CN109749974B - Bacillus amyloliquefaciens strain and application thereof - Google Patents
Bacillus amyloliquefaciens strain and application thereof Download PDFInfo
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Abstract
The invention discloses a bacillus amyloliquefaciens strain and application thereof. The preservation number of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) YB1701 in the China general microbiological culture Collection center is CGMCC No. 16003. The bacillus amyloliquefaciens YB1701 provided by the invention is from the suaeda glauca rhizosphere soil, can effectively antagonize 16 plant pathogenic fungi such as corn black bundle pathogen (Acremonium strictum), yellow Fusarium oxysporum (Fusarium culmorum), Phytophthora capsici Leonian (Phytophthora capsici Leonian), melon Fusarium oxysporum f.sp.melonis, has obvious promotion effect on the growth of rice seedlings, and has wide application prospect in the development of biological pesticides and biological organic fertilizers.
Description
Technical Field
The invention belongs to the technical field of strain cultivation, and particularly relates to a bacillus amyloliquefaciens strain YB1701 and application thereof, in particular to bacillus amyloliquefaciens capable of producing ACC deaminase and IAA auxin and having broad-spectrum antagonism on plant pathogenic fungi and application thereof, and bacteria capable of antagonizing 16 common plant pathogenic fungi and having obvious growth promotion effect on rice seedlings.
Background
Suaeda glauca Bunge, also called Suaeda glauca and sargassum pallidum, mostly grows on saline-alkali lands such as beaches, valleys, roadside, fields and the like, belongs to Suaeda genus of Li family, is a halophyte with extremely strong salt tolerance, and is widely distributed in new, Mongolian, Liao and other provinces of China. At present, suaeda salsa is the first choice plant variety for biologically improving saline soil. Research shows that the soil of the root system of the plant contains rich microorganisms, which is beneficial to the absorption of nutrients by the plant and the promotion of the growth of the plant. The Yuan Ye (2018) is obtained by separating 80 halophilic and alkalophilic bacteria from root soil of Suaeda salsa in the red sea of Panjin by a pure culture method.
Bacillus amyloliquefaciens is a bacterium (wuyijing, etc., 2012) with broad-spectrum bacteriostatic activity, and can produce a large amount of secondary metabolites and various bacteriostatic substances. Qixinxin et al (2004) isolated a strain of endogenous bacillus amyloliquefaciens TB2 with good inhibitory effect on filamentous fungi from tobacco stalks; arrebella et al (2009) isolated Bacillus amyloliquefaciens PPCB004 from fruit surfaces that had antibacterial activity against 7 different citrus postharvest fungal pathogens; wong et al (2010) isolated a bacteriostatic protein from Bacillus amyloliquefaciens NK10 that has inhibitory activity against a variety of pathogens. The Zhagayan and the like (2017) separate a bacillus amyloliquefaciens with obvious inhibiting effect in the prevention and treatment of the root rot of dioscorea opposita, the bacteriostasis rate of the bacillus amyloliquefaciens reaches 89.6 percent and is obviously higher than the treatment of applying chemical medicament fenaminosulf wettable powder. The plum culture medium (2017) separates a bacillus amyloliquefaciens for preventing and treating the gray mold of tomatoes, can prevent and treat diseases of fruit rot, leaf rot, stem rot and flower rot caused by botrytis cinerea, and can increase the yield of the tomatoes.
The microbial fertilizer has an important role in agricultural production, wherein the strain is the application basis of the microbial fertilizer production. On one hand, the microorganism can produce active metabolites which promote the growth of crops; on the other hand, microorganisms antagonize plant pathogenic bacteria, improve disease resistance of crops, and the like.
Disclosure of Invention
The invention provides a bacillus amyloliquefaciens strain YB1701 and application thereof, which have broad-spectrum antibacterial effect, can produce the bacillus amyloliquefaciens for producing ACC deaminase and IAA auxin and have great development value.
The invention is realized by providing a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain, wherein the Bacillus amyloliquefaciens strain is YB1701, the Bacillus amyloliquefaciens strain YB1701 is preserved in the China general microbiological culture Collection center, and the preservation number is CGMCC No. 16003.
Provides a microbial inoculum, the active ingredient of the microbial inoculum is the fermentation liquor of the bacillus amyloliquefaciens YB1701 or the metabolite of the bacillus amyloliquefaciens YB 1701.
The microbial inoculum is any one of the following microbial inoculants:
1) a fungicide for inhibiting plant pathogenic fungi;
2) a fungicide for controlling plant pathogenic fungal diseases;
3) a microbial inoculum for promoting plant growth;
4) a microbial inoculum for improving the stress resistance of plants;
5) a microbial inoculum for reducing the sensitivity of plants to stress;
6) 1-aminocyclopropane-1-carboxylic acid deaminase (ACC) -producing microbial agents;
7) indole-3-acetic acid (IAA) -producing microbial agents;
the stress is drought, high temperature, pest attack or heavy metal pollution.
The plant pathogenic fungi is at least one of the following plant pathogenic fungi:
1) corn black rot (Acremonium strictum);
2) northern leaf blight (Exserohilum turcicum);
3) pythium aphanidermatum (Pythium aphanidermatum);
4) pythium gramincola subbra;
5) corn sphaerica (Bipolaris zeicola);
6) phytophthora capsici Leonian (Phytophthora capsicii Leonian);
7) botrytis cinerea (Botrytis cinerea Pers.);
8) apple moldy core bacteria (Trichothecium roseum));
9) rice blast (Pyricularia grisea);
10) melon Fusarium oxysporum f.sp.melonis;
11) sorghum taraxacum (Bipolaris sorghicola);
12) curvularia lunata (Curvularia lunata);
13) corn ear rot (Fusarium graminearum);
14) fusarium culmorum (Fusarium culmorum);
15) corn top rot fungi (Frumentum subglutinans);
16) helicoverpa zea (sclerotiorum) sp.
Providing a biological organic fertilizer, wherein the active ingredient of the biological organic fertilizer is the bacillus amyloliquefaciens YB1701 or the fermentation liquid of the bacillus amyloliquefaciens YB1701 or the metabolite of the bacillus amyloliquefaciens YB1701 as the claim 1.
There is provided a method for culturing the above-mentioned Bacillus amyloliquefaciens strain, comprising the step of culturing Bacillus amyloliquefaciens YB1701 in a medium for culturing Bacillus amyloliquefaciens.
Specifically, the method comprises the following steps:
1) taking fresh-collected suaeda salsa with soil at the rhizosphere, shaking off the rhizosphere soil, and weighing 10g of the rhizosphere soil;
2) after the root soil is air-dried, placing a soil sample in a conical flask with glass beads and 90mL of sterile physiological saline, and oscillating at room temperature of 120r/min for 30min to prepare a soil suspension;
3) 1mL of soil suspension is taken to be put into a test tube filled with 9mL of sterile normal saline for gradient dilution, and 10 are respectively taken-3，10-4，10-5Dilution 0.1mL was spread onto screening media platesAnd culturing at 37 ℃ for 24h, and selecting a single colony for separation and purification to obtain the bacillus amyloliquefaciens YB 1701.
The screening culture medium comprises the following components: 3g of beef extract, 10g of peptone, 5g of NaCl, 18-20g of agar powder and 1000mL of water, wherein the pH value is 7.2-7.4, and the beef extract is sterilized at 121 ℃ for 20 min.
Specifically, the preparation of the fermentation liquor of the bacillus amyloliquefaciens YB1701 comprises the following steps:
1) inoculating the purified and cultured bacillus amyloliquefaciens YB1701 to a beef extract peptone solid culture medium from a slant, and culturing for 24 hours at 37 ℃ and 180 r/min;
2) inoculating the activated strain in the step 1) into an LB liquid culture medium, and carrying out shake culture in a shaking table at 37 ℃ and 180r/min for 48h to obtain a seed solution;
3) inoculating the seed liquid obtained in the step 2) into a beef extract peptone culture medium according to the inoculation amount of 5%, and carrying out shake culture in a shaking table at the temperature of 37 ℃ and 180r/min for 48h to obtain the bacillus amyloliquefaciens YB1701 fermentation liquid.
The content of the bacillus amyloliquefaciens YB1701 in the fermentation liquid of the bacillus amyloliquefaciens YB1701 is 1.0 multiplied by 106～1.0×108cfu/mL。
The invention relates to a strain preservation as follows:
the strain name is as follows: bacillus amyloliquefaciens
Latin name: bacillus amyloliquefaciens
The strain number is as follows: CGMCC No.16003
The preservation organization: in China general microbiological culture Collection center
The preservation organization is abbreviated as: CGMCC
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 6 and 25 months in 2018
Registration number of the preservation center: CGMCC No.16003
Compared with the prior art, the invention has the advantages that: the bacillus amyloliquefaciens YB1701 is separated from suaeda glauca rhizosphere soil, the strain can produce 1-aminocyclopropane-1-carboxylic Acid (ACC) deaminase and IAA auxin, has obvious bacteriostatic action on 16 plant pathogenic fungi to be tested, and has wide application prospect in the aspects of biological control of plant pathogenic fungi and growth promotion.
Drawings
FIG. 1 shows a colony (a) and a single-stain photograph (b) of Bacillus amyloliquefaciens (B.amyloliquefaciens) YB 1701;
FIG. 2 is a photograph of a transparent circle for amylolysis of Bacillus amyloliquefaciens (B. amyloliquefaciens) YB 1701;
FIG. 3 is a PCR amplification electropherogram of 16S rDNA from Bacillus amyloliquefaciens (B. amyloliquefaciens) YB 1701;
FIG. 4 is a tree phylogenetic from Bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701 constructed based on 16S rDNA;
fig. 5 bacillus amyloliquefaciens (b. amyloliquefaciens) YB1701 optimum ph (a) and growth profile (b);
FIG. 6 photographs of growth of rice seedlings by Bacillus amyloliquefaciens (B.amyloliquefaciens) YB 1701;
fig. 7 is a photograph of the bacterial inhibition of bacillus amyloliquefaciens YB1701 on the pathogenic bacteria of the test plant (in each figure, the front and back photographs of the culture dish are shown in sequence): (a) magnaporthe grisea (b), maize apical rot (c), maize round spot (d), phytophthora capsici (e), botrytis cinerea (f), pythium aphanidermatum (g), sorghum taraxacum (h), fusarium flavum (i), maize ear rot (g), maize curvularia leaf spot (k), sunflower sclerotinia sclerotiorum (l), maize macrophoma (m), maize pythium (n), maize black bundle (o), apple mold core (p) and melon fusarium oxysporum.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The experimental methods used in the following experiments are all conventional methods unless otherwise specified.
Materials, reagents and the like used in the following experiments are commercially available unless otherwise specified.
Beef extract peptone medium: 3g of beef extract, 10g of peptone, 5g of NaCl, 18-20g of agar powder and 1000mL of water, wherein the pH value is 7.2-7.4, and the beef extract is sterilized at 121 ℃ for 20 min.
LB medium: 10g of peptone, 10g of NaCl, 5g of yeast extract and 1000mL of distilled water, pH7.0, and sterilizing at 121 ℃ for 20 min.
Inorganic salt culture medium: MgSO (MgSO)4.7H2O 0.5g，CaCl2 0.02g，KH2PO4 1.0g，NH4NO3 1.0g，FeCl30.05g, 1000mL of distilled water, pH 7.2, and sterilization at 121 ℃ for 20 min.
DF culture medium: KH (Perkin Elmer)2PO4 4g，Na2HPO4 6g，MgSO4·7H20.2g of O, 2g of glucose, 2g of sodium gluconate, 2g of citric acid, (NH)4)2SO42g, 0.1mL each of the fraction 1 and fraction 2 solutions (fraction 1: H)3BO3 10mg，MnSO4·H2O 11.19mg，CuSO4·5H2O 78.22mg，MoO3 10mg，ZnSO4·7H2O124.6 mg, dissolved in 100mL of sterilized distilled water, and stored at-4 ℃; component 2 FeSO4·7H2O100 mg in 10mL of sterile distilled water, stored at-4 ℃), 1000mL of distilled water, pH 7.2.
ADF culture medium: 3mmol/LACC is used to replace (NH) in DF4)2SO4Is the only nitrogen source.
Starch basal medium: 2g of starch, 3g of beef extract, 10g of peptone, 5g of NaCl, 18-20g of agar powder and 1000mL of distilled water, and sterilizing at the pH of 7.2-7.4,121 ℃ for 20 min.
PDA culture medium: 200g of potato, 20g of sucrose (or glucose), 15-20g of agar and 1000mL of distilled water, wherein the pH is natural, and the potato is sterilized for 30min at 121 ℃.
Hoagland nutrient solution: 0.845g of calcium nitrate was added to 1.26g of Hoagland's nutrient solution (powder), the volume was adjusted to 1000mL, and the mixture was sterilized at 115 ℃ for 20 min.
Test phytopathogens: corn black rot (Acremonium strictum); northern leaf blight (Exserohilum turcicum); pythium gramincola subbra; corn sphaerica (Bipolaris zeicola); phytophthora capsici Leonian (Phytophthora capsici Leonian)); botrytis cinerea (Botrytis cinerea Pers.); apple moldy core (Trichothecium roseum)); rice blast (Pyricularia grisea); melon Fusarium oxysporum f.sp.melonis; sorghum taraxacum (Bipolaris sorghicola); curvularia lunata (Curvularia lunata); corn ear rot (Fusarium graminearum); fusarium culmorum (Fusarium culmorum); corn top rot fungi (Frumentum subglutinans); sclerotinia sclerotiorum (sclerotiorum); pythium aphanidermatum (Pythium aphanidermatum). The photographs of the inhibition of the test phytopathogen by bacillus amyloliquefaciens (b. amyloliquefaciens) YB1701 are shown in fig. 7.
Example 1 isolation and characterization of Bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701 of the present invention
1. Isolation of Strain YB1701
The method comprises the steps of taking freshly collected suaeda salsa with soil at the rhizosphere, shaking off the root soil, weighing 10g, air-drying, placing a soil sample in a conical flask with glass beads and 90mL of sterile physiological saline, and oscillating at room temperature of 120r/min for 30min to prepare a soil suspension. 1mL of soil suspension is taken to be put into a test tube filled with 9mL of sterile normal saline for gradient dilution, and 10 are respectively taken-3，10-4，10-5The dilution of 0.1mL is coated on a screening medium plate, cultured for 24h at 37 ℃, and single colony is picked for separation and purification.
2. Identification of Strain YB1701
(1) Morphological identification
The morphological description of a single colony of bacillus amyloliquefaciens (b. amyloliquefaciens) YB1701 in log phase and with stable colony morphology and size includes the colony size, color, transparency, colony surface morphology (flat, wrinkled, etc.), and colony edge morphology (neat, regular, etc.).
The morphology of the single-stained, gram-stained and spore-stained Bacillus amyloliquefaciens YB1701 (in logarithmic growth phase), belonging to gram-negative bacteria or positive bacteria, and having spores were observed under an optical microscope.
The result shows that the bacterial colony of the bacillus amyloliquefaciens YB1701 is milky white and opaque, has folds on the surface and irregular edges and is easy to pick; the thallus is in short rod shape, is gram-positive, has spores, and has a length of 1.6-3.1 μm and a width of 0.9-1.1 μm (see FIG. 1 and FIG. 2).
(2) Physiological and biochemical characteristic analysis
The biochemical and physiological characteristics of the above Bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 are determined by reference to Bergey's Manual (R.E. Bukanan, N.E. Gibbs et al, handbook of Bergey's Manual of bacteria identification, translation of institute of microbiology, national academy of sciences, science publishers, 1984) and microbiology experiments (Shennu, Van Xiu, Liguangwu. microbiology experiments (third edition), Beijing: advanced education publishers, 1999).
The results of the physiological and biochemical characteristic measurement of the bacillus amyloliquefaciens YB1701 are shown in the table 1:
TABLE 1 physio-biochemical characteristics of Bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701
Note: "+" indicates positive, and "-" indicates negative
3.16S rDNA sequence homology analysis
Extracting total DNA of the strain by a colony method to be used as a template for gene amplification, wherein a 16S rDNA universal primer is 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492r:5'-TACGGTTACCTTGTTACGACTT-3', and performing PCR amplification. The reaction system is as follows: 2 XmixTaq (available from TaKaRa Co.) 25uL, 1. mu.L each of two primers (10. mu.M), 2. mu.L of template was amplified using ddH2O make up to 50. mu.L. Reaction procedure: pre-denaturation at 95 deg.C for 1min, annealing at 55 deg.C for 1min, extension at 72 deg.C for 2min, 35 cycles, extension at 72 deg.C for 10min, and storing at 4 deg.C. DNA sequencing was performed by Shanghai, and the base sequences thus determined were subjected to homology analysis using the EzBioCloud database.
The sequence of 16S rDNA of Bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 strain is shown in the sequence table in detail. The results of agarose gel electrophoresis of 16S rDNA of Bacillus amyloliquefaciens strain YB1701 and phylogenetic trees are shown in FIGS. 3 and 4.
4. Determination of starch-resolving ability
And (3) taking 10uL of the suspension of the bacillus amyloliquefaciens YB1701 separated and purified in the step 1 in the logarithmic phase, inoculating the suspension into a culture medium containing starch in a three-point mode, adding iodine tincture into a flat plate after culturing for 2 days, and observing whether a transparent ring appears after 10 min.
The result shows that a clear circle appears on the culture medium (see figure 2), and the bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701 separated and purified in the step 1 is proved to have strong amylolytic capacity.
5. Analysis of growth characteristics
Beef extract peptone liquid medium is adopted as a basic medium, the pH value is set to be 4, 5, 6, 7, 8, 9, 10 and 11, and each group is set for 3 times of repetition. Inoculating to culture medium at 1%, shake culturing at 37 deg.C for 24 hr at 180r/min, sampling, and determining OD value with Nanodrop2000 microspectrophotometer600Plotted as ordinate and different pH as abscissa, and a line graph is shown in fig. 5A.
The result shows that the optimum pH of the separated and purified bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 strain is between pH7.0 and 8.0.
Example 2 growth Curve of Bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701 of the present invention
Taking the content of conventional beef extract peptone bacteria culture medium as substrate, performing shake culture at 37 deg.C 180r/min at physiological salinity (NaCl) 1% according to 1% inoculum size, taking sample at 2h interval, and performing OD600Assay, each set was set up in 3 replicates. Sampling and OD value measurement with Nanodrop2000 microspectrophotometer, OD600The values are plotted from the coordinates and the incubation time is plotted on the abscissa, and a cell growth curve is plotted as shown in FIG. 5B.
Example 3 culture Medium optimization of Bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701 of the present invention
The method comprises the steps of adopting an inorganic salt culture medium as a basic culture medium, respectively using 1% of sucrose, maltose, lactose, glucose, soluble starch and beef extract as unique carbon sources, and respectively using 1% of peptone, tryptone, yeast extract, beef extract, ammonium sulfate, ammonium chloride and potassium nitrate as unique nitrogen sources, and determining the optimal carbon source and nitrogen source. And (3) optimizing the culture medium by adopting an orthogonal test according to the optimal carbon source and nitrogen source screened by the single factor test and the optimal range of the concentration and pH of the monopotassium phosphate as variation factors, and determining the optimal proportion of each component of the culture medium.
TABLE 2 orthogonal assay Medium optimization
The results show that maltose is the most suitable carbon source with the dosage of 0.3 percent, the yeast extract powder is the most suitable nitrogen source with the dosage of 1.5 percent, the beef extract with the dosage of 1 percent, monopotassium phosphate with the dosage of 0.5 percent and the best pH value of 7.5. The size ordering of the factors affecting the growth of the bacillus amyloliquefaciens (b. amyloliquefaciens) YB1701 strain is: yeast extract powder, maltose, beef extract, pH and potassium dihydrogen phosphate.
Example 4 determination of IAA-producing ability and deaminase Activity of Bacillus amyloliquefaciens YB1701 according to the present invention
1. Determination of IAA-producing ability
The ability of the strain of Bacillus amyloliquefaciens YB1701 obtained in this example 1 to produce IAA was measured by a colorimetric method, and the strain was inoculated in DF medium for overnight growth and then transferred to DF medium containing 0.1% L-tryptophan. The strain was cultured at 28 ℃ for 7 days and then centrifuged at 8000rpm for 10 min. 1mL of the suspension was mixed with 2mL of Fe-H2SO4Solution (1mL0.5M FeCl3·6H2O to 75mL of 6.13M H2SO4) Middle and darkStanding for 45 min. The absorbance of the sample at 450nm was measured and the IAA concentration was determined from the standard curve.
The results show that: IAA activity of YB1701 from Bacillus amyloliquefaciens (B. amyloliquefaciens) as described in example 1 was 137.58. mu.g/mL. Therefore, the bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 has the capability of producing IAA.
ACC deaminase activity assay
Bacillus amyloliquefaciens YB1701 obtained in example 1 was inoculated into 5mL beef extract peptone broth and cultured at 28 ℃ for 24 hours. Sucking 0.5mL of the bacterial liquid into 60mL of beef extract peptone liquid medium, performing amplification culture for 36h under the same condition, centrifuging for 10min at 4 ℃ and 8000rpm, removing supernatant, and collecting thalli. 15mL of water was used without addition of (NH)4)2SO4The DF liquid medium (2) was washed twice, resuspended in 24mL of ADF medium with ACC of 3mM final concentration, and cultured at 28 ℃ for 24 hours to induce ACC deaminase production. After the cells were collected, they were washed twice by centrifugation with 20ml of 0.1M Tris-HCl buffer pH 7.6, and the cells were divided into three EP tubes on average and stored at-20 ℃. The stored cells were resuspended in 1mL of 0.1M Tris-HCl buffer pH 7.6, centrifuged at 10000rpm for 5min, the supernatant was discarded, and the cells were disrupted by rapid shaking. 100 mu L of crude enzyme solution is stored at 4 ℃ and is used for measuring the protein concentration; the remaining crude enzyme solutions were immediately assayed for ACC deaminase activity. 200. mu.L of the crude enzyme solution was added to 1.5mL of EP, 33.4. mu.L of 0.3M ACC was added thereto, the mixture was mixed, and the mixture was subjected to water bath at 30 ℃ for 15min, followed by addition of 1mL of 0.56M HCl to terminate the reaction. Comparison: (1) no crude enzyme solution is added; (2) no ACC was added. Centrifuging at 10000rpm for 5min, sucking 1mL of supernatant into a test tube, adding 800 μ L of 0.56M HCl and 300 μ L of 0.2% 2, 4-dinitrophenylhydrazine solution, keeping the temperature at 30 ℃ for 30min, adding 2mL of 2M NaOH solution, and mixing uniformly. And (3) replacing the supernatant with distilled water, performing the same other steps, adjusting zero by using a spectrophotometer, measuring the absorbance value at 540nm, and calculating the content of the alpha-ketobutyric acid according to the standard curve of the alpha-ketobutyric acid. Adding appropriate amount of crude enzyme solution into test tube, adding PBS buffer solution to make up to 75 μ L, adding 2mL Coomassie brilliant blue solution, mixing and shaking, developing for 5min, measuring absorbance value at 595nm, and calculating protein content of thallus according to bovine serum albumin standard curve. ACC deaminaseActivity (μmol. mg)-1·h-1) α -ketobutyric acid content (μmol)/protein content (mg)/reaction time (h). Protein determination was carried out by a colorimetric method using bovine serum albumin as a substrate. The measurement results are an average of 3 times.
The results show that: the ACC deaminase activity of Bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701 as described in example 1 was 3.03. mu. mol. mg-1·h-1. Therefore, the bacillus amyloliquefaciens YB1701 has ACC deaminase activity, and can improve the stress capacities of plants such as drought resistance, high temperature resistance, pest and disease attack resistance or heavy metal pollution resistance.
Example 5 determination of the inhibitory Rate of Bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701 of the present invention against phytopathogenic fungi
The determination of the antifungal bacteriostasis rate of the antagonistic pathogenic fungi of the bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701 obtained in the example 1 is carried out by adopting a three-point confrontation method, which comprises the following specific operations: inoculating fungus cakes of plant pathogenic fungi on three points (in a regular triangle) on a PDA (personal digital assistant) plate, inoculating filter paper sheets of fermentation liquor of a Bacillus amyloliquefaciens (B.amyloliquefaciens strain G81) YB1701 at the middle position, repeating each group for 3 times, culturing in a thermostat at 28 ℃ for 4-5 days, measuring the diameter of each treated colony, calculating the average value and calculating the bacteriostasis rate (%).
The bacteriostatic ratio is (distance between pathogenic bacteria heart and heart (D) — [ distance between test strain heart and pathogenic bacteria heart (D) — (vertical distance between test strain heart and pathogenic bacteria (r) ])/distance between pathogenic bacteria heart and heart (D) × 100%
The result shows that the fermentation liquor of the Bacillus amyloliquefaciens (B.amyloliquefaciens) YB1701 strain has the bacteriostasis rate of more than or equal to 50 percent on the corn black bundle pathogen, the apple mold core pathogen, the corn pythium aphanidermatum, the corn cone spot pathogen, the phytophthora capsici, the tomato gray mold, the apple mold core pathogen, the rice blast pathogen, the melon fusarium wilt pathogen, the sorghum target spot, the corn curvularia leaf spot, the corn ear rot pathogen, the yellow fusarium, the corn top rot pathogen, the sunflower sclerotinia rot and the pythium aphanidermatum in 16 plant pathogenic fungi to be tested; in particular, the bacteriostasis rate of the tested corn northern leaf blight and sunflower nuclear disc bacteria is more than or equal to 80 percent (see table 3).
TABLE 3 antibacterial ratio of Bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701 fermentation broth against pathogenic fungi
Example 6 growth promotion experiment of Bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701 of the present invention on rice seedlings
Respectively selecting rice seeds with similar sizes and plump seeds, soaking for 24h at 28 ℃, then accelerating germination for 24h at 30 ℃, sowing the germinated seeds on 750mL plastic cups which are filled with Hogland nutrient solution and covered with gauze, and repeating each group for 3 times. Placing in a light incubator at a day-night temperature of 28 deg.C/22 deg.C, a light cycle of 12h/12h, a relative air humidity of 80%, and a light intensity of 300 μ M/(M)2.s2) Culturing in medium. And (3) treating the seedlings when the seedlings grow to 2 leaves and one heart stage, and adding 3% of fermentation liquor of bacillus amyloliquefaciens YB1701 in logarithmic phase. Beef extract peptone medium supplemented with 3% was used as a control. Seedling height, root length, seedling and root dry weight were measured at 10d and 16d after treatment and the results are shown in table 4 and fig. 6.
TABLE 4 influence of Bacillus amyloliquefaciens (B. amyloliquefaciens) YB1701 fermentation broth on rice seedlings
The results show that the growth of rice seedlings treated by the fermentation liquor of the bacillus amyloliquefaciens YB1701 is promoted, and the height, the length, the dry weight and the fresh weight of the rest seedlings are obviously increased at 10d and 16d except that the difference between the root length and the dry weight of the seedlings at 10d is not obvious compared with the contrast. It is demonstrated that the bacillus amyloliquefaciens (b. amyloliquefaciens) YB1701 obtained in the example 1 has a significant effect of promoting the growth of rice seedlings.
Sequence listing
<110> Shenyang university
<120> bacillus amyloliquefaciens strain and application thereof
<130> 2019
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cccaccttcc tccggtttgt caccggcagt caccttagag tgcccaactg aatgctggca 360
actaagatca agggttgcgc tcgttgcggg acttaaccca acatctcacg acacgagctg 420
acgacaacca tgcaccacct gtcactctgc ccccgaaggg gacgtcctat ctctaggatt 480
gtcagaggat gtcaagacct ggtaaggttc ttcgcgttgc ttcgaattaa accacatgct 540
ccaccgcttg tgcgggcccc cgtcaattcc tttgagtttc agtcttgcga ccgtactccc 600
caggcggagt gcttaatgcg ttagctgcag cactaagggg cggaaacccc ctaacactta 660
gcactcatcg tttacggcgt ggactaccag ggtatctaat cctgttcgct ccccacgctt 720
tcgctcctca gcgtcagtta cagaccagag agtcgccttc gccactggtg ttcctccaca 780
tctctaacgc atttcaccgc tacacgtgga aattccactc ttctctttct gcactcaagt 840
tccccagttt ccaatgaccc tccccggttg agccgggggc tttcacatca gacttaagaa 900
accgcctgcg agctctttac gcccaataat tcccgacaac gcttgcaccc ttacgtaatt 960
accgcgtctg ctggcacgta ttaaccgtgg cttctggtaa ggaccgttaa ag 1012
Claims (6)
1. The bacillus amyloliquefaciens strain is YB1701, and the bacillus amyloliquefaciens strain YB1701 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC 16003.
2.A microbial inoculum, wherein the active ingredient of the microbial inoculum is the bacillus amyloliquefaciens YB1701 or a fermentation broth of the bacillus amyloliquefaciens YB1701 as claimed in claim 1;
the preparation of the fermentation liquor of the bacillus amyloliquefaciens YB1701 comprises the following steps:
1) inoculating the purified and cultured bacillus amyloliquefaciens YB1701 into a beef extract peptone solid culture medium from a slant, and culturing for 24 hours at 37 ℃ and 180 r/min;
2) inoculating the activated strain in the step 1) into an LB liquid culture medium, and carrying out shake culture in a shaking table at 37 ℃ and 180r/min for 48h to obtain a seed solution;
3) inoculating the seed liquid obtained in the step 2) into a beef extract peptone culture medium according to the inoculation amount of 5%, and carrying out shake culture in a shaking table at the temperature of 37 ℃ and 180r/min for 48h to obtain the bacillus amyloliquefaciens YB1701 fermentation liquid.
3. The microbial inoculum of claim 2, wherein the content of the bacillus amyloliquefaciens YB1701 microbial cells in the fermentation liquid of the bacillus amyloliquefaciens YB1701 is 1.0 x 106 to 1.0 x 108 cfu/mL.
4. The microbial inoculum according to claim 2, which is any one of the following:
1) a fungicide for inhibiting plant pathogenic fungi;
2) a fungicide for controlling plant pathogenic fungal diseases;
3) a microbial inoculum for promoting plant growth;
4) a microbial inoculum for improving the stress resistance of plants;
5) a microbial inoculum for reducing the sensitivity of plants to stress;
6) a microbial inoculum producing 1-aminocyclopropane-1-carboxylic acid deaminase;
7) a indole-3-acetic acid-producing microbial inoculum;
the stress is drought, high temperature, pest attack or heavy metal pollution.
5. The inoculant according to claim 4, wherein the phytopathogenic fungi are at least one of the following phytopathogenic fungi:
1) corn black bunch germs;
2) northern leaf blight of corn;
3) pythium aphanidermatum;
4) pythium species of corn;
5) corn cercospora species;
6) phytophthora capsici;
7) botrytis cinerea;
8) apple moldy core bacteria;
9) rice blast bacteria;
10) melon fusarium wilt bacteria;
11) sorghum taraxacum;
12) curvularia lunata (berk.) Kuntze;
13) corn ear rot;
14) fusarium flavum;
15) corn top rot;
16) helianthus annuus (L.) karst.
6. A method of cultivating a strain of bacillus amyloliquefaciens according to claim 1 comprising the step of cultivating bacillus amyloliquefaciens YB1701 in a medium for cultivating bacillus amyloliquefaciens.
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| CN110317747B (en) * | 2019-06-04 | 2021-04-23 | 华南农业大学 | Bacillus amyloliquefaciens JT68 and application thereof in prevention and treatment of tea anthracnose |
| CN110205273B (en) * | 2019-06-11 | 2021-02-09 | 山东碧蓝生物科技有限公司 | Bacillus amyloliquefaciens with growth promoting and disease resisting effects and application thereof |
| CN110172429A (en) * | 2019-06-13 | 2019-08-27 | 江苏牧之歌生态农业科技有限公司 | A kind of Biocontrol Bacillus that improves is to the culture medium and cultural method of plant pathogenic fungi bacteriostatic activity |
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| CN110819566B (en) * | 2019-11-20 | 2022-09-16 | 东北农业大学 | Bacillus amyloliquefaciens and application thereof |
| CN111567563B (en) * | 2020-06-22 | 2021-11-02 | 上海市农业科学院 | Corn microbial seed coating agent and preparation method thereof |
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| CN112175868B (en) * | 2020-09-30 | 2022-05-17 | 陕西科技大学 | Bacillus amyloliquefaciens strain with antibacterial activity and application thereof |
| CN112175883A (en) * | 2020-10-26 | 2021-01-05 | 上海市农业科学院 | Bacillus amyloliquefaciens with good bacteriostatic ability |
| CN112680377B (en) * | 2021-01-15 | 2022-01-07 | 河北冀微生物技术有限公司 | Bacillus amyloliquefaciens Z-2 strain for preventing and treating root rot of fruits and vegetables and application thereof |
| CN113980859B (en) * | 2021-11-22 | 2023-03-31 | 中国科学院微生物研究所 | Bacillus amyloliquefaciens and application thereof |
| CN115125159B (en) * | 2022-04-24 | 2023-05-23 | 沈阳农业大学 | Bacillus amyloliquefaciens, microbial inoculum thereof, preparation method and application of microbial inoculum |
| CN114908021A (en) * | 2022-06-13 | 2022-08-16 | 中国农业科学院蔬菜花卉研究所 | Bacillus amyloliquefaciens and application thereof in preventing and treating cucumber corynespora leaf spot |
| CN115322928A (en) * | 2022-08-10 | 2022-11-11 | 河北省科学院生物研究所 | Bacillus amyloliquefaciens ZLP-01 and application thereof |
| CN117286059A (en) * | 2023-09-14 | 2023-12-26 | 中化现代农业有限公司 | Composite microbial inoculum, microbial synergist, microbial compound fertilizer and application |
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