Pseudomonas capable of dissolving phosphorus and producing siderophore and application of pseudomonas

Abstract

The invention discloses pseudomonas capable of dissolving phosphorus and producing siderophores and application of the pseudomonas, and relates to the technical field of microorganisms. The bacterial strain P-14-21 is identified as a new species of Pseudomonas sp. And is named as Pseudomonas sp. P-14-21, the bacterial strain is preserved in Guangdong Microbial Culture Collection Center on August 7, 2024, and the preservation number is GDMCC No: 64969. The invention also discloses a preparation method of the Pseudomonas sp. P-14-21, and the bacterial strain is named as Pseudomonas sp. P-14-21. The bacterial strain is named as Pseudomonas sp. P-14-21 and is preserved in Guangdong Microbial Culture Collection Center on August 7, 2024. Besides, the pseudomonas P-14-21 disclosed by the invention has the capacity of dissolving organic phosphorus and high-yield siderophore, the phosphorus dissolving index can reach 2.4, and the activity unit (su) of the siderophore can reach 94.41%, so that the strain has excellent development and application values in agricultural production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a Pseudomonas with the ability of phosphorus solubilization and siderophore production and its application. BACKGROUND

[0002] Phosphorus is one of the three essential nutrients for plant growth and metabolism, and is a key element that determines the primary productivity, multifunctionality and ecological service quality of an ecosystem. The lack of phosphorus can seriously affect the growth and development of crops, such as inhibiting the transformation and metabolism of chloroplast energy and reducing crop yield. However, most of the phosphorus in the soil exists in the form of insoluble phosphorus, and about 95% of the phosphorus is difficult for plants to directly absorb and utilize. In agricultural production, a large amount of phosphorus fertilizer is often applied to meet the supply of phosphorus elements for plants, but the large-scale application of traditional phosphorus fertilizer not only leads to the depletion of phosphorus resources, but also easily causes secondary pollution. Therefore, finding a green and efficient method to improve soil phosphorus utilization rate is still a problem that needs to be solved in current agricultural production.

[0003] Phosphorus-solubilizing bacteria widely exist in soil and can convert insoluble phosphorus in soil into phosphorus that can be absorbed and utilized by plants through the secretion of organic acids, phosphatases and protons, playing an important role in maintaining soil phosphorus balance, promoting crop growth and improving crop yield. In addition, phosphorus-solubilizing bacteria can exchange carbon and phosphorus with arbuscular mycorrhizal fungi to increase the content of available phosphorus in soil. Therefore, screening and utilizing phosphorus-solubilizing bacteria are of great significance for soil phosphorus supply. SUMMARY

[0004] The first object of the present application is to provide a Pseudomonas sp. Pseudomonas P-14-21 with the ability of phosphorus solubilization and siderophore production, which provides a new strain of phosphorus-solubilizing microbial resources. . Preferably, the Pseudomonas P-14-21 is isolated from tomato leaf tissue and preserved in the Guangdong Microbial Culture Collection Center on August 7, 2024, with the preservation number GDMCC No:64969.

[0005] Preferably, the Pseudomonas P-14-21 is a gram-negative bacterium, and the single colony on the NA culture medium is round, light yellow, smooth in surface and regular in edge. With the increase of culture time, the colony gradually turns yellow.

[0006] Preferably, the 16S rRNA gene sequence of the Pseudomonas P-14-21 is (SEQ ID NO.1):

[0007] ​ Preferably, the Pseudomonas P-14-21 is analyzed by sequence alignment analysis through the EzBioCloud (https: / / www.ezbiocloud.net / identify) website, and the strain has the maximum similarity of 98.03% with Pseudomonas meliae CFBP 3225 T Based on the comparative genomic analysis results, the average nucleotide identity of the strain P-14-21 with the type strain of the Pseudomonas genus is 80.57~80.80%, and the digital DNA-DNA hybridization value is 24.10~24.20%. It is shown that the strain P-14-21 represents a new species of the Pseudomonas genus.

[0008] Preferably, the Pseudomonas P-14-21 has the ability to produce siderophores, and the siderophore activity unit (Siderophore Unit, SU) is 94.41%. su

[0009] Preferably, the Pseudomonas P-14-21 has the ability to dissolve organic phosphorus, and the phosphorus-dissolving index is 2.4.

[0010] The second object of the present application is to provide a microbial agent, and the components of the microbial agent include the culture of the above-mentioned Pseudomonas P-14-21 and / or the secondary metabolites produced.

[0011] Preferably, the microbial agent includes a liquid microbial agent or a solid microbial agent.

[0012] The third object of the present application is to provide the use of the above-mentioned Pseudomonas P-14-21 or microbial agent in the preparation of a siderophore-producing product.

[0013] The fourth object of the present application is to provide the use of the above-mentioned Pseudomonas P-14-21 or microbial agent in the dissolution of organic phosphorus or the preparation of a product for degrading phosphorus in soil.

[0014] Preferably, the product includes a bio-fertilizer or a soil remediation agent.

[0015] The fifth object of the present application is to provide a bio-fertilizer containing the above-mentioned Pseudomonas P-14-21 or microbial agent as an active ingredient.

[0016] The sixth object of the present application is to provide a soil remediation agent containing the above-mentioned Pseudomonas P-14-21 or microbial agent as an active ingredient.

[0017] The present application has the following advantages over the prior art: 1) The Pseudomonas P-14-21 is a new species different from other Pseudomonas bacteria.

[0018] ​2) Pseudomonas sp. P-14-21 has the ability to produce siderophores, and the siderophore activity unit ( su ) is 94.41%.

[0019] 3) Pseudomonas sp. P-14-21 has the ability to dissolve organic phosphorus, with a phosphorus solubility index of 2.4.

[0020] Therefore, the Pseudomonas sp. P-14-21 disclosed in the present invention has excellent development and application prospects in preparing biological products producing siderophores and dissolving organic phosphorus or preparing products for degrading phosphorus in soil.

[0021] Pseudomonas ( Pseudomonas sp.) P-14-21, which was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on August 7, 2024, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, deposit number: GDMCC No: 64969. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Colony morphology of Pseudomonas sp. P-14-21 on NA plates.

[0023] Figure 2 : Plates of Pseudomonas sp. P-14-21 producing siderophores (A) and their ability to secrete siderophores (B).

[0024] Figure 3 : Screening plate for organic phosphorus solubility of Pseudomonas P-14-21 (A) and phosphorus solubility index determination plate (B).

[0025] Figure 4 : Phylogenetic tree of the Pseudomonas sp. P-14-21 genome. DETAILED DESCRIPTION

[0026] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0027] Example 1: Isolation of Pseudomonas P-14-21 The Pseudomonas strain P-14-21 was isolated from tomato leaf tissue, which was severely affected by bacterial wilt in Yuanmou County, Chuxiong City, Yunnan Province. The specific isolation steps were as follows: Tomato leaf tissue was rinsed three times with sterile water, then surface-disinfected with 75% anhydrous ethanol for 3 minutes. After disinfection, it was rinsed once with sterile water. The leaf surface was wiped dry with absorbent paper and then minced with sterile scissors. 1 g of sample was weighed in a sterile mortar and pestle, 9 mL of sterile water was added, and the leaf tissue was crushed with a grinding rod. 1 mL of juice sample was taken and added to 9 mL of sterile water. The sample was shaken with a vortex shaker for 3 minutes. The sample was diluted with sterile water to 10% each. -1 , 10-2 , 10 -3 . Absorb the original solution, 10 -1 , 10 -2 , 10 -3 100 μL of the sample was spread on a plate containing TZC medium, and each concentration gradient was repeated 3 times. The plate was placed in a 30°C incubator for inverted culture and observation. After culture for about 1 to 3 days, strains with different colony morphology, color, and size were picked for purification. Pure strains were obtained after 2 to 3 purification cycles.

[0028] TZC medium recipe: 10 g / L bacteriological peptone, 1 g / L acid-hydrolyzed casein, 10 g / L glucose, 15 g / L agar powder, 5 mL of 1% 2,3,5-triphenyltetrazolium chloride, and water as the solvent. Preparation: Weigh the above amounts of bacteriological peptone, acid-hydrolyzed casein, glucose, and agar powder, add 1000 mL of deionized water, and heat with stirring until completely dissolved. Adjust the pH of the solution to 6.8-7.0. Aliquot into Erlenmeyer flasks and autoclave at 121°C for 15 min. After completion, when the culture temperature drops to 55°C, add 5 mL of 1% 2,3,5-triphenyltetrazolium chloride solution, mix thoroughly, and pour into disposable Petri dishes for later use.

[0029] Example 2: Identification and preservation of Pseudomonas sp. P-14-21 Apparent identification of strain P-14-21: The strain was identified based on colony morphology, bacterial color, transparency, glossiness, etc. Figure 1 As shown, the single colonies of Pseudomonas P-14-21 in NA medium (formula: 10 g / L peptone, 3 g / L beef extract powder, 5 g / L sodium chloride, 15 g / L agar powder, solvent is water. Preparation method: weigh the formulated amount of peptone, beef extract powder, sodium chloride, and agar powder, add 1000 mL of deionized water, stir and heat until completely dissolved, adjust the solution pH to 7.2, divide into Erlenmeyer flasks, and autoclave at 121°C for 15 min. After completion, pour into disposable culture dishes for later use) are round, light yellow, with smooth surface and neat edges. With the increase of culture time, the colonies gradually turn yellow.

[0030] 16S rRNA gene sequence analysis of strain P-14-21: Genomic DNA of strain P-14-21 was extracted using the HiPure Bacterial DNA Kit, and the 16S rRNA gene was amplified using universal bacterial primers 27F / 1492R. The PCR product was sent to GeneWeiZ Biotechnology Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO. 1. Sequencing results were compared using EzBioCloud (https: / / www.ezbiocloud.net / identify).

[0031] The results show that the strain P-14-21 has the maximum similarity with the model strain Pseudomonas putida ATCC 49121, and the similarity is 98.03%; the next is Pseudomonas putida FBR 41, Pseudomonas putida CFBP 3225 and Pseudomonas putida ATCC 13522, and the similarities are 98.02% respectively. Pseudomonas meliae CFBP 3225 T The next is Pseudomonas putida FBR 41, Pseudomonas putida CFBP 3225 and Pseudomonas putida ATCC 13522, and the similarities are 98.02% respectively. Pseudomonas savastanoi ATCC 13522 T The next is Pseudomonas putida FBR 41, Pseudomonas putida CFBP 3225 and Pseudomonas putida ATCC 13522, and the similarities are 98.02% respectively. Pseudomonas ficuserectae JCM 2400 T The next is Pseudomonas putida FBR 41, Pseudomonas putida CFBP 3225 and Pseudomonas putida ATCC 13522, and the similarities are 98.02% respectively. The similarity with other model strains of Pseudomonas is less than 98.00%. In the field, the threshold for dividing new species is usually that the similarity of 16S rRNA gene sequence of the strain with the model strain is less than 98.7%, therefore, based on the similarity of 16S rRNA gene sequence, the strain P-14-21 may be a new species in Pseudomonas.

[0032] Whole genome sequence analysis of strain P-14-21: The strain P-14-21 was sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for genome sequencing using the Illumina Novaseq platform. The bacterial genome sequence was spliced using SPAdes v3.15.3 software, and the contig sequence with low quality and less than 500 bp in length was removed. The CheckM v1.1.3 software was used for quality analysis of the integrity and contamination of the bacterial genome. The TYPE software (https: / / tygs.dsmz.de / ) with the whole genome database of the model strain was used to upload the whole genome data of the strain P-14-21 to the database, and the genome phylogenetic tree of the strain P-14-21 was established. In order to further clarify the differences between these bacteria, ANICalculator (http: / / www.ezbiocloud.net / tools / ani) and Genome-to-Genome Distance Calculator v3.0 (https: / / ggdc.dsmz.de / ggdc.php#) were used to calculate the average nucleotide identity (ANI) and digitally DNA-DNA hybridization value (dDDH) between the strain P-14-21 and the genome sequences of the 5 most similar strains.

[0033] Results showed that the genome of strain P-14-21 was 5,210,258 bp in size, with a G+C content of 61.62%, an N50 of 620,010 bp, a genome completeness of 100%, and a contamination level of 0.43%. This is considered a high-quality genome based on a genome completeness of at least 95% and a contamination level of no more than 5%, indicating that the genome of strain P-14-21 is of high quality. Comparison of the ANI and dDDH values ​​between the genome sequences of the five most similar strains revealed that the ANI values ​​of strain P-14-21 and the model strains ranged from 80.57 to 80.80%, and the dDDH values ​​ranged from 24.1 to 24.2% (Table 1). These values ​​are well below the 70% and 95% thresholds for dDDH and ANI, respectively, for distinguishing new bacterial species. (If the ANI between two strains is greater than 95% or the dDDH is greater than 70%, the two strains belong to the same species.) This further confirms that strain P-14-21 is a new species of Pseudomonas. In addition, the genome phylogenetic tree of strain P-14-21 showed that strain P-14-21 formed an independent phylogenetic branch ( Figure 4 ), indicating that strain P-14-21 has an independent taxonomic status.

[0034] In summary, strain P-14-21 belongs to Pseudomonas spp. Pseudomonas sp . ) and named Pseudomonas sp . P-14-21, the strain was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on August 7, 2024, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, the deposit number is: GDMCC No: 64969.

[0035] Table 1 ANI and dDDH between strain P-14-21 and the model strain

[0036] Preservation of Pseudomonas sp. P-14-21: Pick a single identified colony and transfer it to the corresponding test tube slope. Store in a refrigerator at 4°C until use. Simultaneously, prepare a bacterial suspension in 50% distilled glycerol solution and freeze at -80°C.

[0037] Example 3: Preliminary screening of the ability of Pseudomonas sp. P-14-21 to dissolve organophosphate and produce siderophores 1. Strain activation Remove the glycerol tube containing the strain from the -80°C refrigerator, dissolve it at room temperature, and then streak it on a plate containing NA solid medium (the medium formula is the same as in Example 2). Invert the plate and place it in a constant temperature incubator at 30°C for 24 hours to obtain the activated strain.

[0038] 2. Medium preparation Organophosphorus solid medium formula: glucose 10.0 g / L, ammonium sulfate 0.5 g / L, yeast extract 0.5 g / L, sodium chloride 0.3 g / L, potassium chloride 0.3 g / L, magnesium sulfate 0.3 g / L, ferrous sulfate 0.03 g / L, manganese sulfate 0.03 g / L, lecithin 0.2 g / L, calcium carbonate 1.0 g / L, agar powder 15 g / L, solvent is water, pH 7.0-7.5.

[0039] Iron carrier-producing identification solid medium formula: chrome azurol S (CAS) 60.5 mg / L, hexadecyl trimethyl ammonium bromide (HDTMA) 72.9 mg / L, iron chloride hexahydrate 2.645 mg / L, sodium dihydrogen phosphate dihydrate 295.25 mg / L, sodium hydrogen phosphate dodecahydrate 1213.5 mg / L, ammonium chloride 125 mg / L, potassium dihydrogen phosphate 37.5 mg / L, sodium chloride 62.5 mg / L, solvent is water, pH 6.8.

[0040] Medium preparation method: according to the formula, weigh the medium, add 1000 mL of deionized water, stir and heat to completely dissolve, adjust the pH of the solution to the corresponding value, divide into triangular bottles, 121 ℃ high pressure sterilization for 15 min, after completion, pour into disposable culture dishes for standby.

[0041] 3. Inoculation Divide the plates containing organophosphorus and iron carrier-producing identification solid medium into 4 parts, inoculate the activated bacteria respectively, and place the plates in a 30℃ constant temperature incubator upside down for 7 days. Observe the results.

[0042] 4. Results From Figure 2 (A) and Figure 3 (A), it can be seen that strain P-14-21 produces transparent circles around the plate containing organophosphorus and iron carrier-producing identification solid medium. Studies have shown that if a transparent circle appears around a microorganism on a plate containing organophosphorus, it indicates that the microorganism has the ability to dissolve organophosphorus; if an orange-yellow transparent circle is produced on the plate of iron carrier-producing identification solid medium, it indicates that the microorganism has the ability to produce iron carrier. Therefore, strain P-14-21 has the ability to dissolve organophosphorus and produce iron carrier.

[0043] Example 4: Evaluation of the ability of Pseudomonas P-14-21 to dissolve organophosphorus and produce iron carrier 1. Strain activation The same as step 1 of example 3.

[0044] 2. Medium preparation Organic phosphorus solid medium formula is the same as step 2 of example 3.

[0045] NB medium formula is the same as NA medium of example 2 without agar.

[0046] CAS detection solution: A solution, 0.605 g CAS is added to 500 mL distilled water, 0.027 g FeCl3·6H20 is added to 100 mL dilute hydrochloric acid; B solution, 0.729 g HDTMA (hexadecyl trimethyl ammonium bromide) is added to 400 mL distilled water. Then slowly add A solution to B solution to obtain blue CAS detection solution. The detection solution is prepared and used immediately.

[0047] 3. Inoculation The plates containing organic phosphorus and siderophore identification solid medium are divided into 4 equal parts, and the activated bacteria are spotted. Repeat 4 times, and place the plates in a constant temperature incubator at 30℃. The organic phosphorus-containing plates are cultured for 5 days, and the siderophore identification plates are cultured for 7 days. After the culture is completed, the results are observed, the transparent circle diameter mm (HD) and the colony diameter mm (CD) are measured by cross method, and the phosphorus-dissolving index and siderophore-producing index are counted.

[0048] Phosphorus-dissolving index = transparent circle diameter (HD) / colony diameter (CD); Siderophore-producing index = transparent circle diameter (HD) / colony diameter (CD); Siderophore secretion determination: pick single colony and inoculate in NB liquid medium, 30℃, 200 r / min shaking culture for 5 days. After the culture is completed, centrifuge the bacterial suspension at 10000 r / min for 8 min, take 5 mL supernatant and mix well with 5 mL CAS detection solution. Place at room temperature for 1 h in the dark, measure the absorbance value (As) at 630 nm wavelength by spectrophotometer, use double distilled water as control to zero, and use the absorbance value of 5 mL sterile NB liquid medium and 5 mL CAS detection solution reaction as reference value (Ar). Determine the siderophore activity unit (Fe3+) of strain P-14-21. su ).

[0049] su = [(Ar - As) / Ar]×100% 4. Results As can be seen from table 2, strain P-14-21 has better ability to dissolve organic phosphorus and produce siderophore, with phosphorus-dissolving index of 2.4, siderophore-producing index of 2.97, and siderophore activity unit (Fe3+) of 94.41%. su .

[0050] Table 2 Analysis of the ability of Pseudomonas P-14-21 to dissolve organic phosphorus and produce siderophore

[0051] The above detailed description is a specific description of the embodiments of the present application, which is not used to limit the patent scope of the present application. Any equivalent implementation or modification made without departing from the present application shall be included in the patent scope of the present application.

Claims

1. A strain of Pseudomonas ( Pseudomonas sp.) P-14-21, deposit number: GDMCC No: 64969.

2. A bacterial agent, characterized in that The components of the bacterial agent include the culture of Pseudomonas P-14-21 according to claim 1 and / or the secondary metabolites produced.

3. The microbial agent according to claim 2, characterized in that The bacterial agent includes a liquid bacterial agent or a solid bacterial agent.

4. Use of the Pseudomonas sp. P-14-21 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a siderophore-producing product.

5. Use of the Pseudomonas P-14-21 according to claim 1 or the bacterial agent according to claim 2 in dissolving organic phosphorus or preparing products that degrade phosphorus in soil.

6. The use according to claim 4 or 5, characterized in that The products include biofertilizers or soil remediation agents.

7. A biological fertilizer, characterized in that: The invention contains the Pseudomonas P-14-21 according to claim 1 or the bacterial agent according to claim 2 as an active ingredient.

8. A soil remediation agent, characterized in that: The invention contains the Pseudomonas P-14-21 according to claim 1 or the bacterial agent according to claim 2 as an active ingredient.

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