CN113215059B - Bacillus with disease prevention, phosphate dissolution and pesticide residue degradation functions and application thereof - Google Patents
Bacillus with disease prevention, phosphate dissolution and pesticide residue degradation functions and application thereof Download PDFInfo
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- 238000006731 degradation reaction Methods 0.000 title claims description 11
- 229910019142 PO4 Inorganic materials 0.000 title description 17
- 239000010452 phosphate Substances 0.000 title description 17
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 title description 17
- 238000004090 dissolution Methods 0.000 title description 5
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- TVBSSDNEJWXWFP-UHFFFAOYSA-N nitric acid perchloric acid Chemical compound O[N+]([O-])=O.OCl(=O)(=O)=O TVBSSDNEJWXWFP-UHFFFAOYSA-N 0.000 description 1
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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
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2101/00—Agricultural use
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- Virology (AREA)
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- Genetics & Genomics (AREA)
- Pest Control & Pesticides (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Dentistry (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
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- Agronomy & Crop Science (AREA)
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Abstract
The invention provides a bacillus with the functions of preventing diseases, dissolving phosphorus and degrading pesticide residues and application thereof, and relates to the field of microorganisms. Bacillus belgiiBacillus velezensis) SUNO-18S-36 strain with the preservation number of CCTCC NO: m2021099. The microorganism with the functions of dissolving phosphorus, degrading pesticide residues and antagonizing pathogenic bacteria can be developed into a microbial agent, which is not only beneficial to improving the content and the utilization rate of available phosphorus in soil, but also capable of degrading pesticide residues and improving the quality of agricultural products. In addition, the fertilizer also has the effect of preventing diseases for crops, and has very obvious promotion effect on keeping ecological balance, reducing application and improving efficiency and the healthy development of green agriculture.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to bacillus capable of preventing diseases, dissolving phosphorus and degrading pesticide residues and application thereof.
Background
Phosphorus is one of essential elements in the growth and development process of plants, and is extremely easy to fix in soil, so that the effective utilization rate of the phosphorus is reduced. The available phosphorus which can be absorbed and utilized by plants in the soil only accounts for 2% -3% of the total phosphorus, and most of the phosphorus cannot be directly absorbed by the plants and can be finally converted into substances which can be absorbed and utilized by the plants through a series of physical, chemical and biological reactions. The phosphorus-dissolving microorganisms are used for converting phosphorus which is difficult to be absorbed by plants in the soil into a state which is easy to be absorbed and utilized, and the method is an important way for improving the utilization rate of phosphorus elements in the soil at present.
The pesticide applied to the crops is partially attached to the crops, partially scattered in the environment such as soil, atmosphere and water, and part of the pesticide remained in the environment can be absorbed by the plants. The residual pesticide can directly reach the human body and the livestock body through plant fruits or water and atmosphere, or can be finally transferred to the human body and the livestock through the environment and a food chain.
In the prior art, microorganisms which can dissolve phosphorus, can degrade pesticide residues and have antagonistic action on plant pathogenic bacteria are lacked.
Disclosure of Invention
The invention aims to provide a Bacillus subtilis (Bacillus velezensis) and a microbial inoculum thereof, which have the functions of dissolving phosphorus, degrading pesticide residues and antagonizing plant pathogenic bacteria.
The purpose of the invention is realized by adopting the following technical scheme:
a Bacillus velezensis (Bacillus velezensis) SUNO-18S-36 strain with the preservation number of CCTCC NO: m2021099.
The invention also provides a liquid microbial inoculum containing the strain.
In the invention, the Bacillus belgii SUNO-18S-36 strain is activated, inoculated into a culture medium for expansion culture and cultured for 24 to 72 hours at the temperature of between 28 and 32 ℃ to obtain a liquid microbial inoculum.
In the invention, the culture medium contains 10-60 g/L of carbon source, 5-25 g/L of nitrogen source and 0.1-20 g/L of inorganic salt; the carbon source is one or a mixture of two of glucose, starch, rice flour, corn flour, tapioca flour, vinasse and molasses; the nitrogen source is one or a mixture of two of peanut cake powder, soybean meal, peptone, yeast powder, soybean protein powder and silkworm chrysalis powder; the inorganic salt is one or a mixture of two of sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium chloride and calcium carbonate.
The invention also provides application of the Bacillus velezensis SUNO-18S-36 strain in preventing and treating bacterial wilt, soft rot, blight or degrading pesticide residues.
Has the advantages that: the microorganism with the functions of dissolving phosphorus, degrading pesticide residues and antagonizing pathogenic bacteria can be developed into a microbial agent, which is not only beneficial to improving the content and the utilization rate of available phosphorus in soil, but also capable of degrading pesticide residues and improving the quality of agricultural products. In addition, the fertilizer also has the effect of preventing diseases for crops, and has very obvious promotion effect on keeping ecological balance, reducing application and improving efficiency and the healthy development of green agriculture.
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
Example 1 Strain screening
This example describes the isolation and screening of 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 a phosphate solubilizing function to an LB liquid culture medium, culturing for 24h on a shaking table with the temperature of 30 ℃ and the rpm of 150 to obtain a bacterial suspension, adsorbing the bacterial suspension by using a sterile filter paper sheet with the diameter of 5mm, and then placing the sterile filter paper sheet in a sterile blank plate for airing for later use.
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
A plate confronting culture method is used for investigating the antagonistic action of bacteria and fungi with the phosphate solubilizing function on pathogenic bacteria of watermelon fusarium wilt, and the specific method is as follows:
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 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.
Example 2 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 3 tolerance of Bacillus belgii (Bacillus velezensis) SUNO-18S-36 Strain to acidity, 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.
Example 4 preparation of liquid microbial preparation 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.
Example 5 measurement of phosphorus solubilizing ability of Bacillus belgii (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.
Example 6 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).
Adding culture medium added with pesticide into each 250ml triangular flask respectively, and the liquid loading amount is50ml of medium containing each pesticide was set up in 5 replicates. Each flask was inoculated with the Bacillus beijerinckii SUNO-18S-36 strain fermentation broth (15X 10) prepared in example 48cfu/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.
Example 7 inhibitory Effect of Bacillus subtilis SUNO-18S-36 Strain fermentation broth on pathogenic bacteria of watermelon wilt
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.
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> bacillus with disease prevention, phosphate dissolution and pesticide residue degradation functions and application thereof
<130> 202106031
<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 (5)
1. Bacillus belgiiBacillus velezensis) SUNO-18S-36 strain with preservation number of CCTCC NO：M 2021099。
2. A liquid microbial agent comprising the strain of claim 1.
3. The method for preparing a liquid microbial inoculum according to claim 2, which comprises the following steps: activating Bacillus belgii SUNO-18S-36 strain, inoculating the activated Bacillus belgii SUNO-36 strain in a culture medium for expanded culture, and culturing for 24-72 h at 28-32 ℃ to obtain a liquid microbial inoculum.
4. The method according to claim 3, wherein the medium contains 10 to 60g/L of carbon source, 5 to 25g/L of nitrogen source, and 0.1 to 20g/L of inorganic salt; the carbon source is one or a mixture of two of glucose, rice flour, corn flour, tapioca flour, vinasse and molasses; the nitrogen source is one or a mixture of two of peanut cake powder, soybean meal, peptone, yeast powder, soybean protein powder and silkworm chrysalis powder; the inorganic salt is one or a mixture of two of sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, magnesium chloride and calcium carbonate.
5. The Bacillus belgii of claim 1 (b), (c), (d) and d)Bacillus velezensis) The SUNO-18S-36 strain is applied to the prevention and treatment of watermelon fusarium wilt or the degradation of pesticide residues.
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