CN110438026B - Bacillus amyloliquefaciens GLD-191 and microbial inoculum, preparation method and application thereof - Google Patents
Bacillus amyloliquefaciens GLD-191 and microbial inoculum, preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
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Abstract
The invention relates to the field of biological control and microbial fertilizers, in particular to bacillus amyloliquefaciens GLD-191, a microbial inoculum, a preparation method and application thereof. The invention provides bacillus amyloliquefaciens GLD-191, which is preserved in China general microbiological culture Collection center (CGMCC) No.17011 in 12 months and 19 days of 2018. The invention also provides a microbial agent prepared from the bacillus amyloliquefaciens GLD-191 and a preparation method thereof, and application of the bacillus amyloliquefaciens GLD-191 and the microbial agent thereof in plant disease control. According to the invention, the bacillus amyloliquefaciens GLD-191 and the isatis root residue solid medium are subjected to secondary fermentation to prepare the novel microbial agent, so that the utilization rate of nutrient substances in the isatis root residue by plants is improved, the bacillus amyloliquefaciens GLD-191 can generate antibacterial substances with antibacterial activity on plant pathogenic fungi, the beneficial microbial population can be increased, and the disease control spectrum is enlarged.
Description
Technical Field
The invention relates to the field of biological control and microbial fertilizers, in particular to bacillus amyloliquefaciens GLD-191, a microbial inoculum, a preparation method and application thereof.
Background
Bacillus amyloliquefaciens has the capability of widely inhibiting fungi and bacteria, and can change the surrounding flora environment and species, so that the bacillus amyloliquefaciens becomes the microorganism species with the most development potential of biological bactericides and microbial fertilizers. A great deal of research on the world has found that Bacillus amyloliquefaciens can produce antibacterial substances with antibacterial activity on plant pathogenic fungi, and the antibacterial substances mainly comprise antibiotics with low molecular weight such as peptides, lipopeptides, bacteriocins, high molecular weight proteins and the like.
The radix isatidis residue is a byproduct of the antiviral agent after extraction, is rich in organic matters and nutrient substances, but is usually discarded as waste, and is a great waste of plant resources. The isatis root residue still contains a small amount of active products for resisting viruses and diseases, contains a large amount of plant organic matters and nutrient substances, and if the isatis root residue is directly used, plants cannot be effectively utilized, and the isatis root residue contains a large amount of nutrient components such as organic matters, plant proteins and fibers and can be used as a culture medium raw material and an adsorption carrier of biocontrol bacteria. Thus, if the above disadvantages can be overcome by inoculating and fermenting beneficial microorganisms, the plant utilization can be improved.
Disclosure of Invention
According to the existing problems and the defects of the prior art, the invention provides bacillus amyloliquefaciens GLD-191, a microbial agent, a preparation method and application thereof, wherein bacillus amyloliquefaciens GLD-19 can be applied to plant disease control, and the bacillus amyloliquefaciens GLD-191 and a radix isatidis residue solid culture medium are subjected to secondary fermentation to prepare a novel microbial agent with a biocontrol function. The invention has important significance for comprehensively preventing and treating diseases, improving the content of soil organic matters and the population quantity of microorganisms and promoting the recycling of agricultural and sideline products, and has wide application prospect in agricultural production practice, especially in green agricultural development.
In order to achieve the above object, the present invention is realized by the following means:
the invention provides bacillus amyloliquefaciens GLD-191, the bacillus amyloliquefaciens GLD-191 strain is preserved in the China general microbiological culture Collection center (CGMCC) No.17011, the preservation date is 2018, 12 months and 19 days, and the preservation unit address is: no.1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
The 16S rDNA nucleotide sequence of the bacillus amyloliquefaciens GLD-191 is shown as SEQ ID NO.1, the phylogenetic tree is shown as figure 1, the bacillus is a mesophilic, aerobic and spore-producing rod-shaped bacterium, the physiological characteristics of the bacillus are various, the bacillus is widely present in the nature, the bacillus is nontoxic and harmless to human and livestock, does not pollute the environment, can produce various antibiotics and enzymes (originally named as the bacillus can secrete alpha-amylase), and has broad-spectrum antibacterial activity and extremely strong stress resistance.
The invention also provides a microbial agent prepared from the bacillus amyloliquefaciens GLD-191.
Further, the microbial agent is a solid microbial agent.
Further, the solid microbial inoculum is prepared from bacillus amyloliquefaciens GLD-191 seed fermentation liquor and a isatis root solid culture medium.
Further, the preparation method of the microbial agent comprises the following steps:
(1) Inoculating Bacillus amyloliquefaciens GLD-191 into an improved nutrient broth liquid culture medium, and shake-culturing for 48 hours at the temperature of 27-34 ℃ at the rotating speed of 200-350rpm to obtain seed liquid;
(2) Inoculating the seed liquid obtained in the step (1) into an LB liquid culture medium according to the volume ratio of 3-10%, and carrying out liquid fermentation production, wherein the fermentation conditions are as follows: pH is 6.5-7.8, fermentation temperature is 27-34 ℃, stirring speed is 200-350rpm, aeration rate is 0.5:1, fermentation time is 72-96h, and viable spore number of fermentation liquor is more than or equal to 1×10 9 Obtaining bacillus amyloliquefaciens GLD-191 seed fermentation liquor per ml;
(3) Inoculating the seed fermentation liquor of the bacillus amyloliquefaciens GLD-191 seed fermentation liquor obtained in the step (2) into a radix isatidis solid culture medium according to the amount of 60-100ml/Kg, carrying out secondary solid fermentation and decomposition, turning over the pile 1 time every other day in the fermentation process, fermenting for 5-9 days at the fermentation temperature of 20-50 ℃, carrying out ventilation drying at the temperature of not higher than 70 ℃ after the fermentation is finished, controlling the water content to be below 20%, packaging and sealing for standby, and obtaining the solid microbial inoculum.
Further, the isatis root solid culture medium comprises the following components in percentage by mass: lan Genzha 3-25% of plate, 15-40% of bean pulp, 10-35% of wheat bran, 15-30% of yeast extract powder, 0.3-1.5% of urea, 1-5% of NaCl, 0.5-3% of KCl and MgSO 4 0.5-0.8％、CaCO 3 1.2-1.9%, pH is 6.5-8, and radix Isatidis residue is fine powder of granule obtained by pulverizing radix Isatidis diameter, sieving with 200-300 mesh sieve, and is used as culture medium matrix of Bacillus amyloliquefaciens GLD-191 and as adsorption carrier of microbial inoculum.
Further, the modified nutrient broth liquid medium contains: beef extract 0.6%, peptone 0.7%, naCl0.3%, and distilled water for the rest, pH6.3-7.8; the LB liquid medium contained 10g of peptone, 5.0g of NaCl, 10g of yeast extract, 1000ml of distilled water and pH7.2.
The invention provides application of bacillus amyloliquefaciens GLD-191 in plant disease control.
The microbial agent provided by the invention is applied to plant disease control.
The invention has the beneficial effects that:
the bacillus amyloliquefaciens GLD-191 has broad-spectrum antibacterial activity and extremely strong stress resistance, and has good application effect in preventing and controlling plant diseases. According to the invention, the bacillus amyloliquefaciens GLD-191 and the isatis root residue solid medium are subjected to secondary fermentation to prepare the novel microbial agent, and the bacillus amyloliquefaciens GLD-191 can produce antibacterial substances with antibacterial activity on plant pathogenic fungi.
The bacillus amyloliquefaciens GLD-191 and the isatis root residue solid medium are subjected to secondary fermentation, so that the novel microbial agent with the biocontrol function can be prepared. The isatis root residue is a byproduct of the extraction of antiviral agents, is rich in rich organic matters and nutrients and a small amount of antiviral and disease-resistant active products, and is used as a culture medium raw material and an adsorption carrier for biocontrol bacteria, so that the utilization rate of the nutrients in the isatis root residue by plants is improved, the problems that the plants cannot be utilized and have poor effects and even damage seedlings occur when the isatis root residue is directly used are solved, and the problem of agricultural and sideline product waste utilization is solved. The isatis root residue is inoculated and fermented by the bacillus amyloliquefaciens GLD-191, and the obtained microbial agent can be used for preventing and treating watermelon fusarium wilt and can also be applied to promoting plant growth and improving soil.
The invention has important significance for comprehensively preventing and treating diseases, improving the content of soil organic matters and the population quantity of microorganisms and promoting the recycling of agricultural and sideline products, and has wide application prospect in agricultural production practice, especially in green agricultural development.
Drawings
FIG. 1 is a 16S rDNA sequence phylogenetic tree of the Bacillus amyloliquefaciens GLD-191 of the present invention;
FIG. 2 shows the results of a plate-stand test of Bacillus amyloliquefaciens GLD-191 of the present invention against watermelon fusarium wilt.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Isolation of Bacillus amyloliquefaciens GLD-191
Bacillus amyloliquefaciens GLD-191 is obtained by separating from the soil of the original forest of Qinling mountain of Shaanxi province.
The specific method comprises the following steps: the method is characterized in that the original forest soil collected in Taibai county, baiyunxiang, shaanxi province is naturally air-dried and stored at room temperature for antagonistic bacteria separation. 1g of this soil was diluted with sterile water to a gradient of 10-3, 10-4, 10-5. 100. Mu.L of each of the LB medium was aspirated and spread uniformly with a glass spreader. Culturing in a culture box at 28-30 deg.c for 48-72 hr, and separating and purifying bacteria with different colony forms. The watermelon fusarium wilt bacteria are used as indicator bacteria, a flat plate counter method is adopted to screen antagonism of the watermelon fusarium wilt bacteria, and after a growth incubator for 6d at 28 ℃, strains with inhibition zones are selected for further screening.
The strain points after primary screening are connected to 4 corners at a position 3.5cm away from the center of a flat plate by adopting a crisscross method, agar blocks with diameters of 4.5mm and growing watermelon fusarium wilt bacteria are simultaneously moved into the center of the flat plate, and after the strain points are cultured for 6 days at 28 ℃, the strain with a bacteriostasis zone and lasting antagonism is selected. The diameter of the inhibition zone is measured and recorded, and the inhibition rate is calculated. Inhibition ratio (%) = (control group pathogenic bacteria colony diameter-treatment group pathogenic bacteria colony diameter)/control group pathogenic bacteria colony diameter x 100%, as shown in fig. 2.
Example 2
Identification of Bacillus amyloliquefaciens GLD-191
The strain GLD-191 isolated in example 1 was identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) by physiological and biochemical characteristics and 16SrDNA analysis in a conventional manner, and was named as Bacillus amyloliquefaciens GLD-191.
The strain GLD-191 does not generate pigment when grown on LB culture medium, and the colony is flat or round, milky opaque and irregular in edge. The surface has folds. Gram positive, rod-shaped, sporulated, oval or cylindrical. Physiological and biochemical identification of strains was performed by reference to the handbook of common bacteria identification: mannitol test positive, lactose test positive, V-P test positive, 7% NaCl growth test negative, methyl red test negative, indole test positive, pH5.7 growth test positive, citrate utilization test positive, L-arabinose test positive, starch hydrolysis test positive, D-xylose test positive, gelatin liquefaction test positive, casein decomposition test positive, lipase negative, nitrate reduction test positive, malonate utilization negative, cellulose decomposition positive, arginine double hydrolysis negative.
The 16S rDNA amplified product is recovered and purified and then sequenced, and the nucleotide sequence of the 16S rDNA of the bacillus amyloliquefaciens GLD-191 is shown as SEQ ID NO.1, and specifically comprises the following steps:
SEQ ID NO.1:
<110> Shaanxi Lv Feng crop technology Co.Ltd
<120> Bacillus amyloliquefaciens GLD-191 and microbial inoculum, preparation method and application thereof
<210>1
<211>1401
<212>DNA
<213> Bacillus amyloliquefaciens GLD-191
AGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCG GCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGG GATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAAC CGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGA TGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACC AAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACAC TGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTA GGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGC GTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGA AGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTA ACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATA CGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTC GCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACC GGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGG AGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGG AGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACG CTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTG GTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTC CGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGG AGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCC GCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAG AACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGA CGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGT CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC AACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGT GACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAA TCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGA CAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCAC AAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTG AAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAAT ACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGT TTGTAACACCCGAAGTCGGTGAGGTAACCTTT
The sequenced sequences were subjected to BLAST alignment via NCBI database and phylogenetic analysis with model bacteria via MEGA5.2 software. As shown in FIG. 1, the antagonistic strain GLD-191 was closest to Bacillus amyloliquefaciens Bacillus amyloloquefaciens, and strain GLD-191 was identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Bacillus amyloliquefaciens GLD-191 was deposited at the China general microbiological culture Collection center, with the accession number: CGMCC No.17011.
Example 3
Control test of bacillus amyloliquefaciens GLD-191 on watermelon fusarium wilt
Sterile water is used as a negative control, carbendazim is used as a common medicament control, and the control effect of bacillus amyloliquefaciens GLD-191 on potted watermelon fusarium wilt is measured.
The strain GLD-191 isolated in example 1 was placed in a sterile physiological saline-containing tube with a stopper containing glass beads, and then shaken by a shaker to form a bacterial suspension having a concentration of 1X 10 9 CFU/ml。
Respectively spraying leaf surfaces of potted watermelon (total 90 plants, 30 plants each) with sterile water, bacterial suspension and carbendazim (diluted 1000 times according to the instruction), inoculating 1×10 after 48 hr 5 The watermelon fusarium wilt spore suspension is of a single/mL type. Culturing in dark and at 25 deg.C for 72h, and observing the disease condition. Calculating control effect by using morbidity rate, wherein the control effect% = [ (morbidity rate of control area-morbidity rate of treatment area)/morbidity rate of control area]×100。
The test result shows that the control effect of the sterile water control group is 0 (the number of the pathogenic plants is 30), the control effect of the control medicament carbendazim is 63.3% (the number of the pathogenic plants is 11), and the control effect of the bacillus amyloliquefaciens GLD-191 is 86.7% (the number of the pathogenic plants is 4). The test result shows that the bacillus amyloliquefaciens GLD-191 has good control effect on watermelon fusarium wilt and has guiding significance on actual application of agricultural production.
Example 4
The preparation method of the microbial agent prepared by bacillus amyloliquefaciens GLD-191 specifically comprises the following steps:
(1) Bacillus amyloliquefaciens GLD-191 was inoculated into a modified nutrient broth liquid medium containing: beef extract 0.6%, peptone 0.7%, naCl0.3%, distilled water for the rest, pH6.3-7.8, shake flask culturing at 30deg.C at 250rpm for 48 hr to obtain seed solution;
(2) Inoculating the seed liquid obtained in the step (1) into an LB liquid culture medium according to the volume ratio of 5%, wherein the LB liquid culture medium comprises: 10g of peptone, 5.0g of NaC l, 10g of yeast extract, 1000ml of distilled water and pH7.2, and the fermentation conditions are as follows: pH6.5-7.8, fermentation temperature range of 30deg.C, stirring speed of 300rpm, aeration rate of 0.5:1, fermentation time of 84h, and viable spore number of fermentation broth of not less than 1×10 9 Obtaining bacillus amyloliquefaciens GLD-191 seed fermentation liquor per ml;
(3) Inoculating the bacillus amyloliquefaciens GLD-191 seed fermentation liquor obtained in the step (2) into a isatis root solid culture medium according to the amount of 80ml/Kg, and carrying out secondary solid fermentation and decomposition, wherein the isatis root solid culture medium comprises the following components in percentage by mass: 20% of radix isatidis slag, 20% of bean pulp, 30% of wheat bran, 20% of yeast extract powder, 1% of urea, 4% of NaCl, 3% of KCl and MgSO 4 0.5％、CaCO 3 1.5 percent of pH value is 6.5 to 8, the reactor is turned over for 1 time every other day in the fermentation process, the fermentation temperature is 40 ℃, the fermentation is carried out for 6 days, after the fermentation is finished, the ventilation drying is carried out at the temperature of not higher than 70 ℃ to control the water content to be below 20 percent, and the microbial agent is obtained after packaging and sealing for standby.
Example 5
Field effect test for preventing and treating watermelon fusarium wilt and promoting watermelon growth by using isatis root residue microbial agent
In order to show the control effect of the microbial inoculum on watermelon fusarium wilt and the growth promotion effect of watermelon, a field pesticide effect test is performed on the basis of an indoor potting test. In northwest agriculture and forestry science and technology university test fields, two groups of treatments are set: treatment 1 was not applied as a control; treatment 2 the microbial agent prepared in example 4 was applied. Selecting a watermelon field with consistent growth of watermelon seedlings, carrying out soil treatment by combining a watermelon seedling base fertilizer, using 50 Kg/mu, continuously observing for 3 months after treatment, investigating the disease condition in the field, calculating the field prevention effect according to the method of the embodiment 3, measuring the watermelon plant length, and recording the test result.
The test result shows that the disease index of the isatis root residue microbial agent treatment group is obviously lower than that of a blank control group, the disease rate of watermelon fusarium wilt can be effectively reduced, and the field prevention effect reaches 76.7%; meanwhile, the isatis root microbial agent can promote the growth of watermelon seedlings, and the average plant length of a treated group reaches 0.98m and is 0.11m higher than that of a blank control group.
In the description herein, reference to the terms "one embodiment," "example," "specific example," and the like, means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The preferred embodiments of the invention disclosed above are intended only to assist in the explanation of the invention. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise form disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
SEQ ID NO.1:
<110> Shaanxi Lv Feng crop technology Co.Ltd
<120> Bacillus amyloliquefaciens GLD-191 and microbial inoculum, preparation method and application thereof
<210> 1
<211> 1401
<212> DNA
<213> Bacillus amyloliquefaciens GLD-191
agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac 60
gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgg 120
ttgtttgaac cgcatggttc agacataaaa ggtggcttcg gctaccactt acagatggac 180
ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcgacgat gcgtagccga 240
cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300
gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360
aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtgccgtt caaatagggc 420
ggcaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta 480
atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc 540
ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact 600
tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga 660
ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt 720
ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg 780
ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt 840
acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900
tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacaatcct 960
agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg tcgtcagctc 1020
gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag 1080
cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac 1140
gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg acagaacaaa 1200
gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt cggatcgcag 1260
tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt 1320
gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg 1380
aagtcggtga ggtaaccttt 1400
Claims (8)
1. The bacillus amyloliquefaciens GLD-191 is characterized in that bacillus amyloliquefaciens GLD-191 strain is preserved in China general microbiological culture Collection center (CGMCC No. 17011), and the preservation date is 2018, 12 months and 19 days.
2. The bacillus amyloliquefaciens GLD-191 according to claim 1, wherein the 16S rDNA nucleotide sequence of the bacillus amyloliquefaciens GLD-191 is shown in SEQ ID No. 1.
3. The microbial agent prepared by bacillus amyloliquefaciens GLD-191 according to claim 1.
4. A microbial agent according to claim 3, wherein the microbial agent is a solid microbial agent.
5. The microbial agent according to claim 4, wherein the solid microbial agent is prepared from bacillus amyloliquefaciens GLD-191 seed fermentation broth and isatis root solid medium.
6. The microbial agent according to claim 5, wherein the preparation method comprises the following steps:
(1) Inoculating Bacillus amyloliquefaciens GLD-191 into an improved nutrient broth liquid culture medium, and shake-culturing for 48 hours at the temperature of 27-34 ℃ at the rotating speed of 200-350rpm to obtain seed liquid;
(2) Inoculating the seed liquid obtained in the step (1) into an LB liquid culture medium according to the volume ratio of 3-10%, and carrying out liquid fermentation production, wherein the fermentation conditions are as follows: pH6.5-7.8, fermentation temperature range of 27-34 deg.C, stirring speed of 200-350rpm, aeration rate of 0.5:1, fermentation time of 72-96 hr, and viable spore number of fermentation broth of not less than 1×10 9 Obtaining bacillus amyloliquefaciens GLD-191 seed fermentation liquor per ml;
(3) Inoculating the bacillus amyloliquefaciens GLD-191 seed fermentation liquor obtained in the step (2) into a isatis root solid culture medium according to the amount of 60-100ml/Kg, carrying out secondary solid fermentation and decomposition, turning over the pile 1 time every other day in the fermentation process, fermenting for 5-9 days at the fermentation temperature of 20-50 ℃, carrying out ventilation drying at the temperature of not higher than 70 ℃ after the fermentation is finished, controlling the water content to be below 20%, packaging and sealing for standby, and obtaining the solid microbial inoculum.
7. The microbial agent according to claim 6, wherein the isatis root solid culture medium comprises the following components in percentage by mass: 3-25% of radix isatidis residue, 15-40% of soybean meal, 10-35% of wheat bran, 15-30% of yeast extract powder, 0.3-1.5% of urea, 1-5% of NaCl, 0.5-3% of KCl and MgSO 4 0.5-0.8％、CaCO 3 1.2-1.9%, pH is 6.5-8, and the radix Isatidis residue is fine powder of granule obtained by pulverizing radix Isatidis diameter, sieving with 200-300 mesh sieve, and is used as culture medium matrix of Bacillus amyloliquefaciens GLD-191 and as adsorption carrier of microbial inoculum.
8. The microbial inoculant according to claim 6, wherein the modified nutrient broth liquid medium comprises: beef extract 0.6%, peptone 0.7%, naCl0.3%, and distilled water for the rest, pH6.3-7.8; the LB liquid medium contains: 10g of peptone, 5.0g of NaCl, 10g of yeast extract, 1000ml of distilled water and pH7.2.
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| CN105420156A (en) * | 2015-12-19 | 2016-03-23 | 佛山市艳晖生物科技有限公司 | Bacillus amyloliquefaciens with phosphate solubilizing, disease preventing and growth promoting functions and application thereof |
| CN109402027A (en) * | 2018-12-14 | 2019-03-01 | 齐齐哈尔大学 | One plant has the bacillus amyloliquefaciens for inhibiting the Fusarium oxysporum effect of watermelon specialized form and its application |
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| CN105420156A (en) * | 2015-12-19 | 2016-03-23 | 佛山市艳晖生物科技有限公司 | Bacillus amyloliquefaciens with phosphate solubilizing, disease preventing and growth promoting functions and application thereof |
| CN109402027A (en) * | 2018-12-14 | 2019-03-01 | 齐齐哈尔大学 | One plant has the bacillus amyloliquefaciens for inhibiting the Fusarium oxysporum effect of watermelon specialized form and its application |
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