CN113416793B - Method for detecting colonization ability of bacteria in duckweed - Google Patents
Method for detecting colonization ability of bacteria in duckweed Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a method for detecting colonisation ability of bacteria in duckweed, which comprises the following steps: co-culturing bacteria to be detected and sterile duckweed in a 1/2SH culture solution to obtain inoculated duckweed; cleaning the butted duckweed; the duckweed DNA was extracted and PCR amplified. The method for detecting the colonization capability of bacteria in duckweed is based on a function research system of duckweed aquatic model plants, achieves the aim of rapidly detecting the colonization capability of exogenous bacteria, is beneficial to rapidly screening growth-promoting bacteria with strong colonization capability, has important significance for developing efficient microbial agents, effectively purifying water quality and treating water pollution, and enables farmland and urban water environment to keep green sustainable development.
Description
Technical Field
The invention belongs to the technical field of bacterial function research, and particularly relates to a method for detecting bacterial colonization capacity based on duckweed.
Background
In nature, there are various microorganisms in the plant growth environment, and attached to the surface and inside thereof, and these microorganism groups are collectively called a plant microbiome. The interaction between plants and microorganisms is a new hot spot in microbiological research in recent years, and by knowing the interaction between plants and rhizosphere microorganisms, the interaction between peri-foliar microorganisms and endophytes and various biological effects generated by thalli, the microorganisms are better adapted to living environments, the growth promoting mechanism of the microorganisms and the biological control effect on plant diseases are effectively utilized, and the method has important practical significance for improving the yield and quality of crops.
At present, most of microbial colonization researches are focused on root system colonization of terrestrial plants, and researches on root system colonization of aquatic plants are lacking. The variety, quantity and concentration of soil microorganisms are far greater than those of water microorganisms, and the observation and detection aspects are relatively easy. The water body microorganisms have larger mobility along with the water body, and the number of the water body microorganisms which colonize on the root system of the aquatic plant is correspondingly and obviously lower, which causes difficulty in observing the colonization phenomenon of the water body microorganisms, so that a more sensitive technical means is required to detect the colonized water body microorganisms with low concentration.
The duckweed (Spirodela polyrrhiza) genome has been sequenced to a size similar to that of the model plant Arabidopsis (Arabidopsis thaliana) genome (130 Mb). The duckweed is an ideal material for model plant systems because of the advantages of small genome, simple structure, few repeated sequences, short nutrition propagation period (about 30 hours for propagation generation under good nutrition conditions), easy collection and culture, and the like.
In recent years, the interaction research of duckweed and microorganisms shows that the microorganisms can promote the growth rate of duckweed, increase biomass, improve chlorophyll content, accelerate nitrogen and phosphorus absorption of eutrophic water, degrade heavy metals, promote starch and protein content and the like. However, most studies on the growth promoting function of duckweed inoculated bacteria have neglected the detection of bacteria in the colonization space and colonization ability of plant tissues.
In the research of the growth-promoting bacteria, the effective colonization of the growth-promoting bacteria in the plant body is a precondition for the growth-promoting effect, and the colonization characteristics of the growth-promoting bacteria are clear and are important for the industrialized application and field effect of the growth-promoting bacteria. At present, the existing research method of microorganism on aquatic plant colonization has the defects of more chemical reagents, complex preparation, more than 20 minutes for washing aquatic plant tissues, complicated treatment steps, time consumption, rapidness and insensitive detection result.
Disclosure of Invention
Based on this, it is an object of the present invention to provide a method for detecting the ability of bacteria to colonize duckweed, which has few steps, is simple, has few chemical reagents, and can rapidly and sensitively detect the ability of bacteria to colonize duckweed.
The specific technical scheme for realizing the aim of the invention is as follows:
a method of detecting the ability of a bacterium to colonize duckweed, comprising the steps of:
(1) Co-culturing bacteria to be detected and sterile duckweed in a 1/2SH culture solution to obtain inoculated duckweed;
(2) Cleaning the duckweed, wherein the cleaning comprises the following steps: cleaning with sterile water; after cleaning with sterile water, cleaning with chemical solution for 5-10 min, and then flushing with sterile water;
the chemical solution comprises the following components: 127-147 mM NaCl, 30-45% alcohol, 3-5% NaClO, pH 7.0-7.8;
(3) And respectively extracting the DNA of the duckweed after washing, and carrying out PCR amplification.
In some of these embodiments, the chemical solution in step (2) comprises the following components: 135-140 mM NaCl, alcohol with the volume concentration of 35-40 percent, naClO with the mass volume concentration of 4-5 percent, and pH 7.4-7.8.
In some embodiments, the number of times the sterile water is washed in step (2) is 2 to 4, each time for 0.5 to 1min.
In some embodiments, the forward and reverse primers of the PCR amplification reaction in step (3) are shown in SEQ ID No.1 and SEQ ID No.2, respectively.
In some embodiments, the reaction system of the PCR amplification reaction in step (3) is: 2-3 mu L of 10 XPCR buffer and 0.3-0.6 mM MgCl 2 0.1-0.3 mM dNTPs, 0.5-1.0 mu M of forward and reverse primers, 0.3-0.6 mu L of 5U/. Mu.L Taq polymerase, 80-120 ng of DNA template, and adding sterile ddH 2 O to 25. Mu.L.
In some of these embodiments, the sterile duckweed of step (1) is obtained by the following method: washing duckweed with sterile water for 3-5 times, adding 5-10 mL of 45-55% alcohol for 5-10 min, discarding supernatant, adding hypochlorite with mass volume concentration of 5-7% for 1.5-3 min, and washing with sterile water for 2-3 times.
In some of these embodiments, the hypochlorite is present in a mass to volume concentration of 5 to 6% and the washing time is 2 to 3 minutes.
In some embodiments, the hypochlorite is sodium hypochlorite. Sodium hypochlorite solution has strong oxidizing property and strong alkalinity, and can be used for sterilization and disinfection, but has a destructive effect on plants, so that the disinfection concentration and the disinfection time are proper.
In some of these embodiments, the method of determining whether the sterile duckweed is thoroughly sterilized in step (1) is: placing the sterile duckweed on an LB bacterial culture medium, culturing for 12-24 hours at 25-32 ℃, and if the surrounding sterile colonies of the duckweed are produced, thoroughly sterilizing the duckweed.
In some embodiments, the bacteria to be detected in step (1) are co-cultured with sterile duckweed in a bacterial liquid OD 600 =0.1 to 0.3. Preferably OD 600 ＝0.2。
The present invention aims to provide a method for studying the ability of a sterile duckweed plant system to promote colonization by means of exogenous bacteria isolated from an aqueous environment. Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the inventor finds through repeated experiments that when the volume concentration of 30-45% of alcohol is combined with the mass and the volume concentration of 3-5% of NaClO, the effect of thoroughly sterilizing the surface of plant tissues can be achieved, and when the concentration of the alcohol and the NaClO is lower than the concentration of the NaClO disclosed by the invention, the sterilization is difficult to thoroughly sterilize; the problem of incomplete sterilization, especially the problem of incomplete cleaning of fungi, also exists when alcohol with the volume concentration of 30-45% or NaClO with the mass volume concentration of 3-5% is independently selected to clean tissues; during sterilization, naCl physiological saline is properly matched to play a role in protecting cell structures in the cleaning process. The chemical solution only contains salt, alcohol and hypochlorous acid with a certain concentration, the reagent is less to use and is simple to prepare, and the chemical solution can sufficiently sterilize the surface of the duckweed tissue within 5-10 min, and is simple, convenient and time-saving to process, so that the chemical solution can be used for sensitively detecting the colonization capability of bacteria in the duckweed.
2. The method for detecting the colonization capability of bacteria in duckweed is a function research system based on duckweed aquatic model plants, and the aim of rapidly and sensitively detecting the colonization capability of exogenous bacteria is fulfilled by adopting chemical solution with specific components and concentrations to clean duckweed tissues and combining bacteria specific primers and PCR amplification technology.
3. The method for detecting the colonization capability of bacteria in duckweed is beneficial to the rapid screening of growth-promoting bacteria with strong colonization capability, has important significance for developing efficient microbial agents, effectively purifying water quality and treating water pollution, and ensures that farmland and urban water environment keep green sustainable development.
Drawings
FIG. 1 is a chart showing PCR amplification of sterile duckweed, bacteria Rt4c to be detected and positive control Sp245 strain in example 1, respectively, after washing by different washing methods, DNA is extracted;
FIG. 2 is a chart showing PCR amplification of sterile duckweed, bacteria Lm8c to be detected and positive control Sp7 strain in example 1, respectively, after washing by different washing methods, DNA is extracted;
in fig. 1 and 2, rt4c (Aeromonas sp.) and Lm8c (acetobacter sp.) represent two strains to be detected; sp7 and Sp245 are strains of a control group and represent epiphyte Azospirillum brasilense and endophyte Azospirillum brasilense respectively; -represents sterile duckweed; no TC represents PCR blank; + represents the inoculated strain PCR control; lm5576 represents a sterile duckweed PCR negative control; w represents sterile water for cleaning duckweed; CM represents the chemical solution-washed duckweed of the present invention; SD represents sterile water and the existing chemical solution to sequentially clean the sterile duckweed; b represents the prior method for cleaning the sterile duckweed (after the sterile water and SD are washed in sequence, the sodium hypochlorite is washed for 1 to 2 minutes again); IGS: bacterial specific gene fragment 16S-23S rRNA gene intergenic spacer region (. About.1.5 kb);
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
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 invention belongs.
In the present invention, the bacteria to be detected are bacteria isolated from an aqueous environment using conventional methods. Setting blank control and/or positive control at the same time; the blank control is not added with bacterial liquid; the positive control is added with bacterial liquid with known functions. The blank control and the positive control can be selected and set according to the routine skill of those skilled in the art.
The invention is described in further detail below with reference to specific embodiments and figures.
Example 1A method for detecting the ability of bacteria to colonize duckweed
A method of detecting the ability of a bacterium to colonise duckweed in the present embodiment, comprising the steps of:
1. construction of sterile duckweed System
And (3) placing the collected duckweed into sterile water for washing for 3-5 times, taking 5-10 duckweed sheets into a 10mL sterile centrifuge tube, adding 5mL of 50% alcohol for washing for 10min, and discarding the supernatant. 5mL of sodium hypochlorite with the mass volume concentration of 6% is added for washing for 2min, and then sterile water is used for washing for 2-3 times.
Duckweed was transferred to plant medium (1/2SH+0.5% sucrose+0.1% cefotaxime), where SH is an abbreviation for Schenk & Hildebrandt Basal Salt Mixture plant medium. After duckweed growth on the medium for 7 days, duckweed was placed on LB (beef extract peptone) bacteria medium and observed overnight in a 28℃incubator, representing that duckweed was sufficiently sterilized if aseptic drop around duckweed was produced. At this time, some sterile duckweed is transferred to 1L of sterilized 2 XSH culture solution, and then placed in a constant temperature culture room for expansion culture for about 2-3 weeks, so as to expand the biomass of the duckweed and construct a stable sterile duckweed system.
2. Inoculating bacteria into sterile herba Spirodelae
Day1: two strains to be tested Rt4c (Aeromonas Sp.) and Lm8c (Acinetobacter Sp.) (the strain to be tested is the applicant laboratory identification strain, the 16S rDNA sequence of Rt4c is shown as SEQ ID No.3, the 16S rDNA sequence of Lm8c is shown as SEQ ID No. 4) and two positive control strains Sp7 (identified as epiphyte Azospirillum brasilense) and Sp245 (identified as endophyte Azospirillum brasilense) (Jain & Patriquin 1984) were inoculated in 5mL LB medium, incubated at 28℃in an incubator at 250rpm, and shake-cultured overnight;
rt4c 16S rDNA sequence (SEQ ID No. 3):
AATTGCCCAGTCGAGGGGGATAACAGTTGGAAACGACTGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGGACCTTCGGGCCTTGCGCGATTGGATATGCCCAGGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAAGGTTGATGCCTAATACGTATCAACTGTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTGGATAAGTTAGATGTGAAAGCCCCGGGCTCAACCTGGGAATTGCATTTAAAACTGTCCAGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGA
lm8c 16S rDNA sequence (SEQ ID No. 4):
CATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCATGCTTTCGCACCTCAGCGTCAGTCTTTGGCCGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCTCCTCTCCCACACTCTAGCCGGCAGTTTCGAATGCAATTCCCAAGTTGAGCTCGGGGATTTCACATTTGACTTATTTGGCCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCTGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTATTCTGCGAGTAAC
day2: taking 500 mu L to 50mL of LB culture solution from the bacterial culture solution of Day1, and performing amplification culture under the same culture conditions as Day1;
day3: 25mL of the bacterial culture solution of Day2 was taken out into a sterilized centrifuge tube, and centrifuged at 8,000rpm at 4℃for 5min. After discarding the supernatant, 25mL of sterile water was added to mix the precipitate, and the mixture was centrifuged at 8,000rpm for 5min at 4℃and the supernatant was discarded, and this step was repeated. Adding 25mL of 0.5 XSH sterilizing solution, mixing, reading the absorbance of the bacterial solution at 600nm with a spectrophotometer, and diluting the bacterial solution to OD 600 =0.2/50 mL. 50mL of strain Rt4c (Aeromonas sp.) and Lm8c (Acinetobacter sp.) to be detected which are dissolved in 0.5×SH are poured into a sterile bottle, and then the sterile duckweed which is cultured in advance is transferred into a bacteria liquid bottle, and the surface of the bacteria liquid is covered to obtain experimental group-connected bacteria duckweed;
in addition, 50mL of 1/2SH sterilizing solution is arranged and poured into a sterile bottle, and the same amount of duckweed is added to make a blank control; at the same time, 1/2SH is used for diluting the bacterial liquid to OD 600 Sp7 (epiphyte Azospirillum brasilense) and Sp245 (endophyte Azospirillum brasilense) were added to 0.2/50mL of sterile duckweed, respectively, for positive control.
The experimental group bacterial duckweed, the sterile duckweed blank control group and the positive control group bacterial duckweed are transferred into an illumination culture room (16 h illumination/8 h darkness, light intensity 1000 Lx) for culture.
3. Cleaning the duckweed
After one week of incubation, 0.5×sh solution in each bottle was aspirated, discarded, and duckweed from the experimental, placebo, and positive control groups were treated as follows:
a. sterile Water cleaning treatment (labeled W)
Pouring a proper amount of sterile water for washing twice for 1min each time;
b. the chemical solution cleaning treatment of the present invention (labeled CM)
Transferring 10 duckweed treated by W into sterilized CM chemical solution (130 mM NaCl, 30% alcohol, 3% (g/100 ml) NaClO, pH 7.4), washing for 5min, and washing twice with sterile water;
c. the prior art method is cleaning treatment (marked as SD, B)
(1) Selecting 10 duckweed treated with W, transferring to sterilized SD chemical solution (140mM NaCl,2.5mM KCl,10mM Na) 2 HPO 4 ，2mM KH 2 PO 4 ，0.5mM MgSO 4 ，1mM CaCl 2 0.1% (v/v) Triton-X100, ph=7.5), for 20min;
(2) after the step (1), washing with 5% sodium hypochlorite for 1-2 min, and finally using Na 2 S 2 O 3 The solution was rinsed 2 times and labeled B.
4. Extracting duckweed DNA, and performing PCR amplification
The duckweed treated in the step 3 is taken out, transferred into a 2mL microcentrifuge tube pre-filled with 1g of zirconia beads, added with 2x CTAB and chloroform, homogenized in all tissues by a magnetic bead oscillator, and then extracted by a 2x CTAB nucleic acid extraction method.
The DNA of each treatment group was PCR amplified using bacterial specific primer IGS, wherein the forward primer was 16S-e1390f (5 '-TGYACACACCGCCCGTCA-3', SEQ ID No. 1) and the reverse primer 23S-e130R (5 '-GGGTTBCCCCCCACATTCRG-3', SEQ ID No. 2), wherein Y=C/T, R=A/G, B=G/T/C.
The 25. Mu.L PCR reaction system comprises: 2.5. Mu.L of 10 XPCR buffer, 0.5mM MgCl 2 0.2mM dNTPs, 0.8. Mu.M each of forward and reverse primers, 0.5. Mu.L of 5U/. Mu.L Taq polymerase, about 100ng of DNA template, and finally adding sterile ddH 2 O to 25. Mu.L.
The PCR reaction procedure included: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 60℃for 30s, extension at 72℃for 90s,30 cycles; finally, the extension is continued for 5min at 72 ℃.
After ethidium sniffing, the PCR product was electrophoresed through a 1% agarose gel for about one hour, and then the PCR amplification result was detected by a gel imager and photographed.
After cleaning (W) with sterile water, the bacteria to be detected are considered to be colonized in an attached bacteria form, but the colonization capability is weak when the bacteria to be detected are detected to have a fingerprint after washing (CM) with a chemical solution and detecting that the bacteria to be detected have no fingerprint after PCR. If the fingerprint is still present after the cleaning (W) with sterile water and the washing (CM) with chemical solution, the bacteria to be detected are considered to be colonized in the form of endophyte, and the colonization capability is stronger.
After the solution (SD and B) adopting the prior method is cleaned, the judgment basis is as follows: after cleaning (W) with sterile water, the bacteria to be detected are considered to be colonized in the form of epiphyte, and the colonization capacity is weak, if the bacteria to be detected are detected to have a fingerprint after cleaning (W) with sterile water or have a weak fingerprint after cleaning (SD). If the bacteria to be detected have obvious fingerprint patterns after being washed by W, SD and B, the bacteria to be detected are considered to be colonized in an endophyte mode, and the colonization capability is strong.
The results are shown in fig. 1 and 2. As can be seen from fig. 1, compared with the positive control group Sp245 (endophyte), the duckweed inoculated with the strain Rt4c was subjected to sterile water washing (W), the fingerprint was detected by PCR, and after washing with the chemical solution (CM), the fingerprint was still detected by PCR, and it was confirmed that the strain Rt4c was colonized in duckweed tissue in the form of endophyte, and the colonization ability was strong;
as can be seen from fig. 2: compared with the positive control group Sp7 (epiphyte), the duckweed inoculated with the strain Lm8c has a fingerprint after being washed by sterile water (W), but has no fingerprint after being washed by a chemical solution (CM), and the strain Lm8c is determined to be colonized on duckweed tissues in the form of epiphyte and has weak colonization capacity. The method of the invention proves that the colonization ability of bacteria in duckweed can be accurately detected.
However, the prior art chemical solution SD and the SD solution combined with the B solution are used for cleaning the duckweed tissue, and although the colonization capability of bacteria in the duckweed tissue can also be verified, the steps are complicated, the time consumption is long and the sensitivity is not high enough. This is reflected in the following three aspects:
1. the SD solution needs a plurality of reagents, the preparation is complex and time-consuming, the time for washing duckweed tissues by the SD is at least ensured to be more than 20 minutes, the bacteria attached to the surfaces of the tissues can be ensured to be washed away, and then the bacteria attached to the surfaces of the tissues are washed away by the solution B; if the reagent B is used alone, the thorough sterilization of the surface of the plant tissue cannot be ensured; the chemical solution CM combines the sterilization effect of alcohol and hypochlorous acid, and can clean off the bacteria attached to the tissue surface only for 5-10 min, so that the washing time is greatly reduced;
2. when verifying whether the type of the inoculation is endophyte or not, the accuracy of detection cannot be ensured only by the SD solution, and the plant DNA is extracted for PCR detection after the tissue is washed together with the B solution, so that the steps are complicated;
3. when verifying whether the type of the inoculation is the epiphyte or not, the detection results of the PCR after washing with sterile water W and SD are consistent, so that the combination of the SD step and the PCR detection is superfluous.
Example 2A method for detecting the ability of bacteria to colonize duckweed
The method for detecting the colonization ability of bacteria in duckweed of this example is the same as that of example 1 except that the concentration of each component of the chemical solution (CM) of the present invention in step 3b is different from that of example 1; the chemical solution of this example consisted of the following components: 135mM NaCl, 40% alcohol by volume, 5% (g/100 ml) NaClO, pH7.6.
The method for detecting the colonization ability of bacteria in duckweed of this example is the same as that of example 1.
Example 3A method for detecting the ability of bacteria to colonize duckweed
The method for detecting the colonization ability of bacteria in duckweed of this example is the same as that of example 1 except that the concentration of each component of the chemical solution (CM) of the present invention in step 3b is different from that of example 1; the chemical solution of this example consisted of the following components: 140mM NaCl, 35% alcohol by volume, 4% (g/100 ml) NaClO, pH7.8.
The method for detecting the colonization ability of bacteria in duckweed of this example is the same as that of example 1.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> institute of biological engineering at the academy of sciences in Guangdong province
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Claims (9)
1. A method of detecting the ability of a bacterium to colonize duckweed, comprising the steps of:
(1) Co-culturing bacteria to be detected and sterile duckweed in a 1/2SH culture solution to obtain inoculated duckweed; the sterile duckweed is obtained by the following method: washing duckweed with sterile water for 3-5 times, adding 5-10 mL of 45-55% alcohol for 5-10 min, discarding supernatant, adding hypochlorite with mass volume concentration of 5-7% for 1.5-3 min, and washing with sterile water for 2-3 times;
(2) Cleaning the duckweed, wherein the cleaning comprises the following steps: cleaning with sterile water; after cleaning with sterile water, cleaning with chemical solution for 5-10 min, and then flushing with sterile water;
the chemical solution comprises the following components: 130-140 mM NaCl, 30-40% alcohol, 3-5% NaClO, pH 7.0-7.8;
(3) Respectively extracting the DNA of the cleaned duckweed, and carrying out PCR amplification;
if the fingerprint is detected by PCR after the sterile water is washed; however, after cleaning with sterile water, cleaning with chemical solution for 5-10 min, and then cleaning with sterile water, if no fingerprint is detected by PCR, the colonization ability of bacteria to be detected in duckweed is weak;
if the fingerprint is detected by PCR after the sterile water is washed; and after cleaning with sterile water, cleaning with chemical solution for 5-10 min, and then cleaning with sterile water, and detecting by PCR (polymerase chain reaction) to obtain the fingerprint, the bacteria to be detected have strong colonization capability in duckweed.
2. The method for detecting the colonization ability of bacteria in duckweed according to claim 1, wherein the chemical solution in step (2) comprises the following components: 135-140 mM NaCl, 35-40% alcohol, 4-5% NaClO, pH 7.4-7.8.
3. The method for detecting the colonization ability of bacteria in duckweed according to claim 1, wherein the number of times of the sterile water washing in the step (2) is 2 to 4 times, each time for 0.5 to 1min.
4. The method for detecting the ability of bacteria to colonize duckweed according to claim 1, wherein the primers for PCR amplification in step (3) are shown in SEQ ID No.1 and SEQ ID No. 2.
5. According to the weightsThe method for detecting the colonization ability of bacteria in duckweed according to claim 4, wherein the reaction system of the PCR amplification reaction in the step (3) is: 2-3 mu L of 10 XPCRbuffer buffer and 0.3-0.6 mM MgCl 2 0.1-0.3 mM dNTPs, 0.5-1.0 mu M of each primer, 0.3-0.6 mu L of 5U/mu LTaq polymerase, 80-120 ng of DNA template, and adding sterile ddH 2 O to 25. Mu.L.
6. The method for detecting the colonization ability of bacteria in duckweed according to claim 1, wherein the hypochlorite is in a mass-volume concentration of 5 to 6% and the washing time is 2 to 3min.
7. The method of detecting the ability of bacteria to colonize duckweed according to claim 6, wherein said hypochlorite is sodium hypochlorite.
8. The method for detecting the colonization ability of bacteria in duckweed according to any one of claims 1 to 5, wherein in step (1), the method for determining whether the sterile duckweed is thoroughly sterilized is: placing the sterile duckweed on an LB bacterial culture medium, culturing for 12-24 hours at 25-32 ℃, and if the surrounding sterile colonies of the duckweed are produced, thoroughly sterilizing the duckweed.
9. The method for detecting the colonization ability of bacteria in duckweed according to any one of claims 1 to 5, wherein the bacteria to be detected in step (1) are co-cultured with a bacterial liquid OD of sterile duckweed 600 ＝0.1～0.3。
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