CN112662558A - Method for one-time preparation, separation and purification of potato whole-plant microorganism sample - Google Patents
Method for one-time preparation, separation and purification of potato whole-plant microorganism sample Download PDFInfo
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Abstract
The invention discloses a method for one-time preparation, separation and purification of a potato whole plant microorganism sample, which combines microorganism sampling and amplification culture to establish a set of method for separating and purifying the potato whole plant microorganism, can accurately and efficiently explore the flora structure and the main functions of the potato whole plant microorganism, since different media are required for the growth of different microorganisms, it is not desirable to use one or more media to completely isolate all plant microorganisms, therefore, the invention provides a culture medium aiming at different potato leaves and root systems, and can obtain better culture effect, most types of samples related in the current potato microbiome research are separated, collected and extracted at one time, and the types of the samples comprise 7 types of phyllosphere, stem periphery, root zone, rhizosphere, root surface, root inside and tuber attachment, so that the influence and interaction relation of external conditions such as different environments and the like on the growth of the whole potato plant can be completely analyzed.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for one-time preparation, separation and purification of a potato whole plant microorganism sample.
Background
In nature, plants coexist with various microorganisms, and a large number of microorganisms attach to the surface and interior of plants and play an important role in plant growth and disease resistance, and these microorganism groups are collectively called a plant microbiome. The model plant Arabidopsis thaliana has shown great research and application values in the aspects of bacterial colony formation, analytic interaction and the like. The overground part of the plant, called the phyllosphere, is a fixed, open, variable, circadian ecological environment whose microbial habitat is exposed to rapid changes in ultraviolet rays, temperature, moisture, nutrition, and the like. The underground part of the plant is covered by soil, and is mainly influenced by the physicochemical properties of the soil. The epiphytic and endogenous microorganisms of the root system and the soil-borne pathogenic bacteria are root system resident microorganisms which form a root system-related micro-ecological system.
With the popularization of high-throughput sequencing means and the reduction of sequencing cost, high-throughput sequencing becomes the latest means for microbiology research. More and more research has put new insights into the microbiome of different plants using high throughput sequencing. By sequencing microorganisms such as rhizosphere, phyllosphere and the like to generate high-coverage amplicon, genome and transcriptome data so as to estimate the species composition and abundance of the bacterial community, the diversity and complexity of the microbial community in the in-situ environment can be more truly disclosed. These studies open up new ways to explore the composition and structure of plant microbiome and its influencing factors. However, in order to obtain accurate sequencing results, a high-quality microorganism sample is obtained in the first place, so that how to take a sample as complete and real as possible in different environments is a common concern. In addition, most of the members of the group of plant microorganisms are bacteria whose living environment is essentially aerobic, and therefore, the members of the group of plant microorganisms can be isolated and purified. Due to the wide variation in the living environment of different plant microorganisms, no uniform standard method has been used to date for isolating members of the plant microorganism group.
Potatoes are the fourth most important grain crops in the world, and are typical root crops. With the continuous expansion of potato cultivation area and the development lag of high-efficiency cultivation technology in China, the potatoes gradually suffer from the problems of poor seedling growth and development, low yield, high abnormal potato proportion, serious pest and disease damage and the like. The change of related microbial population of the above-ground and underground parts of the potatoes directly or indirectly affects plant photosynthesis and absorption and transformation of soil nutrients, and the unbalance of the microbial population is a main reason for soil quality reduction, yield reduction of the potatoes and frequent pest and disease damage. Although few research reports on potato root microorganisms at home and abroad are provided at present, and researches on phyllospheric and stem periphery microorganism groups are hardly carried out, the research shows that the root microorganism group has important significance on the problems of high-efficiency yield increase of potatoes, prevention and control of plant diseases and insect pests, continuous cropping obstacles and the like. At present, the research of potato microbiology mainly discusses the interaction between microorganisms and potato plants, and relates to sampling different parts of the whole potato plant under different conditions so as to research the influence of factors such as different habitats, different genotypes, different soil types and the like on the growth and development of the overground and underground parts of the potato. However, in the study of potato microbiome, there is no clear method for sampling the rhizosphere, phyllosphere, periphery of stem, surface of potato tuber, etc., and there is no method for obtaining the whole plant microbial sample of potato plant at one time, especially the sequence of sampling different parts and the definition of root zone, rhizosphere, root surface, microbial area in root and their respective sampling methods are rarely reported. Because no related method for rapidly obtaining and separately culturing potato whole-plant microorganism samples exists, the research on potato microbiology and culturism is relatively lagged in progress. Therefore, it is important to solve such problems.
Disclosure of Invention
Aiming at the problems, the invention provides a method for one-time preparation, separation and purification of a potato whole plant microbial sample, which is used for one-time separation, collection and extraction of most types of samples related to the current potato microbiome research, wherein the samples comprise 7 types of phyllosphere, stem periphery, root zone, rhizosphere, root surface, root inside and tuber attachment, and the influence and interaction relation of external conditions such as different environments and the like on the growth of the potato whole plant can be completely analyzed.
In order to realize the technical scheme, the invention provides a one-time preparation, separation and purification method of a potato whole plant microorganism sample, which comprises the following steps:
the method comprises the following steps: dividing sampling parts, namely shearing leaves and stem sections of different parts of a potato plant, and putting the leaves and the stem sections into a numbered sterile container for preparing phyllospheric and stem periphery microorganism samples; cutting off the overground part of the potato plant, sealing the cut with adhesive paper, completely digging the underground part, and respectively putting fibrous roots and potato tubers into numbered sterile plastic bags for preparing root and potato tuber microorganism samples;
step two: transporting the sample, namely putting the taken sample into a storage box according to the classification of leaves, stem segments, roots and tubers, and immediately transporting the sample back to a laboratory for later use under the condition of an ice box;
step three: preparing phyllospheric and stem periphery microorganism samples, taking 10-20g of each of leaves and stem sections, placing the leaves and stem sections into 40mL of sterile PBS buffer solution, shaking the table at 200r/min for 30min, then treating the leaves and stem sections twice by ultrasonic waves at 30s each time, standing the leaves and stem sections for 5-10min, centrifuging the leaves and stem sections at 1500r/min for 1min, transferring the supernatant to another centrifuge tube, centrifuging the supernatant at 12000r/min for 10min to obtain thalli, reserving 10mL of the supernatant, then whirling the supernatant for 30s, subpackaging the supernatant into 2mL or more of EP tubes or cryopreservation tubes, and preparing DNA and RNA of phyllospheric and stem periphery microorganisms;
step four: preparing a root zone microorganism sample, paving numbered sterile paper on an experiment table, and shaking root soil corresponding to the number onto the paper forcibly until the root is invisible to naked eyes and obvious soil is on the root. Weighing 10-25g of shaken soil, sieving with a sterile sieve with the aperture of 2mm, uniformly mixing multiple samples, subpackaging into 2mL or more of EP tubes or freezing tubes, and preparing the DNA and RNA of the microorganisms in the root zone;
step five: rhizosphere microorganism sample preparation, 2-3 sterile centrifuge tubes containing 40mL sterile PBS buffer were prepared. And (3) putting the roots shaken off the soil in the third step into a centrifugal tube, shaking for 2-3min with force, standing for 5-10min, taking out the roots after the soil particles naturally settle, putting the roots into another centrifugal tube, and repeating the step for 1-2 times. Collecting washing liquid containing settled soil particles, centrifuging at 12000r/min for 10min, taking 10mL of supernatant, then vortexing for 30s, and subpackaging into an EP (ethylene propylene) tube or a freezing storage tube with the volume of 2mL or more for preparing rhizosphere microorganism DNA and RNA (ribonucleic acid);
step six: root surface microorganism sample preparation. Putting the washed root system into a sterile centrifuge tube of 10mL sterile PBS buffer solution, treating for 2-3 times by ultrasonic waves with the ultrasonic frequency of 50-60hz and 30s each time, subpackaging into 2mL EP tubes or freezing tubes for preparing the DNA and RNA of the microorganisms on the root surface;
step seven: preparing a microorganism sample in roots, taking out the root sample, washing the root sample with sterile water, carrying out surface disinfection on the root sample for 30s by using 75% ethanol, carrying out disinfection on the root sample for 10min by using 2.5% sodium hypochlorite, and fully washing the root sample with the sterile water; taking 5-10g of disinfected roots, placing the roots in 40mL of sterile PBS buffer solution, treating the roots for 5min by using a crusher, standing the roots for 10min, collecting 12000g of washing solution, centrifuging the washing solution for 10min, reserving 10mL of supernatant, then whirling the supernatant for 30s, and subpackaging the supernatant into 2mL or more of EP tubes or freezing tubes for preparing microorganism DNA and RNA in the roots;
step eight: preparing a tuber-attached microorganism sample, namely forcibly shaking soil on the surface of a potato block, using a cotton swab soaked with sterile physiological saline to roll and wipe the whole area of the surface of the potato block back and forth, cutting off the handheld end of the cotton swab after wiping, putting the cotton swab into a 2mL EP tube filled with sterile PBS buffer solution, carrying out vortex oscillation for 30s twice, fully washing off microorganisms on the cotton swab, removing the cotton swab, and preparing DNA and RNA of tuber-attached microorganisms by using the remainder;
step nine: collecting and preparing samples from different parts in the third step to the eighth step, and backing up the samples by using sterile water 10–2–10–7Dilutions were made, and 100. mu.L of each dilution was taken from different samples and plated on different media plates. The soil-derived microorganism can be isolated by methanol extract culture medium, TSB culture medium, TYG culture medium, YEM culture medium, M715 culture medium, TWYE culture medium, and the phyllospheric and peristemal microorganism can be isolated by MYX culture medium, MM + MeOH culture medium, R2A culture medium.
Step ten: selecting 3 temperature gradients (25 deg.C, 30 deg.C, 35 deg.C) to culture for 2-10 d, and recording colony number and colony characteristics on the plate every day;
step eleven: directly picking single colony with the diameter not less than 200 mu m on the plate and putting the single colony into 1mL of preservation solution, and preserving the single colony at-80 ℃ for later use;
step twelve: for colonies with poor separation on the plate and single colonies smaller than 200 μm, they were streaked on the original medium, respectively, and the same culture conditions were used until single colonies were obtained.
The further improvement lies in that: in this example, the spaces between potato plants were sampled and used as control samples.
The further improvement lies in that: if the potato is already provided with tubers, the tubers are firstly picked off at the beginning of the step two, and then the tuber-attached microorganism sample is prepared according to the operation method of the step eight, so that the tuber skins are not broken during preparation.
The further improvement lies in that: after the microorganism samples are taken in the fourth and fifth steps, the remaining parts, i.e., the soil particles and the sediments, are also used to make a soil extract medium.
The further improvement lies in that: in steps three and seven, after the microorganism sample is taken, the remaining parts, i.e. the sonication liquid and the root precipitate, are also used to make the potato leaf and root extract medium.
The further improvement lies in that: DNA and RNA can be directly extracted from the 7 microorganism samples collected in the third step to the eighth step and are used for identifying bacteria 16S rRNA genes and fungi ITS amplicons or determining sequences of metagenome and macrotranscriptome.
The further improvement lies in that: in the nine to twelve steps, the genome sequence can be further determined and analyzed according to the culture results of different samples. According to the results of genome sequence analysis, if the pure culture is a new species, it can be identified in morphology and various biochemical reactions according to the conventional methods.
The invention has the beneficial effects that:
1. the invention provides a rapid, efficient and convenient method for separating, collecting and extracting most types of samples related to the current potato microbiome research at one time, wherein the samples comprise 7 types of attached leaf lines, stem peripheries, root regions, rhizomes, root surfaces, root interiors and tubers, and the influence and interaction relation of external conditions such as different environments on the growth of the whole potato plant can be completely analyzed. The invention combines microorganism sampling and amplification culture, establishes a set of potato whole plant microorganism separation and purification method, can accurately and efficiently explore the flora structure and main functions of potato whole plant microorganisms, and can not expect to use one or more culture media to separate all plant microorganisms because different microorganisms need different culture media for growth, so that the invention provides culture media aiming at different potato leaves and root systems, and can obtain better culture effect.
2. According to the invention, microbial samples in root areas, rhizospheres and root surfaces are separated and collected by adopting shaking, vibrating and ultrasonic methods respectively, so that the microbial samples in different areas can be classified more accurately and more precisely, and the functions of related microorganisms can be more directly related in the later data analysis process. The microorganisms on the root surface are more greatly influenced by root exudates and have closer interaction relationship with crops, and the microorganisms in the root area are more greatly influenced by the microorganisms in the soil and can better reflect the change of the soil and the environment. If microorganism samples in different areas of the root system cannot be accurately distinguished, the subsequent analysis work of high-throughput sequencing data is disturbed. Previous researches show that microbes attached to root systems are taken as rhizosphere microbe samples by using an aseptic brush, but the method has certain disadvantages in operation, firstly, the moisture of the root systems of potatoes after sampling is heavy, the viscosity of soil attached to the root systems is high, the aseptic brush cannot brush all the potatoes, the sticky soil can block the brush head, and the rhizosphere soil is difficult to separate from the root systems. In the invention, the root system is soaked in PBS buffer solution to vibrate, so that the problem of soil viscosity is solved, the normal PH and osmotic pressure of microbial cells are maintained through the buffer solution, and rhizosphere soil particles attached to the root system are easily shaken off in the vibrating process, so that the purpose of separating rhizosphere microbial samples is achieved.
3. In the previous research, physiological saline is often used for separating and detecting microorganism samples, and PBS buffer solution is selected in the invention, the most essential difference of the two is that the physiological saline mainly has the function of maintaining normal osmotic pressure without buffer capacity, and PBS with a certain concentration has the two capacities at the same time, can buffer when the temperature and gas composition change and is close to the physiological state of cells so as to ensure normal PH and osmotic pressure.
4. When rhizosphere, root surface and root internal samples are collected, the concentration of collected thalli is increased by adopting a mode of centrifuging a PBS buffer solution and then resuspending a precipitate in a small amount of buffer solution so as to ensure that enough microorganisms are used for extracting DNA and RNA.
Detailed Description
In order to further understand the present invention, the following detailed description will be made with reference to the following examples, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
Example one
The embodiment provides a method for preparing, separating and purifying a potato whole plant microorganism sample at one time, which comprises the following steps:
the method comprises the following steps: dividing sampling parts, namely shearing leaves and stem sections of different parts of a potato plant, and putting the leaves and the stem sections into a numbered sterile container for preparing phyllospheric and stem periphery microorganism samples; cutting off the overground part of the potato plant, sealing the cut with adhesive paper, completely digging the underground part, and respectively putting fibrous roots and potato tubers into numbered sterile plastic bags for preparing root and potato tuber microorganism samples;
step two: transporting the sample, namely putting the taken sample into a storage box according to the classification of leaves, stem segments, roots and tubers, and immediately transporting the sample back to a laboratory for later use under the condition of an ice box;
step three: preparing phyllospheric and stem periphery microorganism samples, taking 10-20g of each of leaves and stem sections, placing the leaves and stem sections into 40mL of sterile PBS buffer solution, shaking the table at 200r/min for 30min, then treating the leaves and stem sections twice by ultrasonic waves at 30s each time, standing the leaves and stem sections for 5-10min, centrifuging the leaves and stem sections at 1500r/min for 1min, transferring the supernatant to another centrifuge tube, centrifuging the supernatant at 12000r/min for 10min to obtain thalli, reserving 10mL of the supernatant, then whirling the supernatant for 30s, subpackaging the supernatant into 2mL or more of EP tubes or cryopreservation tubes, and preparing DNA and RNA of phyllospheric and stem periphery microorganisms;
step four: preparing a root zone microorganism sample, paving numbered sterile paper on an experiment table, and shaking root soil corresponding to the number onto the paper forcibly until the root is invisible to naked eyes and obvious soil is on the root. Weighing 10-25g of shaken soil, sieving with a sterile sieve with the aperture of 2mm, uniformly mixing multiple samples, subpackaging into 2mL or more of EP tubes or freezing tubes, and preparing the DNA and RNA of the microorganisms in the root zone;
step five: rhizosphere microorganism sample preparation, 2-3 sterile centrifuge tubes containing 40mL sterile PBS buffer were prepared. And (3) putting the roots shaken off the soil in the third step into a centrifugal tube, shaking for 2-3min with force, standing for 5-10min, taking out the roots after the soil particles naturally settle, putting the roots into another centrifugal tube, and repeating the step for 1-2 times. Collecting washing liquid containing settled soil particles, centrifuging at 12000r/min for 10min, taking 10mL of supernatant, then vortexing for 30s, and subpackaging into an EP (ethylene propylene) tube or a freezing storage tube with the volume of 2mL or more for preparing rhizosphere microorganism DNA and RNA (ribonucleic acid);
step six: root surface microorganism sample preparation. Putting the washed root system into a sterile centrifuge tube of 10mL sterile PBS buffer solution, treating for 2-3 times by ultrasonic waves with the ultrasonic frequency of 50-60hz and 30s each time, subpackaging into 2mL EP tubes or freezing tubes for preparing the DNA and RNA of the microorganisms on the root surface;
step seven: preparing a microorganism sample in roots, taking out the root sample, washing the root sample with sterile water, carrying out surface disinfection on the root sample for 30s by using 75% ethanol, carrying out disinfection on the root sample for 10min by using 2.5% sodium hypochlorite, and fully washing the root sample with the sterile water; taking 5-10g of disinfected roots, placing the roots in 40mL of sterile PBS buffer solution, treating the roots for 5min by using a crusher, standing the roots for 10min, collecting 12000g of washing solution, centrifuging the washing solution for 10min, reserving 10mL of supernatant, then whirling the supernatant for 30s, and subpackaging the supernatant into 2mL or more of EP tubes or freezing tubes for preparing microorganism DNA and RNA in the roots;
step eight: preparing a tuber-attached microorganism sample, namely forcibly shaking soil on the surface of a potato block, using a cotton swab soaked with sterile physiological saline to roll and wipe the whole area of the surface of the potato block back and forth, cutting off the handheld end of the cotton swab after wiping, putting the cotton swab into a 2mL EP tube filled with sterile PBS buffer solution, carrying out vortex oscillation for 30s twice, fully washing off microorganisms on the cotton swab, removing the cotton swab, and preparing DNA and RNA of tuber-attached microorganisms by using the remainder;
step nine: collecting and preparing samples from different parts in the third step to the eighth step, and backing up the samples by using sterile water 10–2–10–7Dilutions were made, and 100. mu.L of each dilution was taken from different samples and plated on different media plates. The soil-derived microorganism can be isolated by methanol extract culture medium, TSB culture medium, TYG culture medium, YEM culture medium, M715 culture medium, TWYE culture medium, and the phyllospheric and peristemal microorganism can be isolated by MYX culture medium, MM + MeOH culture medium, R2A culture medium.
Step ten: selecting 3 temperature gradients (25 deg.C, 30 deg.C, 35 deg.C) to culture for 2-10 d, and recording colony number and colony characteristics on the plate every day;
step eleven: directly picking single colony with the diameter not less than 200 mu m on the plate and putting the single colony into 1mL of preservation solution, and preserving the single colony at-80 ℃ for later use;
step twelve: for colonies with poor separation on the plate and single colonies smaller than 200 μm, they were streaked on the original medium, respectively, and the same culture conditions were used until single colonies were obtained.
Example two
The invention provides a method for one-time preparation, separation and purification of a potato whole plant microorganism sample, which comprises the following steps:
the method comprises the following steps: dividing sampling parts, namely shearing leaves and stem sections of different parts of a potato plant, and putting the leaves and the stem sections into a numbered sterile container for preparing phyllospheric and stem periphery microorganism samples; cutting off the overground part of the potato plant, sealing the cut with adhesive paper, completely digging the underground part, and respectively putting fibrous roots and potato tubers into numbered sterile plastic bags for preparing root and potato tuber microorganism samples;
step two: transporting the sample, namely putting the taken sample into a storage box according to the classification of leaves, stem segments, roots and tubers, and immediately transporting the sample back to a laboratory for later use under the condition of an ice box;
step three: preparing phyllospheric and stem periphery microorganism samples, taking 10-20g of each of leaves and stem sections, placing the leaves and stem sections into 40mL of sterile PBS buffer solution, shaking the table at 200r/min for 30min, then treating the leaves and stem sections twice by ultrasonic waves at 30s each time, standing the leaves and stem sections for 5-10min, centrifuging the leaves and stem sections at 1500r/min for 1min, transferring the supernatant to another centrifuge tube, centrifuging the supernatant at 12000r/min for 10min to obtain thalli, reserving 10mL of the supernatant, then whirling the supernatant for 30s, subpackaging the supernatant into 2mL or more of EP tubes or cryopreservation tubes, and preparing DNA and RNA of phyllospheric and stem periphery microorganisms;
step four: preparing a root zone microorganism sample, paving numbered sterile paper on an experiment table, and shaking root soil corresponding to the number onto the paper forcibly until the root is invisible to naked eyes and obvious soil is on the root. Weighing 10-25g of shaken soil, sieving with a sterile sieve with the aperture of 2mm, uniformly mixing multiple samples, subpackaging into 2mL or more of EP tubes or freezing tubes, and preparing the DNA and RNA of the microorganisms in the root zone;
step five: rhizosphere microorganism sample preparation, 2-3 sterile centrifuge tubes containing 40mL sterile PBS buffer were prepared. And (3) putting the roots shaken off the soil in the third step into a centrifugal tube, shaking for 2-3min with force, standing for 5-10min, taking out the roots after the soil particles naturally settle, putting the roots into another centrifugal tube, and repeating the step for 1-2 times. Collecting washing liquid containing settled soil particles, centrifuging at 12000r/min for 10min, taking 10mL of supernatant, then vortexing for 30s, and subpackaging into an EP (ethylene propylene) tube or a freezing storage tube with the volume of 2mL or more for preparing rhizosphere microorganism DNA and RNA (ribonucleic acid);
step six: root surface microorganism sample preparation. Putting the washed root system into a sterile centrifuge tube of 10mL sterile PBS buffer solution, treating for 2-3 times by ultrasonic waves with the ultrasonic frequency of 50-60hz and 30s each time, subpackaging into 2mL EP tubes or freezing tubes for preparing the DNA and RNA of the microorganisms on the root surface;
step seven: preparing a microorganism sample in roots, taking out the root sample, washing the root sample with sterile water, carrying out surface disinfection on the root sample for 30s by using 75% ethanol, carrying out disinfection on the root sample for 10min by using 2.5% sodium hypochlorite, and fully washing the root sample with the sterile water; taking 5-10g of disinfected roots, placing the roots in 40mL of sterile PBS buffer solution, treating the roots for 5min by using a crusher, standing the roots for 10min, collecting 12000g of washing solution, centrifuging the washing solution for 10min, reserving 10mL of supernatant, then whirling the supernatant for 30s, and subpackaging the supernatant into 2mL or more of EP tubes or freezing tubes for preparing microorganism DNA and RNA in the roots;
step eight: preparing a tuber-attached microorganism sample, namely forcibly shaking soil on the surface of a potato block, using a cotton swab soaked with sterile physiological saline to roll and wipe the whole area of the surface of the potato block back and forth, cutting off the handheld end of the cotton swab after wiping, putting the cotton swab into a 2mL EP tube filled with sterile PBS buffer solution, carrying out vortex oscillation for 30s twice, fully washing off microorganisms on the cotton swab, removing the cotton swab, and preparing DNA and RNA of tuber-attached microorganisms by using the remainder;
step nine: collecting and preparing samples from different parts in the third step to the eighth step, and backing up the samples by using sterile water 10–2–10–7Dilutions were made, and 100. mu.L of each dilution was taken from different samples and plated on different media plates. The soil-derived microorganism can be isolated by methanol extract culture medium, TSB culture medium, TYG culture medium, YEM culture medium, M715 culture medium, TWYE culture medium, and the phyllospheric and peristemal microorganism can be isolated by MYX culture medium, MM + MeOH culture medium, R2A culture medium.
Step ten: selecting 3 temperature gradients (25 deg.C, 30 deg.C, 35 deg.C) to culture for 2-10 d, and recording colony number and colony characteristics on the plate every day;
step eleven: directly picking single colony with the diameter not less than 200 mu m on the plate and putting the single colony into 1mL of preservation solution, and preserving the single colony at-80 ℃ for later use;
step twelve: for colonies with poor separation on the plate and single colonies smaller than 200 μm, they were streaked on the original medium, respectively, and the same culture conditions were used until single colonies were obtained.
In this example, the spaces between potato plants were sampled and used as control samples.
In this example, if potato tubers are formed, the tubers are first removed at the beginning of step two, and then the microorganism samples attached to the tubers are prepared according to the operation method of step eight, so that the tuber skins are not broken during preparation.
In this example, after the microorganism samples were taken in the fourth and fifth steps, the remaining portions, i.e., the soil particles and the sediments, were also used to prepare the soil extract medium.
In this example, in step three and step seven, after the microorganism samples were taken, the remaining parts, i.e., the sonicated liquid and the root precipitate, were also used to make potato leaf and root extract medium.
In this example, the 7 microorganism samples collected in the third to eighth steps can be directly used for extracting DNA and RNA for identification of bacterial 16S rRNA genes, fungal ITS amplicons or determination of metagenome and macrotranscriptome sequences.
In this example, further genomic sequencing and analysis may be performed in steps nine to twelve, depending on the results of the different sample cultures. According to the results of genome sequence analysis, if the pure culture is a new species, it can be identified in morphology and various biochemical reactions according to the conventional methods.
In the whole experiment process, sterile rubber gloves are preferably worn all the time, so that artificial pollution is avoided as much as possible;
when the above experimental procedures are performed, detailed records must be made, especially for each isolated single strain and the corresponding microorganism sample. Therefore, before the experiment is started, the data to be recorded is preferably designed into a table, printed by pages, directly filled in during the experiment, and bound after the experiment is finished. If possible, an applet can be written, entered directly in the fill-in column, and output directly in a tabular form when viewing records.
The invention provides a rapid, efficient and convenient method for separating, collecting and extracting most types of samples related to the current potato microbiome research at one time, wherein the samples comprise 7 types of attached leaf lines, stem peripheries, root regions, rhizomes, root surfaces, root interiors and tubers, and the influence and interaction relation of external conditions such as different environments on the growth of the whole potato plant can be completely analyzed. The invention combines microorganism sampling and amplification culture, establishes a set of potato whole plant microorganism separation and purification method, can accurately and efficiently explore the flora structure and main functions of potato whole plant microorganisms, and can not expect to use one or more culture media to separate all plant microorganisms because different microorganisms need different culture media for growth, so that the invention provides culture media aiming at different potato leaves and root systems, and can obtain better culture effect.
According to the invention, microbial samples in root areas, rhizospheres and root surfaces are separated and collected by adopting shaking, vibrating and ultrasonic methods respectively, so that the microbial samples in different areas can be classified more accurately and more precisely, and the functions of related microorganisms can be more directly related in the later data analysis process. The microorganisms on the root surface are more greatly influenced by root exudates and have closer interaction relationship with crops, and the microorganisms in the root area are more greatly influenced by the microorganisms in the soil and can better reflect the change of the soil and the environment. If microorganism samples in different areas of the root system cannot be accurately distinguished, the subsequent analysis work of high-throughput sequencing data is disturbed. Previous researches show that microbes attached to root systems are taken as rhizosphere microbe samples by using an aseptic brush, but the method has certain disadvantages in operation, firstly, the moisture of the root systems of potatoes after sampling is heavy, the viscosity of soil attached to the root systems is high, the aseptic brush cannot brush all the potatoes, the sticky soil can block the brush head, and the rhizosphere soil is difficult to separate from the root systems. In the invention, the root system is soaked in PBS buffer solution to vibrate, so that the problem of soil viscosity is solved, the normal PH and osmotic pressure of microbial cells are maintained through the buffer solution, and rhizosphere soil particles attached to the root system are easily shaken off in the vibrating process, so that the purpose of separating rhizosphere microbial samples is achieved.
In the previous research, physiological saline is often used for separating and detecting microorganism samples, and PBS buffer solution is selected in the invention, the most essential difference of the two is that the physiological saline mainly has the function of maintaining normal osmotic pressure without buffer capacity, and PBS with a certain concentration has the two capacities at the same time, can buffer when the temperature and gas composition change and is close to the physiological state of cells so as to ensure normal PH and osmotic pressure.
When rhizosphere, root surface and root internal samples are collected, the concentration of collected thalli is increased by adopting a mode of centrifuging a PBS buffer solution and then resuspending a precipitate in a small amount of buffer solution so as to ensure that enough microorganisms are used for extracting DNA and RNA.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A one-time preparation, separation and purification method of a potato whole plant microorganism sample is characterized by comprising the following steps:
the method comprises the following steps: dividing sampling parts, namely shearing leaves and stem sections of different parts of a potato plant, and putting the leaves and the stem sections into a numbered sterile container for preparing phyllospheric and stem periphery microorganism samples; cutting off the overground part of the potato plant, sealing the cut with adhesive paper, completely digging the underground part, and respectively putting fibrous roots and potato tubers into numbered sterile plastic bags for preparing root and potato tuber microorganism samples;
step two: transporting the sample, namely putting the taken sample into a storage box according to the classification of leaves, stem segments, roots and tubers, and immediately transporting the sample back to a laboratory for later use under the condition of an ice box;
step three: preparing phyllospheric and stem periphery microorganism samples, taking 10-20g of each of leaves and stem sections, placing the leaves and the stem sections into 40mL of sterile PBS buffer solution, shaking for 30min at 200r/min in a shaking table, treating the leaves and the stem sections twice by ultrasonic waves for 30s each time, standing for 5-10min, centrifuging for 1min at 1500r/min after removing the leaves and the stem sections, transferring the supernatant to another centrifugal tube, and centrifuging for 10min at 12000r/min to obtain thalli. Taking 10mL of supernatant, vortexing for 30s, and subpackaging into 2mL or more of EP tubes or cryopreservation tubes for preparing phyllospheric and stem periphery microorganism DNA and RNA;
step four: preparing a root zone microorganism sample, namely paving numbered sterile paper on an experiment table, shaking root soil with a corresponding number onto the paper forcibly until obvious soil on roots cannot be seen by naked eyes, weighing 10-25g of the shaken soil, sieving the weighed soil with a sterile sieve with the aperture of 2mm, uniformly mixing a plurality of samples, subpackaging the mixture into an EP (EP) tube or a cryopreservation tube with the volume of 2mL or more, and preparing DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) of the root zone microorganism;
step five: rhizosphere microorganism sample preparation, 2-3 sterile centrifuge tubes containing 40mL sterile PBS buffer were prepared. And (3) putting the roots shaken off the soil in the third step into a centrifugal tube, shaking for 2-3min with force, standing for 5-10min, taking out the roots after the soil particles naturally settle, putting the roots into another centrifugal tube, and repeating the step for 1-2 times. Collecting washing liquid containing settled soil particles, centrifuging at 12000r/min for 10min, taking 10mL of supernatant, then vortexing for 30s, and subpackaging into an EP (ethylene propylene) tube or a freezing storage tube with the volume of 2mL or more for preparing rhizosphere microorganism DNA and RNA (ribonucleic acid);
step six: preparing a root surface microorganism sample, namely putting the washed root system into a sterile centrifuge tube of 10mL sterile PBS buffer solution, treating the root system for 2-3 times by ultrasonic waves with the ultrasonic frequency of 50-60hz and each time for 30s, subpackaging the root system into 2mL EP tubes or cryopreservation tubes, and preparing DNA and RNA of the root surface microorganism;
step seven: preparing a microorganism sample in roots, taking out the root sample, washing the root sample with sterile water, carrying out surface disinfection on the root sample for 30s by using 75% ethanol, carrying out disinfection on the root sample for 10min by using 2.5% sodium hypochlorite, and fully washing the root sample with the sterile water; taking 5-10g of disinfected roots, placing the roots in 40mL of sterile PBS buffer solution, treating the roots for 5min by using a crusher, standing the roots for 10min, collecting 12000g of washing solution, centrifuging the washing solution for 10min, reserving 10mL of supernatant, then whirling the supernatant for 30s, and subpackaging the supernatant into 2mL or more of EP tubes or freezing tubes for preparing microorganism DNA and RNA in the roots;
step eight: preparing a tuber-attached microorganism sample, namely forcibly shaking soil on the surface of a potato block, using a cotton swab soaked with sterile physiological saline to roll and wipe the whole area of the surface of the potato block back and forth, cutting off the handheld end of the cotton swab after wiping, putting the cotton swab into a 2mL EP tube filled with sterile PBS buffer solution, carrying out vortex oscillation for 30s twice, fully washing off microorganisms on the cotton swab, removing the cotton swab, and preparing DNA and RNA of tuber-attached microorganisms by using the remainder;
step nine: collecting and preparing sample backup at different parts in the third step to the eighth stepRun with sterile Water 10–2–10–7Dilutions were made, and 100. mu.L of each dilution was taken from different samples and plated on different media plates. The soil source microorganism can be separated by methanol extract culture medium, TSB culture medium, TYG culture medium, YEM culture medium, M715 culture medium, TWYE culture medium, and the phyllospheric and peristemal microorganism can be separated by MYX culture medium, MM + MeOH culture medium, R2A culture medium;
step ten: selecting 3 temperature gradients of 25 ℃, 30 ℃ and 35 ℃ to culture for 2-10 d, and recording the number and characteristics of colonies appearing on the plate every day;
step eleven: directly picking single colony with the diameter not less than 200 mu m on the plate and putting the single colony into 1mL of preservation solution, and preserving the single colony at-80 ℃ for later use;
step twelve: for colonies with poor separation on the plate and single colonies smaller than 200 μm, they were streaked on the original medium, respectively, and the same culture conditions were used until single colonies were obtained.
2. The method for one-time preparation, separation and purification of a potato whole plant microorganism sample according to claim 1, wherein the method comprises the following steps: the gaps between potato plants were selected for sampling and used as control samples.
3. The method for one-time preparation, separation and purification of a potato whole plant microorganism sample according to claim 1, wherein the method comprises the following steps: if the potato is already provided with tubers, the tubers are firstly picked off at the beginning of the step two, and then the tuber-attached microorganism sample is prepared according to the operation method of the step eight, so that the tuber skins are not broken during preparation.
4. The method for one-time preparation, separation and purification of a potato whole plant microorganism sample according to claim 1, wherein the method comprises the following steps: after the microorganism samples are taken in the fourth and fifth steps, the remaining parts, i.e., the soil particles and the sediments, are also used to make a soil extract medium.
5. The method for one-time preparation, separation and purification of a potato whole plant microorganism sample according to claim 1, wherein the method comprises the following steps: in steps three and seven, after the microorganism sample is taken, the remaining parts, i.e. the sonication liquid and the root precipitate, are also used to make the potato leaf and root extract medium.
6. The method for one-time preparation, separation and purification of a potato whole plant microorganism sample according to claim 1, wherein the method comprises the following steps: DNA and RNA can be directly extracted from the 7 microorganism samples collected in the third step to the eighth step and are used for identifying bacteria 16S rRNA genes and fungi ITS amplicons or determining sequences of metagenome and macrotranscriptome.
7. The method for one-time preparation, separation and purification of a potato whole plant microorganism sample according to claim 1, wherein the method comprises the following steps: in the nine to twelve steps, the genome sequence can be further determined and analyzed according to the culture results of different samples. According to the results of genome sequence analysis, if the pure culture is a new species, it can be identified in morphology and various biochemical reactions according to the conventional methods.
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| CN109136139A (en) * | 2018-09-07 | 2019-01-04 | 中国农业科学院农产品加工研究所 | A kind of potato endophyte and its application |
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| WO2003066880A2 (en) * | 2002-02-08 | 2003-08-14 | Fraunhofer Usa Inc. | Method for accessing microbial diversity |
| CN109136139A (en) * | 2018-09-07 | 2019-01-04 | 中国农业科学院农产品加工研究所 | A kind of potato endophyte and its application |
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