Bacillus velezensis and application thereof in crop planting

Abstract

The invention discloses bacillus velezensis and application thereof in crop planting, the bacillus velezensis QL2 is preserved in the China General Microbiological Culture Collection Center on June 18, 2024, the preservation number is CGMCC No.30997, the preservation address is Institute of Microbiology, China, Beijing, Chinese Academy of Sciences, and the preservation number is CGMCC No.30997. The bacillus velezensis QL2 has the preservation number of CGMCC No.30997. The bacillus velezensis QL2 disclosed by the invention can grow in an acid stress environment with the pH value of 4, an alkali stress environment with the pH value of 9 and a 6% NaCl stress environment, can reduce the content of aflatoxin in soil, and has an obvious antibacterial effect on aspergillus clavatus, so that the growth of peanuts is promoted, and the quality of the peanuts is improved.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to Bacillus Velezii and application thereof in crop planting. Background Art

[0002] Crops refer to various plants cultivated in agriculture, including food crops and cash crops (oil crops, vegetable crops, flowers, grasses, and trees). Edible crops are one of the basic food sources for humans. Peanut is an important oil crop.

[0003] Peanut is a dicotyledonous plant and a leguminous crop. It is rich in fat, protein, dietary fiber, unsaturated fatty acids, carbohydrates and minerals, and is one of the world's five major oil-bearing economic crops. Peanut ranks third in the planting area of ​​major oil crops in my country, and it is also the only oil crop that can achieve self-sufficiency. Due to economic benefits and land restrictions, continuous planting is commonly used in modern agriculture. However, long-term continuous planting can lead to an imbalance in the structure of soil microbial communities, an increase in pathogens and slow crop growth, which is often referred to as "continuous cropping disorder." There are many reasons for the continuous cropping disorder of peanuts, including the accumulation of soil autotoxic substances and the destruction of the soil microbial community structure. Due to the continuous planting of peanut crops on limited arable land and the high frequency of intensive planting, continuous cropping of peanuts is inevitable.

[0004] The problem of continuous cropping of peanuts has harmed the growth and yield of peanuts, and even posed a threat to the ecological health of cultivated soil.

[0005] At present, Bacillus Velez has the prospect of being widely used in biological control of plant diseases and promoting plant growth. For example, CN108949614 A discloses a microbial agent of Bacillus Velez, which can effectively prevent and control peanut root rot and help reduce the use and residue of chemical agents in agricultural products. However, the existing disclosed Bacillus Velez has poor effects in prevention and control and promotion of plant growth, and there is no report on inhibiting the activity of Aspergillus clavatus. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a Bacilus velezensis QL2.

[0009] To solve the above technical problems, the present invention provides the following technical solution: a Bacillus velezensis QL2, deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on June 18, 2024, with a deposit number of CGMCC No.30997, and a deposit address of: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] As a preferred embodiment of the Bacillus Velez subtilis of the present invention, the Bacillus Velez subtilis can tolerate an acid stress environment of pH=4, an alkali stress environment of pH=9 and a 6% NaCl stress environment.

[0011] Another object of the present invention is to overcome the deficiencies in the prior art and provide an application of Bacillus Velezii in crop planting.

[0012] As a preferred embodiment of the application of the present invention, the activated Bacillus velezensis QL2 is prepared into a bacterial suspension;

[0013] The bacterial suspension is used to irrigate the roots of crops at a dosage of 200-300 L / hm2. 2 The concentration of the bacterial agent is 10 5 CFU / mL~10 8 CFU / mL.

[0014] As a preferred embodiment of the application of the present invention, the Bacillus Velezii inhibits the activity of Aspergillus clavatus in the soil where crops are planted, promotes the growth of crops, and improves the quality of crops.

[0015] As a preferred embodiment of the application of the present invention, the crops include peanuts.

[0016] Beneficial effects of the present invention:

[0017] The present invention provides a Bacillus velezensis QL2, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 18, 2024, with a deposit number of CGMCC No. 30997, and a deposit address of Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. The Bacillus velezensis QL2 of the present invention can grow in an acid stress environment of pH=4, an alkaline stress environment of pH=9, and a 6% NaCl stress environment, can reduce the aflatoxin content in the soil, has an obvious antibacterial effect on Aspergillus clavatus, thereby promoting the growth of peanuts and improving the quality of peanuts. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0019] Figure 1 This is a morphological observation diagram of Bacillus Velez in an embodiment of the present invention.

[0020] Figure 2 This is a phylogenetic tree of Bacillus Velezii QL2 based on 16S r DNA in the examples of the present invention.

[0021] Figure 3 Graphs showing the growth data of Bacillus Velez QL2 under stress in the examples of the present invention, wherein the left graph shows the results of a salt tolerance test of QL2, and the right graph shows the results of an acid-base tolerance test.

[0022] Figure 4 This is a test of the growth-promoting properties of Bacillus Velez QL2 in the examples of the present invention.

[0023] Figure 5 These are the growth indicators of peanut seedlings under different treatments in the embodiments of the present invention, wherein CK refers to the potted treatment without the application of microbial agents, and T refers to the potted treatment with the application of microbial agents.

[0024] Figure 6 It is the soil fungi composition (genus) in the embodiments of the present invention.

[0025] Figure 7 It is the aflatoxin content in the soil in the embodiment of the present invention.

[0026] Figure 8 This is a diagram showing the growth-promoting effect of Bacillus Velez on peanuts in an embodiment of the present invention, wherein the left is a control group, the middle is a comparison group, and the right is an experimental group. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0028] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0030] The Bacilus velezensis QL2 of the present invention was deposited in the General Microbiology Center of the China Culture Collection Administration on June 18, 2024, with the deposit number CGMCC No. 30997, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0031] Example 1: Isolation and screening of Bacillus Velez

[0032] The soil came from the peanut planting base in Laiyang City, Shandong Province. According to the S-type 5-point sampling method, 0-20 cm of the tillage layer of soil was collected from each plot and mixed evenly. The collected soil samples were placed in glycerol tubes and transported to the laboratory at low temperature for use.

[0033] 5.0 g of soil sample stored at -80 °C was weighed under sterile conditions, mixed with 100.0 mL of sterile saline, and cultured on a shaking table for 4 h to disperse the microbial cells. -1 Dilutions of different concentrations were prepared and serial dilutions were performed.

[0034] Take 0.1mL of each dilution to 10 -4 , 10 -5 and 10 -6 The dilution was inoculated onto LB solid medium, and the plate was placed in a 28°C constant temperature incubator for 48 hours. Each test was repeated three times. The growth of microorganisms on the plate was observed, and a single bacterial colony was picked and repeatedly streaked to purify the pure strain, which was then stored in a -80°C refrigerator in a glycerol tube for later use.

[0035] Streak the plate on LB medium and incubate at 28℃ for 48h. Observe the colony morphology: colony size, color, shape, colony edge, protrusion, etc. Figure 1 .

[0036] Based on the occurrence of common peanut diseases, the plant pathogen Fusarium oxysporum was used as an indicator bacteria to screen biocontrol bacteria. The cell-free supernatant of the cultured strain was used for the Oxford cup antibacterial test. 10.0 mL of 2% plain agar was used as the bottom culture medium, and the Oxford cup was placed vertically on the plain agar. When the upper culture medium (0.8% agar semi-solid PDA culture medium) was cooled to 40 ± 3 ° C, the indicator bacteria (concentration 10 6 ~10 7 CFU / mL) and mix well, pour 20.0mL onto plain agar to solidify. Take out the Oxford cup, add 200.0μL of cell-free supernatant of the cultured strain into the remaining circular hole, and add 200.0μL of sterile PDA medium to the control group. Place the culture dish in a 4℃ refrigerator for diffusion for 4h and then culture at 37℃ for 72h. Repeat each test three times. Use a vernier caliper to measure the size of the inhibition zone to determine whether it has antibacterial properties, measure and record the results.

[0037] The picked colonies were irregular in shape, with rough wrinkles on the surface, opaque, irregular edges, and dirty white. They were short rod-shaped under a 100x optical microscope and were positive for Gram staining.

[0038] The results of the antibacterial test showed that the diameter of the transparent zone produced by strain QL2 reached 17.91±0.02mm.

[0039] The physiological and biochemical results of the screened strain QL2 showed that it was catalase positive, methyl red negative, and VP test positive. It could hydrolyze starch, did not produce gas, and did not utilize citrate. The screened strain could not ferment galactose, maltose, sucrose, trehalose, melibiose, lactose, cellobiose, and starch; it could assimilate glucose, fructose, sucrose, mannitol, and maltose.

[0040] Example 2: Molecular Biological Identification of Bacillus Velez

[0041] The activated LB solid culture medium of strain QL2 was placed in a clean bench to remove the hyphae, and DNA was extracted according to the instructions of the bacterial genomic DNA kit, and then used for PCR amplification and detection. PCR amplification was performed with universal primers for bacterial 16S rRNA gene sequence (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The PCR amplification product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The PCR reaction program was: 94℃10min; 94℃30s, 55℃30s, 72℃2min, 35 cycles; 72℃5min. The PCR reaction system was: template 1μL,,, Premix Taq (Ex Taq Version 2.0) 25.0μL, Forward Primer (10pmol / μL) 1.0μL, Reverse Primer (10pmol / μL) 1.0μL, ddH2O Up to 50μL. The sequencing results were assembled using ContigExpress and the inaccurate parts at both ends were removed. The assembled sequences were compared in the NCBI database (blast.ncbi.nlm.nih.gov). Figure 2 The identification result showed that strain QL2 was Bacillus velezensis.

[0042] The nucleotide sequence of Bacillus velez in the present invention (Seq-1) is:

[0043] GTGTTAGGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATC CTAGAGATAGGACGTCCCCTTCGGGGGCAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGAC

[0044] Example 3: Determination of stress resistance of Bacillus Velez

[0045] Pour 2 mL of bacterial solution frozen at -80°C into 100 mL of LB liquid culture medium, and culture at 28°C with a shaker at 120 rpm for 48 h. Then, inoculate the cultured bacterial solution into 100 mL of fresh LB liquid at a ratio of 2%, and culture at 28°C with a shaker at 120 rpm until OD600 = 0.8, which is considered to be completed.

[0046] The activated strain was inoculated in batches with 2% inoculation into LB liquid medium containing: 0%, 1%, 2%, 4%, 6%, 8% NaCl (w / w); LB liquid medium with pH of 4, 5, 6, 7, 8, 9. After culturing for 24 hours, the OD 600 The measurement results are as follows Figure 3 .

[0047] The tolerance limit thresholds of NaCl, acid and alkali were found to be 6% (w / w), 4 and 9, respectively.

[0048] Example 4: Determination of growth-promoting properties of Bacillus velez

[0049] In order to clarify the growth-promoting performance of Bacillus Velez QL2, its ability to fix nitrogen, dissolve phosphorus, dissolve potassium, and produce the growth hormone indoleacetic acid (IAA) was determined, and each test was repeated three times. 200uL of strain QL2 culture solution was inoculated on Asbby solid medium (0.2g dipotassium hydrogen phosphate, 0.2g MgSO4·7H2O, 0.2g sodium chloride, 5.0g calcium carbonate, 10.0g mannitol, 0.1g calcium sulfate, 20.0g agar powder, and distilled water was added to make up to 1000.0mL) plates, and the colony growth was checked after culturing at 28°C for 5 days to confirm its nitrogen fixation ability. Inoculate 200uL of strain QL2 culture solution on NBRIP solid medium (glucose 10.0g, ammonium sulfate 0.1g, MgSO4·7H2O0.25g, potassium chloride 0.2g, MgCl2·6H2O 5.0g, Ca3(PO4)5.0g, agar powder 20.0g, add distilled water to make up to 1000.0mL) plate, and after culturing at 28℃ for 5 days, check whether a transparent zone of phosphate solubilization is formed around the colony to determine its phosphate solubilization ability. Inoculate 200uL of strain QL2 culture solution on an Alexander silicate solid medium (sucrose 5.0g, Na2HPO42.0g, MgSO4·7H2O 0.5g, ferric chloride 0.005g, calcium carbonate 0.1g, potassium feldspar 2.0g, agar powder 20.0g, add distilled water to make up to 1000.0mL) plate, and after culturing at 28℃ for 5 days, check whether a potassium-soluble transparent zone is formed around the colony to determine its potassium-soluble ability. Inoculate 200uL of strain QL2 culture medium into YMB liquid medium (yeast powder 1.0g, mannitol 10.0g, K2HPO4 0.5g, MgSO4·7H2O 0.2g, sodium chloride 0.1g, calcium carbonate 1.0g, L-tryptophan 2.0g, add distilled water to make up to 1000.0mL), culture at 28℃ for 48h, centrifuge at 8000r / min for 10min, take out 2.0mL of supernatant and put it into a test tube, add the same amount of Salkowski's reagent (0.50mol / LFeCl3 10.0mL and 35% HClO4 500.0mL), mix and stand in the dark for 30 minutes. Mix YMB liquid medium and Salkowski's reagent as a blank control. If the color turns red, it means that indoleacetic acid (IAA) is produced. The test results are as follows Figure 4 shown.

[0050] Bacillus Velez QL2 can hardly grow normally on Asbby solid medium plates, so it cannot use nitrogen in the air, indicating that it lacks the ability to fix nitrogen. On NBRIP solid medium plates and Alexander silicate medium plates, Bacillus Velez QL2 grows abnormally, and no transparent circles for phosphorus and potassium dissolution are observed, indicating that it does not have the ability to dissolve phosphorus and potassium. The supernatant of Bacillus Velez QL2 grown in YMB medium showed a significant red color after reaction with Salkowski's reagent, indicating that it can use tryptophan to synthesize indoleacetic acid (IAA). Indoleacetic acid (IAA) is a plant growth hormone that is widely present in plants and can promote plant growth and affect plant growth metabolism. It plays an important role in plant growth. IAA can stimulate the growth and development of crop root hairs and lateral roots, thereby increasing the root surface area and increasing the absorption of nutrients by crops. Although Bacillus Velez QL2 cannot fix nitrogen, dissolve phosphorus, or dissolve potassium, it can produce IAA, promote the growth and development of crops, and indirectly improve the disease resistance of crops.

[0051] Example 5: Bacillus Velezii promotes the growth of peanut seedlings

[0052] Luhua No. 28 peanut seeds (provided by Shandong Luhua Group Co., Ltd.) were selected and sown in pots with a diameter of 10 cm filled with peanut continuous cropping soil (obtained from the peanut planting experimental base in Laiyang City, Shandong Province). Three peanut seeds were sown in each pot according to the regular triangle cultivation method.

[0053] The activated strain QL2 was inoculated into LB medium at a 2% inoculum and cultured in a shaker at 28°C and 150 r / min for 24 h. After the culture, centrifuge at 4°C and 8000 r / min for 10 min, discard the supernatant and retain the bacteria, add an appropriate amount of distilled water, and mix well. The effective concentration of the bacterial suspension of strain QL2 was adjusted to 10 6 CFU / mL.

[0054] After the peanuts emerged, pots with uniform growth were selected and two treatments were performed: the control group was potted without the application of fungicide (CK), and the experimental group was potted with the application of fungicide (T).

[0055] 10 ml of bacterial suspension of strain QL2 was used for root irrigation, and an equal volume of sterile distilled water was used as a control. Each treatment was repeated five times, and water was applied every 2 days and the bacterial agent was applied every 5 days.

[0056] The main agronomic indicators of the plants were measured after 30 days of growth, including plant height (cm), leaf area (cm 2 ), stem diameter (cm) and root diameter (cm).

[0057] The leaf area (cm2) of peanut was measured using a portable laser leaf area meter (CI-202, Walz, Camas, USA).2 ); plant height was measured with a ruler, and stem and root diameters of peanut were measured with a chrome-plated vernier caliper (CHILON 3C2370).

[0058] Compared with the control group, the plant height, leaf area, stem diameter and root diameter of the T group increased by approximately 10.87%, 43.77%, 21.74% and 9.31% respectively. Among them, the leaf area of ​​the T group showed a significant difference (P<0.05). Figure 5 , indicating that Bacillus Velez QL2 has a significant promoting effect on the growth of peanut seedlings.

[0059] Example 6: Control of peanut seedling root rot by Bacillus Velez

[0060] Healthy peanut seedlings that had been cultured for 30 days and had uniform growth were selected. After cleaning, they were disinfected with 75% alcohol for 15 seconds, 30% sodium hypochlorite solution for 1 minute, and then washed three times with sterile water.

[0061] Peanut seedlings were transplanted into new pots, with one peanut seedling planted in each pot, and a pot infection test with pathogenic fungus Fusarium oxysporum (purchased from China Center for Type Culture Collection, accession number CCTCCAF 2012001) was conducted.

[0062] Four treatments were performed: the blank group was potted plants not infected with pathogens, the negative group was potted plants infected with pathogens, the treatment group was potted plants infected with pathogens and then treated with microbial agents, and the prevention group was potted plants infected with pathogens after microbial agents were applied. Each treatment was repeated five times, for a total of 20 pots.

[0063] The specific operation was as follows: the blank group was watered with 30.0 mL of sterile distilled water, and the roots of the peanut seedlings in the negative group were watered with 5.0 mL of pathogen suspension (concentration of 10 6 CFU / mL) and 25.0 mL of sterile distilled water. The roots of the peanut seedlings in the treatment group were first watered with 5.0 mL of pathogen suspension for 24 h and then with 25.0 mL of bacterial agent (concentration of 10 6 CFU / mL), the roots of the peanut seedlings in the prevention group were first irrigated with 25.0 mL of the fungal agent (concentration of 10 6 CFU / mL) 24h later, 5.0mL of pathogen suspension was poured in.

[0064] Daily management was watering once every 1-2 days. After 30 days of growth, the disease index and control effect of seedling root rot under different treatments were observed and calculated.

[0065] The disease resistance of strain QL2 to peanut seedling root rot was determined and graded according to the following criteria: Grade 0 means healthy roots with no disease; Grade 1 means surface rot of the roots, with a rot area ≤10%; Grade 2 means the roots are rotten to the inside, but the lateral roots are not rotten, with a rot area of ​​10% to 40%; Grade 3 means the roots are rotten to the inside, and the lateral roots are rotten, with a rot area of ​​40% to 50%; Grade 4 means the roots are rotten to the inside, and the lateral roots are rotten and fall off, with a rot area of ​​50% to 70%; Grade 5 means the roots are rotten to the inside, and the roots are completely rotten, the crops wilt, fall over, or even die, with a rot area ≥70%.

[0066] The disease index was calculated according to the following formula:

[0067]

[0068] The test results are shown in Table 1.

[0069] Table 1

[0070]

[0071] Compared with the negative control group, Bacillus Velez QL2 in both treatment groups could significantly reduce the severity of the disease.

[0072] In the treatment group, the QL2 strain had a therapeutic effect of 36.52% on peanut seedling root rot, and in the prevention group, the QL2 strain had a control effect of 63.09% on peanut seedling root rot. It can be seen that strain QL2 has a good control effect on peanut seedling root rot, and the preventive effect is stronger than the therapeutic effect. This may be because Bacillus Velez QL2 can protect peanut seedlings from pathogenic fungi by secreting a large amount of antifungal organic compounds.

[0073] Example 7: Field trial of Bacillus velez

[0074] The experiment was conducted at the Luhua peanut planting experimental base in Laiyang City, Shandong Province. The field experiment used an isolation belt to separate the control group from the comparison group and the experimental group. The planting area of ​​each group was 0.33hm 2 (5 mu), the peanut variety used in the experiment (Luhua No. 28 peanut seeds, provided by Shandong Luhua Group Co., Ltd.), sowing method and management method were consistent.

[0075] There are three treatment groups: the control group was treated with ordinary fertilizer (commercially available triple compound fertilizer) at a fertilizer dosage of 450 kg / hm2. 2 ;

[0076] The control group applied the same amount of common fertilizer and added 300L / hm 2The amount of microbial agent used (Bacillus velezensis strain Bacilus velezensis, CICC 24099, purchased from China Industrial Microbiological Culture Collection Management Center), the concentration of the agent was 10 6 CFU / mL, 300L / hm 2 Mix the dosage with common base fertilizer and spread it evenly on the soil with machinery;

[0077] The experimental group applied the same amount of common fertilizer and added 300L / hm 2 Dosage of microbial agent (test strain QL2).

[0078] Peanuts were planted on May 15, 2022, and harvested on September 5, 2022. Soil samples were collected at the sowing, flowering, and maturity stages. According to the S-type 5-point sampling method, 0-20 cm of the tillage layer of soil was collected from each plot, mixed, and stored at -80°C for microbial analysis.

[0079] Soil genomic DNA was extracted using the Fast Soil DNA Kit. The final DNA concentration and purification were determined using a Nano Drop NC-2000 UV–vis spectrophotometer, and DNA purity was checked using 1% agarose gel electrophoresis.

[0080] The V3–V4 region of 16S rRNA in DNA samples was amplified using the following bacterial primers: 338F (ACTCCTACGGGGGGCAGCA) and 806R (GGACTACHVGGGTWTCTAAT). Fungal rRNA was amplified in the fungal ITS sequence region using barcoded primers ITS1F (CTTGGTCATTTAGAGGAAGAAGTAA) and ITS2R (GCTGCGTTCTTCATCGATGC).

[0081] PCR amplification program: 95°C pre-denaturation for 3 min, 27 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 45 s), 72°C stable extension for 10 min. The library construction and sequencing of PCR products were completed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0082] The results of the analysis were as follows Figure 7 The abundance of Aspergillus in the soil samples of the flowering period experimental group was significantly lower than that of the control group.

[0083] Aflatoxins are metabolites produced by Aspergillus flavus and Aspergillus parasiticus. The World Health Organization has identified aflatoxins as Class I human carcinogens, of which aflatoxin B1 is the main toxin and the strongest liver cancer carcinogen discovered so far. Peanuts are easily contaminated by aflatoxins, which not only damages the economy but is also harmful to the body.

[0084] Determination of aflatoxin content in soil samples: Take 10g soil sample, grind and sieve, add 20ml 70% methanol solution, shake vigorously for 10 minutes, filter with filter paper, absorb the supernatant and dilute it a certain multiple, and use ELISA kit to detect the toxin content. The results are as follows: Figure 6 As shown. The aflatoxin content in the soil samples of the control group during the flowering and maturity stages was higher than that of the experimental group. The aflatoxin content in the soil samples of the experimental group during the flowering stage (0.225ppb) was lower than that in the soil samples of the control group (0.239ppb), and the aflatoxin content in the soil samples of the experimental group during the maturity stage (0.257ppb) was lower than that in the soil samples of the control group (0.284ppb). The application of Bacillus Velez can reduce the content of aflatoxin, which is beneficial to both the economy and life.

[0085] Patulin is a fungal toxin that is widely found in fruits such as apples and pears, nuts, and Chinese medicinal materials. It has nephrotoxic, hepatotoxic, immunotoxic, and even carcinogenic effects on the human body.

[0086] Determination of the inhibition rate of Bacillus Velez against Aspergillus clavatus.

[0087] Determine the antibacterial rate by plate mixed culture method: Take 100uL 1x10 6 Add 100 μL of 1x10 / mL Velez QL2 suspension to a culture dish (90 mm in diameter). When LB cools to about 45°C, add 10 mL of LB medium to the culture dish with the Velez suspension, shake and mix, and then inoculate 100 μL of 1x10 / mL LB medium in the center of the culture dish containing LB. 6 / mL Aspergillus clavatus spore suspension, and a control group in which an equal amount of sterile water was substituted for the Bacillus velezensis QL2 bacterial suspension;

[0088] A control group was set up in which the commercially available Bacillus Velez subtilis strain CICC 24099 replaced the bacterial suspension of Bacillus Velez subtilis QL2. Each of the above treatments was set up with 4 replicates and cultured at 37°C for 5 days. The inhibition diameter was measured by the cross method.

[0089] The antibacterial rate calculation formula is as follows

[0090]

[0091] The test results are shown in Table 2.

[0092] Table 2

[0093]

[0094] In the plate mixed culture test, Bacillus Velez QL2 had a significant antibacterial effect on Aspergillus clavatus, with an antibacterial rate of 85.3%. Bacillus Velez was evenly distributed on the surface and inside of the plate, showing a sticky white and raised colony morphology. The hyphae of Aspergillus clavatus were yellow-green and white, and the hyphae length was significantly shortened. Bacillus Velez CICC 24099 had no obvious antibacterial effect on Aspergillus clavatus. The hyphae of Aspergillus clavatus were yellow-green and white, and the hyphae grew well. Patulin is a fungal toxin that is widely found in fruits such as apples and pears, as well as nuts and Chinese medicinal materials. It has nephrotoxicity, hepatotoxicity, immunotoxicity, and even carcinogenicity to the human body.

[0095] The abundance of Aspergillus in the soil samples of the experimental group decreased significantly. The application of Bacillus Velez QL2 can reduce the content of aflatoxin in the soil. Bacillus Velez QL2 has a significant antibacterial effect on Aspergillus clavatus. Bacillus Velez QL2 reduces the content of mycotoxins and affects the biosynthesis of mycotoxins, thereby promoting the growth of peanuts and improving the quality of peanuts.

[0096] The growth of peanuts under three different experimental conditions was measured. The results are as follows: Figure 8 , as shown in Table 7.

[0097] Table 7

[0098]

[0099] Compared with the control group, the experimental group was less contaminated by mold and the leaf area increased by 9.5 cm 2 ; Stem diameter increased by 0.07cm; Root length increased by 2.5cm; Fruit number increased by 11; Total fruit weight increased by 93g.

[0100] Compared with the control group, the leaf area of ​​the control group increased by 4.1cm 2 ; Stem diameter increased by 0.02cm; Root length increased by 1.1cm; Fruits increased by 4; Total fruit weight increased by 18.6g. The growth-promoting effect of the experimental group was better than that of the control group, and the growth-promoting effect of Bacillus Velez QL2 was higher than that of the commercially available Bacillus Velez.

[0101] The increase in leaf area helps chlorophyll synthesis, thereby improving photosynthetic efficiency and increasing yield; the increase in stem diameter and root length is conducive to the robust growth of peanut roots, which fully absorbs water and nutrients, and improves peanut yield and quality. The experimental group has increased fruit yield and total fruit. The application of Bacillus Velez QL2 can increase peanut yield and improve peanut quality, and the effect is higher than that of commercially available Bacillus Velez.

[0102] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. Bacillus Velez Bacillus velezensis ) QL2, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 18, 2024, with the deposit number CGMCC No.30997, and the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The Bacillus Velezii of claim 1, characterized in that: The Bacillus Velezii is tolerant to an acid stress environment of pH=4, an alkali stress environment of pH=9, and a 6% NaCl stress environment.

3. Use of the Bacillus Velezii described in claim 1 or 2 in crop planting.

4. The use according to claim 3, characterized in that: Including, activated Bacillus Velez Bacillus velezensis ) QL2 is prepared into a bacterial suspension; The bacterial suspension is used to irrigate the roots of crops at a dosage of 200~300 L / hm 2 , the concentration of the bacterial agent is 10 5 CFU / mL~10 8 CFU / mL.

5. The use according to claim 3, characterized in that: The Bacillus Velezii inhibits the activity of Aspergillus clavatus in the soil where crops are planted, promotes the growth of crops and improves the quality of crops.

6. The use according to claim 5, characterized in that: The crops include peanuts.

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