Bacillus velezensis BLS-27, microbial inoculum comprising same, preparation method and application

Abstract

The invention belongs to the field of microorganisms, and relates to a bacillus velezensis BLS-27 and a fungicide and application thereof, the bacillus velezensis BLS-27 is preserved in China General Microbiological Culture Collection Center on October 21, 2024, the preservation number is CGMCC No.32274, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain BLS-27 has an inhibition effect on wild jujube anthracnose pathogenic bacteria, can effectively inhibit various plant disease pathogenic bacteria, shows broad-spectrum antibacterial activity, is environment-friendly and can be widely applied to plant disease control.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Bacillus Velezii BLS-27 and a bacterial agent comprising the same, as well as a preparation method and application thereof. Background Art

[0002] In current agricultural production, the prevention and control of plant diseases mainly relies on chemical agents. When chemical agents are used properly, they can effectively control diseases to a certain extent and ensure the healthy growth of crops. However, with the widespread use and improper application of chemical substances, a series of problems have gradually emerged. First, due to the large amount of chemical substances in the environment, the resistance and tolerance of pathogens have continued to increase, resulting in the gradual weakening of the prevention and control effect of chemical agents, and the disease problem has become increasingly difficult to control. Secondly, the residues of these chemicals in the environment have caused serious pollution to natural resources such as the atmosphere, soil and water resources, and the ecological environment has been severely damaged, and people's daily life and health have also been threatened.

[0003] In view of the various drawbacks of chemical control, biological control has gradually attracted people's attention as an environmentally friendly and safe alternative method. Studies have found that antagonistic bacteria or fungal strains can be isolated from the inside or rhizosphere of plants and prepared into microbial agents to achieve the effect of biological control. Such microbial agents, including fungi, bacteria, actinomycetes, yeast or viruses, are regarded as the preferred alternative to chemical pesticides due to their high selectivity, safety for humans and animals, environmental friendliness and low drug resistance.

[0004] Among them, Bacillus, as the main biological control bacteria, has broad application prospects. In particular, Bacillus velezensis has attracted much attention due to its strong stress resistance and broad-spectrum antibacterial properties. Bacillus velezensis has a wide variety of species and a wide range of sources, a high natural survival rate, can effectively inhibit a variety of plant disease pathogens, and is environmentally friendly, so it has a broad application space in the field of plant disease control.

[0005] Although Bacillus Velez has shown great potential in biological control, it still faces many challenges in practical application. There are relatively few biological pesticide products on the market, and the mechanism of action is relatively single, resulting in unstable control effects. In addition, the relatively complex research and development and production technology of biological pesticides and their high cost also limit their large-scale promotion and application. Therefore, there is an urgent need to develop more efficient, low-toxic and green disease control strategies to meet the urgent needs of agricultural production for disease control.

[0006] The present invention aims to provide a highly efficient, low-toxic and environmentally friendly Bacillus Velezii strain and its bacterial agent and application method, so as to overcome the defects of the prior art and provide a new solution for the prevention and treatment of plant diseases. Summary of the invention

[0007] In view of the above problems, the present invention provides a strain of Bacillus Velezii BLS-27 with broad-spectrum antibacterial activity. The strain has an inhibitory effect on the pathogen of anthracnose of wild jujube, can effectively inhibit a variety of plant disease pathogens, is environmentally friendly, and can be widely used in plant disease prevention and control.

[0008] In order to solve the above problems in the prior art, the present invention provides the following technical solutions:

[0009] The invention provides a Velezella sp. BLS-27, whose deposit number is CGMCC No.32274.

[0010] The strain was isolated and obtained from rhizosphere soil microorganisms of Jujube jujube, and was numbered BLS-27. The morphological characteristics of strain BLS-27 are: the single colony on LB fermentation medium is milky white, with irregular edges, sticky, opaque, and the surface of the colony is rough and wrinkled; its body is rod-shaped, with a size of 1.23-2.80μm×0.51-0.70μm. Based on the results of various physiological and biochemical experiments and molecular biological analysis, the bacterium was identified as Bacillus Velezii.

[0011] The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 21, 2024, with the deposit number CGMCC No.32274, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0012] The present invention also provides a bacterial agent, comprising the aforementioned Bacillus Velezii BLS-27.

[0013] Based on the broad-spectrum antibacterial activity of Bacillus Velezii BLS-27, it can be used as an active ingredient to prepare bacterial agents.

[0014] Furthermore, the bacterial agent includes the bacterial liquid, bacterial cells, spores or fermentation supernatant of the aforementioned Bacillus Velezii BLS-27.

[0015] In addition, in order to further optimize and improve the bactericidal effect or growth-promoting effect of the microbial agent, the microbial agent may also include other fungicidal active ingredients for use in combination, such as other biocontrol bacteria and antibacterial active substances that have complementary effects in the fungicidal spectrum.

[0016] The present invention also provides the use of the Bacillus Velezii BLS-27 described in the above technical solution or the bacterial agent described in the above technical solution in preventing and controlling plant diseases.

[0017] Preferably, the plant diseases include jujube anthracnose, walnut anthracnose, soapberry wilt, chestnut blight and the like.

[0018] The present invention also provides the use of the Bacillus Velezii BLS-27 described in the above technical solution or the bacterial agent described in the above technical solution in inhibiting plant pathogens.

[0019] Preferably, the plant pathogen is a plant pathogenic fungus; the plant pathogenic fungus is at least one of Colletotrichum gloeosporioides, Colletotrichum frutescens, Fusarium solani, Fusarium oxysporum, Fusarium equisetum, Fusarium fujikura, and Alternaria alternata.

[0020] The present invention provides a method for preparing the above-mentioned bacterial agent, comprising the following steps: inoculating Bacillus Velez subtilis BLS-27 into a fermentation medium to obtain a bacterial liquid of Bacillus Velez subtilis BLS-27.

[0021] The fermentation medium can be selected from conventional microbial fermentation medium, such as LB fermentation medium.

[0022] Preferably, the fermentation medium comprises the following components in mass volume concentrations: 2% glucose, 1% beef extract, and 1% sodium chloride; the pH of the fermentation medium is 7. The use of the fermentation medium can significantly increase the growth of Bacillus Velezii BLS-27.

[0023] Preferably, the Velez subtilis BLS-27 is inoculated into the fermentation medium in the form of seed liquid; the inoculation amount of the seed liquid is 3%. Selecting an inoculation amount of 3% for inoculation can significantly increase the growth amount of Velez subtilis BLS-27.

[0024] Preferably, the fermentation culture conditions are: temperature 30°C, rotation speed 200r / min, and dark culture for 24h. Such fermentation conditions can significantly increase the growth of Bacillus Velezii BLS-27.

[0025] Preferably, in the application of the Bacillus Velez subtilis BLS-27 or the bacterial agent described in the above technical solution in preventing and controlling plant diseases, the fermented bacterial solution of Bacillus Velez subtilis BLS-27 is diluted and then applied. After the fermented bacterial solution of Bacillus Velez subtilis BLS-27 is diluted 200 times, the effect of preventing and controlling plant diseases is the best.

[0026] The present invention has the following beneficial effects:

[0027] The invention screens out a bacillus strain from soil microorganisms of sour jujube, identifies it as Bacillus velezensis through morphological and 16S rDNA comparison analysis, has an inhibitory effect on the pathogen of sour jujube anthracnose, can effectively inhibit various plant disease pathogens, is environmentally friendly, and can be widely used in plant disease prevention and control.

[0028] The present invention optimizes the fermentation medium and fermentation conditions of Bacillus Velezii BLS-27, significantly improves its growth, enhances the prevention and control effect on plant diseases, is conducive to reducing the use of chemical pesticides and fertilizers, and solves the problems of pesticide residues and environmental pollution. The Bacillus Velezii BLS-27 of the present invention and its bacterial liquid, bacterial body, spore or fermentation supernatant, and the bacterial agent including the same can be used to prevent and control plant diseases and inhibit plant pathogens, and is an efficient, low-toxic and green biological control method with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram of the antagonism of Bacillus Velezii BLS-27 to pathogenic bacteria provided by the present invention;

[0030] Figure 2 A schematic diagram of the antagonism of Bacillus Velezii BLS-27 provided by the present invention to 9 pathogenic bacteria;

[0031] Figure 3 A photo of the colony morphology of Bacillus Velezii BLS-27 on LB fermentation medium provided by the present invention;

[0032] Figure 4 A phylogenetic tree constructed based on the 16S rDNA gene sequence of Bacillus Velezii BLS-27 provided by the present invention;

[0033] Figure 5 A schematic diagram of the growth curve of Bacillus Velezii BLS-27 provided by the present invention;

[0034] Figure 6 This is a graph showing the effect of different basic fermentation media on the growth of Bacillus Velezii BLS-27;

[0035] Figure 7 This is a graph showing the effect of different carbon sources on the growth of Bacillus Velezii BLS-27;

[0036] Figure 8 This is a graph showing the effect of the amount of glucose added on the growth of Bacillus Velez BLS-27;

[0037] Fig. 9 This is a graph showing the effect of different nitrogen sources on the growth of Bacillus Velezii BLS-27;

[0038] Fig.10 This is the effect of the amount of beef extract added on the growth of Bacillus Velezii BLS-27;

[0039] Fig.11 This is a graph showing the effect of different inorganic salts on the growth of Bacillus Velezii BLS-27;

[0040] Fig.12 This is the effect of the amount of sodium chloride added on the growth of Bacillus Velez BLS-27;

[0041] Fig.13 This is a graph showing the effect of different inoculation amounts on the growth of Bacillus Velezii BLS-27;

[0042] Fig.14 This is the effect of different pH values ​​on the growth of Bacillus Velezii BLS-27;

[0043] Fig.15 This is a graph showing the effect of different temperatures on the growth of Bacillus Velezii BLS-27;

[0044] Fig.16 This is a graph showing the effect of different rotation speeds on the growth of Bacillus Velezii BLS-27. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and are not limitations of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Unless otherwise specified, the test materials, reagents, methods and equipment used in the following examples are conventional test materials, reagents, methods and equipment in the technical field; unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial channels.

[0047] LB fermentation medium: 10 g tryptone, 5 g yeast powder, 10 g sodium chloride, 20 g agar, distilled water to 1 L;

[0048] PDA fermentation medium: 200 g potato, 20 g agar, 20 g glucose, distilled water to 1 L.

[0049] Example 1: Screening of antagonistic bacteria BLS-27

[0050] The strains isolated from the leaves, fruits and rhizosphere soil of Solanum jujuba were cultured in LB fermentation medium at 28°C and 180r / min for 24h. A sterile punch was used to take a 9mm diameter pathogen cake (Colletotrichum gloeosporioides) cultured for 7d and inoculated in the center of the PDA fermentation medium. 3μL of the strain fermentation liquid was inoculated at 2.50cm from the center of the fermentation medium by the "cross method" and placed in a 28°C constant temperature incubator for 7d. The uninoculated fermentation liquid was used as the control group. Each treatment was repeated 3 times for preliminary screening. The antagonistic strains with antibacterial effect were rescreened and the inhibition rate was calculated.

[0051]

[0052] like Figure 1 As shown, the antagonistic strains with antibacterial effect were screened again by the plate confrontation method, and the strain with the best inhibitory effect on pathogens was screened out. The inhibition rate of this strain on the mycelial growth of pathogens was 80.72%, and it was named BLS-27.

[0053] Example 2: Determination of the broad-spectrum antibacterial activity of antagonistic bacteria BLS-27

[0054] The plate confrontation culture method was used to determine the inhibition rate of the selected antagonistic strains against 9 pathogenic fungi, including Fusarium proliferatum, Alternaria alternata, Fusarium oxysporum, Colletotrichum fructicola, Fusarium equiseti, Fusarium fujikuroi, Fusarium verticillioides, Fusarium incarnatum, and Colletotrichum gloeosporioides. Each treatment was repeated 3 times, and the specific operation was the same as in Example 1. The results are shown in Table 1 and Figure 2 shown.

[0055] Table 1: Growth inhibition effect of antagonistic bacteria BLS-27 on 9 plant pathogens

[0056] deal with Colony diameter (mm) Inhibition rate (%) Fusarium equisetum 32.00±0.20a 62.50±1.78g Fusarium oxysporum 26.73±0.15bc 66.67±1.61ef Fusarium 26.08±0.10bc 69.14±1.04ef Fusarium fujikura 33.70±0.25a 56.63±3.45g Colletotrichum 28.71±0.21b 67.47±2.45f Alternaria 20.70±0.06ef 76.47±0.90bc Fusarium verticillium 25.00±0.10cd 69.51±1.76de Fusarium erythrocyticum 18.00±0.10fg 78.82±1.24ab Colletotrichum gloeosporioides 16.00±0.10g 80.72±80.95a

[0057] Note: The data in Table 1 are mean ± standard deviation. The data with different letters are significantly different at p < 0.05 level by t test, the same below.

[0058] Table 1 and Figure 2The results showed that strain BLS-27 had significant inhibitory effects on 9 different pathogenic fungi, including Fusarium proliferatum, Alternaria alternata, Fusarium oxysporum, Colletotrichum fructicola, Fusarium equiseti, Fusarium fujikuroi, Fusarium verticillioides, Fusarium incarnatum, and Colletotrichum gloeosporioides, and had a broad-spectrum antibacterial effect. After 7 days of confrontation culture, the antagonistic strain BLS-27 had the strongest inhibitory effect on the growth of Colletotrichum gloeosporioides, with a growth inhibition rate of 80.72%; and the weakest inhibitory effect on the growth of Fusarium fujikuroi, with an inhibition rate of 56.63%.

[0059] Example 3: Identification of BLS-27 strain

[0060] (1) Morphological identification

[0061] The colony morphological characteristics of the antagonistic strain BLS-27 were observed and recorded, and then Gram staining technique was used to observe and analyze its bacterial morphology with the help of a biological microscope.

[0062] like Figure 3 As shown, the antagonistic strain BLS-27 is a Gram-positive bacterium. The single colony on the LB fermentation medium is milky white, has irregular edges, is sticky, and opaque, and the surface of the colony is rough and wrinkled; its bacteria are rod-shaped and have a size of 1.23-2.80 μm×0.51-0.70 μm.

[0063] (2) Physiological and biochemical assays

[0064] According to the Manual of Identification of Common Bacterial Systems and the Bergey Manual of Bacterial Identification, the strain BLS-27 was tested for physiological and biochemical characteristics such as catalase, starch hydrolysis, gelatin liquefaction, oxidase, methyl red, VP, citrate utilization and tryptophan deaminase to accurately identify the strain. The identification results are shown in Table 2.

[0065] Table 2: Physiological and biochemical characteristics of strain BLS-27

[0066] project result project result Catalase + VP determination + Starch hydrolysis + Utilization of citric acid + Gelatin liquefaction + Tryptophan deaminase + Oxidase + Indole production － Methyl red + Sportiness +

[0067] The identification results in Table 2 show that the catalase, oxidase, VP assay, starch hydrolysis, methyl red, gelatin liquefaction, citrate utilization, and tryptophan deaminase physiological and biochemical tests of strain BLS-27 are all positive, and the indole production test result is negative, indicating that it has motility. Through the analysis of the morphological characteristics and physiological and biochemical characteristics of strain BLS-27, strain BLS-27 can be preliminarily identified as Bacillus.

[0068] (3) Molecular biological identification

[0069] The DNA of strain BLS-27 was extracted using the bacterial genomic DNA rapid extraction kit of Beijing Coolab Technology Co., Ltd., and the DNA of strain BLS-27 was used as a template to perform PCR amplification of the 16S rDNA gene using universal primers (27F / 1492R). The amplification system was: 2×M5 HiPer plus Taq HiFi PCR mix 10μL, 0.5μL of upstream and downstream primers (27F / 1492R), 1μL of DNA template, and 8μL of ddH2O. The amplification conditions were: pre-denaturation temperature and time 95℃, 3min; denaturation temperature and time 94℃, 20s; annealing temperature and time 64℃, 25s; extension temperature and time 72℃, 15s; final extension temperature and time 72℃, 5min; cycle number 38. After the reaction was completed, the PCR amplification product was detected by 1% agarose gel electrophoresis to determine the target band. The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for gene sequencing.

[0070] Sequencing results of 16S rDNA amplified fragment of strain BLS-27:

[0071]

[0072] The sequencing results were input into the NCBI database for BLAST comparison, and it was found that the homology between strain BLS-27 and Bacillus velezensis strain MFFC11X was 99%.

[0073] like Figure 4 As shown, the phylogenetic tree of strain BLS-27 was constructed using Mega7.0 software. The results showed that strain BLS-27 had the highest homology with Bacillus velezensis strain MFFC11X. Combined with the previous morphological colony observations and morphological and physiological and biochemical characteristics analysis, strain BLS-27 was identified as Bacillus velezensis.

[0074] Based on the above identification results, strain BLS-27 was confirmed to be a strain of Bacillus velezensis and was named BLS-27. The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on October 21, 2024, with the deposit number CGMCC No.32274 and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0075] Example 4: In vitro control effect determination of Bacillus Velezii BLS-27

[0076] Preparation of Bacillus Velez subtilis BLS-27 suspension: Pick a single colony of Bacillus Velez subtilis BLS-27 isolated and purified previously, inoculate it into 50 mL LB fermentation medium, and culture it in a shaking incubator at 28°C and 180 r / min for 24 h. OD 600 The value is adjusted to 1 for subsequent testing.

[0077] Preparation of bacterial cake of pathogenic bacteria strain SZ-1: Use a sterile punch to take bacterial cake from the edge of the colony of strain SZ-1 with a diameter of 9 mm after 7 days of culture.

[0078] Select healthy Solanum jujube hangings, disinfect their surfaces, rinse them three times with sterile water, place them on sterile filter paper to dry, and use an inoculation needle to slightly pierce the hangings.

[0079] Prevention group: 100 μL of BLS-27 bacterial solution was dripped onto the wound of the jujube hanging plant, and then the bacterial cake of strain SZ-1 was inoculated after 24 hours.

[0080] Treatment group: First, the pathogenic bacteria cake of SZ-1 was inoculated on the wound of the jujube hanging, and 100 μL of the bacterial solution of strain BLS-27 was added after 24 hours.

[0081] Positive control group: The pathogenic bacteria strain SZ-1 cake was inoculated on the wound of the jujube hanging.

[0082] Each treatment was repeated 3 times. The plants were cultured in a constant temperature incubator at 28°C. The results were recorded after 7 days. The disease index and control effect of each treatment and the control were calculated according to the disease classification standard.

[0083] Table 3: Disease classification standards

[0084] level Representative value Disease occurrence 0 0 Health Organization 1 1 The lesion area accounts for less than 5% of the entire tissue 2 2 The lesion area occupies 5% to 25% of the whole tissue 3 3 The lesion area occupies 25% to 50% of the entire tissue 4 4 The lesion area occupies 50% to 75% of the entire tissue 5 5 Lesion area is greater than 75% of the entire tissue

[0085]

[0086]

[0087] Table 4: In vitro control effect of Bacillus Velezii BLS-27

[0088] deal with Lesion length (mm) Disease index Control effect (%) Positive Control 51.33±3.51a 76.00±1.52a — prevention 3.33±1.55c 20.00±2.51c 93.51±2.98a treat 12.00±3.08b 44.00±1.53b 76.62±5.84b

[0089] The results in Table 4 show that the average length of the lesions of the preventive treatment of strain BLS-27 was only 3.33 mm, which was significantly smaller than that of the control and treatment treatments; the disease index was 20.00, significantly lower than that of the control and treatment treatments; the control effect was 93.51%, significantly better than the control effect of 76.62% of the treatment treatment.

[0090] Example 5: Fermentation Optimization of Bacillus Velezii BLS-27

[0091] (1) Determination of the growth curve of Bacillus velezensis BLS-27

[0092] The strain BLS-27 cultured at 28°C for 24 h was inoculated into 50 mL LB fermentation medium and cultured in a shaker at 28°C and 180 r / min for 24 h to obtain the seed solution. The growth of the strain BLS-27 seed solution was determined by UV-visible spectrophotometry, and its OD 600 The value was adjusted to 1 for subsequent experiments. The strain BLS-27 seed solution was inoculated with 1% of the inoculation amount into 50 mL of LB fermentation medium, and placed in a shaking incubator at 28°C and 200 r / min for shaking culture. Each treatment was repeated 3 times, and the LB fermentation medium without the strain was used as a blank control. The horizontal axis is the culture time, and the vertical axis is the OD 600 The growth curve of strain BLS-27 was plotted.

[0093] The results are as follows Figure 5 As shown, 0-8h is the growth retardation period of the antagonistic strain BLS-27; 8-24h enters the logarithmic growth period, and the strain reproduces in large quantities; after 24h, it enters the stable period, and the strain reaches the highest value at 24h, OD 600 The value is 2.032, indicating that the bacteria have vigorous metabolism and reproduce quickly.

[0094] (2) Screening and optimization of basic fermentation medium and components

[0095] A. Screening of basic fermentation medium

[0096] The seed solution of strain BLS-27 (OD 600 =1, the same below) was inoculated into sterilized BP, BPY, SOB, NYPD, NB, YT and LB fermentation medium and cultured at 28°C and 180 r / min for 24 h. Each treatment was repeated 3 times and the OD of the fermentation broth was measured. 600 Determine the optimal basic fermentation medium.

[0097] The results are as follows Figure 6 As shown in the figure, different basic fermentation medium had significant effects on the growth of strain BLS-27. When LB fermentation medium was used as the basic fermentation medium, the OD 600 The highest value was 2.08; when YT liquid fermentation medium was used as the basic fermentation medium, the OD 600 The value is the lowest, which is 1.21. Therefore, LB fermentation medium is the best basic fermentation medium.

[0098] B. Screening of the best carbon source and addition amount

[0099] The carbon source in the basic fermentation medium was replaced with equal masses of glucose, sucrose, maltose, lactose and starch, and other conditions were kept unchanged. The 24h-old seed liquid was inoculated for fermentation. The basic fermentation medium was used as a control. Each treatment was repeated 3 times, and the OD of the fermentation liquid of the strain was measured. 600 value.

[0100] The results are as follows Figure 7 As shown in the figure, different carbon sources have different effects on the growth of strain BLS-27. The effect of carbon sources on the growth of strain BLS-27 is glucose> starch> sucrose> maltose> lactose. The OD 600 The highest value is 2.04.

[0101] The best carbon source glucose was screened out, and the fermentation medium was prepared with the addition amount of 0.5%, 1%, 2%, 3%, and 4% by mass volume concentration, respectively. The culture was carried out at 28°C and 180r / min for 24h, and the OD of the fermentation broth of the strain was measured. 600 value.

[0102] The results are as follows Figure 8 As shown in the figure, different addition amounts of glucose have different effects on the growth of strain BLS-27. When the addition amount of glucose is 1%, the growth OD of strain BLS-27 is 600The value is the highest, which is 2.07. Therefore, the optimal addition amount of glucose is determined to be 1%.

[0103] C. Screening of the best nitrogen source and addition amount

[0104] The nitrogen source in the basic fermentation medium was replaced with urea, beef extract, peptone, tryptone and casein of equal mass, and other conditions were kept unchanged. The 24h-old seed liquid was inoculated for fermentation. The basic fermentation medium was used as a control. Each treatment was repeated 3 times, and the OD of the fermentation liquid of the strain was measured. 600 value.

[0105] The results are as follows Fig. 9 As shown in the figure, different nitrogen sources have different effects on the growth of strain BLS-27. The effect of nitrogen sources on the growth of strain BLS-27 is from large to small: beef extract > tryptone > urea > peptone > casein. The OD of beef extract treatment is 600 The highest value is 2.09.

[0106] The best nitrogen source beef extract was screened out, and the fermentation medium was prepared with the addition amount of 0.5%, 1%, 2%, 3%, and 4% by mass volume concentration, and the OD of the fermentation broth of the strain was measured. 600 value.

[0107] The results are as follows Fig.10 As shown in the figure, different amounts of beef extract added have different effects on the growth of strain BLS-27. When the amount of beef extract added is 2%, the growth OD of strain BLS-27 600 The value is the highest, which is 2.11. Therefore, the optimal addition amount of beef extract is determined to be 2%.

[0108] D. Screening of the best inorganic salt and addition amount

[0109] The inorganic salts in the basic fermentation medium were replaced with equal masses of sodium chloride, dipotassium hydrogen phosphate, magnesium sulfate, potassium dihydrogen phosphate and magnesium chloride, and other conditions were kept unchanged. The 24h-old seed liquid was inoculated for fermentation. The basic fermentation medium was used as a control. Each treatment was repeated 3 times, and the OD of the fermentation liquid of the strain was measured. 600 value.

[0110] The results are as follows Fig.11 As shown in the figure, different inorganic salts have different effects on the growth of strain BLS-27. The effect of inorganic salts on the growth of strain BLS-27 is from large to small: sodium chloride > dipotassium hydrogen phosphate > potassium dihydrogen phosphate > magnesium sulfate > magnesium chloride. The OD 600 The highest value is 2.13.

[0111] The best inorganic salt sodium chloride was screened out, and the fermentation medium was prepared with the addition amount of 0.5%, 1%, 2%, 3%, and 4% by mass volume concentration, and the OD of the fermentation broth of the strain was measured. 600 value.

[0112] The results are as follows Fig.12 As shown in Figure 2, different amounts of sodium chloride added have different effects on the growth of strain BLS-27. When the amount of sodium chloride added is 1%, the growth OD of strain BLS-27 600 The value is the highest, which is 2.15, so the optimal addition amount of sodium chloride is 1%.

[0113] E. Orthogonal experiment to optimize the composition of fermentation medium

[0114] With carbon source, nitrogen source and inorganic salt as factors and factor variables as levels, a three-factor three-level orthogonal test table was designed for analysis to determine the optimal proportion of each factor.

[0115] Table 5: Orthogonal experimental design of fermentation medium components

[0116] deal with Carbon source (A) Nitrogen source (B) Inorganic salt (C) 1 0.5％ 1％ 0.5％ 2 1％ 2％ 1％ 3 2％ 3％ 2％

[0117] Table 6: Orthogonal test results of different fermentation medium components

[0118]

[0119]

[0120] The results in Table 6 show that the concentration of glucose has the greatest impact on the growth of strain BLS-27, followed by beef extract and then sodium chloride. 600 The optimal combination was determined to be A3B1C2, and the optimal fermentation medium component combination was obtained to be 2% glucose, 1% beef extract, and 1% sodium chloride.

[0121] (3) Screening and optimization of fermentation conditions

[0122] A. Optimal inoculum size

[0123] The 24h old seed solution was inoculated into the optimized fermentation medium (glucose 2%, beef extract 1%, sodium chloride 1%), and the inoculation volume ratio was 1%, 3%, 5%, 7%, 9% and 11%, respectively. Other conditions remained unchanged. Each treatment was repeated 3 times, and the OD of the fermentation liquid of the strain was measured. 600 value.

[0124] The results are as follows Fig.13 The results show that when the inoculation volume is 3%, the OD 600 The maximum value is 2.20. With the continuous increase of inoculation amount, the bacterial growth OD 600The value is decreasing, therefore, 3% inoculation amount is selected for inoculation.

[0125] B. Optimal pH value

[0126] The optimal inoculum size was selected, and the seed solution with a seed age of 24 h was inoculated for fermentation. The pH value of the fermentation medium was adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 respectively. Other conditions remained unchanged. Each treatment was repeated 3 times, and the OD of the fermentation solution of the strain was measured. 600 value.

[0127] The results are as follows Fig.14 As shown in Figure 2, when the pH value was between 6 and 7, the growth of the strain showed an upward trend, and when the pH value was 7, the growth was the largest, and its OD 600 The value is 2.22; while the growth of the strain shows a downward trend when the pH value is between 7 and 8.5. Therefore, the initial pH value of the fermentation liquid should be controlled at 7.

[0128] C. Optimum speed

[0129] The optimal inoculation amount and pH value were selected, and the seed solution with a seed age of 24h was inoculated for fermentation. The shaking speed was 140, 160, 180, 200, 220, and 240r / min respectively. Other conditions remained unchanged. Each treatment was repeated 3 times, and the OD of the fermentation solution of the strain was measured. 600 value.

[0130] The results are as follows Fig.15 As shown in Figure 2, when the speed was between 140 and 200 r / min, the growth of the strain showed an upward trend. When the speed reached 200 r / min, the growth of the strain was the largest, and its OD 600 The value is 2.38; while the growth of the strain shows a downward trend when the speed is between 200 and 240 r / min. Therefore, the shaking speed of 200 r / min is selected as the optimal speed.

[0131] D. Optimum temperature

[0132] The optimal inoculum size, pH value and rotation speed were selected, and the seed solution with a seed age of 24 h was inoculated for fermentation. The shaking incubator temperature was set to 20°C, 25°C, 28°C, 30°C, 34°C and 37°C, and other conditions remained unchanged. Each treatment was repeated 3 times, and the OD of the fermentation solution of the strain was measured. 600 value.

[0133] The results are as follows Fig.16 As shown in Figure 2, the growth of the strain was the largest when the temperature was 30°C, and its OD 600 The value reached 2.30. The results showed that too high or too low fermentation temperature was not conducive to the growth of the strain. Therefore, 30℃ was selected as the optimal fermentation temperature.

[0134] E. Optimization of fermentation conditions by orthogonal experiment

[0135] With inoculation amount, pH value, temperature, and rotation speed as factors, and each factor variable as a level, a four-factor three-level orthogonal test table was designed for orthogonal design analysis. Several fermentation media were configured for fermentation, and the OD of the fermentation broth of the strain was measured. 600 value.

[0136] Table 7: Orthogonal experimental design of fermentation conditions

[0137] deal with Inoculation amount (A) pH(B) Temperature (C) Speed(D) 1 1％ 6.5 28 180 2 3％ 7 30 200 3 5％ 7.5 34 220

[0138] Table 8: Results of orthogonal test of different fermentation medium components

[0139]

[0140]

[0141] The results in Table 8 show that the culture temperature has the greatest impact on the growth of strain BLS-27, followed by pH value, then inoculum size, and finally rotation speed. 600 The optimal combination was determined to be A2B2C2D1, and the optimal fermentation condition combination was the best inoculum size, pH value, temperature and rotation speed were 3%, 7, 30℃ and 200r / min respectively.

[0142] (4) Comparison before and after fermentation optimization

[0143] A. Determination of growth amount and mycelium growth inhibition effect

[0144] The strain BLS-27 was inoculated into the optimized fermentation medium and cultured under the optimal fermentation conditions for 24 h. 600 The optimized value was obtained by taking LB fermentation medium as control to identify the optimization effect. The optimized fermentation broth and the fermentation broth of the basic fermentation medium were added to the PDA fermentation medium, and the pathogen cake with a diameter of 9 mm after 7 days of culture was inoculated in the center of the PDA fermentation medium. 3 μL of the strain fermentation broth was inoculated at 2.50 cm from the center of the fermentation medium by the "cross method", and the colony diameter was measured. It was placed in a 28°C constant temperature incubator for 7 days, and the fermentation broth was not inoculated as the control group. Each treatment was repeated 3 times, and the inhibitory effect of the optimized fermentation broth and the fermentation broth of the basic fermentation medium on the mycelial growth of the strain BLS-27 was compared and analyzed.

[0145] Table 9: Fermentation result verification

[0146] deal with Colony diameter (mm) Inhibitory effect (%) <![CDATA[OD 600 Value]]> Optimized fermentation broth 10.65±1.53c 87.10±1.85a 2.53±0.04a Basic fermentation medium Fermentation broth 19.67±1.15b 80.21±1.40b 2.03±0.03b Comparison 82.67±2.08a — —

[0147] The results showed that the OD of the fermentation liquid of strain BLS-27 after 24h of fermentation in the optimized fermentation medium was 600The value is 2.53, and the OD value of the fermentation liquid after 24 hours of fermentation in the basic fermentation medium is 600 The value is 2.03, which is quite different. The OD of the fermentation broth after optimization 600 The value increased significantly compared with the basic fermentation medium, increasing by 24.63%, indicating that the optimized fermentation medium is more conducive to the growth of strain BLS-27. The colony diameter of the optimized sterile fermentation broth was 10.65 mm, and the mycelial growth inhibition rate was 87.10%, which was 10.89% higher than that of the sterile fermentation broth with the basic fermentation medium. It can be seen that the optimized fermentation medium is more conducive to the growth of strain BLS-27 (Table 9).

[0148] B. Potted plant control effect determination

[0149] Healthy potted jujube seedlings were selected, and the sterilized jujube branches were wounded on the surface by inoculation. Pathogen cakes were placed on the wounds. The inoculated jujubes were sprayed with clean water and kept moist with cotton. After 24 hours, the antagonistic strain solution was sprayed.

[0150] The experiment set up 6 treatments: A: CK (only pathogens); B: 100 times fermentation broth of antagonistic bacteria; C: 200 times fermentation broth of antagonistic bacteria; D: 400 times fermentation broth of antagonistic bacteria; E: 800 times fermentation broth of antagonistic bacteria; F: 1 600 Each treatment was repeated 3 times. After 15 days, the disease index and control effect of each treatment and the control were calculated according to the disease classification standards in Tables 2 to 5.

[0151] Table 10: Effect of BLS-27 fermentation liquid on the control of pathogenic bacteria strain SZ-1 in potted plants

[0152] deal with Incidence (%) Disease index Control effect (%) Sterile water control 41.10±2.14a 18.53±0.06a — 100 times optimized fermentation broth 16.36±0.96d 9.10±0.07e 50.93±0.07b 200 times optimized fermentation broth 7.69±0.36c 3.73±0.11f 79.83±0.23a 400 times optimized fermentation broth 18.75±0.46c 9.85±0.03d 46.87±0.24c 800 times optimized fermentation broth 22.29±0.18b 10.14±0.20c 45.29±0.10d <![CDATA[1 600 Optimized fermentation broth]]> 22.46±1.45b 11.23±0.05b 39.38±0.15e

[0153] The results in Table 10 show that fermentation broths of different multiples have a certain control effect on strain SZ-1, and the control effects are 50.93%, 79.83%, 46.87%, 45.30% and 39.39% respectively. Among them, the control effect of 200-fold optimized fermentation broth is the best.

[0154] The present invention adopts a plate confrontation method to screen out an antagonistic strain BLS-27 that has an inhibitory effect on the pathogenic bacteria of anthracnose of wild jujube, combines morphological, physiological and biochemical characteristics with molecular biology to identify the antagonistic bacteria, and performs an antibacterial spectrum determination of the antagonistic bacteria, and identifies the strain BLS-27 as Bacillus velez. The strain has high antibacterial activity against 9 plant pathogens, and its antibacterial activity has a broad spectrum, and can be used for biological control of plant diseases.

[0155] The present invention adopts single factor test and orthogonal test to determine liquid fermentation medium and optimize fermentation conditions, which significantly improves its growth amount, enhances the prevention and control effect on plant diseases, is conducive to reducing the use of chemical pesticides and fertilizers, and solves the problems of pesticide residues and environmental pollution.

[0156] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0157] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0158] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

[0159] Sequence Listing Information:

[0160] DTD version: V1_3

[0161] File name: A strain of Bacillus Velezii BLS-27 and a bacterial agent containing the same, a preparation method and application.xml

[0162] Software Name: WIPOSequence

[0163] Software version: 2.3.0

[0164] Date of creation: 2024-12-05

[0165] Basic Information:

[0166] Current application / applicant file name:12130000401707023F

[0167] Applicant's name: Hebei Agricultural University

[0168] Applicant's name / language:zh

[0169] Applicant's name or title / Latin name:Hebei Agricultural University

[0170] Invention Name: A strain of Bacillus Velezii BLS-27 and a bacterial agent containing the same, a preparation method and application (zh)

[0171] Total number of sequences: 1

[0172] sequence:

[0173] Serial number (ID): 1

[0174] Length:1411

[0175] Molecule type: DNA

[0176] Feature Position / Qualifier:

[0177] -source,1..1411

[0178] >mol_type,other DNA

[0179] >organism,Bacillus velezensis

[0180] Residue:

[0181]

[0182]

[0183] END

Claims

1. A strain of Bacillus velezensis BLS-27, characterized in that: The deposit number is CGMCC No.32274.

2. A bacterial agent, characterized in that The invention comprises the Bacillus Velezii BLS-27 as described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The method comprises the bacterial liquid, bacterial body, spore or fermentation supernatant of Bacillus Velezii BLS-27 according to claim 1.

4. Use of the Bacillus Velezii BLS-27 according to claim 1 or the bacterial agent according to any one of claims 2 to 3 in preventing and controlling plant diseases.

5. Use of the Bacillus Velezii BLS-27 according to claim 1 or the bacterial agent according to any one of claims 2 to 3 in inhibiting plant pathogens.

6. A method for preparing the bacterial agent according to any one of claims 2 to 3, characterized in that: The following steps are involved: The Bacillus Velez subtilis BLS-27 was inoculated into a fermentation medium to obtain a bacterial liquid of the Bacillus Velez subtilis BLS-27.

7. The method for preparing the bacterial agent according to claim 6, characterized in that: The fermentation culture conditions are: temperature 30° C., rotation speed 200 r / min, and dark culture for 24 h.

8. The method for preparing the bacterial agent according to claim 6, characterized in that: The fermentation medium comprises the following components in mass volume concentrations: 2% glucose, 1% beef extract, and 1% sodium chloride; the pH value of the fermentation medium is 7.

9. The method for preparing the bacterial agent according to claim 6, characterized in that: The Velez subtilis BLS-27 is inoculated into the fermentation medium in the form of seed liquid; the inoculation amount of the seed liquid is 3%.

10. The use according to claim 4, characterized in that: The fermentation liquid of Bacillus Velez subtilis BLS-27 was diluted and then applied.

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