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

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides bacillus thuringiensis with a disease prevention and growth promotion function and application of the bacillus thuringiensis in agricultural production. The bacillus thuringiensis is preserved in the China Center for Type Culture Collection in Wuhan University, Wuhan, China on April 26, 2023, and the preservation number is CCTCC NO: M20231591. The bacillus thuringiensis has good disease prevention and growth promotion effects, has an inhibition effect on various pathogenic fungi and pathogenic bacteria, can effectively prevent and treat soil-borne diseases caused by continuous cropping planting, promotes crop growth, improves crop yield and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of functional microorganism screening and application, and in particular to a Bacillus thuringiensis strain with disease prevention and growth promotion functions and application thereof. Background Art

[0002] In recent years, the cultivation area of ​​greenhouse vegetables has expanded year by year, and continuous cropping has become more serious, making the occurrence of soil-borne diseases increasingly serious. Generally, the disease leads to a reduction in production of more than 20%, and severe cases even result in a total crop failure, which seriously affects the yield and quality of vegetables. Soil-borne diseases of greenhouse vegetables refer to vegetable diseases caused by pathogens transmitted by the soil. These diseases can infect the roots or stems of vegetables through fungi, bacteria, viruses and other pathogens in the soil, resulting in stunted vegetable growth and reduced yield and quality. The prevention and control of soil-borne diseases of greenhouse vegetables is an important link in vegetable cultivation, which is related to the yield and quality of vegetables, and thus affects the economic benefits of agricultural production. There are many types of soil-borne diseases of greenhouse vegetables, including fungal diseases such as wilt, root rot, stem base rot, and sclerotinia, and bacterial diseases such as soft rot and bacterial wilt.

[0003] In the past, chemical pesticides were the main method of prevention and control. The excessive and unreasonable use of chemical pesticides has led to more serious soil problems, crop health problems, food safety problems, and environmental pollution problems. At the same time, the resulting drug resistance has increased the difficulty of prevention and control. Biological control is green, pollution-free, and sustainable. At the same time, the key to microbial control lies in the screening of functional biocontrol strains. In order to solve the contradiction between the yield and quality of greenhouse vegetables, achieve the purpose of effectively controlling continuous cropping obstacles and reducing the use of chemical pesticides, comprehensive prevention and control can be achieved through biological control, combined with agricultural control and chemical control.

[0004] Biocontrol bacteria can achieve the effect of preventing and controlling crop diseases and promoting crop growth through a variety of action mechanisms. Among them, the competitive effect is mainly reflected in the competition of biocontrol bacteria for nutrients and spatial sites. The purpose of controlling the growth and reproduction of plant pathogenic microorganisms is achieved by effectively colonizing in rhizosphere soil and crops. The stronger the colonization ability, the more significant the biocontrol effect. Producing antagonistic substances, biocontrol bacteria can metabolize and produce a variety of products such as lipopeptides, surfactants, polyketides, polyamino acids, polysaccharides, antibiotics, etc. to effectively inhibit pathogens. Inducing systemic resistance of crops, biocontrol bacteria can induce the increase of enzyme activities of host crop disease resistance-related defense enzymes such as phenylalanine ammonia lyase (PAL), peroxidase (POD), superoxide dismutase (SOD), and polyphenol oxidase (PPO), thereby improving crop resistance to diseases. Biocontrol bacteria improve the absorption and utilization of nutrients by crops through phosphorus dissolution, potassium dissolution, and nitrogen fixation. At the same time, they can secrete plant hormones such as ethylene, cytokinin, auxin, and gibberellin to directly promote crop growth.

[0005] Screening microbial strain resources with excellent performance 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 the present invention is to provide a strain of Bacillus thuringiensi with disease prevention and growth promotion functions and its application in agriculture. The Bacillus thuringiensi has good disease prevention and growth promotion effects, has a significant inhibitory effect on a variety of pathogens, can effectively prevent and control soil-borne diseases caused by continuous cropping, promote crop growth, and increase crop yields.

[0007] On the one hand, the present invention provides a Bacillus thuringiensis, which is Bacillus thuringiensis BT90 (Bacillusthuringiensis BT90), which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on September 4, 2023, with a deposit number of CCTCC NO: M20231591.

[0008] In one aspect, the present invention provides the use of the Bacillus thuringiensis BT90 strain in plant disease prevention and treatment.

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

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

[0011] The invention also provides application of the Bacillus thuringiensis BT90 strain in biofertilizer production.

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

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

[0014] The amount of live bacteria of Bacillus thuringiensis BT90 in the microbial preparation is not less than 10 9 CFU / g.

[0015] The invention also provides application of the microbial preparation in biofertilizer production.

[0016] The present invention also provides application of the microbial preparation in plant disease prevention and treatment.

[0017] The Bacillus thuringiensis BT90 screened and obtained by the present invention has the ability to produce ammonia, dissolve phosphorus, fix nitrogen, and produce siderophore. 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 the growth and development of plants.

[0018] Bacillus thuringiensis BT90 has obvious inhibitory effects on many pathogens such as tobacco black shank pathogen, cotton Verticillium wilt pathogen, bacterial wilt pathogen, strawberry red stylolite pathogen, bayberry root rot pathogen, cucumber powdery mildew pathogen, strawberry gray mold pathogen, apple ring rot pathogen, potato scab pathogen, Fusarium oxysporum pathogen, and Rhizoctonia solani. Among them, it has the best inhibitory effect on tobacco black shank pathogen, bayberry root rot pathogen, and apple ring rot pathogen. The width of the inhibition zone exceeds 23 mm, achieving unexpected technical results.

[0019] Bacillus thuringiensis BT90 can significantly increase the root length and fresh weight of seeds after germination, and improve the plant height, plant weight and yield of cucumber. Compared with the control group, the root length of cucumber seeds in the treatment group treated with Bacillus thuringiensis BT90 increased by 49.09%, and the fresh weight increased by 148.59%; the plant height, plant weight and yield increased by 63.5%, 77.7% and 17.28% respectively. Bacillus thuringiensis BT90 can achieve a control efficiency of up to 82.36% for potted cucumber wilt, and a comprehensive control efficiency of up to 80.20% for field cucumber diseases, with a very significant effect.

[0020] Bacillus thuringiensis BT90 can effectively promote celery germination, promote plant growth, and reduce the occurrence of soft rot. The germination rate, plant height and yield of celery in the treatment group treated with Bacillus thuringiensis BT90 increased by 13%, 34.4% and 16.73% respectively compared with the control group, and the control efficiency of soft rot was as high as 76.88%.

[0021] Bacillus thuringiensis BT90 can significantly promote the growth of tobacco and reduce the occurrence of tobacco black shank disease. The plant height, maximum leaf length, maximum leaf width, number of leaves, and stem circumference of tobacco in the treatment group treated with Bacillus thuringiensis BT90 increased by 9.58%, 16.22%, 28.43%, 11.46%, and 22.67% respectively compared with the control group, with 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 the present invention can be widely used in the planting of facility vegetables and cash crops, effectively improve the crop emergence rate, promote the healthy growth of crops, reduce the occurrence of soil-borne diseases, improve crop yield and quality, improve the increasingly serious continuous cropping obstacles and soil-borne diseases caused by continuous planting, and ensure the yield and quality of crops. Bacillus thuringiensis BT90 can be added to organic fertilizers, chemical fertilizers and other fertilizers for use, and combined with any one or more of other Bacillus, Pseudomonas, Agrobacterium, nitrogen-fixing bacteria, rhizobia, Penicillium, Aspergillus, Rhizopus, and Streptomyces, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the colony morphology of Bacillus thuringiensis BT90;

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

[0025] Figure 3 This is a graph showing the growth promotion index test results of Bacillus thuringiensis BT90;

[0026] Figure 4 This is a comparison chart of the effects of Bacillus thuringiensis BT90 on cucumber seed germination;

[0027] Figure 5 This is a comparison chart of the effects of Bacillus thuringiensis BT90 on cucumber cultivation;

[0028] Figure 6 This is a comparison chart of the effects of Bacillus thuringiensis BT90 on celery cultivation. DETAILED DESCRIPTION

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

[0030] The present invention is further described below in conjunction with specific embodiments.

[0031] Example 1 Isolation and screening of strains

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

[0033] 1.2 Strain Isolation

[0034] Take 10g of soil sample and put it into 90mL sterile water and shake it at 180r / min for 30min to obtain a sample mixture. The sample mixture was gradient diluted and then spread on nutrient agar medium to separate the strains in the sample mixture, and cultured at 37℃ for 48h-96h. Colonies of different shapes, colors and sizes were selected for multiple purifications to obtain 4 strains of bacteria, which were numbered QC1-QC4 and stored in liquid glycerol.

[0035] 1.3 Biocontrol strain screening

[0036] The four isolated strains were evaluated by plate antibacterial experiments, and strains with good antibacterial effects on three plant pathogens, Fusarium oxysporum, Pythium spp., and Rhizoctonia solani, were screened. A 5 mm diameter pathogen cake was inoculated in the center of the PDA medium, and 5 μL of the test bacterial solution was pipetted with a pipette and placed 3 cm away from the bacterial cake. The culture was placed in a 26°C constant temperature incubator, and the antibacterial effect was observed and measured regularly. The specific results are shown in Table 1.

[0037] Table 1 Antibacterial effect of different strains on pathogens

[0038]

[0039] From the results in Table 1, it can be seen that among the four strains screened by the present invention, the QC1 strain has the best antibacterial effect on the three pathogens. The applicant named the strain BT90 and further evaluated it.

[0040] Example 2 Identification of BT90 strain

[0041] 2.1 Colony morphology identification

[0042] The colonies of BT90 strain on nutrient agar medium are as follows Figure 1 As shown, it is milky white in color, round, with neat edges and lighter color, and a raised and sticky surface; the Bacillus bacteria are straight rod-shaped and can produce spores, which are round and positive under Gram staining microscopy.

[0043] 2.2 16S rRNA molecular identification

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

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

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

[0047] The PCR system included: 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°C for 5 min; (2) 94°C pre-denaturation for 30 s; (3) 55°C for 30 s; (4) 72°C for 1 min; perform steps (2) to (4) for 35 cycles; (5) 72°C for 10 min. The amplified PCR product was subjected to 1% agarose gel electrophoresis, and the results showed that the size of the PCR product was about 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 subjected to BLAST comparison in the NCBI database, and it had the highest similarity with Bacillus thuringiensis. Therefore, the BT90 strain was preliminarily determined to be Bacillus thuringiensis.

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

[0050] Take a small amount of BT90 single colony and apply it on the target plate in the form of a film; add 1 μL of the lysis solution in the mass spectrometry sample pretreatment kit and dry it naturally at room temperature; add 1 μL of the matrix solution in the mass spectrometry sample pretreatment kit to cover the sample and dry it naturally at room temperature; put the sample target into the mass spectrometer for identification. The identification results show that the BT90 strain is Bacillus thuringiensis, and its protein spectrum is as follows Figure 2 shown.

[0051] In summary, the applicant used two molecular biological methods, 16S rRNA identification and MALDI-TOF-MS protein spectrum identification, to identify strain BT90, and the identification results were consistent. Combined with the colony morphology of strain BT90, the applicant determined that the strain was Bacillus thuringiensis and named it Bacillus thuringiensis BT90.

[0052] The applicant deposited the above-mentioned Bacillus thuringiensis BT90 (Bacillusthuringiensis BT90) at the China Type Culture Collection of Wuhan University, Wuhan, China on April 26, 2023, with the deposit number CCTCC NO: M20231591.

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

[0054] 3.1 Preparation of bacterial culture

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

[0056] 3.2 Preparation of pathogens

[0057] A total of 9 pathogens (provided by the Plant Protection Institute of Shandong Academy of Agricultural Sciences), including tobacco black shank pathogen, cotton Verticillium wilt pathogen, tomato bacterial wilt pathogen, strawberry red stylol fungus, bayberry root rot pathogen, cucumber powdery mildew pathogen, strawberry gray mold pathogen, apple ring rot pathogen, and potato scab pathogen, were inoculated on PDA culture medium and cultured at 25°C for 5 days before use.

[0058] 3.3 Plate antibacterial test

[0059] A 9-mm diameter bacterial cake of the test pathogen was inoculated in the center of the PDA (chloramphenicol-free) culture medium, and sterilized filter paper was placed on both sides of the bacterial cake 30 mm away from the center of the culture dish. 5 ul of Bacillus thuringiensis BT90 bacterial solution was absorbed to soak the filter paper. The culture dish was then placed in a 25°C incubator and cultured for 5 days before being taken out to measure the width of the inhibition zone. Each treatment was repeated 3 times and the average value was taken.

[0060] Table 2 Antibacterial effect of Bacillus thuringiensis BT90 on different pathogens

[0061] Pathogen name Antibacterial zone width (mm) Phytophthora nicotianae 24.0±1.0 Verticillium dahliae 20.0±0.5 Ralstonia solanacearum 17.0±1.5 Rhizoctonia solani 19.5±0.5 Myrica rubrum 23.5±1.0 Cucumber powdery mildew 22.0±1.0 Botrytis cinerea 22.0±1.0 Apple ring rot 23.5±1.0 Solanum tuberosum 21.5±1.0

[0062] It can be seen from the experimental results in Table 2 that the Bacillus thuringiensis BT90 provided by the present invention has a significant inhibitory effect on the above-mentioned 9 pathogens, among which the antibacterial effect on tobacco black shank pathogen, bayberry root rot pathogen and apple ring rot pathogen is the best, and the width of the antibacterial zone exceeds 23 mm, achieving unexpected technical effects.

[0063] Example 4 Evaluation of the Growth-Promoting Potential of Bacillus thuringiensis BT90

[0064] Microorganisms can secrete growth-promoting substances that directly act on plants, such as auxin (IAA), vitamins or other small molecule metabolites, which directly promote the growth and development of plants; they can also improve the environment of the plant rhizosphere soil through their own metabolism, so that plants can better absorb and utilize the nutrients in the soil. The growth-promoting potential of the strains was evaluated by evaluating their ability to produce ammonia, dissolve phosphorus, fix nitrogen, produce siderophores and plant hormones.

[0065] 4.1 Determination of ammonia production capacity

[0066] The Bacillus thuringiensis BT90 strain was inoculated into LB liquid medium for activation, and 10 μL of the bacterial solution was inoculated into peptone ammoniation medium. The medium without inoculation of bacteria was used as the control. Each treatment was repeated 3 times, and the culture was shaken at 37±1℃ and 200rpm for 5 days. After the culture was completed, centrifuged at 10000rpm for 10 minutes, the supernatant was taken, 1mL of Nessler's reagent was added, and the solution changes were observed. If an orange or yellow precipitate was produced, it means that the strain has the ability to produce ammonia.

[0067] Results: By Figure 3 (A) It can be seen that after adding Nessler's reagent, the blank control did not change, and the supernatant of Bacillus thuringiensis BT90 bacterial solution produced an orange precipitate, indicating that the Bacillus thuringiensis BT90 strain has the ability to produce ammonia, causing amino acids to undergo deamination reactions to produce ammonia and various acids, thereby promoting plant growth and development.

[0068] 4.2 Determination of nitrogen fixation capacity

[0069] The Bacillus thuringiensis BT90 strain was inoculated into LB liquid culture medium for activation. 10 μL of the bacterial solution was inoculated into the center of the Axubei nitrogen-free culture medium plate. The culture medium without antagonistic bacteria was used as the control. Each treatment was repeated 3 times. The plate was placed in a 37±1℃ incubator and cultured for 4 days. Observe regularly. If a transparent circle appears, it means that the strain has the ability to fix nitrogen.

[0070] Results: By Figure 3 (B) It can be seen that after 4 days of cultivation, the Bacillus thuringiensis BT90 strain produced a transparent circle on the plate, indicating that it has a certain nitrogen fixation ability.

[0071] 4.3 Determination of phosphorus solubilizing capacity

[0072] The Bacillus thuringiensis BT90 strain was inoculated into LB liquid culture medium for activation. 10 μL of the bacterial solution was inoculated into the center of the Pikovaskain's culture medium plate. The uninoculated culture medium was used as the control. Each treatment was repeated 3 times and the plates were cultured in a 37±1℃ incubator for 7 days. The plates were observed regularly. If a transparent circle appeared on the edge of the strain, it indicated that the strain had the ability to dissolve phosphate.

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

[0074] 4.4 Determination of siderophore production capacity

[0075] The MSA medium and CAS colorimetric solution were mixed in a ratio of 10:1 to prepare the CAS detection plate. The Bacillus thuringiensis BT90 strain was inoculated into the LB liquid medium for activation, and 10 μL of the bacterial solution was inoculated into the center of the CAS detection plate. The uninoculated medium was used as the control. Each treatment was repeated 3 times and placed in a 37±1℃ incubator for 5 days. The plate was observed regularly. If an orange halo appeared, it indicated that the strain had the ability to produce siderophore.

[0076] Results: By Figure 3 (D) It can be seen that after cultivation, the Bacillus thuringiensis BT90 strain produces a halo on the plate, indicating that the strain has the ability to produce siderophore, which can compete with pathogens for iron and inhibit the normal growth of pathogens. At the same time, it provides iron to plants and promotes plant growth.

[0077] 4.5 Determination of IAA production capacity

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

[0079] The results showed that after 48 hours of fermentation, the IAA content in the fermentation supernatant of Bacillus thuringiensis BT90 could reach 10.6 mg / L. The IAA secreted by Bacillus thuringiensis BT90 can promote the synthesis of IAA in plants, thereby promoting the growth of crops.

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

[0081] 5.1 Preparation of bacterial culture

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

[0083] 5.2 Seed germination experiment

[0084] Select cucumber seeds with full grains and uniform size, use 1% sodium hypochlorite solution to disinfect the seed surface, soak for 1 minute, and then rinse with sterile water three times. The experiment was divided into a BT90 bacterial solution treatment group and a sterile water control group. 2mL BT90 bacterial suspension and sterile water were added to the culture dishes padded with sterile moist filter paper, and 15 cucumber seeds were placed in each dish. Repeat 3 times. The culture dish was placed in a 28℃ constant temperature incubator for 48h, and water was regularly added to maintain humidity. The 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] deal with Germination rate Root length Root length increase rate Fresh weight Fresh weight increase rate CK 100％ 5.5mm - 107mg - BT90 100％ 8.2mm 49.09％ 266mg 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, but can significantly increase the root length and fresh weight of seeds after germination, with the root length increasing by 49.09% and the fresh weight increasing by 148.59%.

[0090] Example 6 Potted experiment of Bacillus thuringiensis BT90 against cucumber wilt

[0091] 6.1 Preparation of bacterial culture

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

[0093] The cucumber wilt pathogen 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 on the plate, and the mycelium of the colony was gently scraped with a scalpel. The mycelium was soaked in water for 30 minutes and then shaken in a test tube, or ground with water and filtered to obtain a mycelium suspension.

[0094] 6.2 Potted plant experiment

[0095] The soil used for potting was sterilized. After the cucumber seedlings were raised, seedlings with basically the same growth were selected. One seedling was placed in each pot, and repeated 5 times. After transplanting, pathogen inoculation and BT90 bacterial solution inoculation were carried out. The specific grouping and treatment were as follows:

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

[0097] Treatment group 1 (BT90-1): potted plants were watered with 100 mL of Bacillus thuringiensis BT90 solution and 100 mL of water, and 100 mL of Fusarium oxysporum mycelium suspension;

[0098] Treatment group 2 (BT90-2): potted plants were watered with 200 mL of Bacillus thuringiensis BT90 solution and 100 mL of Fusarium oxysporum mycelium suspension.

[0099] The growth status of cucumber plants was observed and recorded regularly. Thirty days after transplanting, the incidence, disease index and prevention effect of cucumber wilt in each treatment were investigated.

[0100] Disease classification: Level 0: The plant has no symptoms; Level 1: The leaf edges turn yellow and wrinkled, the base of the stem turns slightly yellow, and the overall growth condition is good; Level 2: The plant grows upright, the leaves wilt slightly, and the base of the stem turns slightly yellow-brown; Level 3: The leaves of the plant wilt obviously, and the base of the stem turns brown and wilts; Level 4: The leaves of the plant wilt severely, the stem wilts and shows obvious yellow-brown symptoms; Level 5: The whole plant wilts, dies, or is obviously diseased after emergence (late emergence, death after emergence, or growth of hyphae).

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

[0102] Relative protective effect (%) = (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 effect of Bacillus thuringiensis BT90 on cucumber wilt

[0105] deal with 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] From the results in Table 4, it can be seen that compared with the control group, the cucumber wilt disease index of the treatment group treated with Bacillus thuringiensis BT90 solution was significantly reduced, and the control efficiency was as high as 82.36%. This shows that the Bacillus thuringiensis BT90 provided by the present invention can effectively control cucumber wilt with significant effect.

[0107] Example 7 Application effect of Bacillus thuringiensis BT90 on cucumber planting

[0108] 7.1 Experimental location and crop varieties:

[0109] Cucumber greenhouse in Jimo District, Qingdao City, variety Cuilong.

[0110] 7.2 Field Experiments:

[0111] The experimental plot area is 10m×4m, and each experimental plot has 8 rows of cucumbers, about 400±10 plants. The treatment group was treated with Bacillus thuringiensis BT90 powder (10 billion CFU / g), and the rest of the fertilizer, water and medicine management remained the same. Each treatment group was repeated 3 times, with a total of 12 experimental plots, and the specific groups are as follows:

[0112] Blank control group (CK): no bacterial agent was used, and water was used;

[0113] Treatment group 1: 0.5 kg / mu was applied in the planting water after transplanting and 10 days after transplanting;

[0114] Treatment group 2: Apply the fertilizer in the planting water after transplanting and 10 days after transplanting, with each application rate of 1kg / mu;

[0115] Treatment group 3: Apply the fertilizer in the planting water after transplanting and 10 days after transplanting, with each application amount of 1.5 kg / mu;

[0116] Disease grading standards: Level 0: no lesions; Level 1: lesion area accounts for less than 5% of the leaf area; Level 3: lesion area accounts for 6%-10% of the leaf area; Level 5: lesion area accounts for 11%-20% of the leaf area; Level 7: lesion area accounts for 21%-40% of the leaf area; Level 9: lesion area accounts for more than 41% of the leaf area.

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

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

[0119] During the cucumber growth period, the incidence of Fusarium wilt was regularly counted and the disease index was investigated; the plant height and plant weight of cucumbers 60 days after transplanting, the Fusarium wilt disease situation 90 days after transplanting, and the yield during the harvest period were counted, and the relative control effect and yield increase effect were calculated. The specific results are shown in Table 5.

[0120] 7.3 Experimental Results

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

[0122] deal with Plant height (cm) Plant weight(g) Output (kg) Yield increase Disease index Comprehensive prevention 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 weight of cucumbers in the treatment group treated with Bacillus thuringiensis BT90 powder were significantly increased, among which the plant height and 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 an increase rate of up to 17.28%.

[0124] In the middle and late stages of cucumber growth, wilt and powdery mildew occurred simultaneously. Both the treatment group and the control group were infected to varying degrees. The application of Bacillus thuringiensis BT90 powder can effectively reduce the occurrence of the disease. With the increase in the amount of powder, the disease index gradually decreased. Among them, the disease prevention effect of treatment group 3 cucumbers was the most significant, with a comprehensive prevention and control efficiency of up to 80.20%, achieving unexpected technical results.

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

[0126] 8.1 Experimental location

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

[0128] 8.2 Field experiments

[0129] The experimental plot area is 2m×20m, divided into two groups, and each group is repeated 5 times. The treatment group applied Bacillus thuringiensis BT90 powder (10 billion CFU / g), 100g / plot was spread when applying base fertilizer during the seedling period, 100 times diluted liquid was used for root treatment during transplanting, and 200g / plot was used for the first water irrigation after transplanting, for a total of three times; the control group (CK) was fertilized conventionally without bacterial agents. The field management measures were kept consistent. The emergence rate was investigated and recorded during the seedling period, the growth of crops and disease conditions were investigated after transplanting, and the yield was investigated during the harvest period to calculate the yield increase effect.

[0130] Celery soft rot disease grading standard: Level 0, the whole plant is disease-free; Level 1, a small number of water-soaked spots appear on a few stems or petioles; Level 2, a few or stems and root bases have serious water-soaked spots; Level 3, most root necks have rot spots; Level 4, most root necks are diseased, causing the plant to rot and eventually die.

[0131] 8.3 Experimental Results

[0132] Table 6 Application effect of Bacillus thuringiensis BT90 on celery cultivation

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

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

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

[0136] 9.1 Experimental location

[0137] Tobacco fields in Jimo District, Qingdao City, Shandong Province.

[0138] 9.2 Field experiments

[0139] The experimental area is 6 mu, divided into 3 treatment groups, each with 2 mu of land, divided into a blank control group and an experimental group treated with Bacillus thuringiensis BT90 powder, and the rest of the fertilizer, water and medicine management remain the same. The specific groups are as follows:

[0140] Blank control group (CK): no bacterial agent was used, and water was used;

[0141] Treatment group 1: Root irrigation was carried out at the transplanting stage and the clustering 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 clustering stage, with a dosage of 2 kg / mu each time;

[0143] In September, when tobacco matured, the parallel line sampling method was used to investigate the growth and disease status of tobacco. 300 tobacco plants were investigated for each treatment. Growth status investigation: the plant height, maximum leaf length, leaf width, number of leaves and stem girth of tobacco were measured, and the average values ​​were calculated; disease status investigation: according to the classification standard of tobacco black shank disease, the disease status of tobacco was recorded, and the disease index and disease prevention effect were calculated.

[0144] Grading standards for tobacco black shank disease: Grade 0, the whole plant is disease-free; Grade 1, stem lesions do not exceed 1 / 3 of the stem circumference, or less than 1 / 3 of the leaves are withered; Grade 3, stem lesions surround 1 / 3 to 1 / 2 of the stem circumference, or 1 / 3 to 1 / 2 of the leaves are slightly withered, or lesions appear on a few leaves at the bottom; 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 are withered; Grade 7, stem lesions completely surround the stem circumference, or more than 2 / 3 of the leaves are withered; 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 plants treated × 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 of tobacco in the field

[0149]

[0150] From the experimental results in Table 7, it can be seen that Bacillus thuringiensis BT90 can significantly promote the growth of tobacco. The plant height, maximum leaf length, maximum leaf width, number of leaves, and stem circumference of the tobacco in the treatment group increased by 9.58%, 16.22%, 28.43%, 11.46%, and 22.67% respectively compared with the control group, and the growth-promoting effect was significant. Bacillus thuringiensis BT90 can also effectively reduce the occurrence of tobacco black shank disease and reduce the disease index, with a control efficiency of up to 70.8%.

[0151] In summary, the Bacillus thuringiensis BT90 provided by the present invention can be widely used in the planting of facility vegetables and cash crops, effectively improve the crop emergence rate, promote the healthy growth of crops, reduce the occurrence of soil-borne diseases, improve crop yield and quality, improve the increasingly serious continuous cropping obstacles and soil-borne diseases caused by continuous planting, and ensure the yield and quality of crops. Bacillus thuringiensis BT90 can be added to organic fertilizers, chemical fertilizers and other fertilizers for use, and combined with any one or more of other Bacillus, Pseudomonas, Agrobacterium, nitrogen-fixing bacteria, rhizobia, Penicillium, Aspergillus, Rhizopus, and Streptomyces, and has broad application prospects.

Claims

1. A Bacillus thuringiensis, characterized in that The preservation number of the Bacillus thuringiensis is CCTCC NO: M20231591.

2. Use of the Bacillus thuringiensis according to claim 1 in preventing and controlling plant diseases.

3. The use according to claim 2, characterized in that The plant disease is any one of tobacco black shank pathogen, cotton verticillium wilt pathogen, tomato bacterial wilt pathogen, strawberry red stylolite pathogen, bayberry root rot pathogen, cucumber powdery mildew pathogen, strawberry gray mold pathogen, apple ring rot pathogen, potato scab pathogen, cucumber wilt pathogen and celery soft rot pathogen.

4. Use of the Bacillus thuringiensis according to claim 1 in the production of biocontrol agents.

5. Use of the Bacillus thuringiensis according to claim 1 in biofertilizer production.

6. A microbial preparation, characterized in that: The microbial preparation comprises the Bacillus thuringiensis according to 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 according to claim 6 or 7, characterized in that The amount of live bacteria of Bacillus thuringiensis in the microbial preparation is not less than 10 9 CFU / g.

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

10. Use of the microbial preparation according to any one of claims 6 to 8 in preventing and controlling plant diseases.

Images