CN110616147A - Process for separating anti-mosaic virus bacteria in soil - Google Patents
Process for separating anti-mosaic virus bacteria in soil Download PDFInfo
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- CN110616147A CN110616147A CN201910981778.8A CN201910981778A CN110616147A CN 110616147 A CN110616147 A CN 110616147A CN 201910981778 A CN201910981778 A CN 201910981778A CN 110616147 A CN110616147 A CN 110616147A
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Abstract
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.
Description
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.
Example 1:
the plant material used in the examples of the present application was a mosaic virus of the blight host triorganophony (nicotiana. tabacum cv. sunnamnn), which was grown by seed culture by the present applicant and cultivated in a greenhouse. Plant culture conditions: the illumination is 16h, the temperature is 25 +/-1 ℃, the illumination intensity is 2000lux, and the relative humidity is 60 percent; the dark time is 8h, the temperature is 25 +/-1 ℃, and the relative humidity is 60%. The nutrient medium is produced by a Ward nutrient soil processing factory in Shouguang city in Shandong province, and additional fertilization is not needed during the growth of the plants.
The mosaic virus source is collected from Qingdao city, Shandong province, and propagated and preserved on common tobacco NC89 cultivated in a greenhouse.
The soil samples are respectively collected from tobacco rhizosphere soil and shallow sea mud.
Preparation of mosaic virus inoculation liquid: the mosaic virus solution was prepared using the method of Gooding & Helbert (1967) with minor modifications. Taking new upper leaves of NC89 infected with mosaic virus, grinding with 10mmol/L phosphate buffer solution (pH 7.4), and homogenizing; then filtered with double layer gauze and centrifuged (1000 Xg, 20 min); the supernatant was taken and treated twice with polyethylene glycol. Centrifuging again (10000 Xg, 30 min); the precipitate was resuspended in 10mmol/L PBS buffer (pH 7.4) to obtain the mosaic virus inoculum. The whole operation is carried out at 4 ℃, then a spectrophotometer is used for measuring the absorbance value of 260nm wavelength, and the virus concentration is calculated according to a formula.
Concentration of virus (mg/mL) ═ A260 Xdilution factor/E0.1％ 1cm260nm
Note: e isExtinction coefficient, i.e. the value of the optical absorption (optical density) at an optical path length of 1crn for a suspension with a concentration of 0.1% (1mg/mL) at a wavelength of 260 nm. E of mosaic Virus0.1％ 1cmThe concentration of the virus mother liquor is 10 mug/mL, and the concentration of the virus mother liquor is 3.1 at 260 nm.
A process for isolating anti-mosaic virus bacteria in soil comprising the steps of:
(1) a1.0 g soil sample was taken, placed in a sterile Erlenmeyer flask, added with 9mL of sterile water (operating in a clean bench), sealed and cultured with shaking for 2 hours (28 ℃,120 rpm).
(2) Taking out, standing in a super clean bench for 30min, and taking supernatant for gradient dilution 100、10-1、10-2、10-3、10-4And 10-5After mixing, 100. mu.L of each concentration was applied to a plate of LB solid medium.
(3) The LB plates were incubated for 16h at 28 ℃ in an incubator.
(4) Taking out, selecting a plate with uniformly distributed bacterial colonies (with moderate colony number), picking a single spot, and placing the single spot into 3mL of LB liquid culture medium for shake culture for 16h (28 ℃,120 rpm).
(5) Taking 1mL of bacterial liquid, centrifuging at 6000 Xg for 10min, taking supernatant, mixing with the mosaic virus inoculation liquid with the same volume, and standing at room temperature for 20 min.
(6) Mixing the mosaic virus inoculation liquid with an equal volume of phosphate buffer solution, and standing at room temperature for 20min to form a control mixed liquid;
(7) the antiviral effect of the bacterial liquid is detected by adopting a 'half-leaf method': uniformly spreading quartz sand on Sansheng tobacco leaves by taking Sansheng tobacco (NN) cultured for 4 weeks as an experimental material, dipping the mixed solution by using a sterile cotton swab, and slightly rubbing half leaves by taking a main vein as a boundary; the other half of the leaves are rubbed and controlled to form seed mixture;
(8) rubbing for 20min, spraying sterile water to clean the leaves, and naturally air drying at room temperature;
(9) isolating (far away from healthy tobacco) in a greenhouse and culturing for 3d, then counting the number of dead spots on leaves, carrying out single-factor variance analysis on experimental data by using an SPSS v18.0 to carry out Duncan new double-range method, carrying out significant difference analysis, and counting the antiviral effect according to the following formula:
mosaic virus.
Example 2:
(1) a1.0 g soil sample was taken, placed in a sterile Erlenmeyer flask, added with 11mL of sterile water (operating in a clean bench), sealed and cultured with shaking for 3 hours (26 ℃,130 rpm).
(2) Taking out, standing in a super clean bench for 50min, and taking supernatant for gradient dilution 100、10-1、10-2、10-3、10-4And 10-5After mixing, 110. mu.L of each concentration was applied to a plate of LB solid medium.
(3) LB plates were incubated for 20h at 26 ℃ in an incubator.
(4) Taking out, selecting a plate with uniformly distributed bacterial colonies (with moderate colony number), picking a single spot, and placing the single spot into 4mL of LB liquid culture medium for shake culture for 20h (26 ℃,130 rpm).
(5) Taking 1mL of bacterial liquid, centrifuging at 6000 Xg for 10min, taking supernatant, mixing with the mosaic virus inoculation liquid with the same volume, and standing at room temperature for 25 min.
(6) Mixing the mosaic virus inoculation liquid with an equal volume of phosphate buffer solution, and standing at room temperature for 25min to form a control mixed liquid;
(7) the antiviral effect of the bacterial liquid is detected by adopting a 'half-leaf method': uniformly spreading quartz sand on Sansheng tobacco leaves by taking Sansheng tobacco (NN) cultured for 4 weeks as an experimental material, dipping the mixed solution by using a sterile cotton swab, and slightly rubbing half leaves by taking a main vein as a boundary; the other half of the leaves are rubbed and controlled to form seed mixture;
(8) rubbing for 25min, spraying sterile water to clean the leaves, and naturally air drying at room temperature;
the other steps were the same as in example 1.
In conclusion, the invention discloses a process for separating mosaic virus-resistant bacteria in soil, which separates bacteria with inhibitory effect on mosaic virus from samples collected from different habitats (shallow sea mud and tobacco rhizosphere soil), enriches the types of beneficial bacteria with antiviral effect, further simply determines the antiviral effect, and lays a foundation for the utilization of the mosaic virus-resistant bacteria.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
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.
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| CN201910981778.8A CN110616147A (en) | 2019-10-16 | 2019-10-16 | Process for separating anti-mosaic virus bacteria in soil |
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| CN201910981778.8A CN110616147A (en) | 2019-10-16 | 2019-10-16 | Process for separating anti-mosaic virus bacteria in soil |
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