CN116622581B - Bacillus bailii HM-3 and application thereof - Google Patents
Bacillus bailii HM-3 and application thereof Download PDFInfo
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- CN116622581B CN116622581B CN202310672007.7A CN202310672007A CN116622581B CN 116622581 B CN116622581 B CN 116622581B CN 202310672007 A CN202310672007 A CN 202310672007A CN 116622581 B CN116622581 B CN 116622581B
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 53
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims abstract description 58
- 240000008067 Cucumis sativus Species 0.000 claims abstract description 49
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims abstract description 43
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 claims abstract description 29
- 241000243785 Meloidogyne javanica Species 0.000 claims abstract description 24
- 238000009335 monocropping Methods 0.000 claims abstract description 15
- 241000193744 Bacillus amyloliquefaciens Species 0.000 claims abstract description 4
- 238000004321 preservation Methods 0.000 claims abstract description 4
- 230000001580 bacterial effect Effects 0.000 claims description 19
- 238000000855 fermentation Methods 0.000 claims description 18
- 230000004151 fermentation Effects 0.000 claims description 18
- 241000244206 Nematoda Species 0.000 claims description 14
- 230000000813 microbial effect Effects 0.000 claims description 13
- 235000001231 Streptopus amplexifolius Nutrition 0.000 claims description 11
- 244000305550 Streptopus amplexifolius Species 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 235000009849 Cucumis sativus Nutrition 0.000 claims description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
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- 229910021536 Zeolite Inorganic materials 0.000 claims description 2
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 claims description 2
- 239000010455 vermiculite Substances 0.000 claims description 2
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- 108020004414 DNA Proteins 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241001143352 Meloidogyne Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 229950008167 abamectin Drugs 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
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- RRZXIRBKKLTSOM-XPNPUAGNSA-N avermectin B1a Chemical compound C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 RRZXIRBKKLTSOM-XPNPUAGNSA-N 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
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- SPFMQWBKVUQXJV-BTVCFUMJSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;hydrate Chemical compound O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O SPFMQWBKVUQXJV-BTVCFUMJSA-N 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
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- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
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- 241001052560 Thallis Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
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- 238000007605 air drying Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 201000003984 candidiasis Diseases 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
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- 230000012447 hatching Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
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- 238000002955 isolation Methods 0.000 description 1
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- 230000001418 larval effect Effects 0.000 description 1
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- 231100000225 lethality Toxicity 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
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- 230000000877 morphologic effect Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000008569 process Effects 0.000 description 1
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- 230000001105 regulatory effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000006283 soil fumigant Substances 0.000 description 1
- 244000000034 soilborne pathogen Species 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000003313 weakening effect Effects 0.000 description 1
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- 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
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- 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
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- A—HUMAN NECESSITIES
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- A01P5/00—Nematocides
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- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
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Abstract
The invention relates to bacillus beleiensis HM-3%Bacillus velezensis) The preservation number is CGMCC No.23739, and the strain is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, has higher field planting capability, can effectively prevent and control cucumber root-knot nematodes, and can degrade p-hydroxybenzoic acid. The invention also relates to application of bacillus beleiensis HM-3 in preventing and treating continuous cropping obstacle of cucumber in a facility.
Description
Technical Field
The invention relates to the technical field of biological control, in particular to bacillus bailii HM-3 and application thereof.
Background
Cucumber is one of important vegetables loved by people in China, and is a main cultivation type in the production of facility vegetables. However, with the contradiction between the limited facility planting area and the annual market demand, the continuous cropping phenomenon of cucumbers is very common, and serious continuous cropping obstacles are caused: namely, under the continuous cropping condition, even if normal cultivation management measures are adopted, the phenomena of weakening of growth vigor and reduction of yield and quality of the cucumber cultivated in facilities occur.
In the current production, the important reasons for causing the continuous cropping obstacle of the facility cucumber are as follows: cucumber root knot nematode hazard and cucumber autotoxicity.
Cucumber root-knot nematode mainly damages cucumber roots, and in recent years, the occurrence area and the occurrence degree of cucumber root-knot nematode disease are obviously increased and aggravated, so that the yield loss reaches 30% -50%, even the whole shed and the room are destroyed, and the cucumber root-knot nematode disease becomes one of the most important soil-borne diseases in the north facility cucumber planting. For the prevention and treatment of cucumber root-knot nematode disease, at present, besides adopting soil fumigant treatment or using chemical agents with a line killing effect, in recent years, reports of applying biocontrol bacteria to control cucumber root-knot nematode are also many, and the biocontrol bacteria control technology has the advantages that: the pesticide residue problem and the pesticide resistance problem of the root knot nematode to the pesticide are avoided. Chinese patent CN110172423A discloses Bacillus belicus and application thereof in preventing and controlling root-knot nematodes, and the fermentation broth of Bacillus belicus Bv-25 strain is used for preventing and controlling root-knot nematodes to 86.0%, but the result is not a test result applied in a facility field but a result measured by a potting test, and the result is often quite different from the field practical application.
Cucumber releases autotoxic substances such as benzoic acid substances through root system to produce autotoxic effect. Parahydroxybenzoic acid has been shown to be the autotoxic substance produced by a variety of plants, especially on cucumber, with the highest content of parahydroxybenzoic acid in root secretions and the strongest autotoxicity.
At present, only a few biocontrol strains are producible as commercial preparations. The method is characterized in that most of the biocontrol bacteria are easy to be interfered by natural environment condition factors when actually applied in fields, the colonization efficiency is low, and the effective viable bacteria number is not up to standard and is unstable. The primary condition of the biological control bacteria capable of exerting the effect of preventing and controlling diseases is whether the biological control bacteria can effectively colonize the rhizosphere, and the colonization capacity of the biological control bacteria at the rhizosphere determines the biological control capacity of the biological control bacteria, so that whether the biological control bacteria effectively colonize the root of plants is an important factor of whether the biological control bacteria play a role in stabilizing and lasting disease prevention effect, is also an important index for screening and evaluating the biological control bacteria of soil-borne diseases, and finally determines whether the biological control bacteria have industrialization prospect.
Under the large environment of agricultural weight-losing and drug-reducing, biological control has become an important means for reducing continuous cropping obstacle gradually. In terms of the production of the facility cucumber, the existing biocontrol bacteria preparation has poor effect of preventing and controlling the continuous cropping obstacle of the facility cucumber, because the existing biocontrol bacteria does not have the performance of antagonizing root-knot nematodes and degrading autotoxic substances, and the colonization capability of many biocontrol bacteria is poor.
Disclosure of Invention
The invention aims to provide bacillus beljalis HM-3 which has higher field planting capability, can effectively prevent and control cucumber root-knot nematodes and can degrade p-hydroxybenzoic acid, and application thereof in preventing and controlling facility cucumber continuous cropping obstacles.
The invention adopts the following technical scheme:
bacillus bailii HM-3%Bacillus velezensis) The microbial strain is preserved in the China general microbiological culture Collection center (CGMCC) with the address of China institute of microbiology (CGMCC No. 23739) with the preservation number of 2021, 11 months and 08 days.
Further, the strain has the capacity of degrading p-hydroxybenzoic acid.
Furthermore, the strain can antagonize cucumber root-knot nematode.
Furthermore, the strain has the functions of antagonizing cucumber root-knot nematode and degrading p-hydroxybenzoic acid.
A microbial agent comprising the bacterial body, spores, fermentation liquor and/or extracellular metabolites of bacillus bailii HM-3.
The microbial agent contains bacillus bailii HM-3 with viable count not less than 10 10 CFU/g。
The microbial agent also comprises diatomite, zeolite, vermiculite, active carbon and biochar.
The preparation method of the microbial agent comprises the following steps:
(1) Preparing an LB liquid culture medium;
(2) Picking bacillus beleiensis HM-3 colony, and carrying out shaking culture at a constant temperature of 200rpm and 30 ℃ for 24 hours in an LB liquid culture medium for activation;
(3) Transferring the activated bacillus bailii HM-3 bacterial liquid into an LB liquid culture medium for continuous culture, and carrying out constant-temperature shaking culture at 200rpm and 30 ℃ for 24 hours to obtain seed liquid;
(4) Inoculating the seed solution into LB liquid culture medium, continuously fermenting, and performing shaking culture at constant temperature of 30 deg.C and 200rpm for 48 hr to obtain fermentation broth, wherein the effective viable count in the fermentation broth is not less than 10 9 CFU/mL；
(5) Uniformly mixing the fermentation liquor and diatomite according to the mass ratio of 10:1, spraying by a freeze dryer to obtain bacillus belicus powder,the number of effective viable bacteria is not less than 10 10 CFU/g。
Application of Bacillus bailii HM-3 in biological control is provided.
Application of Bacillus belicus HM-3 in biological control of cucumber root knot nematode is provided.
An application of the bacillus beleiensis HM-3 in preventing and treating continuous cropping obstacle of cucumber in a facility.
The invention has the beneficial effects that: the bacillus bailii HM-3 has higher colonization capability and better degradation effect on autotoxic substances such as p-hydroxybenzoic acid. The microbial agent prepared by using bacillus belicus HM-3 can effectively control the root-knot nematodes of the cucumber in the facility, the control effect reaches 80.42%, the control effect on the continuous cropping obstacle of the cucumber in a field application test is good, and the cucumber yield is obviously increased.
Drawings
FIG. 1 shows the colony morphology of Bacillus belicus HM-3 cultured 24h on solid LB medium.
FIG. 2 shows the form of Bacillus bailii HM-3 after gram staining under an optical microscope.
FIG. 3 is a phylogenetic tree of Bacillus belay HM-3 constructed based on 16S rDNA.
FIG. 4 shows the poisoning effect of Bacillus belicus HM-3 fermentation broth on root knot nematode.
Detailed Description
The invention is further described below with reference to examples and figures. The scope of the invention is not limited to the examples, and any modifications within the scope of the claims are within the scope of the invention.
The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
EXAMPLE 1 isolation, purification and Primary screening of strains
Soil samples are collected from the cucumber rhizosphere soil of a place where cucumber root knot nematode disease occurs in a greenhouse where cucumbers are planted in Changli county, hebei province, a total of 6 parts are taken, 100g of each part is taken, and the collected 6 parts of soil are uniformly mixed.
Adding 10g of mixed soil sample into 100mL of distilled water, stirring uniformly, standing for 1min, taking supernatant, heating the supernatant in a water bath kettle at 80 ℃ for 30min, and fully stirring the supernatant in the water bath process to uniformly heat the supernatant. After the heating in the water bath was completed, the supernatant was cooled to room temperature, shaken well, and 1mL of the supernatant was taken and diluted 100-fold with sterilized water. And then 0.1mL of diluted clear liquid is coated on an LB plate and uniformly coated, and the plate is placed in a constant temperature incubator at 30 ℃ for 2-4 d, and the growth condition of a bacterial colony is observed at regular time.
After bacterial colonies are grown, 3 times of purification are carried out on LB culture medium, and finally 39 strains are selected and numbered respectively for the next screening.
The strains with different numbers are transferred into a basic culture medium added with 1.0g/L of parahydroxybenzoic acid, and after shaking culture for 3 days at 35 ℃ and 180rpm, 1 strain HM-3 with the best degradation effect on parahydroxybenzoic acid is selected. The basic culture medium comprises the following formula: 5g of peptone, 0.5g of ammonium sulfate, 0.1g of magnesium sulfate, 0.2g of potassium chloride, 0.5g of sodium chloride, 1000mL of water, pH 7.0 and sterilizing at 121 ℃ for 30 minutes.
Example 2 identification of Strain HM-3
(1) Microbiological characteristics
After culturing strain HM-3 on LB plate at 30℃for 3 days, single colony of the strain was milky white, rough in surface and irregular in edge as shown in FIG. 1. Under an optical microscope, the thallus is in a rod shape, the size of the thallus is 0.5 mu m x (1.5-3.5 mu m), the thallus is polymerized together to form short chains or candidiasis arrangement, spores are elliptical, mesogenesis is realized, and the tail end of the thallus expands to form sporangia; gram staining appeared positive as in figure 2.
Morphological identification is carried out on the strain according to the ' Berger's bacteria identification handbook ', and the strain HM-3 is primarily judged to belong to bacillus berryis @Bacillus velezensis）。
(2) Physiological and biochemical characteristics
The physiological and biochemical characteristics of strain HM-3 were evaluated and the results are shown in Table 1.
TABLE 1 physiological and biochemical characterization of strain HM-3
。
(3) Molecular biological Properties
Extracting genome DNA of strain HM-3, using the genome DNA as template, using bacterial 16S rDNA universal primer to make PCR amplification, recovering amplified product and making sequencing so as to obtain the DNA sequence containing 1432 bp as shown in SEQ ID No. 1.
The sequencing result is input into a GeneBank database for BLAST comparison analysis, the NCBI database compares the 16S rDNA sequence, the bacterial belongs to Bacillus bailii on the molecular level, and the result of constructing a phylogenetic tree is shown in figure 3. By homologous comparison, the identity between HM-3 and Bacillus bailii was found to be 99%. Based on strain morphology, sequencing analysis and physiological and biochemical results, HM-3 was identified as Bacillus bailii.
Bacillus behenii HM-3%Bacillus velezensis) The microbial strain is stored in China general microbiological culture Collection center, address: the Beijing city, the Korean district, the North Chen Xiyu No.1, 3, the national academy of sciences microbiological institute, the mail code 100101, the preservation number of the strain CGMCC No.23739.
EXAMPLE 3 Bacillus bailii HM-3 degradation of parahydroxybenzoic acid
Preparation of bacterial suspension: taking the inclined planes of different preserved strains, inoculating colonies into LB liquid medium, placing a constant-temperature shaking table 180r/min for shaking culture for 24 hours, centrifuging bacterial liquid for 10min at 8000r/min, pouring out supernatant, collecting thalli, and re-suspending with sterile water for later use.
Determination of the degradation rate of parahydroxybenzoic acid: taking 2mL of the prepared bacterial suspension, inoculating into 98mL of a liquid culture medium containing 1.0g/L of parahydroxybenzoic acid, shaking and culturing for 3 days at a temperature of 180r/min and 30 ℃ by a shaking table, taking parahydroxybenzoic acid degradation solutions of different strains, centrifuging for 10min at 8000r/min, filtering by a 0.22 mu m filter membrane to obtain clear liquid, taking 98mL of the liquid culture medium containing 2mL of sterile water as a blank control, measuring the content of parahydroxybenzoic acid by an ultraviolet spectrophotometry, calculating the concentration of parahydroxybenzoic acid according to a standard curve, and calculating the degradation rate of the strain on the parahydroxybenzoic acid according to a degradation rate formula.
The formula for calculating the degradation rate of the p-hydroxybenzoic acid is as follows:
p-hydroxybenzoic acid degradation rate (%) = (C) ck -C t ）÷C ck ×100
Wherein C is ck For the control of the content of parahydroxybenzoic acid, C t Is the content of parahydroxybenzoic acid in the treatment.
Standard curve: preparing 100mL of inorganic salt liquid culture medium (0, 0.01, 0.02, 0.03, 0.04, 0.05 mg/mL) containing different concentrations of parahydroxybenzoic acid, and measuring OD values corresponding to the different concentrations of parahydroxybenzoic acid at 247nm wavelength by using an ultraviolet spectrophotometer to obtain a parahydroxybenzoic acid standard curve y=42.15x+0.1161, R 2 = 0.9988 where x is the concentration of p-hydroxybenzoic acid and y is the corresponding OD at this concentration 247 Values.
Table 2 7 absorbance (OD) of bacterial fermentation broth 247 ) Degradation rate calculation result
。
EXAMPLE 4 potted test for determining the ability of Bacillus bailii HM-3 to colonize
5 bacterial strains XJ-16, HG3-X4, HG4-X1, HG1-Z1 and HG4-Z2 which have been used for practical production and are better performing are selected for comparison, and the colonization ability of Bacillus bailii HM-3 is evaluated by a potting test.
(1) Taking soil for potting test from a test base, which is tidal brown soil, and sterilizing at high temperature after air drying to prepare sterile soil;
(2) Bacterial strains XJ-16, HG3-X4, HG4-X1, HG1-Z1, HG4-Z2 and HM-3 are respectively inoculated in LB liquid culture medium, the temperature is 30 ℃, the rotating speed is 160r/min, and the culture is carried out for 48 hours, thus obtaining fermentation liquor, and the fermentation liquor is prepared into bacteriaThe amount is 1.0X10 9 CFU/mL of bacterial suspension;
(3) Mixing sterile soil and solid fermentation culture medium at a ratio of 4:1, placing into flowerpot with diameter of 10cm and height of 11cm to make total volume of the mixture reach 150 cm 3 ；
(4) Pouring 200mL of prepared bacterial suspension, placing the flowerpot into a tray filled with water, sucking the flowerpot to the surface soil through a hole at the bottom of the flowerpot to be wet, removing the water in the tray, placing the flowerpot in a greenhouse, sampling 1g of the soil at a position of 6cm after 15d at the temperature of 20-28 ℃, and detecting the number of effective viable bacteria, wherein each bacterial strain is repeated 5 times. Water was poured every 4d a while.
The solid fermentation medium: bran, chaff, 3% glucose water=7:3:3, urea 2.16%, monopotassium phosphate 2.77%, bagging after uniform mixing, sterilizing at 121 ℃ under high pressure and moist heat for 60 min, and sterilizing for 2 times intermittently.
The number of colonizations and the rate of colonization of each strain per gram of soil were examined. Colonisation rate (%) = colonisation number/(number of vaccinations x 100) the results are shown in table 3.
TABLE 3 results of determination of the colonization rate of the strains
。
As can be seen from Table 3, bacillus belicus HM-3 has excellent colonization ability.
EXAMPLE 5 determination of mortality of Bacillus bailii strain HM-3 to Meloidogyne larvae
(1) Separation of cucumber root knot line worm eggs and culture of second-instar larvae
Collecting cucumber root systems with serious disease in later stage of greenhouse cucumber, washing root soil with clean water, cutting root with a large number of root knots into root segments with length of 1cm, placing the root segments into a triangular flask containing 200mL of 0.5% NaClO solution, sterilizing the surface for 3 min, fully stirring, sequentially passing the stirred suspension through sterile 100-mesh, 300-mesh and 500-mesh sieve, fully washing oversize with sterile water, and finally washing the filter materials on the 500-mesh sieve into a centrifuge tube for centrifugation to obtain egg suspension.
Placing the collected eggs on neutral filter paper, placing on a 200-mesh sieve, placing the sieve in a culture dish containing sterile water, wherein the water level is proper to just wet the filter paper, then placing in a constant temperature box at 28 ℃ for hatching, passing water in the culture dish through a 500-mesh sieve every 24h, collecting the hatched second-instar larvae in a triangular flask, and placing in a refrigerator at 4 ℃ for standby.
(2) Preparation of a sterile fermentation broth of Bacillus belicus strain HM-3: the Bacillus bailii HM-3 fermentation broth obtained in the step (2) of example 4 was centrifuged at 8000r/min for 10min and filtered with a 0.22 μm filter membrane to obtain a sterile supernatant.
(3) Determination of larval mortality of Meloidogyne
The standby nematode larva suspension is taken out and diluted into 1000 nematode larva suspensions per mL, then 0.5mL of bacillus beljalis strain HM-3 sterile clear solution and 0.1mL of suspension are evenly mixed in a 24-well plate, the mixture is placed in a constant temperature incubator at 28 ℃, the number of larva deaths is counted after 24 hours (as shown in figure 4), and the larva death rate is calculated. Mortality (%) = (Ca-Ta)/(ca×100), ca represents the number of non-dead nematodes in the control, ta represents the number of non-dead nematodes in the treatment, the test was repeated 5 times, and the results were averaged, as shown in table 4.
Table 4 HM-3 determination of lethality of sterile supernatants to root knot nematode larvae
。
EXAMPLE 6 preparation of microbial agent
The preparation method of the bacillus belicus HM-3 microbial inoculum by taking diatomite as a carrier comprises the following specific steps:
(1) Preparation of LB liquid Medium: 3g of beef extract, 10g of peptone and 5g of sodium chloride are placed in a 1000mL beaker, 900mL of distilled water is added for heating and dissolving, the pH value is regulated to 7.2-7.4, distilled water is used for fixing the volume to 1L, and sterilization is carried out for 30 minutes at 121 ℃ for standby.
(2) Activating the strain: selecting a loop of bacterial strain HM-3 colony, inoculating the bacterial strain HM-3 colony into a 150mL triangular flask filled with 50mL of LB liquid medium, and performing constant-temperature shaking culture at 160rpm and 37 ℃ for 24 hours for activation;
(3) Preparing seed liquid: inoculating 4mL of activated bacterial liquid into a 1000mL triangular flask filled with 200mL of LB liquid medium, and carrying out constant-temperature shaking culture at 160rpm and 37 ℃ for 24 hours to obtain seed liquid;
(4) Preparation of fermentation liquor: 180mL of the prepared seed solution is inoculated into a 6L small fermentation tank filled with 3.5L of LB liquid medium, and is subjected to constant temperature shaking culture at 160rpm and 37 ℃ for 48 hours to obtain HM-3 fermentation broth with effective viable count of 2.26 multiplied by 10 9 CFU/mL；
(5) Uniformly mixing the fermentation liquor and diatomite according to the mass ratio of 10:1, spraying by a freeze dryer to obtain bacillus belicus powder, and detecting that the effective viable count is 4.12 multiplied by 10 10 CFU/g。
EXAMPLE 7 field application test of Bacillus bailii HM-3 inoculant
Test varieties: tian Jiao No. five.
Test time: 9/5/2022 to 12/31/2022.
Arranging the stubble: the investigation and statistics of all test data of autumn and winter stubble (8 months, 10 days of seedling culture, 9 months, 5 days of field planting, 10 months, 20 days of market starting) are finished before 31 days of 12 months of 2022.
Test site: greenhouse for continuously planting cucumbers for 6 years by a certain farmer in a new village in Changli county in Hebei province, and greenhouse for continuously planting cucumbers for 8 years by a Zhuang Cunmou farmer in Jing' an Guangxi county in Hebei province. Because the soil conditions, the planting years and the like are different, the content of nematodes and parahydroxybenzoic acid in each g of soil in the greenhouse at two places is different, but the application tests are respectively carried out at the two places according to the same test scheme.
Treatment group: the treatment group comprises 1 treatment, 35kg of the bacillus belicus HM-3 microbial inoculum prepared in the example 6 is mixed with soil according to the application amount of 10 per mu kg, holes are punched by a puncher before transplanting and field planting of cucumber seedlings, the depth of each hole is 10cm, and other cultivation management measures are implemented according to the technical specifications of the production of cucumber in a local greenhouse.
Control group (CK): the control group had 2 treatments, CK1, CK2. The specific cultivation management measures of CK1 are: except that lime nitrogen or wilfory are not used for disinfecting soil, other cultivation management measures are implemented according to the technical specifications of the Changli local greenhouse cucumber production. Cucumber is planted in the greenhouse in Changli area, and the soil is generally disinfected by lime nitrogen or wilms. As lime nitrogen is applied to disinfect soil for 15-20 days, and measures such as covering film are also required for applying the wilfordii, the fact that high-temperature covering film or covering film measures are adopted for only a few areas in a greenhouse is difficult to realize in actual production. In addition, nematodes and other soil-borne pathogens can be transmitted through agricultural implements, soles and the like to influence the test effect when entering and exiting the greenhouse frequently. Therefore, the specific cultivation management measures for the control group CK1 treatment were: except that lime nitrogen or wilfory is not used for disinfecting soil, other cultivation management measures are implemented according to the technical specifications of the production of the cucumbers in the local greenhouse in the south of paris or 28390 mu. The CK2 treatment of the control group is avermectin granules with the active ingredient content of 0.5 percent, and 3500g of avermectin granules are applied per mu by a hole application method.
Each treatment of the treatment group and the control group of the present embodiment was repeated 3 times, 1 treatment of the treatment group, 2 treatments of the control group, all treatments were arranged randomly, and the cell area of each treatment was 20m 2 。
Disease grade grading criteria for cucumber root knot nematode disease: the level 0 is that the root system is complete and no root knot exists; the level 1 is that a small number of root knots exist, and the root necrosis is less than 25%; the level 2 is that the root knot number accounts for 26% -50% of the root coefficient; the 3 level is that the root knot number accounts for 51% -75% of the root coefficient; the 4-level is more and larger in root knot, and accounts for 76% -100% of the root coefficient.
The investigation method comprises the following steps: and after the work of investigating and counting cucumber yield is finished, investigating the disease condition of cucumber root-knot nematode at 2022, 12 and 31 days. The surface soil of the rhizosphere is scraped by a tool, and the disease condition of root knot nematodes at the root is observed. And recording the number of the plants suffering from the root knot nematode disease according to the grading standard, and comparing the number with a control CK to determine the disease index and the control effect.
The calculation formula is as follows:
plant incidence (%) = (number of affected plants/total number of investigated plants) ×100
Plant disease index (%) = [ Σ (number of disease plants at each stage x number of corresponding stages)/total number of investigation x highest-grade value ] ×100
Relative control effect (%) = (CK 1 region disease index-treatment region disease index)/CK 1 region disease index×100
Total yield: beginning at 10 at 2022 and ending at 10 and 31 at 12.
The test results are shown in tables 5 and 6. The results in Table 5 clearly show that the biocontrol microbial inoculum prepared by using Bacillus belicus HM-3 can effectively reduce the incidence rate of cucumber nematode disease and reduce the disease index. Wherein the treatment group (the effective viable count in the biocontrol microbial agent is 2.0X10) 10 CFU/mL) is 88.73%, which is significantly higher than the control effect of 63.25% of cucumber root-knot nematode by treatment with abamectin granules, and the disease index of the root-knot nematode is significantly reduced. Meanwhile, the bacillus beljalis HM-3 in the treatment group has better degradation effect on the parahydroxybenzoic acid and other autotoxic substances in the cucumber root secretion, so that adverse factors in the continuous cropping planting of the cucumber are reduced, and the cucumber yield is obviously increased.
TABLE 5 field application test results of different treatments for controlling continuous cropping obstacle of cucumber in a facility (Xiao Ying village)
。
TABLE 6 field application test results of different treatments in the control of facility cucumber continuous cropping obstacle (Western Zhuang Cun)
。
The results in Table 6 clearly show that the biocontrol microbial inoculum prepared by using Bacillus belicus HM-3 can effectively reduce the incidence rate of cucumber nematode disease, reduce disease index and increase melon yield. Wherein the treatment group (the effective viable count in the biocontrol microbial agent is 2.0X10) 10 CFU/mL) is 80.42%, which is significantly higher than 57.98% of the prevention effect of abamectin granules on cucumber root-knot nematode, and the disease index of the root-knot nematode is significantly reduced. Similarly, bacillus bailii HM-3 has an effect on autotoxic substances such as p-hydroxybenzoic acid in cucumber root secretionBetter degradation effect, further improves the cucumber yield and effectively prevents and treats the continuous cropping obstacle of the cucumber.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention. For example, the preparation of the bacillus beijerinckii HM-3 microbial inoculum by taking activated carbon as a carrier can also be carried out by using other common components in the field.
Claims (9)
1. Bacillus bailii HM-3%Bacillus velezensis) The method is characterized in that the preservation number is CGMCC No.23739.
2. Bacillus beleimeris HM-3 as claimed in claim 1, characterised in that it is degradable to p-hydroxybenzoic acid.
3. Bacillus beleimeris HM-3 as claimed in claim 1, characterised in that it antagonises cucumber root knot nematodes.
4. Bacillus beleimeris HM-3 as claimed in claim 1, characterised in that it has both antagonising cucumber root knot nematodes and degrading p-hydroxybenzoic acid.
5. A microbial agent comprising the bacterial cells, spores and/or fermentation broth of bacillus beleiensis HM-3 according to claim 1.
6. The microbial agent according to claim 5, which is characterized in that the viable count of the bacillus beleiensis HM-3 is not less than 10 10 CFU/g。
7. The microbial agent of claim 5, further comprising diatomaceous earth, zeolite, vermiculite, activated carbon, or biochar.
8. Use of bacillus beljalis HM-3 according to claim 1 for biological control of cucumber root-knot nematodes.
9. Use of bacillus belgium HM-3 according to claim 1 for controlling continuous cropping disorders of greenhouse cucumbers.
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