Bacillus velezensis strain bv-3 and application thereof in prevention and treatment of corn leaf spot disease

Abstract

The present invention discloses a Bacillus velezensis BV-3 and its use in preventing and controlling corn leaf spot disease, relating to the technical field of corn leaf spot disease control strains. The strain is deposited with CGMCC No. 30159 and is classified as Bacillus velezensis. The strain is antagonistic to five leaf spot pathogens: Echinococcus nigricans, Echinococcus kaoliang, Helminthosporium zeae, Alternaria alternata, and Helminthosporium maximae. Potted plant experiments show that the strain has a good protective effect against corn leaf spot disease, providing a reference for the biological control of corn leaf spot disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of corn leaf spot disease strain prevention and treatment, and more particularly to a bacillus velezensis strain BV-3 and its application in the prevention and treatment of corn leaf spot disease. BACKGROUND

[0002] Corn is an important food crop and forage crop in China and even in the world, and is also the highest total yield crop in the world. Its planting area is only second to rice and wheat, but it is often harmed by important diseases such as leaf spot disease in the process of corn planting. Corn leaf spot disease is mainly fungal leaf spot disease, and common diseases include large spot disease, small spot disease, curved cell leaf spot disease, gray spot disease, alternaria leaf spot disease and southern rust disease, etc. The above diseases occur frequently or intermittently, causing great losses to corn production in China. Leaf spot disease is very harmful to corn production, and the yield is reduced by about 10% in disease fields in general years, and more than 50% in epidemic years of susceptible varieties, and even heavy ones can lead to absolute yield. At present, the prevention and treatment of leaf spot disease is still mainly based on chemical control and disease-resistant varieties, but due to the complexity of the pathogen, the great variation and differentiation of the pathogenic bacteria, the diversity and difference of corn varieties, and the improper cultivation caused by the expansion of demand, etc., efficient and targeted chemical agents and disease-resistant varieties have not been obtained. Disease-resistant varieties cannot meet the production needs, and chemical control also causes problems such as pesticide residues, environmental pollution and the generation of pathogen resistance.

[0003] Biological control is a field with great development potential and has been attracting attention in recent years. Studies have shown that antagonistic bacteria have significant effects on the prevention and treatment of leaf spot disease, and research on the use of active organic compounds produced by biocontrol bacteria to inhibit corn leaf spot pathogens has also made much progress. However, the available strain resources are still relatively few, and the research on effective inhibitory substances is still lacking, so it is necessary to screen biocontrol strains with high antagonistic activity against corn leaf spot pathogens and explore the inhibitory activity of organic compounds in their metabolites. SUMMARY

[0004] Therefore, the present application is proposed.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] The present application provides a bacillus velezensis strain BV-3, the bacillus velezensis (Bacillus Velezensis) BV-3 was preserved in the China General Microbiological Culture Collection Center on March 27, 2024, the preservation number is CGMCC No. 30159, the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the classification and naming is bacillus velezensis Bacillus Velezensis.

[0007] A second aspect of the embodiments of the present invention provides a use of Bacillus Velez subtilis BV-3 or a sterile fermentation broth of Bacillus Velez subtilis BV-3, wherein the use is in preventing and controlling corn leaf spot.

[0008] In a preferred embodiment, the antibacterial spectrum of the Bacillus Velez subtilis BV-3 or the sterile fermentation broth of Bacillus Velez subtilis BV-3 includes: black Epstein-Barr virus of corn leaf spot, Epstein-Barr virus of corn white spot, flat umbilical cyst of corn small spot, alternator cyst of corn leaf spot, and large umbilical cyst of corn large spot.

[0009] A third aspect of the embodiments of the present invention provides a biocontrol agent for preventing and treating corn leaf spot disease, wherein the biocontrol agent comprises the Bacillus Velezii BV-3.

[0010] A fourth aspect of the embodiments of the present invention provides a preparation for preventing and treating corn leaf spot, the preparation comprising the sterile fermentation broth of the aforementioned Bacillus Velez subtilis BV-3.

[0011] In a preferred embodiment, the preparation process of the sterile fermentation broth is as follows: strain BV-3 is inoculated into LB liquid culture medium, shaken at 28°C and 220r / min for 3 days, refrigerated centrifuged at 10000r / min and 4°C for 20min, and filtered through a 0.22μm filter membrane to obtain sterile fermentation broth.

[0012] Through the above technical solution, it can be seen that compared with the existing technology, the present invention isolates leaf spot pathogenic fungi from diseased leaf samples of the corn planting base in Wuli Village, Chong'an Street, Wuyishan City, Fujian Province, and isolates and screens a strain BV-3 with a good inhibitory effect on corn leaf spot pathogens from the rhizosphere soil. The strain antagonizes five leaf spot pathogens: black Epstein-Barr virus for corn leaf spot, sorghum Epstein-Barr virus for corn white spot, flat-umbilical helminthes for corn small spot, alternator leaf spot for alternator, and large-spotted convex-umbilical helminthes for corn large spot. At the same time, through potted experiments, it can be seen that the strain has a good protective effect on corn leaf spot, providing a reference for the biological control of corn leaf spot. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0014] Figure 1The drawing is a pathogenic bacteria colony characteristic diagram of Epicoccum nigrum, Micrococcus sorghum, Bipolaris maydis, Helminthosporium panicularis and Exserohilum rostratum (A) and the inhibition effect diagram of strain BV-3 on five pathogenic bacteria (B).

[0015] Figure 2 The drawing is a morphological characteristic diagram of strain BV-3.

[0016] Figure 3 The drawing is a phylogenetic tree of strain BV-3 constructed based on 16S rDNA gene sequence neighbor-joining method.

[0017] Figure 4 The drawing is the inhibition effect diagram of BV-3 metabolites on four pathogenic bacteria (5d); A-D: pure culture of Epicoccum nigrum, Bipolaris maydis, Helminthosporium panicularis and Exserohilum rostratum (CK); a-d: flat plate culture containing Bacillus velezensis fermentation liquor. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0019] Test materials

[0020] The test soil sample was collected from Wulivillage, Chong'an Street, Wuyishan City, Fujian Province (latitude: 27°75'116", longitude 118°3'86"). The tested corn pathogenic bacteria were five pathogenic bacteria, including Epicoccum nigrum, Micrococcus sorghum, Helminthodes panicularis, Bipolaris maydis and Exserohilum rostratum, which were provided by the Plant Protection Institute of Fujian Academy of Agricultural Sciences.

[0021] Reagents and culture medium

[0022] PDA medium (peeled potato 200 g, glucose 20 g, agar 20 g, distilled water 1 L), PDB medium (peeled potato 200 g, glucose 20 g, distilled water 1 L), NA medium (agar 18 g, beef extract 3 g, peptone 5 g, glucose 2.5 g), LB liquid medium (tryptone 10 g, yeast extract 5 g, NaCl 10 g), sterile water, methanol (analytical pure), ethyl acetate (analytical pure), etc.

[0023] Data analysis: The test data were entered using Excel 2007, and single factor analysis was performed using DPS 7.05. The significant differences in the inhibition zone width of the antagonistic strain BV-3 against different corn leaf spot pathogens and the seedling stage control effect were analyzed using the Duncan method. Disease index was calculated according to disease grade: disease index (%) = [∑(disease grade spot number x disease grade value)] / (inoculation point number x highest grade value) x 100%; control rate (%) = [(control group disease index - treatment group disease index)] / control group disease index x 100%. The lesion grading standard was referred to (Subramani and Aalbersberg 2012) and adjusted appropriately: 0 grade: wound point disease; 1 grade: diameter less than 5 mm; 2 grade: lesion diameter 5-7 mm; 3 grade: lesion diameter 7-9 mm; 4 grade: lesion diameter 9-14 mm; 5 grade: lesion diameter more than 14 mm.

[0024] Example 1: Screening and isolation of antagonistic strains

[0025] Dilution and spread plate method was used to isolate bacteria from soil collected from corn planting bases. After the soil sample was dried, 1 g of crushed soil was weighed and sequentially diluted 10-fold to obtain 10 -2 , 10 -3 and 10 -4 concentration dilutions. After mixing, 200 μL of soil suspension was taken from each gradient and spread on PDA medium using a flame-sterilized spreader. Three replicates were set for each concentration. The spread medium was placed in a 28°C constant temperature incubator and cultured for 3 days before transfer and purification.

[0026] Primary screening: plate confrontation method was used, the target pathogen was inoculated in the center of the medium, and the biocontrol strain was inoculated 2.5 cm away from the center of the pathogen, a total of four points, with three replicates. Biocontrol strains with good inhibition effect were selected.

[0027] Secondary screening: the primary screening strains were inoculated into LB liquid medium and cultured at 28°C with 220 r / min shaking for 2 days. After freezing and centrifugation (10000 r / min, 4°C) for 20 min, the supernatant was filtered through a 0.22 μm filter to obtain sterile fermentation broth. The sterile fermentation broth was diluted to 1 x 107 CFU / mL of leaf spot pathogen spore suspension 5 mL) on the Oxford cup, select the appropriate spacing, 100 μL of biocontrol bacteria filter liquid injection into the Oxford cup, 30 min after standing, placed in a constant temperature of 28 ℃ for 4 d, measure the diameter of inhibition zone and record the experimental results. Each treatment was repeated 3 times, screening of good strains with strong and long-term inhibition activity for subsequent tests.

[0028] Results are shown in Figure 1 From three gradient soil samples, a total of 15 single colonies were isolated, and one strain with an initial inhibition zone width greater than 5 mm was obtained for re-screening. After 7 days of confrontation culture of the primary screening strain and the pathogen, the inhibition zone width of strain BV-3 was greater than 5 mm. Finally, strain BV-3 was selected for further classification and identification.

[0029] Strain purification was performed by plate streaking method. A sterile gun head was used to pick up the antagonistic bacteria to be purified on the medium with isolated antagonistic bacteria, and parallel streaks were made on the surface of sterile NA medium plates. After 2 days of culture at 28℃, the growth was good, and the strain was stored at 4℃ for standby.

[0030] Example 2 Identification of antagonistic strain

[0031] First, the strain was inoculated into PDA medium and cultured at 28℃ for 2 days. The colony morphological characteristics were observed, and the strain was preliminarily determined. Then the strain was inoculated into NA medium, and the colony color, transparency, edge characteristics, etc. were observed and recorded. Based on the preliminary identification results of morphology, the strain was subjected to Gram staining reaction. The contact enzyme reaction, sodium malonate reaction, methyl red reaction, V-P reaction, citrate utilization, carbohydrate and starch hydrolysis reaction were determined. The pathogenic bacteria were morphologically identified according to the "Common Bacteria System Identification Manual" and "Berger Bacteria Identification Manual".

[0032] The results showed that after inoculation of BV-3 into NA medium and culture at 28℃ for 2 days, the growth was good, the colony was convex, milky white, opaque, the surface was dry, and the edge was irregular. Figure 2 Gram staining of the strain was positive, the cells were rod-shaped, and the size was 0.6-0.8 μm x 2.0-4.0 μm.

[0033] Finally, the genomic DNA of the biocontrol bacteria was extracted by using a bacterial genomic DNA extraction kit (OMEGA), and the BV-3 strain was subjected to molecular identification by 16S rDNA sequence analysis. The 16S rDNA PCR amplification primers were 27-F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO. 1) / 1541-R (5'-AAGGAGGTGATCCAGCCGCA-3', SEQ ID NO. 2). The PCR reaction conditions were as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 45 s, 55°C annealing for 90 s, 72°C extension for 2 min, 35 cycles, and finally 72°C extension for 5 min. The obtained PCR product was sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing. The obtained sequence was spliced and subjected to BLAST analysis in the NCBI database, and was submitted to NCBI to obtain the corresponding GenBank accession number. The 16S rDNA sequences of related strains were downloaded from GenBank based on the comparison and analysis results, and a phylogenetic tree was constructed by using MEGA7.0 Neighbor-joining analysis.

[0034] Results: The 16S rDNA of the strain BV-3 was amplified to obtain a sequence fragment with a length of 1430 bp. The sequencing results were subjected to sequence alignment in GenBank, and it was found that the sequence fragment had a similarity of 99% with the model strain of Bacillus velezensis (GenBank accession number: CP053377). The 16S rDNA gene sequences of related Bacillus strains were selected from the Genbank database to construct a phylogenetic tree (see Figure 3 ). The results showed that the strain BV-3 was clustered with Bacillus velezensis in the phylogenetic tree based on 16S rDNA. Combined with morphological characteristics and physiological and biochemical characteristics, the strain BV-3 was identified as Bacillus velezensis. The strain was preserved in the China General Microbiological Culture Collection Center on March 27, 2024, with a preservation number of CGMCC No. 30159 and a preservation address of No. 3, Beichen West Road, Chaoyang District, Beijing, and was named as Bacillus velezensis.

[0035] Example 3 Antagonistic spectrum of antagonistic strains

[0036] The antagonistic strain BV-3 was selected to determine the inhibition effect on five common maize leaf spot pathogenic fungi, i.e. Clonostachys rosea, Clonostachys cespitosa, Bipolaris spp., Alternaria alternata and Exserohilum turcicum, by means of confrontation culture method, three times of repetition for each treatment, and all the plates were placed in a 28℃ incubator; the inhibition band width and colony diameter of the test group were recorded and measured at 3d, 5d and 7d respectively, and the inhibition band width was the distance between the antagonistic bacteria and the edge of the pathogenic bacteria colony.

[0037] The results of the inhibition spectrum determination are shown in Table 1, and it can be seen that the strain BV-3 has different degrees of inhibition effect on the five common maize leaf spot pathogenic fungi, among which the inhibition effect on Bipolaris spp. is the most obvious, with an inhibition band width of 20.14mm, and it also has good inhibition effect on the other four pathogenic fungi, with an inhibition band width of 6.81-19.28mm. It can be seen that the strain BV-3 has good inhibition effect on a variety of common maize leaf spot pathogenic fungi.

[0038] Table 1 Inhibition band width of strain BV-3 on five maize leaf spot pathogenic fungi (5d)

[0039]

[0040] Example 4 Potting prevention effect of antagonistic strain

[0041] A maize high-resistant variety was selected for the prevention effect determination test, and healthy maize plants with a height of 20cm were selected, and three treatments were set. The purified maize small spot pathogenic fungus was picked in PDB and cultured at 28℃ and 220r / min for 7d, and then filtered to remove mycelium and configured into a pathogenic spore suspension of 1×10 7 CFU / mL, 20mL, inoculated for 2d, and then sprayed with 20mL of sterile water (CK), 1×10 8 CFU / mL of Bacillus velezensis BV-3 fermentation liquid (treatment 1) and 1000 times of 45% prochloraz solution (treatment 2), 10 pots for each treatment, three times of repetition, and routine management during the test. The disease index was counted on the 14th day and the 21st day after inoculation, and the prevention effect was calculated.

[0042] The pot experiment results of the strain BV-3 on corn small spot disease show that the corn plant has good growth, less leaf spot and light disease degree after being inoculated with the fermentation liquor of the strain BV-3. As shown in Table 2, on the 14th day after inoculation, the control effect of the fermentation liquor of the strain BV-3 on corn small spot disease reaches 78.54%; on the 21st day after inoculation, the control effect of the fermentation liquor of the strain BV-3 on corn small spot disease reaches 69.87%, which is higher than the control effect of 1000 times 45% prochloraz. The results show that the antagonistic bacteria have inhibitory effect on corn small spot disease caused by Exserohilum turcicum and have good application potential.

[0043] Table 2 Pot control effect of the strain BV-3 on corn small spot disease

[0044]

[0045] Example 5 Control effect determination of metabolic products of antagonistic strain

[0046] The primary screening strain BV-3 was inoculated into LB liquid medium, and then cultured at 28°C with 220r / min oscillation for 3 days. After being frozen and centrifuged (10000r / min, 4°C) for 20 minutes, the sterile fermentation liquor was filtered through a 0.22μm filter membrane. 1mL, 3mL and 5mL of the fermentation liquor were added into 95mL PDA respectively, and then poured into plates and left to solidify. Four kinds of common pathogenic fungi, i.e. Cladosporium sphaerospermum, Exserohilum turcicum, Alternaria alternata and Alternaria longipes, were inoculated with the pathogenic fungus blocks in the center of the plates containing the metabolic products of the strain BV-3, and then placed in a constant temperature incubator at 28°C. Each treatment was repeated for 3 times. The pathogenic fungi cultured without the sterile fermentation liquor were used as the control. The colony diameters were measured by the cross method at 3d, 5d and 7d respectively, and the inhibition rate of the metabolic products of the antagonistic strain was calculated. Inhibition rate (%) = (control colony diameter - treatment colony diameter) / (control colony diameter - fungus cake diameter) x 100.

[0047] The inhibition effect of the metabolic products of the strain BV-3 on four common corn leaf spot disease pathogenic fungi was determined as shown in Table 3. After constant temperature culture for 5 days, it can be seen that the metabolic products of the strain BV-3 have obvious inhibitory effect on the four common corn leaf spot disease pathogenic fungi. Figure 4

[0048] The colony diameters of CK and the test group were measured by the cross method respectively, and the inhibition rate of the metabolic products of the strain BV-3 was calculated as shown in Table 3. It can be seen that the inhibition rate of the metabolic products of the strain BV-3 on Exserohilum turcicum is the highest, reaching 94.90%, and the inhibition rates on the other three pathogenic fungi are between 92.95% and 94.57%, which are relatively high.

[0049] Table 3 Inhibition rate of the metabolic products of the strain BV-3

[0050] ​

[0051] The various embodiments described in this specification are presented for the purpose of illustration and description. Each of the embodiments described in this specification is presented individually for ease of understanding, and the same or similar elements in the various embodiments are cross-referenced to each other.

[0052] The above description of disclosed embodiments is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of skill in the art upon reading this specification and that the general principles described herein can be applied to other embodiments without departing from the scope of the disclosure. Accordingly, the disclosure is not limited to the embodiments described herein but rather the scope of the disclosure is to be accorded the broadest interpretation of the principles described herein and the claims so as to encompass all reasonable adaptations and modifications.

Claims

1. A Bacillus velezinoffii ( Bacillus Velezensis )BV-3, characterized in that The Bacillus velezinis BV-3 was deposited in the General Microbiology Center of China Culture Collection Administration Committee on March 27, 2024, with the deposit number CGMCC No. 30159, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified as Bacillus velezinis Bacillus Velezensis .

2. The use of the Bacillus Velezii BV-3 according to claim 1, characterized in that The application is application in preventing and controlling corn leaf spot.

3. The use according to claim 2, characterized in that The antibacterial spectrum of the Velez Bacillus BV-3 includes: black Epstein-Barr virus of corn leaf spot, sorghum Epstein-Barr virus of corn white spot, flat umbilical cord of corn small spot, alternaria leaf spot of corn, and large umbilical cord of corn large spot.

4. A biocontrol agent for preventing and treating corn leaf spot, characterized in that: The biocontrol agent comprises the Bacillus Velezii BV-3 according to claim 1.

5. The use of the sterile fermentation liquid of Bacillus Velez subtilis BV-3 according to claim 1, characterized in that: The application is application in preventing and controlling corn leaf spot.

6. The use according to claim 5, characterized in that The antibacterial spectrum of the sterile fermentation broth of Bacillus velezensis BV-3 includes: black coccus of corn leaf spot, Helicoverpa spp. of corn small spot, Alternaria alternata of corn leaf spot and Helicoverpa spp. of corn large spot.

7. A preparation for preventing and treating corn leaf spot, characterized in that: The preparation comprises the sterile fermentation broth of the Bacillus Velez subtilis BV-3 according to claim 1.

8. The preparation according to claim 7, characterized in that The preparation process of the sterile fermentation broth is as follows: the strain BV-3 is inoculated into LB liquid culture medium, shaken and cultured at 28° C. and 220 r / min for 3 days, refrigerated centrifuged at 10,000 r / min and 4° C. for 20 minutes, and filtered through a 0.22 μm filter membrane to obtain the sterile fermentation broth.

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