Lysinibacillus fusiformis KBD-5 antagonizing botrytis cinerea and application of lysinibacillus fusiformis KBD-5

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses lysinibacillus fusiformis KBD-5 for antagonizing botrytis cinerea and application of the lysinibacillus fusiformis KBD-5. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) Whole genome (accession number PRJNA1335659) sequencing reveals that the strain has a triple synergistic antagonistic mechanism for the first time: the strain contains a fengycin-like lipopeptide synthetic gene cluster to destroy pathogenic bacteria cell membranes, and contains GH18 chitinase and GH5 cellulase genes to degrade pathogenic bacteria cell walls. The inhibition rate of the strain on botrytis cinerea reaches 76.42%; the tomato fruit test morbidity is reduced to 25.9%, and the rotting index is reduced by 75.0%; the control effect of greenhouse potting reaches 72.35%, and the control effect is not obviously different from that of procymidone. Biological safety assessment shows that no toxicity factor exists, and no drug-resistant gene horizontal transfer risk exists. The invention further provides a microbial preparation containing the KBD-5 and application of the microbial preparation.

Description

A spindle-shaped lysine-containing Bacillus fusiforme KBD-5 antagonistic to Botrytis cinerea and its application Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a spindle-shaped lysine-containing Bacillus fusiforme KBD-5 that antagonizes Botrytis cinerea and its applications. Background Technology

[0002] Botrytis cinerea, a typical necrotrophic plant pathogen, can widely infect various vegetables, fruits, and ornamental crops such as tomatoes, strawberries, and grapes, causing gray mold. This results in significant pre-harvest yield reductions and post-harvest storage rot, causing enormous economic losses to global agricultural production every year. Currently, the control of gray mold in agricultural production still heavily relies on chemical fungicides. However, long-term, single-agent use of chemical agents has not only led to severe drug resistance in Botrytis cinerea, causing a continuous decline in control effectiveness, but has also caused a series of problems such as soil and water pollution and excessive pesticide residues in agricultural products, seriously hindering the green and sustainable development of agriculture.

[0003] Biological control using beneficial microorganisms and their metabolites has become an important development direction for the green control of gray mold due to its advantages such as environmental friendliness, low likelihood of developing drug resistance, and no negative impact on agricultural product safety. Bacillus bacteria, with their strong resistance, rapid reproduction, abundant metabolites, and ease of industrial development, have become a core resource for the research and development of biocontrol agents. *Lysinibacillus fusiformis* is a widely distributed Bacillus bacteria found in natural environments such as soil and hot springs. Current research on *Lysinibacillus fusiformis* mainly focuses on heavy metal degradation, wastewater purification, and broad-spectrum weak inhibition of common plant saprophytic bacteria. It has not been included in the screening and research scope of biocontrol bacteria for gray mold. No strains of this species have been found to have specific antagonistic activity against *Botrytis cinerea*, and there is a lack of quantitative antibacterial data and verification data on the actual control effect on gray mold.

[0004] Therefore, isolating a new strain of *Bacillus fusiformis* antagonistic to *Botrytis cinerea* with a significant antagonistic effect, a clear biocontrol mechanism, and high biosafety, systematically analyzing its genomic characteristics and biocontrol genetic basis, developing corresponding microbial biocontrol agents, and clarifying its application scheme in the control of gray mold are of great significance and application value. This is crucial for filling the technological gap in the field of biocontrol of gray mold with *Bacillus fusiformis* antagonistic to *Botrytis cinerea*, solving the technical pain points of existing gray mold control, and promoting the development of green control technologies for gray mold. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a spindle-shaped lysine-containing Bacillus lysine KBD-5 that antagonizes Botrytis cinerea and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: On the one hand, the present invention provides a strain of Lysinibacillus fusiformis KBD-5 that antagonizes Botrytis cinerea. This strain was isolated from hot spring soil in Tengchong County, Baoshan City, Yunnan Province, and was deposited at the China General Microbiological Culture Collection Center on November 14, 2022, with the accession number CGMCC No. 26146.

[0007] Furthermore, the complete genome sequence of the aforementioned antagonistic *Botrytis cinerea* KBD-5 contains the nucleotide sequence shown in accession number PRJNA1335659.

[0008] Furthermore, the genome of the aforementioned antagonistic *Botrytis cinerea* KBD-5 contains a cluster of genes related to the synthesis of secondary metabolites associated with fengycin-like lipopeptide synthesis, located at Chr1:415,594-439,817 in its whole genome sequence.

[0009] Furthermore, the genome of the aforementioned antagonistic *Botrytis cinerea* KBD-5 contains GH18 family chitinase genes and GH5 family cellulase genes.

[0010] Furthermore, the genome size of the aforementioned antagonistic *Botrytis cinerea* KBD-5, a lysine-containing bacterium fusiforme, is 4,692,552 bp, with a GC content of 37.28%, and a total of 4,719 protein-coding genes (CDS) were predicted.

[0011] Further bioinformatics analysis revealed the genetic basis of its biocontrol potential: 1. Secondary metabolite synthesis gene clusters: Eight secondary metabolite synthesis gene clusters were predicted, covering types such as beta-actone, NRPS (non-ribosomal peptide synthase), and T3PKS (type III polyketide synthase). Among them, Cluster 1 (betalactone) showed 46% similarity to the fengycin (a fungicide) synthesis gene cluster, which has strong antifungal activity; 2. Carbohydrate-active enzymes: 195 carbohydrate-active enzyme (CAZymes) genes were annotated, including 74 glycoside hydrolases (GH). Among them, GH18 family chitinases and GH5 family cellulases can work synergistically to degrade chitin and cellulose, key components of the fungal cell wall; 3. Biosafety: Virulence factor (PHI) database annotation showed that the vast majority of genes were attenuated or lost in virulence, and no known core virulence genes such as hemolysins and enterotoxins were detected. Analysis of the antibiotic resistance database (CARD) revealed only 7 chromosome-encoded resistance genes, with no plasmid-mediated resistance genes, indicating a low risk of environmental release.

[0012] Furthermore, the aforementioned antagonistic *Botrytis cinerea* fusiforme lysine-containing *Bacillus fusiforme* KBD-5 colonies on LB agar plates were pale yellow, nearly round, and smooth. Using the plate confrontation method, this strain showed a 76.42% inhibition rate on mycelial growth against the tomato gray mold pathogen *Botrytis cinerea* H1 strain.

[0013] On the other hand, the present invention provides a microbial preparation comprising at least one of the above-mentioned live cells of *Bacillus fusiformis* KBD-5, which antagonizes *Botrytis cinerea*, its fermentation metabolites, and an agriculturally acceptable carrier.

[0014] Furthermore, the preparation is a liquid bacterial agent, a solid bacterial agent, or a wettable powder.

[0015] On the other hand, the present invention provides the application of the above-mentioned antagonistic Botrytis cinerea fusiforme Bacillus KBD-5 in the preparation of formulations for the prevention and control of plant fungal diseases.

[0016] Furthermore, the plant fungal disease is caused by Botrytis cinerea.

[0017] Furthermore, the disease in question is tomato gray mold.

[0018] Information on the Preservation of Biological Materials: The antagonistic lysinibacillus fusiformis KBD-5, which relates to this invention, was deposited on November 14, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China (Institute of Microbiology, Chinese Academy of Sciences). The accession number is CGMCC No. 26146.

[0019] The beneficial effects achieved by this invention are as follows: 1. The KBD-5 strain of this invention has specific antagonistic activity against Botrytis cinerea, with a plate confrontation inhibition rate of 76.42%, filling the technical gap in the field of biological control of gray mold and expanding the biocontrol bacterial resource library; 2. This invention systematically reveals the triple synergistic antagonistic mechanism of this strain at the genetic level: it contains a fengycin-like lipopeptide synthesis gene cluster (46% similarity) that destroys the pathogen's cell membrane, and contains GH18 chitinase and GH5 cellulase genes that degrade the pathogen's cell wall, making it difficult for the pathogen to develop resistance; 3. The KBD-5 strain of this invention... The PHI annotation of strain KBD-5 showed no virulence factors and no core virulence genes such as hemolysin or enterotoxin were detected. CARD analysis only found chromosome-encoded resistance genes, with no plasmid-mediated, horizontal transfer risk, and safe environmental release. 4. This invention comprehensively verified the control effect of strain KBD-5 through multi-level experiments: the inhibition rate of Botrytis cinerea on plate confrontation reached 76.42%; the tomato fruit test showed that the incidence rate decreased to 25.9% and the rot index decreased by 75.0%; the greenhouse pot test showed that the control effect on tomato gray mold reached 72.35%, which was not significantly different from the 75.12% of the chemical pesticide iprodione, and has practical application value. Attached Figure Description

[0020] Figure 1 is a morphological diagram of the spindle-shaped lysine-containing Bacillus KBD-5 of the present invention, wherein A: colony morphology on plate; B: morphology observed under electron microscope.

[0021] Figure 2 is a phylogenetic tree of Bacillus spindleii lysine 5.

[0022] Figure 3 shows the inhibitory effect of strain KBD-5 of the present invention on botrytis cinerea H1 of tomato, where A: treatment group; B: control group.

[0023] Figure 4 shows the control effect of strain KBD-5 on gray mold of tomato fruit. In the figure, A: pathogen control; B: KBD-5 treatment; C: blank control; D: disease incidence; E: rot index. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the field. The methods in the following embodiments, unless otherwise specified, are conventional methods in the field. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In this invention, the antagonistic *Botrytis cinerea* fusiforme lysine-containing *Bacillus fusiforme* KBD-5 may be simply referred to as "strain KBD-5" or "KBD-5".

[0026] Example 1: Isolation and Identification of Strain KBD-5 Soil samples were collected from the hot spring area of ​​Tengchong County, Baoshan City, Yunnan Province for isolation. 1g of soil sample was weighed and added to a centrifuge tube containing 9 mL of sterile physiological saline. The mixture was vortexed to prepare a soil suspension. The suspension was then serially diluted (10⁻⁶ ppm). -1 Up to 10 -6 The cultures were spread onto LB agar plates and incubated upside down at 37°C for 24 hours. Single colonies of different morphologies were picked and streaked multiple times on LB plates for purification until pure cultures were obtained. One strain that formed pale yellow, nearly circular, smooth, and opaque colonies on LB plates (Figure 1A) was designated KBD-5.

[0027] Scanning electron microscopy revealed that strain KBD-5 is rod-shaped and produces terminal, spindle-shaped spores (Figure 1B). Genomic DNA was extracted from strain KBD-5, and the 16S rRNA gene was amplified using universal primers 27F and 1492R. The PCR products were sequenced, and the sequences were BLAST-aligned in the NCBI database. The results showed that the sequence had over 99% similarity to several type strains of Lysinibacillus fusiformis. Based on morphological characteristics, this strain was identified as a spindle-shaped Lysinibacillus fusiformis. This strain was deposited on November 14, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 26146.

[0028] Example 2: Phylogenetic analysis of strain KBD-5. To clarify the phylogenetic position of strain KBD-5, genomic DNA of the strain was extracted, its 16S rRNA gene was amplified and sequenced, and the obtained gene sequence was submitted to the NCBI database. Homology analysis was performed by sequence alignment. At the same time, a phylogenetic tree was constructed based on the 16S rRNA gene sequence to analyze its evolutionary relationship with known strains.

[0029] As shown in Figure 2, the 16S rRNA gene sequence of strain KBD-5 showed 100% homology with that of *Lysinibacillus fusiformis* YT41 (GenBank accession number: PQ577987.1). Phylogenetic analysis indicated that strain KBD-5 and YT41 clustered in the same independent branch (Figure 2). Based on these results, strain KBD-5 was preliminarily identified as *Lysinibacillus fusiformis*.

[0030] Example 3: The antagonistic activity of strain KBD-5 against *Botrytis cinerea* was determined using the plate confrontation method. *Botrytis cinerea* H1, the pathogen of tomato gray mold, was activated and cultured on PDA plates at 28°C for 5 days. Mycelial cakes were collected from the edge of the colonies using a sterile punch (7 mm diameter). Strain KBD-5 was cultured in LB liquid medium at 37°C with shaking at 180 rpm for 12 hours until OD... 600 The concentration was approximately 0.3, and a bacterial suspension was prepared. The mycelial side of the *Botrytis cinerea* mycelial cake was inoculated into the center of a PDA plate. Using the cross-cross method, 5 μL of KBD-5 bacterial suspension was spotted at four points 2.5 cm away from the mycelial cake. Plates without KBD-5 suspension served as a blank control. Each treatment was repeated in triplicate. The plates were incubated at 28°C for 5 days. The colony diameter of *Botrytis cinerea* in the control and treatment groups was measured using the cross-cross method.

[0031] Inhibition rate (%) = [(Coronavirus diameter of control group - Coronavirus diameter of treatment group) / (Coronavirus diameter of control group - Coronavirus block diameter)] × 100%.

[0032] The results are shown in Figure 3. The control group colonies (Figure 3B) covered the entire plate (diameter 8.25 cm), while the growth of the treatment group colonies (Figure 3A) was significantly inhibited (diameter 2.48 cm). The inhibition rate was calculated to be 76.42%, demonstrating that KBD-5 has a good antagonistic ability against Botrytis cinerea.

[0033] Example 4: Verification of the control effect of strain KBD-5 on gray mold of tomato fruit. Fresh tomato fruits of uniform size and without visible damage were selected. After surface sterilization by soaking in 1% sodium hypochlorite solution for 30 seconds, they were rinsed 3 times with sterile distilled water. The experiment was set up with 3 treatment groups (15 fruits in each group, repeated 3 times): CK group: fruits were soaked in sterile water for 20 min, dried, and then left uninoculated and without pathogen inoculation; A group (pathogen control): fruits were soaked in sterile water for 20 min, dried, and then a 2 mm × 2 mm wound was made at the equator using a sterile needle, and 20 μL of 1×10⁻⁶ styrene solution was inoculated. 7 cfu mL -1 Botrytis cinerea conidia suspension; Group B (KBD-5 treatment group) fruit soaking 1×10 8 cfu mL -1 After suspending the KBD-5 strain for 20 minutes and drying it, wounds were created according to the method of Group A and inoculated with an equal amount of pathogens.

[0034] After drying, all fruits were placed in sterile plastic containers and cultured for 5 days at 25℃ and 95% relative humidity. The disease severity was assessed according to a 5-level grading standard (Grade 0: no lesions; Grade 1: lesion area ≤ 5%; Grade 2: 5% < lesion area ≤ 10%; Grade 3: 10% < lesion area ≤ 20%; Grade 4: lesion area > 20%). The incidence rate and rot index were calculated using the following formulas: Incidence rate (%) = (number of diseased fruits / total number of fruits) × 100; Rot index (%) = [Σ(number of diseased fruits at each level × corresponding disease level) / (highest disease level × total number of fruits)] × 100. Results showed that 5 days after inoculation, all tomatoes in Group A (Figure 4A) exhibited severe gray mold symptoms, with an incidence rate of 100% and extensive rot in the fruit tissue. In Group B (Figure 4B), the infection severity was significantly reduced, with the incidence rate decreasing to 25.9% (Figure 4D), and the rot index was lower than that in Group A. The CK group showed a 75.0% reduction in disease incidence (Figure 4E); the CK group (Figure 4C) showed no lesions on its fruits. This indicates that the KBD-5 strain can not only inhibit pathogens in vitro, but also effectively protect tomato fruits from gray mold infection in actual postharvest scenarios, demonstrating its potential as a postharvest biological preservative.

[0035] Example 5: Whole-genome sequencing and biocontrol potential analysis of strain KBD-5. KBD-5 bacterial culture was collected, and high-quality genomic DNA was extracted using a DNA extraction kit. Paired-end sequencing was performed using the Illumina NovaSeq 6000 platform. The obtained data underwent quality control, assembly, and error correction. Gene prediction was performed using Prokka. Gene function annotation was performed using databases such as NR, COG, GO, KEGG, CAZy, CARD, and PHI. AntiSMASH was used to predict secondary metabolite synthesis gene clusters.

[0036] The results are shown in Tables 1 to 4. The genome of strain KBD-5 is 4,692,552 bp in size, with a GC content of 37.28%, encoding 4,719 CDS. It contains abundant non-coding RNA and a CRISPR-Cas system, indicating its strong metabolic and environmental adaptability. Eight secondary metabolite synthesis gene clusters (including T3PKS, NRPS, etc.) and 195 carbohydrate-active enzyme genes were predicted. Cluster 1 may be responsible for synthesizing fengycin-like antifungal lipopeptides, which are key substances for inhibiting the growth of *Botrytis cinerea* mycelia. 195 CAZyme genes were annotated, especially GH18 (chitinase) and GH5 (cellulase), which can directly attack and destroy the cell wall of *Botrytis cinerea*. PHI annotation showed that 80.18% of the genes were non-toxic or low-toxic, and CARD analysis found only 7 chromosomal resistance genes, indicating no risk of horizontal transfer and high biosafety of the strain. The genome sequence has been submitted to NCBI, accession number PRJNA1335659.

[0037] Table 1. Genome characteristics of KBD-5

[0038] Note: The number of rRNAs refers to the total number of 5S, 16S, and 23S rRNA genes.

[0039] Table 2. Bacteriocin synthesis and immune-related KEGG pathway in KBD-5.

[0040] Table 3. Annotation of antibiotic resistance genes in KBD-5 fusiform lysine-containing Bacillus fusiformis antagonistic to Botrytis cinerea.

[0041] Table 4. Analysis of secondary metabolite synthesis gene clusters in *Bacillus fusiformis* KBD-5, which antagonizes *Botrytis cinerea*.

[0042] Example 6: Preparation of KBD-5 microbial preparation. KBD-5 strain was inoculated into LB seed culture and cultured at 37℃ for 12 h. A 5% inoculum was then transferred to fermentation medium (composition: glucose 10 g / L, soybean meal 15 g / L, K₂HPO₄ 1 g / L, MgSO₄ 0.5 g / L, pH 7.0), and fermented at 32℃ and 200 rpm for 36-48 h. When the viable cell count reached 5 × 10⁻⁶... 9 When the concentration of CFU / mL exceeds a certain level, fermentation is terminated. The fermentation broth is mixed with a carrier (such as diatomaceous earth or peat moss) at a ratio of 1:1 (v / w) to prepare a solid inoculum; or 1% glycerol is added to the fermentation broth as a preservative to prepare a liquid inoculum, which is then aliquoted and stored.

[0043] KBD-5 solid and liquid inoculants were successfully prepared, with a viable count of 5 × 10⁻⁶. 9 CFU / mL or higher. This formulation has good processing adaptability and can be made into seed coating agents, hole fertilizers, or foliar sprays according to different application scenarios for the control of tomato gray mold, providing diversified product forms for subsequent field applications.

[0044] Example 7: Greenhouse control effect of KBD-5 formulation against tomato gray mold. An experiment was conducted during the flowering stage of tomato seedlings, with three treatment groups (30 tomato plants per group, replicated three times): ① KBD-5 solid inoculant diluted solution (10... 8 ① Foliar spray with CFU / mL; ② Foliar spray with 500 times dilution of iprodione wettable powder (positive control); ③ Foliar spray with an equal amount of water (blank control).

[0045] 24 h after each treatment was sprayed, artificial inoculation was performed with a suspension of Botrytis cinerea spores (concentration 1×10⁻⁶). 6 (number of cells / mL), and cultured in a greenhouse at 22-25℃ and 90% relative humidity. The disease incidence was investigated after 7 days.

[0046] Disease severity grading and index statistics: A disease severity grading standard for tomato gray mold was established: Grade 0 (no lesions), Grade 1 (lesion area ≤ 5%), Grade 2 (lesion area 6%-15%), Grade 3 (lesion area 16%-30%), Grade 4 (lesion area 31%-50%), and Grade 5 (lesion area > 50% or plant death). The number of diseased plants at each grade in each treatment group was counted, and the disease index and control effect were calculated using the formula (Table 5).

[0047] Disease index = ∑ (number of diseased plants at each level × level) / (total number of plants × highest level) × 100%; Control effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0048] Table 5. Control effects of different treatments on tomato gray mold.

[0049] The results are shown in Table 5. The disease index of the KBD-5 preparation group was only 6.72, and the control effect reached 72.35%, which was not significantly different from the control effect of the chemical agent iprodione (75.12%) (p>0.05). Moreover, no plants in the KBD-5 preparation treatment group reached the disease level of 4-5, while the proportion of diseased plants at level 4-5 in the water control group reached 18.89%. This fully demonstrates that the KBD-5 preparation has excellent control effect on tomato gray mold and does not cause phytotoxicity to tomato plants. It can be used as a green alternative to chemical agents for gray mold control.

[0050] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A fusiformis lysinobacillus KBD-5 antagonistic to Botrytis cinerea, characterized by: It was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 14, 2022, with accession number CGMCC No. 26146.

2. The fusiform lysine-containing Bacillus fusiforme KBD-5 antagonistic to Botrytis cinerea according to claim 1, characterized in that: Its whole genome sequence contains the nucleotide sequence shown in accession number PRJNA1335659.

3. The spindle-shaped lysine-containing Bacillus fusiforme KBD-5 antagonistic to Botrytis cinerea according to claim 1 or 2, characterized in that: Its genome contains a gene cluster related to the synthesis of secondary metabolites associated with the synthesis of fengycin-like lipopeptides, the gene cluster being located at Chr1:415,594-439,817 of the whole genome sequence of claim 2.

4. The fusiform lysine-containing Bacillus fusiforme KBD-5 antagonistic to Botrytis cinerea according to claim 1 or 2, characterized in that: Its genome contains GH18 family chitinase genes and GH5 family cellulase genes.

5. The spindle-shaped lysine-containing Bacillus fusiforme KBD-5 antagonistic to Botrytis cinerea according to claim 1 or 2, characterized in that: Its inhibition rate on the mycelial growth of Botrytis cinerea is no less than 76.42%.

6. A microbial preparation, characterized in that: The invention comprises at least one of the following: live cells of *Bacillus fusiformis* KBD-5, which antagonizes *Botrytis cinerea* as described in claim 1, its fermentation metabolites, and an agriculturally acceptable carrier.

7. The microbial preparation according to claim 6, characterized in that: The preparation is a liquid bacterial agent, a solid bacterial agent, or a wettable powder.

8. The use of the spindle-shaped lysine-containing Bacillus fusiformis KBD-5 antagonistic to Botrytis cinerea as described in claim 1 in the preparation of formulations for the prevention and control of plant fungal diseases.

9. The application according to claim 8, characterized in that: The plant fungal disease mentioned is caused by Botrytis cinerea.

10. The application according to claim 9, characterized in that: The disease in question is gray mold of tomatoes.

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