CN111004743A - Method for screening plant rhizosphere soil antagonistic bacteria - Google Patents
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Abstract
A method for screening plant rhizosphere soil antagonistic bacteria belongs to the field of microorganisms. The method comprises the following steps: (1) separating the rhizosphere soil of the sample by using a gradient dilution method to obtain a bacterial strain; (2) purifying the separated strain by using a streak purification method to obtain pure bacterial culture; (3) screening, namely screening antagonistic bacteria by using an improved Oxford cup method, and is characterized in that a semi-solid culture medium and the improved Oxford cup method are used for screening the antagonistic bacteria. The plant rhizosphere soil microorganisms are rich in types, mutual antagonism phenomena possibly exist among bacteria, and the antagonistic bacteria possibly existing are screened out by a quick and simple method. Compared with other existing methods, the method is simpler to operate and more remarkable in effect.
Description
Technical Field
The invention belongs to the field of microbial ecology, and particularly relates to a method for quickly and effectively screening antagonistic bacteria in plant rhizosphere soil.
Background
It is known that plant rhizosphere soil is an environment with an extremely rich microbial population, and various interactions, such as antagonism, parasitism and symbiosis, can exist among different microorganisms.
The significance of studying this antagonistic phenomenon lies in: firstly, the method can provide the most direct experimental evidence to prove that antagonism exists among bacteria in the same habitat, and the method has important significance for researching the interaction among the bacteria in microbial communities; and secondly, the method can be applied to screening bacteria with inhibiting effect on plant pathogenic bacteria, and then the antagonistic bacteria can be applied to the agricultural field. There is a need for a rapid and simple method for screening antagonistic strains.
At present, many scholars are dedicated to research on various bacteriostatic phenomena, but most of the scholars focus on antagonism between fungi or between fungi and bacteria and actinomycetes, and research on mutual antagonism between bacteria is less. Researchers have studied the bacteriostatic action of brevibacillus laterosporus S62-9 on common microorganisms by using the traditional Oxford cup method (Zhang Dan et al, Chinese food science, 2017, 17 (1): 55-61.), Chen Shi Ying et al have studied the interaction relation between antagonistic bacterial strains in a plant pathogenic bacteria biocontrol preparation mixed by a plurality of bacterial strains by using a coating method and a paper filter method and the influence on the biological control effect (Chen Shi et al, plant pathology science, 2005, 35 (6): 539-544.), but most of the studies are to measure the bacteriostatic activity of supernatant by using the traditional Oxford cup method, or use the traditional coating plate method, or dip antagonistic bacteria on a plate by using a paper filter. The methods have some defects, for example, the traditional Oxford cup method uses supernatant liquid with thalli removed to measure the bacteriostatic activity, so that the problems that the indicator bacteria and the antagonistic bacteria are not in direct contact, and the antibacterial substances cannot be continuously generated exist; the flat plate coating method can lead to unclear bacteriostasis phenomenon due to uneven coating of the indicator bacteria; if the filter paper sheet is used, antagonistic bacteria liquid can be diluted, so that the real viable count cannot be determined, and the filter paper sheet is easy to pollute mixed bacteria. The method generally has the problems of complex operation, large workload, poor effect and the like. In addition, these bacteria are obtained from different environments, and studies on the mutual antagonism among bacteria in an environment rich in microorganism species such as plant rhizosphere soil in the same habitat are rarely reported, and few studies on the induction antagonism phenomena possibly existing in the bacteria are also reported.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to provide a method for efficiently screening antagonistic bacteria in plant rhizosphere soil, aiming at the problems of complicated operation and low efficiency of the traditional Oxford cup method. According to the method, a single-layer semi-solid plate is used for replacing a double-layer plate, overnight-cultured bacterial liquid and a fresh culture medium are added into the Oxford cup, and the step of filtering and sterilizing is not needed, so that the operation is simpler. In addition, bacteria grow in the oxford cup, and can synchronously generate antibacterial substances, so that the content of the antibacterial substances is improved, and the screening efficiency of the oxford cup is improved.
The technical measures adopted by the invention are as follows: a method for screening plant rhizosphere soil antagonistic bacteria comprises separating bacteria from a plant rhizosphere soil sample, purifying and screening antagonistic bacteria;
the method for separating the bacteria in the plant rhizosphere soil sample comprises the following steps:
1. treatment of sample soil: after rhizosphere soil is collected, sterile water is added for oscillation treatment to obtain bacterial suspension;
2. dilution coating of the plates: the bacterial suspension was diluted 10-fold in a gradient (10)-1,10-2,10-3，10-4,10-5) Respectively coating the diluent with different gradients on an R2A solid culture medium, and then transferring the flat plate into a constant-temperature incubator at 28 ℃ for culture;
the solid culture medium of R2A: 0.5g of yeast extract powder, 0.5g of peptone, 0.5g of casein hydrolysate, 0.5g of glucose, 0.5g of soluble starch, 0.3 g of dipotassium phosphate, 0.024 g of anhydrous magnesium sulfate, 0.3 g of sodium pyruvate, 15g of agar, 1000 ml of distilled water and pH = 7.4.
3. Separation and purification: according to the color, shape and size characteristics of colonies on the plate, single bacterial colonies with different characteristics are picked out, streaked on an R2A solid culture medium, and then cultured.
And (3) purifying the picked bacterial strains, wherein each bacterial strain is purified at least three times until pure bacterial colonies are obtained, numbering the bacterial colonies in sequence, adding glycerol, and freezing and storing the bacterial colonies.
The antagonistic bacteria screening comprises the following steps:
1. preparing bacterial liquid: respectively inoculating all strains separated from rhizosphere soil into an R2A liquid culture medium, carrying out shake culture at 28 ℃ and 180 rmp, and collecting a bacterial solution for later use when the strains reach the later logarithmic growth stage and the earlier stable stage; selecting any one of bacteria separated from soil as an indicator bacterium; on the flat plate containing the indicator bacteria, the bacteria except the indicator bacteria are all antagonistic bacteria.
The liquid culture medium of R2A: 0.25 g of tryptone, 0.5g of acid hydrolyzed casein, 0.5g of yeast extract powder, 0.5g of soluble starch, 0.3 g of dipotassium phosphate, 0.1 g of magnesium sulfate, 0.3 g of sodium pyruvate, 0.25 g of peptone, 0.5g of glucose and 1000 ml of distilled water, wherein the pH is = 7.4.
2. And (3) indication bacterium treatment: 20 ml of R2A semisolid medium (agar concentration range 6g/L to 10 g/L, pH = 7.4) was used. Adding 20 mu L of indicator bacterium liquid before pouring the flat plate, vibrating and mixing uniformly, and pouring the flat plate;
the R2A semisolid culture medium is prepared by adding agar into R2A liquid culture medium, wherein the agar concentration is 6 g/L-10 g/L.
3. Putting into an oxford cup: directly vertically placing sterilized Oxford cups (the specification is that the inner diameter is 0.78cm, the outer diameter is 0.8cm, and the height is 1 cm) on the surface of the semi-solid culture medium after the culture medium of the mixed bacteria liquid is solidified, and standing for 10 min until the Oxford cups naturally sink;
4. adding antagonistic bacteria: sequentially adding 50 mu L of antagonistic bacteria liquid to be detected and 50 mu L of sterile R2A liquid culture medium into the Oxford cup;
5. standing for 3 h, transferring into an incubator for culturing for 5d, and observing and recording the bacteriostatic phenomenon.
According to the invention, the bacteria liquid of the antagonistic bacteria is directly added into the oxford cup, so that the antagonistic bacteria and the indicator bacteria can be in direct contact to the maximum extent; the addition of the fresh culture medium ensures the growth of the antagonistic bacteria and the continuous production of antagonistic substances; the semi-solid culture medium mixed bacteria liquid is poured into the flat plate, so that the problem of uneven distribution of the indicator bacteria is solved; the operation is simple and quick, pollution is not easy to occur, and the effect is good; meanwhile, after the mutual antagonism phenomenon among bacteria in the same habitat is preliminarily determined, the method lays a foundation for subsequent research. Compared with the effect of the traditional oxford cup method, the effect obtained by using the method is more obvious.
Detailed Description
The method adopted by the present invention will be described in detail below by taking two examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Equivalent modifications and alterations in detail may be made by those skilled in the art without departing from the essential scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are normally commercially available unless otherwise specified.
The media used in the following examples:
1. R2A liquid medium: 0.25 g of tryptone, 0.5g of acid hydrolyzed casein, 0.5g of yeast extract powder, 0.5g of soluble starch, 0.3 g of dipotassium phosphate, 0.1 g of magnesium sulfate, 0.3 g of sodium pyruvate, 0.25 g of peptone, 0.5g of glucose and 1000 ml of distilled water, wherein the pH is = 7.4. .
2. R2A solid medium: 0.5g of yeast extract powder, 0.5g of peptone, 0.5g of casein hydrolysate, 0.5g of glucose, 0.5g of soluble starch, 0.3 g of dipotassium phosphate, 0.024 g of anhydrous magnesium sulfate, 0.3 g of sodium pyruvate, 15g of agar, 1000 ml of distilled water and pH = 7.4.
3. R2A semisolid medium: agar is added into the R2A liquid culture medium, and the concentration of the agar is 6 g/L-10 g/L.
Example a screening of antagonistic bacteria in caragana microphylla rhizosphere soil sample 1.
Firstly, the separation and purification of bacteria in the caragana microphylla rhizosphere soil sample 1 comprises the following steps:
1) collecting a caragana microphylla rhizosphere soil sample from a certain vegetable garden in Ulmus of Shanxi province, and marking the caragana microphylla rhizosphere soil sample as a sample 1;
2) putting the sample 1 into a 50 ml centrifuge tube, adding 30 ml of sterile water, horizontally oscillating for 3 times, 1min each time and 30 s intervals;
3) standing for 10 min, adding 100 μ L of supernatant into 900 μ L of sterile water, shaking, and mixing to obtain a solution; diluting for five times to obtain 6 gradient dilutions, i.e. 0, 10-1、10-2、10-3、10-4、10-5These 6 gradient dilutions;
4) get 10-1、10-3、10-4、10-5Respectively coating 50 mu L of four gradient diluents on an R2A solid culture medium, and placing the culture medium into a constant-temperature incubator at 28 ℃ for standing culture;
5) and respectively picking out single colonies with different colors, forms, textures and sizes on the plates with different gradients, and sequentially numbering the single colonies. Purifying the picked strains by using a plate marking method for at least three times until pure culture is obtained and then preserving the strains; 9 strains are separated from the caragana microphylla rhizosphere soil sample, and are respectively numbered CK01-CK 09.
Further, the screening method of the antagonistic bacterial strain in the caragana microphylla rhizosphere soil sample 1 comprises the following steps:
1. preparing bacterial liquid: respectively inoculating the purified 9 strains of bacteria into an R2A liquid culture medium, performing shake culture at 28 ℃ and 180 rmp until the late logarithmic growth stage, and collecting bacterial liquid for later use when the early logarithmic growth stage is stable;
2. preparing 9 conical flasks, respectively preparing semisolid culture mediums with agar concentration of 10 g/L, sterilizing, and placing in a 55 ℃ oven for later use;
3. treating the indicator bacteria: adding 20 mu L of bacterial liquid into the R2A semisolid culture medium, quickly shaking and uniformly mixing, and pouring into a flat plate. One plate corresponds to one strain, 9 strains of bacteria are separated from the habitat of the sample 1, namely 9 indicator bacterium plates are needed;
4. placing an oxford cup: after the culture medium solidified, the sterilized Oxford cups (with the specification of 0.78cm inside diameter, 0.8cm outside diameter and 1cm high) were placed vertically on the surface of the culture medium with tweezers, and 8 Oxford cups were placed on each plate (except for the Oxford cups, other strains were added to one of the Oxford cups, respectively). Standing for 10 min until the oxford cup naturally sinks;
5. adding antagonistic bacteria liquid: adding 50 mu L of antagonistic bacteria liquid to be detected and 50 mu L of sterile R2A liquid culture medium into the Oxford cup in sequence. The bacterial solution of one strain needs to be continuously added for 8 times. Until the sample adding of all the 9 plates is finished;
6. standing for 3 h, transferring into a constant temperature incubator at 28 ℃, culturing for 5 days, and observing the size of a bacteriostatic zone;
7. the inhibition on each plate was recorded and a table of antagonism was prepared and shown in table 1 for details.
TABLE 1 distribution of antagonistic strains screened in inventive example A
In Table 1, -, indicates no antagonism and + -, indicates antagonism
CK01-CK09 is 9 bacteria isolated from the rhizosphere soil of the twisted shoots. Wherein, CK09 is identified as Ficus microcarpa, and is preserved in China center for type culture Collection with the preservation number of No: CCTCC M20191036; the remaining 8 bacteria were not identified for species.
From Table 1, it can be seen that the habitat is 5 pairs of antagonistic phenomena, namely CK09 antagonism CK02/CK05/CK06/CK07 and CK04 antagonism CK 02.
Example B screening of antagonistic bacteria in corn rhizosphere soil sample 2.
Also, firstly, the isolation and purification of the bacteria in the corn rhizosphere soil sample 2 comprises the following steps:
1) collecting a corn rhizosphere soil sample from a certain vegetable garden in Yuxia City of Shanxi province, and marking the sample as a sample 2;
2) putting the sample into a 50 ml centrifuge tube, adding 30 ml of sterile water, horizontally oscillating for 3 times, each time for 1min, and spacing for 30 s;
3) standing for 10 min, collecting supernatant, performing gradient dilution of 10 times, and diluting for five times to obtain 6 gradient dilutions, 0, 10-1、10-2、10-3、10-4、10-5These 6 gradient dilutions;
4) get 10-1、10-3、10-4、10-5Respectively coating 50 mu L of four gradient diluents on an R2A solid culture medium, and placing the culture medium into a constant-temperature incubator at 28 ℃ for standing culture;
5) respectively picking out single colonies with different characteristics such as color, form, size and the like on different gradient plates, sequentially numbering, and repeatedly purifying the picked strains by a plate marking method until pure culture is obtained and preserving the strains; in this sample, 8 strains were co-isolated, each numbered ZM01-ZM 08.
Further, screening of antagonistic bacterial strains in the corn rhizosphere soil sample 2 comprises the following steps:
1) preparing bacterial liquid: respectively inoculating 8 purified strains into an R2A liquid culture medium, and performing shake culture at 28 ℃ and 180 rmp until the late logarithmic growth stage and the early stable stage;
2) preparing 8 conical flasks, respectively preparing semisolid culture media with agar concentration of 10 g/L, sterilizing, and placing in a 55 ℃ oven for later use;
3) treating the indicator bacteria: adding 20 mu L of bacterial liquid into an R2A semisolid culture medium, quickly oscillating, uniformly mixing, pouring a flat plate, and preparing 8 indicator bacterium flat plates according to the rule that one strain of bacteria corresponds to one flat plate;
4) placing an oxford cup: after the medium had solidified, sterilized oxford cups were placed vertically on the surface of the medium, and 7 oxford cups were placed in each plate (except for itself, the other strains were added to one of the oxford cups). Standing for 10 min until the oxford cup naturally sinks;
5) adding antagonistic bacteria liquid: adding 50 mu L of antagonistic bacteria liquid to be detected and 50 mu L of sterile R2A liquid culture medium into the Oxford cup in sequence. Continuously adding the bacterial liquid of one strain for 7 times until the samples of 8 plates are completely added;
6) standing for 3 h, carefully transferring into a constant-temperature incubator at 28 ℃, and culturing for 5d to observe the bacteriostasis phenomenon;
7) the inhibition on each plate was recorded and tabulated for details in table 2.
TABLE 2 distribution of antagonistic strains screened in inventive example B
In Table 2, -, indicates no antagonism and + -, indicates antagonism
ZM01-ZM08 are 8 bacteria isolated from the rhizosphere soil of maize, none of which have been identified as species.
From Table 2, it can be seen that the habitat is 1 pair antagonistic, namely ZM07 antagonizes ZM 05.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made thereto without departing from the spirit and scope of the invention. Therefore, it is intended that all such modifications and improvements be made without departing from the spirit of the invention.
Claims (2)
1. A method for screening plant rhizosphere soil antagonistic bacteria is characterized by comprising the steps of separating and purifying bacteria in a plant rhizosphere soil sample and screening antagonistic bacteria;
the method for separating the bacteria in the plant rhizosphere soil sample comprises the following steps:
(1) treatment of soil samples: after rhizosphere soil is collected, sterile water is added for oscillation treatment to obtain bacterial suspension;
(2) dilution coating of the plates: diluting the bacterial suspension in a 10-fold gradient manner to obtain diluents with different gradients, respectively coating the diluents with different gradients on an R2A solid culture medium, and then transferring the plate into a constant-temperature incubator at 28 ℃ for culture;
the solid culture medium of R2A: 0.5g of yeast extract powder, 0.5g of peptone, 0.5g of casein hydrolysate, 0.5g of glucose, 0.5g of soluble starch, 0.3 g of dipotassium phosphate, 0.024 g of anhydrous magnesium sulfate, 0.3 g of sodium pyruvate, 15g of agar and 1000 ml of distilled water, wherein the pH is = 7.4;
(3) separation and purification: according to the color, shape and size characteristics of colonies on the plate, single bacterial colonies with different characteristics are picked out, streaked on an R2A solid culture medium, and then bacteria are cultured;
(4) purifying the selected bacterial strains, wherein each bacterial strain is purified for at least three times until pure bacterial colonies are obtained, numbering the bacterial colonies in sequence, adding glycerol, and freezing and storing the bacterial colonies;
the antagonistic bacteria screening comprises the following steps:
(1) preparing bacterial liquid: respectively inoculating all strains separated from rhizosphere soil into an R2A liquid culture medium, carrying out shake culture at 28 ℃ and a rotation speed of 180 rmp, and collecting a bacterial liquid for later use after the strains reach a late logarithmic growth stage and a stable early stage;
the liquid culture medium of R2A: 0.25 g of tryptone, 0.5g of acid hydrolyzed casein, 0.5g of yeast extract powder, 0.5g of soluble starch, 0.3 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate, 0.3 g of sodium pyruvate, 0.25 g of peptone, 0.5g of glucose and 1000 ml of distilled water, wherein the pH is = 7.4;
(2) and (3) indication bacterium treatment: adding 20 mu L of indicator bacterium liquid into 20 ml of R2A semisolid culture medium with agar concentration of 6-10 g/L and pH =7.4, uniformly mixing by shaking, and then pouring the mixture onto a plate; the indicator bacteria are any one selected from bacteria separated from soil;
the R2A semisolid culture medium is prepared by adding agar into R2A liquid culture medium, wherein the agar concentration is 6 g/L-10 g/L;
(3) putting into an oxford cup: after the R2A semisolid culture medium of the mixed bacterial liquid is solidified, directly and vertically placing a sterilized oxford cup on the surface of the R2A semisolid culture medium, standing for 10 min, and waiting for the oxford cup to naturally sink;
(4) adding antagonistic bacteria: sequentially adding 50 mu L of antagonistic bacteria liquid to be detected and 50 mu L of sterile R2A liquid culture medium into the Oxford cup; the antagonistic bacteria are on a flat plate containing the indicator bacteria, and the other bacteria except the indicator bacteria are the antagonistic bacteria;
standing for 3 h, transferring into an incubator for culturing for 5d, and observing whether a bacteriostatic zone exists.
2. The method for screening plant rhizosphere soil antagonistic bacteria according to claim 1, wherein said different gradient of dilution is 0, 10-1、10-2、10-3、10-4、10-5These 6 gradient dilutions.
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| CN112111405A (en) * | 2020-09-27 | 2020-12-22 | 宁德师范学院 | Method for screening biocontrol bacteria of soft rot of betel nut taro |
| CN113957008A (en) * | 2021-10-27 | 2022-01-21 | 福建傲农生物科技集团股份有限公司 | Method for reducing antagonism among different bacterial strains and preparation method of composite microbial inoculum |
| CN115011483A (en) * | 2022-06-10 | 2022-09-06 | 上海市农业科学院 | High-throughput screening method for IAA-producing endophytes of plant tissues |
| CN115011482A (en) * | 2022-06-10 | 2022-09-06 | 上海市农业科学院 | Method for separating plant rhizosphere microorganisms in high flux and culturing and screening IAA-producing strains |
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Application publication date: 20200414 |