CN110669850B - Method for detecting colonization ability of bacteria in salvia miltiorrhiza - Google Patents
Method for detecting colonization ability of bacteria in salvia miltiorrhiza Download PDFInfo
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Abstract
The invention relates to a method for detecting the colonization ability of bacteria in salvia miltiorrhiza, which comprises the following steps: 1) Inoculating bacteria to be detected into the sterilized salvia miltiorrhiza seedlings for culture so as to colonize the inoculated bacteria; 2) The colonized salvia miltiorrhiza seedlings are respectively treated and grouped by the following steps: w) sterile water washing; SD) sterilized saline solution is washed for 10min to 30min and then is washed by sterile water; b) Washing with sterilized saline solution, washing with sodium hypochlorite, and washing with Na 2 S 2 O 3 Washing with a solution; 3) Amplifying the characteristic nucleic acid fragments of the bacteria to be detected which are colonized in each group, dyeing the amplification result, and determining the colonization ability of the bacteria to be detected according to whether target strips appear in W, SD and the group B, wherein the target strip appearing in the group B has the strongest value-fixing ability, the next group SD is the next group, and the last group W is the next group. The method can evaluate the colonization ability of bacteria more objectively.
Description
Technical Field
The invention relates to the field of microorganism-plant interaction research, in particular to a method for detecting the colonization ability of bacteria in salvia miltiorrhiza.
Background
The existing scientific research and the microbial inoculum sold on the market can influence the growth, development and metabolic activity of plants to a certain extent, and especially plant growth-promoting rhizobacteria (PGPR) can promote the growth of plants, prevent and treat diseases, increase crop yield and the like. Successful colonization of bacteria in plant roots plays an extremely important role in bacterial-plant interactions. Because a plurality of microorganisms with remarkable growth promoting effect under laboratory conditions can not play a good role when being applied to a field, the strong and weak colonization ability is the key to influence the action of the microorganisms. Therefore, whether the inoculated beneficial bacteria can successfully colonize the root circumference or the leaf circumference of the host plant or not is realized, and the characteristics of strong colonization capacity, large colonization quantity and the like are realized, so that the key factors for determining the direct action effect, the application dosage, the application conversion and the like of the biocontrol bacteria are realized.
Currently, the most common methods for detecting bacterial colonization include microscopy and colony counting, but are time-consuming, labor-consuming and highly affected by human manipulation. The molecular biology can further reveal the special rule of the colonization of the bacterial roots, the detection result is more accurate, and the existing detection methods for researching the colonization capability of the biocontrol bacteria on the salvia miltiorrhiza by using molecular markers, fluorescent markers, special primers and the like are relatively few.
Disclosure of Invention
The invention relates to a method for detecting the colonization ability of bacteria in salvia miltiorrhiza, which comprises the following steps:
1) Inoculating bacteria to be detected into the sterilized salvia miltiorrhiza seedlings for culture so as to colonize the inoculated bacteria;
2) The colonized salvia miltiorrhiza seedlings are respectively treated and grouped by the following steps:
w) sterile water washing;
SD) sterilized saline solution is washed for 10min to 30min and then is washed by sterile water;
b) Washing with sterilized saline solution for 10-30 min, washing with 3-7% (g/100 ml) sodium hypochlorite for 1.5-2.5 min, and washing with Na 2 S 2 O 3 Washing with a solution;
wherein the salt solution comprises the following components:
127mM～147mM NaCl，1.7mM～3.7mM KCl，5mM～15mM Na 2 HPO 4 ，1.3mM～2.3mM KH 2 PO 4 ，0.3mM～0.7mM MgSO 4 ，0.7mM～1.3mM CaCl 2 0.07-0.13% (v/v) Triton-X100 and the salt solution pH = 7.0-7.8;
3) Amplifying the characteristic nucleic acid fragments of the bacteria to be detected which are colonized in each group, dyeing the amplification result, and determining the colonization ability of the bacteria to be detected according to whether target strips appear in W, SD and the group B, wherein the target strip appearing in the group B has the strongest value-fixing ability, the next group SD is the next group, and the last group W is the next group.
5363 the elution/sterilization capacity of group B and W, SD are different, with group W being the weakest and group B being the strongest, so that if group B has amplification product of target band, it indicates that the colonization capacity of target-corresponding bacteria to be detected is stronger. By the method, the bacteria colonization ability can be evaluated more objectively.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the detection results of the bacteria Sp7 and Sp245 colonizing the root system of Salvia miltiorrhiza Bunge in one embodiment of the present invention;
sp7& Sp245: isolated from wheat, early studies have identified epiphytic and endophytic bacteria; IGS, a 16S-23S rRNA intergenic region of bacteria, sp7 of which is 562bp and Sp245 of which is 547bp; LFY, red sage root LEAFY gene-257 bp; controls: PCR control; no TC: no DNA template; -: sterile red sage root; +: bacterial DNA;
FIG. 2 is a fluorescent staining pattern of two bacteria inoculated to Salvia miltiorrhiza Bunge after three different chemical treatments in one embodiment of the invention; the length of the scale is 32 μm.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.
The invention relates to a method for detecting the colonization ability of bacteria in salvia miltiorrhiza, which comprises the following steps:
1) Inoculating bacteria to be detected into the sterilized salvia miltiorrhiza seedlings for culture so as to colonize the inoculated bacteria;
2) The colonized salvia miltiorrhiza seedlings are respectively treated and grouped by the following steps:
w) sterile water washing;
SD) sterilized saline solution is washed for 10min to 30min and then is washed by sterile water;
b) Washing with sterilized saline solution for 10-30 min, washing with 3-7% (g/100 ml) sodium hypochlorite for 1.5-2.5 min, and washing with Na 2 S 2 O 3 Washing with a solution;
wherein the salt solution comprises the following components:
127mM～147mM NaCl，1.7mM～3.7mM KCl，5mM～15mM Na 2 HPO 4 ，1.3mM～2.3mM KH 2 PO 4 ，0.3mM～0.7mM MgSO 4 ，0.7mM～1.3mM CaCl 2 0.07% to 0.13% (v/v) Triton-X100 and the salt solution pH =7.0 to 7.8;
3) Amplifying the characteristic nucleic acid fragments of the bacteria to be detected which are well colonized in each group, dyeing the amplification result, and determining the colonization ability of the bacteria to be detected according to whether target bands appear in W, SD and group B, wherein the target bands appear in group B with strongest quantification ability, the group SD with the second time, and the group W with the first time.
The invention can be suitable for the research of rhizosphere microorganism colonization condition. Rhizosphere (rhizosphere) refers to an area whose biological and physical characteristics are closely affected by root systems, and the rhizosphere and a microbial community form a very complex ecological system together, which is a habitat for ensuring normal growth and development of plant root systems and is also a main place for exchanging substances and energy between plants and the external environment, wherein microorganisms are rich in diversity and have strong rhizosphere effect. Therefore, the microbial environment formed by rhizosphere microorganisms plays an extremely important role in plant growth.
In some embodiments, the salt solution comprises the following components:
132mM～142mM NaCl，1.9mM～3.5mM KCl，8mM～12mM Na 2 HPO 4 ，1.5mM～2.1mM KH 2 PO 4 ，0.4mM～0.6mM MgSO 4 ，0.9mM～1.1mM CaCl 2 0.08-0.12% (v/v) Triton-X100, and the salt solution pH = 7.2-7.6.
In some embodiments, the nucleic acid fragment characteristic of the bacterium is a fragment in the 16S rDNA of the bacterium.
The 16S rDNA has high conservation in structure and function, is commonly used for bacterial isolation, and can be used as a simple fingerprint sequence of bacterial species in the invention.
In some embodiments, the method of staining the amplification results is selected from the group consisting of silver staining, methylene blue staining, ethidium bromide staining, and acridine orange staining.
In some embodiments, the forward primer and the reverse primer used for amplification of the nucleic acid fragment characteristic of the bacterium are shown in SEQ ID NO 1 and 2, respectively.
The sequences shown in SEQ ID NO 1 and 2 are designed aiming at the evolutionary conserved region of 16S rDNA, and the region can be amplified.
In some embodiments, the method further comprises the step of amplifying a gene whose expression is stable endogenously in salvia miltiorrhiza as an internal reference gene.
In some embodiments, the gene whose endogenous expression of salvia miltiorrhiza is stable is a LEAFY gene.
In some embodiments, the upstream and downstream primers used to amplify the LEAFY gene are set forth in SEQ ID NOS: 3 and 4, respectively.
In some embodiments, the method for preparing the sterilized young salvia miltiorrhiza seedlings comprises the following steps:
and culturing and germinating the salvia miltiorrhiza seeds for 10-15 days to obtain the salvia miltiorrhiza seedlings.
In some embodiments, the salvia miltiorrhiza seeds are subjected to a sterilization cleaning treatment selected from the group consisting of:
70-85% ethanol water solution cleaning, 40-60% (g/100 ml) sodium hypochlorite cleaning and sterile water cleaning.
In some embodiments, the salvia miltiorrhiza seeds are further subjected to low temperature vernalization prior to germination in culture:
culturing in 1/2MS solid culture medium at 0-8 deg.c in dark for 2-4 days.
In some embodiments, the culture conditions for 5-10 days of germination are: the illumination culture is carried out for 12 to 16 hours at the temperature of between 23 and 27 ℃, the dark culture is carried out for 8 to 12 hours at the temperature of between 20 and 24 ℃.
In some embodiments, the method further comprises:
staining the bacteria in the colonized salvia miltiorrhiza seedlings to assist in judging the bacteria colonization ability.
In some embodiments, the dye used to stain the bacteria is SYTO 9 and propidium iodide.
Embodiments of the present invention will be described in detail with reference to examples.
Examples
1. Sterilizing and seedling culturing of salvia miltiorrhiza seeds
Taking 8-10 salvia miltiorrhiza seeds, putting into a centrifuge tube, adding 1mL 80% ethanol, and placing in an oscillator at 1000rpm for shaking for 3min. Discard the supernatant, add 1mL 50% NaClO and shake for 5min. After discarding the supernatant, 1mL of sterile water was added and washed 4 times. The seeds were transferred to 1/2MS solid medium and sealed with Parafilm sealing film and placed in a dark room at 4 ℃ for vernalization at low temperature. After three days, the seeds are moved to a light incubator, the light is carried out at 25 ℃ (14 h), the illumination is carried out, the light is carried out at 22 ℃ (10 h), the darkness is carried out, and the inoculation treatment is carried out after the seeds germinate and grow for one week.
2. Inoculation of bacteria
Using Sp7 and Sp245 isolated from wheat, both of Azospirillum, earlier studies have identified epiphytic bacteria (epiphytic bacteria) and endophytic bacteria (endophytic bacteria), reference strains useful for the study of bacterial colonization characteristics in plant-microorganism interactions.
Two target strains are inoculated in 6mL LB culture solution, and are cultured in a constant temperature incubator at 28 ℃ for 48h by shaking at 250 rpm. Centrifuging the bacterial culture solution at 10,000rpm for 5min, discarding the supernatant, diluting the bacterial solution with sterile water, and measuring the light absorption value of the bacterial solution at 600nm by a spectrophotometer to be about 0.7. 100 mu L of diluted bacterial liquid is evenly coated on a 1/2MS plant culture medium in a clean bench, red sage root seedlings are transferred to square culture dishes (the specification is 9cm multiplied by 9 cm), sealing is carried out by using a sealing film, six seedlings can be placed in each culture medium, and then the culture medium is placed in a constant temperature culture room (25 ℃,12h day and 12h night) for culture for one week. Meanwhile, the medium was coated with 100. Mu.L of sterile water, and six Salvia miltiorrhiza seedlings were placed for blank control.
3. Nucleic acid extraction
For the salvia miltiorrhiza plants inoculated with the bacteria, nucleic acid was extracted seven days after inoculation. Before this, three different types of chemical treatments were used to detect the strength of the ability of the inoculated bacteria to colonize the root system of salvia miltiorrhiza, i.e. to symbiotic with the plant in the form of either epiphytes or endophytes. Three chemical treatments of root system tissue of salvia miltiorrhiza include: 1) Sterile water was washed twice, and the treated sample was labeled as W; 2) Sterile saline solution (137mM NaCl,2.7mM KCl,10mM Na) 2 HPO 4 ,1.8mM KH 2 PO 4 ,0.5mM MgSO 4 ,1mM CaCl 2 0.1% Triton-X100, pH 7.4) for 20min and rinsed twice with sterile water, the sample being scored as SD; 3) The sample was washed with sterile saline solution for 20min, then 5% sodium hypochlorite for 2min, and finally twice with Na2S2O3 solution, and the sample was noted as B.
Transferring 3-5 salvia miltiorrhiza roots into centrifuge tubes marked with W, SD and B in advance, then transferring the salvia miltiorrhiza roots into centrifuge tubes pre-filled with grinding magnetic beads (14 mm silica beads) after the chemical treatment of 1,2 and 3, adding 750 mu L of 2 xCTAB and 300 mu L of chloroform, and fully grinding for 3min by a tissue grinder. Centrifuge at 16,000g for 10min at room temperature and transfer the supernatant to a new centrifuge tube. Add 100. Mu.L ammonium acetate and 750. Mu.L absolute ethanol and centrifuge at 16,000g for 30min at 4 ℃. The supernatant was discarded, 500. Mu.L of 75% ethanol was added, centrifuged at 16,000g at 4 ℃ for 10min, and the procedure was repeated. After discarding the supernatant, the nucleic acid was dried, and the DNA was diluted to 100 ng/. Mu.L with sterile double distilled water, and the purity and concentration of the DNA were examined by Nanodrop 2000 and 1% agarose gel electrophoresis.
4.PCR method for identifying bacterial genes
Bacterial 16S-23S IGS primers, forward primer 16S-e1390f (SEQ ID NO:1:5'-TGYACACACCGCCCGTCA-3', where Y represents T or C), reverse primer 23S-e130R (SEQ ID NO:2:5 '-GGGTCCCATTCRG-3', where B represents G or C or T, and R represents G or A) were used. 25 μ L of PCR reaction included 2.5 μ L of 10 XPCR buffer, 0.5mM MgCl 2 0.2mM dNTPs, 0.8 mu M of forward and reverse primers respectively, 0.5 mu L of 5U/mu L Taq polymerase, about 100ng DNA template, and finally adding sterile ddH 2 O to 25. Mu.L. The PCR reaction procedure included pre-denaturation at 95 ℃ for 5min,30 cycles including denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 90s, and finally extension at 72 ℃ for 5min. After being stained by smelling ethidium, the PCR product is electrophoresed for about one hour through 1% agarose gel, and then the PCR amplification result is detected by a gel imager and photographed. And (3) judging whether the samples W, SD and B have the fingerprint of the target bacteria or not by the result of PCR amplification of the bacterial fingerprint.
Meanwhile, sterile salvia miltiorrhiza bunge which is not inoculated with bacteria is used as a negative control, no bacterial fingerprint is theoretically generated by PCR amplification, if the bacterial fingerprint exists, salvia miltiorrhiza bunge seedlings are polluted, and the experiment needs to be repeated. When the PCR results of the samples W, SD and B have obvious bacterial fingerprint spectra, the bacteria inoculated to the salvia miltiorrhiza are considered to be colonized in an endophyte form, and the colonizing capacity is strong. When only the PCR result of the sample W has a bacterial fingerprint, or the sample SD also has a weak fingerprint, but the sample B does not have the bacterial fingerprint, the bacteria inoculated with the salvia miltiorrhiza is considered to be colonized in the form of epiphyte, and the colonization capability is weak. The results are shown in FIG. 1.
5.PCR method for identifying plant genes
In the PCR amplification, in addition to designing a bacteria-specific primer to amplify a specific bacterial gene, a plant-specific primer is specially designed to amplify a salvia miltiorrhiza specific gene LEAFY gene based on the LEAFY gene of Arabidopsis thaliana. The designed positive and reverse primers of the salvia miltiorrhiza genome are SmLFY-F (SEQ ID NO:3:5 '-GTTCCGGTACGCGAAGAAG-3') and SmLFY-R (SEQ ID NO:4:5 '-GGACGTACCAAATGGAGAGG-3'), respectively. 25 μ L of PCR reaction system comprises 2.5 μ L of 10 XPCR buffer, 0.2mM dNTPs, 0.4 μ M of forward and reverse primers, 0.4 μ L of 5U/. Mu.L Taq polymerase, about 200ng DNA template, and finally sterile ddH 2 O to 25. Mu.L. The PCR reaction program included pre-denaturation at 95 ℃ for 1min,28 cycles including denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 45s, and finally extension at 72 ℃ for 5min. After the dyeing by sniffing ethidium, the PCR product is electrophoresed for about one hour by 1 percent agarose gel, and then the PCR amplification result is detected by a gel imager and photographed and recorded. The results are shown in FIG. 1.
6. Fluorescence microscope double validation
In order to observe the infection condition of bacteria in the interaction between the bacteria and plants, a small part of plant tissue is cut after one week of bacteria inoculation, the plant tissue is washed by sterile water, the plant tissue is combined to pass through three different types of chemical treatment modes, the plant tissue is respectively placed on different glass slides, and about 20 mu L of green fluorescent nucleic acid dye SYTO 9 (SYTO) is dripped TM 9Green Fluorescent Nucleic Acid Stain, thermoFisher, USA) and the Green Red Fluorescent Nucleic Acid dye PI (Propidium iodine Ready probes) TM Reagent, thermo fisher, USA) for 3min, adding a proper amount of sterile water dropwise to wash off the excess dye solution on the glass slide, and finally covering the glass slide. SYTO 9green fluorescent nucleic acid dye was demonstrated to stain both live and dead cells of gram positive and gram negative bacteria. The fluorescent dye PI (propidium iodide) is a nuclear staining reagent which can stain DNA and is commonly used for apoptosis detection. Then, willThe slides were placed under an Olympus (FSX 100) fluorescence microscope and the bacterial colonies stained in the plant tissue were observed looking for a suitable field of view and observed and photographed using magnifications of 10X, 20X and 30X, respectively. The manner in which the bacteria colonize the plant (epiphyte or endophyte) and the strength of the colonization ability were further verified by observing the staining results of the bacteria-infected plants, in contrast to the PCR results of samples W, SD and B. The result of fluorescence microscopy is shown in fig. 2, the fluorescence staining result is consistent with the PCR detection result, and for the epiphytic bacteria Sp7, the salvia miltiorrhiza tissue cannot be detected after being washed by a chemical mode B. For endophytic bacterium Sp245, after the salvia miltiorrhiza tissue is washed by a chemical method B, the bacterium can still be detected. Furthermore, the combination of PCR detection based on two special primers and a specific chemical treatment mode of plant tissues has strong research significance and practical value for the research on the capability of bacteria to colonize salvia miltiorrhiza and the function of bacteria.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
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Claims (8)
1. A method for detecting the colonization ability of bacteria in salvia miltiorrhiza is characterized by comprising the following steps:
1) Inoculating bacteria to be detected into the sterilized salvia miltiorrhiza seedlings for culture so as to colonize the inoculated bacteria; the strain to be detected is Azospirillum Sp7 and Sp 245;
2) The colonized salvia miltiorrhiza seedlings are respectively treated and grouped by the following steps:
w) sterile water washing;
SD) sterilized saline solution is washed for 10min to 30min and then is washed by sterile water;
b) Washing with a sterilized salt solution for 10min to 30min, washing with 3 to 7 percent (g/100 ml) of sodium hypochlorite for 1.5min to 2.5min, and washing with Na 2 S 2 O 3 Washing with a solution;
wherein the salt solution comprises the following components:
127mM~147mM NaCl，1.7mM~3.7mM KCl，5mM~15mM Na 2 HPO 4 ，1.3mM ~2.3mM KH 2 PO 4 ，0.3mM~0.7mM MgSO 4 ，0.7mM~1.3mM CaCl 2 0.07% -0.13% (v/v) Triton-X100, and the salt solution has a pH = 7.0-7.8;
3) Amplifying the characteristic nucleic acid fragments of the bacteria to be detected which are well colonized in each group, dyeing the amplification result, and determining the colonization ability of the bacteria to be detected according to whether target bands appear in W, SD and group B, wherein the colonization ability of the target bands appears in group B is strongest, the group SD is the second, and the group W is the last;
a nucleic acid fragment characteristic of the bacterium is a fragment in the 16S rDNA of the bacterium;
the upstream primer and the downstream primer used for amplifying the characteristic nucleic acid fragment of the bacterium are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.
2. The method for detecting the colonization ability of salvia miltiorrhiza bunge by bacteria according to claim 1, further comprising the step of amplifying a gene with stable endogenous expression of salvia miltiorrhiza bunge as an internal reference gene.
3. The method for detecting the colonization ability of salvia miltiorrhiza bunge by bacteria according to claim 2, wherein the gene with stable endogenous expression of salvia miltiorrhiza bunge isLEAFYA gene.
4. The method of claim 3, wherein the ability of the bacteria to colonize Salvia miltiorrhiza is amplifiedLEAFYThe upstream primer and the downstream primer used by the gene are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4.
5. The method for detecting colonization ability of bacteria in salvia miltiorrhiza bunge according to any one of claims 1~4, wherein the method for preparing the sterilized salvia miltiorrhiza bunge seedling comprises:
and culturing and germinating the salvia miltiorrhiza seeds for 10 to 15 days to obtain the salvia miltiorrhiza seedlings.
6. The method for detecting the colonization ability of salvia miltiorrhiza bunge, according to claim 5, wherein the salvia miltiorrhiza bunge seeds are subjected to a sterilization and cleaning treatment, wherein the sterilization and cleaning treatment is selected from the following treatments:
70-85% ethanol water solution cleaning, 40-60% (g/100 ml) sodium hypochlorite cleaning and sterile water cleaning.
7. The method for detecting the colonization ability of bacteria in salvia miltiorrhiza according to claim 5, wherein the salvia miltiorrhiza seeds are further subjected to low-temperature vernalization treatment before culture and germination:
culturing the cells in a 1/2MS solid culture medium at 0-8 ℃ for 2~4 days in a dark place.
8. The method for detecting the colonization ability of bacteria on salvia miltiorrhiza according to claim 7, wherein the culture conditions for 5 to 10 days of culture germination are as follows: culturing at 12h to 111h under illumination at 23-27 ℃, and culturing at 20-24 ℃ in the dark for 8-12h.
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| CN113406050A (en) * | 2021-06-16 | 2021-09-17 | 广东省科学院生物工程研究所 | Method for identifying bacterial infection type based on duckweed |
| CN113416793A (en) * | 2021-06-16 | 2021-09-21 | 广东省科学院生物工程研究所 | Method for detecting colonization ability of bacteria in duckweed |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110699419A (en) * | 2019-10-24 | 2020-01-17 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Method for researching bacterial function based on duckweed |
| CN113406050A (en) * | 2021-06-16 | 2021-09-17 | 广东省科学院生物工程研究所 | Method for identifying bacterial infection type based on duckweed |
| CN113416793A (en) * | 2021-06-16 | 2021-09-21 | 广东省科学院生物工程研究所 | Method for detecting colonization ability of bacteria in duckweed |
| AU2021104715A4 (en) * | 2021-06-16 | 2021-09-30 | Institute of bioengineering, Guangdong Academy of Sciences | A method for detecting the colonization ability of bacteria in spirodela polyrrhiza |
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| Endophytic fungus Mucor circinelloides DF20 promote tanshinone;Haimin Chen;《Plant Science》;20210630;第307卷;第1-10页 * |
| Host-specific and tissue-dependent orchestration;Weijuan Huang;《Plant Soil》;20200602;第452卷;第379-395页 * |
| Roles of PlantAssociated Microbiota;Weijuan Huang;《trends in plant science》;20180731;第23卷(第7期);第559-562页 * |
| 丹参、黄精内生放线菌的分离及遗传多样性分析;李小林;《微生物学通报》;20100920;第37卷(第9期);第1343页第2.1节、第1342页第1.5节，第1343页第1.5.1和第1.5.2节 * |
| 丹参植株内生菌的分离纯化及其抗病性研究;颜华;《西北植物学报》;20160915;第36卷(第9期);第1813-1818页 * |
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