AU2021104714A4 - A method for identifying bacterial infection types based on duckweed - Google Patents
A method for identifying bacterial infection types based on duckweed Download PDFInfo
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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- Optics & Photonics (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present disclosure discloses a method for identifying bacterial infection types
based on duckweed, which comprises the following steps: co-cultivating bacteria to be
detected and sterile duckweed in 1/2 SH culture solution to obtain inoculated duckweed;
cleaning the duckweed; using the green fluorescent nucleic acid dye DMAO to dye the
duckweed. The method for identifying bacterial infection types based on duckweed of the
present disclosure is based on the function research system of duckweed "aquatic model
plant", which uses chemical solutions of special components and concentrations to clean
duckweed leaf tissues, and then dyes the duckweed tissues with green fluorescent nucleic
acid dye DMAO to quickly identify the types of bacterial infestation; and promotes the
judgment of whether the infecting bacteria is pathogenic to the host plant, whether the
microorganism and the plant establish a symbiotic relationship, and the interaction between
them.
Description
Technical Field
[0001] The present disclosure relates to the bacterial function research technical technology,
in particular to a method for identifying the bacterial infection types in tissues based on
duckweed.
Background Art
[0002] After the bacteria in the natural environment enter into a plant body, due to
physiological adaptation and other factors, some bacteria can survive in the plant body for a
long time, and can be spread between the generations of the plant with seed or asexual
reproduction material, which becomes the main source of endophytic bacteria of offspring
plants. Some bacteria are attached to the surface of plant tissues, which can positively promote
the growth and physiology of plants or cause disease.
[0003] Duckweed is a kind of small floating water plant. Due to its small genome, simple
structure, few repetitive sequences, vegetative reproduction, short genetic cycle (approximately
h for one generation under good nutritional conditions), easy collection and cultivation,
Duckweed is an ideal material for plant model system, which is convenient for studying the
interaction between plants and microorganisms.
[0004] The interaction between plants and microorganisms is a new hot spot in microbiology
research in recent years. By understanding bacteria interaction in the form of epiphytes or
endophytes invading plants and various biological effects by bacteria, the growth-promoting
mechanism of microorganisms and the biological control of plant diseases can be effectively
utilized for improving the yield and quality of crops, which has important practical significance.
[0005] At present, most research methods of bacteria infecting plant tissues labeling
experimental strains with green fluorescent protein GFP gene, can effectively distinguish the
target strains inoculated from the indigenous bacteria existing in the soil and plants, and it is
conducive to real-time and in-situ observe the survival status of the target bacteria. However,
the test results will be affected by factors such as unstable expression of plasmids and unstable
expression of heterologous proteins in Bacillus, which often results in unsatisfactory or even no expression on foreign genes.
[0006] Before bacteria and plants interact, they first infect the surface or body of plants.
However, there is still a lack of simple and quick method to study how bacteria, especially
endophytes infect plants, and to clarify the types of infection in plants tissues. In the current
methods for studying the types of bacterial infections on plant tissues, there are many chemical
reagents involved, complex preparations, and it takes at least 20 minutes to sterilize plant tissues.
The processes are cumbersome, time-consuming, not fast enough, and the detection results are
not sensitive enough.
Summary of the Disclosure
[0007] Based on the above, one of the objects of the present disclosure is to provide a
convenient, quick and sensitive method for identifying the type of bacterial infection in tissues
based on duckweed.
[0008] The specific technical solutions to achieve the above-mentioned objectives are as
follows:
[0009] A method for identifying bacterial infection types based on duckweed, comprising the
following steps:
(1) coculturing the bacteria to be detected and sterile duckweed in 1/2 SH culture medium
to obtain inoculated duckweeds;
(2) cleaning the inoculated duckweeds, which includes: washing with sterile water; then
washing with chemical solution for 5 to 10 minutes, and then washing with sterile water again;
wherein, the chemical solution includes the following components: 127mM-147mM NaCl,
%~45% alcohol by volume concentration, and 3%-5% NaClO by mass volume
concentration with pH 7.0-7.8;
(3) dying the cleaned duckweed separately with the green fluorescent nucleic acid dye
DMAO, and observing with a fluorescence microscope.
[00010] In some embodiments, the number of times of sterile water washing in step (2) is 2 to
4 times, and each time lasts 0.5 min to 1 min. The said chemical solution includes the following
components: 135mM-140mM NaCl, 35-40% alcohol by volume concentration, 4% ~ 5%
NaClO by mass volume concentration with pH 7.4-7.8. Washing the inoculated duckweed with
sterile water and chemical solution in sequence to remove the epiphytic bacteria that may exist on the surface of the duckweed tissue ensures that the target bacteria observed after staining are endophytes.
[00011] In some embodiments, the amount of the green fluorescent nucleic acid dye DMAO in step (3) is 5 L -25 [L, and the dyeing time is 2 min -5 min. Excessive amount of dye or too long of dyeing time will cause the tissue to be stained too dark for observation.
[00012] In some embodiments, the amount of the green fluorescent nucleic acid dye DMAO in step (3) is 5 L -15 L, and the dyeing time is 2 min - 4 min.
[00013] In some embodiments, the sterile duckweed in step (1) is obtained by the following method: washing the duckweeds with sterile water 3 to 5 times, adding 5mL -10 mL of 45%
% alcohol, and washing for 5 min -10 min; discarding the supernatant, adding 5% -7%
hypochlorite by mass volume concentration, and washing for 1.5 min -3 min, and then rinsing with sterile water for 2 to 3 times.
[00014] In some embodiments, the mass volume concentration of the hypochlorite is 5% to 6%,
and the washing time lasts 2 to 3 minutes.
[00015] In some embodiments, the said hypochlorite is sodium hypochlorite. Sodium hypochlorite solution has strong oxidizing properties and is a strong alkalinity at the same time. Although it can be used for sterilization and disinfection, it has a destructive effect on plants, so the concentration of sodium hypochlorite and washing time should be appropriate.
[00016] In some embodiments, the method for determining whether the sterile duckweeds are completely sterilized in step (1) is: culturing the sterile duckweeds on the LB bacterial culture medium for 12h-24h at 25°C-32°C, if there is no colony formed around the duckweed, it indicates the duckweed has been sterilized completely.
[00017] In some embodiments, OD6 0 0 of the bacteria liquid obtained when the bacteria to be tested and the sterile duckweed are co-cultured in step (1) is 0.1-0.3; preferably, OD 60 0 =0.2. OD 6 0 0 is the absorption value of bacteria under UV spectrophotometer. It reflects the concentration of bacteria. If the concentration of inoculated bacteria is too high or too low, it will cause the bacteria colonization on plant tissues to be too dense or too thin, which will affect the observation result of the bacterial population on the plant tissues. Therefore, it is necessary to ensure a proper concentration of bacteria.
[00018] The present disclosure aims to provide a method for studying the infection types by the exogenous bacteria separated from water or soil environment in duckweed tissues based on a sterile duckweed plant system. Compared with the prior art, the present disclosure has the following advantages and beneficial effects:
1. Through all the repeated experiments, the inventor found that after being cleaned with
%-45% alcohol by volume concentration combined with 3% -5% NaClO by mass-volume
concentration, the surface of the plant tissue can be completely sterilized. When the
concentration of both is lower than that of described in the present disclosure, it is difficult to
sterilize thoroughly; if only using the 30%-45% alcohol by volume concentration or 3% to 5%
NaClO by mass-volume concentration separately to clean the tissue, it is difficult to sterilize
thoroughly, too, especially Fungus; During the sterilization process, the proper use of NaCl
saline can protect the cell structure. The chemical solution in the present disclosure only
comprises salt, alcohol and hypochlorous acid with a certain concentration. The preparation is
simple and uses less reagents. In addition, the chemical solution in the present disclosure can
fully sterilize the duckweed tissue surface in 5 min -10 min, and therefore, the processing is
simple, convenient, and time- saving, so that the chemical solution can be used for sensitively
identifying the type of bacterial infection in duckweed tissue;
2. The method of the present disclosure for identifying the type of bacterial infection
based on duckweed is based on the function research system of duckweed "aquatic model plant",
cleaning duckweed leaf tissues with chemical solutions of special components and
concentrations to effectively remove epiphytic bacteria on the surface of the tissue so that
endophytes can be stained and observed. It is convenient and fast, and is not affected by the
bacteria itself. Combine with the green fluorescent nucleic acid dye DMAO for staining, which
stains live and dead cells of Gram-positive bacteria and Gram-negative bacteria, so as to quickly
and sensitively identify the type of bacterial infection, and help determining whether the
infecting bacteria is pathogenic to the host plant, whether the microorganism and the plant
establish a symbiotic relationship, and the interaction between them.
Brief Description of the Drawings
[00019] FIG. 1 shows the results of fluorescence microscopy imaging of duckweed tissues
infected by bacteria after being cleaned with the method in Example 1 of the present disclosure, wherein W represents duckweed tissues cleaned with sterile water; C represents the duckweed tissues cleaned with the chemical solution of the present disclosure after being cleaned with sterile water; the white arrow points to the bacteria.
[00020] FIG. 2 shows the result of fluorescence microscopy imaging of duckweed tissues
infected by bacteria after being cleaned with the existing method in Example 1 of the present
disclosure, wherein W represents duckweed tissues cleaned with sterile water; SD represents
the duckweed tissues cleaned with sterile water and the existing chemical solution in sequence;
B represents duckweed tissues cleaned with the existing method (after washing with sterile
water and SD in sequence, washing with sodium hypochlorite for 1 min-2 min); the white
arrow points to the bacteria;
[00021] FIG. 3 shows the PCR verification results of the duckweed tissues infected by bacteria
after being cleaned with the existing method in Example 1 of the present disclosure, wherein
No TC represents PCR blank control without DNA template; + represents PCR control with
inoculated strains; W represents the duckweed tissue cleaned with the sterile water; C represents
the duckweed tissues cleaned with the chemical solution of the present disclosure after being
cleaned with sterile water;
[00022] FIG. 4 shows the PCR verification results of the duckweed tissues infected by bacteria
after being cleaned by existing method in Example 1 of the present disclosure, wherein No TC
stands for PCR blank control without DNA template; + stands for PCR control with inoculated
strains; SD represents the duckweed tissue cleaned with sterile water and the existing chemical
solution in sequence; B represents the duckweed tissue cleaned with the existing method (after
washing with sterile water and SD in sequence, washing with sodium hypochlorite for 1 min-2
min)
[00023] In FIG.1 to FIG. 4, Lm4a represents the strain to be detected; Aw4b represents the
strain to be detected; - represents the sterile duckweed; Sp245 represents the positive control
strain of endophytes; Sp7 represents the positive control strain of epiphytes.
Description of the Embodiments
[00024] In order to facilitate the understanding of the present disclosure, a more comprehensive
description is given below. The present disclosure can be implemented in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure more thorough and comprehensive.
[00025] In the following embodiment, the experimental methods without specific conditions usually follow conventional conditions, such as those described in Sambrook et al., molecular cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the specific conditions as recommended by the manufacturer. The various common chemical reagents used in the embodiments are commercially available products.
[00026] Unless otherwise defined, all technical and scientific terms used in the present disclosure are the same as commonly understood by those skilled in the art of the present disclosure.
[00027] In the present disclosure, the said bacteria to be detected are the bacteria isolated from water or soil environment with conventional methods. At the same time, a blank control and/or a positive control are set; the said blank control is the sample without adding with bacterial solution; the said positive control is the sample added with a bacterial solution with a known function. The blank control and the positive control can be selected and set according to the routine skills of those skilled in the art.
[00028] The present disclosure will be further described in detail in conjunction with specific embodiments and drawings as below.
Example 1: A method for identifying bacterial infection types based on duckweed
[00029] The method for identifying bacterial infection based on duckweed in this embodiment comprises the following steps: 1. Construction of a sterile duckweed system
[00033] After being washed in sterile water for 3 to 5 times, take 5 to 10 pieces of the collected duckweed and place them into a 10 mL sterile centrifuge tube, add 5 mL of 50% alcohol to wash for 10 minutes, and discard the supernatant. Add 5 mL of 5% sodium hypochlorite to wash for 2 min, then wash with sterile water 2-3 times.
[00034] The duckweeds were transferred to a plant medium (1/2 SH + 0.5% sucrose + 0.1%
cefotaxime), wherein SH is the abbreviation of Schenk & Hildebrandt Basal Salt Mixture Plant
Medium. After growing on the culture medium for 7 days, the duckweeds were placed on the
LB (beef extract peptone) bacterial culture medium and observed overnight in a 28°C incubator.
If there was no colony of bacteria formed around the duckweed, it indicated that the duckweeds
were fully sterilized. At this time, transfer some sterile duckweeds to IL sterilized 2xSH culture
solution, and then place them in a constant temperature culture room to expand the culture for
about 2-3 weeks. The duckweed biomass expanded, forming a stable sterile duckweed system.
2. Inoculation of bacteria in sterile duckweed
[00035] Day 1: The two strains to be detected, Enterobactersp. Lm4a and Pantoea sp. Aw4b
(the strains to be detected were identified by the applicant's laboratory, with the 16S rDNA
sequence of Lm4a shown in SEQ ID No. 3, and the 16S rDNA sequence of Aw4b shown in
SEQ ID No. 4) and the two positive control strains Sp7 (identified as the epiphyte Azospirillum
brasilense) and Sp245 (identified as the endophyte Azospirillum brasilense) (Jain & Patriquin
1984) were inoculated respectively in 5 mL LB culture medium, in a 28°C constant temperature
incubator with 250 rpm shaking for overnight culture;
[00036] 6S rDNA sequence of Lm4a (SEQ ID No. 3):
GGTTAATAACCTCAGCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCG TGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCG TAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCT GGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAgAGGGGGGTAGAATTCC AGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGC CCCCTGGACAAAGA
[00037] 16S rDNA sequence of Aw4b (SEQ ID No. 4):
[00038] Day 2: Take out 500 L bacterial culture solution of Day 1 and place it into 50 mL of LB culture broth for expanding culture. The culture conditions were the same as those of Day 1;
[00039] Day 3: Take out 25 mL of the bacterial culture solution of Day 2 and place it in a sterile centrifuge tube. Centrifuge at 8,000 rpm for 5 min at 4°C. After the supernatant being discarded, the precipitate was added with 25 mL sterile water and mixed well. Centrifuge at 8,000 rpm at 4°C for 5 min. discard the supernatant, and repeat this step. The precipitate was added with 25 mL of 0.5xSH sterile solution and mixed well. The absorbance of the bacterial solution at 600 nm was recorded using a spectrophotometer, and then the bacterial solution was diluted to OD600 = 0.2/50 mL. Pour 50 mL of the tested strains Lm4a (Enterobacter sp.) and Aw4b (Pantoea sp.) dissolved in 0.5xSH into a sterile bottle, and then transfer the pre-cultured sterile duckweeds to the bacterial solution bottle, and cover the surface of the bacterial liquid, then the experimental group of duckweed was obtained;
[00040] In addition, take and place 50 mL of 1/2 SH sterile solution into a sterile bottle, add the same amount of duckweed, as a blank control; at the same time, dilute the bacterial solution with 1/2 SH to a positive control with OD600 = 0.2 / 50 mL Strains Sp7 (Azospirillum
brasilense) and Sp245 (endophytes Azospirillum brasilense), and then respectively add sterile
duckweeds as a positive control.
[00041] Transfer the inoculated duckweed of the experimental group, the sterile duckweed of blank control group, and the inoculated duckweed of positive control group to a light culture room to grow (16h light/8h dark, light intensity OOOLx). 3. Cleaning the inoculated duckweed with sterile water and chemical solution
[00042] After culturing for 2 to 4 days, take out and discard the 0.5xSH solution in each bottle, and cut out a small part of duckweed leaf tissue and perform the following treatments respectively: a. wash the duckweed tissue leaves of the experimental group, blank control group and positive control group with sterile water (mark as W) for 1 min. Repeat the cleaning process once. b. wash the duckweed tissue leaves of the experimental group, blank control group and positive control group with the chemical solution of the present disclosure (mark as C); select and transfer the duckweed samples treated with W to a sterile chemical solution (130mM NaCl, 30% alcohol by volume concentration, 3% NaClO by
mass volume concentration, with pH 7.4) and wash for 5 minutes, and then wash with sterile water twice, 1 min each time; c. clean the duckweed tissue leaves of blank control group and positive control group with existing methods (mark as SD, B);
() select and transfer 10 pieces of the duckweed treated with W to a sterile
SD chemical solution (140mM NaCl, 2.5mM KCl, 10mM Na2HPO4,2mM KH2PO4, 0.5mM MgSO4, 1mM CaC2, 0.1% (v/v) Triton-X100, with PH=7.5), washed for 20 min;
after step (D,wash the duckweed with 5% sodium hypochlorite for 1 min-2
min, then finally rinsed with Na2S203 solution twice, marked as B. 4. Dye the duckweed tissues and identify the type of bacterial infection
[00043] Take out the duckweed samples treated in step 3 and place them on the glass slides, add 5 L -10 L of green fluorescent nucleic acid dye DMAO (Live & Dead Bacterial Staining Kit, #40274ES60) dropwise, dye for 3 to 5 minutes, and then remove the dye solution with paper tower.
[00044] Place the glass slides under the Olympus (FSX100) fluorescence microscope to observe the stained bacterial community in duckweed tissue, using lOx, 20x and 30x magnifications with a green filter, and take photos.
[00045] By observing the staining results of bacteria infected duckweed, it can be determined
whether the duckweed tissues were infected by epiphytic or endophytic bacteria. If the bacteria
are distributed on the surface of leaf tissue, it can be judged that the bacteria infect duckweeds
in the form of epiphytes, and the tissue infection ability is relatively weak; if the bacteria are
distributed inside the leaf tissue, it can be judged that the bacteria infect duckweeds in the form
of endophytes, and the tissue infection ability is strong.
[00046] FIG. 1 shows the results of fluorescence microscopy imaging of duckweed tissues
infected by bacteria after being washed with the chemical solution (W/C) of the present
discourse. After the sterile duckweeds in blank control group are washed with W and C, there
is no bacterial distribution on the surface or inside of the leaf tissue; compared with the positive
control group Sp7, after the duckweeds incubated with the strain Lm4a are washed with solution
W, there are bacteria distributing on the surface of the leaf tissue; however, after the duckweeds
are washed with solution C, there is no bacterial distributing inside the leaf tissues, indicating
that the strain Lm4a infects duckweeds by way of epiphytic bacteria, and its tissue infection
ability is relatively weak. Compared with the positive control group Sp245, after the duckweeds
incubated with the strain Aw4b are washed with solution W, there are bacteria distributing on
the surface of the leaf tissue; after being washed with solution C, there are bacteria distributing
inside the leaf tissue, indicating that the strain Aw4b infects duckweeds in an endophytic manner,
and the tissue infection ability is strong. It is proved that the method of the present disclosure
can well identify the type of bacterial infestation.
[00047] FIG. 2 shows the results of fluorescence microscopy imaging of duckweed tissues
infected by bacteria after being washed with the existing chemical solution (SD/B). After the
sterile duckweeds in the blank control are washed with solutions W, SD and B, there is no
bacterial distributing on the surface and inside of the leaf tissue; after the positive control
epiphyte Sp7 is washed with W and SD, there are still bacterial distribution on the surface and
inside of the leaf tissue; while after being washed with B, there is no bacterial distribution on
the surface and inside of the leaf tissue, indicating that solution SD alone is not enough to clean
the epiphytes, but the combination of SD and B can achieve the effect of removing epiphytes.
Therefore, after the plant tissues are treated with solution W and/or SD, epiphytes can be observed. After the positive control endophytes Sp245 is washed with W, the bacteria are distributed on the surface of the leaf tissue; after being washed with SD and B, the bacteria are distributed inside the leaf tissue, indicating that epiphytic bacteria can be cleaned by the combination of SD and B, but the endophytic bacteria cannot be cleaned, so the distribution of endophytic bacteria can be observed.
5. Verification of bacterial infection of duckweed leaf tissue based on PCR
[00048] The duckweed leaves washed with W and C in step 3 were transferred to a 2mL
microcentrifuge tube which was pre-filled with lg of zirconia beads, add 2 x CTAB buffer and
chloroform, homogenized by a magnetic bead oscillator, and then extract the DNA by 2x CTAB
nucleic acid extraction method.
[00049] The DNA of each treatment group was amplified by primers to amplify the bacterial
16S rDNA gene fragment, wherein the forward primer e9f: 5'-GAGTTT GATCCTGGCTCAG
3'(SEQ ID No. 1), and the reverse primer e926r: 5'-CCGTCAATTCCTTT GAGTTT- 3'(SEQ
ID No. 2).
[00050] The 25 L PCR reaction system includes: 2.5 L of 10 x PCR buffer, 0.5 mM of MgC2,
0.2 mM of dNTPs, 0.8 M each of forward and reverse primers, 0.5 L of 5 U/L Taq
polymerase, 100 ng of DNA template, and finally adding sterile ddH2 0 to 25 L.
[00051] The PCR reaction program includes: 95°C pre-denaturation for 5 min; 95°C
denaturation for 1 min, 50°C annealing for 30 seconds, 72°C extension for 1 min, 25 cycles;
and finally, 72°C extension for 5 min.
[00052] After being stained with ethidium bromide, the PCR products were electrophoresed on
a 1% agarose gel for about half an hour, and then the PCR amplification results were detected
by a gel imager and photographed. If both W and C treatment groups showed bands, it was
endophytic bacteria infecting the duckweed; if W group showed bands, and C treatment group
had no bands, it was epiphytic bacteria infecting the duckweed.
[00053] After washing with the solution (SD and B) of the existing method, the judgment was
based on: after washing with sterile water (W), there was a fingerprint pattern detected by PCR,
or there was a weak fingerprint pattern after washing with solution SD ; but if there was no
fingerprint after washing with solution B, it was considered that the bacteria to be detected are
epiphytes bacteria with weak infectivity. After washing with solutions W, SD and B, if there was a clear fingerprint pattern by PCR detection, it was considered that the bacteria to be detected are endophytic bacteria with strong infectivity.
[00054] The PCR verification result map of the bacteria infection of the duckweed tissue
cleaned by the method of the present disclosure is shown in FIG. 3. It can be seen from FIG. 3
that compared with the positive control Sp245, the W and C treatment groups of the duckweed
tissue inoculated with strain Aw4b, both showed bands, which proved that the duckweed tissues
were infected by endophytes, and the strain Aw4b was endophytic. While compared with the
positive control group Sp7, for the duckweed tissue inoculated with the strain Lm4a, the W
treatment group showed a band, while the C treatment group showed no band, which proved
that the epiphytic bacteria infected the duckweed tissue, and the strain Lm4a was epiphytic
bacteria. It shows that the PCR detection result was consistent with the fluorescent staining
result.
[00055] The PCR verification result map of bacterial infection of the duckweed tissue cleaned
by the existing methods is shown in FIG. 4. It can be seen from FIG. 4 that for the positive
control of endophytes Sp245, the W, SD, and B treatment groups all showed bands, which
proved that the duckweed tissues were infected by endophytes; while for the positive control of
epiphytic bacteria Sp7,and the W treatment groups showed a band and the SD treatment group
also showed the same band as the W treatment group, but there was no band for the B treatment
group, which proved that the duckweed tissue was infected by epiphytic bacteria.
[00056] In summary, using chemical solution SD of the existing technology, or SD solution
combined with B solution to clean duckweed tissues can also verify the type of bacterial
infection in duckweed tissues, but the steps are cumbersome, time-consuming, and not sensitive
enough, which is reflected in the following three aspects: 1. SD solution requires a variety of reagents, which is complicated and time-consuming to prepare, and the time for SD to wash duckweed tissue is at least 20 minutes to ensure that the bacteria attached to the surface of the tissue are cleaned thoroughly, and then followed with cleaning with B solution; if B solution is used alone, it is unable to ensure complete sterilization of the surface of the plant tissue; while the chemical solution CM of the present disclosure combines the sterilization effects of alcohol and hypochlorous acid, and it only takes 5 to 10 min to clean the bacteria attached to the tissue surface, which greatly reduces the washing time;
2. When verifying whether the type of bacterial infested tissue is endophyte infection, cleaning with SD solution alone cannot ensure the accuracy of the. It needs to be combined with B solution to wash the tissue and then extract plant DNA for PCR detection, which is cumbersome; 3. When verifying whether the type of bacterial infested tissue is epiphytic infection, the PCR detection results after washing with sterile water W and SD solution are consistent, so it is unnecessary to carry out the SD step alone and combine PCR detection.
Example 2 A method for identifying bacterial infection types based on duckweed
[00057] The method for identifying the bacterial infection based on duckweed in this embodiment is the same as that of Example 1, except that the concentration of each component of the chemical solution (CM) of the present disclosure in step 3b is different from that of Example 1. The chemical solution in this example comprises the following components: 135mM of NaCl, 40% alcohol by volume, 5% (g/100ml) of NaClO, with pH 7.6. The results of
the method for identifying the bacterial infection types based on duckweed in this example are consistent with that of Example 1. Example 3 A method for identifying bacterial infection types based on duckweed
[00058] The method for identifying bacterial infection types based on duckweed in this example is the same as that of Example 1 except that the concentration of each component of the chemical solution (CM) in step 3b of the present disclosure is different from that of Example 1. The chemical solution of the example comprises the following components: 140mM ofNaCl, % alcohol by volume, 4% (g/100ml) of NaClO, with pH 7.8. The results of the method for
identifying bacterial infection types based on duckweed in this example are consistent with that of Example 1.
[00059] The various technical features in the embodiments above can be combined in various ways. The embodiments above do not describe all the possible combinations of the technical features for a concise description. However, those combinations which are not described should be within the scope of the description as long as no contradiction occurs in the combinations of these technical features.
[00060] The above-described embodiments represent only several embodiments of the present invention, which are described in specific detail but should not be construed as limitations on the scope of the claims. It should be noted that modifications and improvements can be made for those skilled in the art without departing from the spirit of the invention, all of which fall within the scope of the present invention. Accordingly, the scope of the present invention should be subject to the appended claims.
Claims (5)
1. A method for identifying bacterial infection types based on duckweed, comprising the
following steps:
(1) coculturing the bacteria to be detected and sterile duckweeds in 1/2 SH culture
medium to obtain inoculated duckweed;
(2) cleaning the inoculated duckweeds, which includes: washing with sterile water; then
washing with chemical solution for 5 to 10 minutes, and then washing with sterile
water again;
wherein, the chemical solution includes the following components: 127 mM -147
mM NaCl, 30%- 45% alcohol by volume concentration, 3%-5% NaClO by mass
volume concentration, with pH 7.0-7.8;
(3) dying the cleaned duckweed separately with the green fluorescent nucleic acid dye
DMAO.
2. The method for identifying bacterial infection types based on duckweed according to claim
1, wherein the chemical solution in step (2) comprises the following components: 135~140mM
NaCl, 35~40% alcohol by mass volume concentration, 4-5% NaClO by mass volume
concentration, with pH 7.4-7.8.
3. The method for identifying bacterial infection types based on duckweed according to claim
1, wherein the number of times of sterile water washing in step (2) is 2 to 4 times, and each
time lasts 0.5 min to 1 min.
4. The method for identifying bacterial infection types based on duckweed according to any
one of claims 1 to 3, wherein the amount of the green fluorescent nucleic acid dye DMAO in
step (3) is 5 L -25 L, and the dyeing time is 2 min-5 min.
5. The method for identifying bacterial infection types based on duckweed according to claim
1, wherein the sterile duckweeds in step (1) are obtained by the following method: washing the
duckweeds with sterile water 3 to 5 times; adding 5 mL-10 mL of 45-55% alcohol and washing for 5 min to 10 min; discarding the supernatant; adding 5-7% hypochlorite by mass volume concentration and washing for 1.5 min to 3 min, and then washing with sterile water for 2 to 3 times.
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