CN115011483A - High-throughput screening method for IAA-producing endophytes of plant tissues - Google Patents
High-throughput screening method for IAA-producing endophytes of plant tissues Download PDFInfo
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Abstract
A high-throughput screening method for IAA endophytes produced by plant tissues is characterized in that three frequencies, namely low frequency ultrasonic waves, medium frequency ultrasonic waves and high frequency ultrasonic waves, are jointly processed from fresh plant tissues, the types and the number of the released endophytes of the plant tissues are increased, and the obtaining of the endophytes of the plant tissues is increased. 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 IAA tissue endophyte strain with high flux, and provides better strain resource for the function of plant tissue endophyte 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 high-throughput screening method for IAA endophytes produced by plant tissues.
Background
The plant microbiome consists of a wide variety of microorganisms that inhabit different plant tissues and organs, such as seeds, roots, stems, leaves, flowers and fruits. These microbial communities exist outside as epiphytes and inside tissues and organs of plants as endophytes, affecting the vigor and health of the plants. Among these microbial communities, endophytes are microorganisms that live within host plants, do not cause any disease symptoms or negative effects, and have the ability to antagonize plant pathogens. Endophytes play an important role in the operation of host plants by affecting the physiology and development of the host plant. It is well known that endophytic bacteria participate in conferring tolerance or resistance to various biotic and abiotic stresses to host plants by releasing antibacterial metabolites, synthesizing plant hormones, siderophores, competing for space and nutrients with pathogens, and modulating resistance responses in plants. The study of the interaction of microorganisms within plants is of great importance due to the use of beneficial microorganisms as plant growth promoters and biocontrol agents for the control of plant pathogens. Endogenous microorganisms are a possible alternative to reduce the use of pesticides and fertilizers. Endogenous host-associated bacteria are useful tools for developing biocontrol agents for controlling plant pathogens. The composition of bacterial endophytes varies from plant to plant, tissue to organ to genotype to breed to soil to breed.
The research of the plant tissue endophyte group is entering a rapid development stage, the next stage necessarily focuses on researching the function of the plant tissue endophyte group in the plant, and gradually reveals the molecular mechanism of the interaction process of the plant and the plant tissue endophyte group.
The most active auxin in plants is indole-3-acetic acid (IAA). IAA is the most versatile and essential metabolite, inducing plant growth and anatomical improvement in cell elongation, apical dominance maintenance, prevention of cellular senescence, and also regulating the growth and development of plant tissues. IAA acts as an effector molecule in plant and microbial interactions, in the aerial parts, and it enhances photosynthesis, pigment synthesis and early and delayed growth processes in seeds, flowers, fruits and leaves. IAA can affect plant cell division, nodule formation, seed sowing and tuber germination, xylem and root development, photosynthesis, and can also increase plant resistance to various stress conditions.
At present, the key factor hindering the endophyte function of plant tissues and the interaction research between plants and microorganisms is the endophyte resource of the plant tissues. 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 in-situ microorganisms from given plant tissues, and provide better strain resources for the function of endophytes in the plant tissues and the interaction research with plants.
The current method for screening microbial strains mainly separates and screens endophytes in plant tissues by a plate dilution method:
chinese patent 202110634318.5 discloses a method for screening endophytes for promoting the growth of marsdenia tenacissima; chinese patent 201980013968.9 discloses "endophyte screening"; the methods for obtaining endophytes described in the above two patents require multiple cultivations and streaking to purify the 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.
Isolated culture of microorganisms in situ is important to reveal the function of microorganisms in plant growth and health. The combination of the microorganism which is separately cultured and a sterile system promotes the important technology of the development of the endophyte group of the plant tissue from description to functional research.
Disclosure of Invention
The invention provides a high-throughput screening method for IAA endophytes produced by plant tissues, which is used for screening the IAA-producing plant tissue endophyte in-situ strains in a high-throughput manner, so that the obtaining rate of the obtained single strain is increased to 100 percent, the screening efficiency is greatly improved, better strain resources are provided for the function of the plant tissue endophytes and the interaction research with plants, and the method is suitable for in-situ separation culture of microorganisms of the plant tissues such as green vegetables, tomatoes, lettuce, spinach and the like.
In order to achieve the purpose, the invention provides the following technical scheme:
a high-throughput screening method for IAA endophytes produced by plant tissues comprises the following steps:
1) obtaining plant tissue sample microorganism suspension
Cleaning collected crop tissue samples growing healthily, sterilizing and disinfecting the outside, carrying out ultrasonic treatment for 20-50s at room temperature by using an active release type buffer solution, filtering and collecting filtrate to obtain a plant tissue suspension, and storing at 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 20-40 KHz, 40-50 KHz and 80-100 KHz respectively;
the active release buffer comprises ammonium chloride: 0.2-0.7g/L, dipotassium hydrogen phosphate: 0.4-1.0g/L, potassium dihydrogen phosphate: 0.3-0.6g/L, MgSO 4 -7H 2 O：0.6-1.2g/L，CaCl 2 -2H 2 O： 0.1-0.5g/L，FeSO 4 -7H 2 O：2-8mg/L，ZnSO 4 -7H 2 O：50-150ug/L，MnCl 2 -4H 2 O: 15-45ug/L, boric acid: 100-500ug/L CoCl 2 -6H 2 O：100-400ug/L，CuCl 2 -2H 2 O： 5-15ug/L，NiCl 2 -6H 2 O: 15-30ug/L, KCl: 2-3mM, 3-7 wt% of microcystis algae polysaccharide, pH 7.2-7.4;
2) determining the optimal dilution concentration for isolation culture
Adding the plant tissue suspension into a high-throughput separation liquid culture medium, respectively diluting the plant tissue dilution in a gradient manner, respectively transferring each gradient dilution into different cell culture plate holes under aseptic operation for culture, taking 5-15% of TSB solution without adding the plant tissue suspension as negative control, repeating more than three times for each gradient, sealing a 96-hole cell culture plate, placing the 96-hole cell culture plate on a shaking table, and culturing for 2-10 days at the temperature of 28-37 ℃ at 80-180 r/min;
20-35% of the wells in the 96-well cell culture plate are in a turbid state visible to naked eyes, and the concentration of the plant tissue diluent corresponding to the 96-well cell culture plate is used as the optimal dilution concentration for separation culture;
the high-throughput separation liquid culture medium comprises 50-150 mg/L of L-tryptophan, 10-30 g/L of casein polypeptide, 10-30 g/L of soybean protein polypeptide, 2-7g/L of sodium chloride and dipotassium phosphate: 0.5-1.0g/L, potassium dihydrogen phosphate: 0.3-0.7g/L, MgSO 4 -7H 2 O：0.6-1.3g/L， CaCl 2 -2H 2 O：0.1-0.3g/L，FeSO 4 -7H 2 O：2.0-5.0mg/L，ZnSO 4 -7H 2 O：50-150 ug/L，MnCl 2 -4H 2 O: 20-50ug/L, boric acid: 200-400ug/L of CoCl 2 -6H 2 O： 100-300ug/L，CuCl 2 -2H 2 O：5-15ug/L，NiCl 2 -6H 2 O：10-30ug/L， Na 2 MoO 4 -2H 2 O: 40-60mg/L, methanol: 2-7ml/L, 1.0-3.5mM potassium myristate, 2-5 wt% strigolactone, 0.5-1 wt% methyl jasmonate, and pH of 6.0-8.0;
3) screening a single strain for producing IAA;
transferring the plant tissue diluent with the optimal dilution concentration into the holes of the cell culture plate under the 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, turbid bacterial liquid in the cell culture plate holes is sucked into the white ceramic plate, an alkowski color developing solution is added for color development reaction, and meanwhile, IAA standard solution is used as a positive control; a high-flux separation liquid culture medium solution without tissue sample homogenate is used as a negative control;
placing the white ceramic plate at room temperature in a dark condition for 20-40min, and observing, wherein the white ceramic plate is red in color and shows that the strain can generate IAA; strains in IAA-producing cell culture plates were laboratory-numbered and glycerol-deposited as a result of the IAA color reaction.
Preferably, in step 1), the sonication time is between 20 and 50 s.
Preferably, in the step 3), the concentration of the IAA in the IAA standard solution is 10-100 mg/L.
Preferably, the plant variety is a green vegetable, tomato, lettuce or spinach.
The invention collects crop tissue samples which grow healthily and cleans the crop tissue samples by using a multi-frequency ultrasonic auxiliary activity release type buffer solution to obtain a suspension of the endophyte samples in plant tissues, wherein ultrasonic is an elastic mechanical wave in a matter medium and is used as a physical energy form. The microorganism species and the quantity of the suspension liquid of the endophyte sample of the plant tissue obtained by cleaning the multi-frequency combined ultrasonic auxiliary active release type buffer solution are superior to those of single-frequency ultrasonic.
Filtering the plant tissue endophyte sample suspension and collecting the plant tissue endophyte suspension; performing gradient dilution by using a high-flux separation liquid culture medium containing L-tryptophan; the soil microbial community has the highest diversity, and the second is the endophyte community of the plant tissue. During the reproductive growth of the plant, the flora in the plant tissue is gradually stabilized, and the number of the flora is in a relatively stable interval. The gradient setting is set according to the number of endophytes in plant tissues, and the optimal dilution concentration can be rapidly and accurately screened through the gradient setting, namely 20-35% of the pores in a 96-pore cell culture plate are in a visible turbid state under the optimal dilution concentration.
In the high-flux bacteria separation step, plant tissue suspensions with different concentrations are added into a cell culture plate for culture, 20-35% of wells in a 96-well cell culture plate present plant tissue suspension dilution concentrations corresponding to a macroscopic turbid state and serve as optimal dilution concentrations, and the proportion of single microorganisms obtained is increased by using a gradient dilution method. Through hundreds of experiments of bacteria with optimal dilution concentration, statistical analysis shows that when 20-35% of wells show a turbid state visible to the naked eye, the bacteria in the wells can be regarded as pure bacteria.
The invention adopts bidirectional synchronization to respectively carry out IAA color reaction and strain preservation, and carries out numbering preservation on the IAA-producing crop tissue endophyte strain according to the result of the IAA color reaction, thereby greatly saving the strain breeding time. The IAA tissue-producing endophyte strain is screened in a high-throughput manner, and better strain resources are provided for the function of the plant tissue endophyte and the interaction research with plants.
Compared with the prior art, the invention has the following beneficial effects:
the method uses low, medium and high frequency ultrasonic waves to jointly process fresh plant tissues, increases the types and the quantity of released endophytes of the plant tissues, and increases the obtaining of the endophytes of the plant tissues. 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 two-way synchronous IAA color reaction and strain preservation, screens IAA tissue endophyte strain with high flux, and provides better strain resource for the function of the plant tissue endophyte 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.
Strigolactone (SL) is a novel phytohormone synthesized at the root of a plant, regulates the growth and development relationship between the plant and endophytes, and is added with the strigolactone and other components so as to achieve the aim of culturing and screening the endophytes of the root system. In the patent, the activity of the endogenous microorganism is sufficiently released and maintained by adding an activity release buffer assisted by ultrasound. Then, the optimum dilution concentration for isolation culture was determined and high-throughput screening of endophyte microorganisms was achieved using 96-well plates in combination with the Salkowski method.
Drawings
FIG. 1 is a schematic diagram of a method for high-throughput isolation of plant tissue endophyte culture and screening of IAA-producing strains in an embodiment of the invention.
FIG. 2 shows the results of the IAA-producing color reaction of the culture screen of endophyte in high-throughput isolated plant tissue in the example of the present invention.
FIG. 3 is a comparison chart of the IAA-producing strains screened by the invention and CK on the vegetable growth promotion effect.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1
Referring to fig. 1, the high-throughput screening method for IAA-producing endophytes of plant tissues comprises the following steps:
1) obtaining plant tissue sample microorganism suspension
Collecting a fresh healthy plant tissue sample from a field, washing the collected tissue sample 10g with 100mL of phosphate buffer solution for three times, sequentially soaking with 75% ethanol for 2min, 100PPM sodium hypochlorite for 7min, 75% ethanol for 30s, putting into a 50mL sterile centrifuge tube, adding 30mL of sterilized active release buffer solution into the tube, wherein the active release buffer solution contains ammonium chloride: 0.5g, dipotassium hydrogen phosphate: 0.7g, potassium dihydrogen phosphate: 0.54g, MgSO 4 -7H 2 O：1.0g，CaCl 2 -2H 2 O：0.2g， FeSO 4 -7H 2 O：4.0mg，ZnSO 4 -7H 2 O：100ug，MnCl 2 -4H 2 O: 30ug of boronAcid: 300ug, CoCl 2 -6H 2 O：200ug，CuCl 2 -2H 2 O：10ug，NiCl 2 -6H 2 O: 20ug, KCl: 2.6mM, 4% of microspherical algal polysaccharide, the pH value of 7.2, adopting three frequencies of 15KHz, 60KHz and 100KHz to carry out ultrasonic treatment for 30s together to obtain a plant tissue endophyte sample suspension containing abundant microorganisms, then filtering the plant tissue endophyte sample suspension by adopting 4 layers of gauze to remove impurities such as root systems and the like, collecting the filtered plant tissue endophyte suspension, and placing the filtered plant tissue endophyte suspension in a 100mL sterile centrifuge tube for later use;
2) determining the optimal dilution concentration for isolation culture
Respectively putting the plant tissue endophyte suspension into 6 reagent bottles, adding a high-flux separation liquid culture medium, and respectively diluting into gradient root soil diluents which are 100 times, 300 times, 900 times, 2700 times, 9100 times and 27300 times; respectively transferring each gradient diluent into different cell culture plate holes under aseptic operation for culture, and taking a liquid culture medium without adding plant tissue endophyte suspension as a negative control;
and (2) filling the plant tissue endophyte diluent into 96-hole cell culture plates, shaking each bottle of the plant tissue endophyte diluent in a super clean bench, pouring 50mL of the plant tissue endophyte diluent into a 13cm sterile square dish, sucking the liquid by a discharge gun, transferring the liquid into each hole of the 96-hole cell culture plates, wherein each hole is 160 mu L, and each bottle of the diluent transfers 3 cell culture plates. Liquid is prevented from splashing outside the hole as much as possible during liquid transferring, otherwise, the liquid is easy to cause 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 the diluent of the endophyte in the plant tissue corresponding to the 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 the plant tissue endophyte diluent with the optimal dilution concentration into 1-100 holes of a cell culture plate under the aseptic operation, sealing the cell culture plate, placing the cell culture plate on a shaking table, and culturing for 3-10 days at 35 ℃ and 150 r/min; after the culture is finished, turbid bacterial liquid in the cell culture plate holes is sucked into the white ceramic plate, the same amount of Salkowski developing solution is added for developing reaction, and meanwhile IAA standard solution is used as a positive control; the standard solution containing 50 μ L IAA (50mg/L) was used as a positive control, and the 10% TSB solution (containing 100 mg/L-tryptophan) without adding the bacteria solution was used as a negative control; standing the white ceramic plate at room temperature in a dark condition for 20-40min, and observing, wherein the white ceramic plate can generate IAA when the color turns red; as a result, referring to FIG. 2, the strains corresponding to the red-shifted wells were stored in numbers, and the IAA production amount of each strain was further quantitatively measured, after which the obtained strains were stored.
The proportion of wells in a macroscopic turbid state in a 96-well cell culture plate is counted, and the statistical results are shown in table 1:
TABLE 1
As can be seen from Table 1, when a 20-35% of the pores of the 96-pore cell culture plate are in a turbid state, the colonies corresponding to the turbid state are single colonies, namely the colonies generated under the concentration of the diluent of the endophyte in the plant tissue corresponding to the 96-pore cell culture plate are single colonies, the IAA-producing strains can be screened under the concentration, and the screened colonies can be guaranteed to be the single colonies.
1.1 Effect of ultrasound conditions on the type and quantity of microorganisms in tissue sample suspensions:
under the condition that other conditions are not changed, the endophyte microorganisms of the plant root system are respectively extracted by ultrasonic waves with different frequencies for treatment, plant root system tissue sample suspension liquid containing abundant endophyte microorganisms is obtained, and the types and the quantity of the microorganisms in the plant root system tissue 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 suspension of the plant root tissue sample were the largest when the triple-frequency ultrasonic treatment was performed.
1.2 whether the IAA-producing crop plant tissue endophyte strain has a growth-promoting effect on plants
The IAA-producing crop plant tissue endophyte strains are subjected to laboratory numbering according to the IAA color reaction result, and the IAA producing capability of partial strains is shown in a table 3.
TABLE 3
In order to verify whether the plant tissue endophyte producing IAA has the growth promoting effect on plants, a pot experiment of lettuce is developed in the research, and the specific operation steps are as follows:
soaking lettuce seeds in warm soup for germination acceleration: soaking the seeds in warm water at 40 ℃, then dispersing the seeds in a culture dish filled with a wet filter paper sheet, accelerating germination at 26 ℃, and transplanting the seeds into a seedling culture substrate after exposing the seeds to white (vermiculite: substrate: 1.5: 1). Normally culturing under normal sunshine condition at 26 deg.C without applying any fertilizer, and transplanting to pots when lettuce seedling grows to four-leaf stage, 1 seedling per pot.
Experimental and control groups were set up separately, with 3 replicates per group.
The treatment group was treated with the IAA-producing plant tissue endophyte inoculant obtained by screening of the present invention, and the control group was treated with 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.
Endophyte strains with higher IAA production capacity are selected for vegetable crop growth-promoting pot culture experiments, and specific results are shown in a table 4. Wherein the content of the first and second substances,
as can be seen from table 4, the number of the strain having a significant growth promoting effect on green vegetables is 11, the strain is deposited by registration and preservation of Bacillus belgii TXM-1Bacillus velezensis, the strain is deposited in the common microorganism center of the china committee for culture collection of microorganisms, the preservation number is CGMCC No.19548, the preservation date is 2020, 04 and 02 days, the preservation institution address is: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North. The strain has obvious growth promoting effect on vegetables, and the yield is improved by more than 78.53%. The growth of green vegetables is shown in figure 3.
TABLE 4
Claims (4)
1. A high-throughput screening method for IAA endophytes produced by plant tissues comprises the following steps:
1) obtaining a plant tissue sample microorganism suspension
Cleaning collected crop tissue samples growing healthily, sterilizing and disinfecting the outside, carrying out ultrasonic treatment for 20-50s at room temperature by using an active release type buffer solution, filtering and collecting filtrate to obtain a plant tissue suspension, and storing at 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 20-40 KHz, 40-50 KHz and 80-100 KHz;
the active release buffer comprises ammonium chloride: 0.2-0.7g/L, dipotassium hydrogen phosphate: 0.4-1.0g/L, potassium dihydrogen phosphate: 0.3-0.6g/L, MgSO 4 -7H 2 O：0.6-1.2g/L，CaCl 2 -2H 2 O：0.1-0.5g/L，FeSO 4 -7H 2 O：2-8mg/L，ZnSO 4 -7H 2 O：50-150ug/L，MnCl 2 -4H 2 O: 15-45ug/L, boric acid: 100-500ug/L CoCl 2 -6H 2 O：100-400ug/L，CuCl 2 -2H 2 O：5-15ug/L，NiCl 2 -6H 2 O: 15-30ug/L, KCl: 2-3mM, 3-7 wt% of microcystis algae polysaccharide, pH 7.2-7.4;
2) determining the optimal dilution concentration for isolation culture
Adding the plant tissue suspension into a high-throughput separation liquid culture medium, respectively diluting the plant tissue dilution in a gradient manner, respectively transferring each gradient dilution into different cell culture plate holes under aseptic operation for culture, taking 5-15% of TSB solution without adding the plant tissue suspension as negative control, repeating more than three times for each gradient, sealing a 96-hole cell culture plate, placing the 96-hole cell culture plate on a shaking table, and culturing for 2-10 days at the temperature of 28-37 ℃ at 80-180 r/min;
20-35% of the wells in the 96-well cell culture plate are in a turbid state visible to naked eyes, and the concentration of the plant tissue diluent corresponding to the 96-well cell culture plate is used as the optimal dilution concentration for separation culture;
the high-throughput separation liquid culture medium comprises 50-150 mg/L of L-tryptophan, 10-30 g/L of casein polypeptide, 10-30 g/L of soybean protein polypeptide, 2-7g/L of sodium chloride and dipotassium phosphate: 0.5-1.0g/L, potassium dihydrogen phosphate: 0.3-0.7g/L, MgSO 4 -7H 2 O：0.6-1.3g/L，CaCl 2 -2H 2 O：0.1-0.3g/L，FeSO 4 -7H 2 O：2.0-5.0mg/L，ZnSO 4 -7H 2 O：50-150ug/L，MnCl 2 -4H 2 O: 20-50ug/L, boric acid: 200-400ug/L of CoCl 2 -6H 2 O：100-300ug/L，CuCl 2 -2H 2 O：5-15ug/L，NiCl 2 -6H 2 O：10-30ug/L，Na 2 MoO 4 -2H 2 O: 40-60mg/L, methanol: 2-7ml/L, 1.0-3.5mM potassium myristate, 2-5 wt% strigolactone, 0.5-1 wt% methyl jasmonate and pH of 6.0-8.0;
3) screening a single strain for producing IAA;
transferring the plant tissue diluent with the optimal dilution concentration into the holes of the cell culture plate under the 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, turbid bacterial liquid in the holes of the cell culture plate is sucked into the white ceramic plate, and an alkwski developing solution is added for developing reaction, and meanwhile, an IAA standard solution is used as a positive control; a high-flux separation liquid culture medium solution without tissue sample homogenate is used as a negative control;
placing the white ceramic plate at room temperature in a dark condition for 20-40min, and observing, wherein the white ceramic plate is red in color and shows that the strain can generate IAA; strains in IAA-producing cell culture plates were laboratory-numbered and glycerol-deposited as a result of the IAA color reaction.
2. The high-throughput screening method for the IAA-producing endophytes of the plant tissues according to claim 1, wherein in the step 1), the ultrasonic treatment time is 20-50 s.
3. The method for high-throughput screening of IAA-producing endophytes from plant tissues according to claim 1, wherein in step 3), the concentration of IAA in the IAA standard solution is 10 to 100 mg/L.
4. The high-throughput screening method for IAA-producing endophytes of plant tissues as claimed in any one of claims 1 to 3, wherein the plant variety is selected from the group consisting of green vegetables, tomatoes, lettuce and spinach.
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