CN115161240A - Bacillus velezensis strain and application thereof - Google Patents
Bacillus velezensis strain and application thereof Download PDFInfo
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- CN115161240A CN115161240A CN202210875837.5A CN202210875837A CN115161240A CN 115161240 A CN115161240 A CN 115161240A CN 202210875837 A CN202210875837 A CN 202210875837A CN 115161240 A CN115161240 A CN 115161240A
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- 241000193744 Bacillus amyloliquefaciens Species 0.000 title claims abstract description 37
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- 244000005700 microbiome Species 0.000 claims abstract description 6
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- 241000894006 Bacteria Species 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000000642 acaricide Substances 0.000 claims description 7
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- UOAMTSKGCBMZTC-UHFFFAOYSA-N dicofol Chemical compound C=1C=C(Cl)C=CC=1C(C(Cl)(Cl)Cl)(O)C1=CC=C(Cl)C=C1 UOAMTSKGCBMZTC-UHFFFAOYSA-N 0.000 description 1
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- YYJNOYZRYGDPNH-MFKUBSTISA-N fenpyroximate Chemical compound C=1C=C(C(=O)OC(C)(C)C)C=CC=1CO/N=C/C=1C(C)=NN(C)C=1OC1=CC=CC=C1 YYJNOYZRYGDPNH-MFKUBSTISA-N 0.000 description 1
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- 239000002581 neurotoxin Substances 0.000 description 1
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- 229930000223 plant secondary metabolite Natural products 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
- 230000002265 prevention Effects 0.000 description 1
- ZYHMJXZULPZUED-UHFFFAOYSA-N propargite Chemical compound C1=CC(C(C)(C)C)=CC=C1OC1C(OS(=O)OCC#C)CCCC1 ZYHMJXZULPZUED-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- C12N1/205—Bacterial isolates
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
- A01P7/02—Acaricides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
Abstract
The invention discloses a bacillus velezensis strain and application thereof, wherein the bacillus velezensis strain is preserved in common microorganisms of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.24996. In indoor bioassay experiments, the acaricidal effect reaches 96%.
Description
Technical Field
The invention relates to the field of agriculture, in particular to a bacillus velezensis strain and application thereof.
Background
Among the current agricultural pest control, phytophagous mites are one of the most difficult pest groups recognized in the world, the main group of which is spider mites, commonly known as red spiders, belonging to the phylum Arthropoda (arthopoda), arachnida (Arachnida), acarina (Acari), euacarina (Acariformes), spider mite superfamily (Tetranychoidea). Tetranychus urticae is a worldwide harmful mite, is extremely easy to generate drug resistance and extremely difficult to control due to small size, short generation, short development period, quick propagation, strong adaptability and high mutation rate, so that the growing situation of the small mites causing the crop mites to become increasingly harmful is caused, the agricultural production is greatly lost, the agricultural production increase and the farmer income increase are influenced, and the Tetranychus urticae is an important major problem to be solved in the agricultural production at present.
Chemical control is still the main strategy for controlling mite damage in the production of horticultural crops in China, but the abuse and misuse of acaricides easily cause the generation of drug resistance of tetranychus urticae, and the field control effect of chemical pesticides is greatly reduced. Statistics in 2008 show that spider mites have drug resistance to 92 active ingredients in the current medicament, including neurotoxin insecticides such as organic 24976. The drug resistance of the tetranychus urticae is much more serious than that of other field pests such as diamondback moth, aphid and the like, so that the tetranychus urticae is regarded as the most drug resistant species by scientific personnel. The drug resistance of Tetranychus Urticae Koch is higher than that of other mites on greenhouse vegetables and open fruit trees. For example, the resistance times of the tetranychus urticae to abamectin, dicofol, propargite and the like are more than 10 times of those of other mites. The whole genome DNA research of two-spotted spider mite found that its internal detoxification gene was 3 times as many as other animals, and contained 39 genes of a drug resistance gene family, while the number of insects and vertebrates was only 14. Therefore, development of control strategies based on biological control is an important approach to sustainable control.
Among various control means of pest mites, biological control has been a focus and hot spot of research, playing an important role in pest control strategies. At present, the biological control research on the aspects of pest mite natural enemies, botanical pesticides, tetranychus urticae and the like has made some progress and results. The natural enemies of the tetranychus urticae are various, phytoseiidae and predatory mite are two types which are more researched and applied, and particularly, the phytoseiidae is industrially produced on a large scale in many countries. The netherlands have 60% of greenhouses that use phytoseiid persimmons to control tetranychus urticae on cucumbers, whereas british, finland, sweden and danish have exceeded 70%. The plant protection institute of agricultural academy of sciences of Fujian province initially establishes the first mass breeding base of predatory mites in China, and the popularization and application area of the plant protection base in China reaches 13.3 ten thousand hectares. However, the use of natural enemies is limited in application and development because of the disadvantages that dominant populations are not easy to establish, the cost for maintaining the natural enemy populations is high, the chemical control cannot be carried out together with the chemical control, and the like. The active ingredients of the botanical pesticide are safe and low in toxicity, most of the botanical pesticide is a plant secondary metabolite with acaricidal activity, more than 200 plants which are measured for the acaricidal activity are reported, but the active ingredients with low activity are not clear, so that the botanical pesticide has little development and utilization value.
Arthropods are widely distributed with various cytoplasmic-inherited intracellular symbionts, which are classified as primary symbionts (primary sympositions) and secondary symbionts (secondary sympositions). The nascent symbiotic bacteria are necessary for the survival of the host, are co-evolved with the host, are widely distributed in the histiocyte of the host, are mainly vertically transmitted through a mother system, and can be horizontally transmitted at the same time. Secondary symbionts are not essential for host survival, may be beneficial or harmful to the host, and harmful may be referred to as spider mite pathogens. The tetranychus pathogenic bacteria and tetranychus urticae have long evolutionary processes, and interdependence and restriction relationships are formed between the tetranychus pathogenic bacteria and the tetranychus urticae. How to utilize the pathogenic bacteria and bioactive substances thereof has become a main attack direction of many laboratories in developed countries in recent years.
Tetranychus urticae is relatively few in pathogenic bacteria in nature, and is recorded mainly by Wollbachia (Wollachia), and is originally found in the ovary of Culex pipiens (Culexpipiens) and can infect various arthropods such as insects and mites. The bacteria can trigger abnormal host reproduction behaviors, such as cytoplasm incompatibility, parthenogenesis, emasculation, feminization and the like. The strain with similar activity also comprises Candidatus cardium, which is called cardium for short, is found in parasitoids of Encarsia, and can cause the feminization of spider mites, induce parthenogenesis, induce cytoplasm incompatibility, influence the fitness of hosts and change the oviposition behavior of the hosts. Verticillium lecanii Zimm can be used for preventing and treating aphid, whitefly and mite. The Acremonium hansfordii strain is obtained by separating larvae of naturally infected peach aphids (Myzuspicate Sulzer), has infection effects on cabbage caterpillar, peach aphid and two-spotted spider mite, consumes proteins in vivo after host infects the Acremonium, destroys tissues and organs of synthetic proteins, and makes detoxification enzymes and protective enzymes out of balance in vivo. The research cases and the utilization of the pathogenic bacteria of the tetranychus urticae are relatively few, but the pathogenic bacteria of the tetranychus urticae are utilized to 'control mites with bacteria', so that the population density can be safely and effectively regulated, the tetranychus urticae plays an important role in biological control, and the tetranychus urticae is an important component of biological control work.
Under the current premise that the pests generally generate drug resistance and the persistence and the harmlessness of comprehensive pest control are emphasized, the microbial resources are developed, strains with high pathogenicity are screened, and the use amount of chemical pesticides is reduced, so that the development direction of the current advanced agriculture is provided.
Disclosure of Invention
Based on the problems, the invention provides a bacillus velezensis strain which is used for preventing and controlling plant mites, and the mite killing effect is 96% in indoor bioassay experiments.
The bacillus velezensis strain is preserved in common microorganisms of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.24996.
The bacillus velezensis strain is named as LMFSSMJ-1, and the morphological characteristics of the LMFSSMJ-1 are as follows: gram-positive bacteria, wherein the bacteria are rod-shaped, the two ends of the bacteria are blunt, the bacteria appear singly, in pairs or in strings, the bacteria have spores, the spores are oval or circular, and can move from the middle end to the secondary end, and the flagellum is generated circumferentially; the strain is in a round shape on a nutrient agar culture medium, has irregular edges, obvious folds, a diameter of 2-3 mm, slight bulges, white color and non-transparency.
Based on the bacillus velezensis strain, the application also provides a method for detecting the genetic stability of the strain.
The method for detecting the genetic stability of the bacillus velezensis strain comprises the following steps:
s1, carrying out shake flask fermentation culture on a strain with the preservation number of CGMCC No.24996 and the name of bacillus velezensis, and then carrying out an indoor bioassay acaricidal effect experiment;
and S2, continuously passaging the strain with the preservation number of CGMCC No.24996 and the name of bacillus velezensis for more than five times, and repeating the step of S1 each time to perform an acaricidal effect experiment.
In one or more embodiments of the present application, the culture medium is peptone 2%, glucose 2.5%, potassium dihydrogen phosphate 0.1, calcium carbonate 0.02%, magnesium sulfate heptahydrate 0.005%, manganese sulfate 0.002%, sodium chloride 0.002%, and water in balance.
Based on the bacillus velezensis strain, the application also provides a fermentation liquid.
A fermentation liquor is prepared from the bacillus velezensis strain.
Based on the bacillus velezensis strain, the application also provides a preparation method of the fermentation liquor.
A preparation method of fermentation liquor adopts the bacillus velezensis strain.
Based on the bacillus velezensis strain, the application also provides an acaricide.
An acaricide comprising the above Bacillus velezensis strain.
Based on the fermentation liquor, the application also provides an acaricide.
An acaricide comprising a fermented liquid as an active ingredient, the fermented liquid comprising the above fermented liquid.
Based on the bacillus velezensis strain, the application also provides application of the bacillus velezensis strain.
The bacillus velezensis strain is applied to control plant mites.
The invention principle and the beneficial effects are as follows:
the bacillus velezensis strain can be used for preventing and treating citrus mites, and the mite killing rate is 96% in indoor bioassay experiments.
Description of biological preservation
The deposited bacteria are classified and named Latin, namely bacillus velezensis, and are preserved in China general microbiological culture Collection center (the address: no. 3 of West Lu No. 1 of Beijing, chaoyang, north Chen, china institute of microbiology, japan, postal code: 1000101) in 30 months 05 and 2022, and the preservation number is CGMCC No.24996.
Detailed Description
The present invention will be further explained below.
Example 1
An original strain is separated from orange leaves in Qingshen county of Meishan city (the preservation number of the original strain is CGMCC No.24996, and the name of the original strain is bacillus velezensis).
The preservation number is CGMCC No.24996, the bacterial strain named Bacillus velezensis (Bacillus belgii) is named LMFSSMJ-1, and the morphological characteristics of the bacterial strain of LMFSSMJ-1 are as follows: gram-positive bacteria, which are rod-shaped, have two blunt ends, appear singly, in pairs or in strings, have spores, are oval or circular, grow from the middle end to the secondary end, can move, and grow around flagellum. The strain is in a round shape on a nutrient agar culture medium, has irregular edges, obvious folds, a diameter of 2-3 mm, slight bulges, white color and non-transparency.
Example 2 genetic stability test
The genetic stability test comprises the following steps:
S1:
activating strains: picking a ring of a glycerol tube storing the strain with the preservation number of CGMCC No.24996 and the name of Bacillus velezensis (Bacillus belgii) in example 1, streaking the glycerol tube on an NA solid medium plate, and culturing the glycerol tube for 2 days at 30 ℃;
seed culture: picking out the single colony subjected to the strain activation culture, inoculating into a 500ml baffle triangular shake flask filled with 100ml culture solution, and culturing for 16h under the conditions that the temperature is 30 ℃ and the rotation speed is 200rpm to obtain seed solution;
fermentation culture: inoculating the cultured seed solution into 1000ml baffle triangular flask containing 300ml culture solution at an inoculum size of 5%, and culturing at 30 deg.C and 200rpm for 72h to obtain fermentation broth (first generation).
S2, continuously passaging the strain with the preservation number of CGMCC No.24996 and the name of Bacillus velezensis (Bacillus belgii) in the embodiment 1 for five times, and repeating the step of S1 once per passage to respectively obtain fermentation liquor (second generation), fermentation liquor (third generation), fermentation liquor (fourth generation) and fermentation liquor (fifth generation).
In the present embodiment, the first and second electrodes are,
the NA culture medium comprises the following components: 10g of peptone, 3g of beef extract, 5g of sodium chloride, 15-20g of agar and 1000ml of distilled water;
the culture solution is: 2% of peptone, 2.5% of glucose, 0.1% of monopotassium phosphate, 0.02% of calcium carbonate, 0.005% of magnesium sulfate heptahydrate, 0.002% of manganese sulfate, 0.002% of sodium chloride and the balance of water.
Example 3
Indoor acaricidal activity test: the fermentation broths of example 2 were diluted 100-fold, three replicates were set, and clear water was used as a control.
The double-sided adhesive tape is cut into 2-3cm long pieces, the pieces are attached to one end of a microscope slide, paper pieces on the adhesive tape are removed by tweezers, female adult mites with consistent size, bright body color and lively action are selected by a zero-size brush pen, the backs of the female adult mites are stuck on the double-sided adhesive tape (not sticking mite feet, mite whiskers and mouthparts), 3 lines are stuck on each piece, and 10 heads are stuck on each line. Then placing the mixture into a biochemical incubator with the temperature of 26 ℃ and the relative humidity of about 80 percent, standing for 4 hours, observing by using a microscope, and removing dead or inactive individuals.
And immersing one end of the glass slide with the mites into the diluted fermentation liquor or contrast clear water, slightly shaking for 5s, taking out, and quickly sucking away the mites and redundant liquid medicine around the mites by using absorbent paper.
And (5) placing the culture medium in the biochemical incubator again, and checking the result after 24 hours. The mites were lightly touched with a writing brush, and the mites died in the absence of sufficient mites. The results are given in table 1 below.
Calculating the prevention effect: mortality = (number of dead insects/total number of treated insects) × 100%, corrected mortality = (treatment mortality-blank mortality)/(1-blank mortality) × 100%. If the control mortality is < 5%, no correction is required, and if the control mortality is between 5% and 20%, correction is required. If the control mortality is > 20%, the test is invalid.
TABLE 1
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. The bacillus velezensis strain is preserved in common microorganisms of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.24996.
2. The bacillus velezensis strain as claimed in claim 1, wherein the strain named as LMFSSMJ-1, LMFSSMJ-1 has the morphological characteristics of: gram-positive bacteria, wherein the bacteria are rod-shaped, the two ends of the bacteria are blunt and round, the bacteria appear singly, in pairs or in strings, the bacteria have spores, the spores are oval or round, and the bacteria can move and generate flagellum peritomatically from the middle end to the secondary end; the strain is in a round shape on a nutrient agar culture medium, has irregular edges, obvious folds, a diameter of 2-3 mm, slight bulges, white color and non-transparency.
3. A method for the detection of the genetic stability of a strain of bacillus velezensis according to any one of claims 1-2, comprising the steps of:
s1, carrying out shake flask fermentation culture on a strain with the preservation number of CGMCC No.24996 and the name of bacillus velezensis, and then carrying out an indoor bioassay acaricidal effect experiment;
and S2, continuously passaging the strain with the preservation number of CGMCC No.24996 and the name of bacillus velezensis for more than five times, and repeating the step of S1 each time to develop an indoor bioassay acaricidal effect experiment.
4. The method for detecting the genetic stability of the bacillus velezensis strain as claimed in claim 3, wherein the cultured culture solution is peptone 2%, glucose 2.5%, potassium dihydrogen phosphate 0.1, calcium carbonate 0.02%, magnesium sulfate heptahydrate 0.005%, manganese sulfate 0.002%, sodium chloride 0.002%, and the balance of water.
5. A fermentation broth produced from the bacillus velezensis strain of any one of claims 1-2.
6. A method for producing a fermentation broth, characterized in that the Bacillus velezensis strain of any one of claims 1-2 is used.
7. An acaricide comprising the Bacillus velezensis strain of any one of claims 1 to 2.
8. An acaricide comprising a fermentation broth as an active ingredient, characterized in that the fermentation broth comprises the fermentation broth of claim 5.
9. The use of a strain of bacillus velezensis according to any of claims 1-2 for controlling spider mites of plants, mainly citrus mites.
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