CN113667732A - Plant-mediated-based screening method for microbial composite strains - Google Patents
Plant-mediated-based screening method for microbial composite strains Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganism screening, and particularly relates to a screening method of a plant-mediated microorganism composite bacterial line. The invention takes 9 strains and plants with plant rhizosphere growth promoting function screened in a laboratory as research objects, obtains a plant high-affinity growth promoting compound bacterial line from a microorganism area system gathered by a plant root system through host-mediated selection, and realizes the conversion of strain compatibility from artificial combination to natural plant screening.
Description
Technical Field
The invention belongs to the technical field of microorganism screening, and particularly relates to a screening method of a plant-mediated microorganism composite bacterial line.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
With the increasing of the yield of crops by using chemical fertilizers and pesticides, people focus on the aspects of green and sustainable agricultural development and ecological stability. Diversified soil composition and structure affect crop yield and agricultural product quality from multiple aspects. Meanwhile, plant growth-promoting rhizobacteria have been widely noticed by people because of their stable colonization characteristics, and have growth-promoting effects of promoting plant nutrient absorption, inhibiting pathogenic bacteria from invading plants, and the like. On the basis of environment-friendly type, plant rhizosphere growth-promoting bacteria become the basis for developing green sustainable agriculture and establishing green food safety system, and as a biological fertilizer, the plant rhizosphere growth-promoting bacteria, organic fertilizer and chemical fertilizer form plant nutrient resources in the 21 st century.
On one hand, compared with single bacteria, the composite bacteria has various types of microorganisms and different growth promoting functions, can provide sufficient nutrition and resist stress for plants under the condition of limited resources and space, and has higher stability; on the other hand, the interaction and survival mechanism also exist among the microorganisms of the compound bacterial system, and through the interaction among the microorganisms, a plurality of functions are executed together or the complex task is decomposed into a plurality of steps, so that the complex biochemical process which cannot be carried out by a single species is effectively completed, and the growth promotion function of the complex biochemical process is more fully exerted.
The field experiment is used for exploring the assembly and variation of the root system microorganism group under natural conditions, and environmental factors such as temperature, wind, rain, the microorganism group, soil nutrients and the like cannot be controlled. Experiments conducted in controlled greenhouses using natural soil can avoid environmental changes and provide reproducible results, but using this technique, there is no deep profiling of microbiota members and soil nutrients.
Synthetic community (SynCom) refers to a rhizosphere flora synthesized by artificial synthesis or natural screening of host plants. The culture and recombination of the microorganisms play an extremely important role in the process, the living environment of the plant root system is furthest reduced under the controllable condition, the plant high-affinity growth-promoting compound bacterial system is obtained through host-mediated selection, the transformation from artificial combination to natural screening of the plant of the bacterial strain compatibility is realized, and the full research on the interaction between the plant and the microorganisms related to the plant by people becomes possible.
For a long time, people mostly take the growth promotion of single bacteria to plants and the growth promotion of artificial combined floras to plants as main research contents, and although partial research results are obtained, the single bacterial strain of the single bacterial strain and the artificial combined floras of the single bacterial strain cannot stably colonize plant rhizosphere for a long time, and a key link for mutual selection of plants and rhizosphere microorganisms is lacked, so that the microorganisms cannot fully exert the growth promotion effect, and the expected growth promotion effect is not achieved.
Disclosure of Invention
In order to better understand and reasonably apply rhizosphere microorganisms and screen effective plant rhizosphere growth promoting compound strains, the invention provides a screening method of a plant-mediated microorganism compound strain system.
In order to achieve the above object, the present invention provides in a first aspect a plant-mediated microorganism-based complex bacterial line comprising: bacillus subtilis GQJK61, Bacillus altitudinis GQYP101, Enterobacter laterosgii I42, Lekikurougheri (YokennellargesburgeiBAC 068), Bacillus parachromobacter (Bacillus paranthracePR 1), Bacillus subtilis (Bacillus subtilisPR10), Enterobacter laterosgii (Enterobacter laterosgiJP 6), Enterobacter laterosgii (Enterobacter laterosgiJP 9), Bacillus velezii (Bacillus velezensis FKM10), wherein the core strain is Enterobacter laterosgii (JP6, 9, I42), Bacillus parachromobacter (PR1), Bacillus cerebellis (QQYP 101), Bacillus subtilis FKM 10);
further, enterobacter ledebensis (JP6, JP9, I42): bacillus parachromobacter (PR 1): geobacillus altitudinis (GQYP 101): the ratio of Bacillus belgii (FKM10) was 1.53:0.27:0.16: 0.04.
The second aspect of the present invention provides a method for screening a composite microbial strain based on plant-mediated microorganisms, comprising: and mixing the bacterial liquids with equal bacterial quantities, then colonizing the rhizosphere of the plant, and analyzing sequencing results of plant rhizosphere bacteria amplicons and metagenomics in different periods by utilizing a high-throughput sequencing technology to obtain the dominant species and proportion of the rhizosphere of the plant.
One or more embodiments of the present invention have at least the following advantageous effects:
(1) the screening method provided by the invention obtains the plant high-affinity growth-promoting composite bacterial line from the microbial flora gathered by the plant root system through host-mediated selection, and realizes the conversion of strain compatibility from artificial combination to natural screening of plants.
(2) The composite bacterial line obtained by screening by the screening method provided by the invention is applied to a plant pot experiment according to the proportion of dominant bacteria, has obvious increase in plant height, stem thickness and fresh and dry weight of plants compared with a control, and shows good growth promoting effect.
(3) The screening method can be operated repeatedly under the condition of a controllable laboratory, the influence of abiotic and irrelevant biological factors in the field and the greenhouse environment is avoided, the rhizosphere dominant bacteria and the proportion thereof of plants in different periods can be screened by using the screening method, the dynamic change of the rhizosphere flora in different periods can be explored, and the natural screening and the assembly of different crops and microorganisms in different soils can be realized.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a Venn diagram of a bacterial community; wherein, Y is the original mixed sample, 0 is the sampling on the day after the bacteria dipping, 1 is the sampling 5 days after the bacteria dipping, 2 is the sampling 10 days after the bacteria dipping, and 3 is the sampling 15 days after the bacteria dipping.
FIG. 2 is species level metagenomic bacterial species distribution; wherein 3 represents the collection of corn rhizosphere bacteria samples 15 days after the first test dip, and 3.1, 3.2 and 3.3 are three replicates; for a second trial to repeat the experimental results, D3 represents the collection of a sample of corn rhizobacteria 15 days after the second trial dip, with D3.1, D3.2 and D3.3 being three replicates of the second trial).
FIG. 3 is the effect of complex bacterial lines on agronomic traits and biomass of maize; wherein, a is a comparison graph of TreatA, TreatB and CK in plant height, b is a comparison graph of TreatA, TreatB and CK in stem thickness, c is a comparison graph of TreatA, TreatB and CK in overground fresh weight and overground dry weight, and d is a comparison graph of TreatA, TreatB and CK in underground fresh weight and underground dry weight.
FIG. 4 is a diagram showing the growth promoting effect of a composite bacterial line potted plant;
FIG. 5 is a schematic diagram of a plant sterile culture apparatus;
note in the figure:
(a) plant sterile culture bottle: bottle height: 30cm, outside diameter of bottle mouth: 8cm, outer diameter of the bottle body: 7.5cm, inner diameter: 7cm, the bottle mouth protrudes 0.5cm outwards from the bottle body, the rubber band at the sealing position is strengthened for fixation, and microorganisms outside the bottle are prevented from entering.
(b) Sealing gauze: four layers with side length of 18 cm.
(c) Sealing the glass paper: two layers, the side length is 18cm, 10 round holes with the diameter of about 3mm are scattered in the central area (the tweezers are heated above the flame of the alcohol lamp and then the round holes are pricked on the glass paper).
(d) Sealing the newspaper: four layers with side length of 18 cm.
(e) Rubber band: two, respectively winding 2-3 circles to tighten the bottle mouth (sequentially stacking gauze, cellophane and newspaper on the bottle mouth)
(f) The whole picture of the plant sterile culture device with the culture medium (after sterilization);
FIG. 6 shows the process of screening complex strains;
FIG. 7 is a technical route of the screening process.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As described in the background art, in the prior art, when studying the interaction between plants and microorganisms related to plants, the main study contents are that single bacteria promote the growth of plants and artificial combined floras promote the growth of plants, and although some research results have been obtained, the former bacterial strain is single, the latter artificial combined floras cannot stably colonize in plant rhizosphere for a long time, and a key link of mutual selection between plants and rhizosphere microorganisms is lacked, so that the microorganisms cannot fully exert the growth promotion effect, and the expected growth promotion effect is not achieved.
In order to solve the above technical problems, a first aspect of the present invention provides a plant-mediated microorganism-based complex bacterial line, comprising: bacillus subtilis GQJK61, Bacillus altitudinis GQYP101, Enterobacter laterosgii I42, Lekikurougheri (YokennellargesburgeiBAC 068), Bacillus parachromobacter (Bacillus paranthracePR 1), Bacillus subtilis (Bacillus subtilisPR10), Enterobacter laterosgii (Enterobacter laterosgiJP 6), Enterobacter laterosgii (Enterobacter laterosgiJP 9), Bacillus velezii (Bacillus velezensis FKM10), wherein the core strain is Enterobacter laterosgii (JP6, 9, I42), Bacillus parachromobacter (PR1), Bacillus cerebellis (QQYP 101), Bacillus subtilis FKM 10);
further, enterobacter ledebensis (JP6, JP9, I42): bacillus parachromobacter (PR 1): geobacillus altitudinis (GQYP 101): the ratio of Bacillus belgii (FKM10) was 1.53:0.27:0.16: 0.04.
The second aspect of the present invention provides a method for screening a composite microbial strain based on plant-mediated microorganisms, comprising: and mixing the bacterial liquids with equal bacterial quantities, then colonizing the rhizosphere of the plant, and analyzing sequencing results of plant rhizosphere bacteria amplicons and metagenomics in different periods by utilizing a high-throughput sequencing technology to obtain the dominant species and proportion of the rhizosphere of the plant.
The screening method of the microbial composite bacterial system provided by the invention obtains the plant high-affinity growth-promoting composite bacterial system from the microbial flora gathered by the plant root system through host-mediated selection, and realizes the conversion of strain compatibility from artificial combination to natural plant screening.
The method is used for screening under the non-infectious condition, the dominant strains of the rhizosphere of the corn in 15 days of the colonized bacteria liquid are Enterobacter ludwigii (Enterobacter ludwigii), Bacillus paratyphenicus (Bacillus paranthraceis), Bacillus altitudinis (Bacillus altitudinis) and Bacillus belgii (Bacillus velezensis), and the average relative abundances are 66.39%, 11.66%, 6.80% and 1.80% respectively. The functional annotation analysis of the metagenomic histone shows that the corn rhizosphere growth promoting compound strain screened by the method has related genes, proteins and enzymes by utilizing plant rhizosphere secretion and inorganic ions in the surrounding environment. The pot experiment result shows that the composite bacterial strain liquid obtained by the screening method is applied to the rhizosphere of the early-stage corn seedling, can generate a good growth promoting effect on the corn, and has obvious increase in the height, stem thickness and fresh and dry weight of the corn compared with a control group, thereby achieving obvious or extremely obvious difference. The screening method of the plant rhizosphere microorganism composite bacterial line provided by the invention avoids the influence of abiotic and irrelevant biological factors in natural environment, and can be repeatedly operated under the condition of a controllable laboratory.
Moreover, the screening method provided by the invention can be repeatedly operated under the controllable laboratory condition, so that the influence of abiotic and irrelevant biological factors in fields and greenhouse environments is avoided; the screening method can be used for screening and obtaining the plant rhizosphere dominant bacteria and the proportion thereof in different periods, and simultaneously can explore the dynamic change of rhizosphere flora in different periods. The composite bacterial line obtained by screening by the screening method is applied to a plant pot experiment according to the proportion of dominant bacteria, has obvious increase in plant height, stem thickness and fresh and dry weight of plants compared with a control, and shows good growth promoting effect.
Further, before mixing the bacterial liquids, the test strains are required to be adjusted to the absorbance corresponding to the unit bacterial quantity, specifically, the number of each bacterial strain is required to be adjusted to 108OD corresponding to cfu/mL600The value is obtained.
Further, sterilizing and carrying out aseptic germination acceleration treatment on plant seeds to form plant seedlings, and carrying out bacterial liquid colonization;
the method comprises the following specific steps:
s1: strain culture is carried out on an LB culture medium;
specifically, solid LB culture medium is adopted for activation, and then the activated single colony is transferred into liquid LB culture medium for culture.
The solid LB medium comprises: 5g of yeast powder, 10g of peptone, 10g of sodium chloride, 15-20g of agar, 1000mL of deionized water, pH7.0 and autoclaving at 121 ℃ for 20 min;
liquid LB medium: 5g of yeast powder, 10g of peptone, 10g of sodium chloride, 1000mL of deionized water, pH7.0 and autoclaving at 121 ℃ for 20 min.
S2: uniformly mixing each bacterial solution in a 1:1 equal volume manner to prepare a mixed bacterial solution;
specifically, activating the bacteria for experiment, inoculating and shaking culture, transferring and fermenting, collecting the bacteria, diluting with PBS buffer solution and adjusting the concentration; then regulating the number of each bacterium to 108OD corresponding to cfu/mL600And (4) respectively sucking the bacterial liquid into a sterilization collection bottle, and uniformly mixing in an equal volume of 1: 1.
S3: selecting seeds, disinfecting and accelerating germination in sequence to obtain sterile seedlings;
further, the method for disinfecting the seeds comprises the following steps:
(1) shaking and soaking with 75% ethanol for 5 min;
(2) shaking and soaking in 5% sodium hypochlorite solution for 10 min;
(3) shaking and soaking with sterile water for 5min, repeating for three times, and sealing the conical flask;
(4) detoxification: and (4) putting the sterilized seeds at room temperature, and detoxifying for 20h for later use.
Further, accelerating germination of seeds is carried out in an aseptic 1/2MS tissue culture bottle; the seed germination accelerating method comprises the following steps: in an aseptic operation environment, the detoxified seeds are clamped into an aseptic 1/2MS tissue culture bottle by using aseptic forceps, one seed is taken from each bottle, a cover is tightly sealed, and the bottle is placed in an illumination incubator for accelerating germination for 5-7 days.
S4: placing the sterile seedlings into the mixed bacterial liquid for bacterial liquid colonization, and culturing in 1/2MS seedling culture medium after colonization;
further, washing the roots of the aseptic seedlings with aseptic water, washing off the culture medium attached to the surfaces, and then putting the roots of the aseptic seedlings into the mixed bacterial liquid to ensure that the roots of the seedlings are completely immersed in the mixed bacterial liquid for bacterial liquid colonization.
Further, the time of the bacterial liquid colonization process is 30-60 min;
further, after colonization, transplanting the seedlings into an aseptic seedling culture bottle, clamping the stem parts of the seedlings by using aseptic tweezers, enabling root systems not to touch the bottle wall, vertically suspending the seedlings, and enabling the seedlings to stand on a bottom 1/2MS seedling culture medium;
further, the conditions for cultivation after colonization are as follows: the illumination condition is as follows: the illumination intensity is 60 percent at 25 ℃ for 16 h; dark conditions: the illumination intensity is 0% at 25 ℃ for 8 h.
S5: shearing roots of the seedlings in different cultivation periods, and oscillating in sterile water to extract plant rhizobacteria;
s6: screening of a compound strain: and (3) carrying out high-throughput sequencing on the collected plant rhizobacteria, carrying out metagenome sequencing analysis on the rhizobacteria, and screening out dominant bacteria.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Experimental materials:
1. main apparatus and equipment
Vertical pressure steam sterilization pot, superclean bench, visible spectrophotometer, centrifuge, magnetic stirrer, vortex oscillator, pH meter, shaking table, electrothermal blowing drying oven, electrothermal constant temperature incubator, illumination constant temperature incubator, ultra-low temperature refrigerator, electrophoresis apparatus, PCR apparatus, water bath, electronic balance, pipetting gun, culture dish, erlenmeyer flask, tissue culture bottle, sterile seedling culture bottle, tweezers, 50mL centrifuge tube, 5mL centrifuge tube, coating rod, inoculating loop
2. Primary drug agent
75% ethanol, 5% sodium hypochlorite, sterile water, deionized water, phosphate buffer solution, peptone, yeast powder, sodium chloride, agar powder, sucrose, ammonium nitrate, calcium superphosphate, potassium feldspar powder, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate, zinc sulfate heptahydrate, boric acid, potassium iodide, sodium molybdate, copper sulfate, cobalt chloride, disodium ethylenediamine tetraacetate, ferric sulfate heptahydrate, inositol, glycine, nicotinic acid, pyridoxine hydrochloride, thiamine hydrochloride
3. Test strains
The test strains are selected from 9 plant rhizosphere growth-promoting bacteria with growth-promoting effect which are screened and preserved in a laboratory, and are specifically shown in table 1.
TABLE 1 test strains
4. Test plant
Corn variety: zhengdan 958 corn hybrid, culturing to two cotyledons, selecting healthy and consistent corn seedling for standby, and repeating each treatment for a plurality of times.
Culture medium:
1. conventional culture medium
Solid LB medium: 5g of yeast powder, 10g of peptone, 10g of sodium chloride, 15-20g of agar, 1000mL of deionized water, pH7.0 and autoclaving at 121 ℃ for 20 min;
liquid LB medium: 5g of yeast powder, 10g of peptone, 10g of sodium chloride, 1000mL of deionized water, pH7.0 and autoclaving at 121 ℃ for 20 min.
2. Improved 1/2MS culture medium
①NH4NO3 1.2g/L，Ca3(PO4)2(superphosphate) 660mg/L, potassium feldspar powder 3g/L, MgSO4·7H2O 185mg/L；
②CuSO4 0.0125mg/L，MnSO4·4H2O 11.15mg/L，ZnSO4·7H2O4.3 mg/L, KI 0.415mg/L, boric acid 3.1mg/L, cobalt chloride 0.0125mg/L and sodium molybdate 0.125 mg/L;
③ disodium ethylenediaminetetraacetate (Na)2EDTA·2H2O)37.3mg/L，FeSO4·7H2O 27.8mg/L；
100mg/L inositol, 15g/L sucrose, 2mg/L glycine, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride and 0.1mg/L thiamine hydrochloride.
Note:
(1) dissolving ammonium nitrate and magnesium sulfate heptahydrate in a formula I by using deionized water to prepare 50 multiplied mother liquor I, and fixing the volume to 1000 mL; calcium superphosphate and potassium feldspar powder are insoluble and need to be subpackaged;
(2) dissolving all the medicines in the formula II by using deionized water to prepare 1000 multiplied mother liquor II, and fixing the volume to 1000 mL;
(3) formula (III) iron salt mother liquor III (preparing iron salt solution: solution A: weighing 3.73g Na)2EDTA·2H2Dissolving O in 200mL of deionized water, and preheating at 70 ℃; solution B: 2.78g of FeSO are weighed out4·7H2Dissolving O in 200mL of deionized water; and slowly pouring the solution B into the solution A, mixing while stirring, placing the mixture into a water bath kettle at the temperature of 70 ℃ for chelation for two hours, and cooling the mixture completely to reach the constant volume of 1000 mL. ) (ii) a
(4) Dissolving glycine, nicotinic acid, pyridoxine hydrochloride and thiamine hydrochloride in the formula IV by using deionized water to prepare 1000 multiplied mother liquor IV, and fixing the volume to 1000 mL; inositol and sucrose are weighed and added after the four mother solutions are mixed;
(5) the prepared four mother solutions are placed in a conical flask, sealed by a sealing film and stored in a refrigerator at 4 ℃ for later use.
(6) The components of the germination medium are shown in Table 2.
TABLE 21/2 MS Germination media
Absorbing the four mother solutions by using a liquid transfer gun, adding 0.1g of inositol and 30g of sucrose after the mother solutions are completely mixed, adjusting the pH value to 6.5, fixing the volume to 1000mL, filling 0.033g of calcium superphosphate, 0.15g of potassium feldspar powder and 0.4g (0.8%) of agar in each tissue culture bottle, filling the solution with the fixed volume, and filling 50mL in each tissue culture bottle. After subpackaging, autoclaving at 116 deg.C for 30 min.
The preparation of the seedling culture medium and the component contents are shown in table 3.
TABLE 31/2 MS culture medium for seedlings
Absorbing the four mother solutions by using a liquid transfer gun, adding 0.1g of inositol and 15g of sucrose after the mother solutions are completely mixed, adjusting the pH value to 6.5, fixing the volume to 1000mL, filling 0.165g of calcium superphosphate, 0.75g of potassium feldspar powder and 1.5g (0.6%) of agar in each sterile seedling culture bottle, subpackaging the solutions with the fixed volumes, and subpackaging 250mL of each sterile seedling culture bottle. After the split charging, four layers of gauze, two layers of glassine paper (10 holes with diameter of about 3mm are poked by tweezers for convenient ventilation) and four layers of newspaper are used for sealing in sequence, and autoclaving is carried out for 30min at 116 ℃.
Experimental procedure (scheme shown in fig. 6 and 7):
the plant sterile culture device used in the method is shown in figure 5;
1. preparation of mixed bacterial liquid
a cultivation of the strains
(1) Respectively sucking 2 mu L of thallus preservation solution from a glycerol preservation tube in an aseptic operation environment to one side edge of a solid LB culture medium flat plate, carrying out three-zone lineation by using a sterilization loop, inverting the flat plate to culture for 12h in an electrothermal constant-temperature incubator at 37 ℃, culturing to separate and purify a single bacterial colony, respectively transferring the single bacterial colony to a new solid LB culture medium flat plate to carry out secondary activation, and carrying out the same steps as the first step.
(2) Selecting activated single colonies by using an inoculating loop in a sterile operation environment, respectively transferring the single colonies into 50mL of liquid LB culture medium to serve as seed liquid, and placing the seed liquid at 37 ℃ for shake culture for 10 hours in a shaking table at 180 r/min;
(3) after 10h, respectively inoculating the bacterial liquid into a new 50mL liquid LB culture medium with the inoculation amount of 10 percent, using the bacterial liquid as a fermentation liquid, placing the fermentation liquid in a shaking table at 37 ℃ and 180r/min for shaking culture for 4h, and taking out the fermentation liquid for later use.
b, determination of viable count of bacteria
(1) After shaking culture for 4h, collecting thalli for multiple times by using a 50mL centrifuge tube in an aseptic operation environment, washing for three times by using 15mL PBS buffer solution, centrifuging for three times, pouring out supernate, adding 5mL PBS buffer solution, and then oscillating and whirling by using a vortex oscillator to uniformly mix the collected thalli in the PBS buffer solution for later use;
(2) diluting the bacterial liquid with PBS buffer solution in sterile operation environment, and measuring OD with spectrophotometer600Diluting with sterile water to 10-5、10-6Two gradients, coating 100 μ L on LB plate, arranging three gradients in parallel, culturing in 37 deg.C incubator for 12 hr, taking out, counting, calculating viable bacteria amount in each ml of fermentation liquid according to dilution gradient, and performing gradient 10-5Hundreds of bacteria per plate coated or gradient 10-6The number of the bacteria on each flat plate is more than ten, and the number of the viable bacteria reaches 108OD corresponding to cfu/mL600Values, recorded in a table.
c: mixing of bacterial liquid
(1) Respectively activating 9 strains of experimental bacteria, inoculating and shaking culture, transferring and fermenting, collecting bacteria, and diluting with PBS buffer solution to adjust concentration;
(2) adjusting the number of each bacterium to 108OD corresponding to cfu/mL600Respectively sucking the bacterial liquid into a sterilization collection bottle, and uniformly mixing in an equal volume of 1:1 for later use.
2. Sterile seedling culture
(1) Seed selection
The corn variety is a first generation hybrid corn new variety bred by Zhengdan 958 (Zheng58/Chang 7-2 (selected)) hybridization, and the corn seeds with plump seeds and consistent appearance are selected to be used in a sterilized conical flask for later use.
(2) Seed disinfection
1) Shaking and soaking with 75% ethanol for 5 min;
2) shaking and soaking in 5% sodium hypochlorite solution for 10 min;
3) shaking and soaking with sterile water for 5min, repeating for three times, and sealing the conical flask;
4) detoxification: and (4) putting the sterilized seeds at room temperature, and detoxifying for 20h for later use.
(3) Seed germination acceleration
In an aseptic operation environment, the detoxified corn seeds are clamped into an aseptic 1/2MS tissue culture bottle by using aseptic forceps, one seed is taken from each bottle, a cover is tightly sealed, and the bottle is placed in an illumination incubator for pregermination for 5-7 d.
The illumination condition is as follows: 16h, 25 ℃ and 60% of illumination intensity; dark conditions: the illumination intensity is 0% at 25 ℃ for 8 h.
3. Colonization with bacterial liquid
(1) In an aseptic operation environment, pouring the mixed bacterial liquid into a large sterile plate, clamping the seedlings which are successfully germinated (germinate for 7d) by using aseptic tweezers, washing the roots of the seedlings for three times by using aseptic water, washing off the culture medium attached to the surface, putting the seedlings into the mixed bacterial liquid, completely immersing the roots of the seedlings into the mixed bacterial liquid, and colonizing for 30-60 min;
(2) transplanting the seedlings into an aseptic seedling culture bottle by using aseptic tweezers after colonization, clamping stem parts of the seedlings by using the tweezers, preventing root systems from touching the bottle wall, vertically suspending and placing the seedlings to enable the seedlings to stand on a bottom 1/2MS seedling culture medium, sampling and processing 10 bottles respectively by 0d, 5d, 10d and 15d, sequentially sealing the bottle mouth by using four layers of gauze with the side length of 18cm, two layers of glass paper (the central area is dispersed with 10 round holes with the diameter of about 3mm to facilitate ventilation) and four layers of newspaper, tightening the bottle mouth by winding 2-3 circles of rubber bands, and culturing the sealed bottle mouth in a light culture box.
The illumination condition is as follows: 16h, 25 ℃ and 60% of illumination intensity; dark conditions: the illumination intensity is 0% at 25 ℃ for 8 h.
4. Sampling and thallus enrichment
(1) And (3) sucking 9 strains of bacteria mixed bacteria with equal proportion in a certain volume into a 50mL centrifuge tube, centrifugally collecting at 8000rpm for 2min, and washing with PBS buffer solution for three times to obtain the original mixed bacteria with the number of Y.
(2) When the corn plants grow to 0d, 5d, 10d and 15d in an aseptic seedling culture flask, selecting 10 bottles to be treated in each period, taking out the corn plants in an aseptic operation environment by using aseptic tweezers, cutting off the roots of the corn plants by using aseptic scissors, placing the corn plants in a collecting bottle filled with glass beads and 200mL of aseptic water, fully vibrating the corn plants, using an ultrasonic oscillator to fully release plant rhizobacteria in the aseptic water if necessary, collecting thalli by using a 50mL centrifugal tube, centrifuging and collecting the thalli for multiple times at 8000rpm for 2min, numbering collected thalli samples as 0, 5, 10 and 15 according to the time sequence, taking out and cleaning the roots in the bottle when the thalli are completely collected, sucking water on the surface of the roots by using absorbent paper, and bagging stems, leaves and roots respectively for storage.
5. Screening of Complex strains
(1) Sequencing analysis of amplicons of rhizobacteria
The collected original mixed thalli, 0d, 5d, 10d and 15d corn rhizosphere thalli are numbered as Y, 0, 5, 10 and 15, sent to Haipahi Senno bioscience company for high-throughput Sequencing, a Second generation Sequencing technology (SGS) is used, an Illumina MiSeq Sequencing platform is taken as a basis, V3-V4 region of 16S rDNA is sequenced, the specific composition structure of the community is surveyed, the diversity change rule is mastered, the dominant species in the community are revealed, and the succession change of the corn rhizosphere bacterial community composition at different periods is summarized.
(2) Rhizosphere bacterium metagenome sequencing analysis
The collected part 15d of corn rhizosphere thallus is sent to Shanghai Senno biotechnology company for metagenome sequencing, and a bird gun sequencing technology (Shotgun sequencing) is used in combination with a strategy of full-Microbiome Association exploration (Microbion-Wide Association Studies, MiWAS) to more comprehensively and finely display the functional metabolic spectrum and the expression spectrum of the whole community from the DNA and RNA levels respectively, so that the principle of the role played by the microbial community in an ecosystem is revealed through the research on the action mechanism.
6. Pot experiment
(1) Design of experiments
Activating the dominant bacterial strain with LB culture medium, inoculating, shake-flask culture, transferring fermentation, collecting thallus, diluting with PBS buffer solution, adjusting concentration until the bacterial count of each strain reaches 108OD corresponding to cfu/mL600And uniformly mixing the bacteria liquid according to the proportion obtained by screening for later use.
Uniformly mixing soil to be tested, removing obvious large soil blocks, putting the soil into flowerpots with the diameter of 15cm and the height of 20cm, wherein the soil filling amount of each pot is about 1.5kg, selecting two healthy corn seeds with the same size for each pot, selecting corn seedlings with the same growth vigor when the corn sprouts to grow two cotyledons, only reserving one strain for each pot, diluting the mixed bacteria liquid into 200mL of water to irrigate the roots of the corn, keeping the same watering amount in the later management process, keeping the humidity between 40 and 50 percent, and repeating three times for each treatment.
(2) Index measurement and method
1) Agronomic traits were determined after significant differences in maize at day 45: high plant height and thick stem. The specific operation is as follows: firstly, measuring the height of a marked maize seedling: and measuring the distance from the labeled seedling pot soil to the heart leaves by using a meter ruler, and recording. Measuring the stem thickness of the marked maize seedlings: the stem circumference of the 3cm part from the ground without root hair was measured with a vernier caliper and recorded.
2) And (3) measuring the fresh and dry weight of the marked maize seedlings, separating the collected roots and stems of the maize seedlings, cleaning root soil, sucking water by using filter paper, and weighing the fresh weight on the ground and the fresh weight under the ground respectively. And then putting the mixture into an oven, deactivating enzyme at 105 ℃, drying at 85 ℃, and weighing the above-ground dry weight and the underground dry weight.
All statistical calculations were performed using the span 19.0 software. To compare the mean, the Duncan multi-range test was used at a probability level of 5%. Finally, the graph was drawn using GraphPad Prism6 software.
Results and analysis
1. Sequencing analysis of corn rhizosphere bacterium amplicon and metagenome
As shown in FIG. 1, OTU cluster analysis can obtain a plant rhizosphere bacterial community which is gradually simplified and tends to be stable along with the change of time, and can show that the screening method of the microorganism composite bacterial line can explore the dynamic change of rhizosphere bacterial communities in different periods while screening the composite bacterial line. Species annotation and abundance analysis were performed on each sample at the classification level (fig. 2), and the core strains of rhizosphere samples sampled 15 days under the screening method were enterobacter ledebarkii (enterobacter lutedwigii), Bacillus parachromicus (Bacillus parathratus), Bacillus altitudinis (Bacillus altitudinis), and Bacillus belgii (Bacillus velezensis), and the average relative abundances were 66.39%, 11.66%, 6.80%, and 1.80%, respectively. The ratio is enterobacter ledwigii (JP6, JP9, I42): bacillus parachromobacter (PR 1): geobacillus altitudinis (GQYP 101): bacillus belgii (FKM10) ═ 1.53:0.27:0.16: 0.04. The metagenome analysis shows that the plant rhizosphere dominant complex strain line obtained by the screening method of the plant-mediated microorganism complex strain line has related genes, proteins and enzymes which utilize plant rhizosphere secretion and surrounding environment inorganic ions, and is an important reason that the plant rhizosphere dominant complex strain line can stably colonize on the plant rhizosphere, interact with plants to play a role in promoting growth and can be screened by the method.
2. Composite bacterial system corn pot effect analysis
The corn rhizosphere dominant bacteria strain and the proportion thereof are Enterobacter ludwigii (JP6, JP9 and I42) obtained by screening based on a plant-mediated microorganism composite bacteria line screening method: bacillus parachromobacter (PR 1): geobacillus altitudinis (GQYP 101): bacillus belgii (FKM10) ═ 1.53:0.27:0.16: 0.04. The pot test was designed based on the screening results as shown in table 4.
TABLE 4 potted plant experimental design for growth promoting effect of composite bacterial line
As can be seen from FIG. 3a, the composite bacterial line screened by the screening method of the invention has significantly increased plant height compared with CK, and TreatA and TreatB have 29.46% and 28.94% respectively higher plant height than CK, and have significant difference (p <0.01) with CK.
As can be seen from fig. 3b, the stem thickness of the maize seedlings treated by the composite bacterial line is increased to different degrees compared with CK, treataa and TreatB are respectively increased by 13.05% and 7.88% compared with CK, and although the difference is not significant compared with CK, the stem thickness is still greatly increased.
As shown in fig. 3c, the maize seedlings treated by the composite bacterial line were significantly increased in the fresh weight and dry weight on the ground compared with CK, TreatA and TreatB were increased by 33.96% and 47.65% respectively compared with CK on the ground, and both achieved significant differences (p <0.05), wherein TreatB achieved very significant differences (p <0.01) compared with CK. Compared with the above-ground dry weight of CK, the dry weight of TreatA and TreatB are respectively increased by 48.37 percent and 50.61 percent, and the dry weight of TreatB are both very different from CK (p is less than 0.01).
As shown in fig. 3d, the maize seedlings treated by the composite bacterial line were increased to different degrees in the fresh underground weight and the dry underground weight compared with CK, and TreatA and TreatB were increased by 14.67% and 8.07% respectively in comparison with CK fresh underground weight, but were not significantly different from CK. Treataa and TreatB increased by 51.20% and 35.20% respectively compared to CK underground dry weight, wherein treataa showed significant differences (p <0.05) compared to CK.
Data corresponding to fig. 3 are shown in table 5.
FIG. 4 shows the growth promoting effect of the composite bacterial line potted plant, and the growth advantages of the corn seedlings treated by the composite bacterial line relative to CK in the aspects of stem thickness and plant height can be clearly seen.
TABLE 5 growth promoting effect of composite strain potted plant
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A screening method based on a plant-mediated microorganism composite bacterial line is characterized by comprising the following steps: the method comprises the following steps: and mixing the bacterial liquids with equal bacterial quantities, then colonizing the rhizosphere of the plant, analyzing the sequencing results of the amplicons and the metagenome of the rhizosphere of the plant in different periods by utilizing a high-throughput sequencing technology, and screening out the dominant bacteria.
2. The screening method according to claim 1, wherein: before mixing the bacterial liquid, the test strain needs to be adjusted to the absorbance corresponding to the unit bacterial quantity.
3. The screening method according to claim 1, wherein: and (3) sterilizing and carrying out aseptic germination acceleration treatment on the plant seeds to form plant seedlings, and carrying out bacterial liquid colonization.
4. The screening method according to claim 1, wherein: the method comprises the following specific steps:
s1: strain culture is carried out by adopting an LB culture medium;
s2: uniformly mixing each bacterial solution in a 1:1 equal volume manner to prepare a mixed bacterial solution;
s3: selecting seeds, disinfecting and accelerating germination in sequence to obtain sterile seedlings;
s4: placing the sterile seedlings into the mixed bacterial liquid for bacterial liquid colonization, and culturing in 1/2MS seedling culture medium after colonization;
s5: shearing roots of the seedlings in different cultivation periods, and oscillating in sterile water to extract plant rhizobacteria;
s6: screening of a compound strain: and (3) carrying out high-throughput sequencing on the collected plant rhizobacteria, carrying out metagenome sequencing analysis on the rhizobacteria, and screening out dominant bacteria.
5. The screening method according to claim 4, wherein: in the step S3, the seed germination is carried out in a sterile 1/2MS tissue culture bottle.
6. The screening method according to claim 4, wherein: in the step S4, the roots of the aseptic seedlings are washed with aseptic water, the surface-attached culture medium is washed away, and then the roots of the aseptic seedlings are placed into the mixed bacterial liquid, so that the roots of the seedlings are completely immersed in the mixed bacterial liquid for bacterial liquid colonization.
7. The screening method according to claim 6, wherein: the time of the bacterial liquid colonization process is 30-60 min.
8. The screening method according to claim 4, wherein: in the step S4, after colonization, the seedlings are transplanted into a sterile seedling culture bottle, and the stem part of the seedlings is gripped by sterile tweezers, and the root system is vertically suspended without touching the bottle wall, so that the seedlings stand upright on the bottom 1/2MS seedling culture medium.
9. The screening method according to claim 4, wherein: in step S4, the conditions for post-colonization incubation are: the illumination condition is as follows: the illumination intensity is 60 percent at 25 ℃ for 16 h; dark conditions: the illumination intensity is 0% at 25 ℃ for 8 h.
10. The screening method according to claim 4, wherein: and (3) carrying out metagenome determination on the collected plant rhizobacteria, and preparing a microorganism compound bacterial line according to the microorganism strains according to the metagenome result, namely the plant-mediated microorganism compound bacterial line.
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