Bacillus velezensis and application thereof in disease prevention and growth promotion

Abstract

The invention discloses a bacillus velezensis strain and an application thereof in disease prevention and growth promotion, the strain is preserved in the China General Microbiological Culture Collection Center on May 14, 2024, the preservation number is CGMCC No.30619, and the strain is named as bacillus velezensis. The invention further discloses a preparation method of the bacillus velezensis strain, and the application of the bacillus velezensis strain in disease prevention and growth promotion of the bacillus velezensis strain in disease prevention and growth promotion of the bacillus velezensis strain in disease prevention and growth promotion of the bacillus velezensis strain. When the strain is applied to the field of agricultural biological prevention and control, a biological membrane can be formed and stably colonized at the rhizosphere of a plant, a relatively strong and broad-spectrum inhibition effect can be generated on various plant pathogenic fungi, and substances such as IAA and siderophores can be generated to promote plant growth. The bacillus velezensis suspension has a prevention effect of 56.36% on cucumber fusarium wilt potted plants, has a certain growth promoting effect on cucumbers, is a biocontrol strain with excellent performance, and has good development and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural biological control, and specifically relates to a strain of Bacillus velezensis W-107 with disease prevention and growth promotion functions and application thereof. Background Art

[0002] Plant diseases have posed a major threat to global food production and security. Over-reliance on the use of chemical pesticides and fertilizers has long led to increasingly prominent problems such as increased environmental pollution, enhanced bacterial resistance, and imbalance in soil microecology. Therefore, there is an urgent need to explore microbial products that can replace chemical pesticides and fertilizers. Although there are many microbial products on the market, they often face problems such as low colonization ability and short effective period in field applications, which limits their actual application effect. Therefore, exploring microorganisms with broad-spectrum and high-efficiency characteristics from plant-related microbial communities such as the rhizosphere and root endophytes has become a hot topic for scholars at home and abroad.

[0003] In recent years, Bacillus has attracted widespread attention for its unique advantages. Bacillus is a Gram-positive bacterium, which usually has the characteristics of broad antibacterial spectrum, rapid growth, easy cultivation, strong stress resistance and high biosafety. These characteristics make Bacillus have broad application prospects in agriculture, food, industry, medicine and other fields. Among them, Bacillus Velezii is widely distributed in nature and can be isolated from a variety of environments. This strain is harmless to humans and animals, does not pollute the environment, and can produce secondary metabolites that have antagonistic effects on a variety of pathogenic microorganisms. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of Bacillus velezensis W-107 with disease prevention and growth promotion functions and application thereof in agricultural production.

[0005] The Bacillus velezensis W-107 provided by the present invention was deposited in the General Microbiology Center of the China Culture Collection Committee (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​May 14, 2024, with a deposit number of CGMCC No. 30619.

[0006] The fermentation broth or fermentation product of Bacillus velezensis W-107 is also within the scope of protection of the present invention. Preferably, the fermentation medium is LBGM medium or LBM medium.

[0007] The stable colonization of the above-mentioned Bacillus velezensis W-107 in soil and plant roots is also within the scope of protection of the present invention.

[0008] The use of the above-mentioned Bacillus velezensis W-107 fermentation liquid or fermentation product in the preparation of plant antibacterial agents is also within the scope of protection of the present invention. In the application of the preparation of the antibacterial agent, the relevant pathogens are corn blight (Helminthosporium turcicum Pass), pepper phytophthora (Phytophthora capsici), apple anthracnose (Glomerella ciningulaia), cotton wilt (Verticillium dahliae), wheat fusarium (Fusarium graminearum), sorghum black spot (Setosphaeria turcica (Luttr.) Leonard & Suggs), rice sheath blight (Rhizoctonia solani), apple ring pattern (Botryospuaeria berengeriana), potato dry rot (Fusarium oxysporum Schlecht.), tomato early blight (Alternaria solani), tomato gray mold (Botrytis cinerea), red date black spot (Isariopsis imdica var. Ziziphi), tobacco red star (Alteraria alternata), apple rot (Valsa mali Miyabe et al. Yamada), Sclerotiniasclerotiorum, Geratobasidium cornigeru (Bourd.) Rogers, Fusarium oxysporum f.sp.niveum or Fusarium oxysporum.sp.cucumebriumOwen.

[0009] The application of the fermentation liquid or fermentation product of Bacillus velezensis W-107 in the preparation of a plant growth promoting agent is also within the scope of protection of the present invention. In this application, the growth promoting properties are IAA and siderophore.

[0010] The use of the above-mentioned Bacillus velezensis W-107 fermentation liquid or fermentation product in the preparation of plant disease prevention and growth promotion preparations or organic fertilizers is also within the scope of protection of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the developmental tree of Bacillus velezensis W-107 provided in Example 1 of the present invention.

[0012] Figure 2 It is the antibacterial activity of Bacillus velezensis W-107 provided in Example 2 of the present invention against 18 pathogenic fungi; A represents corn blight; B represents pepper phytophthora; C represents apple anthrax; D represents cotton yellow board; E represents wheat fusarium; F represents sorghum brown spot; G represents rice sheath blight; H represents apple ring spot; I represents potato dry rot; J represents tomato early blight; K represents tomato gray mold; L represents red date black spot; M represents tobacco red star; N represents apple rot; O represents rape sclerotia; P represents wheat sheath blight; Q represents watermelon wilt; R represents cucumber wilt.

[0013] Figure 3 This is the standard curve of IAA production by Bacillus velezensis W-107 provided in Example 3 of the present invention.

[0014] Figure 4 It is the production of iron carriers by Bacillus velezensis W-107 provided in Example 3 of the present invention; A, B, C, D, and E represent the ability of the strain to decompose inorganic phosphorus, the ability of the strain to decompose organic phosphorus, the ability of the strain to decompose potassium, the ability of the strain to fix nitrogen, and the ability of the strain to produce iron carriers, respectively.

[0015] Figure 5 The biofilm formation of Bacillus velezensis W-107 in different culture media provided in Example 4 of the present invention; A, B, C, and D represent the biofilm of the strain in culture media LB, LBG, LBM, and LBGM, respectively.

[0016] Figure 6 This is the potted plant protection effect of Bacillus velezensis W-107 provided in Example 6 of the present invention on cucumber wilt. DETAILED DESCRIPTION

[0017] Unless otherwise specified, the scientific and technical terms used herein are understood according to the knowledge of ordinary technicians in the relevant fields.

[0018] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0019] Example 1: Isolation, purification and molecular biological identification of Bacillus Velezii (also known as strain W-107) of the present invention

[0020] Weigh 25g of humus organic fertilizer and add it to 225ml of sterile water in a conical flask and shake it evenly. Heat the conical flask in a water bath at 80℃ for 20min to kill the nutrient cells. After the sample is allowed to stand for 20min and return to room temperature, perform gradient dilution and take 10 -4 , 10 -5 , 10 -6 100 μL of the diluted sample was spread on LB agar plates, and 3 plates were spread in parallel for each dilution; the plates were left at room temperature for 20 min, and after the bacterial solution was absorbed, they were placed in a constant temperature incubator at 30°C for overnight culture, and the growth of the colonies was observed the next day.

[0021] DNA extraction was performed according to the procedure of the column bacterial DNA extraction kit of Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. The universal bacterial primer pair was selected: F27 5'-AGA GTT TGA TCC TGG CTC AGG-3', P1541 5'-AAG GAG GTG GTG ATC CAG CCG CA-3'. The reaction conditions were: 94℃ denaturation for 45s, 50℃ annealing for 45s, 72℃ extension for 75s, 50μL reaction system for 30 cycles; PCR products were detected by agarose gel electrophoresis, and the sequence results were obtained by cloning and sequencing. The obtained 16S rDNA full sequence was compared with the 16S rDNA sequence obtained from databases such as Genbank, and the phylogenetic tree was constructed using the Mega 6.0 software package.

[0022] The strain W-107 was identified by molecular biological methods. The results of the 16S rDNA base sequence determination of the strain W-107 are shown in SIQ ID NO.1. A total of 1434 bases of the 16S rDNA of the strain W-107 were determined.

[0023] The fragment size of the 16S rDNA obtained by amplification from the genomic DNA of the strain was compared with the 16S rDNA sequence obtained from the Genbank database and the accession number was W-107 (OR577620). Construction of phylogenetic tree: 10 model strains were selected and the Neighbor-Joining method in MEGA 6 software was used for phylogenetic analysis. The constructed phylogenetic tree is shown in Figure 1 shown.

[0024] Example 2: Determination of antibacterial effect of strain W-107

[0025] The plate confrontation method was used to detect Helminthosporium turcicum Pass, Phytophthora capsici, Glomerella ciningulaia, Verticillium dahliae, Fusarium graminearum, Setosphaeria turcica (Luttr.) Leonard & Suggs, Rhizoctonia solani, Botryospuaeria berengeriana, Fusarium oxysporum Schlecht., Alternaria solani, Botrytis cinerea, Isariopsis imdica var. Ziziphi, Alteraria alternata, Valsa mali Miyabe et Yamada, Sclerotinia sclerotiorum, and other pests. sclerotiorum), wheat stripe blight (Geratobasidium cornigeru (Bourd.) Rogers), watermelon wilt (Fusarium oxysporum f.sp.niveum), cucumber wilt (Fusariumoxysporum.sp.cucumebrium Owen) and a total of 18 plant pathogenic fungi (all provided by Shaanxi Biopesticide Engineering Technology Research Center, Northwest Agriculture and Forestry University). First, with the center of the culture dish as the center point, cross lines were drawn on the bottom of the culture dish, and the strain activated on the LB medium was inoculated 2.5 cm away from the center point; then, the activated plant pathogenic fungi were punched with a 5mm puncher and inoculated in the center of the PDA medium plate, the culture dish was sealed, and cultured at 28°C until the pathogen control grew to two-thirds of the culture dish, and the radius of the pathogen and the width of the transparent circle formed were counted.

[0026] The "plate confrontation" method was used to evaluate the antibacterial spectrum of strain W-107 with 18 pathogenic fungi as targets. The results showed that strain W-107 had a broad spectrum of inhibitory activity against 18 pathogenic fungi ( Figure 2). Among them, strain W-107 had the highest inhibitory activity against the pathogen of corn leaf blight, with an inhibition rate of 93.20%; the inhibitory activity against the pathogen of wheat eyebrow blight was relatively obvious, with an inhibition rate of 82.34%; the inhibition rates of apple anthracnose, wheat fusarium, sorghum black spot, rice eyebrow blight, apple ring spot, potato dry rot, tomato early blight, tomato gray mold, tobacco red star, apple rot, rape sclerotium, watermelon wilt and cucumber wilt were between 60.00% and 70.00%; the inhibition rate against pepper phytophthora pathogen was 52.94% (Table 1).

[0027] Table 1 Inhibitory effects of strains on 18 pathogenic fungi

[0028]

[0029]

[0030] Example 3: Determination of growth-promoting indexes of strain W-107

[0031] First, the strain stored at -80℃ was streaked on LB medium and activated by culturing in a 28℃ incubator for 24h; then the activated strain was inoculated with a bacterial needle in a 250mL conical flask containing 100mL LB liquid medium, and 1mL L-tryptophan was added to the conical flask to make a final concentration of 100μg / mL, and cultured at 28℃ and 180r / min for 5d, with two biological replicates for each strain; 8mL of the strain fermentation broth was taken and centrifuged at 8000r / min and 4℃, and the supernatant was aspirated for quantitative determination of the strain IAA content.

[0032] For the quantitative determination of IAA content, 60 μL of supernatant was aspirated into a 2 mL centrifuge tube, and then 120 μL of Salkowski reagent was added for color development in the dark, and the absorbance of each treatment was measured at 530 nm using a UV spectrophotometer; at the same time, a standard curve of IAA content and absorbance was prepared using a gradient diluted IAA solution (50, 10, 5, 2.5, 1.25, 0.625 and 0 μg / mL), and the IAA production ability of the strain was evaluated based on the standard curve.

[0033] The activated strain was cultured at 28°C for 5 days on an inorganic phosphorus culture medium ((NH4)2SO4 0.5g, NaCl 0.3g, KCl 0.3g, MgSO4·7H2O 0.3g, Ca3(PO4)2 25g, FeSO4·7H2O 0.03g, MnSO4·H2O 0.03g, glucose 10g, PH 7.4-7.6) using the spot inoculation method. The strain's ability to degrade inorganic phosphorus was evaluated based on whether a transparent zone was formed around the strain and the size of the transparent zone.

[0034] The activated strain was cultured in an organophosphorus medium ((NH4)2SO4 0.5g, MgSO4·7H2O0.3g, NaCl 0.3g, KCl 0.3g, CaCO3 3.5g, glucose 10.0g, MnSO4·H2O 0.03g, FeSO4·7H2O0.03g, lecithin 0.2g, agar 17-20g, sterilized at 121℃ and high temperature and high pressure for 25min) at 28℃ for 5 days by using the spot inoculation method. The ability of the strain to degrade organophosphorus was evaluated based on whether a transparent zone was formed around the strain and the size of the transparent zone.

[0035] The activated strain was inoculated on Alek-sandrov medium (5 g glucose, 0.5 g MgSO4·7H2O, 0.0005 g FeCl3, 0.1 g CaCl2, 0.2 g sodium potassium aluminosilicate, 0.2 g Ca3(PO4)2, PH 7.0) by the spot inoculation method and cultured at 28°C for 5 days. The potassium-solubilizing ability of the strain was evaluated based on whether a transparent zone was formed around the strain and the size of the transparent zone.

[0036] The activated strain was inoculated into Ashby medium (nitrogen fixation selective medium) (g / L) by the spot inoculation method: KH2PO4 0.2g, CaCO3 5.0g, MgSO4·7H2O 0.2g, NaCl 0.2g, CaSO4·2H2O 0.1g, mannitol 10.0g, pH 7.4-7.6, agar 17-20g, and sterilized at 121℃ for 25min; the nitrogen fixation ability of the strain was evaluated based on whether a transparent zone was generated around the strain and the size of the transparent zone.

[0037] The activated strain was inoculated on chrome azure culture medium (CAS) using the dot inoculation method. The inoculated plates were cultured at 28°C for 7 days, and the ability of the strain to produce iron carriers was evaluated based on the appearance and size of the yellow halo around the colonies.

[0038] The growth-promoting indexes of the isolated strain W-107, such as IAA production, inorganic phosphorus decomposition, organic phosphorus decomposition, potassium decomposition, nitrogen fixation and iron carrier production, were determined. Figure 3 ), strain W-107 was found to produce 17.65 μg / mL of IAA; it has the ability to produce siderophores (Siderophores) (Table 2, Figure 4 ), but it does not have the ability to decompose inorganic phosphorus (OP), decompose organic phosphorus (IOP), decompose potassium (K) and fix nitrogen (N) (Table 2).

[0039] Table 2 Evaluation of growth-promoting properties of strain W-107

[0040] Strain IAA (μg / mL) OP IOP K N Siderophores W-107 17.65 - - - - +

[0041] Example 4: Strain W-107 biofilm formation assay

[0042] A single colony was picked and inoculated into a 250mL conical flask containing 100mL LB liquid culture medium, and cultured overnight at 160rpm / min and 30℃; 200μL LB, LBG (containing 1% glycerol (v / v)), LBM (containing 0.1mM MnSO4) and LBGM (containing 1% glycerol (v / v) and 0.1mM MnSO4) culture media were added to a 16-well plate, 2μL of overnight cultured bacterial solution was gently dripped on the liquid surface of different culture media, and the 16-well plate was cultured at 30℃ for 3d, and the biofilm formation was observed and recorded.

[0043] The results of biofilm determination of strain W-107 showed that the strain could form obvious biofilm in LBGM medium, followed by LBM medium, and no obvious biofilm was formed in LBG and LB medium ( Figure 5 ).

[0044] Example 5: Potted plant protection efficacy of strain W-107 against cucumber wilt

[0045] Cucumber seeds were sown in seedling trays for seedling raising. After one week of seedling raising, they were transplanted into seedling pots, with one seedling per pot. After transplanting, when the cucumbers grew to the 2-leaf and 1-heart stage, the root irrigation method was used for the prevention and efficacy test. The experimental treatments included: 1) inoculation of only 25 mL of cucumber wilt and an equal amount of sterile water (FOC); 2) inoculation of 25 mL of cucumber wilt and an equal amount of bacterial suspension (10 8 CFU / mL) (W-107-FOC); 3) Inoculate only the bacterial suspension (10 8 CFU / mL) (W-107) and 4) inoculated with 25 mL of sterile water (CK); each treatment was repeated 4 times. The disease index, plant height, and fresh weight were counted one month after inoculation, and the control effect was calculated based on the disease index.

[0046] Cucumber wilt disease index:

[0047] Level 0: no symptoms;

[0048] Level 1: Less than 1 / 4 of the leaves of the plant show wilting symptoms, the stem base is asymptomatic, and the plant grows normally;

[0049] Level 2: 1 / 4-1 / 2 of the leaves of the plant show wilting symptoms, and the lower 1 / 2 of the stem base turns brown, and the plant becomes dwarfed;

[0050] Level 3: More than 1 / 2 of the leaves of the plant show wilting symptoms, more than 1 / 2 of the stem base turns brown, and the plant is obviously dwarfed;

[0051] Level 4: The whole plant wilts and dies.

[0052]

[0053] The fermented liquid of strain W-107 has a significant protective effect on cucumber wilt, with a protective effect of 42.27% (Table 3). Compared with the blank control, the chlorophyll content of strain W-107 fermented liquid treatment (23.00) is higher than the chlorophyll content of the control (21.38); the plant height of cucumber plants treated with strain W-107 fermented liquid (60.24cm) is significantly higher than the plant height of the control (42.54cm); the fresh weight of cucumber plants treated with strain W-107 fermented liquid (12.84g) is significantly higher than the fresh weight of blank control cucumber plants (10.06g); the dry weight of cucumber plants treated with strain W-107 fermented liquid (6.40g) is higher than the dry weight of blank control cucumber plants (5.10g). It can be seen that the fermented liquid of strain W-107 has a certain growth-promoting effect on cucumber plants (Table 3).

[0054] Table 3 Disease prevention and growth promotion effect of strain W-107 on cucumber wilt

[0055]

[0056] Nucleotide sequence listing

[0057]

[0058]

Claims

1. A strain of Bacillus velezensis, which is named Bacillus velezensis and has a deposit number of CGMCC No. 30619.

2. A fermentation product, characterized in that The fermentation product is produced by fermentation with the Bacillus Velez subtilis according to claim 1.

3. The fermentation product according to claim 3, characterized in that The fermentation medium is LBGM medium or LBM medium.

4. Use of the Bacillus Velez or its fermentation product according to claim 1 in preparing an antibacterial agent for plant pathogens.

5. The use according to claim 4, characterized in that: The plant pathogens are Helminthosporium turcicum Pass, Phytophthora capsici, Glomerella ciningulaia, Verticillium dahliae, Fusarium graminearum, Setosphaeria turcica (Luttr.) Leonard & Suggs, Rhizoctonia solani, Botryospuaeria berengeriana, Fusarium oxysporum Schlecht., Alternaria solani, Botrytis cinerea, Isariopsis imdica var. Ziziphi, Alteraria alternata, Valsa mali Miyabe et al. Yamada), rape sclerotium (Sclerotiniasclerotiorum), wheat stripe blight (Geratobasidiumcornigeru(Bourd.)Rogers), watermelon wilt (Fusarium oxysporum f.sp.niveum), cucumber wilt (Fusarium oxysporum.sp.cucumebriumOwen) pathogens.

6. Use of the Bacillus Velez or its fermentation product according to claim 1 for preparing a plant growth promoting agent.

7. The use according to claim 6, characterized in that: The plant growth promoter promotes plant growth by producing IAA and siderophore.

8. Use of the Bacillus Velez or its fermentation product according to claim 1 in preparing a plant biocontrol agent.

9. Use of the Bacillus Velez subtilis or its fermentation product according to claim 1 in preparing organic fertilizer.

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