Pseudomonas sp. with phosphorus solubilizing and siderophore producing ability and application thereof

Abstract

This invention discloses a *Pseudomonas* strain with phosphate-solubilizing and siderophore-producing capabilities and its applications, relating to the field of microbial technology. The strain P-14-21 has been identified as belonging to the genus *Pseudomonas* (…). Pseudomonas A new species of sp., and named Pseudomonas sp. P-14-21, this strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on August 7, 2024, with accession number GDMCC No:64969. Furthermore, the Pseudomonas P-14-21 disclosed in this invention possesses the ability to dissolve organic phosphorus and produce high levels of siderophores, with a phosphorus solubility index reaching 2.4 and a siderophore activity unit (…). su The efficiency can reach 94.41%, therefore this strain has excellent development and application value 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 energy conversion and metabolism of chloroplasts, 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, thereby increasing the content of available phosphorus in soil. Therefore, it is of great significance to screen and utilize phosphorus-solubilizing bacteria for soil phosphorus supply. SUMMARY

[0004] The first object of the present application is to provide a Pseudomonas new species (Pseudomonas sp Pseudomonas P-14-21) with the ability of phosphorus solubilization and siderophore production, which provides a new strain of phosphorus-solubilizing microorganism 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 of 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 the edge is neat. 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]

[0008] ​

[0009] Preferably, the Pseudomonas P-14-21 is analyzed by sequence alignment through the EzBioCloud (https: / / www.ezbiocloud.net / identify) website, and the strain has the maximum similarity of 98.03% with Pseudomonas sp. CFBP 3225. 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 genus Pseudomonas 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 genus Pseudomonas.

[0010] Preferably, the Pseudomonas P-14-21 has the ability to produce siderophores, and the siderophore activity unit (Siderophore Unit, SU) is 94.41%. su

[0011] Preferably, the Pseudomonas P-14-21 has the ability to dissolve organic phosphorus, and the phosphorus-dissolving index is 2.4.

[0012] 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 Pseudomonas P-14-21 and / or the secondary metabolites produced by the Pseudomonas P-14-21.

[0013] Preferably, the microbial agent includes a liquid microbial agent or a solid microbial agent.

[0014] The third object of the present application is to provide the use of the Pseudomonas P-14-21 or the microbial agent in the preparation of a product for producing siderophores.

[0015] The fourth object of the present application is to provide the use of the Pseudomonas P-14-21 or the microbial agent in the preparation of a product for dissolving organic phosphorus or degrading phosphorus in soil.

[0016] Preferably, the product includes a bio-fertilizer or a soil remediation agent.

[0017] The fifth object of the present application is to provide a bio-fertilizer containing the Pseudomonas P-14-21 or the microbial agent as an active ingredient.

[0018] The sixth object of the present application is to provide a soil remediation agent containing the Pseudomonas P-14-21 or the microbial agent as an active ingredient.

[0019] The present application has the following advantages over the prior art:

[0020] 1) The Pseudomonas P-14-21 is a new species different from other Pseudomonas bacteria. ​

[0021] 2) Pseudomonas P-14-21 has the ability to produce siderophores, with siderophore activity units ( su The percentage was 94.41%.

[0022] 3) Pseudomonas P-14-21 has the ability to dissolve organic phosphorus, with a phosphorus solubility index of 2.4.

[0023] Therefore, the Pseudomonas P-14-21 disclosed in this invention has excellent prospects for development and application in the preparation of siderogenic biological products and the dissolution of organic phosphorus or the preparation of products that degrade phosphorus in soil.

[0024] Pseudomonas ( Pseudomonas sp.) P-14-21, which was deposited on August 7, 2024 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No:64969. Attached Figure Description

[0025] Figure 1 Colony morphology of Pseudomonas P-14-21 on NA plates.

[0026] Figure 2 Siderophore-producing plate (A) and siderophore-secreting capacity (B) of Pseudomonas P-14-21.

[0027] Figure 3 Primary screening plate (A) and phosphorus solubility index determination plate (B) of Pseudomonas P-14-21 for dissolving organophosphates.

[0028] Figure 4 Phylogenetic tree of Pseudomonas P-14-21 genome. Detailed Implementation

[0029] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0030] Example 1: Isolation of Pseudomonas P-14-21

[0031] Pseudomonas P-14-21 strain is isolated from tomato leaf tissue of tomato bacterial wilt outbreak in Yuanmou County, Chuxiong City, Yunnan Province. The specific isolation steps are as follows: the tomato leaf tissue is washed with sterile water for 3 times, then the surface is sterilized with 75% anhydrous ethanol for 3 min, then washed with sterile water for 1 time, the water on the surface of the leaf is wiped dry with a water-absorbing paper, then the leaf is cut into pieces with sterile scissors, 1 g sample is weighed in a sterile mortar, 9 mL sterile water is added, the leaf tissue is crushed with a grinding rod, 1 mL juice sample is taken in 9 mL sterile water, and the sample is shaken for 3 min with a vortex shaker, and then the sample is diluted with sterile water to 10 -1 、10 -2 、10 -3 The original liquid, 10 -1 、10 -2 、10 -3 samples are taken 100 μL, respectively, and are coated on plates containing TZC medium, each concentration gradient is repeated 3 times, and is placed in a 30℃ incubator for inverted culture observation, and is cultured for 1-3 days, and the strains with different colony morphology, color and size are picked and purified, and the pure strain is obtained after 2-3 times of purification.

[0032] The formula of TZC culture medium is: 10 g / L bacteriological peptone, 1 g / L acid hydrolysis casein, 10 g / L glucose, 15 g / L agar powder, 1% 2,3,5-chlorinated triphenyl tetrazole 5 mL, and the solvent is water. The preparation method is as follows: the bacteriological peptone, acid hydrolysis casein, glucose and agar powder are weighed according to the above amount, 1000 mL deionized water is added, stirred and heated to completely dissolve, the pH of the solution is adjusted to 6.8-7.0, and the solution is divided into triangular bottles, 121℃ high pressure sterilization for 15 min, after completion, when the medium temperature decreases to 55℃, 5 mL 1% 2,3,5-chlorinated triphenyl tetrazole solution is added, mixed uniformly, and then poured into disposable culture dishes for standby.

[0033] Example 2: Identification and preservation of Pseudomonas P-14-21

[0034] The apparent identification of strain P-14-21: the strain is identified according to the colony morphology, color, transparency and luster of the strain. For example Figure 1As shown, the Pseudomonas P-14-21 single colony on NA medium (formula: 10 g / L peptone, 3 g / L beef infusion powder, 5 g / L sodium chloride, 15 g / L agar powder, solvent is water. Preparation method: weigh the formula amount of peptone, beef infusion powder, sodium chloride, agar powder, add 1000 mL of deionized water, stir and heat to completely dissolve, adjust the pH of the solution to 7.2, distribute in a flask, autoclave at 121°C for 15 min, then pour into a disposable petri dish for standby) is round, light yellow, smooth surface, and the edge is neat. With the increase of culture time, the colony gradually turns yellow.

[0035] 16S rRNA gene sequence analysis of strain P-14-21: Genomic DNA of strain P-14-21 was extracted using HiPure Bacterial DNA Kit kit, and 16S rRNA gene was amplified using bacterial universal primers 27F / 1492R. The PCR product was sent to Jinweizhi Biotechnology Co., Ltd. for sequencing, and the sequence is SEQ ID NO. 1. The sequencing results were compared by EzBioCloud (https: / / www.ezbiocloud.net / identify) website.

[0036] The results show that the strain P-14-21 has the maximum similarity with the model strain Pseudomonas meliae CFBP3225 T 98.03%; followed by Pseudomonas savastanoi ATCC13522 T 98.02%; Pseudomonas ficuserectae JCM 2400 T 98.02%. The 16S rRNA gene sequence similarity with other model strains of Pseudomonas is less than 98.00%. In the art, a similarity of less than 98.7% between the 16S rRNA gene sequence of a bacterial species and a model strain is generally used as a threshold for dividing a new species, therefore, based on the 16S rRNA gene sequence similarity results, strain P-14-21 may be a new species within the genus Pseudomonas.

[0037] Strain P-14-21 whole genome sequence analysis: Strain P-14-21 was sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for genome sequencing using the Illumina Novaseq platform. SPAdes v3.15.3 software was used for bacterial genome sequence assembly, and contig sequences with low quality and less than 500 bp in length were removed. 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 / ) comes with a whole genome database of model strains. The whole genome data of strain P-14-21 was uploaded to the database to establish the genome phylogenetic tree of strain P-14-21. At the same time, 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 strain P-14-21 and the genome sequences of the 5 most similar bacterial species.

[0038] The results showed that the genome size of strain P-14-21 was 5210258 bp, the G+C content was 61.62%, the N50 was 620010 bp, the genome integrity was 100%, and the contamination was 0.43%. According to the genome integrity of not less than 95% and the contamination of not more than 5%, it is considered as a high-quality genome, which indicates that the genome of strain P-14-21 is a high-quality genome. By counting the ANI and dDDH values between strain P-14-21 and the genome sequences of the 5 most similar bacterial species, the results showed that the ANI value between strain P-14-21 and the model bacterial species was 80.57~80.80%, and the dDDH value was 24.1~24.2% (Table 1), which was much lower than the threshold of 70% and 95% for distinguishing new bacterial species by dDDH and ANI (if the ANI between the genomes of two strains is >95% or dDDH is >70%, then the two strains belong to the same bacterial species), which further indicates 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 (Fig. 1), Figure 4 which indicates that strain P-14-21 has an independent taxonomic status.

[0039] In summary, strain P-14-21 is a new species of Pseudomonas (P-14-21), which was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on August 7, 2024, at 100 Xianlie Road, Guangzhou, Guangdong, China, Postcode: 510070, and the deposit number is GDMCC No:64969. Pseudomonas sp . ) and is named P-14-21. The strain was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on August 7, 2024, at 100 Xianlie Road, Guangzhou, Guangdong, China, Postcode: 510070, and the deposit number is GDMCC No:64969. Pseudomonas sp . P-14-21, which was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on August 7, 2024, at 100 Xianlie Road, Guangzhou, Guangdong, China, Postcode: 510070, and the deposit number is GDMCC No:64969.

[0040] Table 1 ANI and dDDH between strain P-14-21 and type strain

[0041]

[0042] Preservation of Pseudomonas P-14-21: The identified single colony was transferred to the corresponding test tube slant and stored in a 4°C refrigerator for standby. At the same time, the bacterial suspension was prepared by using 50% glycerol distilled water solution and stored in a -80°C ultra-low temperature freezer.

[0043] Example 3: Preliminary screening of Pseudomonas P-14-21 for its ability to dissolve organic phosphorus and produce siderophores

[0044] 1. Strain activation

[0045] The glycerol tube containing the strain was taken out from the -80°C refrigerator, dissolved at room temperature, and streaked on the NA solid medium (medium formula same as in Example 2). The plate was inverted and placed in a 30°C constant temperature incubator for 24 h to obtain activated strains.

[0046] 2. Medium preparation

[0047] Organic phosphorus solid medium formula: glucose 10.0 g / L, ammonium sulfate 0.5 g / L, yeast extract powder 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 water, pH 7.0-7.5.

[0048] Siderophore-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 phosphate monobasic dihydrate 295.25 mg / L, sodium phosphate dibasic dodecahydrate 1213.5 mg / L, ammonium chloride 125 mg / L, potassium phosphate monobasic 37.5 mg / L, sodium chloride 62.5 mg / L, solvent water, pH 6.8.

[0049] Preparation of the medium: according to the formula, the medium was weighed, 1000 mL of deionized water was added, stirred and heated to complete dissolution, the pH of the solution was adjusted to the corresponding value, and then the solution was divided into flasks, sterilized at 121°C for 15 min, and then poured into disposable petri dishes for standby.

[0050] 3. Inoculation

[0051] The plates containing the solid medium for identification of organophosphorus and siderophore production were divided into four parts, and the activated bacterial cells were inoculated, respectively. The plates were placed in a constant temperature incubator at 30°C and cultured for 7 days. The results were observed.

[0052] 4. Results

[0053] From Figure 2 (A) and Figure 3 (A), it can be seen that strain P-14-21 produces transparent circles around the plate containing the solid medium for identification of organophosphorus and siderophore production. Studies have shown that if a transparent circle appears around the microorganism on a plate containing organophosphorus, it indicates that the microorganism has the ability to dissolve organophosphorus; if an orange-yellow transparent circle appears on the plate containing the solid medium for identification of siderophore production, it indicates that the microorganism has the ability to produce siderophore. Therefore, it is concluded that strain P-14-21 has the ability to dissolve organophosphorus and produce siderophore.

[0054] Example 4: Evaluation of the ability of Pseudomonas P-14-21 to dissolve organophosphorus and produce siderophore

[0055] 1. Activation of the strain

[0056] The same as step 1 of Example 3.

[0057] 2. Preparation of the medium

[0058] The formula of the organophosphorus solid medium is the same as step 2 of Example 3.

[0059] The formula of NB medium is to remove agar from NA medium in Example 2.

[0060] CAS detection solution: A solution, 0.605 g CAS was added to 500 mL distilled water, 0.027 g FeCl3·6H20 was added to 100 mL dilute hydrochloric acid; B solution, 0.729 g HDTMA (hexadecyl trimethyl ammonium bromide) was added to 400 mL distilled water. Then A solution was slowly added to B solution to obtain blue CAS detection solution. The detection solution was prepared and used immediately.

[0061] 3. Inoculation

[0062] The plate containing organic phosphorus and siderophore-producing identification solid culture medium is four-divided, and the activated bacterial body is spotted, four times repeated, and the plate is placed in a constant temperature incubator at 30 DEG C in an inverted manner, cultured for 5 days for the plate containing organic phosphorus, and cultured for 7 days for the siderophore-producing identification plate, and the observation result is taken out after the culture is finished, the cross method is used to measure the transparent circle diameter (HD) and the colony diameter (CD) in mm, and the phosphorus-dissolving index and the siderophore-producing index are counted.

[0063] Phosphorus-dissolving index = transparent circle diameter (HD) / colony diameter (CD) ;

[0064] Siderophore-producing index = transparent circle diameter (HD) / colony diameter (CD) ;

[0065] Siderophore secretion determination: a single colony is picked and inoculated in NB liquid medium, 30 DEG C, 200 r / min oscillation culture for 5 days, after the culture is finished, the bacterial suspension is centrifuged at 10000 r / min for 8 min, 5 mL of supernatant is mixed with 5 mL of CAS detection liquid, and the mixture is fully mixed, and the mixture is placed at room temperature for 1 h, the absorbance value (As) is measured at 630 nm wavelength by spectrophotometer, and the absorbance value is measured by using sterile NB liquid medium 5 mL and CAS detection liquid 5 mL as the reference value (Ar), and the siderophore activity unit (U) of the strain P-14-21 is determined. su

[0066] su = [(Ar - As) / Ar] × 100%

[0067] 4、Results

[0068] As shown in Table 2, the strain P-14-21 has better ability to dissolve organic phosphorus and produce siderophore, the phosphorus-dissolving index is 2.4, the siderophore-producing index is 2.97, and the siderophore activity unit (U) reaches 94.41%. su

[0069] Table 2 Analysis of the ability of Pseudomonas P-14-21 to dissolve organic phosphorus and produce siderophore

[0070]

[0071] The above detailed description is a specific description of the embodiments of the present application, and the embodiments are not used to limit the patent scope of the present application, and any equivalent implementation or change without departing from the present application should be included in the patent scope of the present application.​​

Claims

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

2. A microbial agent, characterized in that, The bacterial agent comprises a culture of Pseudomonas P-14-21 as described in claim 1.

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

4. The use of the Pseudomonas P-14-21 of claim 1 or the bacterial agent of claim 2 in the preparation of products that produce iron carriers and dissolved organophosphates.

5. The application according to claim 4, characterized in that, The products include bio-fertilizers or soil remediation agents.

6. A bio-fertilizer, characterized in that, The active ingredient contains either the Pseudomonas P-14-21 of claim 1 or the bacterial agent of claim 2.

7. A soil remediation agent, characterized in that, The active ingredient contains either the Pseudomonas P-14-21 of claim 1 or the bacterial agent of claim 2.

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