoots dying. The browning of branches extends to the main trunk, plant growth stops, and the entire plant may even be on the verge of death.

[0073] Disease index (%) = ∑(Grade number × Number of plants in the same grade) / (Total number of plants × 4) × 100%. Control effect (%) = (Disease index of blank control group – Disease index of experimental group) / Disease index of blank control group × 100%.

[0074] II. Results and Analysis

[0075] The results of the experiment are shown in Table 4 below.

[0076] Table 4. Control efficacy of LH-BLS012 against eucalyptus scorch.

[0077]

[0078] The experimental results showed that there were significant differences in the control effects of different treatment groups on eucalyptus scorch disease. The control group (CK) had the highest average disease index, reaching 81.10±1.64%, and the disease index of each replicate was above 79%, indicating that eucalyptus scorch disease was extremely severe and posed a serious threat to plant growth when no control measures were taken.

[0079] Treatment with different concentrations of Bacillus belyceta LH-BLS012 bacterial suspension all showed certain control effects, exhibiting a significant concentration effect. Specifically, the average disease index in the 0.1 billion CFU / mL treatment group was 54.16±2.07%, with a control efficacy of 33.22%; the average disease index in the 1 billion CFU / mL treatment group decreased to 38.10±1.32%, while the control efficacy increased to 53.03%, nearly 20 percentage points higher than the 0.1 billion CFU / mL treatment group; the 1 billion CFU / mL treatment group showed the best control efficacy, with an average disease index of only 25.52±3.52% and a control efficacy as high as 68.53%.

[0080] The average disease index of the positive control group treated with benzoylpyrazole was 26.99±1.84%, with a control efficacy of 66.73%. Statistically, the 1 billion CFU / mL bacterial suspension treatment group and the benzoylpyrazole treatment group both belonged to grade d, with no significant difference. Notably, the average disease index of the 1 billion CFU / mL bacterial suspension treatment group was slightly lower than that of the chemical control group, and the control efficacy was also slightly higher, fully demonstrating the excellent biocontrol potential of this strain and making it an effective alternative for the green control of eucalyptus scorch disease.

[0081] Example 5: Biocontrol effect of Bacillus belyceta var. berberis LH-BLS012 against rice false smut.

[0082] I. Experimental Materials and Methods

[0083] 1. Test materials

[0084] The control agent was tebuconazole, 430 g / L, commercially available; the strain was Bacillus berberis HL-BLS012, a proprietary strain; the test plant was rice; and the target disease was rice false smut.

[0085] 2. Test methods

[0086] The experiment was conducted at Hongxin Farm, Hongxin Town, Chuzhou City, Anhui Province. Fields with a history of severe and relatively uniform rice false smut infection were selected for the trial. A blank control without any treatment was set up, and tebuconazole was used as a positive control at the recommended concentration. Three treatments were applied: HL-BLS012 bacterial suspension at final concentrations of 0.1, 1, and 1 billion CFU / mL. Each treatment was replicated four times, with each replicate measuring 30 square meters. The solution was sprayed evenly on both sides of the plant leaves until it just dripped. Starting with the first application, applications were made every seven days, for a total of three applications.

[0087] The investigation begins 7 days after the last application of pesticide. Grading standards: Grade 0: No rice grains with blast disease on the entire panicle; grains are normally developed, without deformities, discoloration, or mycelial clumps; plant growth is good. Grade 1: Mild disease, with diseased grains accounting for less than 5% of the total. Only a few small blasted grains appear on the panicle, with minimal impact on the overall appearance and grain filling; most grains still mature normally. Grade 2: Moderate disease, with diseased grains accounting for 6%-15% of the total. Multiple blasted grains are visible in the lower part of the panicle; some blasted grains enlarge and begin to compress surrounding normal grains, causing some normal grains to be hindered in development or fall off, but the overall grain filling rate of the plant remains high. Grade 3: Severe disease, with diseased grains accounting for 16%-30% of the total. The number of rice blast balls on the rice panicle increases significantly, expanding to the middle and upper parts of the panicle. Most of these blast balls are large, significantly compressing normal grains and causing many normal grains to become deformed, shriveled, or fall off, significantly impacting yield. Level 4 indicates severe disease, with diseased grains accounting for more than 30% of the total grains. The entire panicle is covered with blast balls, leaving very few normal grains. The blast balls often cluster together, increasing panicle weight, increasing lodging susceptibility, and drastically reducing the seed setting rate, severely affecting yield and quality.

[0088] Disease index (%) = ∑(Grade number × Number of plants in the same grade) / (Total number of plants × 4) × 100%. Control effect (%) = (Disease index of blank control group – Disease index of experimental group) / Disease index of blank control group × 100%.

[0089] II. Results and Analysis

[0090] The results of the experiment are shown in Table 5 below.

[0091] Table 5. Control efficacy of LH-BLS012 against rice false smut.

[0092]

[0093] The experimental results show that:

[0094] Different treatments showed significant differences in their control effects on rice false smut. Bacillus berberis LH-BLS012 bacterial suspension had a clear biocontrol effect on rice false smut, and the control efficacy showed a regular change with increasing concentration.

[0095] From the perspective of disease index, the average disease index of the blank control group (CK) was the highest, reaching 66.10±4.31%, and the disease index of each replicate was above 60%, indicating that the natural incidence of rice false smut in the experimental field was relatively severe, providing a reliable background for evaluating the control effect.

[0096] Among the treatment groups, the average disease index of the 0.1 billion CFU / mL LH-BLS012 bacterial suspension treatment group was 43.91±4.53%, with a control efficacy of 33.57%. Although it could inhibit the disease to some extent, the control effect was relatively limited. When the bacterial suspension concentration was increased to 100 million CFU / mL, the average disease index decreased to 32.26±0.83%, and the control efficacy increased to 51.2%, showing a trend of increasing control efficacy with increasing concentration. The control efficacy of the 1 billion CFU / mL bacterial suspension treatment group was further improved, with an average disease index of 25.30±3.24% and a control efficacy of 61.72%, significantly better than the low concentration treatment group.

[0097] The average disease index of the positive control group treated with benzoylpyrazole was 21.35±2.82%, with a control efficacy of 67.7%, demonstrating good chemical control effects. Statistical analysis showed that the 100 million CFU / mL and 1 billion CFU / mL bacterial suspension treatment groups, along with the benzoylpyrazole treatment group, belonged to grade c, significantly lower than the 0.1 billion CFU / mL bacterial suspension treatment group (grade b) and the blank control group (grade a), indicating that high-concentration bacterial suspension treatment and chemical treatment were at the same level in controlling the disease index.

[0098] It is worth noting that while the control efficacy of the 1 billion CFU / mL bacterial suspension treatment (61.72%) was slightly lower than that of benzoylpyrazole (67.7%), the difference was small, and this concentration of bacterial suspension effectively controlled the disease index at a low level. In summary, Bacillus berberis LH-BLS012 exhibits a significant concentration effect in the control of rice false smut, with the 1 billion CFU / mL concentration treatment demonstrating efficacy approaching that of chemical agents, indicating its potential application as a green control technology for rice false smut.

[0099] Example 6: Growth-promoting and yield-increasing effects of Bacillus belye LH-BLS012 on rice.

[0100] I. Experimental Setup

[0101] The experiment was conducted from May to October 2024 in Zhetang Village, Fengyang County, Anhui Province. Rice paddies with high production levels and relatively uniform growth in previous years were selected. The experimental fields totaled 20 mu (approximately 1.3 hectares), of which 10 mu (approximately 0.67 hectares) served as a conventional production control, grown entirely according to previous years' pesticide, fertilizer, and water application patterns. The other 10 mu (approximately 0.67 hectares) were treated with Bacillus thuringiensis microbial fertilizer, with HL-BLS012 microbial fertilizer granules evenly spread into the field at a dosage of 10 kg / mu (approximately 0.67 hectares) at rice transplanting. Apart from this, the two fields were identical. Subsequent field management, agricultural practices, and all operations related to water, fertilizer, and pesticide application were performed in the same manner.

[0102] When harvesting rice, the two fields are harvested separately, and the yield is measured.

[0103] II. Results and Analysis

[0104] Field harvest data at the end of October showed that the conventional production field yielded 16,675 jin (approximately 8,883 catties), equivalent to 1,667.5 jin (approximately 8,883 catties) per mu (approximately 0.067 hectares); the field treated with HL-BLS012 microbial fertilizer yielded 20,250 jin (approximately 10,867 catties), equivalent to 2,025 jin (approximately 1,025 kg) per mu (approximately 0.067 hectares). The average yield increase was 357.5 jin (approximately 178.5 catties) per mu, representing a yield increase of 21.4%.

[0105] The HL-BLS012 microbial fertilizer treatment added extra costs to the conventional agricultural production inputs. According to the experimental setup, using 10 kg of HL-BLS012 microbial fertilizer per mu (approximately 0.067 hectares) incurs a cost of about 50 yuan. The total cost of microbial fertilizer for a 10-mu experimental field is 50 × 10 = 500 yuan. In 2024, the national average purchase prices for early indica rice, medium and late indica rice, and japonica rice were 2808 yuan / ton, 2850 yuan / ton, and 2809 yuan / ton, respectively. For ease of calculation, the average value is taken as (2808 + 2850 + 2809) ÷ 3 = 2822.33 yuan / ton, which translates to approximately 1.41 yuan per jin (approximately 0.5 kg). The conventional production field yielded 16675 jin (approximately 838.85 catties), which, calculated at 1667.5 jin (approximately 838.85 catties) per mu, results in a total revenue of 16675 × 1.41 = 23511.75 yuan. The field treated with HL-BLS012 microbial fertilizer yielded 20,250 jin (approximately 10,165 catties), or 2,025 jin (approximately 1,025 kg) per mu (approximately 0.067 hectares), resulting in a total revenue of 20,250 × 1.41 = 28,552.5 yuan. The microbial fertilizer-treated field yielded an additional 5,040.75 yuan compared to conventionally grown fields, a difference of 28,552.5 - 23,511.75 = 5,040.75 yuan. After deducting the total cost of 500 yuan for the 10 mu of microbial fertilizer, the actual increase in revenue due to its use was 4,540.75 yuan, averaging 454.08 yuan per mu, representing a 19.31% increase per mu. From an input-output perspective, every 50 yuan invested in microbial fertilizer resulted in an additional 454.08 yuan in revenue, achieving an input-output ratio of 1:9.08. Even under the national average rice price system, the use of Bacillus baijis HL-BLS012 microbial fertilizer still brings significant economic benefits to farmers.

[0106] Example 7: Growth-promoting and yield-increasing effects of Bacillus belyssus LH-BLS012 on wheat.

[0107] I. Experimental Setup

[0108] The experiment was conducted from November to May 2024 in Damiao Town, Fengyang County, Anhui Province. Wheat fields with high production levels and relatively uniform growth in previous years were selected. The experimental field comprised 10 mu (approximately 1.65 acres), of which 5 mu (approximately 0.8 acres) served as a conventional production control, grown entirely according to previous years' pesticide, fertilizer, and water application patterns. The other 5 mu (approximately 0.85 acres) were treated with Bacillus thuringiensis microbial fertilizer, with HL-BLS012 microbial fertilizer granules evenly mixed into the field at a dosage of 10 kg / mu (approximately 1.65 acres) before wheat sowing. Apart from this, the two fields were identical. Subsequent field management, agricultural practices, and all operations related to water, fertilizer, and pesticide application were performed in the same manner.

[0109] When the wheat is harvested, the two fields are harvested separately and the yield is measured.

[0110] II. Results and Analysis

[0111] Field harvest data at the end of May showed that the conventional production field yielded 6107.5 jin (approximately 345.75 catties), equivalent to 1221.5 jin (approximately 614.75 kg) per mu (approximately 0.067 hectares); the field treated with HL-BLS012 microbial fertilizer yielded 7617.5 jin (approximately 334.75 catties), equivalent to 1523.5 jin (approximately 761.75 catties) per mu (approximately 0.067 hectares). The average yield increase was 302 jin (approximately 150.75 catties) per mu, representing a yield increase of 24.7%.

[0112] The HL-BLS012 microbial fertilizer treatment field incurred additional costs on top of conventional agricultural inputs. According to the experimental plan, 10 kg of HL-BLS012 microbial fertilizer was used per mu (approximately 0.067 hectares), costing about 50 yuan. The total cost of microbial fertilizer for a 5-mu experimental field was 50 × 5 = 250 yuan. The national average wheat price in the first half of 2025 was 2429 yuan / ton, equivalent to approximately 1.21 yuan per jin (approximately 0.5 kg). The conventional production field yielded 6107.5 jin (approximately 345.75 kg), which, calculated at 1221.5 jin (approximately 614.75 catties) per mu, yielded a total revenue of 6107.5 × 1.21 = 7390.075 yuan. The HL-BLS012 microbial fertilizer treatment field yielded 7617.5 jin (approximately 334.75 catties), which, at 1523.5 jin (approximately 771.75 catties) per mu, yielded a total revenue of 7617.5 × 1.21 = 9217.175 yuan. The field treated with microbial fertilizer yielded an additional 1827.1 yuan (9217.175 - 7390.075 = 1827.1 yuan) compared to conventionally grown fields. After deducting the total cost of 250 yuan for the microbial fertilizer on 5 mu (approximately 0.33 hectares), the actual increase in income due to the use of microbial fertilizer was 1577.1 yuan, averaging 315.42 yuan per mu, representing an average increase of 21.34% per mu. From an input-output ratio perspective, every 50 yuan invested in microbial fertilizer resulted in an additional 315.42 yuan in revenue, resulting in an input-output ratio of 6.3:1. This indicates that, at the wheat price level in the first half of 2025, the use of Bacillus baijis HL-BLS012 microbial fertilizer can significantly improve the economic benefits for farmers.

[0113] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. The application of Bacillus belye LH-BLS012 in the biological control and growth promotion of plant diseases, characterized in that, This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) under the number CGMCC No. 34146 and is used for the biological control of plant diseases and the promotion of plant growth.

2. As described in claim 1, the application of Bacillus belyss LH-BLS012 includes not only its direct fermentation culture, bacterial powder and / or secondary metabolites made from the fermentation broth, but also fermentation broth, bacterial powder and / or secondary metabolites of engineered bacteria modified through natural mutagenesis or gene editing such as CRISPR / Base Editing / Prime Editing.

3. As described in claim 1, the plant diseases are selected from fungal diseases of forest plants, fungal diseases of landscaping plants, and fungal diseases of crops. Promoting plant growth mainly includes applications in promoting the growth of forest trees, landscaping plants, and crops.

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