A strain of Pseudomonas aeruginosa S46 and its application

Abstract

The present invention discloses a strain of Pseudomonas aeruginosa S46 and its application. The strain is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee, with a deposit address of Beijing, China, a deposit number of CGMCC NO.28278, and a deposit date of August 28, 2023. The classification name is Pseudomonas aeruginosa. The strain S46 has a strong colonization ability in the rhizosphere of poplars, can significantly improve the salt resistance of poplars, and promote the growth and development of poplars; it is a bacterium used to improve the salt resistance of poplars, with a small amount of active trays, low cost, and significant effect; it can effectively alleviate the salt stress reaction of poplars, promote the growth of poplars under salt stress, and achieve the purpose of effectively improving the salt resistance of poplars.

Description

Technical Field

[0001] The invention relates to a strain of Pseudomonas aeruginosa S46 and application thereof, belonging to the technical field of forestry production. Background Art

[0002] Poplar is an important tree species for afforestation in my country. It has the characteristics of fast growth rate and high timber value, and is an important tree species to solve the problem of timber supply in my country. my country's poplar plantation area ranks first in the world. Poplars are widely distributed in saline-alkali areas such as the northwest, north China, and the coastal areas of east China. However, unreasonable species configuration and aging of forests in many areas have led to prominent problems of poplar plantation degradation, which seriously restricts the resource utilization of saline-alkali soil. Therefore, exploring the salt-alkali tolerance mechanism of poplars and developing more efficient and environmentally friendly poplar resistance regulation technologies are of great significance to ensuring the sustainable management of poplar plantations and improving the utilization rate of saline-alkali land.

[0003] Improving the salt resistance of forest trees by breeding new salt-tolerant varieties is currently a common method. Traditional breeding techniques, such as hybrid breeding and induced breeding, can improve the salt resistance of forest trees to a certain extent, but have the disadvantages of long breeding cycle, poor predictability and low efficiency. Molecular breeding technology can introduce salt-tolerant genes into new varieties, and can aggregate multiple genes to significantly improve the salt tolerance of plants, but the safety of transgenic plants is difficult to evaluate in field trials. Salt stress induces enhanced synthesis of secondary metabolites in plants, among which phenolic substances, such as phenolic acids and flavonoids, can be secreted into the rhizosphere as root secretions. Phenolic acids can change the composition of plant rhizosphere microorganisms, can serve as a carbon source for rhizosphere bacteria, and recruit specific beneficial bacteria to enrich in the rhizosphere. Soil beneficial microorganisms can regulate plant growth and development and improve tolerance to salt stress by improving plant nutrient absorption, regulating endogenous hormone levels, osmotic regulating substances, and blocking stress factors. In addition, microorganisms come from the soil, have no negative impact on the environment, and respond quickly to environmental stimuli. Therefore, developing biological agents based on beneficial microorganisms to improve plant salt resistance is an environmentally friendly, efficient and fast means of regulating plant resistance.

[0004] At present, the application of microbial agents has made certain progress in production practice. More than 20 species of rhizosphere microorganisms have been proven to have growth-promoting, antibacterial, and stress-resistant effects, and have been intensively applied to herbaceous plants. However, there are still problems such as unstable effects and poor environmental adaptability during promotion. Therefore, the rhizosphere microorganisms enriched in resistant poplar varieties under salt stress are screened. They are easy to form a stable symbiosis with plants, and the development of agents with them as the core functional components can avoid the survival problems of microorganisms under adversity and improve the stability of the agents. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a strain of Pseudomonas aeruginosa S46 and its application, so as to effectively alleviate the damage of salt stress to poplars and improve the salt resistance of poplars. The method of the present invention has strong specialization and stable effect.

[0006] Technical solution: In order to solve the above technical problems, the present invention provides a Pseudomonas aeruginosa S46, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit address of CGMCC NO.28278 and a deposit date of August 28, 2023. Its classification name is Pseudomonas aeruginosa, and the 16s rDNA base sequence of the S46 strain is shown in SEQ ID No: 1.

[0007] The present invention also provides the use of the S46 strain in improving the salt resistance of poplar:

[0008] (1) Obtain rhizosphere soil of salt-tolerant poplar species planted in saline soil.

[0009] (2) Weigh 0.1 g of rhizosphere soil and add 0.9 mL of sterile water. Dilute twice to obtain a dilution of 10. -3 0.1 mL of the soil dilution was spread on a TSA medium plate (medium ingredients: 5 g soy peptone, 15 g tryptone, 5 g NaCl, 15 g agar, 1000 mL distilled water), and cultured at 26°C for 1-3 days.

[0010] (3) Pick a single colony and streak it on a plate on the isolation medium for purification. Perform three rounds of purification.

[0011] (4) Activate the isolated and purified strains, select a single colony, and culture it in TSB medium at 160 rpm and 26°C for 16 to 18 h. Centrifuge the bacterial solution at 6000 rpm for 5 min, discard the supernatant, resuspend the bacterial strain in sterile water, and calculate the OD 600 Adjust to 0.2 to obtain the desired bacterial solution. Culture bacteria in a carbon-free liquid medium (medium formula: tryptone 15g, NaCl 5g, distilled water 1000mL) containing different concentrations of sodium benzoate (0, 1μM, 50μM, 100μM, 1mM, 10mM, 25mM, 50mM) and measure the OD of the bacterial solution after 48h of culture. 600 When the concentration of sodium benzoate in the culture medium was 1 mM, it could significantly promote the growth of strain S46, indicating that S46 could utilize phenolic acids in the culture medium.

[0012] (5) When the S46 bacterial solution was inoculated into the roots of aseptic poplar seedlings grown in salt medium for 5 to 6 days, the damage to 84K poplar was reduced and the salt resistance was improved compared with that without inoculation. Strain S46 can chelate sodium ions and reduce the availability of sodium ions to plants by producing extracellular polysaccharides and biofilms, thus protecting plant roots from the effects of salt stress.

[0013] The present invention also provides a bacterial agent or preparation, which contains one or more of the Pseudomonas aeruginosa S46 or its fermentation liquid and sterile supernatant.

[0014] The present invention also provides the use of the Pseudomonas aeruginosa S46 or the bacterial agent or preparation in preparing an active tray.

[0015] The present invention also provides an active tray, which is obtained by inducing and strengthening the Pseudomonas aeruginosa S46 with phenolic acid and colonizing it in a mixture of rapeseed cake fertilizer and rice husk charcoal. The active tray has strong adaptability in the field and can be used to improve the salt tolerance of poplars.

[0016] Among them, the complex composed of beneficial bacteria and the substrate that carries their activity is called an active tray.

[0017] Wherein, the content of Pseudomonas aeruginosa S46 is 1×10 9 cfu / g~1×10 12 cfu / g.

[0018] The Pseudomonas aeruginosa S46 active tray preparation technology provided by the present invention is:

[0019] (1) Prepare fortified culture medium: 1.5% (g / L) tryptone, 0.5% (g / L) NaCl, 1 mM sodium benzoate.

[0020] (2) Strain stability enhancement: the strain was added to the above culture medium, the rotation speed was 160 r / min, the pH value was adjusted to 7.0-7.6, the fermentation temperature was 26° C., the fermentation time was 48 h, and the OD value of the bacterial solution was measured to be about 1.0;

[0021] (3) Preparation of active trays: After the rapeseed cake fertilizer and rice husk carbon are mixed evenly in a mass ratio of 1:2, sterilize at 121°C for 40 minutes to obtain a sterile composite carrier, and use an appropriate amount of 121°C, 20 minutes sterile water / pure water to adjust the moisture of the composite carrier to moist without caking. The above-mentioned enhanced fermentation liquid is inoculated into the above-mentioned sterile carrier at a ratio of 10% (mL / g), and placed in a constant temperature box (26°C) for 7 days; after the second step of strengthening the strain, the active tray can efficiently colonize in the rapeseed cake fertilizer and rice husk carbon mixture, and the bacterial amount reaches 1×10 9 cfu / g~1×10 12 cfu / g. Finally, S46 solid active tray was obtained.

[0022] The active tray application method of the present invention is to sow the strains at the base of the poplars when the poplars are cut; in this way, the strains can be effectively colonized in the rhizosphere of the poplars for a long time, thereby alleviating the salt stress reaction of the poplars and promoting the growth of the poplars under salt stress.

[0023] The present invention also provides application of the Pseudomonas aeruginosa S46 or the bacterial agent or preparation or the active tray in improving the salt resistance of poplar.

[0024] The method comprises the following steps: applying the Pseudomonas aeruginosa S46 or the bacterial agent or preparation or the active tray to the poplar.

[0025] Wherein, the usage of the active tray is 15-25g / plant.

[0026] The present invention also provides a method for obtaining a poplar with high salt resistance, comprising the following steps: applying the Pseudomonas aeruginosa S46 or the bacterial agent or preparation or the active tray to the poplar.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The strain S46 has a strong ability to colonize in the rhizosphere of poplars, can significantly improve the salt resistance of poplars, and promote the growth and development of poplars; 2. The strain S46 is a bacterium used to improve the salt resistance of poplars, with a small amount of active trays, low cost, and significant effect; 3. Saline-alkali land is an important resource for artificial afforestation of poplars. If the active tray prepared by the above method is applied to the roots of poplar cuttings, the salt stress response of poplars can be effectively alleviated, and the growth of poplars under salt stress can be promoted, thereby achieving the purpose of effectively improving the salt resistance of poplars. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 OD of S46 strain after growing in medium containing different concentrations of sodium benzoate for 48 h 600 value;

[0029] Figure 2 The growth of Pseudomonas S46 strain on the culture medium. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0031] Embodiment 1:

[0032] The rhizosphere soil of salt-tolerant poplar species planted in saline soil was collected for relevant experiments, and the specific steps were as follows:

[0033] (1) Soil sample acquisition: Saline soil with a salt content of 0.35% was collected from Haibin, Dongtai City, Yancheng City, Jiangsu Province (120.84°E, 32.90°N), crushed and sieved, and mixed with perlite in a volume ratio of 4:1. Each pot was filled with 1 kg for use as a planting medium. The roots of poplar trees planted in the above medium for 120 days in the laboratory were shaken until the soil stopped shaking. The roots of the soil stopped shaking were clamped with tweezers in a 100 mL conical flask filled with 50 mL 0.1 M phosphate buffer, and then placed in a shaking table at 150 rpm and 20.5°C for 30 min to allow the soil on the surface of the roots to be completely leached. The rhizosphere soil suspension was obtained. The roots in the conical flask were picked out with sterile tweezers, and the remaining suspension was centrifuged at high speed (8000 rpm) for 8 min. The precipitate was the rhizosphere soil.

[0034] (2) Spreading separation: Weigh 0.1 g of rhizosphere soil and add 0.9 mL of sterile water. Dilute it twice to obtain a dilution of 10 -3 0.1 mL of the soil dilution was spread on a TSA medium plate (medium ingredients: 5 g soy peptone, 15 g tryptone, 5 g NaCl, 15 g agar, 1000 mL distilled water), and cultured at 26°C for 1-3 days.

[0035] (3) Cultivation and purification: Pick a single colony and draw a line on the isolation medium for purification and cultivation. Perform three rounds of purification ( Figure 2 The strain obtained after purification and culture was used as the test strain and inoculated onto a TSA plate for temporary storage.

[0036] Experimental Example 2: Co-culture experiment of strains and phenolic acid

[0037] The purified strain of Experimental Example 1 was used to perform a co-culture experiment with phenolic acid:

[0038] The isolated and purified strain was activated, and a single colony was selected. After shaking culture in TSB medium at 160 rpm and 26 °C for 16 to 18 h, the bacterial solution was centrifuged at 6000 rpm for 5 min, the supernatant was discarded, the bacterial cell was resuspended in sterile water, and the OD 600Adjust to 0.2 to obtain the desired bacterial solution. Culture bacteria in a carbon-free liquid medium containing different concentrations of sodium benzoate (medium formula: tryptone 15g, NaCl 5g, distilled water 1000mL) and measure the OD of the bacterial solution after 48h of culture. 600 value.

[0039] There are 8 treatments in the experiment.

[0040] CK: strains were cultured in medium without sodium benzoate;

[0041] 1 μM: strains were cultured in medium containing 1 μM sodium benzoate;

[0042] 50 μM: strains were cultured in medium containing 50 μM sodium benzoate;

[0043] 100 μM: strains were cultured in medium containing 100 μM sodium benzoate;

[0044] 1mM: strains were cultured in medium containing 1mM sodium benzoate;

[0045] 10mM: strains were cultured in medium containing 10mM sodium benzoate;

[0046] 25mM: strains were cultured in medium containing 25mM sodium benzoate;

[0047] 50 mM: The strain was grown in a medium containing 50 mM sodium benzoate.

[0048] Each strain and each treatment were replicated 5 times.

[0049] pass Figure 1 It can be seen that when the concentration of sodium benzoate in the culture medium is less than or equal to 1mM, the growth promotion effect of prebiotics on S46 is significantly enhanced as the treatment concentration increases. When the concentration of sodium benzoate is greater than 1mM, the growth of the strain is significantly inhibited, indicating that S46 can utilize low concentrations of phenolic acid prebiotics in the culture medium.

[0050] Example 3: Experiment on the effect of strain S46 on salt resistance of poplar

[0051] (1) Activate the S46 strain, select a single colony, and culture it in TSB medium at 160 rpm and 26°C for 16 to 18 h. Centrifuge the culture solution at 6000 rpm for 5 min, discard the supernatant, resuspend the cells in sterile water, and calculate the OD 600Adjust to 1.0 to obtain the bacterial solution required for inoculation. After the sterile seedlings of Populus davidiana×Populusbolleana, SXY and Populus alba×Populus glandulosa'84K', 84K were propagated by terminal bud propagation, the seedlings were transferred to salt culture medium (1 / 2MS+0.65% agar+160mmol / L NaCl) after 20 days of growth. After 5 days of growth, the isolated and purified bacterial solution was inoculated into the poplar roots, 40μL per root, and the same amount of sterile water was inoculated as the control (CK). After 10 days of growth, the damage of the two treated plants was compared.

[0052] Statistical standard for stem browning: If the browning part of the stem accounts for 25% to 75% of the total stem length of the plant, it is counted as 0.5 tree; if the browning part of the stem accounts for more than 75% of the total stem length of the plant, it is counted as 1 tree.

[0053] Leaf damage grading standards: Level 1, the area of ​​necrotic part of the leaf accounts for 12.5% ​​to 37.5% of the total leaf area; Level 2, the area of ​​necrotic part of the leaf / total leaf area is 37.5% to 62.5%; Level 3, the area of ​​necrotic part of the leaf accounts for 62.5% to 87.5% of the total leaf area; Level 4, the area of ​​necrotic part of the leaf accounts for more than 87.5% of the total leaf area.

[0054] Table 1 Browning rate of plant stems

[0055]

[0056] Table 2 Plant leaf damage index

[0057]

[0058] Note: Leaf damage index = (D1*25%+D2*50%+D3*75%+D4*100%) / T*100%

[0059] D1 (number of leaves damaged at level 1), D2 (number of leaves damaged at level 2), D3 (number of leaves damaged at level 3), D4 (number of leaves damaged at level 4). T (total number of leaves).

[0060] It can be seen from Tables 1 and 2 that compared with the sterile water inoculation, the damage of SXY inoculated with S46 was significantly reduced, the stem browning rate decreased by 25%, and the leaf damage index decreased by 14.36%. Compared with the sterile water inoculation, the stem browning rate of 84K inoculated with S46 decreased by 25%, and the leaf damage index decreased by 12.31%. Therefore, the S46 strain has a significant effect on improving the salt resistance of SXY and 84K.

[0061] (2) Determination of taxonomic status: DNA of S46 was extracted and 16S rDNA sequencing was performed to determine the taxonomic status of the strain. The 16S rDNA base sequence of Pseudomonas aeruginosa S46 is shown in SEQ ID No:1.

[0062] (3) Through the above experiments, a strain that can significantly alleviate the salt stress response of poplar was screened and expanded for culture. The strain belongs to the Proteobacteria, γ-Proteobacteria, Pseudomonas, Pseudomonas, and was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on August 28, 2023, with a deposit number of CGMCC NO.28278.

[0063] Example 4: Application effect of active tray

[0064] Pseudomonas aeruginosa S46 was added to a fortified culture medium (tryptone 1.5%, NaCl 0.5%, sodium benzoate 1 mM), the rotation speed was 160 r / min, the fermentation temperature was 26° C., liquid fermentation was carried out for 48 hours, and the OD value of the bacterial liquid was measured to be about 1.0.

[0065] Rapeseed cake fertilizer (Ningbo Huacai Horticulture Co., Ltd., item number: 002) and rice husk carbon (Ningbo Huacai Horticulture Co., Ltd., item number: D575864245120) were mixed evenly according to a mass ratio of 1:2, sterilized at 121°C for 40 min to obtain a sterile composite carrier, and an appropriate amount of sterile water sterilized at 121°C for 20 min was used to adjust the moisture of the composite carrier to be moist without caking. The fermentation liquid was inoculated into the above sterile composite carrier at a ratio of 10% (mL / g), placed in a constant temperature box (26°C) for 7 days, and used as an active tray for standby.

[0066] The dilution plate method was used to determine the number of viable bacteria in the active tray. Accurately weigh 2 g of the active tray and add it to a conical flask containing 20 mL of sterile water. After fully shaking at room temperature for 2 hours, the resulting bacterial suspension was 10 -1 The bacterial suspension was diluted in gradients, and 0.1 mL of each dilution was applied to a solid TSA medium plate. Three replicates were set for each concentration, and colonies were counted after 2 days of culture. The content of Pseudomonas aeruginosa S46 in the active tray was 1×10 9 cfu / g.

[0067] Saline soil with a salt content of 0.20% was collected from the seaside of Dongtai City (120.84°E, 32.90°N) under the jurisdiction of Yancheng City, Jiangsu Province. The soil was crushed and sieved, and the saline soil and perlite (brand: Bohanmei) were mixed in a volume ratio of 4:1. 1 kg was placed in each pot as a cutting medium.

[0068] Cut one-year-old poplar seedlings into 12-15cm cuttings with a flat incision. Select 10 cuttings with the same location, uniform thickness and 2-3 full buds. Divide them into 2 groups of 5 for later use.

[0069] When cutting, the cuttings are directly inserted into the substrate, with the upper cut slightly higher than the surface of the substrate.

[0070] The experiment has two treatments:

[0071] Synbiotics-: Salty soil, no active trays applied during cuttings;

[0072] Synbiotics+: In saline soil, apply active trays at the base of the cuttings at the same time as cuttings, 20g per plant;

[0073] Each treatment had 15 pots, and the cuttings were watered regularly after transplantation to observe the growth status. The survival rate was calculated every 5 days.

[0074] Table 3 Survival rate of cuttings on the 5th, 10th and 15th days

[0075] deal with Survival rate on day 5 Survival rate on day 10 Survival rate on day 15 Synbiotics- 100％ 73.33％ 66.67％ Synbiotics+ 100％ 100％ 100％

[0076] According to Table 3, on the 15th day, the survival rate increased by 33.33% when the active tray was applied to the base of the cuttings at the same time as the cuttings, compared with the case where no active tray was applied during the cuttings, which showed a significant improvement. This indicated that the active tray improved the salt resistance of the cuttings.

Claims

1. A strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) S46, characterized in that, It is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address: Beijing, China, the deposit number is CGMCCNO.28278, the deposit date is August 28, 2023, and its classification name is: Pseudomonas aeruginosa ( Pseudomonas aeruginosa ).

2. A bacterial agent or preparation, characterized in that: The bacterial agent or preparation contains the Pseudomonas aeruginosa according to claim 1 ( Pseudomonas aeruginosa ) S46 or one or both of its fermentation broth.

3. The Pseudomonas aeruginosa according to claim 1 ( Pseudomonas aeruginosa ) S46, use of the bacterial agent or preparation described in claim 2 in the preparation of active trays.

4. An active tray, characterized in that: The active tray is made of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) S46 was induced and enhanced with phenolic acid and colonized in a mixture of rapeseed cake fertilizer and rice husk charcoal.

5. The active tray according to claim 4, characterized in that: The Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) The content of S46 is 1× 10 9 cfu / g ~1× 10 12 cfu / g.

6. The Pseudomonas aeruginosa according to claim 1 ( Pseudomonas aeruginosa ) S46, use of the bacterial agent or preparation described in claim 2 or the active tray described in claim 4 or 5 to improve the salt resistance of poplar.

7. The use according to claim 6, characterized in that: The following steps are involved: Applying the Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) S46, the bacterial agent or preparation as described in claim 2 or the active tray as described in claim 4.

8. The use according to claim 6 or 7, characterized in that: The usage of the active tray is 15-25 g / plant.

9. A method for obtaining a poplar tree with high salt resistance, characterized in that: The following steps are involved: Applying the Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) S46, the bacterial agent or preparation as described in claim 2, and the active tray as described in claim 4.

10. The method according to claim 9, characterized in that: The usage of the active tray is 15-25 g / plant.

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