Bacillus velezensis and application thereof in improving disease resistance of barley

Abstract

The present application provides a bacillus velezensis strain, taking the above-mentioned strain separated in the laboratory as an example, the bacillus velezensis strain is used in barley planting, the bacillus velezensis and the fermentation liquor and metabolites thereof can significantly inhibit the pathogenic bacteria vibrio parahaemolyticus, improve the disease resistance of barley, and have good production application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Bacillus belye that antagonizes barley pathogens. Background Technology

[0002] Bacillus spp., as biocontrol bacteria, has been widely used in the prevention and control of diseases in crops, livestock, poultry, and aquatic animals, and is currently a research hotspot in microbial ecological control. Bacillus velezensis, a new species in the genus Bacillus, is widely used in the biological control of plant and animal diseases due to its ability to produce various secondary metabolites and its broad-spectrum antibacterial activity and growth-promoting effects. Bacillus velezensis can secrete various antibacterial substances, such as lipopeptides, antibacterial proteins, and polyketides.

[0003] my country is a major barley-producing country, and barley is widely used as a grain, feed, and brewing ingredient. Bacterial diseases severely restrict yield and grain quality during barley production. Barley black luster, bacterial leaf spot, and bacterial streak are the main bacterial diseases affecting barley, caused by *Xanthomonas hygroscopicus*, *Pseudomonas syringae*, and *Acidophilus spp.*, respectively. These pathogens can be spread through seeds and by rain splash, easily causing outbreaks under high humidity conditions. Disease outbreaks can reduce barley yield by 10-35% and severely degrade the quality of brewing barley. Currently, production mainly relies on chemical agents for disease control. However, long-term and excessive use of pesticides can lead to increased pathogen resistance, pesticide residues, and damage to the farmland microbial community. Therefore, developing biocontrol strains adapted to the barley growing environment is of significant practical importance for the green control of barley diseases. Research has found that *Bacillus belye* has good antagonistic potential against various plant pathogens and can be used for the biological control of bacterial diseases in barley. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus velezensis LHZY6-14, which can inhibit the transparent pathogenic strain of Xanthomonas velezensis, the pathogen causing barley black rim disease, and also inhibit Pseudomonas syringae and Acidophilus spp., showing great promise for green control of barley diseases in agriculture.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The present invention relates to Bacillus bellis LHZY6-14, the fermentation broth and metabolites of the above-mentioned Bacillus bellis, and the application of the above-mentioned Bacillus bellis and its fermentation broth and metabolites in inhibiting barley pathogenic bacteria and improving the disease resistance of barley. The barley pathogenic bacteria include, but are not limited to, Xanthomonas hyalinus, Pseudomonas syringae, and Acidophilus olfleurone.

[0006] Extraction of antibacterial active substances from Bacillus vesiculosus: In a clean bench, Bacillus vesiculosus LHZY6-14 was inoculated into 2216E liquid medium and cultured at 28℃ and 150rpm for 72h to obtain fermentation broth; then ammonium sulfate precipitation with 70% ammonium sulfate saturation was carried out overnight at 4℃, centrifuged at 10000rpm and 4℃ for 20 minutes, and the precipitate was collected; the precipitate was dialyzed in a 3000Da dialysis bag until Nessler's reagent test showed no further yellowing, and then freeze-dried to obtain crude antibacterial protein; in a clean bench, a suspension of Xanthomonas hygroscopic pathogenic strain VP-HL was prepared, adjusted to OD=1, and 100μL was taken to make an antibacterial plate. After punching holes, 100μL of the above four Bacillus strains was added to the wells. The main antibacterial substances are proteins. The effect of Bacillus vesiculosus on the control of bacterial diseases of barley under pot conditions was investigated in three periods: seedling establishment period, biocontrol treatment period, and pathogen challenge period.

[0007] Barley seedlings were randomly divided into four groups: a positive control group and a negative control group. Seedling establishment period: After grouping, cultivate for 7 days, water regularly and in measured amounts, and maintain a stable cultivation environment; Biocontrol treatment period: No biocontrol bacteria were added to either the positive or negative control groups. The experimental group was irrigated with LHZY6-14 bacterial solution, with a final concentration of 3×10⁻⁶ in the soil. 5 CFU / g soil, biocontrol treatment period lasts three days; During the pathogen challenge period: the positive control group and the experimental group were inoculated with Xanthomonas hyalinus transparent pathogenic strains; the negative control group was not inoculated with pathogens; the disease incidence of barley plants was observed and recorded regularly. According to data analysis, the disease severity in the experimental group was significantly lower than that in the positive control group. Therefore, the fermentation broth of strain LHZY6-14 can effectively control barley black awn disease. Attached Figure Description

[0008] Figure 1 shows the effect of fermentation broth of strain LHZY6-14 on inhibiting the transparent pathogenic strain of Xanthomonas hyalinus; Figure 2 shows the effect of crude antibacterial protein extracted from strain LHZY6-14 on inhibiting the transparent pathogenic species of Xanthomonas hygroscopicus; Figure 3 shows the statistical results of disease incidence in barley when strain LHZY6-14 was used as a biocontrol agent in artificially inoculated barley pots. Detailed Implementation

[0009] Example 1: Isolation, purification and identification of Bacillus belye The colony morphology and strain morphology of the above strains were observed. Strain LHZY6-14 was cultured on 2216E solid medium at 28℃ for 15h. The colony morphology characteristics were rod-shaped cells, 0.4-0.6μm×0.9-4.0μm, arranged singly or in pairs, with nearly round spores, apical, and enlarged sporangia. It belongs to Bacillus belye and is Gram-positive.

[0010] Physiological and biochemical characteristics of strain LHZY6-14 were analyzed and are shown in Table 1. The 16S rDNA sequence of this strain was determined, yielding a 1392 bp gene sequence fragment with the sequence registration number OL990396. Comparison with EZ website confirmed that it is identified as Bacillus velezensis.

[0011] Based on the morphological, physiological, and biochemical characteristics of strain LHZY6-14, as well as the results of 16S rDNA sequence homology comparison, strain LHZY6-14 was identified as Bacillus velezensis. Its physiological and biochemical characteristics are shown in the table below:

[0013]

[0014]

[0015] Example 2: Determination of growth conditions for Bacillus belyssus strain Determination of suitable pH for growth: The activated Bacillus belye LHZY6-14 was adjusted to a bacterial suspension with an OD of approximately 0.4. 5 μL of the suspension was added to a 96-well plate containing 2216E liquid culture medium with different initial pH values. The plate was incubated at 28°C and 150 rpm for 7 days. Using 2216E liquid culture medium as a blank, the optical density of the bacterial suspension was measured at a wavelength of 630 nm at regular intervals. The suitable pH range for the growth of Bacillus belye was found to be 3-12.

[0016] Determination of suitable growth temperature: The activated Bacillus belye culture was adjusted to a bacterial suspension with an OD concentration of approximately 0.4. After mixing, 5 μL was evenly dropped onto 2216E solid medium and cultured at 4℃, 15℃, 28℃, 35℃, 45℃, 60℃ and 80℃ for 24 h. The colony growth was observed, and the suitable temperature range for Bacillus belye growth was found to be 15℃-45℃.

[0017] Example 3 Antimicrobial susceptibility test of Bacillus belyssus The above-mentioned *Bacillus belyssioides* LHZY6-14 was cultured on 2216E solid medium at 28℃ for 15 h. The culture was then diluted with sterile seawater to form a bacterial suspension. 100 μL of this suspension was evenly spread onto 2216E solid medium. Using sterile forceps, discs containing 21 antibiotics, including ampicillin, were evenly placed on agar plates. After incubation at 28℃ for 15 h, the plates were removed for observation and the results were recorded as follows:

[0018] Example 4 Anaerobic Experiment with Bacillus belyssus Bacillus belye LHZY6-14 was cultured on 2216E solid medium at 28℃ for 15 h. The culture was then diluted with sterile seawater to form a bacterial suspension. 10 μL of the suspension was evenly distributed onto 2216E solid medium, sealed, and placed in an anaerobic bag. The suspension was then cultured at 28℃ for 120 h. Afterward, it was observed that the Bacillus belye had grown obvious colonies. Given that oxygen may remain on the culture medium plates, Bacillus belye LHZY6-14 can survive even under microaerobic conditions.

[0019] Example 5: Antagonistic effect of Bacillus belyssus on Xanthomonas hyaline pathogenic strain VP-HL In a clean bench, *Xanthomonas hygroscopicus* strain with clear pathogenicity was inoculated into 2216E liquid medium and cultured at 28°C and 150 rpm for 12 h to obtain a seed culture. Then, 100 μL of the seed culture was inoculated into 2216E solid medium and cultured at 28°C for 15 h for later use. *Bacillus belyssus* LHZY6-14 was inoculated into 2216E liquid medium and cultured at 28°C and 150 rpm for 72 h to obtain a fermentation broth for later use. A suspension of *Xanthomonas hygroscopicus* strain VP-HL was prepared and adjusted to OD600=1. 100 μL of the suspension was used to make an inhibition plate. After punching holes, 100 μL of the fermentation broth prepared by the above strain was added to the wells. The diameter of the inhibition zone was observed after 10 h at 28°C.

[0020] Experimental results:

[0021] Example 6 Extraction of antibacterial active substances from Bacillus belyssus In a clean bench, Bacillus berberis LHZY6-14 was inoculated into 2216E liquid medium and cultured at 28℃ and 150 rpm for 72 h to obtain fermentation broth. Then, ammonium sulfate precipitation with 70% ammonium sulfate saturation was carried out overnight at 4℃, followed by centrifugation at 10000 rpm and 4℃ for 20 minutes to collect the precipitate. The precipitate was dialyzed in a 3000 Da dialysis bag until Nessler's reagent test showed no further yellowing, and then freeze-dried to obtain antibacterial crude protein.

[0022] In a clean bench, a suspension of Xanthomonas hyalinus VP-HL, a transparent pathogenic strain, was prepared and adjusted to OD=1. 100 μL of the suspension was used to make an antibacterial plate. After punching holes, 100 μL of the above four Bacillus strains was added to each hole. The main antibacterial substance is protein. Example 7: Effect of Bacillus belye on the control of bacterial diseases in barley under pot cultivation conditions The experiment was divided into three phases: the seedling establishment period, the biocontrol treatment period, and the pathogen challenge period.

[0023] Barley seedlings were randomly divided into four groups: a positive control group, a negative control group, and an experimental group, with three replicates in each group. Seedling establishment period: After grouping, cultivate for 7 days, water regularly and in measured amounts, and maintain a stable cultivation environment; Biocontrol treatment period: No biocontrol bacteria were added to either the positive or negative control groups. The experimental group was irrigated with LHZY6-14 bacterial solution, with a final concentration of 3×10⁻⁶ in the soil. 5 CFU / g soil, biocontrol treatment period lasts three days; During the pathogen challenge period: the positive control group and the experimental group were inoculated with Xanthomonas hyalinus transparent pathogenic strains; the negative control group was not inoculated with pathogens; the disease incidence of barley plants was observed and recorded regularly. According to data analysis, the disease severity in the experimental group was significantly lower than that in the positive control group. Therefore, the fermentation broth of strain LHZY6-14 can effectively control barley black awn disease.

Claims

1. A strain of Bacillus velezensis and its application in improving barley disease resistance, namely Bacillus velezensis, whose fermentation broth and crude metabolites can inhibit the barley pathogenic bacterium Xanthomonas translucens, the fermentation broth and metabolites of the aforementioned Bacillus velezensis, and the application of the aforementioned Bacillus velezensis and its fermentation broth and metabolites in inhibiting barley pathogenic bacteria and improving barley disease resistance, wherein the barley pathogenic bacteria include, but are not limited to, Xanthomonas translucens, Pseudomonas syringae, and Acidophilus velezensis.

2. The Bacillus berberis described in claim 1 can be used as a biocontrol bacterium in barley cultivation.

3. The biocontrol bacteria described in claim 3 can be used as a barley disease control agent.

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