Bacillus thuringiensis with disease prevention and growth promotion functions and application thereof

Abstract

The present application relates to the technical field of functional microorganism screening and application, and specifically provides a bacillus thuringiensis with disease prevention and growth promotion functions Bacillus thuringiensis ) and application thereof in agricultural production. The bacillus thuringiensis has been preserved in the China Center for Type Culture Collection of Wuhan University in Wuhan, China, on April 26, 2023, with a preservation number of CCTCC NO: M20231591. The bacillus thuringiensis has good disease prevention and growth promotion effects, has inhibitory effects on various pathogenic fungi and pathogenic bacteria, can effectively prevent and control soil-borne diseases caused by continuous cropping, promote crop growth, increase crop yield, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of functional microbial screening and application technology, specifically to a strain of Bacillus thuringiensis with disease prevention and growth promotion functions and its application. Background Technology

[0002] In recent years, the cultivation area of ​​greenhouse vegetables has been expanding year by year, and continuous cropping has intensified, leading to increasingly serious soil-borne diseases. These diseases generally result in yield reductions of over 20%, and in severe cases, even total crop failure, seriously affecting vegetable yield and quality. Soil-borne diseases in greenhouse vegetables refer to vegetable diseases caused by pathogens transmitted through the soil. These diseases can infect the roots or stems of vegetables through fungi, bacteria, viruses, and other pathogens in the soil, hindering vegetable growth and reducing yield and quality. The prevention and control of soil-borne diseases in greenhouse vegetables is a crucial aspect of vegetable cultivation, affecting vegetable yield and quality, and consequently impacting the economic benefits of agricultural production. There are many types of soil-borne diseases in greenhouse vegetables, including fungal diseases such as wilt, root rot, stem base rot, and sclerotinia rot, and bacterial diseases such as soft rot and bacterial wilt.

[0003] Previously, pest control primarily relied on chemical pesticides. However, the excessive and inappropriate use of chemical pesticides has led to more serious soil problems, crop health issues, food safety problems, and environmental pollution. Furthermore, the resulting pesticide resistance has increased the difficulty of control. Biological control is characterized by being green, pollution-free, and sustainable. The key to microbial inoculant control lies in the screening of functional biocontrol strains. To resolve the contradiction between yield and quality in greenhouse vegetables, and to effectively control continuous cropping obstacles while reducing the use of chemical pesticides, a comprehensive approach combining biological control with agricultural and chemical control methods can be adopted.

[0004] Biocontrol bacteria can achieve the effects of controlling crop diseases and promoting crop growth through multiple mechanisms of action. Among these, the competitive effect is mainly reflected in the competition of biocontrol bacteria for nutrients and space sites. By effectively colonizing the rhizosphere soil and crops, they control the growth and reproduction of plant pathogenic microorganisms; the stronger the colonization ability, the more significant the biocontrol effect. They also produce antagonistic substances, metabolizing into various products such as lipopeptides, surfactants, polyketides, polyamino acids, polysaccharides, and antibiotics to effectively inhibit pathogens. Furthermore, they induce systemic resistance in crops, increasing the activity of disease-resistant defense enzymes in host crops, such as phenylalanine ammonia-lyase (PAL), peroxidase (POD), superoxide dismutase (SOD), and polyphenol oxidase (PPO), thereby enhancing crop resistance to diseases. Finally, biocontrol bacteria improve the absorption and utilization of nutrients by crops through phosphorus solubilization, potassium solubilization, and nitrogen fixation. They also secrete plant hormones such as ethylene, cytokinins, auxins, and gibberellins, directly promoting crop growth.

[0005] Screening high-performance microbial strains is of great significance for the promotion and application of microbial agents in agricultural production and is also a research hotspot in this field. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Bacillus thuringiensis with disease prevention and growth-promoting functions and its application in agriculture. The Bacillus thuringiensis exhibits good disease prevention and growth-promoting effects, significantly inhibits various pathogens, effectively prevents soil-borne diseases caused by continuous cropping, promotes crop growth, and increases crop yield.

[0007] In one aspect, this invention provides a Bacillus thuringiensis strain, specifically Bacillus thuringiensis BT90, which was deposited on September 4, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, with accession number CCTCC NO: M20231591.

[0008] This invention provides, in one aspect, the application of the Bacillus thuringiensis BT90 strain in the prevention and control of plant diseases.

[0009] The plant diseases mentioned include any one of the following: tobacco black shank fungus, cotton verticillium wilt fungus, tomato bacterial wilt fungus, strawberry red stele fungus, waxberry root rot fungus, cucumber powdery mildew fungus, strawberry gray mold fungus, apple ring rot fungus, potato scab fungus, cucumber wilt, and celery soft rot.

[0010] The present invention also provides the application of the Bacillus thuringiensis BT90 strain in the production of biocontrol agents.

[0011] The present invention also provides the application of the Bacillus thuringiensis BT90 strain in bio-fertilizer production.

[0012] The present invention also provides a microbial preparation comprising Bacillus thuringiensis strain BT90.

[0013] The microbial preparation also includes any one or more combinations of Bacillus, Pseudomonas, Agrobacterium, nitrogen-fixing bacteria, Rhizobium, Penicillium, Aspergillus, Rhizopus, and Streptomyces.

[0014] The viable count of Bacillus thuringiensis BT90 strain in the microbial preparation is not less than 10. 9 CFU / g.

[0015] This invention also provides the application of the above-mentioned microbial preparations in the production of bio-fertilizers.

[0016] This invention also provides the application of the above-mentioned microbial agents in the prevention and control of plant diseases.

[0017] The Bacillus thuringiensis BT90 obtained by screening in this invention has the ability to produce ammonia, solubilize phosphorus, fix nitrogen, and produce iron. After fermentation culture for 48 hours, the indoleacetic acid (IAA) content in the fermentation supernatant can reach 10.6 mg / L, which can effectively promote plant growth and development.

[0018] Bacillus thuringiensis BT90 exhibits significant inhibitory effects against a variety of pathogens, including tobacco black shank fungus, cotton verticillium wilt fungus, bacterial wilt fungus, strawberry red columnar rot fungus, waxberry root rot fungus, cucumber powdery mildew fungus, strawberry gray mold fungus, apple ring rot fungus, potato scab fungus, Fusarium oxysporum, and Rhizoctonia solani. Among these, the inhibitory effect is the best against tobacco black shank fungus, waxberry root rot fungus, and apple ring rot fungus, with an inhibition band width exceeding 23 mm, achieving unexpected technical results.

[0019] Bacillus thuringiensis BT90 significantly increased the root length and fresh weight of seeds after germination, thereby improving cucumber plant height, plant weight, and yield. Compared with the control group, the cucumber seed root length in the Bacillus thuringiensis BT90 treatment group increased by 49.09%, and the fresh weight increased by 148.59%; plant height, plant weight, and yield increased by 63.5%, 77.7%, and 17.28%, respectively. Bacillus thuringiensis BT90 showed a control efficiency of up to 82.36% against Fusarium wilt in potted cucumbers and an integrated control efficiency of up to 80.20% against diseases in field-grown cucumbers, demonstrating highly significant effects.

[0020] Bacillus thuringiensis BT90 can effectively promote celery emergence, enhance plant growth, and reduce the incidence of soft rot. In the treatment group treated with Bacillus thuringiensis BT90, the celery emergence rate, plant height, and yield increased by 13%, 34.4%, and 16.73%, respectively, compared to the control group, with a control efficiency of up to 76.88% against soft rot.

[0021] Bacillus thuringiensis BT90 significantly promotes tobacco growth and reduces the incidence of tobacco black shank disease. In the treatment group treated with Bacillus thuringiensis BT90, the plant height, maximum leaf length, maximum leaf width, number of leaves, and stem circumference increased by 9.58%, 16.22%, 28.43%, 11.46%, and 22.67% respectively compared to the control group, demonstrating a significant growth-promoting effect. Bacillus thuringiensis BT90 can also effectively reduce the disease index, with a control efficiency of up to 70.8%.

[0022] The Bacillus thuringiensis BT90 provided by this invention can be widely used in the cultivation of greenhouse vegetables and cash crops. It effectively improves crop emergence rate, promotes healthy crop growth, reduces soil-borne diseases, increases crop yield and quality, and alleviates the increasingly serious replanting obstacles and soil-borne diseases caused by continuous cropping, thus ensuring crop yield and quality. Bacillus thuringiensis BT90 can be added to organic fertilizers, chemical fertilizers, and other fertilizers, and can be combined with any one or more of other Bacillus, Pseudomonas, Agrobacterium, nitrogen-fixing bacteria, Rhizobium, Penicillium, Aspergillus, Rhizopus, and Streptomyces, showing broad application prospects. Attached Figure Description

[0023] Figure 1 This is a colony morphology diagram of Bacillus thuringiensis BT90.

[0024] Figure 2 The protein spectrum of Bacillus thuringiensis BT90;

[0025] Figure 3 The graph shows the results of the growth-promoting indicators of Bacillus thuringiensis BT90.

[0026] Figure 4 Comparative graph showing the effects of Bacillus thuringiensis BT90 on cucumber seed germination;

[0027] Figure 5 Comparison of the effects of Bacillus thuringiensis BT90 on cucumber cultivation;

[0028] Figure 6 Comparison of the effects of Bacillus thuringiensis BT90 on celery cultivation. Detailed Implementation

[0029] For the specific methods or materials used in the embodiments, those skilled in the art can make conventional substitutions based on existing technologies, and not be limited to the specific descriptions in the embodiments of the present invention.

[0030] The present invention will be further described below with reference to specific embodiments.

[0031] Example 1: Isolation and Screening of Strains

[0032] 1.1 Sample source: Rhizosphere soil of celery from Majiagou, Pingdu City, Qingdao.

[0033] 1.2 Strains Isolation

[0034] Take 10g of soil sample and place it in 90mL of sterile water. Shake at 180rpm for 30min to obtain a sample mixture. After serial dilution, the sample mixture is spread on nutrient agar medium for isolating bacterial strains. Incubate at 37℃ for 48-96h. Select colonies of different morphology, color, and size for multiple purifications to obtain 4 strains, which are numbered QC1-QC4 and preserved in liquid glycerol.

[0035] 1.3 Screening of biocontrol strains

[0036] Four isolated strains were evaluated using a plate inhibition test to screen for strains with good inhibitory effects against three plant pathogens: Fusarium oxysporum, Pythium spp., and Rhizoctonia solani. A 5 mm diameter bacterial cake was inoculated in the center of a PDA medium. 5 μL of the test bacterial solution was pipetted onto the cake 3 cm away and incubated at 26°C. The inhibitory effect was observed and measured periodically. Specific results are shown in Table 1.

[0037] Table 1. Antibacterial effects of different strains against pathogens.

[0038]

[0039] As shown in Table 1, among the four strains screened in this invention, strain QC1 showed the best antibacterial effect against all three pathogens. The applicant named this strain BT90 and conducted further evaluation on it.

[0040] Example 2 Identification of BT90 strain

[0041] 2.1 Colony morphology identification

[0042] Colonies of BT90 strain on nutrient agar medium as shown Figure 1 As shown, the color is milky white, round, with neat edges and a lighter color, and the surface is raised and viscous; the Bacillus cells are straight rod-shaped and can produce spores, which are round and Gram stain positive under microscopic examination.

[0043] 2.2 Molecular identification of 16S rRNA

[0044] The genome of strain BT90 was extracted using a kit. Then, using this genome as a template, its 16S rRNA was amplified using specific primers 27F and 1492R.

[0045] 27F: 5'-AGAGTTTGATCATGGCTCAG-3';

[0046] 1492R: 5'-TAGGGTTACCTTACGACTT-3'.

[0047] The PCR system consisted of: 0.7 μl 27F, 0.7 μl 1492R, 4 μl template DNA, 17.5 μl SuperMiX, and 12.1 μl water. The PCR reaction conditions were set as follows: (1) 94℃ for 5 min; (2) 94℃ pre-denaturation for 30 s; (3) 55℃ for 30 s; (4) 72℃ for 1 min; and steps (2) to (4) were repeated for 35 cycles; (5) 72℃ for 10 min. The amplified PCR products were detected by 1% agarose gel electrophoresis. The results showed that the PCR product size was approximately 1500 bp, which met the requirements.

[0048] The PCR amplification product was sent to a sequencing company for sequencing. The obtained 16S rRNA sequence SEQ ID NO: 1 was compared with the NCBI database using BLAST, and it showed the highest similarity to Bacillus thuringiensis. Therefore, strain BT90 was preliminarily identified as Bacillus thuringiensis.

[0049] 2.3 MALDI-TOF-MS protein spectroscopy identification

[0050] A small amount of BT90 single colony was coated onto a target plate in the form of a thin film; 1 μL of lysis buffer from the mass spectrometry sample pretreatment kit was added, and the sample was allowed to air dry at room temperature; 1 μL of matrix solution from the mass spectrometry sample pretreatment kit was added to cover the sample, and the sample was allowed to air dry at room temperature; the sample target was then placed in a mass spectrometer for identification. The identification results showed that the BT90 strain was *Bacillus thuringiensis*, and its protein spectrum is shown below. Figure 2 As shown.

[0051] In summary, the applicant used two molecular biology methods—16S rRNA identification and MALDI-TOF-MS protein spectrometry—to identify strain BT90, and the results were consistent. Combined with the colony morphology characteristics of strain BT90, the applicant identified the strain as Bacillus thuringiensis and named it Bacillus thuringiensis BT90.

[0052] The applicant deposited the aforementioned Bacillus thuringiensis BT90 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China on April 26, 2023, with accession number CCTCC NO: M20231591.

[0053] Example 3: Evaluation of the antibacterial effect of Bacillus thuringiensis BT90

[0054] 3.1 Preparation of bacterial culture

[0055] Bacillus thuringiensis BT90 was activated, and the activated Bacillus thuringiensis BT90 was picked and inoculated into nutrient broth medium and cultured at 37℃ and 220 r / min for 18 h to obtain a viable count of 10. 8 -10 9 CFU / ml bacterial culture.

[0056] 3.2 Preparation of pathogens

[0057] Nine pathogens, including tobacco black shank fungus, cotton verticillium wilt fungus, tomato bacterial wilt fungus, strawberry red columnar rot fungus, waxberry root rot fungus, cucumber powdery mildew fungus, strawberry gray mold fungus, apple ring rot fungus, and potato scab fungus (provided by the Plant Protection Institute of Shandong Academy of Agricultural Sciences), were inoculated onto PDA medium and incubated at 25°C for 5 days before use.

[0058] 3.3 Plate Antibacterial Test

[0059] Inoculate the center of a 9 mm diameter bacterial cake of the test pathogen into the PDA (chloramphenicol-free) medium. Place sterilized filter paper discs on both sides of the bacterial cake, 30 mm from the center of the culture dish. Soak the filter paper discs with 5 μL of Bacillus thuringiensis BT90 bacterial solution. Then, place the culture dish in a 25°C incubator for 5 days and remove it to measure the width of the inhibition zone. Each treatment is repeated 3 times, and the average value is taken.

[0060] Table 2. Antibacterial effects of Bacillus thuringiensis BT90 against different pathogens.

[0061] Pathogen name Inhibition zone width (mm) Tobacco black shank pathogen 24.0±1.0 Cotton verticillium wilt pathogen 20.0±0.5 Tomato bacterial wilt pathogen 17.0±1.5 Strawberry red center stem pathogen 19.5±0.5 Waxberry root rot pathogen 23.5±1.0 Cucumber powdery mildew pathogen 22.0±1.0 Strawberry gray mold pathogen 22.0±1.0 Apple ring rot pathogen 23.5±1.0 Potato scab pathogen 21.5±1.0

[0062] As shown in Table 2, the Bacillus thuringiensis BT90 provided by this invention has a significant inhibitory effect on all nine pathogens mentioned above. Among them, the inhibitory effect on Tobacco Black Shank Bacterium, Myrica rubra rot Bacterium, and Apple Ring Spot Bacterium is the best, with an inhibition band width of more than 23 mm, achieving unexpected technical results.

[0063] Example 4: Evaluation of the growth-promoting potential of Bacillus thuringiensis BT90

[0064] Microorganisms can secrete growth-promoting substances that directly affect plants, such as auxins (IAA), vitamins, or other small-molecule metabolites, thereby directly promoting plant growth and development. They can also improve the rhizosphere soil environment through their own metabolic processes, enabling plants to better absorb and utilize nutrients from the soil. The growth-promoting potential of microorganisms is evaluated by assessing their ability to produce ammonia, solubilize phosphorus, fix nitrogen, produce hematophiles, and generate plant hormones.

[0065] 4.1 Ammonia production capacity determination

[0066] Bacillus thuringiensis BT90 strain was activated by inoculation into LB liquid medium. 10 μL of the bacterial culture was then inoculated into peptone-ammoniated medium, with uninoculated medium serving as a control. Each treatment was repeated three times. The culture was carried out at 37±1℃ with shaking at 200 rpm for 5 days. After incubation, the culture was centrifuged at 10000 rpm for 10 min, and the supernatant was collected. 1 mL of Nessler's reagent was added, and the solution changes were observed. The formation of an orange or yellow precipitate indicated that the strain possessed ammonia-producing ability.

[0067] Result: From Figure 3 (A) It can be seen that after adding Nessler's reagent, the blank control showed no change, and the supernatant of Bacillus thuringiensis BT90 bacterial solution produced an orange precipitate, indicating that Bacillus thuringiensis BT90 strain has the ability to produce ammonia, which causes amino acids to undergo deamination reaction to generate ammonia and various acids, thereby promoting plant growth and development.

[0068] 4.2 Nitrogen fixation capacity determination

[0069] Bacillus thuringiensis BT90 strain was inoculated into LB liquid medium for activation. 10 μL of bacterial suspension was inoculated into the center of Assoube nitrogen-free medium plates. Medium without antagonistic bacteria was used as a control. Each treatment was repeated 3 times. The plates were placed in an incubator at 37±1℃ for 4 days. Regular observation was performed. If a clear zone appeared, it indicated that the strain had nitrogen-fixing ability.

[0070] Result: From Figure 3 (B) It can be seen that after 4 days of culture, Bacillus thuringiensis BT90 strain produced a clear zone on the plate, indicating that it has a certain nitrogen-fixing ability.

[0071] 4.3 Phosphorus solubility determination

[0072] Bacillus thuringiensis BT90 strain was activated by inoculating it into LB liquid medium. 10 μL of bacterial solution was then inoculated into the center of a Pikovaskain's medium plate, with uninoculated medium as a control. Each treatment was repeated 3 times. The plates were incubated at 37±1℃ for 7 days. The plates were observed regularly. If a clear zone appeared at the edge of the strain, it indicated that the strain had phosphate-solubilizing ability.

[0073] Result: From Figure 3 (C) It can be seen that after cultivation, Bacillus thuringiensis BT90 strain produced a transparent zone on the plate, indicating that the strain has the ability to dissolve insoluble phosphorus in the soil, which is beneficial to the absorption of nutrients by plants and promotes plant growth.

[0074] 4.4 Determination of Ferrophilic Production Capacity

[0075] Prepare CAS detection plates by mixing MSA medium and CAS chromogenic solution at a ratio of 10:1. Inoculate Bacillus thuringiensis BT90 strain into LB liquid medium for activation. Inoculate 10 μL of the bacterial suspension into the center of the CAS detection plate, using uninoculated medium as a control. Repeat each treatment three times. Incubate at 37±1℃ for 5 days. Observe the plates regularly; the appearance of an orange-yellow halo indicates that the strain has the ability to produce heptaphiles.

[0076] Result: From Figure 3 (D) It can be seen that after cultivation, the Bacillus thuringiensis BT90 strain produced a halo on the plate, indicating that the strain has the ability to produce iron, which can compete with pathogens for iron, inhibit the normal growth of pathogens, and at the same time provide iron for plants and promote plant growth.

[0077] 4.5 IAA Production Capacity Determination

[0078] Bacillus thuringiensis BT90 strain was inoculated at a 5% inoculum into LB liquid medium containing 5% NaCl and cultured at 30℃ and 180 r / min for 48 h. The fermentation broth was centrifuged at 10000 rpm for 10 min to remove the bacterial cells. 2 mL of the supernatant was mixed with an equal volume of Salkowski reagent, and the mixture was placed in the dark. After 30 min, the absorbance of the solution was measured at a wavelength of 530 nm. The IAA content in the fermentation broth was calculated according to the IAA standard curve.

[0079] As a result, after 48 hours of fermentation culture with Bacillus thuringiensis BT90, the IAA content in the fermentation supernatant reached 10.6 mg / L. The IAA secreted by Bacillus thuringiensis BT90 can promote the synthesis of IAA in plants, thereby promoting crop growth.

[0080] Example 5: Effect of Bacillus thuringiensis BT90 on cucumber seed germination

[0081] 5.1 Preparation of bacterial culture

[0082] Bacillus thuringiensis BT90 was activated, and the activated Bacillus thuringiensis BT90 was picked and inoculated into nutrient broth medium and cultured at 37℃ and 220 r / min for 18 h to obtain a viable count of 10. 8 -10 9 CFU / ml bacterial culture.

[0083] 5.2 Seed germination experiment

[0084] Select plump, uniformly sized cucumber seeds and disinfect their surface with a 1% sodium hypochlorite solution, soaking for 1 minute, followed by rinsing three times with sterile water. The experiment was divided into a BT90 bacterial suspension treatment group and a sterile water control group. 2 mL of BT90 bacterial suspension and sterile water were added to petri dishes lined with sterile, moistened filter paper, respectively, with 15 cucumber seeds placed in each dish, repeated three times. The petri dishes were placed in a 28℃ constant temperature incubator for 48 hours, with water added periodically to maintain humidity. Root length, fresh weight, and germination rate of the cucumber seeds were observed and recorded.

[0085] Germination rate (%) = (Number of germinated seeds / Total number of seeds) * 100%.

[0086] 5.3 Experimental Results

[0087] Table 3 Effects of Bacillus thuringiensis BT90 on cucumber seed germination

[0088] Treatment Germination rate Root length Root length increase rate Fresh weight Fresh weight increase rate CK 100％ 5.5 mm - 107 mg - BT90 100％ 8.2 mm 49.09％ 266 mg 148.59％

[0089] From Table 3 and Figure 4 The experimental results show that Bacillus thuringiensis BT90 has no inhibitory effect on cucumber seed germination, and can significantly increase the root length and fresh weight of the seeds after germination, with the root length increasing by 49.09% and the fresh weight increasing by 148.59%.

[0090] Example 6: Pot experiment on cucumber wilt caused by Bacillus thuringiensis BT90

[0091] 6.1 Preparation of bacterial culture

[0092] Bacillus thuringiensis BT90 was activated, and the activated Bacillus thuringiensis BT90 was picked and inoculated into nutrient broth medium and cultured at 37℃ and 220 r / min for 18 h to obtain a viable count of 10. 8 -10 9 CFU / ml bacterial culture.

[0093] The pathogen of cucumber wilt, Fusarium oxysporum, was purified and cultured on PDA medium. After culturing at 25°C for 5 days, a small amount of sterile water was poured onto the plate, and the colony mycelium was gently scraped off with a scalpel. The mycelium was then soaked in water for 30 minutes and then shaken in a test tube, or ground with water and filtered to obtain a mycelial suspension.

[0094] 6.2 Potted Plant Experiment

[0095] The soil used for potted plants was sterilized. After cucumber seedling cultivation, seedlings with similar growth were selected, one seedling per pot, repeated 5 times. After transplanting, pathogen inoculation and BT90 bacterial suspension inoculation were performed. The specific grouping and treatment are as follows:

[0096] Control group (CK): Potted plants were watered with 200 mL of water and 100 mL of Fusarium oxysporum mycelium suspension;

[0097] Treatment group 1 (BT90-1): Potted plants were irrigated with 100 mL of Bacillus thuringiensis BT90 bacterial solution and 100 mL of water, and 100 mL of Fusarium oxysporum mycelial suspension.

[0098] Treatment group 2 (BT90-2): Potted plants were irrigated with 200 mL of Bacillus thuringiensis BT90 bacterial solution and 100 mL of Fusarium oxysporum mycelial suspension.

[0099] Regularly observe and record the growth status of cucumber plants. 30 days after transplanting, investigate the incidence, disease index, and control efficacy of cucumber wilt in each treatment.

[0100] Disease grading: Grade 0: No symptoms of disease; Grade 1: Leaf edges turn yellow and wrinkled, stem base turns slightly yellow, overall growth is good; Grade 2: Plants grow upright, leaves show slight wilting, stem base turns slightly yellowish-brown; Grade 3: Plant leaves wilt significantly, stem base turns brown and wilts; Grade 4: Plant leaves wilt severely, stems wilt and show obvious yellowish-brown symptoms; Grade 5: The whole plant wilts, dies, or is obviously diseased after emergence (late emergence, death after emergence, or mycelium growth).

[0101] Disease index = [∑(disease grade value × number of diseased leaves at that grade) / (total number of leaves surveyed × highest disease grade value)] × 100.

[0102] Relative efficacy (%) = (Disease index of control group - Disease index of treatment group) / Disease index of control group × 100%.

[0103] 6.3 Experimental Results

[0104] Table 4. Control efficacy of Bacillus thuringiensis BT90 against cucumber wilt.

[0105] Treatment Disease incidence (%) Disease index Control efficiency CK 96.67 82.2 - Treatment group 1 66.5 21.6 73.72％ Treatment group 2 60.2 14.5 82.36％

[0106] As shown in Table 4, compared with the control group, the disease index of cucumber wilt was significantly reduced in the treatment group treated with Bacillus thuringiensis BT90 bacterial solution, with a control efficiency as high as 82.36%. This demonstrates that the Bacillus thuringiensis BT90 provided by this invention can effectively control cucumber wilt with significant results.

[0107] Example 7: Application effect of Bacillus thuringiensis BT90 in cucumber cultivation

[0108] 7.1 Experimental site and crop varieties:

[0109] Cucumber greenhouses in Jimo District, Qingdao City, featuring the Cuilong variety.

[0110] 7.2 Field Experiment:

[0111] The experimental plots were 10m × 4m in size, with 8 rows of cucumbers and approximately 400 ± 10 plants per plot. The treatment groups were treated with Bacillus thuringiensis BT90 inoculum powder (10 billion CFU / g), while other fertilizer, water, and pesticide management remained consistent. Each treatment group was replicated three times, for a total of 12 experimental plots, as follows:

[0112] Blank control group (CK): No bacterial agent was used; the bacteria were applied by watering.

[0113] Treatment Group 1: Apply 0.5 kg / mu each time by watering after transplanting and by irrigation 10 days after transplanting.

[0114] Treatment Group 2: Apply 1 kg / mu each time by watering after transplanting and by irrigation 10 days after transplanting.

[0115] Treatment group 3: Apply 1.5 kg / mu each time by watering after transplanting and by irrigation 10 days after transplanting.

[0116] Disease grading standards: Grade 0: No lesions; Grade 1: Lesions cover less than 5% of the leaf area; Grade 3: Lesions cover 6%-10% of the leaf area; Grade 5: Lesions cover 11%-20% of the leaf area; Grade 7: Lesions cover 21%-40% of the leaf area; Grade 9: Lesions cover more than 41% of the leaf area.

[0117] Disease index = [∑(disease grade value × number of diseased leaves at that grade) / (total number of leaves surveyed × highest disease grade value)] × 100.

[0118] Relative efficacy = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0119] During the cucumber growing season, the incidence of Fusarium wilt was regularly recorded, and the disease index was investigated. Plant height and weight were recorded 60 days after transplanting, Fusarium wilt disease status was recorded 90 days after transplanting, and yield was recorded at harvest. The relative control effect and yield increase were calculated. Specific results are shown in Table 5.

[0120] 7.3 Experimental Results

[0121] Table 5. Application effects of Bacillus thuringiensis BT90 on cucumber.

[0122] Treatment Plant height (cm) Plant weight (g) Yield (kg) Yield increase rate Disease index Comprehensive control effect CK 134.1 130 644.5 - 44.5 - Treatment group 1 189.5 170 698.9 8.44％ 16.6 62.71％ Treatment group 2 212.0 210 724.3 12.38％ 11.2 74.83％ Treatment group 3 219.3 231 755.9 17.28％ 8.8 80.20％

[0123] From Table 5 and Figure 5 The results showed that, compared with the control group, the plant height and plant weight of cucumbers treated with Bacillus thuringiensis BT90 powder were significantly increased. In particular, the plant height and plant weight of cucumbers in treatment group 3 increased by 63.5% and 77.7%, respectively. At the same time, the yield of cucumbers in the treatment group was also greatly increased, with a yield increase rate as high as 17.28%.

[0124] During the mid-to-late stages of cucumber growth, wilt and powdery mildew occurred simultaneously. Both the treatment group and the control group showed varying degrees of disease infection. Applying Bacillus thuringiensis BT90 powder effectively reduced the occurrence of the disease. Furthermore, as the amount of powder increased, the disease index gradually decreased. The disease control effect was most significant in treatment group 3 cucumbers, with a comprehensive control efficiency of up to 80.20%, achieving unexpected technical results.

[0125] Example 8: Evaluation of the application effect of Bacillus thuringiensis BT90 in celery cultivation

[0126] 8.1 Experimental Location

[0127] Celery greenhouses in Majiagou, Pingdu City, Qingdao.

[0128] 8.2 Field Experiment

[0129] The experimental plots were 2m × 20m in size, divided into two groups, with each group replicated five times. The treatment group received Bacillus thuringiensis BT90 powder (10 billion CFU / g). 100g / plot was applied during the seedling stage when tilling and applying basal fertilizer, a 100-fold diluted solution was applied to the roots at transplanting, and 200g / plot was applied as a root drench during the first irrigation after transplanting, for a total of three applications. The control group (CK) received conventional fertilization without any inoculant. Field management practices were kept consistent. Emergence rate was recorded during the seedling stage, crop growth and disease incidence were monitored after transplanting, and yield was assessed at harvest to calculate the yield increase.

[0130] Grading standards for celery soft rot: Level 0: No disease in the whole plant; Level 1: A few stems or petioles show a small number of water-soaked lesions; Level 2: A few stems or roots show severe water-soaked lesions; Level 3: Most root collars show rotten lesions; Level 4: Most root collars are infected, causing the plant to rot and eventually die.

[0131] 8.3 Experimental Results

[0132] Table 6. Application effects of Bacillus thuringiensis BT90 in celery cultivation

[0133] Group Emergence rate Plant height Disease index Control effect Yield CK 84％ 12.8 cm 37.2 - 125.5 kg Treatment group 97％ 17.2 cm 8.6 76.88％ 146.5 kg

[0134] From Table 6 and Figure 6 The experimental results show that Bacillus thuringiensis BT90 can effectively promote celery seedling emergence, promote plant growth, and reduce the occurrence of soft rot. The seedling emergence rate, plant height, and yield of celery in the treatment group increased by 13%, 34.4%, and 16.73%, respectively, and the control efficiency against soft rot was as high as 76.88%.

[0135] Example 9: Application effect of Bacillus thuringiensis BT90 in tobacco cultivation

[0136] 9.1 Experimental Location

[0137] Yantian, Jimo District, Qingdao City, Shandong Province.

[0138] 9.2 Field Experiment

[0139] The experimental plot covered a total area of ​​6 mu (approximately 0.4 hectares), divided into 3 treatment groups, each with 2 mu (approximately 0.3 hectares) of land. One group was a blank control group, and the other was treated with Bacillus thuringiensis BT90 powder. All other fertilizer, water, and pesticide management practices remained consistent. The specific groupings are as follows:

[0140] Blank control group (CK): No bacterial agent was used; the bacteria were applied by watering.

[0141] Treatment Group 1: Root irrigation was carried out at the transplanting stage and the seedling stage, with a dosage of 1 kg / mu each time;

[0142] Treatment Group 2: Root irrigation was carried out at the transplanting stage and the seedling stage, with a dosage of 2 kg / mu each time;

[0143] During the tobacco ripening period in September, a parallel line sampling method was used to investigate the growth status and disease incidence of tobacco plants. 300 tobacco plants were surveyed for each treatment. Growth status survey: Plant height, maximum leaf length, leaf width, number of leaves, and stem circumference were measured, and average values ​​were calculated. Disease incidence survey: According to the tobacco black shank disease grading standard, the disease incidence was recorded, and the disease index and disease control effect were calculated.

[0144] Grading standards for tobacco black shank disease: Grade 0: No disease on the whole plant; Grade 1: Stem lesions do not exceed 1 / 3 of the stem circumference, or less than 1 / 3 of the leaves wither; Grade 3: Stem lesions surround 1 / 3 to 1 / 2 of the stem circumference or 1 / 3 to 1 / 2 of the leaves with slight wilting, or a few leaves on the lower part of the plant have lesions; Grade 5: Stem lesions exceed 1 / 2 of the stem circumference, but do not completely surround the stem circumference, or 1 / 2 to 2 / 3 of the leaves wither; Grade 7: Stem lesions completely surround the stem circumference, or more than 2 / 3 of the leaves wither; Grade 9: The plant is basically dead.

[0145] Disease index = (∑(number of diseased plants at each level × representative value at each level) × 100) / (total number of treated plants × highest representative level)

[0146] Disease prevention effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0147] 9.3 Experimental Results

[0148] Table 7. Effects of different treatments on the growth and disease status of field tobacco.

[0149]

[0150] Table 7 shows that Bacillus thuringiensis BT90 significantly promoted tobacco growth. Compared to the control group, the plant height, maximum leaf length, maximum leaf width, number of leaves, and stem circumference of the treated tobacco plants increased by 9.58%, 16.22%, 28.43%, 11.46%, and 22.67%, respectively, demonstrating a significant growth-promoting effect. Bacillus thuringiensis BT90 also effectively reduced the incidence of tobacco black shank disease, lowering the disease index, with a control efficiency of up to 70.8%.

[0151] In summary, the Bacillus thuringiensis BT90 provided by this invention can be widely used in the cultivation of greenhouse vegetables and cash crops, effectively improving crop emergence rate, promoting healthy crop growth, reducing soil-borne diseases, increasing crop yield and quality, and mitigating the increasingly serious replanting obstacles and soil-borne diseases caused by continuous cropping, thus ensuring crop yield and quality. Bacillus thuringiensis BT90 can be added to organic fertilizers, chemical fertilizers, and other fertilizers, and can be combined with any one or more of other Bacillus, Pseudomonas, Agrobacterium, nitrogen-fixing bacteria, Rhizobium, Penicillium, Aspergillus, Rhizopus, and Streptomyces, showing broad application prospects.

Claims

1. A Bacillus thuringiensis strain, characterized in that, The Bacillus thuringiensis has the accession number CCTCC NO: M20231591.

2. The application of Bacillus thuringiensis as described in claim 1 in the prevention and control of plant diseases.

3. The application as described in claim 2, characterized in that, The plant diseases mentioned are any one of the following: tobacco black shank fungus, cotton verticillium wilt fungus, tomato bacterial wilt fungus, strawberry red stele fungus, waxberry root rot fungus, cucumber powdery mildew fungus, strawberry gray mold fungus, apple ring rot fungus, potato scab fungus, cucumber wilt, and celery soft rot.

4. The application of Bacillus thuringiensis as described in claim 1 in the production of biocontrol agents.

5. The application of Bacillus thuringiensis as described in claim 1 in the production of bio-fertilizer.

6. A microbial preparation, characterized in that, The microbial preparation comprises Bacillus thuringiensis as described in claim 1.

7. The microbial preparation according to claim 6, characterized in that, The microbial preparation further comprises any one or more combinations of Bacillus, Pseudomonas, Agrobacterium, nitrogen-fixing bacteria, Rhizobium, Aspergillus, Rhizopus, and Streptomyces.

8. The microbial preparation as described in claim 6 or 7, characterized in that, The viable count of Bacillus thuringiensis in the microbial preparation is not less than 10. 9 CFU / g.

9. The application of the microbial preparation according to any one of claims 6-8 in the production of biofertilizer.

10. The use of the microbial preparation according to any one of claims 6-8 in the prevention and control of plant diseases.

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