CN113322206A - Bacillus belgii SUNO-18S-36-containing microbial agent and application thereof - Google Patents
Bacillus belgii SUNO-18S-36-containing microbial agent and application thereof Download PDFInfo
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- CN113322206A CN113322206A CN202110639503.3A CN202110639503A CN113322206A CN 113322206 A CN113322206 A CN 113322206A CN 202110639503 A CN202110639503 A CN 202110639503A CN 113322206 A CN113322206 A CN 113322206A
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Abstract
The present invention provides a Bacillus belgiiBacillus velezensis) A fermentation method of SUNO-18S-36 strain, relating to micro-fermentationThe field of biology. The fermentation method adopts the following culture media: 4-10 g/L of glucose, 5-20 g/L of corn steep liquor, 5-25 g/L of yeast powder, 5-10 g/L of silkworm chrysalis powder, 0-10 g/L of calcium carbonate, 0-30 g/L of calcium gluconate, 0.1-1 g/L of magnesium sulfate heptahydrate, 0-0.1 g/L of manganese sulfate, 0-10 g/L of calcium phosphate and pH 7.0-7.5. The microorganism involved in the fermentation method has the functions of dissolving phosphorus and degrading pesticide residue, and has antagonistic action on pathogenic bacteria.
Description
Technical Field
The invention relates to the field of microbial preparations, in particular to a microbial inoculum containing Bacillus belgii SUNO-18S-36 and application thereof.
Background
In the prior art, the application of Bacillus belgii in straw degradation and sewage ammonia nitrogen degradation is reported, the application in aluminum-toxicity soil remediation, aluminum-toxicity stress alleviation and plant aluminum-toxicity stress resistance improvement is also reported, and the Bacillus belgii capable of preventing and treating peanut root rot is also reported. However, the prior art lacks microorganisms which can simultaneously dissolve phosphorus, degrade pesticide residues and have antagonistic action on plant pathogenic bacteria, and further lacks efficient microbial agents of the microorganisms.
Disclosure of Invention
The invention aims to provide a microbial inoculum containing Bacillus velezensis SUNO-18S-36, which is easy to recover and has high survival rate.
The purpose of the invention is realized by adopting the following technical scheme:
a solid microbial inoculum containing Bacillus velezensis SUNO-18S-36 strain is prepared by adding adsorption carrier and adjuvant into Bacillus velezensis SUNO-18S-36 fermentation liquid, adsorbing, drying, and pulverizing. The content of Bacillus belgii SUNO-18S-36 in the microbial inoculum is not less than 1000 hundred million cfu/g.
In the invention, the adsorption carrier is one or a mixture of more than two of kaolin, light calcium carbonate, diatomite, white carbon black and biomass carbon.
In the present invention, the auxiliary agents include a wetting agent, a nutrient, an ultraviolet protective agent and a water-retaining agent.
In the invention, the wetting agent is selected from one or two of sodium dodecyl benzene sulfonate and sodium lignin sulfonate; the nutrient is selected from one or more of glucose, sucrose, starch, yeast powder, amino acid powder and bean cake powder; the ultraviolet protective agent is selected from one or more of flavone, lecithin, fluorescent whitening agent, Robinia pseudoacacia toxin and hemp seed oil; the water-retaining agent is selected from one or a mixture of more than two of polyacrylamide, sodium polyacrylate, potassium polyacrylate, ammonium polyacrylate, starch grafted acrylate and sodium carboxymethyl cellulose. .
In the invention, the carrier is a mixture of diatomite and biomass charcoal, the mass ratio of the diatomite to the biomass charcoal is 1: 0.1-0.5, and the using amount of the carrier is 10-30% of the mass of the fermentation liquor.
In the invention, the nutrient substances comprise 0.1-2% of glucose, 0.05-2% of amino acid powder, 0.5-5% of sodium lignosulfonate, 0.02-0.5% of fluorescent whitening agent, 0.2-2% of polyacrylamide and 0.2-2% of sodium carboxymethylcellulose by mass of the fermentation liquor.
In the present invention, the fluorescent whitening agent is 2,2- (4, 4-distyryl) dibenzoxazole.
In the invention, the fermentation liquor is added with diatomite accounting for 25% of the mass of the fermentation liquor, biomass charcoal accounting for 5%, glucose accounting for 0.5%, amino acid powder accounting for 0.25%, sodium lignosulfonate accounting for 2.5%, fluorescent whitening agent accounting for 0.1%, polyacrylamide accounting for 1.25% and sodium carboxymethylcellulose accounting for 0.75%.
The invention also provides application of the microbial inoculum in the aspects of disease prevention, pesticide residue reduction, rooting and seedling strengthening.
The bacterial agent of the invention takes Bacillus belgii SUNO-18S-36 fermentation liquor with high viable bacteria content and high spore rate as raw materials, and is dried and crushed after adding an adsorption carrier and an auxiliary agent (glucose, amino acid, sodium lignosulfonate, a fluorescent whitening agent, polyacrylamide and sodium carboxymethylcellulose) to obtain the Bacillus belgii SUNO-18S-36 bacterial agent with viable bacteria content not less than 1000 hundred million cfu/g. The addition of the auxiliary agent is beneficial to protecting thalli, accelerating recovery and promoting survival. The microbial inoculum is used for the treatment of seed soaking, seedbed splashing and root irrigation during transplanting. Has the functions of preventing diseases and rooting and strengthening seedlings for crops.
Drawings
FIG. 1 is a photograph of SUNO-18S-36 strain observed with a microscope (1000X).
FIG. 2 colony morphology of SUNO-18S-36 strain.
Detailed Description
The sources of reagents in the examples are as follows:
amino acid powder, purchased from Peng Generation Limited liability company, Mianzhu, Sichuan; sodium lignosulfonate, CAS: 8061-51-6, available from Dalochi chemical industries, Tianjin; 2,2- (4, 4-distyryl) bisbenzoxazole (optical brightener), CAS: 1533-45-5, available from Yino chemical technology, Inc., Guangzhou; polyacrylamide, CAS: 9003-05-8, available from Texas Deshipchemistry Engineers, Inc.; sodium carboxymethylcellulose, CAS: 9004-32-4, available from Qinghui Biotech, Zhejiang.
The solvent used in the medium of the present invention is water. The aeration ratio is the amount of aeration per minute (m)3) Volume of fermentation broth (m)3) The ratio of. The inoculum size is the ratio of the volume of the inoculated seed solution to the volume of the culture medium.
The viable bacteria content in the fermentation liquor adopts a dilution plating method, and is specifically shown in related content in the industry standard NY/T2321-2013.
The method for measuring the spore rate in the fermentation liquor or the suspension of the microbial inoculum comprises the following steps:
(1) and (3) taking the fermentation liquid or the suspension of the microbial inoculum after the fermentation is finished, and subpackaging the fermentation liquid or the suspension of the microbial inoculum in 250mL sterile triangular flasks, wherein each flask is 30mL, and 6 flasks are ready for use.
(2) The temperature of the constant temperature water bath (with oscillation) is set to be 80 +/-1 ℃, and the constant temperature water bath is reserved after the temperature is stable.
(3) Shaking the 6 Erlenmeyer flasks at room temperature to obtain 3 Erlenmeyer flasksOscillating for 20min, diluting and coating on flat plate to determine viable bacteria content, and taking average value as VCK。
(4) Placing the other 3 triangular flasks in 80 deg.C water bath, shaking at constant temperature for 20min (the shaking frequency is the same as that of control treatment at room temperature), taking out, cooling tap water to room temperature, diluting, coating on flat plate to detect viable bacteria content, and taking average value as VTreatment of。
(5) The spore rate was calculated using the following formula: the spore rate is as follows: is equal to VTreatment of/VCK×100％。
The method for measuring the content of the antibacterial active substances in the fermentation liquor comprises the following steps:
(1) activating watermelon fusarium wilt pathogen (F.oxysporum) stored in laboratory on PDA slant culture medium at 28 deg.C for 24 hr (culturing at 28 deg.C); then streaking and inoculating PDA plate culture medium, and culturing at constant temperature of 28 ℃ until spore grows out.
(2) Washing the spores with sterile normal saline to obtain watermelon fusarium wilt pathogen spore liquid, counting the number of the hemocyte plates, and refrigerating the cytopenia liquid in a refrigerator for later use.
(3) Centrifuging fermentation liquor to be tested of SUNO-18S-36 strain, taking supernatant, and passing through a sterile filter membrane of 0.22um to obtain sterile clear liquid for later use.
(4) Diluting pathogenic bacteria spore liquid of watermelon wilt with PDA liquid culture medium to prepare 1 × 105Per mL of watermelon wilt pathogen spore suspension;
(5) taking 15 sterile test tubes (one group of test tubes), arranging in a row, adding 2mL of 1 × 10 concentration into each tube5one/mL of spore suspension of pathogenic bacteria of blight disease.
(6) And adding 2mL of sterile clear liquid obtained after treatment of the fermentation stock solution to be detected into the 1 st tube, uniformly mixing, sucking 2mL of mixed solution into the 2 nd tube, continuously diluting to the 14 th tube in the way, uniformly mixing, sucking 2mL of mixed solution from the 14 th tube, and discarding. Tube 15 served as control. During the dilution process, the pipette is replaced after each 2mL pipette into the next tube.
(7) At this time, the fermentation tubes from 1 st to 14 th are diluted by 2% respectively in comparison with the original fermentation sterilized clear solution1、22、23……214And (4) doubling.
(8) The above experiment was repeated for 3 groups.
(9) The mixed tubes were stoppered and incubated at 28 ℃ for 36 h.
If the growth condition of the pathogenic bacteria of the fusarium wilt of the Chinese and western melons in the control tube is checked to be good, the tube with the largest dilution factor grows through visual observation, the largest dilution factor is the MIC (minimum inhibitory concentration) of the sterile clear liquid of the fermentation liquid, and the quantity of the bacteriostatic active substances in the dilution liquid corresponding to the largest dilution factor is defined as 1 activity unit (1U). The content of the antibacterial active substance in the original sterile clear liquid of the fermentation liquid is the maximum dilution times multiplied by 1U, and the unit is U/ml.
Example 1 Strain screening and identification
Firstly, separating and screening strains.
1. Materials and methods
1.1 sample
2 diseased tomato plants in a common field and 3 rhizosphere soil samples;
2 watermelon field diseased plants in a common field and 3 rhizosphere soil samples;
3 tomato diseased plants and 5 rhizosphere soil samples in the salinized field land;
the total number of samples was 18.
1.2 culture Medium
LB liquid medium: 10g of peptone, 5g of yeast extract and 10g of sodium chloride, dissolving with water, fixing the volume to 1L, and adjusting the pH to 7.0 to obtain the LB liquid culture medium. 15g of agar powder was added to the LB liquid medium to obtain an LB solid medium.
PDA liquid culture medium: adding 800mL of water into 200g of peeled and cut potatoes, heating to boil, maintaining for 20-30 min, filtering while hot by using gauze, and supplementing the volume of the filtrate to 1000mL by using water to obtain the PDA liquid culture medium. 15g of agar powder is added into the PDA liquid culture medium to obtain a PDA solid culture medium.
Phosphate solubilizing plate medium: 10g of glucose, 5g of calcium phosphate, 0.3g of sodium chloride, 0.5g of ammonium sulfate and 15g of agar powder, dissolving the mixture with water, fixing the volume to 1L, and adjusting the pH value to 7.0-7.5.
1.3 Strain isolation
Treating diseased plants: cutting 2 segments of 1cm long stem from the diseased part of each diseased plant with a sterile knife, washing the epidermis with sterile water, placing into a triangular flask filled with 100mL sterile physiological saline (10 sterile glass beads are pre-filled in the triangular flask), shaking the triangular flask at 30 ℃ and 100rpm for 2h at a constant temperature.
Rhizosphere soil sample: after each soil sample is uniformly mixed, 1g of the mixture is put into a triangular flask filled with 100mL of sterile physiological saline (10 sterile glass beads are pre-filled in the triangular flask), and the mixture is shaken for 2 hours at the constant temperature of 30 ℃ and 100rpm by a shaking table.
Taking the supernatant obtained after each diseased plant and the soil sample are subjected to vibration treatment, carrying out continuous gradient dilution, respectively and uniformly coating on an LB solid culture medium flat plate and a PDA solid culture medium flat plate, culturing for 24-36 h in an incubator at constant temperature of 30 ℃, selecting a single bacterial colony which grows and propagates quickly, then respectively carrying out streak separation and purification culture for 2 generations, selecting a purified single bacterial colony, carrying out slant culture and numbering, and storing in a refrigerator for later use.
1.4 bacterial Strain rescreening
1.4.1 phosphate solubilizing bacteria screening
And (3) respectively inoculating the strains preserved in the refrigerator to a phosphate solubilizing plate culture medium (each plate is inoculated with 2 bacteria or 1 fungus), culturing for 48-72 h in an incubator at the constant temperature of 30 ℃, and observing whether a phosphate solubilizing transparent ring appears.
1.4.2 antagonistic screening against plant pathogenic bacteria
And (3) carrying out antagonistic screening on the phytopathogen on the strain with the transparent circle on the phosphate solubilizing culture medium in the step 1.4.1.
1.4.2.1 preparation of microbial samples with phosphate solubilizing function
Inoculating bacteria with phosphate solubilizing function to LB liquid culture medium, culturing for 24h at 30 deg.C and 150rpm shaking table to obtain bacterial suspension, adsorbing the bacterial suspension with sterile filter paper sheet with diameter of 5mm, and air drying in sterile blank plate. And (5) standby.
Inoculating fungi with phosphate solubilizing function to PDA solid culture medium, culturing at 28 deg.C until mycelium covers the whole plate, selecting plate with good growth and uniform mycelium coverage, and punching circular block with diameter (d) of 5mm with sterile punch.
1.4.2.2 antagonism against bacterial pathogens
The antagonistic action of bacteria with the phosphate-solubilizing function on pathogenic bacteria of tomato bacterial wilt and soft rot of Chinese cabbage is examined by adopting a bacteriostatic circle method, which comprises the following specific steps: after the pathogenic bacteria suspension is diluted and coated with LB solid culture medium, a filter paper sheet which adsorbs the bacterial suspension to be detected in 1.4.2.1 is placed, and after the filter paper sheet is cultured for 36 hours in an incubator at the constant temperature of 30 ℃, whether a bacteriostatic zone appears is observed.
Adopting a plate culture method to investigate the antagonistic action of the fungi with the phosphate-solubilizing function on pathogenic bacteria of tomato bacterial wilt and soft rot of Chinese cabbage, and the specific method comprises the following steps: after the pathogenic bacteria suspension is diluted and coated with LB solid culture medium, a prepared bacteria block in 1.4.2.1 is placed in the middle, the bacteria block is placed in a constant temperature incubator at 28 ℃ for culture, and whether a bacteriostasis zone appears or not is observed.
1.4.2.3 antagonistic action against fungal pathogens
The antagonism of bacteria/fungi with the phosphate-solubilizing function to pathogenic bacteria of watermelon fusarium wilt is investigated by a plate confronting culture method, and the specific method comprises the following steps:
preparing a PDA solid culture medium, inoculating watermelon fusarium wilt pathogenic bacteria on one side:
(1) if the antagonism of the bacteria with the phosphate-solubilizing effect is examined, a filter paper sheet which adsorbs the bacterial suspension to be detected in 1.4.2.1 is placed on the other side, and after the filter paper sheet is cultured for 48 hours in an incubator at the constant temperature of 30 ℃, whether the antagonism is generated or not is observed.
(2) If the antagonism of the fungus with phosphate-solubilizing function is examined, the prepared fungus block in 1.4.2.1 is placed on the other side, and cultured in a constant temperature incubator at 28 ℃ to observe whether the antagonism is generated.
2. Results
2.1 isolation of the Strain
A total of 122 strains were isolated, 78 of the bacteria and 44 of the fungi. Numbering said strains, originating from the diseased strain, with a P-prime mark; soil-derived, marked with an S start.
2.2 phosphate solubilizing bacteria screening results
The 122 strains are screened by the phosphate solubilizing plate culture medium in the first step to obtain 14 strains with transparent circles, namely microorganisms with the phosphate solubilizing function. Among them, the bacteria are: p-4, P-7, P-19, S-07, S-11, S-15, S-22, S-25, S-33, S-36 and S-45. The fungi are as follows: p-37, S-58 and S-62.
2.3 antagonistic screening results of plant pathogenic bacteria
And carrying out an antagonistic test on 14 microbial strains with plant pathogenic bacteria. The results show that most strains have no antagonism or weak antagonism or have antagonism only on individual pathogenic bacteria to tomato bacterial wilt pathogenic bacteria, Chinese cabbage soft rot pathogenic bacteria and watermelon fusarium wilt pathogenic bacteria, only S-36 strain has antagonism to the above 3 kinds of pathogenic bacteria, especially has obvious antagonism to tomato bacterial wilt pathogenic bacteria, watermelon fusarium wilt pathogenic bacteria and watermelon fusarium wilt pathogenic bacteria. The S-36 strain was designated as SUNO-18S-36 strain.
The experimental result shows that the SUNO-18S-36 strain not only has obvious bacteriostatic action on plant pathogenic bacteria, but also has the capability of converting insoluble phosphate in soil into soluble phosphorus which is easy to absorb by plants. This not only increases soil fertility, but also improves soil structure and environment. Has great development prospect in agriculture.
Identification of SUNO-18S-36 Strain
Dyeing: gram staining was performed on SUNO-18S-36 strain according to a method conventional in the art, and the results showed that the strain was a gram-positive bacterium.
Morphological characteristics: the cells were cultured on LB plate medium at 30 ℃ for 2 days, and the morphology and color of colonies were observed. Taking a thallus smear, and observing the shape of the thallus after staining. The results were: the bacterial colony is white and opaque, the early bacterial colony is more moist, and the later stage is wrinkled. The thallus is straight or nearly straight rod-shaped, only contains one spore and has motility. As shown in fig. 1 and 2.
The 16S rDNA sequence of SUNO-18S-36 strain was PCR-amplified. The sequencing result is shown in SEQ ID NO. 1. The similarity between the 16S rDNA sequence of the SUNO-18S-36 strain and the Bacillus velezensis CR-502 strain reaches 99.85528% through BLAST analysis, so that the SUNO-18S-36 strain is Bacillus velezensis (Bacillus velezensis).
Bacillus belgii (Bacillus velezensis) SUNO-18S-36 strain was deposited.
The preservation information is as follows:
the name of the depository: china Center for Type Culture Collection (CCTCC).
The classification is named as: bacillus belgii SUNO-18S-36
Bacillus velezensis SUNO-18S-36。
The address of the depository: china, wuhan university.
The preservation date is as follows: 18/1/2021.
The preservation number is: CCTCC NO: m2021099.
Example 2 Properties of Bacillus belgii (Bacillus velezensis) SUNO-18S-36 Strain
Tolerance to acidity or alkalinity and salinity
1. Materials and methods
1.1 activation of SUNO-18S-36 Strain: a part of lawn is picked from a slant of a Bacillus beleisi SUNO-18S-36 strain by using a sterile inoculating needle into a shake flask filled with an LB liquid culture medium (the liquid content in the shake flask is 50mL) and the LB liquid culture medium turns turbid after being cultured for 24 hours at 30 ℃ and 150r/min for standby.
1.2 taking LB culture medium as basic culture medium, respectively adjusting pH with hydrochloric acid or NaOH aqueous solution to: 3.4, 5, 6, 7 (control), 8, 9, 10, 11, in a 250mL Erlenmeyer flask, 50mL of the above pH medium was filled, 3 replicates for each pH experiment. Sterilizing the culture medium with various pH values, cooling to room temperature, adjusting the pH value to a value close to that before sterilization by adding acid or alkali if the pH value changes due to sterilization, and recording the actually measured pH value. Adding 1 mL/bottle of the bacterial solution cultured in the title 1.1 of the embodiment into a sterilized triangular flask with the adjusted pH value, culturing at 30 ℃ and 150r/min for 36h, observing whether the culture medium is turbid, streaking and inoculating an LB solid culture medium, culturing in an incubator at constant temperature of 30 ℃ for 24-36 h, and observing whether viable bacteria grow out.
1.3 taking LB solid culture medium without sodium chloride as basic culture medium, respectively preparing solid culture medium with sodium chloride content of 0%, 1% (contrast), 4%, 8%, 9%, 10%, 10.5%, 11%. On LB solid medium containing sodium chloride of each concentration, SUNO-18S-36 strain was streaked, cultured in a constant temperature 30 ℃ incubator, and it was observed whether viable bacteria grew out.
2. Results and discussion
The culture results are shown in tables 1 and 2.
TABLE 1 Effect of different pH on the growth of SUNO-18S-36 Strain
| Design pH value | 3 | 4 | 5 | 6 | 7 (control) | 8 | 9 | 10 | 11 |
| Actually measuring pH after sterilization and adjustment | 3.03 | 3.98 | 5.09 | 6.02 | 7.03 | 7.95 | 9.06 | 10.03 | 11.0 |
| Whether the culture solution is turbid | - | + | ++ | +++ | +++ | +++ | ++ | + | - |
| Whether the plate has bacteria growing out | - | + | ++ | +++ | +++ | +++ | ++ | + | - |
TABLE 2 Effect of different salinity on the growth of SUNO-18S-36 strains
Remarking: in tables 1 and 2, "+" indicates growth of bacteria, and a larger amount of + indicates a better growth; "-" indicates no growth.
As is clear from Table 1, Bacillus belgii SUNO-18S-36 strain has a wide tolerance range for acid and base, and can grow at a pH of 4 to 10.
As is clear from Table 2, viable bacteria were observed only in the plates with a salt content of 9% or less after 24 hours of culture, and in the plates with a salt content of 10% or 10.5% after 96 hours of culture. However, in 11%, no growth of viable bacteria was observed. Therefore, the Bacillus belgii SUNO-18S-36 strain can resist the salinity of 10.5 percent at most and has very high salinity resistance.
The salt content of the heavy saline-alkali soil is over 0.6 percent, the pH value is over 9.5, and the emergence rate is lower than 50 percent. The Bacillus belgii SUNO-18S-36 strain can grow between the pH4 and the pH10 and can resist the salinity of 10.5 percent at most, so that the Bacillus belgii SUNO-18S-36 strain can survive and play a role in heavy saline-alkali soil.
In addition, the Bacillus belgii SUNO-18S-36 strain has wide tolerance range of pH and salinity, can be applied to high-salt sewage treatment, purifies water quality by aeration and reduces pollution.
Secondly, preparing a liquid microbial inoculum of Bacillus velezensis (Bacillus velezensis) SUNO-18S-36 strain
Activation of Bacillus belgii (Bacillus velezensis) SUNO-18S-36 Strain: a part of lawn is picked by a sterile inoculating needle from a slant of a Bacillus velezensis SUNO-18S-36 strain into a shake flask filled with LB liquid culture medium (the volume of the shake flask is 250mL, and the liquid loading is 50mL), and the seed liquid of the SUNO-18S-36 strain is obtained after the cultivation is carried out for 24 hours at 30 ℃ and 150 r/min.
The fermentation medium contains: peptone 12g/L, yeast powder 4g/L, glucose 50g/L, NaCl 12g/L, MgCl g20.46g/L、KH2PO4 0.7g/L、CaCO3 3g/L，pH7.2。
Placing the fermentation medium in a fermentation tank, wherein the volume of the fermentation tank is 50L, the liquid loading capacity is 35L, and sterilizing.
Inoculating Bacillus velezensis (Bacillus velezensis) SUNO-18S-36 strain seed liquid into a fermentation tank according to the inoculation amount of 1% (V/V), and introducing the seed liquid into the fermentation tank at the air flow rate of 1:1 (V/V)Ventilation volume/min：VLiquid loading amount) And culturing for 36h at the stirring speed of 150r/min and the temperature of 30 ℃ to obtain the liquid microbial inoculum. The content of viable bacteria in the liquid microbial inoculum is 15 multiplied by 108cfu/mL。
In the course of fermentation tank culture, the fermentation culture condition can be properly regulated according to fermentation characteristics of strain, and in order to control its metabolic process, different aeration quantities and stirring rotation speeds can be adopted in different growth stages of strain so as to obtain the viable bacteria content of 1X 107～2×109cfu/mL of liquid bacterial liquid.
Determination of phosphate solubilizing ability of Bacillus velezensis (Bacillus velezensis) SUNO-18S-36 Strain
Activation of Bacillus belgii (Bacillus velezensis) SUNO-18S-36 Strain: a part of lawn is picked by a sterile inoculating needle from a slant of a Bacillus velezensis SUNO-18S-36 strain into a shake flask filled with LB liquid culture medium (the volume of the shake flask is 250mL, and the liquid loading is 50mL), and the seed liquid of the SUNO-18S-36 strain is obtained after the cultivation is carried out for 24 hours at 30 ℃ and 150 r/min.
Phosphate solubilizing liquid culture medium: taking 10g of glucose, 5g of calcium phosphate, 0.3g of sodium chloride and 0.5g of ammonium sulfate, adding water to a constant volume of 1L, and adjusting the pH value to 7.0. The liquid loading amount of a phosphate-solubilizing liquid culture medium in a 250mL triangular flask is 50mL, 2mL of SUNO-18S-36 strain seed liquid is inoculated in each triangular flask, culture is carried out for 72h and 96h respectively under the conditions of 30 ℃ and the stirring speed of 150r/min, fermentation liquid is taken for centrifugation, supernatant liquid is taken, after digestion is carried out by adopting nitric acid-perchloric acid (GB 11893), the content of phosphorus in the supernatant liquid is determined by a molybdenum-antimony colorimetric method, the repetition is carried out for 3 times, and the average value is calculated. The non-inoculated medium was used as a Control (CK).
The phosphorus dissolution rate was calculated by the following formula: the phosphorus dissolution rate is the phosphorus content (mg/L)/total phosphorus content (mg/L) in the supernatant × 100%.
The determination of total phosphorus was carried out according to GB 11893.
The results are shown in Table 3.
TABLE 3 determination of inorganic phosphorus decomposing ability of Bacillus belgii
As can be seen from the table, compared with a control, the phosphorus-solubilizing rate of the Bacillus belgii SUNO-18S-36 strain can reach 62.98% at most, and the phosphorus-solubilizing rate of the control group is only 2.07-2.14%, so that the phosphorus-solubilizing effect of the SUNO-18S-36 strain is obvious.
Therefore, the Bacillus belgii SUNO-18S-36 strain can improve the solubility of insoluble phosphorus salt, so that the content of available phosphorus in soil is increased, the use amount of phosphate fertilizer is reduced, phosphorus elements in the soil can be fully utilized, and the aim of improving the soil is fulfilled.
Degradation of pesticide residue by Bacillus velezensis (Bacillus velezensis) SUNO-18S-36 strain
1. Chemical pesticide:
(1)2.5 percent of high-efficiency cyhalothrin emulsifiable concentrate;
(2) 50% carbendazim suspension;
(3) a41% aqueous solution of glyphosate isopropylamine salt.
2. The test method comprises the following steps:
the chemical pesticide was added to the LB liquid medium according to the dilution concentrations of the three agents used in the field, and the final concentrations were controlled as follows: 2.5% lambda-cyhalothrin emulsifiable concentrate: 25 ppm; 50% carbendazim suspension: 500 ppm; 41% glyphosate isopropylamine salt aqua: 4000ppm (acid content).
The culture medium added with the pesticide is added into each 250ml triangular flask, the liquid loading is 50ml, and the culture medium containing each pesticide is respectively provided with 5 times of repetition. Each flask was inoculated with the Bacillus beijerinckii SUNO-18S-36 strain fermentation broth (15X 10) prepared in the second preparation of the example8cfu/mL)1 mL; the blank was prepared by using LB liquid medium containing the chemical pesticide (final concentration of each pesticide is the same as above) without the inoculum solution, and repeating the steps for 5 times. Continuously culturing for 4 days after inoculation, taking culture solution every day, centrifuging, taking supernatant, and determining the content of effective components of the chemical pesticide by HPLC.
The degradation rate of the chemical pesticide is calculated by adopting the following formula: degradation rate is (1-C)1/Co)×100％。
In the formula: c1: the content of the effective components of the chemical pesticide after the inoculation of the live bacteria for culture; c0: the content of the effective ingredient of the chemical pesticide after blank control culture.
3. Results and discussion
The results are shown in Table 4 below.
TABLE 4 content of chemical pesticides in culture Medium when SUNO-18S-36 strain was cultured for various periods of time
As can be seen from the results in Table 4, after 4 days of culture, the degradation rate of the effective components of the 2.5% lambda-cyhalothrin emulsifiable concentrate can reach 94.65%, the degradation rate of the effective components of the 50% carbendazim suspending agent can reach 92.96%, and the degradation effect is very obvious. The degradation rate of the effective components of the 41 percent glyphosate isopropylamine salt aqueous solution is slightly low, and is only 27.12 percent.
From the data, it is known that Bacillus velezensis SUNO-18S-36 strain is capable of degrading the above 3 kinds of chemical pesticides, and has remarkable effect particularly on lambda-cyhalothrin and carbendazim. Therefore, the Bacillus velezensis SUNO-18S-36 strain is used in the field, so that the residue of the 3 chemical pesticides can be obviously reduced, the pesticide residue in soil and agricultural products is reduced, the healthy and green development of agriculture is facilitated, the food safety is improved, and the health of people is facilitated to be guaranteed.
Inhibition of Bacillus velezensis (Bacillus velezensis) SUNO-18S-36 strain fermentation liquid on watermelon fusarium wilt pathogenic bacteria
The method for measuring the inhibition effect of Bacillus velezensis (Bacillus velezensis) SUNO-18S-36 strain on watermelon Fusarium oxysporum pathogenic bacteria by using a plate confronting culture method comprises the following steps:
(1) PDA plates were prepared.
(2) Inoculating watermelon Fusarium wilt pathogenic bacteria (Fusarium oxysporum) on a PDA (personal digital assistant) plate, culturing at 28 ℃ until hypha covers the whole plate, selecting a plate with good growth and uniform hypha coverage, and punching a circular fungus block with the diameter (d) of 5mm by using a sterile puncher for later use.
(3) Activating Bacillus velezensis (Bacillus velezensis) SUNO-18S-36 strain on LB solid plate, collecting thallus to prepare 1.5 × 108cfu/mL of bacterial suspension, then sucking 2mL of bacterial suspension and uniformly mixing with PDA culture medium (100mL) cooled to about 45 ℃ (without scalding hands), immediately spreading a plate, and cooling and solidifying for later use.
(4) Placing the bacterium block in step (2) in the middle of the solid plate containing SUNO-18S-36 strain prepared in step (3), culturing at 28 deg.C in a constant temperature incubator for 3 days, measuring the diameter of the bacterial colony of pathogenic bacteria, repeating for 3 times, and taking average value (D)Treatment of). Placing a pathogenic bacteria block in the middle of a solid plate without adding Bacillus beleisis SUNO-18S-36 bacteria liquid as a control, culturing at 28 deg.C in a constant temperature incubator for 3 days, measuring the diameter of pathogenic bacteria colony, repeating for 3 times, and taking an average value (D)CK)。
(5) Inhibition rate: (D)CK─DTreatment of)/DCKX 100%, wherein DTreatment ofIs the average diameter of pathogenic bacteria colony on a solid plate containing Bacillus belgii SUNO-18S-36 bacteria liquid, DCKMean diameter of pathogen colonies for control.
The specific results are shown in Table 5 below.
TABLE 5 inhibitory Effect of Bacillus velezensis (Bacillus velezensis) SUNO-18S-36 Strain on pathogenic bacteria
As can be seen from the data in Table 5, Bacillus velezensis SUNO-18S-36 strain has good inhibitory action on pathogenic bacteria of watermelon fusarium wilt and has the potential of being developed into a fungicide for preventing and treating plant diseases.
Example 3 preparation of Bacillus belgii (Bacillus velezensis) SUNO-18S-36 Strain
This example describes the preparation of a Bacillus belgii SUNO-18S-36 strain.
1. Preparation of Bacillus beleisi SUNO-18S-36 strain fermentation liquor with high viable bacteria content and high spore rate
Activation of Bacillus belgii SUNO-18S-36 Strain: from the slant of Bacillus belgii SUNO-18S-36 strain, a part of lawn was picked up with a sterile needle into a shake flask containing LB liquid medium (50 mL in 250mL shake flask), and after 24 hours of culture at 30 ℃ and 150r/min, the LB medium became turbid. And taking the bacterial liquid to an LB solid culture medium for streak culture for 30h to obtain the activated Bacillus belgii SUNO-18S-36 strain.
Preparing a seed solution: the liquid loading of LB liquid medium in a 500mL seed shake flask was 100 mL. And selecting the activated single colony on the LB solid culture medium, inoculating the single colony in a seed shake flask, and culturing the seed shake flask at 30 ℃ and 150r/min for 24h to obtain a seed solution.
The basal medium at the previous stage was charged in a 50L fermenter at a liquid charge of 60%. The early basal medium contains: 5g/L of glucose, 5g/L of corn steep liquor, 8g/L of yeast powder, 7.5g/L of silkworm chrysalis powder, 15g/L of calcium gluconate, 0.5g/L of magnesium sulfate heptahydrate, 5g/L of calcium phosphate and pH 7.2.
Before the inoculation is prepared, the temperature of a fermentation tank is controlled to be 30 ℃, the stirring speed is 250r/min, and the aeration ratio is 1:1, the tank pressure is 0.05MPa, and after stabilization, the Dissolved Oxygen (DO) is calibrated to be 100 percent. 350mL of seed solution of Bacillus subtilis SUNO-18S-36 was inoculated into a fermenter.
In the fermentation process, controlling the aeration ratio to be 1: 1. the stirring speed is 250r/min, the tank pressure is 0.05MPa, and the culture temperature is 30 ℃. In the process of culturing the Bacillus belgii SUNO-18S-36 strain, when DO is greater than 14.5%, feeding the culture medium automatically, and when DO is less than or equal to 14.5%, stopping feeding. Until the feed medium is consumed. After the feed supplement is finished, the fermentation condition is maintained until the fermentation time is 36 hours, and the fermentation is finished.
Wherein the volume of the feed medium is 5L, and the feed medium comprises: 75g/L of corn steep liquor, 36g/L of yeast powder and 0.525g/L of manganese sulfate.
The fermentation broth was treated as follows: introducing the fermentation liquid into coil, heating at 37 deg.C for 5min, heating at 45 deg.C for 10min, and measuring viable bacteria content and spore rate. Heating to obtain viable bacteriaThe content is 446X 108cfu/mL, the spore rate of 96.24 percent and the content of bacteriostatic active substances of 2048U/mL.
2. Preparation of Bacillus velezensis (Bacillus velezensis) SUNO-18S-36 strain microbial inoculum
An adsorption carrier accounting for 30% of the mass of the fermentation broth is added into the fermentation broth of the Bacillus belgii SUNO-18S-36 strain obtained in the title 1 of the example after the coil treatment, and after the mixture is fully adsorbed and stirred uniformly, raw powder of the Bacillus belgii SUNO-18S-36 is obtained after drying. Wherein the adsorption carrier is a mixture of diatomite and biomass charcoal in a mass ratio of 1: 0.2. The moisture content in the raw powder was 8.0%.
EXAMPLE 4 Effect of Water-retaining Agents on Bacillus belgii SUNO-18S-36 in microbial Agents
(1) Effect of Water-loss Agents on viable and spore content in suspensions
To the Bacillus belgii SUNO-18S-36 raw powder prepared in example 3, 0.5% by mass of glucose, 0.25% by mass of amino acid powder, 2.5% by mass of sodium lignosulfonate, and 0.1% by mass of fluorescent whitening agent (2,2- (4, 4-distyryl) dibenzoxazole) were added and mixed uniformly to obtain a semi-finished product.
And adding 2g of diatomite into 100g of the semi-finished product, and uniformly mixing to obtain the microbial inoculum 1.
And adding 2g of polyacrylamide into 100g of the semi-finished product, and uniformly mixing to obtain the microbial inoculum 2.
And adding 2g of sodium carboxymethylcellulose into 100g of the semi-finished product 100g, and uniformly mixing to obtain the microbial inoculum 3.
1.25g of polyacrylamide and 0.75g of sodium carboxymethylcellulose are added into 100g of the semi-finished product and are uniformly mixed to obtain the microbial inoculum 4.
Respectively taking 10g of samples from the 4 bactericides, putting 100mL of sterile water into a sterile triangular flask, performing shake culture at 30 ℃ and 100r/min for 24h to obtain suspension of each bactericide, measuring the viable bacteria content and spore content of the suspension of each bactericide in shake culture for 0h and 24h, and calculating the spore rate. And (5) investigating the influence of substances added into the semi-finished product subsequently on the content of viable bacteria and spore in the suspension. The results are shown in Table 6. Wherein, the viable bacteria content and spore content detection method and the spore rate calculation method are the same as those of the fermentation liquor.
TABLE 64 suspension measurements of the inoculum
As can be seen from the data in Table 6, when cultured in a liquid for 24 hours, polyacrylamide and sodium carboxymethylcellulose have no significant promoting effect on the viable cell content, spore content and spore ratio of Bacillus beijerinckii SUNO-18S-36 strain.
(2) Influence of water-retaining agent on viable bacteria content and spore content in sandy soil environment
Selecting soft sandy soil, drying in the sun, sieving, removing impurities such as stones, rhizomes, large particles and the like, mixing uniformly, and sieving. Each Erlenmeyer flask (20 bottles in total) was filled with 50g of sieved sand, sterilized at 121 ℃ for 30min, left at room temperature for 2 days, and then sterilized at 121 ℃ for 30 min. Drying in an oven to constant weight, cooling, and shaking.
10g of the microbial inoculum 1 is put into a sterile Erlenmeyer flask, 100mL of sterile water is poured into the sterile Erlenmeyer flask, after uniform mixing, 12.5mL of homogeneous suspension is put into the Erlenmeyer flask filled with 50g of sterile sandy soil, after uniform shaking, the Erlenmeyer flask is placed in a constant temperature incubator at 30 ℃ (a fan is started to enhance air circulation), and 5 times of repeated operation are carried out. The microbial inoculum 2, 3 and 4 are also treated by the same method.
Samples were taken at 0d, 1d, 2d, 4d, and 8d of culture (all Erlenmeyer flasks were mixed again before each sampling), and the water content and viable cell content of the samples were measured. Wherein the culture time of 0d is the time when the suspension of the microbial inoculum is inoculated to the sterilized sandy soil.
And (3) measuring the water content of the inoculated sandy soil by each microbial inoculum: a10 g sample (to the nearest 0.01g, noted W) was taken from the sandy soil inoculated with the microbial inoculum0) Putting the dried mixture into a drying dish which is accurately weighed, drying the dried mixture to constant weight, cooling the dried mixture, weighing the dried mixture and recording the weight as W1Calculation method of water contentThe method comprises the following steps: (W)0—W1)/W0×100％。
The viable bacteria content detection method comprises the following steps: after the sandy soil inoculated with the microbial inoculum is uniformly mixed, 10g of the sandy soil is taken to be put into 100mL of sterile water, and after shaking for 30min on a shaking bed at the temperature of 30 ℃ and the rpm of 100, the viable bacteria content in the sandy soil is measured by taking the suspension of the bacteria, and the unit is cfu/g.
The method for calculating the content of the dry viable bacteria comprises the following steps: dry viable bacteria content-measured viable bacteria content/(1-water content).
The survival rate of 8d is equal to the content of live dry bacteria after 8 d/the content of live dry bacteria at 0d multiplied by 100 percent.
The results of the measurements are shown in Table 7 below.
TABLE 7 influence of Water-retaining agent on viable bacteria content of Bacillus belgii SUNO-18S-36 strain
As can be seen from the data in Table 7, the water content of the 3 microbial inoculum (microbial inoculum 2, 3 and 4) samples added with the water retention agent is obviously higher than that of the microbial inoculum 1 within 8 days.
The sand treated by the microbial inoculum 1 has the dry weight viable bacteria content of 26.11 multiplied by 10 from the beginning8cfu/g reduced to 1.42X 10 of 8d8The survival rate of cfu/g, 8d was only 5.44%. The sand treated by the microbial inoculum 2 has the dry weight viable bacteria content of 26.52 multiplied by 10 from the beginning8cfu/g reduced to 6.06X 10 of 8d8cfu/g, survival rate 22.85%. The sand treated by the microbial inoculum 3 has the dry weight viable bacteria content of 27.70 multiplied by 10 from the beginning88.62X 10 cfu/g down to 8d8cfu/g, survival rate 31.12%. The sand treated by the microbial inoculum 4 has the dry weight viable bacteria content of 26.49 multiplied by 10 from the beginning817.08X 10 cfu/g down to 8d8cfu/g, survival rate 64.48%. The dry weight viable bacteria content of the microbial inoculum 4 at 8 days is respectively 12.03 times, 2.82 times and 1.98 times of that of the microbial inoculum 1, 2 and 3, and the survival rates are respectively 1 time, 2 time and 3 time of the microbial inoculum11.85 times, 2.82 times and 2.07 times of the total weight of the composition, and the effect is obvious. As can be seen from the data in Table 7, the result of the microbial inoculum prepared by adding polyacrylamide and sodium carboxymethylcellulose shows that the dry weight viable bacteria content at 8d is obviously superior to that of other microbial inoculants.
Example 5 Effect of directly adding an auxiliary agent to the fermentation broth on the SUNO-18S-36 of Bacillus beijerinckii in the microbial inoculum
This example examined the effect of drying after addition of the auxiliary directly to the fermentation broth on Bacillus belgii SUNO-18S-36.
And (5) microbial inoculum: 250kg of diatomite and 50kg of biomass charcoal were added to 1000kg of the fermentation broth of Bacillus belgii SUNO-18S-36 strain obtained after the coil heat treatment in example 3, and the mixture was stirred uniformly, adsorbed sufficiently, and spray-dried to obtain 312.5kg of fungal powder.
And (6) microbial inoculum: 250kg of diatomite, 50kg of biomass charcoal, 1.56kg of glucose, 0.78kg of amino acid powder, 7.8kg of sodium lignin sulfonate, 0.3125kg of a fluorescent whitening agent (2,2- (4, 4-distyryl) dibenzoxazole), 3.9kg of polyacrylamide and 2.344kg of sodium carboxymethylcellulose are added to 1000kg of the Bacillus belgii SUNO-18S-36 fermentation broth obtained after the coil heating treatment in example 3, and after being uniformly mixed, the mixture is spray-dried to obtain bacterial powder, and then the bacterial powder is jet-pulverized to obtain the bacterial agent 6.
Respectively taking 10g of each microbial inoculum, adding 100mL of sterile water, and carrying out shake culture at 30 ℃ and 100r/min to obtain suspension of each microbial inoculum. Each inoculum was run in 5 replicates. Taking bacterial suspensions (uniformly stirring before sampling) when the bacteria are cultured for 0h, 12h, 24h and 48h respectively by shaking, and determining the viable bacteria content and the spore content in the suspension of the microbial inoculum. Calculating the ratio of spores to nutrients.
The nutrient ratio (viable bacteria content of the microbial agent 6-spore content of the microbial agent 6)/(viable bacteria content of the microbial agent 5-spore content of the microbial agent 5).
The results are shown in Table 8 below.
TABLE 8 Effect of adjuvant addition on Bacillus belgii SUNO-18S-36 survival and Resuscitation
The results in Table 8 show that the viable bacteria content in the suspension is always reduced and the spore rate is always higher without the aid added microbial inoculum 5. The microbial inoculum 6 added with the auxiliary agent has the viable bacteria content gradually increased in the suspension within 24 hours, and the spore rate is only 8.97 percent within 12 hours, namely most of the bacteria exist in the form of reproducible nutrient bodies. At 24h and 48h, the content of viable bacteria in the suspension of the microbial inoculum 6 is respectively 20.22 times and 34.22 times of that of the microbial inoculum 5, and the content of nutrients in the suspension of the microbial inoculum 6 is respectively 51.76 times and 76.92 times of that of the microbial inoculum 5. Namely: the addition of the auxiliary agent accelerates the recovery of Bacillus belgii SUNO-18S-36, and the existence of nutrient substances is favorable for the reproduction of thalli.
According to the method in (2) in example 4, the influence of the auxiliary agent on the viable bacteria content and spore content of the microbial inoculum in a sandy soil environment is examined. The results are shown in Table 9 below.
TABLE 9 investigation of the viability of Bacillus belgii SUNO-18S-36
As shown in Table 9, the survival rate of viable bacteria in sandy soil inoculated with the microbial inoculum 6 is 80.43% in 8d, which is higher than 64.48% in sandy soil inoculated with the microbial inoculum 4 in Table 7. In the sandy soil inoculated by the microbial inoculum 5, the survival rate of viable bacteria of 8d is only 8.57 percent. Namely: the addition agent is directly added into the fermentation liquor, so that the survival rate of the thalli in a sandy soil environment is improved.
The uv resistance comparative test design is set forth in table 10 below.
TABLE 10 sample treatment
A low ozone straight tube ultraviolet lamp (30W, irradiance of 78 μ W/cm2) was placed above the sterile chamber. Opening the ultraviolet lamp tube for 5min, opening the culture dish containing the sample after the ultraviolet lamp tube is stabilized, placing the culture dish under a desktop ultraviolet lamp, irradiating for 10min, and then closing the ultraviolet lamp. And taking a sample which does not irradiate the ultraviolet lamp as a blank Control (CK), measuring the viable bacteria content of the blank control and the viable bacteria content after ultraviolet irradiation, and calculating the survival rate.
The survival rate calculation method is as follows: viable bacteria content after ultraviolet irradiation/viable bacteria content of blank control multiplied by 100%.
TABLE 11 results of measurement of ultraviolet resistance
As can be seen from the data in Table 11, the viable cell survival rates of the bacterial agent 5 and the bacterial suspension thereof after ultraviolet irradiation are respectively 36.29% and 7.95%, and the viable cell survival rates of the bacterial agent 6 are respectively 62.19% and 45.52%. Particularly, for the bacterial suspension, the survival rate of the bacterial agent 6 is 5.73 times of that of the bacterial agent 5.
Example 6 prevention of watermelon wilt disease by Bacillus subtilis SUNO-18S-36 Strain
Control agents: 30% hymexazol aqua.
SUNO-18S-36 microbial inoculum: bacterial preparation 6 in example 5, the content of viable bacteria in the bacterial preparation was measured before the test (1220X 10)8cfu/g)。
Clear water was set as a blank control.
Objects of use and disease control: watermelon fusarium wilt.
The using method comprises the following steps: see table 12 below.
TABLE 12 prevention and cure method of watermelon fusarium wilt by different drugs
| Time of use and method | SUNO-18S-36 microbial inoculum | Control agent |
| For the first time: seed soaking and residual liquid splashing onOn the seedbed | 100g, 100 times diluted | 20mL, 500-fold dilution |
| And (3) second use: irrigating roots during transplanting and field planting | 150g, diluted 400 times | 100mL, 500 times diluted |
| For the third use: irrigating roots 30 days after planting | 200g, 500 times diluted | 200mL, 500 times diluted |
Setting a test cell: the area of a single cell is about 35-50 square meters, 9 cells are arranged in total, 3 test schemes (contrast medicament, microbial inoculum and blank contrast) are adopted, and 3 cells are tested in each test. The treatment methods of the agents are shown in Table 12, and the blank control group is clear water.
Field management: except for different medicaments for preventing and treating the blight, the field management of other water, fertilizers, medicaments and the like is the same. However, if the other agents are strong acids, strong bases or bactericides, it is preferable to use them separately and in a staggered manner, so that the simultaneous use or mixing is avoided.
The main symptom of watermelon wilt in seedling stage is that leaves gradually wilting from the base upwards, which looks like water shortage. In the early stage, the water shortage will wither obviously, and the normal state can be recovered in the morning and at night; after several days, the plant will die slowly without wilting. In many cases, the whole plant is attacked, and some diseased plants are attacked by only partial stem vines. The base of the stem and vine of the diseased plant is slightly atrophied, the diseased part is longitudinally cracked, the colloidal liquid from light red to pink overflows, the root is rotten and discolored, the rhizome is longitudinally cut, and the vascular bundle part is browned.
Investigation method and grading standards: the disease condition is investigated and counted in the whole district of each district by taking the plant as a unit according to the following grading method:
level 0: watermelon plants are healthy and have no disease symptoms;
level 1: slight scab appears on the watermelon plant stem, but the growth is normal;
and 3, level: the diseased spots on the watermelon plant stem are obvious, and the growth is slightly inhibited;
and 5, stage: the disease spots on the stem of the watermelon plant are obvious, the top of the plant is wilted, and the growth is slow or rigid;
and 7, stage: browning and withering less than 50% of watermelon plants;
and 9, stage: over 50% of watermelon plants become brown, withered or withered.
And (4) investigating a result: day 30 after the third dose. Calculating disease index and preventing and treating effect.
The efficacy calculation was performed according to the following formula:
disease index (%) (Σ [ number of diseased plants at each stage × relative stage ])/(× 9 for total investigated plants) × 100
Control effect (%) - (CK disease index-treatment disease index) ÷ CK disease index × 100, where CK is a blank control. The treatment disease index refers to the disease index under the condition of treatment by using a control medicament or SUNO-18S-36 microbial inoculum.
The results are shown in Table 13 below.
TABLE 13 control of watermelon fusarium wilt by different treatments
As can be seen from Table 13, the control of the bacterial agent containing Bacillus belgii SUNO-18S-36 against the watermelon wilt disease can reach 83.42%, which is obviously superior to that of hymexazol by 60.36%.
The microbial inoculum prepared by the invention can be applied to root irrigation in seed soaking, seedbed splashing and transplanting and field planting. Has the functions of preventing diseases and rooting and strengthening seedlings for crops. The microbial inoculum can be developed not only on microbial pesticides, but also on microbial fertilizers.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
SEQUENCE LISTING
<110> Sunong (Guangde) Biotech Co., Ltd
<120> bacterial agent containing Bacillus belgii SUNO-18S-36 and application thereof
<130> 202106033
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1433
<212> DNA
<213> Bacillus belgii (Bacillus velezensis) SUNO-18S-36 Strain
<400> 1
tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60
acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120
atggttgtct gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat 180
ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag 240
ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360
gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata 420
gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540
tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660
tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720
gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780
agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960
tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020
gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080
ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140
tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200
caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260
gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380
cccgaagtcg gtgaggtaac ctttatggag ccagccgccg aagggacaga ggt 1433
Claims (9)
1. A bacterial agent containing Bacillus velezensis (Bacillus velezensis) SUNO-18S-36 strain, which is characterized in that: adding an adsorption carrier and an auxiliary agent into the Bacillus belgii SUNO-18S-36 fermentation liquor, and drying and crushing after adsorption to obtain the solid microbial inoculum. The content of Bacillus belgii SUNO-18S-36 in the microbial inoculum is not less than 1000 hundred million cfu/g.
2. The microbial inoculum of claim 1, wherein: the adsorption carrier is one or a mixture of more than two of kaolin, light calcium carbonate, diatomite, white carbon black and biomass carbon.
3. The microbial inoculum according to claim 1 or 2, wherein the auxiliary agents comprise wetting agents, nutrients, uv protection agents and water retention agents.
4. The microbial inoculum according to claim 3, wherein the wetting agent is selected from one or two of sodium dodecyl benzene sulfonate and sodium lignin sulfonate; the nutrient is selected from one or more of glucose, sucrose, starch, yeast powder, amino acid powder and bean cake powder; the ultraviolet protective agent is selected from one or more of flavone, lecithin, fluorescent whitening agent, Robinia pseudoacacia toxin and hemp seed oil; the water-retaining agent is selected from one or a mixture of more than two of polyacrylamide, sodium polyacrylate, potassium polyacrylate, ammonium polyacrylate, starch grafted acrylate and sodium carboxymethyl cellulose.
5. The microbial inoculum according to claim 4, wherein the carrier is a mixture of diatomite and biomass charcoal, the mass ratio of the diatomite to the biomass charcoal is 1: 0.1-0.5, and the using amount of the carrier is 10-30% of the mass of the fermentation liquor.
6. The microbial inoculum according to claim 5, wherein the nutrient substances comprise 0.1-2% of glucose, 0.05-2% of amino acid powder, 0.5-5% of sodium lignosulfonate, 0.02-0.5% of fluorescent whitening agent, 0.2-2% of polyacrylamide and 0.2-2% of sodium carboxymethylcellulose by mass of the fermentation liquor.
7. The microbial inoculum according to claim 6, wherein the fluorescent whitening agent is 2,2- (4, 4-distyryl) dibenzoxazole.
8. The microbial inoculum according to claim 7, wherein the fermentation liquor is added with 25% of diatomite, 5% of biomass charcoal, 0.5% of glucose, 0.25% of amino acid powder, 2.5% of sodium lignosulfonate, 0.1% of fluorescent whitening agent, 1.25% of polyacrylamide and 0.75% of sodium carboxymethylcellulose.
9. The use of the bacterial agent of claim 1 in preventing diseases, reducing pesticide residues, rooting and strengthening seedlings.
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| CN114891514A (en) * | 2022-04-15 | 2022-08-12 | 西南石油大学 | Shale gas development platform soil reclamation enhancer and preparation method thereof |
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| CN114891514A (en) * | 2022-04-15 | 2022-08-12 | 西南石油大学 | Shale gas development platform soil reclamation enhancer and preparation method thereof |
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