CN116790412B - Pseudomonas stutzeri XN05-1, application thereof and obtained plant salt-resistant microbial agent - Google Patents
Pseudomonas stutzeri XN05-1, application thereof and obtained plant salt-resistant microbial agent Download PDFInfo
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- 241000589614 Pseudomonas stutzeri Species 0.000 title claims abstract description 36
- 150000003839 salts Chemical class 0.000 title claims abstract description 15
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- 238000009629 microbiological culture Methods 0.000 claims abstract description 11
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- 238000004321 preservation Methods 0.000 claims abstract description 6
- 244000068988 Glycine max Species 0.000 claims description 20
- 235000010469 Glycine max Nutrition 0.000 claims description 19
- 241000196324 Embryophyta Species 0.000 claims description 18
- 239000004480 active ingredient Substances 0.000 claims description 2
- 244000046052 Phaseolus vulgaris Species 0.000 claims 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims 1
- 241000589516 Pseudomonas Species 0.000 claims 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 abstract description 8
- 230000012010 growth Effects 0.000 abstract description 8
- 239000011574 phosphorus Substances 0.000 abstract description 8
- 229910052698 phosphorus Inorganic materials 0.000 abstract description 8
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- 239000002363 auxin Substances 0.000 abstract description 4
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- 238000005259 measurement Methods 0.000 abstract description 3
- 239000002068 microbial inoculum Substances 0.000 abstract description 3
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Abstract
The invention provides pseudomonas stutzeri XN05-1, application thereof and an obtained plant salt-resistant microbial agent, and belongs to the technical field of microorganisms. The pseudomonas stutzeri Pseudomonas stutzeri XN-1 provided by the invention has a preservation number of CGMCC No.26683 and is preserved in China general microbiological culture collection center (China general microbiological culture collection center) for the year 2023, month 02 and day 27. According to the invention, the obtained pseudomonas stutzeri is subjected to salt tolerance and growth promotion capability measurement, and the pseudomonas stutzeri has the activity of dissolving organic phosphorus, has strong capability of producing auxin, can produce extracellular polysaccharide, has good salt tolerance, and can effectively solve the problems of low environmental adaptability and low salt tolerance of the existing microbial inoculum.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to pseudomonas stutzeri Pseudomonas stutzeri XN-1, application thereof and an obtained plant salt-resistant microbial agent.
Background
The high-salinity environment may cause osmotic stress, ionic stress and oxidative stress to plants, thereby inhibiting the growth and development of plants. The salinization of soil seriously damages the growth of crops, and although the existing microbial agents improve the salt tolerance of plants to a certain extent, the microbial agents have low environmental adaptability and low salt tolerance. More and more researches show that rhizosphere microorganisms can increase the salt resistance of plants and co-evolve with hosts thereof to resist external stress, while salt-tolerant plants can resist higher salt stress, and besides the physiological mechanism of the rhizosphere microorganisms, the rhizosphere microorganisms play an important role. Therefore, compared with common soil microorganisms, rhizosphere microorganisms of wild salt-tolerant plants can tolerate higher salinity, have stronger environmental adaptability and need to be developed and utilized.
Disclosure of Invention
The invention provides pseudomonas stutzeri Pseudomonas stutzeri XN-1, application thereof and an obtained plant salt-resistant microbial inoculum, and the obtained pseudomonas stutzeri is subjected to salt resistance and growth promotion capability measurement, so that the pseudomonas stutzeri has the activity of dissolving organic phosphorus, has stronger capability of generating auxin and can generate extracellular polysaccharide (0.26 mg/L), and meanwhile, has good salt resistance, and the problems of low environmental adaptability and low salinity resistance of the existing microbial inoculum can be effectively solved.
In order to achieve the aim, the invention provides pseudomonas stutzeri Pseudomonasstutzeri XN-1 which is characterized in that the preservation number is CGMCC No.26683 and is preserved in China general microbiological culture collection center (China general microbiological culture Collection center) at the year 2023 and the month 27.
Preferably, the Pseudomonas stutzeri Pseudomonas stutzeri XN-1 has a nucleotide sequence shown in SEQ ID NO. 1.
The invention also provides a PCR amplification method of pseudomonas stutzeri Pseudomonas stutzeri XN-1 according to the technical scheme, which comprises the following steps:
1) Extracting genomic DNA isolated from a bacterial strain in a soil suspension;
2) Taking genome DNA of a bacterial strain as a template, and carrying out PCR amplification by rTaq enzyme by using an upstream primer and a downstream primer;
wherein: the PCR reaction system is as follows: 2 XTaq Master Mix 12.5. Mu.L, forward and reverse primers (10. Mu.M) 1. Mu. L, DNA template 1. Mu. L, ddH each 2 O9.5. Mu.L, 25. Mu.L overall.
Preferably, the amplification conditions are: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1.5min, and circulation for 30 times; finally, the extension is carried out for 10min at 72 ℃.
Preferably, the primer pair is:
forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3';
reverse primer: 5'-GGTTACCTTGTTACGACTT-3'.
The invention also provides an application of the pseudomonas stutzeri Pseudomonas stutzeri XN-1 in improving the salt tolerance of wild soybeans.
Preferably, the Pseudomonas stutzeri XN05-1 is inoculated at a concentration of 10 per pot 7 At CFU/g, root length was increased by 114.01% and root weight was increased by 24.38% compared to unvaccinated wild soybeans.
The invention also provides a plant salt-resistant microbial agent, which takes pseudomonas stutzeri Pseudomonas stutzeri XN-1 as a main active ingredient.
Preferably, the plant is wild soybean.
Compared with the prior art, the invention has the advantages and positive effects that:
the invention provides a Pseudomonas stutzeri Pseudomonas stutzeri XN-1,by measuring the salt tolerance and the growth promoting ability of the obtained Pseudomonas stutzeri, it was found that it had an activity of dissolving organic phosphorus, had a strong ability to produce auxin (170.31. Mu.g/mL), was able to produce extracellular polysaccharide (0.26 mg/L), and it was proposed for the first time that the Pseudomonas stutzeri could be used to increase the salt tolerance of wild soybean, had a good salt tolerance, still grown at a NaCl concentration of 6%, with an optimum NaCl concentration of 3.0%, and an inoculation concentration of 10 per pot 7 At CFU/g, compared with the root length of unvaccinated wild soybean, the root length can be increased by 114.01%, and the root weight can be increased by 24.38%. The Pseudomonas stutzeri can enrich the variety of the existing microbial preparation, and effectively solves the problems of low environmental adaptability and low salinity resistance of the existing microbial preparation.
Drawings
FIG. 1 shows colony morphology of strain XN05-1 on NA medium according to the examples of the present invention;
FIG. 2 shows the salinity tolerance range of strain XN05-1 provided in the examples of the present invention;
FIG. 3 shows colonization of a wild soybean root system by a strain XN05-1 under a salt stress environment provided by the embodiment of the invention; the left side is a control, and the right side is an inoculated strain XN05-1;
FIG. 4 shows the growth of wild soybeans after inoculation of strain XN05-1 under salt stress provided by the embodiment of the invention; * Represents P <0.001; * Represents P <0.01.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 soil sample acquisition
The wild soybean plants after salt stress are pulled up continuously, loose-adhered soil at the roots is shaken off, a tightly adhered rhizosphere soil sample is put into a sterile self-sealing bag, and the sterile self-sealing bag is transported back to a laboratory at low temperature for strain separation culture.
EXAMPLE 2 screening of Pseudomonas stutzeri
2.1 reagent:
NA medium (g/L): peptone 10.0, beef extract 3.0, sodium chloride 5.0, agar powder 20.0.
NB medium (g/L): peptone 10.0, beef extract 3.0, sodium chloride 5.0.
Salinity test medium (g/L): 10.0 g of peptone, 3.0 g of beef extract and 0-60g of sodium chloride with different concentrations.
PKO inorganic phosphorus medium (g/L): glucose 10.0, (NH) 4 ) 2 SO 4 0.5，MgSO 4 ·7H 2 O0.1，NaCl 0.2，KCl 0.2，FeSO 4 ·7H 2 O 0.003，MnSO 4 ·4H 2 O 0.03，Ca 3 (PO 4 ) 2 5.0, yeast powder 0.5 and agar 20.0.
Organophosphorus bacteria culture medium: purchased from Qingdao sea Bo biotechnology Co.
Modified CAS agar medium: purchased from beijing cool pacing technologies limited.
Other biochemical reagents and common chemical reagents are imported or homemade analytically pure.
2.2 isolation culture of bacteria:
the rhizosphere soil samples were thoroughly mixed with 0.85% normal saline (1:9, w/v) in a sterile environment. Standing, sucking 100 μl of supernatant, adding into 900 μl of physiological saline, and gradient diluting to 10 -4 . Respectively draw 10 -2 -10 -4 A100. Mu.L concentration of the dilution was spread on Nutrient Agar (NA) medium. Culturing at 28deg.C for 3d, picking different single colonies according to colony color, morphology, surface smoothness, etc., scribing and purifying for 2-3 times, inoculating to NB liquid culture medium, shake culturing for 24h to logarithmic phase, mixing the bacterial liquid with 50% glycerol 1:1, and freezing and preserving at-80deg.C.
A dominant strain (colony count: 30%) was found, and a single colony was randomly picked and numbered XN05-1. After 48h of cultivation on NA medium, the colonies were characterized by pale yellow, moist, smooth and clean edges, and the colony diameters reached 2mm, as shown in FIG. 1.
2.3 screening and molecular characterization of Pseudomonas stutzeri:
bacterial 16S rRNA gene sequences were amplified using colony PCR techniques using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The specific amplification system adopted is as follows: 2 XTaq Master Mix 12.5. Mu.L, forward and reverse primers (10. Mu.M) 1. Mu. L, DNA template 1. Mu. L, ddH each 2 O9.5. Mu.L, 25. Mu.L overall; the amplification conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 95 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1.5min, and circulation for 30 times; finally, the extension is carried out for 10min at 72 ℃.
The PCR products were sent to Qingdao Rui Biotechnology Inc. for sequencing, and the resulting bacterial sequences were aligned and uploaded to the EzBioCloud database (https:// www.ezbiocloud.net /). As shown in FIG. 1, the 16S rRNA gene sequence of the strain XN05-1 has the highest similarity with Pseudomonas stutzeri Pseudomonas stutzeri and is 99.67%, so that the strain XN05-1 is primarily identified as Pseudomonas stutzeri and has the nucleotide sequence shown in SEQ ID NO. 1. The preservation number of the pseudomonas stutzeri Pseudomonas stutzeri XN-1 is CGMCC No.26683, and the pseudomonas stutzeri is preserved in China general microbiological culture collection center (China general microbiological culture collection center) at the month of 02 and 27 of 2023.
Example 3 salt tolerance and growth promoting index of Pseudomonas stutzeri XN05-1
3.1 salt tolerance of Pseudomonas stutzeri XN05-1
Preparing culture mediums with different NaCl concentrations (0-6%), autoclaving, packaging in 96-well culture plates, inoculating XN05-1 bacteria solution according to 1% inoculum size, and measuring OD value of the culture solution with an enzyme-labeled instrument after 24 hr.
3.2 growth promoting index of Pseudomonas stutzeri XN05-1
The phosphorus-solubilizing activity, extracellular polysaccharide-producing ability and siderophore-producing ability of Pseudomonas stutzeri XN05-1 were measured using different indicator media.
3.2.1 determination of phosphorus-solubilizing Activity: will 10 μL OD 600 Bacterial liquid of =0.6 was inoculated onto PKO inorganic phosphorus and organic phosphorus bacterial medium, cultured at 28℃for 3 days, and observed for the formation of a transparent ring around the colony, and the solubilization of the strain was evaluated by using the Solubilization Index (SI), and the results were countedThe calculation formula is as follows: dissolution index (SI) = (halo+colony diameter)/colony diameter.
3.2.2 measurement of extracellular polysaccharide production ability: strain XN05-1 was inoculated into NB medium, cultured overnight, and then allowed to stand for 48 hours (OD 600 =1.5). Then 200. Mu.L of the bacterial liquid was mixed with 200. Mu.L of phenol (5%) and 1mL of concentrated sulfuric acid, and the OD value was measured at 485 nm. Simultaneously, 200 mu L of glucose with different concentrations is taken as a standard curve, and the OD of the bacterial liquid is calculated 485 The values were taken into a standard curve and the extracellular polysaccharide production was calculated.
3.2.3 determination of siderophore production Capacity: the XN05-1 strain was inoculated into CAS agar medium, cultured at 30℃for 7d, and the color change around the bacterial colony was observed, and the color was changed from blue to orange or deep yellow, indicating that the strain could produce siderophores.
As a result, it was found that the strain XN05-1 was still able to grow at a NaCl concentration of 6%, with an optimum NaCl concentration of 3.0% (FIG. 2); has the activity of dissolving organic phosphorus and has strong ability of producing auxin
(170.31. Mu.g/mL), capable of producing extracellular polysaccharide (0.26 mg/L); does not have the ability to dissolve inorganic phosphorus and iron-producing carriers.
Example 4 colonization ability of Pseudomonas stutzeri XN05-1 on the rhizosphere of Glycine max
The wild soybean seeds were sterilized with 3% sodium hypochlorite solution, then germinated on wet filter paper for three days, then transferred to 96-well plates for short-term culture, transferred to test tubes until the plant height reached about 3cm, each tube was filled with 1/2 Hoagland culture medium, and NaCl was added at a final concentration of 200 mM. Pseudomonas stutzeri XN05-1 labeled with green fluorescent protein was inoculated into NB medium and shake-cultured at 28℃and 180r/min for 24 hours. 1ml OD is taken 600 Bacterial solution=0.5 was added to the culture tube, and 1ml of sterile water was added to the control group. After the culture is continued for 7 days, the colonization condition of the strain XN05-1 on the wild soybean root system is observed by using a laser confocal microscope.
As shown in FIG. 3, the surface of the wild soybean root system shows a large number of green light spots after the strain XN05-1 is added, but the control group does not show green light spots, which indicates that the strain XN05-1 can successfully colonize the wild soybean root system.
EXAMPLE 5 Pseudomonas stutzeri XN05-1 to improve salt tolerance in wild soybeans
Fresh Pseudomonas stutzeri XN05-1 was inoculated into NB medium and shake cultured at 28℃and 180r/min for 24h. Centrifuging at 4deg.C at 6000r/min for 5min, and re-suspending with sterile water to OD 600 =0.5. The wild soybean seeds were sterilized with 3% sodium hypochlorite solution and then germinated on wet filter paper for three days. 200g of field soil is filled in each small basin, 4 germinated wild soybean seeds are placed in each basin, and the seeds are placed in a climatic chamber for culture. Stress treatment with 200mM NaCl was performed 3 days after transplanting, 20ml XN05-1 bacteria solution was irrigated simultaneously, and the control group was irrigated with an equal amount of sterile water, and 6 replicates were set for each group. The plant height, root length, plant weight and root weight of wild soybeans were measured after 15 days of cultivation.
As shown in FIG. 4, it was found that although strain XN05-1 had little effect on the plant height and the plant weight of wild soybeans under salt stress, it was able to significantly increase the root length (increase 114.01%) and root weight (increase 24.38%), indicating that strain XN05-1 helps to increase salt tolerance by promoting root elongation of wild soybeans.
Claims (3)
1. Pseudomonas stutzeriPseudomonas stutzeri) XN05-1, wherein the preservation number is CGMCC No.26683, and the CGMCC is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) at the year 2023, month 02 and day 27.
2. Pseudomonas stutzeri according to claim 1Pseudomonas stutzeri) The application of XN05-1 in improving the salt tolerance of wild soybeans is characterized in that the preservation number is CGMCC No.26683 and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the year of 2023 and the month of 02.
3. A plant salt-resistant microbial agent, characterized in that the Pseudomonas stutzeri according to claim 1 is usedPseudomonas stutzeri) XN05-1 is a main active ingredient, and the preservation number is CGMCC No.26683, and is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for the year 2023, the month 02 and the day 27; the plant is wild plantAnd (5) beans.
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