CN115011482A - Method for separating plant rhizosphere microorganisms in high flux and culturing and screening IAA-producing strains - Google Patents
Method for separating plant rhizosphere microorganisms in high flux and culturing and screening IAA-producing strains Download PDFInfo
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Abstract
A method for separating plant rhizosphere microorganisms at high flux and culturing and screening IAA-producing strains is disclosed, which uses ultrasonic waves with three frequencies of low frequency, medium frequency and high frequency to jointly treat the plant rhizosphere from a fresh plant rhizosphere, so as to increase the variety and the number of the plant rhizosphere microorganisms released from roots and increase the obtaining of the rhizosphere microorganisms. The bacteria were then cultured using 96-well plates for high-throughput isolation, and the ratio of single bacteria obtained was increased using a gradient dilution method. The invention adopts bidirectional synchronous IAA color reaction and strain preservation, screens the IAA rhizosphere in-situ strain with high flux, and provides better strain resources for the function of rhizosphere microorganisms and the interaction research with plants. The obtaining rate of the obtained single strain is improved to 100 percent, the strain is not required to be purified by conventional plate streaking, and the screening efficiency is greatly improved.
Description
Technical Field
The invention belongs to the technical field of environmental microbiology, and particularly relates to a method for separating plant rhizosphere microorganisms at high flux and culturing and screening IAA-producing strains.
Background
The root system is the main organ for crops to absorb nutrition and moisture, and is also the main place for interaction of plants and microorganisms. Naturally-growing crops have their root surfaces enriched with a large and diverse number of microorganisms, collectively known as the rhizosphere microbiome, from the soil.
Rhizosphere microorganisms, including beneficial, harmful and neutral microorganisms, play an important role in the growth process of plants. At present, the research of plant rhizosphere microbiome mainly focuses on descriptive work, and mainly utilizes high-throughput amplicon and metagenome sequencing technologies to describe species classification and genetic composition of various plant rhizosphere microbiomes growing in natural soil.
The research of plant rhizosphere microbiome is entering into a rapid development stage, and the next stage is bound to focus on the research of the function of the rhizosphere microbiome in plants and gradually reveal the molecular mechanism of the interaction process of the plants and the rhizosphere microbiome.
At present, the key factor hindering rhizosphere microbial functions and plant-microorganism interaction research is rhizosphere microbial resources. Although the international reserve center has stored thousands of microorganisms, these microorganisms come from a variety of environments and species. The colonization ability of microorganisms in a specific niche is likely to generate differentiation in different habitats and different species roots, so that it is necessary to separate and culture the microorganisms in situ from given soil and hosts, and provide better strain resources for the function of rhizosphere microorganisms and the interaction research with plants.
Chinese patent 201811048405.7 discloses "Jujun grass rhizosphere growth-promoting bacteria and application thereof", Chinese patent 202111203354.2 discloses "a plant rhizosphere growth-promoting bacteria and application thereof", and the method for screening microbial strains disclosed in the above patent is to separate and screen the plant rhizosphere growth-promoting bacteria by a plate dilution method. The current plate dilution method requires multiple incubations and streaking of the purified strain to obtain a single pure strain. The obtained strain is subjected to functional verification, the whole screening process is long in period and complicated to operate.
Chinese patent 202122887945.8 discloses a device for Salkowski legal screening of IAA-secreting strains, which is mainly composed of a reaction plate and a light shield device, and can realize simultaneous experiment of multiple samples, effectively shield light and prevent the color reaction in the detection process of Salkowski method from being influenced by the existence of light.
In summary, rhizosphere microorganisms are mainly separated and screened by a plate dilution method at present, multiple times of culture are needed, strains are required to be streaked and purified to obtain a single pure strain, the obtained strain is subjected to functional verification, and the whole screening process is long in period and complicated in operation.
Isolated culture of microorganisms in situ is important to reveal the function of microorganisms in plant growth and health. The combination of the isolated and cultured microorganisms and a sterile system promotes important technology for the development of rhizosphere microbiome from description to functional research.
Disclosure of Invention
The invention provides a method for separating plant rhizosphere microorganisms at high flux and culturing and screening IAA-producing strains, which is used for screening IAA-producing rhizosphere in-situ strains at high flux, so that the yield of the obtained single strains is increased to 100%, the screening efficiency is greatly improved, better strain resources are provided for the function of the rhizosphere microorganisms and the interaction research with plants, and the method is suitable for separating and culturing the microorganisms at the rhizosphere of plants such as green vegetables, tomatoes, lettuce, spinach and the like.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for separating plant rhizosphere microorganisms in high flux and culturing and screening IAA-producing strains comprises the following steps:
1) obtaining microbial suspension of rhizosphere soil sample
Carrying out ultrasonic treatment on the collected healthy growing crop rhizosphere sample room for 10-40 s by using an activity-maintaining buffer solution at the temperature to obtain a rhizosphere microorganism sample suspension liquid containing abundant microorganisms, then filtering the rhizosphere microorganism sample suspension liquid, collecting filtrate to obtain a rhizosphere soil suspension liquid, and storing the obtained rhizosphere soil suspension liquid at the temperature of 2-7 ℃ for later use;
the ultrasonic condition is that three ultrasonic waves with different frequencies are adopted for processing at the same time, and the ultrasonic frequencies are respectively 15-30 KHz, 45-60 KHz and 80-100 KHz;
the activity-maintaining buffer solution contains 5-9.5 mM Na 2 HPO 4 ，110～140mM NaCl， 1～3mM KH 2 PO 4 1-3.5 mMKCl, 3-7% of microcystis polysaccharide, and the pH is 6.5-7.5;
2) determining the optimal dilution concentration for isolation culture
Adding the rhizosphere soil suspension into a high-flux separation liquid culture medium, diluting the rhizosphere soil suspension in a gradient manner to obtain rhizosphere soil diluents with different concentrations, respectively transferring the rhizosphere soil diluents with different concentrations into holes of different 96-hole cell culture plates under aseptic operation for culture, taking 5-15% of TSB solution without rhizosphere soil suspension as negative control, setting more than three times for each sample, sealing the 96-hole cell culture plates on a shaking bed, and culturing for 2-10 days at the temperature of 28-37 ℃ and 80-180 r/min;
20-35% of the cells in the 96-well cell culture plate are in a turbid state visible to naked eyes, and the concentration of rhizosphere soil diluent corresponding to the 96-well cell culture plate is used as the optimal dilution concentration for separation culture;
the high-throughput isolation liquid medium contains: 50-150mg/L of L-tryptophan, 10-30g/L of casein polypeptide, 10-30g/L of soybean protein polypeptide and 2-7g/L of sodium chloride, wherein the pH value is 6.0-8.0;
3) screening a single strain for producing IAA;
transferring rhizosphere soil diluent with the optimal dilution concentration into the holes of the cell culture plate under aseptic operation, sealing the cell culture plate, placing the cell culture plate on a shaking table, and culturing for 2-10 days at the temperature of 28-37 ℃ and at the speed of 80-180 r/min; after the culture is finished, sucking turbid bacterial liquid in the cell culture plate holes into the white ceramic plate, adding Salkowski developing solution for developing reaction, and taking IAA standard solution as positive control; liquid culture medium solution containing 50-150 mg/L-tryptophan, 10-30g/L casein polypeptide, 10-30g/L soybean protein polypeptide, 2-7g/L sodium chloride and pH of 6.0-8.0 is used as negative control; placing the white ceramic plate at room temperature, standing in a dark condition for 20-40 min, and observing, wherein the condition that the color turns red indicates that IAA can be generated; the strains in the IAA-producing cell culture plates were deposited under the accession number based on the results of the IAA color reaction.
Preferably, in the step 1), the ultrasonic treatment time is 20-40 s.
Preferably, in the step 3), the concentration of the IAA in the IAA standard solution is 10-100 mg/L.
Preferably, the crop is a green vegetable, tomato, lettuce or spinach.
The invention collects crops rhizosphere samples which grow healthily and cleans them in activity-keeping buffer solution by using multi-frequency ultrasound to obtain rhizosphere microorganism sample suspension, the ultrasonic is an elastic mechanical wave in a substance medium, as a physical energy form, when cleaning rhizosphere microorganism samples, ultrasonic research on different frequency characteristics finds that the multi-frequency ultrasound fully exerts the characteristics of the ultrasonic with different frequencies, eliminates standing waves, makes a sound field more uniform, and the multi-frequency combined ultrasound has frequency doubling waves, sum waves and difference waves besides fundamental frequency. The microorganism species and the quantity of the rhizosphere microorganism sample suspension obtained by washing the activity-maintaining buffer solution assisted by multi-frequency combined ultrasound are superior to single-frequency ultrasound.
In the invention, rhizosphere microorganism sample suspension is filtered and then rhizosphere microorganism suspension is collected; performing gradient dilution by using a high-flux separation liquid culture medium containing L-tryptophan; the soil microbial community has the highest diversity, followed by the rhizosphere microbial community. During the reproductive growth of plants, the rhizosphere microbial community becomes stable, and the number of microbial communities is in a relatively stable interval. The gradient setting of the invention is set according to the interval of the number of rhizosphere microorganisms, and the optimal dilution concentration of the invention can be rapidly and accurately screened through the gradient setting, namely 20-35% of the pores in a 96-pore cell culture plate under the optimal dilution concentration are in a turbid state visible to naked eyes.
In the high-flux bacteria separation step, rhizosphere soil diluents with different concentrations are added into a cell culture plate for culture, 20-35% of holes in a 96-hole cell culture plate are in a turbid state visible to the naked eye, the concentration of the rhizosphere soil diluents is used as the optimal dilution concentration, the proportion of single microorganisms is increased by using a gradient dilution method, and the optimal dilution concentration can be rapidly and accurately screened through the gradient arrangement. Through hundreds of times of optimal dilution concentration bacteria division experiments, statistical analysis shows that when 20-35% of the pores are in a turbid state visible to the naked eye, the bacteria in the pores can be regarded as pure bacteria. The preferred setting of the dilution concentration is based on our data analysis in a number of experiments.
The invention adopts bidirectional synchronization to respectively carry out IAA color reaction and strain preservation, and carries out numbering preservation on the rhizosphere in-situ strains of crops producing IAA according to the result of the IAA color reaction, thereby greatly saving the time for strain breeding. The method screens the IAA-producing rhizosphere in-situ strains at high flux, and provides better strain resources for the function of rhizosphere microorganisms and the interaction research with plants.
Compared with the prior art, the invention has the following beneficial effects:
the method utilizes the low, medium and high frequency ultrasonic waves to carry out the common treatment in the rhizosphere of the fresh plant, thereby increasing the release of rhizosphere microorganisms of the plant from the root and increasing the acquisition of the rhizosphere microorganisms. The bacteria were then grown in 96-well plates for high throughput isolation and the gradient dilution method was used to increase the proportion of single bacteria obtained.
The invention adopts bidirectional synchronous IAA color reaction and strain preservation, screens the IAA rhizosphere in-situ strain with high flux, and provides better strain resources for the function of rhizosphere microorganisms and the interaction research with plants. The yield of the obtained single strain is improved to 100 percent, the strain is not required to be purified by conventional plate streaking, and the screening efficiency is greatly improved.
The rhizosphere microorganism is fully released and maintains the activity by using an ultrasonic-assisted activity maintaining buffer solution. Then, the optimal dilution concentration for isolation culture is determined, and the rhizosphere microorganisms are screened in a high-throughput manner by using a 96-well culture plate and combining a Salkowski method.
Drawings
FIG. 1 is a schematic diagram of a method for screening IAA-producing strains by culturing high-throughput isolated plant rhizosphere microorganisms in the embodiment of the invention.
FIG. 2 shows the results of the IAA-producing chromogenic reaction of the culture screen for high-throughput isolation of plant rhizosphere microorganisms in the examples of the present invention.
FIG. 3 is a comparison chart of the influence of IAA-producing strains and CK on vegetable growth promotion.
Detailed Description
The present invention is further illustrated by the following specific examples.
Examples
Referring to fig. 1, a method for high throughput isolation of plant rhizosphere microorganisms and culture screening of IAA-producing strains, comprising the steps of:
1) obtaining microbial suspension of rhizosphere soil sample
Collecting a healthy root sample of fresh green vegetables from a field, removing soil particles and untight-connected soil on the surface of the root sample of the green vegetables, and collecting the root sample of the green vegetables10g of this product was placed in a 50mL sterile centrifuge tube, to which was added 30mL of sterilized activity-maintaining buffer containing 8.1mM Na 2 HPO 4 ，135mM NaCl，2mM KH 2 PO 4 2.6mM KCL and 4% microspherical algal polysaccharide, wherein the pH is 7.2, the three frequencies of 15KHz, 60KHz and 100KHz are adopted for ultrasonic treatment for 30s together to obtain rhizosphere microorganism sample suspension containing abundant microorganisms, then the rhizosphere microorganism sample suspension is filtered by 4 layers of gauze to remove impurities such as roots and the like, and the filtered rhizosphere soil suspension is collected and placed in a 100mL sterile centrifuge tube for later use;
2) determining the optimal dilution concentration for isolation culture
Respectively putting the rhizosphere soil suspension into 6 reagent bottles, adding a high-flux separation liquid culture medium, and respectively diluting into gradient rhizosphere soil dilution liquid of which the volume is 100 times, 300 times, 900 times, 2700 times, 9100 times and 27300 times; transferring each gradient diluent into different cell culture plate holes under aseptic operation for culture, wherein the high-flux separation liquid culture medium comprises: 100mg/L of L-tryptophan, 20g/L of casein polypeptide, 20g/L of soybean protein polypeptide, 5g/L of sodium chloride and pH 7.0; taking a separation liquid culture medium solution without rhizosphere soil diluent as a negative control;
packing rhizosphere soil diluent into 96-hole cell culture plates, shaking each bottle of rhizosphere soil diluent uniformly in a super clean bench, pouring 50mL of rhizosphere soil diluent into a 13cm sterile square dish, sucking liquid by a row gun, transferring the liquid into each hole of the 96-hole cell culture plates, wherein each hole is 160 mu L, and 3 cell culture plates are transferred in each bottle of diluent; transferring 3 cell culture plates as negative controls to a high-flux separation liquid culture medium without adding rhizosphere soil diluent, and avoiding liquid from splashing outside the holes as much as possible during liquid transfer, otherwise easily causing pollution; sealing each cell culture plate with Parafilm;
sealing a 96-hole cell culture plate on a shaking table, and culturing at 35 ℃ at 150r/min for 10 days; observing the growth condition of bacteria in a 96-hole cell culture plate, and taking the concentration of rhizosphere soil diluent corresponding to a cell culture plate with 20-35% of holes in a turbid state visible to naked eyes as the optimal dilution concentration for separation culture;
3) screening a single strain for producing IAA;
transferring rhizosphere soil diluent with the optimal dilution concentration into 1-100 holes of a cell culture plate under aseptic operation, sealing the cell culture plate, placing the cell culture plate on a shaking table, and culturing for 3-10 days at 35 ℃ at 150 r/min; after the culture is finished, sucking turbid bacterial liquid in the cell culture plate holes into the white ceramic plate, adding the same amount of Salkowski developing solution for developing reaction, and taking an IAA standard solution as a positive control; a liquid culture medium solution containing 100mg/L of L-tryptophan, 20g/L of casein polypeptide, 20g/L of soybean protein polypeptide, 5g/L of sodium chloride and pH of 6.0-8.0 is used as a negative control; the white ceramic plate is placed at room temperature in a dark condition for 20-40 min and then observed, and the IAA can be generated when the color turns red, and the result is shown in figure 2; and numbering and storing the strains which are changed into red holes, further quantitatively measuring the IAA yield of each strain, and then storing the obtained strains.
The proportion of wells in the 96-well cell culture plate which are in a visible turbid state is counted, and the statistical results are shown in table 1:
TABLE 1
As can be seen from the table 1, when 20-35% of the wells of the 96-well cell culture plate have a visible turbid state, the corresponding colonies are single colonies, namely the colonies generated under the rhizosphere soil diluent concentration corresponding to the 96-well cell culture plate are single colonies, the IAA-producing strains can be screened under the concentration, and the colonies obtained by screening can be guaranteed to be single colonies.
1.1 Effect of ultrasound conditions on the type and quantity of microorganisms in sample suspensions:
under the condition that other conditions are not changed, the microorganism varieties of the plant root systems are respectively extracted by ultrasonic waves with different frequencies for treatment, rhizosphere microorganism sample suspension liquid containing abundant microorganisms is obtained, and the types and the amounts of the microorganisms in the rhizosphere microorganism sample suspension liquid corresponding to different ultrasonic treatment conditions are shown in a table 2.
TABLE 2
As can be seen from table 2, the treatment was performed using single-, double-and triple-frequency ultrasonic waves, respectively, and the types and amounts of microorganisms in the rhizosphere microorganism sample suspension were the largest when the triple-frequency ultrasonic treatment was used.
1.2 production of IAA screening strains whether or not they have a plant growth promoting effect
The crop plant rhizosphere microbial strains capable of producing IAA are subjected to laboratory numbering according to the IAA color reaction result, and the IAA producing capability of part of the strains is shown in Table 3:
TABLE 3
In order to verify whether the screened strains capable of producing IAA have the growth promoting effect on plants, the study develops a potting test of green vegetables, and the specific operation steps are as follows:
soaking seeds of the green vegetables in warm soup for germination acceleration: soaking the seeds in warm water at 40 ℃, then dispersing the seeds in a culture dish filled with wet filter paper sheets, accelerating germination at 26 ℃, transplanting the seeds after white emergence into a seedling culture substrate (vermiculite: substrate: 1.5: 1), normally culturing under the condition of normal sunshine at 26 ℃ without applying any fertilizer, and transplanting the seedlings into pots when the green vegetable seedlings grow to the four-leaf stage, wherein 1 seedling is planted in each pot.
Experimental and control groups were set up separately, with 3 replicates per group.
The treatment group is treated by adding the IAA-producing microbial inoculum obtained by screening, and the control group is treated by using the practical CK.
Preparing an IAA-producing microbial inoculum: the prepared bacterial liquid is centrifuged for 10min under the condition of 5000rmp, and is resuspended by sterile water, and the OD600 of the bacterial liquid is adjusted to 1 for later use.
And (3) starting microbial inoculum treatment after transplanting for 3 days, treating the treated group with the microbial inoculum (5 mL/plant), treating the control group with the same amount of clear water, pouring the control group once every 7 days for 4 times, and measuring the fresh weight, the plant height, the root thickness, the root length and the leaf number of the green vegetables after 30 days.
We select the strains with higher IAA production ability to carry out the vegetable crop growth promoting pot experiment, and the specific results are shown in Table 4.
As can be seen from table 4, the number of the strain having a significant growth promoting effect on green vegetables is 11, and the strain is registered and stored, the strain is Bacillus amyloliquefaciens AR7Bacillus amyloliquefaciens, and is stored in the common microorganism center of the china committee for culture collection of microorganisms, the storage number is CGMCC No.20623, the storage date is 2020, 09 months and 09 days, the storage institution address is: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North. The strain has obvious growth promoting effect on green vegetables, and the yield is improved by more than 29%. The growth of green vegetables is shown in figure 3.
TABLE 4
Claims (4)
1. A method for separating plant rhizosphere microorganisms at high flux and culturing and screening IAA-producing strains is characterized by comprising the following steps:
1) obtaining microbial suspension of rhizosphere soil sample
Carrying out ultrasonic treatment on the collected healthy growing crop rhizosphere sample room for 10-40 s by using an activity-maintaining buffer solution at the temperature to obtain a rhizosphere microorganism sample suspension liquid containing abundant microorganisms, then filtering the rhizosphere microorganism sample suspension liquid, collecting filtrate to obtain a rhizosphere soil suspension liquid, and storing the obtained rhizosphere soil suspension liquid at the temperature of 2-7 ℃ for later use;
the ultrasonic condition is that three ultrasonic waves with different frequencies are adopted for processing at the same time, and the ultrasonic frequencies are respectively 15-30 KHz, 45-60 KHz and 80-100 KHz;
the activity-maintaining formThe buffer solution contains 5-9.5 mM Na 2 HPO 4 ，110～140mM NaCl，1～3mM KH 2 PO 4 1-3.5 mKCl, 3-7% of microspherococcus polysaccharide, and the pH value is 6.5-7.5;
2) determining the optimal dilution concentration for isolation culture
Adding the rhizosphere soil suspension into a high-flux separation liquid culture medium, diluting the rhizosphere soil suspension in a gradient manner to obtain rhizosphere soil diluents with different concentrations, respectively transferring the rhizosphere soil diluents with different concentrations into holes of different 96-hole cell culture plates under aseptic operation for culture, taking 5-15% of TSB solution without rhizosphere soil suspension as negative control, setting more than three times for each sample, sealing the 96-hole cell culture plate, placing the 96-hole cell culture plate on a shaker, and culturing for 2-10 days at the temperature of 28-37 ℃ at 80-180 r/min;
20-35% of the cells in the 96-well cell culture plate are in a turbid state visible to naked eyes, and the concentration of rhizosphere soil diluent corresponding to the 96-well cell culture plate is used as the optimal dilution concentration for separation culture;
the high-throughput isolation liquid medium contains: 50-150mg/L of L-tryptophan, 10-30g/L of casein polypeptide, 10-30g/L of soybean protein polypeptide and 2-7g/L of sodium chloride, wherein the pH value is 6.0-8.0;
3) screening a single strain for producing IAA;
transferring rhizosphere soil diluent with the optimal dilution concentration into the holes of the cell culture plate under aseptic operation, sealing the cell culture plate, placing the cell culture plate on a shaking table, and culturing for 2-10 days at the temperature of 28-37 ℃ and at the speed of 80-180 r/min; after the culture is finished, sucking turbid bacterial liquid in the cell culture plate holes into the white ceramic plate, adding Salkowski developing solution for developing reaction, and taking IAA standard solution as positive control; liquid culture medium solution containing 50-150 mg/L-tryptophan, 10-30g/L casein polypeptide, 10-30g/L soybean protein polypeptide, 2-7g/L sodium chloride and pH of 6.0-8.0 is used as negative control; placing the white ceramic plate at room temperature, standing in a dark condition for 20-40 min, and observing, wherein the white ceramic plate can generate IAA when the color turns red; strains in IAA-producing cell culture plates were deposited with the accession number based on the results of the IAA color reaction.
2. The method for separating plant rhizosphere microorganisms at high throughput and culturing and screening the IAA-producing strains according to claim 1, wherein in the step 1), the ultrasonic treatment time is 20-40 s.
3. The method for separating plant rhizosphere microorganisms at high throughput and culturing and screening IAA-producing strains according to claim 1, wherein in the step 3), the concentration of IAA in the IAA standard solution is 10-100 mg/L.
4. The method for high-throughput isolation of plant rhizosphere microorganisms and culture screening of IAA-producing strains according to claim 1, wherein the crop is cabbage, tomato, lettuce or spinach.
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