CN116694501A - Pseudomonas orientalis and application thereof - Google Patents
Pseudomonas orientalis and application thereof Download PDFInfo
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- CN116694501A CN116694501A CN202310221797.7A CN202310221797A CN116694501A CN 116694501 A CN116694501 A CN 116694501A CN 202310221797 A CN202310221797 A CN 202310221797A CN 116694501 A CN116694501 A CN 116694501A
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- 241001291513 Pseudomonas orientalis Species 0.000 title claims description 21
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- 238000002360 preparation method Methods 0.000 claims abstract description 38
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- 241000429405 Pseudomonas extremorientalis Species 0.000 claims abstract description 14
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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
- C12N1/205—Bacterial isolates
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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
- A01N63/27—Pseudomonas
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- 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
- A01P5/00—Nematocides
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses an extreme oriental pseudomonas and application thereof. The invention separates soil samples of the suburban wetland from the south and west, and obtains the extremely eastern pseudomonas (Pseudomonas extremorientalis) SZSY-02 with high mortality rate for pine wood nematodes and sweet potato stem nematodes through line killing activity screening, the preservation number is CGMCC No.25551, the liquid preparation of the strain has 79.12 percent of prevention effect on pine wood nematodes and 75.51 percent of prevention effect on sweet potato stem nematodes, can effectively prevent the occurrence and diffusion of pine wood nematode diseases and sweet potato stem nematode diseases, is easy to ferment and produce, has simple use method, and can effectively relieve environmental pollution caused by the use of chemical pesticides.
Description
Technical Field
The invention relates to an extreme pseudomonas orientalis and application thereof, belonging to the technical field of microbial pesticides.
Background
Pine wood nematode disease (Bursaphelench xylophilus) is known as "pine tree cancer", and once pine trees are infected, there is no effective treatment, and the mortality rate is extremely high. At present, pine trees lost by pine wood nematode disease in China accumulate up to billions of plants, and the direct economic loss and ecological service value loss caused by the pine wood nematode disease are billions of yuan. If the epidemic situation of pine wood nematode is not effectively controlled, not only does the landscape mainly comprising pine trees not exist, but also serious damage to the ecological environment is caused.
Sweet potato stem nematode disease is a disease caused by sweet potato stem nematodes (Ditylenchus dipsaci) and occurring in sweet potatoes. The onset is serious in China in Shandong provinces and Hebei provinces. The disease mainly damages potato blocks, so that the bran core of the potato blocks is damaged, and secondly, the potato seedlings and the root parts of potato vines are damaged, the yield is generally reduced by 15%, and the yield can be reduced by 60% even in serious cases.
Pine wood nematode disease and sweet potato stem nematode disease are two kinds of nematode disease which are seriously harmful and difficult to control in forestry and agriculture at present. At present, chemical and physical control is mainly used for controlling the two nematode diseases, such as: perforation and drug injection and cleaning of affected trees are still main prevention and treatment means of pine wood nematode diseases; the application of chemical pesticides is a common control means for sweet potato stem nematode disease. Although the control modes have a certain control effect, the control modes bring heavy burden to the ecological environment; at the same time, the time and the labor are wasted, and the time and the labor are not long-term-! Therefore, the research on how to find a safe, environment-friendly and efficient biological control method for controlling pine wood nematodes on the premise of protecting environment and biodiversity is of great significance to protecting forest ecological safety in China, and the search for a safe, environment-friendly and efficient biological control method for controlling sweet potato stem nematode diseases is of great significance to improving sweet potato yield and agricultural production safety.
The extremely oriental pseudomonas (Pseudomonas extremorientalis) is a new strain which has relatively little function research and has potential development value. Patent CN110144302A discloses a high-yield ferrite-addicted extremely-eastern pseudomonas, a microbial agent and application, wherein the strain is SWNY 4-5, the microorganism preservation number is CGMCC No.16563, and related experiments show that the microbial agent prepared from the strain can effectively prevent and treat plant physiological iron-deficiency yellow diseases and effectively prevent and treat plant pathological yellow diseases caused by root knot nematodes; however, the research on the root-knot nematodes in the patent is not verified by field tests, the popularization is insufficient, and the prevention and control effects of the strain on pine wood nematodes and sweet potato stem nematodes are not researched. At present, little research is carried out on the extremely-oriental pseudomonas, and no report on prevention and control of pine wood nematodes and sweet potato stem nematodes by the extremely-oriental pseudomonas is seen.
Disclosure of Invention
Aiming at the problems, the invention provides the extremely oriental pseudomonas and the application thereof, wherein the extremely oriental pseudomonas is SZSY-02 (Pseudomonas extremorientalis), can effectively prevent and treat pine wood nematode diseases and sweet potato stem nematode diseases, can reduce environmental pollution caused by chemical pesticide use, and is easy to prepare microbial inoculants by fermentation, so that the extremely oriental pseudomonas is wide in application prospect.
The above object of the present invention is achieved by the following technical solutions: the invention separates from the mud sample of suburban wetland in the south of the Ji and West and obtains the extremely oriental pseudomonas (Pseudomonas extremorientalis) SZSY-02 with high mortality rate to pine nematodes and sweet potato stem nematodes through line killing activity screening, the strain is preserved in the China general microbiological culture Collection center (CGMCC No. 25551) on the day of 08 months of 2022 and the preservation address is North Chen West road No.1 and No. 3 in the North YangKorea of Beijing city. The strain grows on an NA culture medium, is colorless and transparent in the early stage, gradually turns into milky white, has neat edges and smooth and moist surfaces, is round and does not produce pigment; a large amount of yellow-green pigment is produced in the late stage, and the medium is caused to change color.
Another object of the present invention is to provide the use of Pseudomonas orientalis (Pseudomonas extremorientalis) SZSY-02 for controlling pine wood nematode disease and/or sweet potato stem nematode disease.
The invention also provides an extreme pseudomonas orientalis (Pseudomonas extremorientalis) SZSY-02 liquid preparation.
The preparation method of the extremely-oriented pseudomonas (Pseudomonas extremorientalis) SZSY-02 liquid preparation is characterized in that extremely-oriented pseudomonas SZSY-02 seed liquid is inoculated into a large amount of fermentation culture medium according to the volume ratio of 3-8%, and is fermented and cultured for 16-20 h at 25-30 ℃ to obtain the extremely-oriented pseudomonas SZSY-02 liquid preparation with the viable count of 800-1000 hundred million/ml.
A large amount of fermentation medium formula (mass ratio): 1.8 to 2.2 percent of edible glucose, 0.4 to 0.6 percent of fish peptone, 0.2 to 0.4 percent of molasses, 1.2 to 1.8 percent of yeast extract powder, 0.008 to 0.015 percent of magnesium sulfate, 0.02 to 0.04 percent of monopotassium phosphate, 0.4 to 0.6 percent of calcium carbonate and the balance of water, and the pH value is regulated to 7.2 to 7.3.
Preferably, the mass fermentation medium formula (mass ratio): 2% of edible glucose, 0.5% of fish peptone, 0.3% of molasses liquid, 1.5% of yeast extract powder, 0.01% of magnesium sulfate, 0.03% of monopotassium phosphate, 0.5% of calcium carbonate and the balance of water, and regulating the pH to 7.2-7.3.
The preparation method of the extremely oriental pseudomonas SZSY-02 seed solution comprises the following steps: inoculating the activated extreme Pseudomonas orientalis SZSY-02 into a seed liquid culture medium, and carrying out constant-temperature shaking culture for 10-15 h at 25-30 ℃ to prepare the extreme Pseudomonas orientalis SZSY-02 seed liquid. Wherein, the formula (mass ratio) of the seed liquid culture medium is as follows: glucose 0.5%, peptone 0.5%, beef powder 0.3%, sodium chloride 0.5% and water in balance, and adjusting pH 7.3+ -0.1.
The invention has the technical effects that:
1. the invention separates and screens mud samples of the suburban wetland in the south of the self-care, and obtains the extremely oriental pseudomonas (Pseudomonas extremorientalis) SZSY-02 with high mortality rate for pine wood nematodes and sweet potato stem nematodes, the liquid preparation of the strain has 79.12 percent of prevention effect on pine wood nematodes among forests, 75.51 percent of prevention effect on sweet potato stem nematodes in fields, and can effectively prevent the occurrence and diffusion of pine wood nematode diseases and sweet potato stem nematode diseases;
2. the extremely oriental pseudomonas SZSY-02 liquid preparation is easy to ferment and produce, has a simple use method, can effectively relieve environmental pollution caused by the use of chemical pesticides, and has wide application prospect in green control of pine wood nematodes and sweet potato stem nematodes.
Drawings
FIG. 1 shows the effect of SZSY-02 and SZSY-05 standard bacterial liquid on the correction of mortality of pine wood nematodes;
FIG. 2 is a colony morphology of Pseudomonas orientalis (Pseudomonas extremorientalis) SZSY-02;
FIG. 3 is a schematic representation of the morphology of Pseudomonas orientalis (Pseudomonas extremorientalis) SZSY-02 cells, wherein Panel A is a simple stain; panel B shows the result of gram staining.
Detailed Description
The following description of the embodiments of the present invention will clearly and fully describe the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1: isolation and purification of Pseudomonas orientalis (Pseudomonas extremorientalis) Strain SZSY-02
1. Bacterial strain separation and purification method
(1) Separating the source: the extremely oriental pseudomonas (Pseudomonas extremorientalis) SZSY-02 is separated from a suburban wetland mud sample in the south of the Henan province.
(2) Separation time and place: was isolated from the laboratory of the Ministry of research and development of Santa agriculture and forestry science, inc. in Saint North America, 4 th year 2021.
(3) Separation and purification steps: the preparation method is separated by adopting a dilution coating method, and comprises the following specific steps:
(1) sample collection: collecting a proper amount of mud blocks around the wetland and within the depth range of 10-20 cm in the center of the wetland by adopting a five-point sampling method in suburban wetland blocks in Jinan, shandong province, sub-packaging the mud blocks in a fresh-keeping bag, placing the fresh-keeping bag in a foam box containing a biological ice bag, and transporting the fresh-keeping bag back to a laboratory; each freshness protection package notes information such as collection time, collection place, collection person and the like;
(2) preparing mud sample suspension: 1g of mud sample is weighed and put into a 500mL triangular flask containing 100mL of sterile physiological saline, and is put into a constant-temperature shaking incubator at 30 ℃ and evenly shakes for 10-20 min at 150rpm, so that thalli, spores or spores in river mud are evenly dispersed, and mud sample suspension is prepared;
(3) and (3) dilution coating: sequentially diluting the mud sample suspension into 10 by using a 10-fold dilution method -1 ～10 -5 A diluted bacterial sample; respectively from 10 -3 、10 -4 And 10 -5 100 mu L of each diluted bacterial sample is respectively coated on NA and a chloramphenicol-containing PDA plate, and each gradient is respectively coated with three parallel media;
(4) culturing: sealing the coated flat plate, placing the flat plate in a 30 ℃ incubator for culturing for 2-3 d, and observing the growth condition of the flat plate colony at regular time;
(5) purifying: according to different colony forms on the flat plate, single bacteria are selected and streaked by an inoculating loop and an inoculating hook, and the culture is inverted at the constant temperature of 30 ℃ until a single colony grows on the flat plate;
(6) and (3) preserving: the purified single colony is inoculated in a corresponding culture medium test tube, and is preserved in a refrigerator at the temperature of 4 ℃ for standby after being kept at the constant temperature of 30 ℃ for 2-3 d.
2. Analysis of results
According to the method, 12 strains of bacteria are co-separated from a suburban wetland mud sample in the south of the Henan and the West, and the number is SZSY-01 to SZSY-12; the fungus 5 strain is separated, and the number is SZSY-21 to SZSY25.
Example 2: screening of highly efficient nematicidal strains of Pseudomonas orientalis SZSY-02
1. Experimental method
(1) Preparation of standard bacterial solutions of different strains to be tested
Under the aseptic condition, streaking 12 bacteria to be detected on an NA culture medium, performing activation culture at 30 ℃, and observing the presence or absence of bacteria; inoculating the first loop of lawn to NB culture at 27 deg.c and 120r/min shaking culture for 12 hr to obtain seed liquid of different strain; inoculating the seed solution into NB culture medium according to 1% of inoculum size by volume ratio, and performing constant-temperature shaking culture at 27deg.C and 120r/min for 20h to obtain different strain fermentation broths; preparing the fermentation liquid of the strain to be tested into a concentration of 1.0X10 by dilution method 9 CFU·mL -1 Standard bacterial liquid.
(2) Preparation of pine wood nematode and sweet potato stem nematode solution
Respectively transferring pine wood nematodes and sweet potato stem nematodes stored in the botrytis cinerea flat plates into new botrytis cinerea flat plates, sealing with a preservative film, and culturing in an incubator at 25 ℃ for 5-10 d; after the nematode culture is completed, separating the nematodes by utilizing a Bellman funnel method under aseptic conditions, sterilizing by using 0.002% cycloheximide and 0.1% streptomycin sulfate, and rinsing by using sterile water to prepare an aseptic nematode extract with the concentration of about 5000 heads/mL.
(3) Detection of killing activity of standard bacterial solutions of different strains to be detected on pine wood nematodes and sweet potato stem nematodes
Mixing standard bacterial liquid of the strain to be tested and nematode extracting solution (shaking uniformly) in 96-well plates according to the volume ratio of 1:1, wherein each 100 mu L is obtained, and the nematodes are stabilized at 2500 heads/mL; taking sterile water treatment as a blank control; standing at 25 ℃ for 24 hours; after the treatment is finished, the mixed solution is centrifuged for 180 seconds in a 2000r/min centrifuge, the supernatant is removed, 200 mu L of sterile water is used for resuspension, 50 mu L of resuspension is taken on a glass slide, the nematode death condition is observed through microscopic examination, the stiff fixed nematodes are used as a standard for killing the nematodes, and the standard bacterial solution treatment nematode mortality rate and the correction mortality rate of each strain to be detected are calculated.
Mortality (%) =number of nematode deaths/total number of nematodes x 100;
corrected mortality (%) = (treatment mortality-placebo mortality)/(1-placebo mortality) ×100.
2. Analysis of results
Experiments prove that only standard bacterial solutions of SZSY-02 and SZSY-05 in the 12 detected bacteria show nematicidal activity, and the standard bacterial solutions have nematicidal activity on pine wood nematodes and sweet potato stem nematodes (refer to table 1).
TABLE 1 statistics of Standard bacterial solutions of different strains on killing Activity against two nematodes
Note that: "+, +" means "having killing activity against both nematodes", "-" means "having no killing activity against both nematodes".
Through calculation of the corrected mortality, the corrected mortality of the pine wood nematodes and the sweet potato stem nematodes is respectively 100% and 96.97% after the treatment of SZSY-02 standard bacterial liquid for 24 hours; after 24 hours of treatment with SZSY-05 standard bacterial liquid, the corrected mortality of pine wood nematodes and sweet potato stem nematodes is 25.66% and 50.91%, respectively (see FIG. 1). Further analysis shows that the killing activity of the SZSY-02 standard bacterial liquid on pine wood nematodes and sweet potato stem nematodes is obviously higher than that of the SZSY-05 standard bacterial liquid. The result shows that the SZSY-02 bacterial liquid can effectively kill pine wood nematodes and sweet potato stem nematodes, and the SZSY-02 can be used as an excellent bacterial strain for biological control of the pine wood nematodes and the sweet potato stem nematodes for subsequent research.
Example 3: identification of Strain SZSY-02
(1) Colony culture characteristics: the strain grows on NA culture medium (beef extract 0.3%, peptone 1%, sodium chloride 0.5% and agar 1.5-2%), is colorless and transparent in the early stage and gradually changes into milky white, has neat edges and smooth and moist surfaces, is round and does not produce pigment; in the latter stage (incubation at constant temperature of 27-30℃for 3 d), a large amount of yellow-green pigment is produced, and the medium is caused to change color (see FIG. 2).
(2) Microscopic characteristics observation: the bacteria have a rod shape or slightly curved shape (refer to FIG. 3A) for microscopic examination, and the size is 1.8-2.2X10.2-0.4 μm; no spores are produced; gram staining was negative (see fig. 3B).
(3) Molecular biological properties:
the 16S rDNA gene sequence of this strain was determined as follows (SEQ-1):
GAGAAGCTTGCTTCTCTTGAGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGATAACGTTCGGAAACGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTTACCTAATACGTGATTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGACTGACTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTAATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAAGCCTTGAGCTTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTCATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGC
the strain SZSY-02 is identified as extremely oriental pseudomonas (Pseudomonas extremorientalis) through microbiological characteristic and molecular biological characteristic researches, and is preserved in China general microbiological culture Collection center (CGMCC) No.25551 in the year 08 and 19 of 2022.
Example 4: determination of virulence of Pseudomonas orientalis SZSY-02 on pine wood nematodes and sweet potato stem nematodes
1. Experimental method
Firstly, preparing fermentation liquor of pseudomonas orientalis SZSY-02 with different concentrations: the concentration of the Pseudomonas orientalis SZSY-02 fermentation broth prepared in example 2 was adjusted to 0.4X10, respectively 8 CFU·mL -1 、0.8×10 8 CFU·mL -1 、1.6×10 8 CFU·mL -1 、3.2×10 8 CFU·mL -1 And 6.4X10 8 CFU·mL -1 。
The nematicidal activity of the Pseudomonas orientalis SZSY-02 fermentation broth of different concentrations was examined by the method described in example 2, two nematode deaths were observed after 24h treatment, the corrected mortality of each nematode was calculated, and the LC of 24h was determined 50 . After treatment, the final concentration of the fermentation liquor of the extremely oriental pseudomonas SZSY-02 is 0.2 multiplied by 10 8 CFU·mL -1 、0.4×10 8 CFU·mL -1 、0.8×10 8 CFU·mL -1 、1.6×10 8 CFU·mL -1 And 3.2X10 8 CFU·mL -1 。
2. Analysis of results
The experimental result analysis shows that 3.2×10 8 CFU·mL -1 After the extremely oriental pseudomonas SZSY-02 fermentation broth is used for treating nematodes for 24 hours, the corrected mortality of pine wood nematodes and sweet potato stem nematodes respectively reaches 95.56 percent and 87.76 percent; we further obtain the virulence regression equation, and respectively obtain LC of SZSY-02 fermentation broth for 24h through the calculation of the virulence regression equation 50 Value, i.e. LC 50 (pine wood nematode) =0.73x10 8 CFU·mL -1 ，LC 50 (sweet potato stem nematode) =0.97x10 8 CFU·mL -1 . The results further demonstrate that SZSY-02 fermentation broths have higher nematicidal activity (see Table 2).
TABLE 2 killing effect of fermentation broths of SZSY-02 with different concentrations on pine wood nematodes and sweet potato stem nematodes
Example 5: preparation process of extremely oriental pseudomonas SZSY-02 liquid preparation
Seed liquid culture medium formula (mass ratio): glucose 0.5%, peptone 0.5%, beef powder 0.3%, sodium chloride 0.5% and water in balance, and adjusting pH 7.3+ -0.1.
A large amount of fermentation medium formula (mass ratio): 2% of edible glucose, 0.5% of fish peptone, 0.3% of molasses liquid, 1.5% of yeast extract powder, 0.01% of magnesium sulfate, 0.03% of monopotassium phosphate, 0.5% of calcium carbonate and the balance of water, and regulating the pH to 7.2-7.3. The molasses liquid is a byproduct of sugar industry, and the main components of the molasses liquid are sugar, solid matters are about 50%, sucrose is about 24% -36%, and other sugar is about 12% -24%; 3% -6% of protein; in addition, the water-soluble colloid also contains 3 to 4 percent of soluble colloid and 8 to 10 percent of mineral matters.
The preparation process of the liquid preparation of the pseudomonas orientalis SZSY-02 comprises the following steps:
(1) Seed liquid obtaining: under aseptic conditions, streaking the extremely oriental pseudomonas SZSY-02 on an NA culture medium, performing activation culture at 30 ℃, and observing the presence or absence of bacteria; inoculating the annular lawn into a seed solution culture medium by an inoculating loop after activation is completed, and carrying out constant-temperature shaking culture for 12 hours at the temperature of 27 ℃ and the constant temperature of 120r/min to prepare SZSY-02 seed solution;
(2) And (3) carrying out mass fermentation culture: injecting water according to 60-70% of the volume of the tank body, feeding according to a large amount of fermentation medium formula, adjusting pH to 7.2-7.3 after the raw materials are completely dissolved, adding a small amount of defoaming agent, and sterilizing for 30min at 121 ℃ under 0.1-0.12 MPa; after sterilization, inoculating the seed liquid into a fermentation tank (500L) according to the inoculation amount of 5% by volume ratio, and fermenting and culturing for 16-20 h under the conditions of 27 ℃ and 120r/min and ventilation of 1.2V/V.min, wherein the bacterial count can reach 800-1000 hundred million/ml, and the liquid preparation is the extremely oriental pseudomonas SZSY-02 liquid preparation.
Example 6: forest pesticide effect test of extremely oriental pseudomonas SZSY-02 liquid preparation
The embodiment provides a related experiment of the extremely oriental pseudomonas SZSY-02 liquid preparation on controlling pine wood nematode woodland.
1. Test method
(1) Test site
And (5) selecting a pine seedling production base in the Wired loop green region Zhang Cunzhen experimentally. The test area is arranged in the pine nursery land, has flat topography and is easy to observe and survey. The cultivation conditions in the test area are consistent, the damage degree of the pine wood nematodes is equivalent, and the test requirements are met.
(2) Test agent
The SZSY-02 liquid preparation prepared in the embodiment 5 of the invention is 300 times diluted solution.
(3) Test crop and control object
The test crop is black pine; the control object is pine wood nematode.
(4) Test method
The test consisted of 1 application treatment: 300 times of SZSY-02 liquid preparation is used in an amount of 400 kg/mu (two times of application are carried out, each time of application is 200 kg/mu), 71 mu is used, and 3789 plants are treated; the control is clear water, the dosage is 400 kg/mu, 78 mu is added, and 3923 plants are treated. We developed unmanned aerial vehicle spray tests at the production base of pine seedlings in the Wiggaihui green region Zhang Cunzhen on day 1, 6, 2021, and performed a second application 20 days after application. After the two applications are completed, the death condition of the black pine is counted in the next year, and the disease index and the prevention and treatment effect of each treatment are calculated.
The disease stage of pine wood nematode disease is shown in Table 3.
TABLE 3 grading Standard for disease incidence of pine wood nematode disease
Disease index DI (%) = [ Σ (disease grade) x number of plants at the grade) ]/[ (highest value of disease grade x total number of investigation) ]x100;
control effect (%) = (control disease index-treatment disease index)/control disease index×100.
2. Analysis of results
The experimental result analysis shows that after the liquid preparation of SZSY-02 is sprayed 300 times, the disease index of the pine wood nematode disease is reduced to 0.57 percent and is obviously lower than that of clear water control; further, the control effect was calculated to find that the control effect of the 300-time SZSY-02 liquid preparation is 79.79% (refer to Table 4). The result shows that the SZSY-02 liquid preparation can effectively prevent and treat pine wood nematode diseases in woodlands, and can be popularized and applied as a biological control preparation for the pine wood nematode diseases.
TABLE 4 disease index and control effect of each treatment
Example 7: field efficacy test of extremely oriental pseudomonas SZSY-02 liquid preparation
The embodiment provides a related experiment of the extremely oriental pseudomonas SZSY-02 liquid preparation on the field control of the sweet potato stem nematodes.
1. Test method
(1) Test site
And selecting a mountain town sweet potato planting area in the Laiwu area in Jinan city in a test area. The sweet potato stem nematodes in the plot of the test area are serious in disease occurrence from year to year, and meet the test requirements.
(2) Test agent
The SZSY-02 liquid preparation prepared in the embodiment 5 of the invention is 300 times diluted solution.
(3) Test crop and control object
The test crop is sweet potato, and the variety is the common market variety "Longshu No. 9"; the control object is sweet potato stem nematode.
(4) Test method
The test was carried out in 2022, 5 and 15, at which point the sweet potato planting was completed. The test consisted of 1 application treatment: SZSY-02 liquid preparation is applied in an amount of 4 kg/mu of land (two times of application, each time of application is 2 kg/mu), and the total application area is 10 mu. The seedling stage is irrigated once with water drops, and the second time of the application is carried out by the same method after 30d of application. The control area is 5 mu of clear water control. The disease conditions of all cells are investigated in the middle and late 10 months of the current year, and the disease rate, disease index and prevention and treatment effects of potato blocks are calculated.
Incidence = number of diseased potatoes/total number of potato blocks x 100%;
disease index (%) = [ Σ (number of potato pieces at each level×number of corresponding levels)/(total number of potato pieces×number of highest levels) ]×100;
control effect (%) = [ (control area disease index-treatment area disease index)/control area disease index ] ×100.
The disease stage numbers of the sweet potato stem nematode disease are shown in Table 5.
TABLE 5 disease grading Standard for sweet potato stem nematodes
2. Analysis of results
Experimental results show that after the 300-time SZSY-02 liquid preparation is applied, the incidence rate of the sweet potato stem nematode disease is reduced to 17.96%, the disease index is reduced to 6.29%, and the two data are obviously lower than that of the clear water control; further, the control effect was calculated to find that the control effect of the 300-time SZSY-02 liquid preparation was 74.34% (refer to Table 6). The result shows that the SZSY-02 liquid preparation can effectively prevent and treat the diseases of the sweet potato stem nematodes in the field, and can be popularized and applied as a biological control preparation for the sweet potato stem nematode diseases.
TABLE 6 morbidity, index of disease and controlling effect of each treatment
Claims (8)
1. An extreme pseudomonas orientalis (Pseudomonas extremorientalis) SZSY-02, wherein the preservation number of the strain is CGMCC No.25551.
2. Use of pseudomonas extreme eastern of claim 1 SZSY-02 for controlling pine wood nematode disease and/or sweet potato stem nematode disease.
3. A liquid formulation of pseudomonas extreme eastern style SZSY-02 comprising pseudomonas extreme eastern style SZSY-02 as defined in claim 1 as an active ingredient.
4. The preparation method of the extreme oriental pseudomonas SZSY-02 liquid preparation of claim 3, which is characterized in that the extreme oriental pseudomonas SZSY-02 seed liquid is inoculated into a large amount of fermentation culture medium according to the volume ratio of 3-8 percent and is fermented and cultured for 16-20 hours at 25-30 ℃ to obtain the extreme oriental pseudomonas SZSY-02 liquid preparation.
5. The method according to claim 4, wherein,
the mass fermentation culture medium comprises the following components in percentage by mass: 1.8 to 2.2 percent of edible glucose, 0.4 to 0.6 percent of fish peptone, 0.2 to 0.4 percent of molasses, 1.2 to 1.8 percent of yeast extract powder, 0.008 to 0.015 percent of magnesium sulfate, 0.02 to 0.04 percent of monopotassium phosphate, 0.4 to 0.6 percent of calcium carbonate and the balance of water, and the pH value is regulated to 7.2 to 7.3.
6. The method according to claim 5, wherein,
the mass fermentation culture medium comprises the following components in percentage by mass: 2% of edible glucose, 0.5% of fish peptone, 0.3% of molasses liquid, 1.5% of yeast extract powder, 0.01% of magnesium sulfate, 0.03% of monopotassium phosphate, 0.5% of calcium carbonate and the balance of water, and regulating the pH to 7.2-7.3.
7. The preparation method of the Pseudomonas orientalis SZSY-02 seed liquid according to claim 4, wherein the preparation method of the Pseudomonas orientalis SZSY-02 seed liquid comprises the following steps: inoculating the activated extreme Pseudomonas orientalis SZSY-02 into a seed liquid culture medium, and carrying out constant-temperature shaking culture for 10-15 h at 25-30 ℃ to prepare the extreme Pseudomonas orientalis SZSY-02 seed liquid.
8. The method according to claim 7, wherein,
the seed liquid culture medium comprises the following components in percentage by mass: glucose 0.5%, peptone 0.5%, beef powder 0.3%, sodium chloride 0.5% and water in balance, and adjusting pH 7.3+ -0.1.
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