CN113040140A - Auxiliary penetrating agent suitable for bacteriophage to be immersed into lianas and woody plants and preparation method and application thereof - Google Patents

Auxiliary penetrating agent suitable for bacteriophage to be immersed into lianas and woody plants and preparation method and application thereof Download PDF

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CN113040140A
CN113040140A CN202110118427.1A CN202110118427A CN113040140A CN 113040140 A CN113040140 A CN 113040140A CN 202110118427 A CN202110118427 A CN 202110118427A CN 113040140 A CN113040140 A CN 113040140A
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phage
percent
auxiliary
penetrant
bacteriophage
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CN113040140B (en
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于浩
丛郁
乔欢
徐旭凌
肖逍
丁良
何四龙
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Phagelux Nanjing Biotechnology Co ltd
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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
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/30—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests characterised by the surfactants
    • 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
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • 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
    • A01N63/40—Viruses, e.g. bacteriophages
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • 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
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Agronomy & Crop Science (AREA)
  • Plant Pathology (AREA)
  • Dentistry (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Virology (AREA)
  • Toxicology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

The application relates to the technical field of phage biopesticides, in particular to an auxiliary penetrating agent suitable for phage to be immersed into lianas and woody plants, and a preparation method and an application method of the phage auxiliary penetrating agent. The auxiliary osmotic agent components described herein include sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol polyoxyethylene ether, dioctyl sodium sulfosuccinate, thioxanthone, calcium stearate, and mannitol. The auxiliary penetrating agent has reasonable components, easily obtained materials, low production cost and no harm to human bodies. The auxiliary penetrating agent can increase the number of the phages entering the cane and woody plants during immersion treatment, improve the utilization efficiency of the phages and enhance the control effect of the phages on the bacterial diseases of the plants.

Description

Auxiliary penetrating agent suitable for bacteriophage to be immersed into lianas and woody plants and preparation method and application thereof
Technical Field
The application relates to the technical field of phage biopesticides, in particular to an auxiliary penetrating agent suitable for phage to be immersed into lianas and woody plants, and a preparation method and application of the phage auxiliary penetrating agent.
Background
Crop bacterial diseases are popular diseases in the world, and the harm degree of the bacterial diseases is more serious than that of fungal diseases and is more difficult to control. The pesticide for preventing and treating crop bacterial diseases has less varieties for a long time, and the prevention and treatment effect of the existing pesticide on the bacterial diseases is also poor.
Compared with traditional pesticides, the phage has the following advantages: bacteriophages are self-replicating viruses that survive and replicate only in the presence of a host; in the absence of host, the phage will rapidly die; the bacteriophage has no toxicity to the environment, does not cause pollution to the environment, and meets the requirement of green and pollution-free agriculture advocated in the present era; the bacteriophage has strong specificity, only aims at corresponding pathogenic bacteria, and does not destroy normal flora. Therefore, the prevention and treatment of bacterial diseases by using bacteriophage to prepare biopesticide is gradually valued and developed by people. However, there are many limitations to the use of bacteriophages for bacterial disease control, and climate and field environmental factors, etc. need to be considered. For example: the application effect of the phage may be influenced by factors such as the pH value of the soil and physical obstacles in the soil, as well as factors such as air temperature, rain water and ultraviolet rays. Among them, the soaking treatment before sowing vine and woody plant is very important, and the current chemical soaking method has many disadvantages. Therefore, the use of phage immersion is attempted, which not only ensures biosafety but also effectively kills pathogenic bacteria.
However, the current bacteriophage has poor plant penetration effect, can not be effectively attached to the surfaces of vines and woody plants, and is more difficult to enter the bodies of the vines and the woody plants.
Content of application
In view of the existing technical situation, the application provides an auxiliary penetrating agent suitable for bacteriophage to be immersed into lianas and woody plants, and a preparation method and application thereof.
The first purpose of the application is to provide an auxiliary penetrant suitable for bacteriophage to be immersed in lianas and woody plants, the technical scheme is as follows, and the auxiliary penetrant at least comprises: one or more of sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thioxanthone, calcium stearate and mannitol.
As one embodiment, the auxiliary penetrant comprises triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thiaketone, calcium stearate and mannitol, and the content of each component is as follows: the content of the sorbitan laurate is 0 to 0.04 percent by weight; 0 to 0.04 percent of polyoxyethylene sorbitan monopalmitate; the dosage of the triton is 0.8-1.6%; the dosage of the fatty alcohol-polyoxyethylene ether is 2-2.4%; the dosage of the dioctyl sodium sulfosuccinate is 1-2.6%; the dose of the thiaketone is 1-3%; the dosage of the calcium stearate is 1.2-2.4%; the dosage of the mannitol is 4-8%; the balance being deionized water.
The auxiliary penetrant of the present application can be used for purposes including, but not limited to, a Ralstonia solanacearum phage or a Xanthomonas carpi phage or a Pseudomonas syringae Actinidia pathovar.
As one embodiment, the auxiliary penetrant for a ralstonia solanacearum bacteriophage described herein comprises the following components in dosage: based on the weight percentage of the components,
sorbitan laurate: 0.4 percent;
polyoxyethylene sorbitan monopalmitate: 0.4 percent;
triton: 1.4 percent;
fatty alcohol polyoxyethylene ether: 2.3 percent;
dioctyl sodium sulfosuccinate: 1.5 percent;
and (3) thiaketone: 2.5 percent;
calcium stearate: 2.2 percent;
mannitol: 7 percent;
the balance of deionized water;
or a secondary penetrant for xanthomonas carpi phages, comprising the following components in the following dosage: based on the weight percentage of the components,
sorbitan laurate: 0.35 percent;
polyoxyethylene sorbitan monopalmitate: 0.3 percent;
triton: 1.4 percent;
fatty alcohol polyoxyethylene ether: 2.3 percent;
dioctyl sodium sulfosuccinate: 1.5 percent;
and (3) thiaketone: 2.5 percent;
calcium stearate: 2.2 percent;
mannitol: 7 percent;
the balance being deionized water.
Or an auxiliary penetrant for pseudomonas syringae kiwi pathovar phage, comprising the following components in dosage: based on the weight percentage of the components,
sorbitan laurate: 0.3 percent;
polyoxyethylene sorbitan monopalmitate: 0.3 percent;
triton: 1.4 percent;
fatty alcohol polyoxyethylene ether: 2.3 percent;
dioctyl sodium sulfosuccinate: 1.5 percent;
and (3) thiaketone: 3 percent;
calcium stearate: 2.2 percent;
mannitol: 8 percent;
the balance being deionized water.
Or a secondary penetrant for use in a phage composition, comprising the following components in dosages: based on the weight percentage of the components,
sorbitan laurate: 0.35 percent;
polyoxyethylene sorbitan monopalmitate: 0.3 percent;
triton: 1.4 percent;
fatty alcohol polyoxyethylene ether: 2.3 percent;
dioctyl sodium sulfosuccinate: 1.5 percent;
and (3) thiaketone: 2.5 percent;
calcium stearate: 2.2 percent;
mannitol: 7 percent;
the balance being deionized water.
The phage-assisted penetrant component of the present application is a preferred component.
As one embodiment, the auxiliary osmotic agent may be stored for 18 months at 4 ℃; can be stored for 12 months at 25 ℃; can be stored at 37 deg.C for 6 months.
The auxiliary penetrating agent of the scheme has good stability at various temperatures.
A second object of the present application is to provide a method for preparing an auxiliary penetrant suitable for bacteriophage impregnation into vines and woody plants, comprising the following steps:
(1) adding 0-0.04% of sorbitan laurate, 0-0.04% of polyoxyethylene sorbitan monopalmitate, 0.8-1.6% of triton, 2-2.4% of fatty alcohol-polyoxyethylene ether and 1-2.6% of dioctyl sodium sulfosuccinate into 1/2 weight of water, heating to dissolve, and uniformly stirring by magnetic force for later use;
(2) adding 1-3% of thiazone, 1.2-2.4% of calcium stearate and 4-8% of mannitol into 1/2 weight of water, dissolving at normal temperature, and uniformly stirring by magnetic force for later use;
(3) adding the mixed solution obtained in the step (1) into the solution obtained in the step (2) for mixing, and uniformly stirring by magnetic force;
(4) and (4) sterilizing the mixed solution obtained in the step (3) at 121 ℃ for 20min, and storing at 4 ℃ for later use to obtain the phage auxiliary penetrant.
As one embodiment, the auxiliary penetrant for a ralstonia solanacearum bacteriophage is prepared as follows: weighing 4g of sorbitan laurate, 4g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 25g of thiaketone, 22g of calcium stearate and 70g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the mixture is fully dissolved, and adding deionized water to reach the constant volume of 1000 mL; sterilizing at 121 deg.C for 20min to obtain bacteriophage auxiliary penetrant, and storing at 4 deg.C;
or the preparation method of the auxiliary penetrant for the xanthiomonas carpet grass bacteriophage is as follows: weighing 3.5g of sorbitan laurate, 3g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 25g of thiaketone, 22g of calcium stearate and 70g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the mixture is fully dissolved, and adding the deionized water to the volume of 1000 mL; sterilizing at 121 deg.C for 20min to obtain bacteriophage auxiliary penetrant, and storing at 4 deg.C;
or the preparation method of the auxiliary penetrant for the pseudomonas syringae kiwi pathopoiesia variety phage comprises the following steps: weighing 3g of sorbitan laurate, 3g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 30g of thiaketone, 22g of calcium stearate and 80g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the mixture is fully dissolved, and adding the deionized water to a constant volume of 1000 mL; sterilizing at 121 deg.C for 20min to obtain bacteriophage auxiliary penetrant, and storing at 4 deg.C;
or the auxiliary penetrant for phage composition is prepared as follows: weighing 35g of sorbitan laurate, 3g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 25g of thiaketone, 22g of calcium stearate and 70g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the mixture is fully dissolved, and adding deionized water to reach the constant volume of 1000 mL; sterilizing at 121 deg.C for 20min to obtain bacteriophage auxiliary penetrant, and storing at 4 deg.C.
The preparation and preservation method of the phage auxiliary penetrant is simple and feasible in process, high in efficiency, low in application cost, simple to operate, easy to realize and convenient to apply.
The third purpose of the application is to provide an application method of the auxiliary penetrant suitable for the bacteriophage to be immersed into the liana and the woody plant, wherein the auxiliary penetrant is mixed into the bacteriophage solution with the final concentration of 12% and stirred uniformly.
As one embodiment, the penetrant is added into the phage solution under aseptic conditions, and the phage solution and the penetrant are uniformly mixed according to the volume ratio of 1:9 to obtain the phage preparation with the penetrant.
As an embodiment, the bacteriophage is a ralstonia solanacearum bacteriophage or a xanthomonas carpi bacteriophage or a pseudomonas syringae actinidia pathovar phage or a combination of the three bacteriophages.
The phage-assisted penetrants of the present application can be applied to different types of phage, including, but not limited to, a Ralstonia solanacearum phage or a Xanthomonas carpi phage or a Pseudomonas syringae Actinidia pathovar. The solanaceous Ralstonia phage includes, but is not limited to, solanaceous Ralstonia solanacearum phage GP1(Ralstonia solanacearum phage GP1), solanaceous Ralstonia phage GP2(Ralstonia solanacearum phage GP2), or solanaceous Ralstonia phage GP3(Ralstonia solanacearum phage GP 3).
The Xanthomonas carpi phage includes, but is not limited to, Xanthomonas carpi phage YHC5(Xanthomonas axonopodis phage YHC 5).
Pseudomonas syringae Actinidia pathovar typa phage includes, but is not limited to Pseudomonas syringae Actinidia phage PSA-P1(Pseudomonas syringae pv. Actinidiae phage PSA-P1).
The solanaceae Ralstonia phage GP1(Ralstonia solanacearum phase GP1) with the preservation number of M2016633, the preservation unit is China center for type culture Collection with the preservation date of 2016, 11 and 10 days;
the solanaceae Ralstonia phage GP2(Ralstonia solanacearum phase GP2) with the preservation number of M2016634, the preservation unit is China center for type culture Collection with the preservation date of 2016, 11 and 10 days;
solanaceae Ralstonia phage GP3(Ralstonia solanacearum phase GP3), with a accession number of CCTCC NO: m2016635, the preservation unit is China center for type culture Collection, the preservation date is 2016, 11 and 10 days;
the Xanthomonas campestris phage YHC5(Xanthomonas axonopodis phase YHC5) has the preservation number of CCTCC NO of M2018579, the preservation unit is China center for type culture Collection, and the preservation date is 2018, 08 months and 30 days.
Pseudomonas syringae kiwi fruit pathogenic variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1) with preservation number of CCTCC NO: M2020252, preservation unit of China center for type culture Collection, and preservation date of 2020, 06, 30 days.
As one embodiment, the optimal treatment concentration for phage infusion into vines and woody plants is 12%.
The phage auxiliary penetrant is convenient to use and can be used for various phages and target lianas and woody plants. The phage auxiliary penetrant has good adaptability, can be operated at room temperature, and has extremely high application prospect.
Compared with the prior art, the method has the following advantages:
(1) the phage auxiliary penetrating agent has the advantages of reasonable components, wide raw material source, easiness in obtaining and low cost, can effectively increase the quantity of phage entering rattans and woody plants during immersion treatment, and can improve the speed of phage entering the rattans and woody plants.
(2) The preparation and storage method of the phage auxiliary penetrating agent is reasonable, simple and feasible in process, high in efficiency, low in application cost, simple to operate, easy to realize, good in long-term stability (more than 6m) at different temperatures, and capable of being used for multiple times after being prepared once.
(3) The application combines active microorganisms with chemical reagents to form a brand new non-toxic and safe osmotic agent.
(4) The phage auxiliary penetrant has a very high application prospect, has low requirements on storage conditions, and is convenient to apply when accelerating the penetration process of phage under different environmental conditions.
Detailed Description
The following examples and experimental examples serve to further illustrate the present application, but do not limit the effective scope of the present application in any way.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Generally, the nomenclature used herein is those well known or commonly used in the art, and the components of the osmotic agent reagents used in the examples herein are commercially available, unless otherwise specified.
In the following examples, the reference numbers of the strains involved are numbered in the manner of the company's name.
In the following examples, the reagents used are as follows:
the LB liquid culture medium has the formula: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride and 1000mL of distilled water.
The LB solid culture medium has the formula: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar and 1000mL of distilled water.
The adopted equipment comprises the following steps: 0.22 μm sterile filter (Millpore);
sterile centrifuge tubes (Eppendorf).
Sorbitan laurate, which is a commercially available product, has a purity of 99.0% or more and has a molecular formula of C18H34O6And the molecular weight is 346.459.
Polyoxyethylene sorbitan monopalmitate which is a commercially available product, has a purity of 99.0% or more and a molecular formula of C12H18O11And the molecular weight is 338.264.
The triton is a commercial product, the purity is more than or equal to 99.5 percent, and the molecular formula C34H62O11And the molecular weight is 646.86.
The fatty alcohol-polyoxyethylene ether is a commercial product, the purity is more than or equal to 99.0 percent, and the molecular formula is C12H25O.(C2H4O)nAnd the molecular weight is 1199.55.
Sodium dioctyl sulfosuccinate is a commercial product, has a purity of 99.0% or more, and has a molecular formula of C20H37O7SNa, molecular weight 444.25.
The thiaketone is a commercial productPurity of 97.0% or more, molecular formula C11H13O3SN, molecular weight 239.29.
Calcium stearate is a commercial product, and has purity of 99.0% or more and molecular formula C36H70CaO4And the molecular weight is 607.01.
Mannitol is a commercial product with purity of 99.0% or more, and molecular formula C6H14O6And the molecular weight is 182.17.
The solanaceae Ralstonia phage GP1(Ralstonia solanacearum phase GP1) has the preservation number of M2016633, the preservation unit is China center for type culture Collection, and the preservation date is 2016, 11 and 10 days. The preservation address is Wuhan university in China.
The solanaceae Ralstonia phage GP2(Ralstonia solanacearum phase GP2) has the preservation number of M2016634, the preservation unit is China center for type culture Collection, and the preservation date is 2016, 11 and 10 days. The preservation address is Wuhan university in China.
Solanaceae Ralstonia phage GP3(Ralstonia solanacearum phase GP3), with a accession number of CCTCC NO: m2016635, the preservation unit is China center for type culture Collection, and the preservation date is 2016, 11 and 10 days. The preservation address is Wuhan university in China.
The Xanthomonas campestris phage YHC5(Xanthomonas axonopodis phase YHC5) has the preservation number of CCTCC NO of M2018579, the preservation unit is China center for type culture Collection, and the preservation date is 2018, 08 months and 30 days. The preservation address is Wuhan university in China.
Pseudomonas syringae kiwi fruit pathogenic variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1) with preservation number of CCTCC NO: M2020252, preservation unit of China center for type culture Collection, and preservation date of 2020, 06, 30 days. The preservation address is Wuhan university in China.
EXAMPLE 1 preparation of eight reagents formulations of different concentrations
1g, 2.5g, 3g, 3.5g and 4g of sorbitan laurate are accurately weighed, and a proper amount of deionized water is respectively added and stirred until the sorbitan laurate is fully dissolved, and then the deionized water is added to the mixture until the volume is up to 1000 mL; five 1000mL solutions obtained were sterilized at 121 ℃ for 20min and stored at 4 ℃ until use.
Accurately weighing 1g, 2.5g, 3g, 3.5g and 4g of polyoxyethylene sorbitan monopalmitate, respectively adding a proper amount of deionized water, stirring until the deionized water is fully dissolved, and then adding the deionized water to a constant volume of 1000 mL; five 1000mL solutions obtained were sterilized at 121 ℃ for 20min and stored at 4 ℃ until use.
Accurately weighing 8g, 10g, 12g, 14g and 16g of triton, respectively adding a proper amount of deionized water, stirring until the deionized water is fully dissolved, and then adding the deionized water to a constant volume of 1000 mL; five 1000mL solutions obtained were sterilized at 121 ℃ for 20min and stored at 4 ℃ until use.
Accurately weighing 20g, 21g, 22g, 23g and 24g of fatty alcohol-polyoxyethylene ether, respectively adding a proper amount of deionized water, stirring until the deionized water is fully dissolved, and then adding the deionized water to a constant volume of 1000 mL; five 1000mL solutions obtained were sterilized at 121 ℃ for 20min and stored at 4 ℃ until use.
Accurately weighing 10g, 15g, 18g, 22g and 24g of dioctyl sodium sulfosuccinate, respectively adding a proper amount of deionized water, stirring until the deionized water is fully dissolved, and then adding the deionized water to a constant volume of 1000 mL; five 1000mL solutions obtained were sterilized at 121 ℃ for 20min and stored at 4 ℃ until use.
Accurately weighing 10g, 15g, 20g, 25g and 30g of thiazone, respectively adding a proper amount of deionized water, stirring until the mixture is fully dissolved, and then adding deionized water to a constant volume of 1000 mL; five 1000mL solutions obtained were sterilized at 121 ℃ for 20min and stored at 4 ℃ until use.
Accurately weighing 12g, 15g, 18g, 22g and 24g of calcium stearate, respectively adding a proper amount of deionized water, stirring until the calcium stearate is fully dissolved, and then adding the deionized water to a constant volume of 1000 mL; five 1000mL solutions obtained were sterilized at 121 ℃ for 20min and stored at 4 ℃ until use.
Accurately weighing 40g, 50g, 60g, 70g and 80g of mannitol, respectively adding a proper amount of deionized water, stirring until the mannitol is fully dissolved, and then adding the deionized water to a constant volume of 1000 mL; five 1000mL solutions obtained were sterilized at 121 ℃ for 20min and stored at 4 ℃ until use.
1000mL of deionized water was sterilized at 121 ℃ for 20min as a blank and stored at 4 ℃ for further use.
Example 2 preparation of phage-assisted penetrant
(1) The preparation method of the auxiliary penetrating agent suitable for the solanaceae ralstonia phage comprises the following steps: accurately weighing 4g of sorbitan laurate, 4g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 25g of thiaketone, 22g of calcium stearate and 70g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the mixture is fully dissolved, and adding the deionized water to a constant volume of 1000 mL; sterilizing at 121 deg.C for 20min, and storing at 4 deg.C for use.
(2) The preparation method of the auxiliary penetrant suitable for the xanthiomonas carpet grass bacteriophage is as follows: accurately weighing 3.5g of sorbitan laurate, 3g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 25g of thiaketone, 22g of calcium stearate and 70g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the mixture is fully dissolved, and adding the deionized water to a constant volume of 1000 mL; sterilizing at 121 deg.C for 20min, and storing at 4 deg.C for use.
(3) The preparation method of the auxiliary penetrant suitable for the pseudomonas syringae kiwi fruit pathopoiesia phage comprises the following steps: accurately weighing 3g of sorbitan laurate, 3g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 30g of thiaketone, 22g of calcium stearate and 80g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the deionized water is fully dissolved, and adding the deionized water to a constant volume of 1000 mL; sterilizing at 121 deg.C for 20min, and storing at 4 deg.C for use.
(4) The preparation method of the auxiliary penetrant suitable for the phage composition is as follows: accurately weighing 3.5g of sorbitan laurate, 3g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 25g of thiaketone, 22g of calcium stearate and 70g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the mixture is fully dissolved, and adding the deionized water to a constant volume of 1000 mL; sterilizing at 121 deg.C for 20min, and storing at 4 deg.C for use.
Example 3 preparation of Ralstonia solanacearum phage GP1 solution
The preserved single colony of the Ralstonia solanacearum is inoculated into an erlenmeyer flask containing 500mL of LB culture medium, and when the culture is performed with shaking at 240rpm at 28 ℃ until the OD value is 0.2, 1000PFU/mL of the Ralstonia solanacearum phage GP1(Ralstonia solanacearum phage GP1, preservation number CCTCC NO: M2016633) 100 mu L is added, and the culture is performed with shaking at 240rpm at 28 ℃ for 18 h. The fermentation broth was centrifuged at 8000rpm for 15min, and the supernatant was filtered through a 0.22 μm filter to obtain a solution of phage GP1 of Ralstonia solanacearum.
Example 4 preparation of Ralstonia solanacearum phage GP2 solution
The preserved single colony of the Ralstonia solanacearum is inoculated into an erlenmeyer flask containing 500mL of LB culture medium, and when the culture is performed with shaking at 240rpm at 28 ℃ until the OD value is 0.2, 1000PFU/mL of the Ralstonia solanacearum phage GP2(Ralstonia solanacearum phage GP2, preservation number CCTCC NO: M2016634) 100 mu L is added, and the culture is performed with shaking at 240rpm at 28 ℃ for 18 h. The fermentation broth was centrifuged at 8000rpm for 15min, and the supernatant was filtered through a 0.22 μm filter to obtain a solution of phage GP2 of Ralstonia solanacearum.
Example 5 preparation of Ralstonia solanacearum phage GP3 solution
The preserved single colony of the Ralstonia solanacearum is inoculated into an erlenmeyer flask containing 500mL of LB culture medium, and when the culture is performed with shaking at 240rpm at 28 ℃ until the OD value is 0.2, 1000PFU/mL of the Ralstonia solanacearum phage GP3(Ralstonia solanacearum phage GP3, preservation number CCTCC NO: M2016635) 100 mu L is added, and the culture is performed with shaking at 240rpm at 28 ℃ for 18 h. The fermentation broth was centrifuged at 8000rpm for 15min, and the supernatant was filtered through a 0.22 μm filter to obtain a solution of phage GP3 of Ralstonia solanacearum.
Example 6 preparation of Xanthomonas campestris phage YHC5 solution
The phage YHC5 host strain was inoculated into an Erlenmeyer flask containing 500mLLB medium, and when the culture was shaken at 240rpm at 25 ℃ until the OD value became 0.2, 1000PFU/mL Xanthomonas carpet phage YHC5(Xanthomonas axonopodis phage YHC5, accession No. CCTCC NO: M2018579) was added thereto, and the culture was shaken at 240rpm at 25 ℃ for 18 hours. The fermentation liquor is centrifuged for 15min at 8000rpm, and the supernatant is filtered by a filter membrane of 0.22 μm to obtain the xanthomonas carpi phage YHC5 solution.
Example 7 preparation of Pseudomonas syringae Kiwi fruit pathogenic variety phage PSA-P1 solution phage PSA-P1 host bacteria were inoculated into Erlenmeyer flask containing 500mL LB medium, shake-cultured at 240rpm at 28 ℃ until OD value was 0.2, 1000PFU/mL Pseudomonas syringae Kiwi phage PSA-P1(Pseudomonas syringae pv. Actinidiae phage PSA-P1, preservation No. CCTCC NO: M2020252) were added thereto, and shake-cultured at 240rpm at 28 ℃ for 18 h. Centrifuging the fermentation liquid at 8000rpm for 15min, and filtering the supernatant with 0.22 μm filter membrane to obtain PSA-P1 solution.
Example 8 preparation of a Single phage preparation with auxiliary penetrant
Taking the three solanaceae ralstonia phage solutions, the xanthiomonas carpropoides phage YHC5 solution and the pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 solution obtained in the embodiments 3-7, and respectively adding the phage auxiliary penetrants prepared in the embodiment 2 into the 5 phage solutions under an aseptic condition. Each phage was mixed with the auxiliary penetrant homogeneously in a volume ratio of 9: 1. Finally, a preparation containing auxiliary penetrating agent of solanaceae ralstonia phage GP1, a preparation of solanaceae ralstonia phage GP2, a preparation of solanaceae ralstonia phage GP3, a preparation of xanthomonas carpi phage YHC5 and a preparation of pseudomonas syringae kiwi pathopoiesia variant phage PSA-P1 are obtained.
Example 9 preparation of phage composition
Providing a Ralstonia solanacearum phage GP1(Ralstonia solanacearum phase GP1) with the preservation number of CCTCC NO: M2016633; the solanaceae Ralstonia solanacearum phage GP2(Ralstonia solanacearum phase GP2) with the preservation number of CCTCC NO: M2016634; solanaceae Ralstonia phage GP3(Ralstonia solanacearum phase GP3) with the preservation number CCTCC NO: M2016635; xanthomonas carpi phage YHC5(Xanthomonas axonopodis phase YHC5) with the preservation number of CCTCC NO: M2018579; pseudomonas syringae kiwi fruit pathopoiesia variant phage PSA-P1(Pseudomonas syringae pv. Actinidiae phase PSA-P1), with the preservation number of CCTCC NO: M2020252, and prepared into a phage composition according to the volume ratio of 1:1:1:1: 1: 1.
Example 10 preparation of phage composition preparation with auxiliary penetrant
The phage composition auxiliary penetrant prepared in example 2 was added to the phage composition of example 9 under aseptic conditions and mixed well, wherein the volume ratio of the auxiliary penetrant to the phage composition was 1:9, resulting in a preparation of phage composition with auxiliary penetrant.
Experimental example 1 toxicological test
Taking 40 experimental mice, each half of the experimental mice are subjected to adaptive feeding for three days, and then the experimental mice are randomly divided into four groups: the phage composition-treated group (treatment 1), the phage penetrant preparation-treated group (treatment 2), the phage composition-treated group with phage penetrant preparation (treatment 3), and the control group, each group consisting of 10 mice (5 mice each male and female). Respectively give treatment 1 a dose of 1010PFU/kg of phage composition (prepared in example 9); treatment 3 was given a dose of 1010PFU/kg of phage composition with phage penetration preparation (prepared in example 10); treatment 2 was given a 10-fold dilution of an equivalent amount of the phage penetration preparation (prepared in example 2); the control group was given an equal amount of physiological saline. After 15 days of continuous administration, the rats were sacrificed by neck-off and examined for visceral status.
The experimental results show that the treatment has no influence on the daily behaviors of the mice, and the viscera of the mice are not abnormal by anatomical examination. The phage auxiliary osmotic agent and the phage composition have biological safety, and the mixed phage auxiliary osmotic agent and the phage composition have no biological toxicity, and can be applied to vine and woody plant immersion.
Experimental example 2 detection of auxiliary permeation effect of eight reagents on Ralstonia solanacearum phage GP1 Single-factor experiments were performed on 5 concentration gradients of each of the reagent solutions prepared in example 1, such as sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thiaketone, calcium fatty acid, mannitol, and the like.
450mL of each of 41 bottles of 450mL of the solution of the phage GP1 for L.solanacearum (4.3X 10) was prepared as in example 36PFU/mL), respectively adding 50mL of reagent solution preparations such as sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thikone, calcium fatty acid, mannitol and the like into 40 bottles, and uniformly mixing; adding 50mL of sterile deionized water into a 41 th bottle as a reference 1, and uniformly mixing; while 500mL of sterile deionized water was used as control 2.
The liquid in 42 bottles was used to irrigate the cucumber vine. After 3h of root irrigation treatment, taking 3 samples of the roots of the cucumber vines, measuring the titer of the phage in the samples by a double-layer plate method, and calculating the increase rate of the phage in the samples. The experiment was repeated 3 times.
The phage increase rate (phage amount in plants after the addition of auxiliary penetrant-phage amount in plants after control 1 treatment)/phage amount in plants after control 1 treatment × 100%.
The measurement results are shown in table 1, in the treatment of sorbitan laurate with different dosages, 4g of treatment group is added, so that the penetration amount of the phage GP1 of the Ralstonia solanacearum is increased by 31.03 percent, and therefore 4g of dosage is selected as the optimal dosage;
in the treatment of polyoxyethylene sorbitan monopalmitate with different dosages, 4g of treatment group is added to improve the penetration of the solanaceae ralstonia phage GP1 by 34.48 percent, so 4g of dosage is selected as the optimal dosage;
in the triton treatment of different dosages, the addition of 14g of treatment group increases the penetration of the solanaceae ralstonia phage GP1 by 34.48 percent, so that 14g of dosage is selected as the optimal dosage;
in the treatment of fatty alcohol-polyoxyethylene ether with different dosages, the penetration amount of the solanaceae ralstonia phage GP1 is improved by 31.03% by 23g of treatment groups, so that 23g of treatment groups is selected as the optimal treatment group;
in the treatment of dioctyl sodium sulfosuccinate with different dosages, the penetration amount of the solanaceae ralstonia phage GP1 is improved by 58.62% by the treatment group of 15g, so that the dosage of 15g is selected as the optimal dosage;
in the treatment of different dosages of the thiaketone, the 25g treatment group improves the penetration amount of the solanaceae ralstonia phage GP1 by 61.53 percent, so that the dosage of 25g is selected as the optimal dosage;
in the treatment of calcium stearate with different doses, the 22g treatment group improves the penetration amount of the solanaceae ralstonia phage GP1 by 62.06 percent, so 22g is selected as the optimal dose;
in the treatment of mannitol with different dosages, 70g of treatment groups improve the penetration amount of the solanaceae ralstonia phage GP1 by 58.62 percent, so 70g is selected as the optimal dosage;
in conclusion, sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol polyoxyethylene ether, dioctyl sodium sulfosuccinate, thidone, calcium stearate, and mannitol are all essential components of the phage auxiliary penetrant, and the optimal dosages are 4g, 4g,14g, 23g, 15g, 25g, 22g, and 70g, respectively.
TABLE 1 penetration results of various concentrations of eight reagents on the solution of the Ralstonia solanacearum phage GP1
Figure BDA0002921592010000121
Figure BDA0002921592010000131
Experimental example 3 detection of auxiliary permeation effect of eight reagents on Ralstonia solanacearum phage GP2
5 concentration gradients of the reagent solutions of sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thikone, calcium stearate, mannitol and the like prepared in example 1 were selected to perform single-factor experiments respectively.
450mL of each of 41 bottles of 450mL of the solution of the phage GP2 for L.solanacearum (6.5X 10) was prepared as in example 46PFU/mL), 50mL of sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, and fatty alcohol polyoxy ether were added to 40 bottles of the solutionPreparing reagent solution preparations such as vinyl ether, dioctyl sodium sulfosuccinate, thioxanthone, calcium fatty acid, mannitol and the like, and uniformly mixing; adding 50mL of sterile deionized water into a 41 th bottle as a reference 1, and uniformly mixing; while 500mL of sterile deionized water was used as control 2.
The liquid in 42 bottles was used to irrigate the cucumber vine. And after root irrigation for 3 hours, taking a sample of the root of the cucumber vine, measuring the titer of the phage in the sample by a double-layer plate method, and calculating the increase rate of the phage in the sample. The experiment was repeated 3 times.
The increase rate of the cucumber rattan section phage with the diameter of 1cm is (phage amount in the plant body after the auxiliary penetrating agent is added-phage amount in the plant body after the control 1 is treated)/phage amount in the plant body after the control 1 is treated multiplied by 100%.
The measurement results are shown in table 2, in the treatment of different doses of sorbitan laurate, the addition of 4g of the treatment group increases the penetration of the solanaceae ralstonia phage GP1 by 39.13%, so that 4g of the dose is selected as the optimal dose;
in the treatment of polyoxyethylene sorbitan monopalmitate with different dosages, 4g of treatment group is added to improve the penetration amount of the solanaceae ralstonia phage GP1 by 41.30 percent, so 4g of dosage is selected as the optimal dosage;
in the treatment of the triton with different dosages, the treatment groups added with 14g and 16g both improve the penetration of the solanaceae ralstonia phage GP2 by 41.30 percent, so that the minimum dosage of 14g is selected as the optimal dosage;
in the treatment of fatty alcohol-polyoxyethylene ether with different dosages, 22g and 23g of treatment groups are added to improve the penetration of the solanaceae ralstonia phage GP2 by 28.26 percent, so that the minimum dosage of 22g is selected as the optimal dosage;
in the treatment of dioctyl sodium sulfosuccinate with different dosages, the penetration amount of the solanaceae ralstonia phage GP2 is improved by 28.26% by the treatment group of 15g, so that the dosage of 15g is selected as the optimal dosage;
in the treatment of different dosages of the thiaketone, the penetration amount of the phage GP2 of the Ralstonia solanacearum is improved by 50% by a treatment group of 25g, so that the dosage of 25g is selected as the optimal dosage;
in the treatment of calcium stearate with different dosages, 22g of treatment groups improve the penetration of the solanaceae ralstonia phage GP2 by 21.73 percent, so 22g is selected as the optimal dosage;
in the treatment of mannitol with different dosages, 70g of treatment groups improve the penetration of the solanaceae ralstonia phage GP2 by 45.65 percent, so 70g is selected as the optimal dosage;
in conclusion, sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thionone, calcium stearate and mannitol are all necessary components of the phage auxiliary penetrant, and the optimal dosages are respectively 4g, 4g,14g, 22g, 15g, 25g, 22g and 70 g.
TABLE 2 penetration results of various concentrations of eight reagents on the solution of the Ralstonia solanacearum phage GP2
Figure BDA0002921592010000151
Figure BDA0002921592010000161
Experimental example 4 detection of auxiliary permeation effect of eight reagents on Ralstonia solanacearum phage GP3
5 concentration gradients of the reagent solutions of sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thikone, calcium stearate, mannitol and the like prepared in example 1 were selected to perform single-factor experiments respectively.
31 parts of 450mL of each of the Klebsiella pneumoniae GP3 solutions of Solanaceae (5.0X 10) were prepared as in example 56PFU/mL), respectively adding 50mL of reagent solution preparations such as sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thikone, calcium ester and mannitol into a 40 th bottle, adding 50mL of sterile deionized water into a 41 th bottle as a reference 1, and uniformly mixing; while 500mL of sterile deionized water was used as control 2.
The liquid in 42 bottles was used to irrigate the cucumber vine. And after root irrigation for 3 hours, taking a sample of the root of the cucumber vine, measuring the titer of the phage in the sample by a double-layer plate method, and calculating the increase rate of the phage in the sample. The experiment was repeated 3 times. The increase rate of the cucumber rattan section phage with the diameter of 1cm is (phage amount in the plant body after the auxiliary penetrating agent is added-phage amount in the plant body after the control 1 treatment)/phage amount in the plant body after the control 1 treatment multiplied by 100%
The measurement results are shown in table 3, in the treatment of different doses of sorbitan laurate, 4g of the treatment group is added, so that the penetration of the solanaceae ralstonia phage GP1 is improved by 19.74%, and therefore 4g of the treatment group is selected as the optimal dose;
in the treatment of polyoxyethylene sorbitan monopalmitate with different dosages, 4g of treatment group is added to improve the penetration amount of the solanaceae ralstonia phage GP1 by 20.51 percent, so 4g of dosage is selected as the optimal dosage;
in the treatment of the triton with different dosages, the treatment groups added with 14g and 16g both improve the penetration of the solanaceae ralstonia phage GP3 by 17.94 percent, so that the minimum dosage of 14g is selected as the optimal dosage;
in the treatment of fatty alcohol-polyoxyethylene ether with different dosages, 23g of treatment group is added to improve the penetration of the solanaceae ralstonia phage GP3 by 20.51 percent, so 23g of dosage is selected as the optimal dosage;
in the treatment of dioctyl sodium sulfosuccinate with different dosages, the 14g treatment group improves the penetration amount of the solanaceae ralstonia phage GP3 by 22.5 percent, so that the dosage of 14g is selected as the optimal dosage;
in the treatment of different dosages of the thiaketone, the 30g treatment group improves the penetration amount of the solanaceae ralstonia phage GP3 by 25.64 percent, so that the dosage of 30g is selected as the optimal dosage;
in the treatment of calcium stearate with different dosages, 22g of treatment groups improve the penetration of the solanaceae ralstonia phage GP3 by 33.33 percent, so 22g is selected as the optimal dosage;
in the treatment of mannitol with different dosages, 70g of treatment groups improve the penetration amount of the solanaceae ralstonia phage GP3 by 30.76 percent, so 70g is selected as the optimal dosage;
in conclusion, sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol polyoxyethylene ether, dioctyl sodium sulfosuccinate, thidone, calcium stearate, and mannitol are all essential components of the phage auxiliary penetrant, and the optimal dosages are 4g, 4g,14g, 23g, 14g, 30g, 22g, and 70g, respectively.
TABLE 3 penetration results of various concentrations of eight reagents on the solution of the Ralstonia solanacearum phage GP3
Figure BDA0002921592010000181
Figure BDA0002921592010000191
Experimental example 5 auxiliary penetration effect detection of eight reagents on Xanthomonas carpi phage YHC5
5 concentration gradients of the reagent solutions of sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thikone, calcium stearate, mannitol and the like prepared in example 1 were selected to perform single-factor experiments respectively.
31 portions of 450mL of Xanthomonas campestris YHC5(3.3X 10) were prepared as described in example 66PFU/mL), respectively adding 50mL of reagent solution preparations such as sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thikone, calcium ester and mannitol into a 40 th bottle, adding 50mL of sterile deionized water into a 41 th bottle as a reference 1, and uniformly mixing; while 500mL of sterile deionized water was used as control 2.
The liquid in 42 bottles was used to irrigate the cucumber vine. And after root irrigation for 3 hours, taking a sample of the root of the cucumber vine, measuring the titer of the phage in the sample by a double-layer plate method, and calculating the increase rate of the phage in the sample. The experiment was repeated 3 times.
The increase rate of the cucumber rattan section phage with the diameter of 1cm is (phage amount in the plant body after the auxiliary penetrating agent is added-phage amount in the plant body after the control 1 treatment)/phage amount in the plant body after the control 1 treatment multiplied by 100%
The measurement results are shown in table 4, in the treatment of sorbitan laurate with different dosages, the addition of 3.5g of the treatment group increases the penetration of the phage GP1 of Ralstonia solanacearum by 25%, so that the dosage of 3.5g is selected as the optimal dosage;
in the treatment of polyoxyethylene sorbitan monopalmitate with different dosages, the addition of 3g of the treatment group improves the penetration amount of the solanaceae ralstonia phage GP1 by 34.09 percent, so that 3g of the dosage is selected as the optimal dosage;
in the triton treatment of different dosages, the 14g treatment group is added to improve the infiltration amount of the xanthomonas carpi phage YHC5 by 34.09 percent, so that 14g is selected as the optimal dosage;
in the treatment of fatty alcohol-polyoxyethylene ether with different dosages, 23g of treatment group is added to improve the permeation quantity of the solanaceae ralstonia phage GP3 by 31.81 percent, so 23g of dosage is selected as the optimal dosage;
in the treatment of dioctyl sodium sulfosuccinate with different dosages, the addition of 15g of treatment group improves the penetration amount of the Ralstonia solanacearum phage GP3 by 20.54 percent, so 23g of dosage is selected as the optimal dosage;
in the treatment of different dosages of the thiaketone, the penetration of the xanthomonas carpi phage YHC5 was improved by 20.54% for the treatment group of 25g, so that the optimal dosage was selected as 25 g;
in the treatment of different doses of calcium stearate, 22g of the treatment group improves the penetration amount of the xanthomonas carpi phage YHC5 by 29.54%, so 22g is selected as the optimal dose;
in the treatment of different doses of mannitol, 70g of the treatment group improves the penetration amount of the xanthomonas carpi phage YHC5 by 31.81 percent, so 70g is selected as the optimal dose;
in conclusion, sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol polyoxyethylene ether, dioctyl sodium sulfosuccinate, thidone, calcium stearate, and mannitol are all essential components of the phage auxiliary penetrant, and the optimal dosages are 3.5g, 3g, 14g, 23g, 15g, 25g, 22g, and 70g, respectively.
TABLE 4 results of permeation of different concentrations of eight reagents on the Xanthomonas campestris YHC5 solution
Figure BDA0002921592010000211
Figure BDA0002921592010000221
Experimental example 6 auxiliary permeation effect detection of eight reagents on Pseudomonas syringae Kiwi berry pathovar phage PSA-P1 Single factor experiments were performed on 5 concentration gradients of each of the reagent solutions prepared in example 1, such as sorbitan laurate, polyoxyethylene sorbitan monopalmitate, Triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thidanone, calcium stearate, mannitol, and the like.
31 parts of 450mL each of Pseudomonas syringae Actinidia var typhimurium phage PSA-P1 (5.1X 10) were prepared as in example 76PFU/mL), respectively adding 50mL of reagent solution preparations such as sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thikone, calcium ester and mannitol into a 40 th bottle, adding 50mL of sterile deionized water into a 41 th bottle as a reference 1, and uniformly mixing; while 500mL of sterile deionized water was used as control 2.
The liquid in 42 bottles was used to irrigate the cucumber vine. And after root irrigation for 3 hours, taking a sample of the root of the cucumber vine, measuring the titer of the phage in the sample by a double-layer plate method, and calculating the increase rate of the phage in the sample. The experiment was repeated 3 times.
Phage increase rate (phage amount in plant after adding auxiliary penetrant-phage amount in plant after control 1 treatment)/phage amount in plant after control 1 treatment × 100%
The measurement results are shown in table 5, in the treatment of different doses of sorbitan laurate, the addition of 3g of the treatment group increases the penetration of the solanaceae ralstonia phage GP1 by 19.44%, so that 3g of the dose is selected as the optimal dose;
in the treatment of polyoxyethylene sorbitan monopalmitate with different dosages, the addition of 3g of treatment group leads the penetration amount of the solanaceae ralstonia phage GP1 to be improved by 27.77 percent, so the dosage of 3g is selected as the optimal dosage;
in the treatment of the triton with different dosages, the addition of 14g of treatment groups improves the permeation quantity of PSA-P1 of the pseudomonas syringae kiwi pathopoiesia variety phage by 27.77 percent, so 14g is selected as the optimal dosage;
in the treatment of fatty alcohol-polyoxyethylene ether with different dosages, the permeation quantity of pseudomonas syringae kiwi fruit pathopoiesia variety phage PSA-P1 is improved by 22.22% by 23g of treatment groups, so that 23g of treatment groups is selected as the optimal treatment group;
in the treatment of different doses of dioctyl sodium sulfosuccinate, the penetration of the pseudomonas syringae kiwi fruit pathopoiesia phage PSA-P1 is improved by 22.2% by 15g of treatment groups, so that the optimal dose is selected as 15g of treatment groups;
in the treatment of different dosages of the thiaketone, the penetration amount of the pseudomonas syringae kiwifruit pathopoiesia variety phage PSA-P1 is improved by 33.3% by 30g of treatment groups, so that the optimal dosage is selected as 30 g;
in the treatment of calcium stearate with different dosages, 22g of treatment groups improve the permeation quantity of PSA-P1, so that 22g is selected as the optimal dosage;
in the treatment of mannitol with different dosages, 80g of treatment groups improve the infiltration capacity of pseudomonas syringae kiwifruit pathopoiesia variety phage PSA-P1 by 38.88 percent, so 80g is selected as the optimal dosage;
in conclusion, sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol polyoxyethylene ether, dioctyl sodium sulfosuccinate, thidone, calcium stearate, and mannitol are all essential components of the phage auxiliary penetrant, and the optimal dosages are 3g, 3g, 14g, 23g, 15g, 30g, 22g, and 80g, respectively.
TABLE 5 penetration results of various concentrations of eight reagents into Pseudomonas syringae Kiwi pathovar phage PSA-P1 solution
Figure BDA0002921592010000241
Figure BDA0002921592010000251
Experimental example 7 Effect of concentration of phage-assisted penetrant on the effect of phage-immersing in cucumber rattan
Each of the phages GP1, GP2, GP3, YHC5, PSA-P1 solution and phage composition solution prepared in example 3 to example 7 and example 9 was taken in 5 parts. To this was added the auxiliary penetrants (prepared in example 2) at final concentrations of 5%, 12%, 15%, 20% and 25%, respectively (v/v ratio to phage) and mixed well, making the total volume of the solution 500mL and the titer 8.2X104PFU/mL; 500mL of sterile deionized water was also used as a control. The cucumber stems are irrigated with roots respectively. And (3) after root irrigation treatment for 3 hours, taking a sample of the root of the cucumber vine, and measuring the titer of the phage in the sample by a double-layer plate method. The experiment was repeated 3 times.
The results are shown in table 6, and in the six treatments, each phage in the root of cucumber vine has higher titer at final concentrations of auxiliary penetrant of 12% and 15%. The optimal working concentration of the phage auxiliary penetrant for immersing the roots of the cucumber vines is 12% (the concentration is calculated by the volume ratio of the auxiliary penetrant to the phage to obtain v/v) by combining comprehensive factors such as production cost and the like.
TABLE 6 phage titer in different concentrations of phage-assisted penetrant treatment in cucumber rattan roots
Figure BDA0002921592010000261
Experimental example 8 Effect of concentration of phage-assisted penetrant on penetration of phage into citrus leaves
The phages GP1, GP2, GP3, YHC5 and GP1 prepared in example 3-example 7 and example 9, respectively,The PSA-P1 solution and the phage composition solution were each 5 parts. Phage-assisted permeabilizers (prepared in example 2) were added to final concentrations of 5%, 12%, 15%, 20%, and 25% (v/v volume ratio to phage) respectively and mixed well such that the total volume of the solution was 500mL and the titer was 8.2X104PFU/mL; 500mL of sterile deionized water was also used as a control. And 3mL of the liquid is taken, respectively and uniformly sprayed on the surface of citrus leaves with the diameter of about 5cm, and after standing for 2h at room temperature, the titer of the phage in the citrus leaves in each treatment is measured. The experiment was repeated 3 times.
The results are shown in table 7, where phage in citrus leaves had higher titers at both the final concentrations of the auxiliary penetrant of 12% and 15% in the six treatments. The optimal working concentration of the phage auxiliary penetrant for dipping the treated citrus leaves is 12% (v/v) by combining comprehensive factors such as production cost and the like.
TABLE 7 bacteriophage potency in treatment of citrus leaves with varying concentrations of bacteriophage-assisted penetrant
Figure BDA0002921592010000262
Figure BDA0002921592010000271
Experimental example 9 Effect of storage conditions on Effect of phage-assisted penetrant
The phage auxiliary penetrant (prepared in example 2) was placed at 4 deg.C, 25 deg.C and 37 deg.C, respectively, and removed every 6 months until 18 months. This was compared to a titer of 8.2 × 10 prepared from example 3-example 7 and example 9, respectively4The PFU/mL phage GP1, GP2, GP3, YHC5, PSA-P1 solution and the phage composition solution were mixed well to give a final concentration of the auxiliary penetrant of 12% (v/v ratio to phage). The above solutions were prepared in 8 portions each of 500 mL. Uniformly spraying 3mL of the above liquid onto surface of citrus leaf with diameter of about 5cm, standing at room temperature for 2 hr, measuring bacteriophage titer in each citrus leaf, and repeating the experiment for 3 times, wherein the juice is used as citrus leafAnd (5) sheet processing I. The cucumber stems are irrigated with roots respectively. After 3h of root irrigation treatment, taking a sample of the cucumber vine root, measuring the titer of phage in the sample by a double-layer plate method, and repeating the experiment for 3 times, wherein the experiment is used as cucumber vine root treatment I.
The titres obtained from example 8 and example 10 were taken as 8.2X10 respectively4PFU/mL phage GP1, GP2, GP3, YHC5, PSA-P1 solution and phage composition solution with auxiliary penetrant were each 8 parts, 500mL each. Taking 3mL of each of the above liquids, uniformly spraying the liquid on the surface of citrus leaf with the diameter of about 5cm, standing at room temperature for 2h, measuring the titer of phage in the citrus leaf in each process, and repeating the experiment for 3 times, wherein the experiment is used as a control I of the citrus leaf. The cucumber stems are irrigated with roots respectively. After 3h of root irrigation treatment, a sample of the cucumber vine root is taken, the phage titer in the sample is determined by a double-layer plate method, and the experiment is repeated for 3 times, wherein the sample is used as a cucumber vine root control I. The auxiliary penetrant used in control I is a freshly prepared auxiliary penetrant.
The titres of 8.2X10 from example 3 to example 7 and example 9, respectively, were taken48 portions of each of the phage GP1, GP2, GP3, YHC5, PSA-P1 solution and the phage composition solution of PFU/mL, each of 440mL, to which 60mL of sterile deionized water was added and mixed well. Taking 3mL of each of the above liquids, uniformly spraying the liquid on the surface of citrus leaf with diameter of about 5cm, standing at room temperature for 2h, measuring the titer of phage in the citrus leaf in each process, and repeating the experiment for 3 times, wherein the result is used as a control II of the citrus leaf. The cucumber stems are irrigated with roots respectively. After 3h of root irrigation treatment, a sample of the cucumber vine root is taken, the phage titer in the sample is determined by a double-layer plate method, and the experiment is repeated for 3 times, wherein the sample is used as a cucumber vine root control II.
And (3) taking 8 parts of 3mL sterile deionized water, uniformly spraying the sterile deionized water on the surfaces of citrus leaves with the diameter of about 5cm, standing at room temperature for 2h, measuring the titer of the phage in the citrus leaves in each process, and repeating the experiment for 3 times, wherein the experiment is used as a citrus leaf control III.
And 8 parts of 500mL sterile deionized water is taken and used for respectively irrigating roots of cucumber vines. After 3h of root irrigation treatment, a sample of the cucumber vine root is taken, the phage titer in the sample is determined by a double-layer plate method, and the experiment is repeated for 3 times, wherein the sample is used as a cucumber vine root control III.
The effect of long-term standing of the phage-assisted penetrant at 4 ℃ on the permeation effect is shown in tables 8-1 to 8-3: after the phage auxiliary penetrating agent is stored for 18 months at 4 ℃, the auxiliary penetrating effect is similar to that of the newly configured penetrating agent, and the titer of the phage in the citrus leaf and the cucumber vine root is close to that of the phage spraying liquid.
The effect of long-term standing of the phage-assisted penetrant at 25 ℃ on permeation is shown in tables 8-4 to 8-6: after the phage auxiliary penetrant is stored for 12 months at 25 ℃, the auxiliary penetrant effect is similar to the effect (contrast I) of the newly configured penetrant, and the titer of the phage in the citrus leaves and the roots of the cucumber vines are close to the titer of the phage spraying liquid; after 18 months of storage, the auxiliary permeation effect is weakened, the titer of the phage in the citrus leaves and the roots of the cucumber vines is the same as the magnitude order of the titer of the phage spraying liquid, but the auxiliary permeation effect is still far better than the permeation effect of a pure phage solution (control II).
The effect of long-term standing of the phage-assisted penetrant at 37 ℃ on penetration is shown in tables 8-7 to 8-9: after the phage auxiliary penetrant is stored for 6 months at 37 ℃, the auxiliary penetrant effect is similar to the effect (contrast I) of the newly configured penetrant, and the titer of the phage in the citrus leaves and the roots of the cucumber vines is close to the titer of the phage spraying liquid; the auxiliary permeation effect is weakened after the storage for 12 months; the titer of the phage in the citrus leaves and the roots of the cucumber vines stored for 18 months is the same as the titer of the phage spray solution, but the auxiliary permeation effect of the phage spray solution is still far better than that of the pure phage solution (control II).
The test results show that the phage auxiliary penetrant has good stability at 4 ℃, and the auxiliary penetrant can be stored for 18 months without influencing the auxiliary penetration effect; the stable storage time of the phage auxiliary penetrant at 25 ℃ and 37 ℃ is 12 months and 6 months, respectively. The phage auxiliary osmotic preparation is easy to store, can be placed in various environments, can be used for multiple times after being prepared once, and is convenient and fast to apply.
TABLE 8-1 phage titer (PFU/mL) in citrus leaf and cucumber vine root after penetration by different phage treatment
Figure BDA0002921592010000281
Figure BDA0002921592010000291
TABLE 8-2 phage titer in Citrus leaves in tubers after immersion with different phage treatment (PFU/mL)
Figure BDA0002921592010000292
Figure BDA0002921592010000301
TABLE 8-3 phage titer in Citrus leaf after immersion of different phage treatments (PFU/mL)
Figure BDA0002921592010000302
TABLE 8-4 phage titer in Citrus leaves after penetration by different phage treatment (PFU/mL)
Figure BDA0002921592010000303
Figure BDA0002921592010000311
TABLE 8-5 phage titer in Citrus leaf after immersion for different phage treatments (PFU/mL)
Figure BDA0002921592010000312
Figure BDA0002921592010000321
TABLE 8-6 phage titer in Citrus leaf after immersion for different phage treatments (PFU/mL)
Figure BDA0002921592010000322
TABLE 8-7 phage titer in Citrus leaf after immersion for different phage treatments (PFU/mL)
Figure BDA0002921592010000323
Figure BDA0002921592010000331
TABLE 8-8 phage titer in Citrus leaves after immersion for different phage treatments (PFU/mL)
Figure BDA0002921592010000332
Figure BDA0002921592010000341
TABLE 8-9 phage titer in Citrus leaves after immersion for different phage treatments (PFU/mL)
Figure BDA0002921592010000342
The results of the above experimental examples show that the phage-assisted penetrant preparation has good long-term stability (> 6m) at different temperatures and is easy to store. Meanwhile, the phage auxiliary osmotic preparation can continuously and effectively help the phage to be attached to the bodies of the vines and the woody plants, so that the effective concentration of the phage in the bodies of the vines and the woody plants is maintained.
The auxiliary penetrating agent can obviously accelerate the speed of the bacteriophage entering the bodies of the vines and the woody plants, and simultaneously obviously increase the quantity of the bacteriophage entering the bodies of the vines and the woody plants in unit time.
The method greatly saves the time of researchers for processing samples, and can reduce the influence of long-time immersion on the quality of the vines and the woody plants, thereby being beneficial to preventing the foreign bacterial diseases of the vines and the woody plants.
The auxiliary penetrant of the present application is applicable to other lianas or woody plants in addition to the above-mentioned cucumber or citrus, and should not be limited herein.
The above embodiments are all preferred embodiments of the present application, and the scope of protection of the present application is not limited thereby, so: all equivalent changes made according to the principles of the present application should be covered by the protection scope of the present application.

Claims (10)

1. An auxiliary penetrant suitable for use in bacteriophage impregnation of lianas and woody plants, wherein the auxiliary penetrant comprises at least: one or more of sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol-polyoxyethylene ether, dioctyl sodium sulfosuccinate, thioxanthone, calcium stearate and mannitol.
2. The auxiliary osmotic agent suitable for phage infusion into lianas and woody plants according to claim 1 wherein said auxiliary osmotic agent comprises sorbitan laurate, polyoxyethylene sorbitan monopalmitate, triton, fatty alcohol polyoxyethylene ether, dioctyl sodium sulfosuccinate, thixene, calcium stearate and mannitol in the following amounts: based on the weight percentage of the components,
0% -0.04% of sorbitan laurate;
0% -0.04% of polyoxyethylene sorbitan monopalmitate;
the dosage of the triton is 0.8-1.6%;
the dosage of the fatty alcohol-polyoxyethylene ether is 2-2.4%;
the dosage of the dioctyl sodium sulfosuccinate is 1-2.6%;
the dose of the thiaketone is 1-3%;
the dosage of the calcium stearate is 1.2-2.4%;
the dosage of the mannitol is 4-8%;
the balance being deionized water.
3. The auxiliary penetrant for phage immersion in lianas and woody plants according to claim 2, wherein said auxiliary penetrant comprises:
an auxiliary penetrant for a ralstonia solanacearum bacteriophage comprising the following components in dosage: based on the weight percentage of the components,
sorbitan laurate: 0.4 percent;
polyoxyethylene sorbitan monopalmitate: 0.4 percent;
triton: 1.4 percent;
fatty alcohol polyoxyethylene ether: 2.3 percent;
dioctyl sodium sulfosuccinate: 1.5 percent;
and (3) thiaketone: 2.5 percent;
calcium stearate: 2.2 percent;
mannitol: 7 percent;
the balance of deionized water;
or a secondary penetrant for xanthomonas carpi phages, comprising the following components in the following dosage: based on the weight percentage of the components,
sorbitan laurate: 0.35 percent;
polyoxyethylene sorbitan monopalmitate: 0.3 percent;
triton: 1.4 percent;
fatty alcohol polyoxyethylene ether: 2.3 percent;
dioctyl sodium sulfosuccinate: 1.5 percent;
and (3) thiaketone: 2.5 percent;
calcium stearate: 2.2 percent;
mannitol: 7 percent;
the balance of deionized water;
or an auxiliary penetrant for pseudomonas syringae kiwi pathovar phage, comprising the following components in dosage: based on the weight percentage of the components,
sorbitan laurate: 0.3 percent;
polyoxyethylene sorbitan monopalmitate: 0.3 percent;
triton: 1.4 percent;
fatty alcohol polyoxyethylene ether: 2.3 percent;
dioctyl sodium sulfosuccinate: 1.5 percent;
and (3) thiaketone: 3 percent;
calcium stearate: 2.2 percent;
mannitol: 8 percent;
the balance of deionized water;
or a secondary penetrant for use in a phage composition, comprising the following components in dosages: based on the weight percentage of the components,
sorbitan laurate: 0.35 percent;
polyoxyethylene sorbitan monopalmitate: 0.3 percent;
triton: 1.4 percent;
fatty alcohol polyoxyethylene ether: 2.3 percent;
dioctyl sodium sulfosuccinate: 1.5 percent;
and (3) thiaketone: 2.5 percent;
calcium stearate: 2.2 percent;
mannitol: 7 percent;
the balance being deionized water.
4. The auxiliary penetrant for phage immersion in vines and woody plants according to claim 3, wherein said auxiliary penetrant can be stored for 18 months at 4 ℃; can be stored for 12 months at 25 ℃; can be stored at 37 deg.C for 6 months.
5. The method for preparing the auxiliary penetrant suitable for bacteriophage to be impregnated into lianas and woody plants according to any one of claims 1 to 4, comprising the steps of:
(1) adding 0-0.04% of sorbitan laurate, 0-0.04% of polyoxyethylene sorbitan monopalmitate, 0.8-1.6% of triton, 2-2.4% of fatty alcohol-polyoxyethylene ether and 1-2.6% of dioctyl sodium sulfosuccinate into 1/2 weight of water, heating to dissolve, and uniformly stirring by magnetic force for later use;
(2) adding 1-3% of thiazone, 1.2-2.4% of calcium stearate and 4-8% of mannitol into 1/2 weight of water, dissolving at normal temperature, and uniformly stirring by magnetic force for later use;
(3) adding the mixed solution obtained in the step (1) into the solution obtained in the step (2) for mixing, and uniformly stirring by magnetic force;
(4) and (4) sterilizing the mixed solution obtained in the step (3) at 121 ℃ for 20min, and storing at 4 ℃ for later use to obtain the phage auxiliary penetrant.
6. The method for preparing an auxiliary penetrant suitable for bacteriophage to be impregnated into lianas and woody plants according to claim 5, wherein the auxiliary penetrant for the bacteriophage of Ralstonia solanacearum is prepared as follows: weighing 4g of sorbitan laurate, 4g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 30g of thiaketone, 22g of calcium stearate and 70g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the mixture is fully dissolved, and adding deionized water to reach the constant volume of 1000 mL; sterilizing at 121 deg.C for 20min to obtain bacteriophage auxiliary penetrant, and storing at 4 deg.C;
or the preparation method of the auxiliary penetrant for the xanthiomonas carpet grass bacteriophage is as follows: weighing 3.5g of sorbitan laurate, 3g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 25g of thiaketone, 22g of calcium stearate and 70g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the mixture is fully dissolved, and adding the deionized water to the volume of 1000 mL; sterilizing at 121 deg.C for 20min to obtain bacteriophage auxiliary penetrant, and storing at 4 deg.C;
or the preparation method of the auxiliary penetrant for the pseudomonas syringae kiwi pathopoiesia variety phage comprises the following steps: weighing 3g of sorbitan laurate, 3g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 30g of thiaketone, 22g of calcium stearate and 80g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the mixture is fully dissolved, and adding deionized water to reach the constant volume of 1000 mL; sterilizing at 121 deg.C for 20min to obtain bacteriophage auxiliary penetrant, and storing at 4 deg.C;
or the auxiliary penetrant for phage composition is prepared as follows: weighing 35g of sorbitan laurate, 3g of polyoxyethylene sorbitan monopalmitate, 14g of triton, 23g of fatty alcohol-polyoxyethylene ether, 15g of dioctyl sodium sulfosuccinate, 25g of thiaketone, 22g of calcium stearate and 70g of mannitol, adding a proper amount of deionized water, heating to 20-30 ℃, magnetically stirring until the mixture is fully dissolved, and adding deionized water to reach the constant volume of 1000 mL; sterilizing at 121 deg.C for 20min to obtain bacteriophage auxiliary penetrant, and storing at 4 deg.C.
7. The method for applying the auxiliary penetrant suitable for immersing the bacteriophage into the lianas and the woody plants according to any one of claims 1 to 4, wherein the auxiliary penetrant is mixed into the bacteriophage solution at a final concentration of 12%, and is stirred uniformly.
8. The method of using the auxiliary penetrant appropriate for bacteriophage to penetrate lianas and woody plants according to claim 7, wherein the auxiliary penetrant is added to the bacteriophage solution under aseptic conditions and mixed uniformly in a volume ratio of 1:9 to obtain the bacteriophage preparation with the auxiliary penetrant.
9. The method of claim 8, wherein the phage is a Ralstonia solanacearum phage or a Xanthomonas carpi phage or a Pseudomonas syringae Actinidia var. syringae phage or a combination of the three phages.
10. The method of applying a secondary penetrant suitable for bacteriophage immersion in lianas and woody plants according to claim 9, wherein said application method comprises: the optimum treatment concentration of the auxiliary penetrant for bacteriophage immersion in vines and woody plants is 12%.
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