阅读说明：本技术 一种分离土壤中抗花叶病毒细菌的工艺 (Process for separating anti-mosaic virus bacteria in soil ) 是由 张国基 张希兰 汤燕雯 赵甜 于 2019-10-16 设计创作，主要内容包括：本发明公开了一种分离土壤中抗花叶病毒细菌的工艺,包括以下步骤：(1)取1.0g土样,放入瓶内,加入无菌水,密封后震荡培养1-3h；(2)将步骤(1)中震荡培养后的溶液静置20-60min后,取上清液梯度稀释,混合均匀后每个浓度取80-120μL分别均匀涂布于培养基的平板上；(3)将平板在25-30℃恒温培养箱中培养15-20h；(4)取出培养后的平板,从中选择细菌菌落分布均匀的平板,挑取单斑放至2-5mL培养液中震荡培养10-20h,形成菌液。本发明公开了从不同生境(浅海海泥和烟草根际土壤)采集的样品中分离对花叶病毒病毒具有抑制作用的细菌,丰富抗病毒有益细菌的种类,并进一步简单测定抗病毒效果,为抗花叶病毒细菌的利用奠定了基础。(The invention discloses a process for separating mosaic virus-resistant bacteria in soil, which comprises the following steps: (1) taking 1.0g of soil sample, placing into a bottle, adding sterile water, sealing, and culturing for 1-3h with shaking; (2) standing the solution subjected to shake culture in the step (1) for 20-60min, taking supernatant for gradient dilution, uniformly mixing, and respectively uniformly coating 80-120 mu L of each concentration on a flat plate of a culture medium; (3) culturing the flat plate in a constant temperature incubator at 25-30 ℃ for 15-20 h; (4) and taking out the cultured plate, selecting a plate with uniformly distributed bacterial colonies from the plate, picking a single spot, placing the single spot into 2-5mL of culture solution, and performing shake culture for 10-20h to form a bacterial solution. The invention discloses a method for separating bacteria with inhibiting effect on mosaic virus from samples collected from different environments (shallow sea mud and tobacco rhizosphere soil), enriching the types of beneficial antiviral bacteria, further simply determining antiviral effect and laying a foundation for the utilization of the mosaic virus resisting bacteria.)

1. A process for the isolation of anti-mosaic virus bacteria in soil, comprising the steps of:

(1) taking 1.0g of soil sample, placing into a bottle, adding sterile water, sealing, performing shake culture for 1-3h, performing shake culture, standing the culture solution for 20-60min, taking supernatant, diluting in gradient, and uniformly mixing, and uniformly coating 80-120 mu L of each concentration on a plate of a culture medium;

(2) selecting a flat plate with uniformly distributed bacterial colonies from the flat plate, picking a single spot, placing the single spot into 2-5mL of culture solution, and performing shake culture for 10-20h to form a bacterial solution;

(3) centrifuging 1mL of bacterial liquid for 8-12min, mixing the supernatant with the mosaic virus inoculation liquid with the same volume, and standing at room temperature for 15-30min to form a mixed liquid;

(4) mixing the mosaic virus inoculation liquid with an equal volume of phosphate buffer solution, and standing at room temperature for 15-30min to form a control mixed liquid;

(5) uniformly spreading quartz sand on the Sansheng tobacco leaves by taking the Sansheng tobacco as an experimental material, and slightly rubbing the mixed liquid on half leaves by taking the main veins of the Sansheng tobacco leaves as a boundary; the other half of the leaves are rubbed to contrast the mixed solution;

(6) rubbing the mixed solution and the control mixed solution for 15-30min, spraying sterile water to clean the leaves, and naturally air-drying at room temperature;

(7) carrying out isolation culture in a greenhouse for 2-4d, then counting the number of dead spots on leaves, carrying out one-factor variance analysis on experimental data, carrying out significant difference analysis, and counting the antiviral effect according to the following formula:

mosaic virus.

2. The process for the isolation of anti-mosaic virus bacteria in soil according to claim 1, wherein sterile water is added in the amount of 7-12mL in step (1).

3. The process for separating mosaic virus-resistant bacteria in soil according to claim 1, wherein step (1) and/or step (4) is/are shaken at 140rpm and 100 ℃ to 30 ℃.

4. Process for the isolation of anti-mosaic-virus bacteria in soil according to claim 1, characterized in that the supernatant of step (2) is diluted 10 in gradient⁰、10^-1、10^-2、10^-3、10^-4And 10^-5。

5. The process for separating anti-mosaic virus bacteria in soil according to claim 1, wherein said culture medium is LB solid medium.

6. The process for separating anti-mosaic virus bacteria in soil according to claim 1, wherein said culture solution is LB liquid medium.

7. The process for the isolation of anti-mosaic virus bacteria in soil according to claim 1, wherein said soil sample is taken from shallow sea mud or tobacco rhizosphere soil.

8. The process for the isolation of anti-mosaic virus bacteria in soil according to claim 1, wherein said triclosan is cultured under conditions of 16 hours of light duration, 2000lux of light intensity, 8 hours of dark duration, 25 ± 1 ℃ of temperature, 60% of relative humidity.

9. The process for the isolation of anti-mosaic virus bacteria in soil according to claim 8, wherein said step (7) employs three-week-old tobacco cultivation.

10. The process for the isolation of anti-mosaic virus bacteria in soil according to claim 1, wherein the preparation of the mosaic virus inoculation solution comprises the steps of:

(1) grinding part of tobacco leaves infected with mosaic virus with 10mmol/L, pH 7.4.4 phosphate buffer solution, and homogenizing;

(2) filtering the serous fluid generated in the step (1) by using double-layer gauze, and centrifuging;

(3) taking the supernatant centrifuged in the step (2), treating the supernatant with polyethylene glycol twice, and then centrifuging again;

(4) and (4) after the final centrifugation in the step (3), taking the precipitate, and carrying out heavy suspension by using 10mmol/L, pH 7.4.4 phosphate buffer solution to obtain the mosaic virus inoculation solution.

Technical Field

The invention relates to the technical field of microbial ecology, in particular to a process for separating mosaic virus-resistant bacteria in soil.

Background

At present, modes such as breeding of antiviral varieties, control of virus-carrying media, cross protection and the like are mainly adopted in the treatment of virus diseases, and few effective chemical pesticides aiming at plant virus diseases need to be solved by agricultural scientific researchers. The Mosaic Virus (TMV) is a pathogen of tobacco mosaic disease and the like, has wide host range, can infect at least 9 125 plants of families such as cruciferae, solanaceae and cucurbitaceae, has high occurrence frequency, is quick in prevalence, is serious in harm and difficult to treat, cannot be effectively controlled once the plants are infected, is very easy to cause the prevalence of the virus disease, and causes serious economic loss. Economic losses due to mosaic virus are as high as billions of dollars per year.

It has been found that many bacteria and fungi and their metabolites have a certain ability to inactivate the particles of the mosaic virus, but there is no good way to isolate the anti-mosaic virus bacteria. After plant viruses are contaminated by bacteria, the sap thereof can lose the infection activity quickly, and then some researchers are engaged in the research of the antiviral activity of microorganisms and metabolites thereof. Therefore, it is necessary to provide a further solution to the above problems.

Disclosure of Invention

The invention aims to provide a process for separating mosaic virus-resistant bacteria in soil, so as to overcome the defects in the prior art.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a process for isolating anti-mosaic virus bacteria in soil comprising the steps of:

(1) taking 1.0g of soil sample, placing into a bottle, adding sterile water, sealing, and culturing for 1-3h with shaking;

(2) standing the solution subjected to shake culture in the step (1) for 20-60min, taking supernatant for gradient dilution, uniformly mixing, and respectively uniformly coating 80-120 mu L of each concentration on a flat plate of a culture medium;

(3) culturing the flat plate in a constant temperature incubator at 25-30 ℃ for 15-20 h;

(4) taking out the cultured plate, selecting a plate with uniformly distributed bacterial colonies from the plate, picking out a single spot, placing the single spot into 2-5mL of culture solution, and performing shake culture for 10-20h to form a bacterial solution;

(5) centrifuging 1mL of bacterial liquid for 8-12min, mixing the supernatant with the mosaic virus inoculation liquid with the same volume, and standing at room temperature for 15-30min to form a mixed liquid;

(6) mixing the mosaic virus inoculation liquid with an equal volume of phosphate buffer solution, and standing at room temperature for 15-30min to form a control mixed liquid;

(7) uniformly spreading quartz sand on the Sansheng tobacco leaves by taking the Sansheng tobacco as an experimental material, and slightly rubbing the mixed liquid on half leaves by taking the main veins of the Sansheng tobacco leaves as a boundary; the other half of the leaves are rubbed to contrast the mixed solution;

(8) rubbing the mixed solution and the control mixed solution for 15-30min, spraying sterile water to clean the leaves, and naturally air-drying at room temperature;

(9) carrying out isolation culture in a greenhouse for 2-4d, then counting the number of dead spots on leaves, carrying out one-factor variance analysis on experimental data, carrying out significant difference analysis, and counting the antiviral effect according to the following formula:

mosaic virus.

Preferably, 7-12mL of sterile water is added in step (1).

Preferably, the step (1) and/or the step (4) is/are oscillated at 100-140rpm at 25-30 ℃.

Preferably, the supernatant liquid obtained in the step (2) is diluted by a gradient 10⁰、10^-1、10^-2、10^-3、10^-4And 10^-5。

Preferably, the culture medium adopts LB solid culture medium.

Preferably, the culture solution is an LB liquid medium.

Preferably, the soil sample is taken from shallow sea mud or tobacco rhizosphere soil.

Preferably, the three-generation tobacco is cultured under the conditions of illumination time of 16h, illumination intensity of 2000lux, dark time of 8h, temperature of 25 +/-1 ℃ and relative humidity of 60%.

Preferably, step (7) employs three-generation tobacco cultured for four weeks.

Preferably, the preparation method of the mosaic virus inoculation liquid comprises the following steps:

(1) grinding part of tobacco leaves infected with mosaic virus with 10mmol/L, pH 7.4.4 phosphate buffer solution, and homogenizing;

(2) filtering the serous fluid generated in the step (1) by using double-layer gauze, and centrifuging;

(3) taking the supernatant centrifuged in the step (2), treating the supernatant with polyethylene glycol twice, and then centrifuging again;

(4) and (4) after the final centrifugation in the step (3), taking the precipitate, and carrying out heavy suspension by using 10mmol/L, pH 7.4.4 phosphate buffer solution to obtain the mosaic virus inoculation solution.

Preferably, the preparation of the mosaic virus inoculum is operated at 4 ℃.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses a process for separating mosaic virus-resistant bacteria in soil, which separates bacteria with inhibiting effect on mosaic virus from samples collected from different habitats (shallow sea mud and tobacco rhizosphere soil), enriches the types of beneficial bacteria for resisting virus, further simply determines the antiviral effect and lays a foundation for the utilization of the mosaic virus-resistant bacteria.

Detailed Description

The conjunction "consisting of …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "optional" or "any" means that the subsequently described event or events may or may not occur, and that the description includes instances where the event occurs and instances where it does not.