Bacillus orientalis and application thereof

Abstract

The invention discloses bacillus orientalis and application thereof, and relates to the technical field of microorganisms, the bacillus orientalis is bacillus orientalis ZB-201, the bacillus orientalis ZB-201 is preserved in China General Microbiological Culture Collection Center (CGMCC) on February 25, 2025, the classification name of the bacillus orientalis ZB-201 is bacillus orientalis, and the preservation number of the bacillus orientalis ZB-201 is CGMCC NO.33653. According to the invention, a strain of bacillus orientalis ZB-201 with a remarkable antagonistic effect on meloidogyne incognita is successfully screened and identified from a soil sample. Compared with chemical prevention and control, the bacillus orientalis biological prevention and control has the advantages of being environmentally friendly, not prone to generating drug resistance, safe to non-target organisms and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a strain of Bacillus orientalis and application thereof. Background Art

[0002] Southern root-knot nematode ( Meloidogyne incognita ) belongs to the family Meloidogyneidae, superfamily Heterodera, order Meloidogyne, and is one of the most destructive plant parasitic nematodes worldwide. Its host range is extremely broad, encompassing vegetables, fruits, flowers, and grain crops. According to statistics, it harms over 2,000 plant species. Common vegetables such as cucumbers, tomatoes, eggplants, and peppers are particularly vulnerable; fruits such as citrus, grapes, bananas, and strawberries are also susceptible; and flowers such as chrysanthemums, roses, and carnations are also susceptible to infestation by southern root-knot nematodes.

[0003] When the southern root-knot nematode infects crops, it primarily invades through the root tips as second-instar larvae. They continuously migrate and feed within the root system, stimulating abnormal division and expansion of root cells, resulting in root knots of varying sizes. These knots severely hinder the root system's absorption of water and nutrients. Infected crops exhibit obvious growth problems above ground, with stunted growth, yellowing and wilting of leaves, and stunted growth, severely impacting photosynthesis and metabolism. Southern root-knot nematodes pose a significant threat to crop yields, typically causing a 20%-40% reduction in crop yield. In severely affected areas, yield reductions can reach as high as 70%-80%, with some plots even experiencing total crop failure. For example, in cucumber cultivation, infection with the southern root-knot nematode can significantly reduce both yield and quality, leading to deformed fruits and a poorer taste. Moreover, crops infected by southern root-knot nematodes have significantly reduced stress resistance due to damaged root systems, making them more susceptible to secondary infection by other pathogens, such as fungal wilt and bacterial wilt, further aggravating the severity of crop diseases and dealing a heavy blow to agricultural production.

[0004] Chemical control is the primary means of controlling the southern root-knot nematode. Chemical pesticides such as methyl bromide, aldicarb, and carbofuran, due to their rapid nematicidal effects, were for a time a key weapon in controlling the southern root-knot nematode. Methyl bromide quickly penetrates the soil and directly affects the nematode's nervous system, paralyzing and killing it. Aldicarb kills the nematode by inhibiting acetylcholinesterase activity, disrupting nerve conduction. However, over time, the drawbacks of chemical control have become increasingly apparent. Long-term and extensive use of chemical pesticides has led to the gradual development of resistance in the southern root-knot nematode. Studies have shown that in fields treated with the same chemical nematicide for multiple years, the southern root-knot nematode's resistance can be several times or even dozens of times greater, significantly reducing the effectiveness of chemical control. Furthermore, chemical pesticide residues pose a serious threat to food safety and the ecological environment. These residues not only accumulate in agricultural products and enter the human body through the food chain, endangering human health, but also disrupt the structure of soil microbial communities and affect the balance of the soil ecosystem. Long-term use of chemical pesticides may also lead to soil compaction and decreased fertility, further affecting the sustainable development of agriculture.

[0005] In contrast, biological control, as a green and sustainable pest control method, has gradually gained widespread attention. Biological control utilizes beneficial organisms and their metabolites to control pest populations and thereby prevent and control diseases. It is environmentally friendly, less susceptible to pesticide resistance, and safe for non-target organisms. It effectively controls the damage caused by southern root-knot nematodes while protecting the ecological environment and biodiversity, aligning with the principles of modern sustainable agricultural development.

[0006] Among numerous biocontrol resources, Bacillus stands out for its unique advantages, making it a highly promising biocontrol agent. Bacillus is a Gram-positive bacterium widely distributed in various ecological environments, including soil, plant rhizospheres, and water bodies. It possesses strong stress tolerance, capable of surviving and reproducing in harsh environmental conditions, including high and low temperatures, high salinity, and drought. Under high temperatures, Bacillus forms spores, which have thick walls that protect against heat damage. When environmental conditions are favorable, spores germinate into vegetative cells, resuming growth and metabolism. Bacillus has a diverse metabolic profile, producing a variety of bioactive substances, including antimicrobial peptides, lipopeptides, degradative enzymes, non-sugar antibiotics, macrolides, and polyenes. These substances can inhibit the growth and reproduction of southern root-knot nematodes through various pathways, such as dissolving the nematode's cuticle, inhibiting hatching, and interfering with its metabolic processes. The antimicrobial peptides produced by Bacillus subtilis can disrupt the cell membrane structure of the southern root-knot nematode, causing the cell contents to leak out and killing the nematode. The chitinase secreted by Paenibacillus polymyxa can degrade the chitin in the nematode eggshell, inhibiting egg hatching. Bacillus can also establish a symbiotic relationship with plant roots, colonizing the rhizosphere and forming a biofilm. This not only protects plant roots from southern root-knot nematode infestation but also promotes root growth and development, enhancing plant resistance to stress.

[0007] Based on this, the screening and identification of highly effective new antagonistic bacteria Bacillus provides new strategies and resources for the biological control of southern root-knot nematodes, which helps reduce the use of chemical pesticides, reduce environmental pollution, ensure the quality and safety of agricultural products, and promote the green and sustainable development of agriculture. Summary of the Invention

[0008] In order to solve the above problems, the present invention provides a strain of Bacillus orientalis and its application.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a strain of Bacillus orientalis, wherein the Bacillus orientalis is Bacillus orientalis ZB-201, which was deposited in the General Microbiology Center of China Culture Collection Administration Committee on February 25, 2025, and is classified and named Bacillus toyonensis , the deposit number is CGMCC NO.33653.

[0010] The present invention also provides application of a strain of Bacillus toyotii in preventing and controlling root-knot nematodes.

[0011] The present invention also provides the use of a strain of Bacillus toyotii in preparing a biological preparation for preventing and treating root-knot nematodes.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The present invention successfully screened and identified a strain of Bacillus orientalis ZB-201 from soil samples that exhibits significant antagonism against the southern root-knot nematode. Compared to chemical control, biological control with Bacillus orientalis offers advantages such as environmental friendliness, resistance to drug resistance, and safety against non-target organisms. The strain identified in this invention, Bacillus orientalis ZB-201, has demonstrated promising potential for the control of southern root-knot nematodes, providing a new resource for biological control of the disease and possessing significant theoretical and practical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the lethality of the fermentation liquid in Example 2 of the present invention to the second-instar larvae of the southern root-knot nematode; Figure 2 (a) Colony morphology, (b) Gram stain (10×40), and (c) Gram stain (10× oil immersion lens) of strain ZB-201 in Example 3 of the present invention; Figure 3 This is a diagram of the catalase test in Example 3 of the present invention; Figure 4 This is the methyl red test in Example 3 of the present invention (the left picture is before addition, and the right picture is after addition); Figure 5 This is a diagram of a starch hydrolysis test in Example 3 of the present invention; Figure 6 This is a diagram of the protease production test in Example 3 of the present invention; Figure 7 This is a diagram of a cellulase production detection test in Example 3 of the present invention; Figure 8 This is a graph showing the VP determination test in Example 3 of the present invention; Figure 9 This is a diagram of the oxidase test in Example 3 of the present invention; Figure 10 This is the phylogenetic tree of the antagonistic strain ZB-201 constructed based on the 16S rDNA sequence in Example 3 of the present invention; Figure 11 This is the result of the gyrb gene sequence alignment in Example 3 of the present invention.

[0014] The Bacillus subtilis ZB-201 of the present invention was deposited in the General Microbiology Center of China Culture Collection Administration Committee on February 25, 2025, and was classified and named Bacillus toyonensis , the deposit number is CGMCCNO.33653. DETAILED DESCRIPTION

[0015] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0016] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0017] Example 1 Materials 1. Soil sample collection Soil samples were collected from greenhouse vegetable fields in Taigu District, Jinzhong City, Shanxi Province, from May to July 2024, where southern root-knot nematode infestation is prevalent. The warm and humid greenhouse climate is ideal for the growth and reproduction of southern root-knot nematodes. Furthermore, root-knot nematode infestations are common and typical of vegetable cultivation. At each sampling location, a five-point sampling method was used, with five different sampling points selected. Surface soil was collected at a depth of 0-20 cm at each sampling point. Soil samples were carefully collected using a sterile spatula to avoid contamination. Approximately 500 g of soil was collected from each sampling point. After thoroughly mixing the soil from all five sampling points, approximately 1000 g was collected as the soil sample for that site. The samples were placed in sterile ziplock bags and labeled with information such as the sampling location, time, and crop type. A total of 70 soil samples were collected and immediately stored in a refrigerator at 4°C. They were then brought back to the laboratory for subsequent processing as soon as possible.

[0018] 2. Test nematodes Second-instar larvae (J2) of the southern root-knot nematode were isolated, purified, and preserved in our laboratory from the rhizosphere soil of infested melons. The preservation method involves chopping the diseased melon roots containing southern root-knot nematode eggs into small pieces, placing them in a Behmann funnel, adding an appropriate amount of water, and incubating them in the dark at 25-28°C for 24-48 hours. The nematode suspension at the bottom of the funnel is collected, washed multiple times with sterile water, and then adjusted to the desired concentration. The suspension is then stored in a refrigerator at 4°C until needed. During experiments, the preserved nematode suspension is removed and allowed to rest at room temperature for a period of time to allow it to recover before use in subsequent experiments.

[0019] 3. Test culture medium (1) Basic culture medium: NA medium: 10 g peptone, 3 g beef extract, 5 g sodium chloride, 15 g agar powder, 1000 mL distilled water, pH 7.0.

[0020] NB medium: NA medium without agar powder is NB medium.

[0021] (2) Starch hydrolysis test medium: Add 0.2% soluble starch to the general carpone agar medium.

[0022] (3) Methyl red test medium: 5 g peptone, 5 g glucose, 5 g K2HPO4, 1000 mL distilled water, pH 7.0-7.2.

[0023] (4) VP determination test medium: same as methyl red test.

[0024] 4. Test reagents and instruments (1) Physiological and biochemical identification reagents: Catalase reaction reagent: 3% hydrogen peroxide.

[0025] VP determination reagents: creatine 0.3% or original powder, 40% NaOH solution.

[0026] Methyl red test reagent: Weigh 0.04g of methyl red and dissolve it in 60mL of 95% ethanol, then add 40mL of distilled water.

[0027] Starch hydrolysis test reagent (Lugol's iodine solution): iodine 1g, potassium iodide 2g, distilled water 300mL.

[0028] Oxidase reaction reagent: N,N-dimethyl-p-phenylenediamine dihydrochloride.

[0029] (2) Bacterial genomic DNA extraction kit was purchased from Jinsha Biotechnology Co., Ltd.; PCR amplification reagents, including Taq DNA polymerase, dNTPs, 10× PCR Buffer, etc., were purchased from Shanghai Sangon Biotechnology Co., Ltd.; primers were also synthesized by Shanghai Sangon Biotechnology Co., Ltd.; other biochemical reagents such as Gram staining solution and 3% hydrogen peroxide solution were purchased from Beijing Solebau Technology Co., Ltd.

[0030] (3) The main instruments and equipment include: clean bench; constant temperature incubator; high-speed refrigerated centrifuge; PCR instrument; gel imaging system; electronic balance; microscope; oscillating incubator; high-pressure steam sterilizer.

[0031] 5. 16SrDNA sequence analysis and phylogenetic tree construction website and software Blast online analysis software (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) NCBI database (https: / / www.ncbi.nlm.nih.gov / ) GenBank database (http: / / www.ncbi.nlm.nih.gov / genbank) Clustalw2 online analysis software (http: / / www.ebi.ac.uk / Tools / msa / clustalw2 / ) Bioedit software, PAUP software, Treeview software Example 2 Strain isolation and screening Prepare a dilution series by placing 0.02 g of soil in 25 mL of 10 mmol magnesium chloride for 15 minutes with shaking. Transfer 4500 μL, 1500 μL, 500 μL, 167 μL, 56 μL, and 19 μL of the soil mixture to six vials containing 1 L of 10% TSB to generate a 222×, 666×, 2000×, 6000×, 18000×, and 54000× dilution series. Dispense 180 μL of the dilutions into 96-well plates. Seal the plates with parafilm and incubate in the dark for one week to determine the optimal dilution (visible bacterial growth in 30% of the plates). Streak the 96-well plates containing the optimal dilution to obtain a single colony. Inoculate the single colony into NB fermentation medium and incubate at 30°C and 180 rpm for 24 hours to obtain fermentation broth. First, observe and compare the mortality rate of fermentation liquid to the second-instar larvae of southern root-knot nematode under a microscope, select the strain with a mortality rate of more than 80%, and then dilute it 10 times to make the concentration 10 7 CFU / mL, and then the in vitro nematocidal activity was compared again to screen out strains with high lethality and stable activity against nematodes for subsequent studies.

[0032] A total of 177 strains, 67 species, were isolated and screened from 70 soil samples. The initial screening results showed that 18 antagonistic strains with antibacterial effects were isolated and screened. After purification and rescreening, the antagonistic strain ZB-201 achieved 100% mortality of second-instar larvae in just 3 hours before dilution. The concentration of 10 7 CFU / mL, the mortality rate can reach 95% at 6h ( Figure 1 , stiffness indicates death of the root-knot nematode, while bending indicates survival).

[0033] Example 3 Strain Identification 1. Morphological identification Inoculate the strain onto a NA plate and incubate at 30°C for 24-48 hours. Observe the colony's morphology, size, color, margins, surface texture, and transparency. At the same time, select a single colony for Gram staining. The specific steps are as follows: place a drop of saline on a glass slide, use an inoculating loop to pick a small amount of bacteria, spread evenly in the saline, allow to dry naturally, and then fix with a flame. Next, stain with crystal violet for 1 minute, rinse with water; mordant with iodine solution for 1 minute, rinse with water; decolorize with 95% ethanol for 20-30 seconds, rinse with water; and finally, counterstain with safranin for 1 minute, rinse with water. After drying, observe the color and morphology of the bacteria under a microscope to determine the Gram stain result.

[0034] The selected strains were inoculated onto NA plates and cultured for 48 hours. The colonies were round with slightly wavy edges, 2-4 mm in diameter, milky white with a slight yellowish tint, opaque, and slightly raised in the center. Figure 2 a); Gram staining was performed simultaneously ( Figure 2 b, 2c) are Gram-positive bacteria. Observing the bacterial morphology under a microscope, it was preliminarily determined to be Bacillus.

[0035] 2. Physiological and biochemical identification The specific test methods for determining physiological and biochemical indicators refer to the Bergey Manual of Bacterial Identification by RE Buchanan et al. and the Manual of Identification of Common Bacterial Systems by Dong Xiuzhu et al.

[0036] According to the conventional physiological and biochemical identification method, the biochemical characteristics of the strain are: when 3% hydrogen peroxide is added to the glass slide with the strain suspension in the presence of enzyme, a large number of bubbles are generated, which is positive (see Figure 3 In the methyl red test, a drop of methyl red reagent is added to the culture medium after the bacteria have been cultured for 2 days and 6 days, and the bacteria turn red, indicating that the methyl red test is positive (see Figure 4 In the starch hydrolysis reaction, when iodine solution is added to the plate, the plate turns blue-black and a transparent circle that does not change color appears around the colony, indicating that starch hydrolysis is positive (see Figure 5 ); In the protease reaction and cellulase reaction, clear transparent circles appeared around the colonies, indicating that the strain produced protease and cellulase (see Figure 6 、 7 In the VP assay, the indicator turns red after the strain is cultured for 2 days, 6 days, or for an additional 2 days, indicating a positive reaction (see Figure 8 ); No color change on the filter paper in the oxidase assay indicates a negative result (see Figure 9 The physiological and biochemical identification results of the strains are shown in Table 1.

[0037] Table 1 Physiological and biochemical identification results of the strains

[0038] 3. Molecular biology identification (1) Extraction of genomic DNA from antagonistic strains Use a bacterial genomic DNA extraction kit to extract genomic DNA from the strain. Follow the kit instructions: Take 1-2 mL of bacterial culture medium grown to the logarithmic growth phase, centrifuge at 12,000 rpm for 2 minutes, and discard the supernatant. Add 200 μL of buffer GA to the precipitate and shake until the bacteria are completely suspended. Add 20 μL of proteinase K solution, mix thoroughly, and incubate in a 56°C water bath for 1-3 hours until the solution becomes clear. Add 200 μL of buffer GB, mix thoroughly, and incubate in a 70°C water bath for 10 minutes until the solution becomes clear. Add 200 μL of anhydrous ethanol and mix thoroughly; a flocculent precipitate may form. Add the mixture to adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. Add 500 μL of buffer GD to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. Add 600 μL of rinse buffer PW to the adsorption column and centrifuge at 12,000 rpm for 30 seconds. Discard the filtrate and repeat this step once. Return the adsorption column CB3 to the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove any remaining rinse buffer. Place the adsorption column CB3 in a clean centrifuge tube and dropwise add 50-100 μL of elution buffer TE to the center of the adsorption membrane. Incubate at room temperature for 2-5 minutes. Centrifuge at 12,000 rpm for 2 minutes. Collect the eluted DNA solution and store at -20°C until needed.

[0039] (2) PCR amplification of 16S rDNA of antagonistic strains 16S rDNA sequences were amplified using extracted genomic DNA as a template. Universal primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were used. The PCR reaction system (25 μL) consisted of 1 μL template DNA, 2.5 μL 10× PCR Buffer, 2 μL dNTPs (2.5 mM each), 1 μL primer 27F (10 μM), 1 μL primer 1492R (10 μM), 0.2 μL Taq DNA polymerase (5 U / μL), and 17.3 μL ddH2O. PCR reaction conditions included 94°C denaturation for 5 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, followed by extension at 72°C for 10 min. After PCR amplification products were examined by 1% agarose gel electrophoresis, the target bands were excised and purified using a gel recovery kit. The purified PCR products were then sent to a sequencing company for sequencing. The obtained 16S rDNA sequence (SEQ ID NO: 1) was compared against the NCBI database using BLAST analysis. Model strain sequences with high similarity to this sequence were selected and a phylogenetic tree was constructed using the neighbor-joining method using MEGA 7.0 software to determine the taxonomic status of the strains.

[0040] (3) Analysis of 16S rDNA sequence similarity and construction of phylogenetic tree The measured 16S rDNA sequences were compared with the NCBI database by BLAST analysis. The model strain sequences with high similarity to the sequences were selected. The phylogenetic tree was constructed using MEGA7.0 software and the neighbor-joining method to determine the taxonomic status of the strains.

[0041] The genomic DNA of the strain was extracted and the 16S rRNA gene was amplified by PCR using universal primers and gyrb gene primers. The amplified product was sequenced to obtain a 1137 bp 16S rDNA sequence. The 16S rDNA was compared with the 16S rDNA sequences registered in the NCBI database using Blast software, and a phylogenetic tree was constructed (see Figure 10 ). The 16S sequence alignment results of the antagonistic strain ZB-201 are Bacillus toyonensis BCT-7112, sequencing results for comparison see Figure 11 .

[0042] Table 2 Primer sequences used

[0043] Note: Among them, M=C / A, Y=C / T, R=A / G, N=A / G / C / T.

[0044] Example 4 Field control efficacy test Experimental location: solar greenhouse in Dongshandi Village, Taigu District, Jinzhong City, Shanxi Province (37°22′N, 112°34′E).

[0045] Test varieties: Melon seedlings were provided by Shanxi Juxin Weiye Agricultural Science and Technology Development Co., Ltd.

[0046] Preparation of biocontrol bacteria fermentation broth: Inoculate the screened and identified Bacillus subtilis strain into NB medium and culture in a shaker at 30°C and 180 rpm for 24 hours. After the strain is cultured, take an appropriate amount of fresh bacterial broth and measure its OD on a spectrophotometer. 600 value, diluted to 1×10 7 CFU / mL was reserved and 200 mL was used to irrigate the roots of each seedling.

[0047] Chemical control: 5% avermectin microemulsion, applied according to the dosage recommended on the packaging bag at 500 ml / mu.

[0048] 1. Experimental Design The experiment set up 3 treatments, each with 2 plots, for a total of 6 plots. The plots were arranged in random blocks, with 24 melon seedlings planted in each plot × 1 row = 24 melon seedlings. A: 5% avermectin microemulsion (chemical control group), B: Biocontrol bacteria fermentation liquid treatment group, C: water control group.

[0049] Each treatment was repeated twice, and guard rows were set between adjacent plots in a completely random arrangement.

[0050] One week after the melon seedlings are transplanted and survive, root irrigation is carried out. After 30 days, the melons in all plots are graded and investigated for their condition. Root irrigation is carried out a second time, and the condition is investigated again 30 days later.

[0051] 2. Data collection and indicator measurement (1) Plant growth indicators: Plant height: vertical height from base to growth point (cm), measured once a week, and the final data was taken; Stem diameter: diameter of the fifth internode measured with a vernier caliper (mm); Leaf number: Count fully expanded leaves (≥3 cm).

[0052] (2) Disease assessment: Disease classification: Based on the degree of leaf yellowing and wilting: Level 0: healthy, no visible symptoms; Level 1: Yellowing area of leaf margins <30%; Level 2: Yellowing between veins >50% or apical wilting; Level 3: The whole plant is dead.

[0053] Incidence rate: Calculate the proportion of plants in each grade (healthy = grade 0, slightly yellowed = grade 1, severely wilted = grades 2+3).

[0054] (3) Physiological and yield indicators: Survival rate: the percentage of plants that survived at the end of the period; Yield per plant: The fruits harvested at maturity were weighed (kg).

[0055] (4) Statistical analysis SPSS 26.0 software was used for data analysis; Continuous variables such as plant height and stem diameter were analyzed using one-way analysis of variance (ANOVA), and differences between groups were analyzed using Duncan's multiple comparison test ( P <0.05); categorical variables such as incidence were analyzed using the chi-square test (χ²).

[0056] 3. Field test results (1) Plant growth indicators There were no significant differences in plant height, stem diameter, and leaf number between the biocontrol bacteria fermentation liquid treatment group and the chemical agent control group ( P >0.05, but both groups were significantly better than the water control group ( P <0.01). The plant height of the chemical control group was 37.4% higher than that of the water control group, and the plant height of the biocontrol bacteria fermentation liquid treatment group was 39.7% higher, indicating that both treatments had a growth-promoting effect. Specific growth indicators are shown in Table 3.

[0057] Table 3 Plant growth indicators

[0058] Note: All stem diameters are measured from the 5th internode. ** The difference with the water control group was extremely significant ( P <0.01).

[0059] (2) Disease incidence and resistance performance The proportion of healthy plants in the biocontrol bacteria fermentation liquid treatment group was significantly higher than that in the water control group (χ²=40.2, P <0.001), but there was no significant difference between the healthy plants in the biocontrol bacteria fermentation liquid treatment group and the chemical control group (χ²=0.346, P >0.05). In the water control group, 40% of the plants were completely inactivated due to root necrosis.

[0060] Table 4 Disease status of plants

[0061] Note: ** The difference with the water control group was extremely significant ( P <0.01), and no mark indicates no difference between the groups.

[0062] (3) Physiological and yield indicators There was no statistical difference in the survival rate and yield between the biocontrol bacteria fermentation broth treatment group and the chemical control group ( P >0.05, but both groups were significantly higher than the water control group ( P <0.01).

[0063] Table 5 Physiological and yield indicators

[0064] Note: ** The difference with the water control group was extremely significant ( P <0.01), and no mark indicates no difference between the groups.

[0065] In summary, the present invention successfully screened and identified a strain of Bacillus thuringiensis from soil samples that has a significant antagonistic effect on southern root-knot nematodes. Bacillus toyonensis ZB-201 has demonstrated promising potential for the control of southern root-knot nematodes. In the future, this strain is expected to be widely used in agricultural production, providing a new and effective means for the green control of southern root-knot nematodes. Based on the significant control efficacy demonstrated by this strain in laboratory and field trials, its application in vegetable cultivation could be further expanded beyond melons to include tomatoes, cucumbers, eggplants, and other vegetable varieties severely affected by southern root-knot nematodes. This would ensure safe vegetable production, reduce the use of chemical pesticides, lower agricultural production costs, and protect the ecological environment. Furthermore, bionematicides developed based on this strain offer advantages such as being environmentally friendly, environmentally friendly, and less susceptible to drug resistance. They are expected to become effective alternatives to chemical nematicides on the market, promoting the widespread use and application of biocontrol products in agriculture. Bacillus also promotes plant growth, with plants inoculated with Bacillus showing significant increases in plant height and fresh weight. Compared to chemical control, Bacillus biocontrol offers advantages such as environmental friendliness, resistance to drug resistance, and safety against non-target organisms. The Bacillus toyotii ZB-201 strain screened and identified by the present invention provides a new resource for biological control of southern root-knot nematodes, and has important theoretical and practical significance.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A strain of Bacillus toyotii, characterized by: The Bacillus orientalis is Bacillus orientalis ZB-201, which was deposited in the General Microbiology Center of China Culture Collection Administration Committee on February 25, 2025, and is classified and named Bacillus toyonensis , the deposit number is CGMCC NO.33653.

2. Use of the Bacillus toyotii strain according to claim 1 in preventing and controlling root-knot nematodes.

3. Use of the Bacillus toyotii strain according to claim 1 in preparing a biological agent for controlling root-knot nematodes.

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