A strain of Bacillus atrophaeus and its application

Abstract

The present invention provides a strain of Bacillus atrophaeus ( Bacillus atrophaeus ), the Bacillus atrophaeus is deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M2021736. The present invention also provides its use in preparing products of pathogens of spring black stem disease of alfalfa, pathogens of ryegrass leaf spot disease, pathogens of anthracnose of arrow pea, pathogens of Fusarium spp. root rot disease of highland barley, pathogens of root rot disease of purple alfalfa and / or pathogens of cabbage wilt. The present invention studies the morphology, physiological and biochemical characteristics and liquid fermentation conditions of Bacillus atrophaeus ZY1825 strain, and provides technical parameters for the biological control application of Bacillus atrophaeus. In the next step of research, the exploration of the mechanism of action of biocontrol bacteria and other aspects should be strengthened to lay a theoretical foundation for the development and research of new biocontrol agents.

Description

Technical Field

[0001] The invention relates to a strain of Bacillus atrophaeus and application thereof. Background Art

[0002] Plant diseases affect all stages of forage production, reduce forage nutrients, reduce yields, produce harmful substances, and thus affect the development of animal husbandry. Anthracnose commonly occurs on peas, seriously affecting their yields and limiting the development of agriculture and animal husbandry in pea planting areas. Lolium leaf spot seriously affects forage yields and reduces the turf value of ryegrass. Alfalfa spring black stem disease is one of the most common alfalfa fungal diseases in my country and is a worldwide disease. It affects the wintering of alfalfa and reduces its yield by about 50%. Highland barley root rot is common, reducing highland barley yield by 15%-30%. For a long time, chemical control has been the main measure for the prevention and control of forage diseases, but the large-scale use of chemical pesticides will pollute the ecological environment and affect the health of livestock. The long-term use of a single type of pesticide is also prone to increasing the resistance of pathogens, resulting in the continuous degradation of the disease resistance of the variety. Microbial control is a control measure that uses beneficial living organisms to prevent and control pests and diseases. It is in line with the development direction of modern sustainable agriculture and animal husbandry, and green ecological agriculture.

[0003] Bacillus (B. atrophaeus ) is an important bacterium that can form spores and has strong resistance to external harmful factors. It is widely present in soil, water, and air, does not pollute the environment, is harmless to humans and animals, and has significant biocontrol potential. In recent years, it has been widely used in plant disease prevention and control. The spores produced by Bacillus can interfere with the infection and colonization of plant pathogenic microorganisms to a certain extent, and have a good biocontrol effect on a variety of soil-borne plant diseases. Biocontrol agents developed using Bacillus have been widely used in plant disease prevention and control. The antibacterial activity of biocontrol bacteria is not only related to the physiological characteristics of the strain itself, but also closely related to the fermentation medium and culture conditions. This is not only crucial to improving the fermentation level of the strain, but also the key to determining whether it can be successfully commercialized. Microbial fermentation is a step that must be taken in the process of biocontrol microorganisms from laboratory culture research to field application practice. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a strain of Bacillus atrophaeus and application thereof.

[0005] The present invention provides a strain of Bacillus atrophaeus ( Bacillus atrophaeus ), the Bacillus atrophaeus was deposited in the China Center for Type Culture Collection on June 21, 2021, and the preservation address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M2021736.

[0006] The Bacillus atrophaeus ZY1825 strain of the present invention is an extreme habitat microorganism isolated and screened from the desert sand of the Hexi Corridor. The control effect of the indoor root-irrigated potted plant on cabbage wilt is 53.03%, and the strain is a biocontrol bacterial strain with development potential. The present invention determines the antagonistic effect of the strain on four pathogenic bacteria of forage diseases, and studies the morphological, physiological and biochemical characteristics of the Bacillus atrophaeus ZY1825 strain in order to clarify its liquid fermentation conditions. The research results can provide a technical parameter theoretical basis for further using Bacillus atrophaeus ZY1825 to prepare biocontrol agents for preventing and controlling forage diseases.

[0007] The present invention also provides a bacillus atrophaeus bacterial liquid, which is obtained by fermenting and culturing the above-mentioned bacillus atrophaeus strain.

[0008] Preferably, during the fermentation, the fermentation medium is composed of the following components in parts by weight: 200 parts of potatoes, 10-20 parts of sucrose, 10-16 parts of yeast extract powder, 2-10 parts of magnesium sulfate, and 1000 parts of water.

[0009] As further preferred, during the fermentation, the fermentation medium consists of the following components in parts by weight: 200 parts of potatoes, 20 parts of sucrose, 14 parts of yeast extract powder, 5 parts of magnesium sulfate, and 1000 parts of water.

[0010] Preferably, the fermentation conditions during the fermentation are as follows: fermentation temperature is 22-35°C, rotation speed is 120-240r / min, liquid volume is 20-40 mL per 150 mL shake flask, initial inoculum volume is 0.2-1%, initial pH value is 5-7, and fermentation time is 22-24h.

[0011] As further preferred, the fermentation conditions during the fermentation are as follows: fermentation temperature is 32° C., rotation speed is 210 r / min, liquid volume is 20 mL per 150 mL shake flask, initial inoculum volume is 1%, initial pH value is 7, and fermentation time is 22 h.

[0012] The present invention also provides a biological control agent, wherein the active ingredient of the biological control agent is the above-mentioned Bacillus atrophaeus or the above-mentioned Bacillus atrophaeus bacterial liquid.

[0013] The present invention also provides the use of the above-mentioned Bacillus atrophaeus, Bacillus atrophaeus bacterial liquid or biological control preparation in preparing products for preventing and controlling alfalfa spring black stem disease, ryegrass leaf spot disease, arrow pea anthracnose, highland barley Fusarium root rot, alfalfa root rot and / or cabbage wilt disease.

[0014] Preferably, the pathogen of spring black stem disease of alfalfa is Phyllostomia lucerne ( Phoma medicaginis ), the ryegrass leaf spot pathogen is Helminthosporium root decay ( Bipolaris sorokiniana); the pathogen of pea anthracnose is spinach anthracnose ( Colletotrichum spinaciae ), the pathogen of highland barley Fusarium root rot is Fusarium avenae ( Fusarium avenaceum ), the alfalfa root rot pathogen is Fusarium chlamydophorum ( Fusarium chlamydosporum ), the cabbage wilt pathogen is Fusarium oxysporum ( Fusarium oxysporum ).

[0015] The present invention studies the morphology, physiological and biochemical characteristics and liquid fermentation conditions of Bacillus atrophaeus ZY1825 strain, and provides technical parameters for the biological control application of Bacillus atrophaeus. In the next step of research, the exploration of the mechanism of action of biocontrol bacteria should be strengthened to lay a theoretical foundation for the development and research of new biocontrol agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0017] Figure 1 This is an observation of the morphological characteristics of Bacillus atrophaeus ZY1825 strain.

[0018] Figure 2 This is the effect of different culture media on the growth of Bacillus atrophaeus ZY1825 strain.

[0019] Figure 3 This is the effect of different carbon sources, nitrogen sources and inorganic salt culture media on the growth of Bacillus atrophaeus ZY1825 strain.

[0020] Figure 4 This paper shows the effects of different sucrose, yeast extract powder and magnesium sulfate contents on the growth of Bacillus atrophaeus ZY1825 strain.

[0021] Figure 5 The effects of initial pH, temperature and rotation speed on the growth of Bacillus atrophaeus ZY1825 strain.

[0022] Figure 6 This is the effect of liquid volume and inoculum size on the growth of Bacillus atrophaeus ZY1825 strain.

[0023] Figure 7 This is the growth curve of Bacillus atrophaeus ZY1825 strain.

[0024] Figure 8 The antagonistic effect of the biocontrol bacterium Bacillus atrophaeus ZY1825 against Fusarium oxysporum. DETAILED DESCRIPTION

[0025] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0026] Example 1

[0027] 1 Materials and methods

[0028] 1.1 Test strains

[0029] 1.1 Pathogen of spring blackstem disease of alfalfa, Stemostomyces luciensis Phoma medicaginis ); Lolium perenne leaf spot pathogen Helminthosporium radix ( Bipolaris sorokiniana ); Colletotrichum sphaeroides, the pathogen of pea anthracnose Colletotrichum spinaciae ) provided by the College of Grassland Agriculture Science and Technology, Lanzhou University; Fusarium spp., the pathogen of barley Fusarium root rot, Fusarium spp. ( Fusarium avenaceum ) is deposited in the Institute of Plant Protection, Gansu Academy of Agricultural Sciences; Fusarium chlamydophorum, the pathogen of alfalfa root rot ( Fusarium chlamydosporum ) provided by the College of Plant Protection, Gansu Agricultural University; Fusarium oxysporum, the pathogen of cabbage wilt disease ( Fusarium oxysporum f.sp.conglutinans) , provided by the Institute of Vegetable and Floriculture, Chinese Academy of Agricultural Sciences.

[0030] 1.2 Bacillus atrophaeus strain ZY1825 was isolated and purified from the desert sandy soil of Huangbawanlang, Minqin County, Wuwei City, Gansu Province by the Institute of Plant Protection, Gansu Academy of Agricultural Sciences using the gradient dilution method, plate coating method and plate streak method. After mixing the soil sample evenly, 1.0 g of the soil sample was weighed and placed in a test tube containing 9 mL of sterile water, and the gradient dilution was 10 -5 , 100 μL was taken from the last three concentrations of the test tube and spread on LB solid medium. After culturing for 72 hours, colonies of different morphologies were picked and streaked on LB solid medium to purify the colonies. The plate confrontation method was used to confront the screened bacterial culture with Fusarium oxysporum, the pathogen of cabbage wilt, on two plates. Bacteria with antibacterial effect were selected for rescreening. The strain culture obtained from the initial screening was streaked on the LB plate for 48 hours, and then confronted with Fusarium oxysporum on two plates to screen out the strain with the best antibacterial effect on Fusarium oxysporum, Bacillus atrophaeus ZY1825, and the Bacillus atrophaeus ZY1825 was selected. Bacillus atrophaeus The ZY1825 strain was sent to the China Center for Type Culture Collection (CCTCC) for preservation on June 21, 2021. The preservation address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M2021736.

[0031] 1.2 Test culture medium

[0032] LB medium: 10 g sodium chloride, 13 g agar, 5 g yeast extract powder, 10 g tryptone, 1000 mL distilled water; used for activation and preservation of Bacillus atrophaeus. Medium A to G are used for basic medium screening.

[0033] A: 5 g beef extract, 10 g peptone, 20 g sucrose, 1000 mL water;

[0034] B: 200 g potato, 10 g peptone, 20 g sucrose, 1000 mL water;

[0035] C: beef extract 3 g, peptone 5 g, glucose 8 g, NaCl 5 g, water 1000 mL;

[0036] D: 8 g beef extract, 5 g yeast extract, 10 g glucose, 1000 mL water;

[0037] E: peptone 10 g, yeast extract 5 g, NaCl 10 g, water 1000 mL;

[0038] F: 10 g sodium chloride, 5 g yeast extract powder, 10 g tryptone, 1000 mL distilled water;

[0039] G: 5 g beef extract, 5 g yeast extract, 10 g peptone, 5 g glucose, 5 g NaCl, 1000 mL water.

[0040] PDA medium.

[0041] 1.3 Main test instruments and equipment

[0042] UV / visible spectrophotometer, produced by Shanghai Hongji Instrument Equipment Co., Ltd.; shaker, produced by Beijing SaiBiao Technology Co., Ltd.; sterile operating table, produced by Suzhou Antai Air Technology Co., Ltd.; autoclave, produced by Guangzhou Huayuexing Instrument Co., Ltd.; constant temperature incubator, produced by Ningbo Jiangnan Instrument Factory.

[0043] 1.4 Morphological observation

[0044] The activated Bacillus atrophaeus strain ZY1825 was streaked and inoculated on LB solid medium, and the growth, color and morphological characteristics of the colonies were observed after culturing at 32°C for 24h and 48h. The morphological characteristics of the strain were observed by scanning electron microscopy, and the size of Bacillus atrophaeus ZY1825 was measured.

[0045] 1.5 Determination of physiological and biochemical indicators

[0046] Physiological and biochemical test indicators mainly include Gram staining, VP (Voges-Proskauer) test, methyl red test, gelatin liquefaction test, glucose fermentation test, starch hydrolysis test, indole test, nitrate reduction test, urease test, hydrogen sulfide test, etc. The specific test methods refer to the methods in the "Handbook of Common Bacterial System Identification".

[0047] 1.6 Antagonistic effect of Bacillus atrophaeus ZY1825 on pathogens

[0048] The bacterial liquid of Bacillus atrophaeus ZY1825 was streaked on an LB plate for 48 h, the pathogens of anthracnose of arrow pea, ryegrass leaf spot, spring black stem disease of alfalfa, Fusarium spp. root rot of highland barley, and root rot of alfalfa were inoculated on PDA plates for 5 d, respectively. A bacterial cake with uniform growth and a diameter of 0.6 mm was punched out from the edge of the colony with a sterile puncher, and the pathogens of these forage diseases and a single colony of Bacillus atrophaeus strain ZY1825 were inoculated at a relative position of about 2 cm from the edge of a PDA plate with a diameter of 90 mm. This was repeated 3 times, and the plates were cultured in a constant temperature incubator at 28°C. The pathogen cultured alone was used as a control, and the growth of the colonies was observed continuously. The pathogen colony diameter in the control treatment was ≥4.5 cm, the radius of the pathogen and the width of the inhibition zone in each treatment were recorded, and the inhibition rate was calculated. Inhibition rate (%) = (control pathogen diameter - treatment group pathogen diameter) / control pathogen colony diameter × 100%.

[0049] 1.7 Screening of antagonistic bacterial fermentation conditions

[0050] 1.7.1 Preparation of seed solution

[0051] The preserved Bacillus atrophaeus ZY1825 strain was inoculated on LB medium and activated in a 28°C incubator, then inoculated into LB liquid medium and cultured at 28°C and 180 r / min for 24 hours to prepare seed solution for use (the liquid culture medium was filled with 50 mL in a 150 mL conical flask).

[0052] 1.7.2 Effects of fermentation medium on bacteria

[0053] The culture medium A to G in step 1.2 was divided into conical flasks (100 mL / 150 mL), and the pH was adjusted to 7 and sterilized. Then 1 mL of seed solution was added and cultured in a constant temperature oscillator (28°C, 180 r / min). LB liquid medium was used as the control and repeated 3 times. After culturing for 24 h, the OD value of the fermentation liquid was measured. 600nm Determine the biomass to determine the optimal basal medium.

[0054] 1.7.3 Effects of carbon source, nitrogen source and inorganic salt on bacterial growth and determination of optimal concentration

[0055] Sucrose, corn starch, soluble starch, maltose, lactose, glucose, mannitol and fructose were used to replace the carbon source in the best basal medium, and the culture solution was placed in conical flasks (50 mL / 150 mL) with no carbon source added as the control. The best carbon source screened was set to 0, 5, 10, 15, 20, 25 and 30 g / L, respectively, to determine the optimal concentration of the carbon source.

[0056] The nitrogen source in the best basal medium was replaced with beef extract, ammonium nitrate, yeast extract powder, potassium nitrate, magnesium sulfate, peptone, sodium nitrate and ammonium chloride, respectively, and the culture solution was divided into conical flasks (50 mL / 150 mL) with no nitrogen source added as the control. The best nitrogen source screened was set to 0, 6, 8, 10, 12, 14 and 16 g / L, respectively, to determine the optimal concentration of the nitrogen source.

[0057] 0.1% disodium hydrogen phosphate, sodium citrate, magnesium sulfate, sodium chloride, calcium chloride, zinc sulfate, manganese sulfate and copper sulfate were added to the best basal culture medium obtained by screening, and the culture solution was divided into conical flasks (50 mL / 150 mL). The weight percentages of the best inorganic salts obtained by screening were set to 0, 0.1%, 0.2%, 0.5%, 1%, 1.5%, 2% and 2.5%, respectively, to determine the optimal concentration of the inorganic salt.

[0058] 1.7.4 Orthogonal test of culture medium components

[0059] According to the screening results, L9(3 3 ) orthogonal test (Table 1), the absorbance of the fermentation broth was measured after culturing at 28°C and 180 r / min for 24 h (OD 600nm ), and the results were analyzed by variance test to determine the best culture medium combination.

[0060] Table 1 L9 (3 3 ) Orthogonal Experimental Design

[0061]

[0062] 1.7.5 Screening of shake flask culture conditions

[0063] The pH was set to 4, 5, 6, 7, 8, 9, 10, and 11, the temperature was set to 22, 25, 28, 32, and 35°C, the speed of the thermostatic oscillator was set to 120, 150, 180, 210, and 240 r / min, the oxygen supply of the shake flask was set to 20, 40, 60, 80, and 100 mL of the medium in the 150 mL conical flask, and the inoculation volume was set to 0.2%, 0.4%, 1%, 2%, and 4%. Using the above culture conditions, on the basis of the optimal culture medium combination in step 1.7.4, the biocontrol strain Bacillus atrophaeus ZY1825 was cultured using the single factor test method, and the absorbance of the fermentation broth (OD 600 ) determine the biomass and screen for the optimal liquid fermentation culture conditions.

[0064] 1.7.6 Effect of fermentation time on bacteria

[0065] The fermentation parameters were set using the optimal culture medium combination and optimal culture conditions. The strain of Bacillus atrophaeus ZY1825 was cultured on a shaker. Samples were taken every 4 h for 3 times to determine the number of viable bacteria and biomass (OD 600nm ) until the number of live bacteria stabilizes, then stop fermentation and put the tank in place, and collect the fermentation liquid for later use.

[0066] 1.8 Antagonistic effect of biocontrol bacteria ZY1825 against Fusarium wilt pathogen of cabbage

[0067] The antagonistic bacteria solution was streaked on LB plates for 48 h, and the cabbage wilt pathogen Fusarium oxysporum ( Fusarium oxysporum ) Cultured on a PDA plate for 5 days, a bacterial cake with consistent growth and a diameter of 0.6 mm was punched out from the edge of the colony with a sterile puncher, and single colonies of the cabbage wilt pathogen and Bacillus atrophaeus ZY1825 strain were inoculated at a relative position of about 2 cm from the edge of a 90 mm diameter PDA plate, respectively. Repeated 3 times, and cultured in a 28°C constant temperature incubator. The cabbage wilt pathogen was cultured alone as a control, and the colony growth was continuously observed. The pathogen colony diameter in the control treatment was ≥4.5 cm, and the radius of the pathogen and the width of the inhibition zone of each treatment were recorded to calculate the inhibition rate. The bacterial solution before and after the optimization of the culture medium and fermentation parameters was confronted with the cabbage wilt pathogen, and the inhibition rate was calculated. Inhibition rate (%) = (control pathogen diameter - treatment group pathogen diameter) / control pathogen colony diameter × 100%

[0068] 1.9 Data Processing

[0069] DPS9.50 software was used to perform statistical analysis on the measured data, and the measurement results were expressed as mean values ​​and standard errors. One-way analysis of variance was performed on different carbon sources, nitrogen sources, inorganic salts and different shake flask culture conditions, and the Duncan method was used for multiple comparisons of the measured data. Excel2021 and Origin were used to make graphs.

[0070] 2 Results and Analysis

[0071] 2.1 Morphological characteristics and physiological and biochemical properties of Bacillus atrophaeus ZY1825 strain

[0072] After culturing Bacillus atrophaeus ZY1825 on LB solid medium at 32°C for 48h, the colonies formed were 0.85-5.8mm in diameter, light yellow, opaque, with wrinkles on the surface and irregular edges ( Figure 1 -A), after 96 hours of culture, the colonies and culture medium gradually turned dark brown, and pigments were produced ( Figure 1 -B). Under a scanning electron microscope, the bacteria are rod-shaped ( Figure 1 -C), no capsule, size 2.3 ~ 3.91 × 0.87 ~ 0.94μm, produce oval spores, Gram stain positive ( Figure 1 -D).

[0073] Figure 1 This is an observation of the morphological characteristics of Bacillus atrophaeus ZY1825. A: morphological characteristics of the colony; B: 96h colony and culture medium characteristics; C: individual morphology of the strain under scanning electron microscope (×30000); D: Gram staining results (10×100).

[0074] As shown in Table 2, the VP test, methyl red test and gelatin liquefaction test of Bacillus atrophaeus ZY1825 strain were all positive, and it could hydrolyze starch, utilize urea, reduce nitrate, but could not produce indole or hydrogen sulfide.

[0075] Table 2 Physiological and biochemical characteristics of Bacillus atrophaeus strain ZY1825

[0076]

[0077] Among them, "+" indicates positive, and "-" indicates negative.

[0078] 2.2 Antagonistic effect of the biocontrol bacterium Bacillus atrophaeus ZY1825 on several forage disease pathogens

[0079] The test results (Table 3) show that Bacillus atrophaeus ZY1825 has a good inhibitory effect on five forage diseases, with the inhibition rate reaching more than 40%. It has the best inhibitory effect on alfalfa stem mildew, with an inhibition rate of 50.78% and an inhibition zone width of 4.35 cm, which is significantly larger than other strains.

[0080] Table 3 Inhibitory effect of Bacillus atrophaeus strain ZY1825 on five forage diseases

[0081]

[0082] 2.3 Fermentation condition optimization results

[0083] 2.3.1 Effect of culture medium on the growth of Bacillus atrophaeus ZY1825 strain

[0084] like Figure 2 As shown in the figure, Bacillus atrophaeus ZY1825 strain can grow in all eight culture media, and there are significant differences. After 24h shaking culture, Bacillus atrophaeus ZY1825 strain grew fastest in medium B (200 g potato, 10 g peptone, 20 g sucrose, 1000 mL water), with an OD of 0. 600nm The value reached 1.935; followed by A, D, C, F, G, and E medium, and the slowest growth was in LB (CK) medium. Therefore, the best basal medium for Bacillus atrophaeus ZY1825 strain is B medium.

[0085] Figure 2 This is the effect of different culture media on the growth of Bacillus atrophaeus ZY1825 strain.

[0086] 2.3.2 Effects of carbon, nitrogen sources and inorganic salts on the growth of Bacillus atrophaeus ZY1825

[0087] Fix the above test results, and use the formula of 200 g potato, 10 g peptone, and 1000 mL water in the medium B to screen the best carbon source. The results are as follows Figure 3 -a as shown.

[0088] like Figure 3 -a, it can grow on all 8 carbon sources, but sucrose is the most suitable for the growth of Bacillus atrophaeus ZY1825 strain, OD 600nm The value can reach 2.191, which is significantly higher than other carbon source culture media. Glucose can also promote the growth of Bacillus atrophaeus ZY1825 strain well. When corn starch is used as a carbon source, the absorbance value of the fermentation liquid is the lowest, which indicates that the strain's ability to decompose corn starch is weaker than that of the other seven carbon sources. Therefore, sucrose was selected as the carbon source for the fermentation medium of Bacillus atrophaeus ZY1825 strain.

[0089] Fix the above test results, and use the formula of 200 g potato, 20 g sucrose and 1000 mL water in the medium B to screen the best nitrogen source. The results are as follows Figure 3 -b as shown.

[0090] like Figure 3 -b shows that the eight nitrogen sources can promote the growth of the strain well. When yeast extract powder is used as the nitrogen source, the concentration of Bacillus atrophaeus ZY1825 in the fermentation broth is the highest, reaching 2.358. When peptone is used as the nitrogen source, the concentration of Bacillus atrophaeus ZY1825 is the lowest, only 2.119, which means that the ability of the strain to decompose peptone is weaker than that of the other seven nitrogen sources. Therefore, yeast extract powder is selected as the nitrogen source for the fermentation medium of Bacillus atrophaeus ZY1825.

[0091] Fix the above test results, add different inorganic salts to the formula of medium B, which contains 200 g potato, 20 g sucrose, 10 g yeast extract powder, and 1000 mL water. The results are as follows Figure 3 -c.

[0092] like Figure 3 -c shows that Bacillus atrophaeus ZY1825 strain can grow in 8 different inorganic salts. When magnesium sulfate is used as the inorganic salt, the fermentation liquid OD 600nm The highest is 2.308. When copper sulfate, manganese sulfate, zinc sulfate and calcium chloride are inorganic salts, OD 600nm The values ​​were significantly lower than those of the control, zinc sulfate and copper sulfate. 600nm A value lower than 1 indicates that Mn 2+ and Cu 2+ It has an inhibitory effect on Bacillus atrophaeus ZY1825 strain, so magnesium sulfate was selected as the inorganic salt of the fermentation medium.

[0093] Figure 3 The effect of different carbon sources, nitrogen sources, and inorganic salt culture media on the growth of Bacillus atrophaeus ZY1825 strain. In figure a, A: sucrose; B: corn starch; C: soluble starch; D: maltose; E: lactose; F: glucose; G: mannitol; H: fructose; CK. In figure b, A: beef extract; B: ammonium nitrate; C: yeast extract powder; D: potassium nitrate; E: magnesium sulfate; F: peptone; G: sodium nitrate; H: ammonium chloride; CK. In figure c, A: disodium hydrogen phosphate; B: sodium citrate; C: magnesium sulfate; D: sodium chloride; E: calcium chloride; F: zinc sulfate; G: manganese sulfate; H: copper sulfate; CK.

[0094] The results of screening of carbon, nitrogen source and inorganic salt concentrations showed that the OD of Bacillus atrophaeus ZY1825 strain in medium with different contents of sucrose, yeast extract powder and magnesium sulfate was significantly increased. 600nmwere significantly higher than the control, indicating that different sucrose, yeast extract powder, and magnesium sulfate contents can promote the growth of Bacillus atrophaeus ZY1825 strain, among which the concentrations of sucrose, yeast extract powder, and magnesium sulfate were 15 g / L, 12 g / L, and 5 g / L, respectively ( Figure 4 ), the bacterial concentration is the largest; therefore, the most suitable concentrations of carbon, nitrogen source and inorganic salt in the fermentation medium of Bacillus atrophaeus ZY1825 strain are selected as 15 g / L, 12 g / L and 5 g / L, respectively.

[0095] Figure 4 This paper shows the effects of different sucrose, yeast extract powder and magnesium sulfate contents on the growth of Bacillus atrophaeus ZY1825 strain.

[0096] That is, the optimal fermentation medium for Bacillus atrophaeus ZY1825 strain is: 200 g potato, 15 g sucrose, 12 g yeast extract powder, 5 g magnesium sulfate, and 1000 mL water.

[0097] 2.3.3 Screening of culture medium components

[0098] As shown in the orthogonal test result table (Table 4), according to the R value in the table, the R value of sucrose is the highest at 0.0883, followed by yeast extract powder at 0.0603, and the R value of magnesium sulfate is the smallest at 0.0320. The order of influence of the three factors on bacterial growth is sucrose> yeast extract powder> magnesium sulfate. According to the size of the K value in the table, K3 is the highest for sucrose, yeast extract powder, and magnesium sulfate. However, overall, treatment 9 has the highest biomass. The optimal combination of culture medium levels is sucrose (20 g), yeast extract powder (14 g), and magnesium sulfate (5 g), that is, the optimized culture medium formula is 200 g potato, 20 g sucrose, 14 g yeast extract powder, 5 g magnesium sulfate, and 1000 mL water.

[0099] Table 4 Orthogonal test results

[0100]

[0101] 2.3.4 Effect of initial pH on the growth of Bacillus atrophaeus ZY1825

[0102] like Figure 5 -a, the initial pH had a significant effect on the strain of Bacillus atrophaeus ZY1825. The strain has a wide range of adaptability to pH and can grow in the range of pH 5-9. There is no significant difference in the OD of Bacillus atrophaeus ZY1825 at pH 5.0, 6.0 and 7.0. 600nm The values ​​were 2.439, 2.431 and 2.451 respectively. When the pH was 8 and 9, there was a significant difference, and the bacterial concentration was relatively low; when the pH was 4.0 and 10.0, the strain of Bacillus atrophaeus ZY1825 basically did not grow, and the OD600nm Only 0.18 and 0.1. This shows that the overly acidic or alkaline environmental conditions have some inhibitory effects on the growth of the strain, and it can grow well under weak acid and weak alkaline conditions. Therefore, the optimal pH range for Bacillus atrophaeus ZY1825 strain is 5-7.

[0103] Figure 5 The effects of initial pH, temperature and rotation speed on the growth of Bacillus atrophaeus ZY1825 strain.

[0104] 2.3.5 Effects of temperature and rotation speed on the growth of Bacillus atrophaeus ZY1825

[0105] like Figure 5 -b shows that under the temperature environment of 22-35℃, there are significant differences in the effects of different temperatures on bacterial growth. The strain of Bacillus atrophaeus ZY1825 can grow normally. When the temperature is 32℃, the strain ZY1825 grows relatively fast. When the temperature increases from 22℃ to 32℃, the concentration of bacterial liquid generally shows a gradual upward trend, reaching the highest at 32℃, and OD 600nm The value was 2.469; then the ambient temperature was gradually raised to 35℃, and the bacterial concentration gradually decreased. Too high or too low temperature has an adverse effect on the growth of Bacillus atrophaeus ZY1825 strain, so the optimal fermentation temperature of Bacillus atrophaeus ZY1825 strain is 32℃.

[0106] like Figure 5 -c shows that the strain can grow normally when the speed is between 120 and 240 r / min, but the effect of different shaker speeds on the growth of Bacillus atrophaeus ZY1825 strain is significantly different. When the shaker speed is 210 r / min, the bacterial concentration is the highest after 24 hours of culture, and OD 600nm The value is 2.681, that is, the optimal fermentation speed of Bacillus atrophaeus ZY1825 strain is 210 r / min.

[0107] 2.3.6 Effect of liquid volume on the growth of Bacillus atrophaeus ZY1825 strain

[0108] like Figure 6 -a, there are significant differences in the effects of different liquid volumes on bacterial growth. When the liquid volume in the 150 mL triangular flask was 20 mL, the bacterial concentration of Bacillus atrophaeus ZY1825 strain was the highest after 24 hours of shaking culture, and OD 600nm The value is 2.623. As the volume of liquid increases, bacterial growth is affected and the concentration of bacterial solution gradually decreases. When the volume of liquid is 100mL, OD 600nm The value is 2.259. This shows that Bacillus atrophaeus ZY1825 is an aerobic bacterium, and the higher the dissolved oxygen content in the liquid culture medium, the better the growth of the bacteria.

[0109] Figure 6 This is the effect of liquid volume and inoculum size on the growth of Bacillus atrophaeus ZY1825 strain.

[0110] 2.3.7 Effect of initial inoculum dose on the growth of Bacillus atrophaeus ZY1825 strain

[0111] like Figure 6 -b, when the initial inoculum amount was between 0.2% and 1%, the bacterial concentration of Bacillus atrophaeus ZY1825 strain increased with the increase of inoculum amount. When the initial inoculum amount was 1%, the bacterial concentration was the highest, and OD 600nm The value was 2.47, but when the inoculation amount exceeded 1%, the bacterial concentration decreased. Therefore, the optimal initial inoculation amount of Bacillus atrophaeus ZY1825 was 1%.

[0112] 2.3.8 Effect of fermentation time on the growth of Bacillus atrophaeus ZY1825

[0113] like Figure 7 As shown, from 0 to 6 hours, the number of viable bacteria increased slowly, OD 600nm The value increased logarithmically. The number of viable bacteria and OD value increased logarithmically from 6 to 18 hours. Bacillus atrophaeus ZY1825 grew fastest during this period. From 18 to 22 hours, the number of viable bacteria increased to the maximum, with a maximum value of 9.98×10 9 cfu / mL, OD 600nm The value tends to be stable. After 22 hours, the number of viable bacteria tends to decrease, and the OD 600nm The value tends to be stable, and this period of time is the decay period of bacterial growth. After optimizing the liquid fermentation conditions of Bacillus atrophaeus ZY1825 strain, the number of viable bacteria of Bacillus atrophaeus ZY1825 strain cultured for 24 hours increased from 1.57×10 9 cfu / mL increased to 9.28×10 9 cfu / mL, which increased by 5.91 times, laying a foundation for the production and application of biocontrol agents for Bacillus atrophaeus ZY1825 strain.

[0114] Figure 7 This is the growth curve of Bacillus atrophaeus ZY1825 strain.

[0115] 2.3.9 Antagonistic effect of biocontrol agent ZY1825 against Fusarium oxysporum

[0116] Test results ( Figure 8) showed that Bacillus atrophaeus ZY1825 had a good inhibitory effect on Fusarium oxysporum, with an inhibition rate of 43.8%. The inhibition rate of the bacterial solution against Fusarium oxysporum reached 30.79% before optimization, and reached 41.09% after optimization, which was significantly higher than before optimization. The inhibition rate against Fusarium wilt of cabbage was increased by 33.45% after optimization.

[0117] Figure 8 The antagonistic effect of the biocontrol bacterium Bacillus atrophaeus ZY1825 on Fusarium oxysporum. A: biocontrol bacterium ZY1825 and Fusarium oxysporum are cultured in confrontation; B: Fusarium oxysporum.

[0118] Table 5 Confrontation effect before and after optimization

[0119] deal with Antibacterial rate (%) Before optimization 30.79±0.007a After optimization 41.09±0.008b

[0120] 3 Conclusion and discussion

[0121] Bacillus atrophaeus ( Bacillus atrophaeus ) is an important industrial strain. The present invention measured the morphological, physiological and biochemical characteristics of the Bacillus atrophaeus ZY1825 strain isolated from the desert sand in the Hexi Corridor. The results showed that the single bacterial body of the Bacillus atrophaeus ZY1825 strain was 2.3-3.91×0.67-0.94 μm, which was consistent with the basic morphological characteristics of Bacillus atrophaeus, namely, 2.0-4.0 um in width and 0.5-1.0 μm in length, uniform in color, and opaque on the culture medium. The strain was positive for Gram staining and gelatin liquefaction, could reduce nitrate and hydrolyze starch, which was the same as the Bacillus atrophaeus ATCC9372 screened by Ma Dinghong et al.; the Bacillus atrophaeus ZY1825 strain could utilize urea, reduce nitrate and ferment glucose, which was basically the same as the physiological and biochemical characteristics of the cherry leaf spot antagonist strain QH-588 isolated from the rhizosphere soil of wolfberry by Zhu Jie et al.; the VP test and methyl red test of strain ZY1825 were both positive, and it could not produce indole and hydrogen sulfide, which was basically consistent with the Bacillus isolated by Lu Yanhong et al. from the soil of the Xinjiang pear planting area and the existing strains in the laboratory. Preliminary studies have shown that the ZY1825 strain of Bacillus atrophaeus has a significant inhibitory effect on Fusarium oxysporum and has a broad antibacterial spectrum. The present invention has determined the antagonistic effect of the bacterium on four forage diseases: alfalfa stem spot mildew, root rot helminth, spinach anthracnose, and avenae Fusarium. The inhibition rate of the bacterium on these four forage diseases is more than 40%. The bacterium is a very good forage disease antagonist with potential for development and application.

[0122] In order to improve the yield, the present invention adopts single factor and orthogonal experimental design method to optimize the basic culture medium and fermentation conditions of Bacillus atrophaeus ZY1825 strain. The results show that the optimal fermentation medium formula is 200 g potato, 20 g sucrose, 14 g yeast extract powder, 5 g magnesium sulfate, and 1000 mL water; the optimal fermentation conditions are temperature 32 ° C, oscillator speed 210r / min, optimal liquid volume 150 mL shake flask liquid 20 mL, medium initial pH 7, and optimal inoculum size 1%. The optimal fermentation conditions of Bacillus atrophaeus ZY1825 strain were compared with those of Bacillus atrophaeus XW2 isolated from healthy leaves of poplar by Zhu Junmiao and Bacillus atrophaeus E20303 isolated from the mud of Qarhan Salt Lake in Qinghai by Qiao Jiahuizi et al. It can be found that the fermentation temperature of the three strains is concentrated between 28-32℃, which is consistent with the optimal growth temperature of Bacillus spp. between 28-37℃; the optimal speed is between 200-210r / min; but the optimal fermentation pH of ZY1825 and XW2 is 7, which is neutral, while the initial pH of E20303 is 8.64, which is slightly alkaline, which may be due to excessive starch content. The optimal speed of Bacillus subtilis MC4-2 is 210 r / min and the temperature is 32℃, which is similar to the results of this study. In summary, it can be found that even if they are all Bacillus spp., the fermentation conditions are different due to different sources and habitats of the strains.

[0123] The carbon source, nitrogen source, inorganic salt and other components of the culture medium, the culture temperature, liquid volume, inoculation volume, rotation speed, initial pH value and other factors have a great influence on the growth and fermentation of the strain. Through the indoor fermentation test, the culture and fermentation conditions of Bacillus atrophaeus ZY1825 were preliminarily clarified, and the target strain can be obtained in large quantities under laboratory conditions. Optimizing the fermentation conditions is a prerequisite for the successful development of microbial agents, and lays the foundation for large-scale industrial production and fermentation culture. For the optimization test of the liquid fermentation conditions of the present invention, there are still aspects that need to be slightly improved. Later experiments can be based on this single factor and orthogonal experimental design, and the carbon and nitrogen sources of the fermentation medium and the fermentation conditions are appropriately supplemented by experimental methods such as the response surface method, so as to increase the amount of live bacteria in the fermentation liquid of Bacillus atrophaeus ZY1825.

[0124] The present invention studies the morphology, physiological and biochemical characteristics and liquid fermentation conditions of Bacillus atrophaeus ZY1825 strain, and provides technical parameters for the biological control application of Bacillus atrophaeus. In the next step of research, the exploration of the mechanism of action of biocontrol bacteria should be strengthened to lay a theoretical foundation for the development and research of new biocontrol agents.

[0125] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Bacillus atrophaeus ( Bacillus atrophaeus ), use of Bacillus atrophaeus liquid or biological control agent in the preparation of products for preventing and controlling spring black stem disease of alfalfa, ryegrass leaf spot disease, arrow pea anthracnose, barley fusarium root rot and / or alfalfa root rot; The pathogen of spring black stem disease of alfalfa is Stemophyton lucerne ( Phoma medicaginis ), the ryegrass leaf spot pathogen is Helminthosporium root decay ( Bipolaris sorokiniana ); the pathogen of pea anthracnose is spinach anthracnose ( Colletotrichum spinaciae ), the pathogen of highland barley Fusarium root rot is Fusarium avenae ( Fusarium avenaceum ), the alfalfa root rot pathogen is Fusarium chlamydophorum ( Fusarium chlamydosporum ); The Bacillus atrophaeus ( Bacillus atrophaeus ) is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO: M2021736; The Bacillus atrophaeus bacterial solution is the Bacillus atrophaeus ( Bacillus atrophaeus ) obtained by fermentation culture of strains; The active ingredient of the biological control agent is the Bacillus atrophaeus ( Bacillus atrophaeus ) or the Bacillus atrophaeus liquid.

2. The use according to claim 1, characterized in that: During the fermentation, the fermentation medium is composed of the following components in parts by weight: 200 parts of potatoes, 10-20 parts of sucrose, 10-16 parts of yeast extract powder, 2-10 parts of magnesium sulfate, and 1000 parts of water.

3. The use according to claim 2, characterized in that: During the fermentation, the fermentation medium is composed of the following components in parts by weight: 200 parts of potatoes, 20 parts of sucrose, 14 parts of yeast extract powder, 5 parts of magnesium sulfate, and 1000 parts of water.

4. The use according to any one of claims 1 to 3, characterized in that: The fermentation conditions during the fermentation are as follows: fermentation temperature is 22-35°C, rotation speed is 120-240r / min, liquid volume is 20-40 mL per 150 mL shake flask, initial inoculum volume is 0.2-1%, initial pH value is 5-7, and fermentation time is 22-24h.

5. The use according to claim 4, characterized in that: The fermentation conditions during the fermentation are as follows: fermentation temperature is 32° C., rotation speed is 210 r / min, liquid volume is 20 mL per 150 mL shake flask, initial inoculum volume is 1%, initial pH value is 7, and fermentation time is 22 h.

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