Bacillus pumilus WYH-1275 and application thereof in plant disease prevention and growth promotion

Abstract

The invention discloses a bacillus pumilus WYH-1275 strain and application thereof in plant disease prevention and growth promotion, the strain is preserved in the China General Microbiological Culture Collection Center on November 1, 2023, the preservation number is CGMCC No.1. 60181, and the preservation name is bacillus pumilus WYH-1275. The invention further discloses a preparation method of the bacillus pumilus WYH-1275 strain. When being applied to the field of agricultural biological prevention and control, the strain can form a biological membrane to be stably colonized at the rhizosphere of a plant, and can generate substances such as IAA and siderophores to promote the growth of the plant. A broad-spectrum inhibition effect can be generated on various plant pathogenic fungi and oomycetes. Compared with a blank control potted plant, the bacillus pumilus has the advantages that the height of the potted cucumber plant is increased by 48.71%, the prevention effect of the cucumber fusarium wilt potted plant is as high as 44.59%, and the bacillus pumilus 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 pumilus WYH-1275 with disease prevention and growth promotion functions and application thereof. Background Art

[0002] Plant diseases pose a serious threat to the world's food production and security. For a long time, the use of chemical pesticides and fertilizers has aggravated environmental pollution, bacterial resistance, and soil microecological imbalance. It is urgent to explore microbial products that can replace chemical pesticides and fertilizers. Although there are a large number of microbial products, due to their low colonization ability and short effective period in the field, it has become a research hotspot for scholars at home and abroad to explore broad-spectrum and highly efficient microorganisms from plant-related microbial communities such as rhizosphere and root endophytes. Summary of the invention

[0003] Based on new research findings, the purpose of the present invention is to provide a strain of Bacillus pumilus WYH-1275 with disease prevention and growth promotion functions and its application in agricultural production.

[0004] The Bacillus pumilus WYH-1275 provided by the present invention has been deposited on November 1, 2023 in the General Microbiology Center of the China Culture Collection Administration (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), Postal Code: 100101, and the deposit number is CGMCC No.1.60181.

[0005] The bacterial suspension, fermentation liquid or fermentation product of the above-mentioned Bacillus pumilus WYH-1275 is also within the scope of protection of the present invention.

[0006] The use of the bacterial suspension, fermentation liquid or fermentation product of the above-mentioned Bacillus pumilus WYH-1275 in antibacterial treatment is also within the scope of protection of the present invention.

[0007] The use of the bacterial suspension or fermentation liquid or fermentation product of the above-mentioned Bacillus pumilus WYH-1275 in promoting plant growth is also within the scope of protection of the present invention.

[0008] The use of the bacterial suspension, fermentation liquid or fermentation product of the above-mentioned Bacillus pumilus WYH-1275 in preventing and controlling plant diseases is also within the scope of protection of the present invention.

[0009] In the above application, the pathogen is rice sheath blight (Rhizoctonia solani), mango anthracnose (Colletotrichum gloeosporioides), tomato early blight (Alternaria solani (Ell. et Mart.) Jones et Grout), potato dry rot (Fusarium solani (Mart.) Sacc), corn leaf blight (Exserohilum turcicum (Pass.) Leonay et Suggs), cucumber wilt (Fusariumoxysporum.sp.cucumebrium Owen), tomato gray mold (Botrytis cinerea) or wheat take-all (Gaeumannomyces graminsis (sacc.) Arx & Olivier Var tritici J.walker).

[0010] The application of the bacterial suspension or fermentation liquid or fermentation product of Bacillus pumilus WYH-1275 in the preparation of plant disease prevention and growth promotion preparations is also within the scope of protection of the present invention. In the above application, the growth promotion characteristics are the production of IAA and siderophore. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the developmental tree of Bacillus pumilus WYH-1275 provided in Example 1 of the present invention.

[0012] Figure 2 The invention discloses the antibacterial activity of Bacillus pumilus WYH-1275 against 8 pathogenic fungi provided in Example 2; wherein A (pathogenic fungi) and E (pathogenic fungi + 1275) represent tomato gray mold; B (pathogenic fungi) and F (pathogenic fungi + 1275) represent tomato early blight; C (pathogenic fungi) and G (pathogenic fungi + 1275) represent cucumber wilt; D (pathogenic fungi) and H (pathogenic fungi + 1275) represent mango anthrax; I (pathogenic fungi) and M (pathogenic fungi + 1275) represent potato root rot; J (pathogenic fungi) and N (pathogenic fungi + 1275) represent rice blight; K (pathogenic fungi) and O (pathogenic fungi + 1275) represent wheat total eclipse; L (pathogenic fungi) and P (pathogenic fungi + 1275) represent corn leaf blight.

[0013] Figure 3 This is the standard curve of IAA production by Bacillus pumilus WYH-1275 provided in Example 3 of the present invention.

[0014] Figure 4 It is the formation of the biofilm of Bacillus pumilus WYH-1275 provided in Example 3 of the present invention; A, B, C, and D represent the biofilm of strain WYH-1275 in culture medium LBG, LBM, LBGM, and LB culture medium, respectively.

[0015] Figure 5 This is the ability of Bacillus pumilus WYH-1275 to produce siderophore provided in Example 3 of the present invention.

[0016] Figure 6 This is the potted plant disease prevention and growth promotion effect of Bacillus pumilus WYH-1275 on cucumber wilt provided in Example 5 of the present invention. 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] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0019] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0020] Example 1: Isolation, purification and molecular biological identification of strain WYH-1275 (also referred to herein as 1275)

[0021] Weigh 10 g wheat rhizosphere soil (the soil was obtained from the wheat experimental field of Northwest Agriculture and Forestry University, 34°17′N, 108°04′E) and add 200 mL 0.01 M sterile PBS buffer. Shake at room temperature and 180 r / min for 30 min to obtain a rhizosphere soil suspension, which was then diluted to 10 with sterile water. -1 , 10 -2 and 10 -3, use a spreading rod to evenly spread it on TYG medium (TryptoneYeast extract Glucose Medium, TYG), whose composition is: tryptone 1g, yeast extract 1g, glucose 0.5g, KCl 6.34g, NaCl 1.2g, MgSO4·7H2O 0.25g, K2HPO4 0.13g, CaCl2·2H2O 0.22g, K2SO40.17g, Na2SO4 2.4g, NaHCO3 0.5g, Na2CO3 0.09g, FeEDTA 0.07g, agar 15g, distilled water 1000mL, pH=7.2-7.4; after constant temperature culture at 28℃ for 2d, pick a single colony for purification culture.

[0022] DNA extraction was performed according to the procedure of the column bacterial DNA extraction kit of Shanghai Sangon Biotechnology (Shanghai) Co., Ltd. The bacterial universal 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; the PCR product was detected by agarose gel electrophoresis, and the sequence result was obtained by cloning and sequencing; the obtained 16S rDNA full sequence was compared with the 16S rDNA sequence obtained from Genbank and other databases, and the phylogenetic tree was constructed using the Mega 6.0 software package.

[0023] The strain WYH-1275 was identified by molecular biological methods. The results of the 16S rDNA base sequence determination of the strain WYH-1275 are shown in SIQ ID NO.1. A total of 1436 bases of the 16S rDNA of the strain WYH-1275 were determined.

[0024] The fragment size was amplified from the genomic DNA of the strain. The obtained 16S rDNA full sequence was compared with the 16S rDNA sequence obtained from the Genbank database and the accession number was obtained: WYH-1275 (OR574219). Construction of phylogenetic tree: 10 model strains were selected for phylogenetic analysis, and the constructed phylogenetic tree was as follows Figure 1 shown.

[0025] Example 2: Determination of antibacterial effect of strain WYH-1275

[0026] The plate confrontation method was used to determine the inhibitory effect on eight plant pathogenic fungi (all provided by Shaanxi Biopesticide Engineering Technology Research Center of Northwest A&F University), including rice sheath blight (Rhizoctonia solani), mango anthracnose (Colletotrichum gloeosporioides), tomato early blight (Alternaria solani (Ell. et Mart.) Jones et Grout), potato dry rot (Fusarium solani (Mart.) Sacc), corn leaf blight (Exserohilum turcicum (Pass.) Leonay et Suggs), cucumber wilt (Fusarium oxysporum. sp. cucumebrium Owen), tomato gray mold (Botrytis cinerea) and wheat take-all (Gaeumannomyces graminsis (sacc.) Arx & Olivier Var tritici J. walker).

[0027] The specific plan is: first, with the center of the culture dish as the center point, cross lines are drawn on the bottom of the culture dish, and the strain that has been activated on the LB medium is inoculated 2.5 cm away from the center point; then, the activated plant pathogenic fungi are punched with a 5mm puncher and inoculated in the center of the PDA medium plate, the culture dish is sealed, and the control pathogen is cultured at 28°C. When the growth reaches two-thirds of the culture dish, the radius of the pathogen and the width of the transparent circle formed are counted.

[0028] The "plate confrontation" method was used to evaluate the antibacterial spectrum of strain WYH-1275 with 8 pathogenic fungi as targets. The results showed that strain WYH-1275 had a broad spectrum of inhibitory activity against 7 pathogenic fungi ( Figure 2 Among them, strain WYH-1275 had the highest inhibitory activity against the pathogen of corn leaf blight, with an inhibition rate of 61%; the inhibitory activity against wheat take-all and tomato gray mold pathogens was more obvious, with inhibition rates of 51% (Table 1).

[0029] Table 1 Inhibitory effect of strain WYH-1275 on 12 pathogenic fungi

[0030]

[0031] Note: The data in the table are mean ± standard deviation.

[0032] Example 3: Determination of growth-promoting indexes of strain WYH-1275

[0033] First, the strain stored at -80℃ was streaked on LB medium and activated in a 28℃ incubator for 24h; then the activated strain was inoculated into a 250mL conical flask containing 100mL LB liquid medium, and 1mL L-tryptophan was added to make the final concentration 100μg / mL, and cultured at 28℃ and 180r / min for 5d, with 2 biological replicates for each strain. 8mL of the strain fermentation liquid was taken and centrifuged at 8000r / min and 4℃, and the supernatant was aspirated for quantitative determination of the strain IAA content.

[0034] For the quantitative determination of IAA content, 60 μL of supernatant was taken 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 dilution of IAA solution (50, 10, 5, 2.5, 1.25, 0.625 and 0 μg / mL), and the IAA production capacity of the strain was evaluated based on the standard curve.

[0035] 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.

[0036] The activated strain was inoculated into DF medium (glucose 2.0g, gluconic acid 2.0g, citric acid 2.0g, trace element solution (CaCl2 200mg, FeSO4·7H2O 200mg, H3BO3 15mg, ZnSO4·7H2O 20mg, Na2MoO4 10mg, KI 10mg, NaBr 10mg, MnCl2 10mg, COCl2 5mg, CuCl2 5mg, AlCl3 2mg, NiSO4 2mg, deionized water 1,000mL) 10mL, deionized water 990mL) with 3mMACC as the sole nitrogen source by spot inoculation method, DF culture with 0.2% (w / v) (NH4)2SO4 as the sole nitrogen source as positive control, and DF medium as negative control. The inoculated plates were cultured at 28℃ for 5 days, and the ACC production capacity of the strains was comprehensively evaluated based on the growth conditions of the strains on the three culture media.

[0037] The activated strain was inoculated on chrome azure culture medium (CAS) by 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 WYH-1275, such as IAA production, phosphate solubilization, ACC production and iron carrier production, were determined. Figure 3 ), strain WYH-1275 was found to produce 97.38 μg / mL of IAA at 100 μg / mL; and its ability to produce siderophore (Sid) was positive ( Figure 5 ); but it does not have the ability to decompose organic phosphorus (OP) and inorganic phosphorus (IOP), decompose potassium (K), fix nitrogen (N) and produce ACC (ACC) (Table 2).

[0039] Table 2 Evaluation of growth-promoting properties of strain WYH-1275

[0040]

[0041] Note: IAA means the ability to produce auxin, OP means the ability to decompose organic phosphorus, IOP means the ability to decompose inorganic phosphorus, K means the ability to decompose potassium, N means the ability to fix nitrogen, ACC means the ability to produce ACC, and Sid means the ability to produce siderophore.

[0042] Example 4: Biofilm formation assay of strain WYH-1275

[0043] Pick a single colony and inoculate it in a 250mL conical flask containing 100mL LB liquid medium, and culture it overnight at 160rpm / min and 30℃. Add 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 medium to a 16-well plate, take 2μL of overnight cultured bacterial solution and gently drop it on the liquid surface of different culture media, and culture the 16-well plate at 30℃ for 3d, observe the biofilm formation, and record it.

[0044] The results of biofilm determination of strain WYH-1275 showed that the strain could form obvious biofilm in LB medium, followed by LBM medium, and no obvious biofilm was formed in LBG and LBGM medium ( Figure 4 ). Biofilm is an important form of existence for biocontrol bacteria to occupy ecological niches, which can help them resist adverse environments. Moreover, the formation of biofilm of biocontrol bacteria significantly affects their colonization in plants, which in turn directly affects the biological control effect.

[0045] Example 5: Potted plant protection efficacy of strain WYH-1275 against cucumber wilt

[0046] Commercially available 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 with 25 mL of cucumber wilt plant pathogen and 25 mL of sterile water (FOC); 2) inoculation with 25 mL of cucumber wilt and 25 mL of bacterial suspension (10 8 CFU / mL)(1275-FOC); 3) inoculate only bacterial suspension (10 8 CFU / mL)(1275); 4) inoculate with 25 mL of sterile water (CK); each treatment was repeated 4 times; one month after inoculation, the disease index, plant height, and fresh weight were counted, and the protection effect was calculated based on the disease index.

[0047] Cucumber wilt disease index:

[0048] Level 0: no symptoms;

[0049] 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;

[0050] 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;

[0051] 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;

[0052] Level 4: The whole plant wilts and dies.

[0053]

[0054] The strain WYH-1275 has a significant protective effect on cucumber wilt, with a protection rate of 44.59%. Compared with the blank control, the plant height of cucumber plants treated with the strain WYH-1275 suspension (63.26 cm) was significantly higher than that of the control (42.54 cm); the fresh weight of cucumber plants treated with the strain WYH-1275 suspension (16.00 g) was significantly higher than that of the blank control (10.01 g); it can be seen that the strain WYH-1275 has a certain growth-promoting effect on cucumber plants (Table 3).

[0055] Table 3 Disease prevention and growth promotion effect of strain WYH-1275 on cucumber wilt

[0056]

[0057] Note: The data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the 0.05 level.

[0058]

[0059]

Claims

1. A strain of Bacillus pumilus WYH-1275, named Bacillus pumilus, with a deposit number of CGMCC No. 1.60181.

2. A fermentation product, characterized in that The fermentation product is produced by fermenting the Bacillus pumilus WYH-1275 according to claim 1.

3. The fermentation product according to claim 2, characterized in that The fermentation starter is selected from LB medium or LBG medium containing glycerol.

4. Use of the Bacillus pumilus WYH-1275 or its fermentation product according to claim 1 for preparing an antibacterial agent for plant pathogens.

5. The use according to claim 4, characterized in that: The plant pathogens are rice sheath blight (Rhizoctonia solani), mango anthracnose (Colletotrichum gloeosporioides), tomato early blight (Alternaria solani (Ell. et Mart.) Jones et Grout), potato dry rot (Fusarium solani (Mart.) Sacc), corn leaf blight (Exserohilum turcicum (Pass.) Leonay et Suggs), cucumber wilt (Fusarium oxysporum.sp.cucumebrium Owen), tomato gray mold (Botrytis cinerea) or wheat take-all (Gaeumannomyces graminsis (sacc.) Arx & Olivier Var tritici J. walker).

6. Use of the Bacillus pumilus WYH-1275 or its fermentation product according to claim 1 for preparing a plant growth promoter.

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 pumilus WYH-1275 or its fermentation product according to claim 1 for preparing a plant growth promoter.

9. Use of the Bacillus pumilus WYH-1275 or its fermentation product according to claim 1 for preparing a plant biocontrol agent.

10. Use of the Bacillus pumilus WYH-1275 or its fermentation product according to claim 1 for preparing organic fertilizer.

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