CN114480222B - Paenibacillus kriging spaceflight mutant and application thereof - Google Patents

Abstract

The invention discloses a Paenibacillus clarkii space mutant and application thereof, and provides a Paenibacillus clarkii space mutant strain DeltaPS 04-17 which is preserved in the microorganism strain preservation center of Guangdong province in 3 months of 2022, wherein the preservation number is as follows: GDMCC NO. 62233. The research of the invention shows that the mutant strain DeltaPS 04-17 has larger forward variation, and the antibacterial effect of the mutant strain DeltaPS 04-17 is obviously better than that of a wild PS04 strain, and has wider antibacterial property; the growth vigor of the strain is also stronger than that of a wild PS04 strain, and the strain has better saline-alkali tolerance than that of the wild strain, can relieve the saline-alkali stress of tomatoes, promote tomatoes to grow under the saline-alkali stress, and provide important strain resources for the microbial remediation of soil salinization.

Description

Paenibacillus kriging spaceflight mutant and application thereof

Technical Field

The invention belongs to the technical field of microorganisms. More particularly, relates to a Paenibacillus clarkii aerospace mutant and application thereof.

Background

The soil salinization problem is a worldwide difficult problem, is particularly seriously distributed in arid and semiarid regions of China, and has the characteristics of wide saline-alkali soil distribution range, high salinization degree, complex and diversified types of saline-alkali soil and the like. Plants grown on saline-alkali soil are difficult to absorb water from the soil, thereby causing difficulty in plant root growth. In agricultural production, the yield of grains per unit area of cultivated land is low, and even the grains cannot be harvested. The development of saline-alkali soil is very necessary for the ecological environment and the safety of grain crops under the pressure of increasingly serious environmental pollution and shortage of important resources such as fresh water resources.

The existing soil salinization improvement method mainly comprises three methods of physical improvement, chemical improvement and biological improvement. Compared with the traditional soil improvement technology, the biological improvement technology has the advantages of high efficiency, cleanness and safety, and has been widely popularized and applied in recent years. The technological measures of biological improvement include breeding, planting and microbial utilization of salt tolerant plant variety. After researchers apply the microbial inoculum to saline-alkali soil, the yield and starch accumulation of cassava can be obviously improved after the cassava is planted. The influence of the microbial fertilizer on the physiological characteristics of the oat in the saline-alkali soil and the available nutrients in the soil is also found, and the growth of the oat is good and the available nutrients in the soil are obviously improved after the microbial fertilizer is applied. Therefore, the microbial soil restoration is used, the ecological balance of the saline-alkali soil can be maintained, and the microbial soil restoration method has good application prospect and economic value.

With the development of the current scientific technology, in recent years, the development of the aerospace technology is rapid, a return satellite or a spacecraft is utilized to carry biological materials into a space, and the special environment of space is utilized: is in a microgravity state (10) ^-3 -10 ^-6 g) Under the environmental conditions of strong radiation, ultra-vacuum, ultra-clean and the like, the biological material is subjected to mutagenesis under the action of factors, and then returns to the ground, so that the biological breeding technology for breeding new materials and cultivating new varieties is called space mutation. Space mutation can lead to favorable mutation of strains, but due to randomness of mutation, strains with excellent properties can be obtained by screening under certain conditions.

In the prior art, it has been reported that the microorganism strain utilizes space mutation technology, carries China Shenzhou No. eight airship to screen mutant strains with excellent properties, such as Pochonia chlamydosporia (Pochonia chamydosporia), paecilomyces lilacinus (Paecilomyces lilacinus), saccharomyces cerevisiae (Saccharomyces cerevisiae), beauveria bassiana (Beauveria bassiana) and the like, has high mutation rate of space mutation and rich mutation types, provides a new way for breeding biological control excellent strains, and provides more opportunities for breeding excellent strains. However, recently, there are reports about the breeding and research of excellent strains of Paenibacillus kriging (Paenibacillus kribbensis), and in order to provide more excellent strain resources, it is necessary to adopt more advanced aerospace technology to breed more excellent strains.

Disclosure of Invention

The invention aims to overcome the defects and the shortcomings of the existing problems and provide a Paenibacillus kriging spaceflight mutant and application thereof.

The first object of the invention is to provide a Paenibacillus clarkii space mutagenesis mutant DeltaPS 04-17.

A second object of the present invention is to provide the use of said DeltaPS 04-17 strain.

The third object of the invention is to provide a microbial soil restoration agent for improving soil salinization.

The fourth object of the invention is to provide a method for improving soil salinization.

The above object of the present invention is achieved by the following technical scheme:

the invention divides the wild strain PS04 of the Paenibacillus clarkii (Paenibacillus kribbensis) into two parts, and one part is reserved on the ground for conventional preservation and used as a control sample; the other part is carried and lifted off at the Wenchang launching base in the south China in the 3 month of 2020, and the mutant library of the wild type strain PS04 of the Paenibacillus clarkii is obtained after a series of space condition mutagenesis for 5 months. The wild strain PS04 of the Paenibacillus clarkii is used as a control, and the variability of the space mutation strain in the aspects of bacteriostasis effect, phenotype, growth speed, saline-alkali tolerance and the like is compared, so that the mutant strain with obviously improved bacteriostasis effect and saline-alkali tolerance is obtained through screening.

Firstly, screening mutant strains with a better antibacterial effect than wild type through the opposition of flat plates, respectively inoculating the screened mutant strains to the flat plates containing 10% of NaCl for screening, finally screening to obtain the strain delta PS04-17 with the strongest growth capacity, and carrying out subsequent research by using the strain delta PS04-17.

According to the invention, the phenotype of the Paenibacillus clarkii space mutation strain and the phenotype of the original strain under different conditions are compared, a large amount of polysaccharide is covered on the surface of a PS04 colony on an LB plate compared with DeltaPS 04-17, and the colony viscosity is high. PS04 bacterial liquid is observed to be white in the liquid culture medium, and the bacterial liquid has high viscosity; delta PS04-17 bacterial liquid is yellow, and the bacterial liquid viscosity is weaker than PS 04. Comparing PS04 with DeltaPS 04-17 by electron microscope, it can be seen that DeltaPS 04-17 cell (1.04 μm-3.33 μm 0.29 μm-0.64 μm) is shorter than PS04 cell (2.59 μm-6.72 μm 0.34 μm-0.50 μm).

The invention compares the growth capacities of space mutation mutant strain and original strain, respectively coats the space mutation mutant strain and the original strain in a gradient plate containing saline and alkaline, PS04 can not grow at all under the condition of 75mmol/L mixed saline and alkaline, and DeltaPS 04-17 strain can still grow normally, so that the colony number is reduced only. The two strains are respectively fermented in a liquid culture medium containing high-concentration mixed saline and alkaline, and the result shows that the delta PS04-17 strain can grow under the conditions of 125mmol/L and 150mmol/L, and the PS04 can not grow at all, which proves that the saline and alkaline tolerance of the mutant strain delta PS04-17 is obviously improved. Shows that the salt and alkali tolerance of the mutant delta PS04-17 is obviously higher than that of the wild type.

According to the invention, a tomato potting experiment is further carried out, and the mutant strain delta PS04-17 has a remarkable growth promoting effect on plants, and can remarkably relieve the inhibition effect of saline alkali on plant growth.

After space mutation of the Paenibacillus kriging (Paenibacillus kribbensis) in general, the mutant strain DeltaPS 04-17 obtained by screening has obvious forward mutation on the antibacterial effect, the growth capacity and the saline-alkali tolerance. Is a strain with excellent mutation property and has wide application prospect in improving soil salinization. The mutant strain DeltaPS 04-17 of Paenibacillus clarkii (Paenibacillus kribbensis) was deposited at 3 months 17 of 2022 with the Guangdong province microbiological culture Collection center (GDMCC) under the accession number: GDMCC NO 62233, accession number: the collection address of the microorganism strain collection in Guangdong province: building 5 is a No. 59 building of 100 universities in Mitrex, guangdong province.

Therefore, the invention provides application of the space mutation mutant strain DeltaPS 04-17 in soil remediation or preparation of a microbial soil remediation agent.

Preferably, the soil is restored to improve soil salinization.

The invention provides a microbial soil restoration agent for improving soil salinization, which contains the space mutation mutant strain DeltaPS 04-17 strain and/or bacterial liquid.

Preferably, the bacterial liquid is fermentation liquid.

Preferably, the fermentation conditions of the fermentation broth are: the DeltaPS 04-17 seed solution is inoculated into a fermentation culture medium with an inoculum size of 1-10 percent, and is fermented for 3-4 days at 25-30 ℃ and 150-200 rpm.

The invention provides a method for improving soil salinization, which adopts the mutant strain DeltaPS 04-17 or the microbial soil restoration agent to dilute and then uses the diluent to treat soil.

The invention provides application of the space mutation mutant strain DeltaPS 04-17 in plant growth promotion or plant growth promoter preparation.

Preferably, the plant growth promotion is a tomato plant growth promotion.

The invention provides application of a space mutation mutant DeltaPS 04-17 in promoting plant growth under saline-alkali stress or in preparing a plant saline-alkali resistant growth promoter.

The invention also provides application of the space mutation mutant strain DeltaPS 04-17 in pathogen inhibition or pathogen bactericide preparation.

Preferably, the pathogenic bacteria are one or more of soybean anthracnose (Colletotrichum truncatum), white gourd fusarium (Fusarium oxysporum f.sp.cuarmorium), banana fusarium (Fusarium oxysporum f.sp.cube), cabbage black spot (Alternaria oleracea) and rice sheath blight (Rhizoctonia solani AG 1-IA).

Those skilled in the art can prepare DeltaPS 04-17 fermentation broth according to various methods disclosed in the literature; for example, a two-stage culture method is employed, namely: seed culture and liquid fermentation culture to obtain fermentation liquor. Wherein, during liquid fermentation culture, fermentation culture can be performed by adopting a fermentation tank fermentation or a shake flask culture mode. Wherein, the components of the liquid fermentation culture medium comprise a carbon source and a nitrogen source; the carbon source is sucrose or soluble starch. The nitrogen source includes, but is not limited to, any one or more of sodium nitrate, ammonium chloride or peptone.

The invention has the following beneficial effects:

the invention carries out space mutation on the Paenibacillus kriging PS04 strain, the mutant strain DeltaPS 04-17 obtained by screening is a strain with excellent mutation property, the antibacterial effect of the mutant strain DeltaPS 04-17 is obviously superior to that of a wild PS04 strain, and the mutant strain DeltaPS 04-17 has wider antibacterial property; the growth vigor of the strain is also stronger than that of a wild PS04 strain, and the strain also has better saline-alkali tolerance, can relieve the saline-alkali stress of tomatoes, and promotes tomatoes to grow under the saline-alkali stress. The DeltaPS 04-17 strain can be used for restoring soil, treating saline-alkali soil so as to eliminate stress on plants, and has important application significance for restoring and utilizing the saline-alkali soil.

Drawings

FIG. 1 is a graph of the effect of rescreening, A being wild-type and B being mutant;

FIG. 2 shows the antibacterial radii of 30 space mutation mutants;

FIG. 3 shows the growth pattern of the strain on LB plates (the growth pattern of PS04 on LB plates on the left and the growth pattern of DeltaPS 04-17 on LB plates on the right);

FIG. 4 shows the growth state of the strain in the liquid medium of Czochralski (the growth state of PS04 on the left side in the liquid medium of Czochralski, and the growth state of DeltaPS 04-17 on the right side in the liquid medium of Czochralski);

FIG. 5 shows the morphology of the strain under electron microscopy (PS 04 on the left side and DeltaPS 04-17 on the right side);

FIG. 6 shows the comparison of the bacteriostatic effect of PS04 and DeltaPS 04-17 on part of pathogenic bacteria (A: soybean anthracnose B: white gourd fusarium C: banana fusarium D: cabbage black spot);

FIG. 7 shows the radius of the circle of inhibition of the DeltaPS 04-17 strain against soybean anthracnose, white gourd fusarium, cabbage black spot, banana fusarium and rice sheath blight;

FIG. 8 shows the growth of DeltaPS 04-17 strain on saline-alkali culture medium with certain concentration gradient (DeltaPS 04-17: PS04; left-to-right concentration of 25mmol/L, 50mmol/L, 75mmol/L, 100mmol/L, respectively);

FIG. 9 is a growth curve of DeltaPS 04-17 strain in liquid medium;

FIG. 10 is a graph showing the growth of DeltaPS 04-17 strain in medium-high concentration saline-alkaline liquid medium;

FIG. 11 shows the growth of DeltaPS 04-17 strain in liquid medium;

FIG. 12 is a graph showing the effect of growth of a salt-tolerant pot of tomato (a first pot from left to right is a control group, only clear water is applied; a second pot is a salt-tolerant treatment group, 75mmol/L of saline-alkali solution is applied; a third pot is a mutant strain group, deltaPS 04-17 bacterial liquid is added and clear water is applied; a fourth pot is a salt-tolerant + mutant strain group, deltaPS 04-17 bacterial liquid is added and 75mmol/L of saline-alkali solution is applied);

FIG. 13 shows experimental results of the height of a saline-alkali tolerant pot plant of tomato (A: control group B: saline-alkali treated group C: mutant strain group D: saline-alkali + mutant strain group).

Detailed Description

The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

LB solid medium: the ingredients of the composition are 10g/L of tryptone, 5g/L of yeast extract, 5g/L of sodium chloride and 17g/L of agar powder.

Improved liquid culture medium: the components of the fertilizer are 20.0g/L of sodium nitrate, 0.7g/L of monopotassium phosphate, 0.3g/L of dipotassium phosphate, 0.5g/L of potassium chloride, 0.5g/L of magnesium sulfate, 0.01g/L of ferrous sulfate, 30.0g/L of sucrose and pH value of 7.0-7.2.

The bacteria above soybean anthracnose (Colletotrichum truncatum), white gourd fusarium (Fusarium oxysporum f.sp.cuarmeriamm), banana fusarium (Fusarium oxysporum f.sp.cube), cabbage black spot (Ahernaria oleracea) and rice sheath blight (Rhizoctonia solani AG 1-IA) are provided by Tropical subtropical fungus research laboratory of agricultural university of south China.

EXAMPLE 1 screening of space mutation mutants

1. Test method

(1) Test strain:

paenibacillus kriging (Paenibacillus kribbensis) PS04, accession number: CGMCC No.7996, preservation time: and the microorganism is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms (China) on the 8 th and 13 th days of 2013. After the test strain Paenibacillus kriging PS04 is subjected to space carrying mutagenesis and returned to the ground, the mutant library is preserved at the temperature of minus 80 ℃ for standby.

(2) Space flight carrying conditions:

taking Paenibacillus kriging PS04 as a starting strain, carrying out lift-off on a Wenchang launching base in the south China in 3 months of 2020, and carrying out on-orbit flight for 67 hours under normal pressure; a large elliptical orbit, with a height ranging from 324 to 7970Km, a near location of 324Km, a far location of 7970Km, traversing a Fan Ailun radiation band; inclination angle is 42.5; the mutant library of the Paenibacillus kriging PS04 is obtained in 5 months by mutagenesis under the conditions of 20+/-5 ℃ of the position of a carried object in the rail cabin.

(3) Screening of mutants:

3.1 preliminary screening of space mutation mutants

100. Mu.L of mutant library bacteria liquid is coated on an LB plate and cultured for 3d at 28 ℃ until single colony is grown. Single colonies were inoculated onto new LB plates with sterilized toothpicks and mutant strain numbers were recorded. The rhizoctonia solani (Rhizoctonia solani AG 1-IA) is used as indicator bacteria, and mutant strains with better antibacterial effect than the wild strains are screened one by a counter culture method. Rhizoctonia solani AG1-IA fungus blocks (8 mm diameter) are inoculated at the center of a PDA flat plate, 4 bacterial colonies, namely a wild type mutant and three mutants, are respectively inoculated at positions which are 2.5cm away from the bacterial colonies in the vertical direction around the bacterial colonies by using a sterilizing toothpick. Only Rhizoctonia solani AG1-IA was inoculated as a control. The plate was inverted and incubated in the dark at 28℃for 2 days, and the mutant strain with a zone of inhibition greater than that of the wild type was selected as the candidate mutant strain for the next experiment.

The mutant strains obtained by primary screening are respectively inoculated in LB liquid culture medium, and are cultured for 48 hours at the temperature of 28 ℃ and under the environment of 200r/min to obtain fermentation liquor, and 50 percent of glycerol is added to preserve strains at low temperature.

3.2 rescreening of space mutation mutants

The 30 mutant strains obtained by the preliminary screening were inoculated into new LB plates, respectively, and cultured at 28℃for 3 days, followed by re-screening by the counter experiment. Rhizoctonia solani AG1-IA fungus blocks (8 mm diameter) are inoculated at the center of a PDA flat plate, and 4 mutant colonies are respectively inoculated at the positions which are 2.5cm away from the colonies in the vertical direction around the colonies by using a sterilizing toothpick. The inoculated wild-type colonies were used as controls. The plates were incubated upside down and the radius of the zone of inhibition was measured after incubation in the dark at 28℃for 2 days. Screening forward mutant strains with antibacterial rate, and shaking and preserving each strain.

(4) Evaluation of saline-alkali tolerance of space mutation mutant:

the wild type strain PS04 of Paenibacillus clarkii is used as a control, 30 mutant strains obtained by wild type and preliminary screening are inoculated on a flat plate containing salt (10% NaCl) respectively by using a sterilizing toothpick according to the serial number sequence, and are cultured for 48 hours under the environment of 28 ℃, and the growth conditions of the wild type and mutant bacterial colonies are compared.

2. Test results

(1) Re-screening results for high antibacterial rate mutant strains:

the result of the antagonism of the mutant strain to the rhizoctonia solani AG1-IA is shown in the figure 1, the antibacterial effect of the mutant strain obtained by primary screening is stable, the radius of a wild type antibacterial circle is 0.8 cm, the radius of the antibacterial circle of the mutant is 1.1 cm, and the antibacterial effect of the mutant is obviously better than that of the wild type. The results of the bacteriostatic radii of the different mutants are shown in figure 2, wherein the radius of the bacteriostatic circle of 26 mutants is obviously increased (p < 0.05) compared with that of the wild type, and 11 strains with the bacteriostatic circle radius larger than 1.2cm are numbered as mutants 2, 4, 5, 6, 7, 14, 17, 21, 25, 28 and 30 respectively.

(2) Growth of mutants on drug-containing plates:

the Paenibacillus clarkii wild-type strain PS04 was unable to grow on plates containing 10% NaCl (pH 9.0). The mutants grew on plates of 10% NaCl (pH 9.0) as follows: mutants 12, 13, 17, 27 showed the strongest growth and the rest were relatively weak. The growth of a part of space mutation strains on a salt-containing plate is shown in table 1, and the research shows that mutant strain No. 17 (delta PS04-17 strain) has better salt tolerance and is selected as a subsequent study object.

TABLE 1 growth of partial space mutation strains on drug-containing plates

Note that: "-" means no growth; "+" indicates growth ability

Experimental example 2 identification of mutant Strain DeltaPS 04-17

1. Test method

The Paenibacillus clarkii DeltaPS 04-17 screened in example 1 was placed in a modified liquid medium, cultured in a shaker at 28℃for 48 hours, and after DNA was extracted using OMEGA Bacterial DNA Kit and PCR amplification was performed with the primers 1492R (5'-TACCTTGTTACGACTT-3') and 27F (5'-AGAGTTTGATCCTGGCTCAG-3'). The PCR reaction system is as follows: 25. Mu.L of PCR premix, 1. Mu.L of primer each, 1. Mu.L of DNA, and 22. Mu.L of sterile water. The PCR reaction procedure was: 94 ℃ for 5min;94℃45s,65℃45s,72℃90s (35 cycles); 72℃for 10min. And after the reaction is finished, the PCR product is placed at 4 ℃ for electrophoresis detection.

The PCR products were electrophoresed on a 1% mass fraction agarose gel, and the electrophoresis bands were observed in a gel electrophoresis imager and recorded by photographing. The PCR product was sequenced by the Biochemical company to obtain the ITS sequence of the DeltaPS 04-17 strain. The obtained sequences were Blast and submitted to the nucleic acid database at NCBI (www.ncbi.nlm.nih.gov).

2. Test results

Sequencing results show that the mutant strain DeltaPS 04-17 is still Paenibacillus krigineus (Paenibacillus kribbensis). This sequence has been submitted for NCBI GenBank accession number DeltaPS 04-17: SUB10959124 sequence OM278265.1. The mutant strain DeltaPS 04-17 of Paenibacillus clarkii (Paenibacillus kribbensis) was deposited at 3 months 17 of 2022 with the Guangdong province microbiological culture Collection center (GDMCC) under the accession number: GDMCC NO 62233, accession number: the collection address of the microorganism strain collection in Guangdong province: building 5 is a No. 59 building of 100 universities in Mitrex, guangdong province.

Experimental example 3 phenotypic analysis of ΔPS04-17 Strain

1. Comparison of Strain morphology

Growth morphology on LB plates: 100. Mu.L of PS04 and DeltaPS 04-17 bacterial solutions are respectively taken and coated on LB plates, and after the coating is completed, the plates are respectively placed in a 28 ℃ incubator for 48 hours for cultivation and then observed.

Growth morphology in liquid medium of young: 100ml of the liquid culture medium was scraped with an inoculating loop on a plate containing PS04 and DeltaPS 04-17, and cultured in a shaking table at 28 ℃.

2. Shape of bacteria under electron microscope

And (3) collecting a sample by single cells, respectively taking bacterial liquid in the Nahnsonian liquid culture medium, and centrifugally collecting soybean-sized bacterial cells. Cells were collected after gentle and brief centrifugation of the culture using a 1.5mL centrifuge tube, fresh buffer was added, centrifugation was performed at 5000rpm/min for 5min at low temperature, and the supernatant was discarded. Dripping fresh phosphate buffer solution, re-suspending for 15min, centrifuging at 5000rpm/min at low temperature for 5min, discarding supernatant, and repeating for 2 times. The sample was resuspended after addition of glutaraldehyde fixative and was subjected to electron microscopy after overnight fixation at 4 ℃. And observing the bacterial forms of the wild type and the mutant under an electron microscope.

3. Experimental results

As shown in FIG. 3, the morphology of the Paenibacillus krigineus PS04 strain and the mutant strain DeltaPS 04-17 on the LB plate is compared, and the colony surface of PS04 is covered with a large amount of polysaccharide and has higher colony viscosity compared with DeltaPS 04-17. Under the growth form in the liquid culture medium of the Nahniki, as shown in fig. 4, the PS04 bacterial liquid is observed to be white, and the bacterial liquid has larger viscosity; delta PS04-17 bacterial liquid is yellow, and the bacterial liquid viscosity is smaller than PS 04. As shown in FIG. 5, the morphology of the cells under the electron microscope was observed by the electron microscope to show that the morphology of PS04 and DeltaPS 04-17 cells (1.04 μm-3.33 μm, 0.29 μm-0.64 μm) were shorter than that of PS04 cells (2.59 μm-6.72 μm, 0.34 μm-0.50 μm).

Experimental example 4 broad-spectrum antibacterial Effect of DeltaPS 04-17

1. Experimental method

Pathogenic bacteria are prepared by the following steps: soybean anthracnose (Colletotrichum truncatum), white gourd fusarium (Fusarium oxysporum f.sp.curcurcurmer), banana fusarium (Fusarium oxysporum f.sp.cube), cabbage black spot (Alternaria oleracea), rice sheath blight (Rhizoctonia solani AG 1-IA) and other bacterial blocks (8 mm diameter) are inoculated at the center of a PDA plate, 5 mu L of bacterial liquid are respectively inoculated at positions which are 2cm away from the bacterial colony in the vertical direction on two sides of the bacterial colony by a liquid-transferring gun, and a wild type bacterial liquid and a mutant are respectively inoculated. The radius of the zone of inhibition was measured after incubation in dark at 28℃for 3d with the control inoculated with pathogen alone, with the plate inverted.

2. Experimental results

The antibacterial effect of PS04 and DeltaPS 04-17 strains on pathogenic bacteria is shown in figure 6, and the result shows that the antibacterial effect of DeltaPS 04-17 on soybean anthracnose, banana fusarium and rice sheath blight is obviously better than that of DeltaPS 04, the radius of the antibacterial circle of different pathogenic bacteria is shown in figure 7, and DeltaPS 04-17 also has better antibacterial effect on white gourd fusarium and cabbage black spot.

Experimental example 6. DELTA.PS 04-17 determination of saline-alkali tolerance

1. Experimental method

The preparation method of the saline-alkali Charles culture medium comprises the following steps: 3.5g of liquid Soxhlet medium powder was added, and 50mL of deionized water was added to the mixture to sterilize the mixture in a sterilizer. NaCl and Na are added according to the molar ratio of 1:9:9:1 ₂ SO ₄ 、NaHCO ₃ And Na (Na) ₂ CO ₃ Mixing and adding 50mL deionized water to dissolve to prepare a saline-alkali solution. The saline-alkali solution is filtered to a sterile 50mL of Nahnia medium by a 0.45 μm bacterial filter, and the volume is fixed to 100mL.

Concentration screening of strain salt and alkali resistance: the wild type and mutant strains were plated for activation and single colony shaking was picked 2d. 1mL of the bacterial liquid is taken and added into 100mL of modified Boehringer's medium, and the bacterial liquid is obtained after shaking for 3 d. The absorbance of the bacterial liquid was measured, and the modified schiff medium was added to dilute to od=1.0. Saline-alkali with different concentrations is added into each 5ml modified Bosch's plate according to a certain gradient, 50 mu L of wild type strain bacterial liquid and mutant strain bacterial liquid (prepared according to the method) are respectively coated on the modified Bosch's plate (25, 50, 75, 100, 125, 150 and 200 mmol/L7 concentrations are set) for culture at 28 ℃, and the growth condition is observed.

Drawing growth curves of strains under the saline-alkali concentration of 125mmol/L and 150 mmol/L: 1mL of mutant fermentation broth is taken and added with saline and alkaline solution containing 125mmol/L and 150mmol/L (NaCl and Na are added according to the molar ratio of 1:9:9:1) ₂ SO ₄ 、NaHCO ₃ And Na (Na) ₂ CO ₃ Mix) modified Bosch culture medium 100mL, wild type was treated in the same manner. The OD value of 600nm was measured by a spectrophotometer at intervals of 48h at 30℃and 180r/min using a saline-alkali free medium as a blank. Growth was plotted against the OD values measured.

2. Experimental results

As shown in figure 8, the growth conditions of the strain on a saline-alkali gradient plate are good in PS04-17 bacterial liquid in saline-alkali Boehmeria culture medium with different concentrations of 25mmol/L, 50mmol/L, 75mmol/L and 100mmol/L, and the growth conditions of PS04 and DeltaPS 04-17 are good in 25 mmol/L; at 50mmol/L, PS04 can only grow sporadic colonies, and delta PS04-17 colonies are more; at 75mmol/L, PS04 can not grow at all, and the number of DeltaPS 04-17 colonies is reduced, but still can grow; at 100mmol/L, PS04 and DeltaPS 04-17 bacteria were unable to grow.

As shown in FIG. 9, the OD value of the mutant strain DeltaPS 04-17 is found to grow faster than that of the wild strain, which indicates that the DeltaPS 04-17 has stronger growth activity than that of the wild strain. The growth curve of the strain at the medium and high saline-alkali concentration is shown in fig. 10, and the OD value of the wild strain at the high concentration is a flat line, which indicates that the PS04 strain cannot grow well at the medium and high saline-alkali concentration. The delta PS04-17 strain grows exponentially at 125mmol/L and 150mmol/L, and the growth trend under the condition of 150mmol/L is better than that of 125 mmol/L. The growth state of the strain in the saline-alkali liquid culture medium for 8 days is shown in figure 11, wherein the bacterial liquid of PS04 is transparent and has small viscosity; and DeltaPS 04-17 (bacterial liquid is relatively yellow and turbid, and has relatively high viscosity), and the saline-alkali tolerance of the mutant DeltaPS 04-17 is obviously higher than that of a wild strain.

Experimental example 7 the ability of DeltaPS 04-17 strain to restore saline-alkali stress to soil was evaluated by potting experiments

1. Experimental method

The saline-alkali tolerance of the fermentation broth of the mutant strain DeltaPS 04-17 to tomatoes is evaluated by using a potting experiment. According to the early-stage experiment result, determining 75mmol/L as the concentration of the salt damage stress of the tomatoes, grouping and transplanting four young tomato plants with equal heights, recording the original height, and performing grouping experiments. The experiment is divided into: control group A (wet sterile water only), treatment group B1 (wet 300mL 75mmol/L saline solution only), treatment group C2 (wet 300mL 100-fold diluted bacteria solution only), treatment group D3 (wet 300mL 75mmol/L saline solution and 300mL 100-fold diluted bacteria solution). Each group of plants was watered with an appropriate amount of water every 7 days, and after one month of cultivation, the growth of the plants was observed and the data was recorded.

2. Experimental results

The experimental result of the salt and alkali of the potted tomato is shown in figure 12, and the growth vigor of the tomato plant treated by the delta PS04-17 bacterial liquid is obviously better than that of the plant treated by the salt and alkali; the plant height change of the plants after the saline-alkali treatment of the potted tomatoes is shown in fig. 13, and the result shows that the tomatoes in the control group A grow normally, the plant height is increased from 40cm to 52cm, the plant height of the tomatoes in the group B is increased from 40cm to 45cm after the 75mmol/L saline-alkali treatment, and the growth of the tomatoes in the saline-alkali treatment is inhibited; after the group C is treated by singly using DeltaPS 04-17 bacterial liquid, the tomato plant height is increased from 40cm to 83cm, and the tomato growth is obviously promoted after the treatment; and after the group D is subjected to 75mmol/L saline-alkali stress and treated by adopting DeltaPS 04-17 bacterial liquid, the plant height of tomatoes is increased from 40cm to 78cm, and the growth of tomatoes is obviously superior to that of the group B and the group A which only grow normally under the saline-alkali stress, which shows that the application of DeltaPS 04-17 bacterial liquid can relieve the inhibition effect of saline-alkali on plant growth and promote plant growth, and the application of DeltaPS 04-17 bacterial liquid alone can also promote tomato plant growth.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (9)

1. Paenibacillus kriging @Paenibacillus kribbensis) The space mutation mutant strain DeltaPS 04-17 is characterized in that the strain is deposited in the microorganism strain collection of Guangdong province at 3-17 of 2022, and the deposit number is: GDMCC NO. 62233.

2. Use of the space mutation mutant strain Δps04-17 according to claim 1 for soil remediation or for the preparation of a microbial soil remediation agent, characterized in that the soil remediation is an improvement of soil salinization.

3. A microbial soil restoration agent for improving soil salinization, which is characterized by comprising the mutant strain DeltaPS 04-17 and/or bacterial liquid according to claim 1.

4. A microbial soil remediation agent according to claim 3 wherein the bacterial liquid is a fermentation broth.

5. The microbial soil conditioner according to claim 4, wherein the fermentation conditions of the fermentation broth are: inoculating the DeltaPS 04-17 bacterial liquid into a fermentation culture medium at an inoculum size of 1-10%, and fermenting for 3-4 days at 25-30 ℃ and at 150-200 rpm.

6. A method for improving soil salinization, characterized in that the mutant Δps04-17 according to claim 1 or the microbial soil restoration agent according to any one of claims 3 to 5 is used for dilution and then the soil is treated with the dilution liquid.

7. Use of the space mutation mutant Δps04-17 according to claim 1 for promoting plant growth or for the preparation of a plant growth promoter, characterized in that the plant is tomato.

8. The use of the space mutation mutant Δps04-17 according to claim 1 for promoting plant growth under salt and alkali stress or for preparing a plant salt and alkali tolerant growth promoter, characterized in that the plant is tomato.

9. The use of the space mutation mutant DeltaPS 04-17 as defined in claim 1 for inhibiting pathogenic bacteria or preparing pathogenic bacteria bactericide for the purpose of non-disease diagnosis and treatment, wherein the pathogenic bacteria isColletotrichum truncatum、Fusariumoxysporum f. sp. cucurmerimum、Fusarium oxysporum f.sp. cubense、Alternaria oleracea、Rhizoctonia solaniOne or more of AG 1-IA.

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