Wild tamarix chinensis rhizosphere Prussiella aryabilis TC214 and phosphate solubilizing application thereof under saline-alkali stress

Abstract

The invention discloses wild tamarix chinensis rhizosphere Prussiella aryabilis TC214 and phosphate solubilizing application thereof under saline-alkali stress, and belongs to the technical field of phosphate solubilizing microbial fertilizers. The preservation number of the wild tamarix chinensis rhizosphere prelisteria aryi TC214 disclosed by the invention is CGMCC (China General Microbiological Culture Collection Center) No.31822, and the wild tamarix chinensis rhizosphere prelisteria aryi TC214 is preserved in the China General Microbiological Culture Collection Center on September 2, 2024. The wild Chinese tamarisk rhizosphere Prussiella aryabilis TC214 disclosed by the invention can tolerate saline-alkaline conditions of 10% NaCl with the pH value of 9, and also has relatively high phosphate solubilizing capacity, and the phosphate solubilizing amount reaches 106.94 mg / L after being cultured for 84 hours; and after the strain is inoculated under a saline-alkali condition, the germination rate of the suaeda salsa seeds is greatly improved, and the strain has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of phosphate-solubilizing microbial fertilizers, and more specifically, to an Arthrobacter arilaitensis strain TC214 in the rhizosphere of wild Tamarix and its application in phosphate solubilization under saline-alkali stress. Background Art

[0002] The attenuation of phosphorus availability caused by saline-alkali stress has become a key limiting factor for the growth of halophytes. Although the total phosphorus reserve in the soil is abundant, the phosphorus that can be absorbed by plants only accounts for 0.1% of the total phosphorus; while the high-saline-alkali environment further inhibits the biological availability of phosphorus through mechanisms such as ion toxicity and pH perturbation, forming a nutrient supply-demand contradiction of "high reserve - low activation". The core scientific problem derived from this is: how to awaken the "dormant" state of the soil phosphorus pool through biological activation technology, convert insoluble phosphorus in situ into plant-available phosphorus, and then reconstruct the phosphorus availability balance in the saline-alkali habitat.

[0003] As the biological engine for soil phosphorus activation, significant progress has been made in recent years in studying the functional mechanisms of phosphate-solubilizing microorganisms. These microorganisms can effectively dissociate insoluble phosphorus such as iron / aluminum-bound phosphorus by secreting metabolites such as organic acids and phosphatases, and mineralize inorganic phosphate and organic phosphorus into plant-available forms. For saline-alkali areas, salt-tolerant phosphate-solubilizing bacteria are of great significance for solving the phosphorus limitation problems of regional soils and vegetation. The phosphate-solubilizing ability of each phosphate-solubilizing bacterium is significantly affected by strain characteristics and environmental conditions. At present, the salt tolerance of the screened phosphate-solubilizing strains is generally not high, and the phosphate-solubilizing effect in saline-alkali soil is poor. As a microbial inoculant for improving saline-alkali soil, it has not been successfully and comprehensively applied. Based on this, screening phosphate-solubilizing bacteria that can tolerate both high salt and high alkali environments is of great significance for developing phosphate-solubilizing bacterial fertilizers that are stably and efficiently applied to saline-alkali soils.

[0004] Therefore, providing an Arthrobacter arilaitensis strain TC214 in the rhizosphere of wild Tamarix and its application in phosphate solubilization under saline-alkali stress is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides an Arthrobacter arilaitensis strain TC214 in the rhizosphere of wild Tamarix and its application in phosphate solubilization under saline-alkali stress. The Arthrobacter arilaitensis strain TC214 can increase the content of available phosphorus and promote seed germination.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A wild Priestia aryabhattai strain TC214 from the rhizosphere of Tamarix ramosissima, with the preservation number CGMCC No. 31822, has been deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, abbreviated as CGMCC, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was September 2, 2024, and the taxonomic name is Priestia aryabhattai.

[0008] Furthermore, the application of the wild Priestia aryabhattai strain TC214 from the rhizosphere of Tamarix ramosissima in phosphorus solubilization.

[0009] Furthermore, the application of the wild Priestia aryabhattai strain TC214 from the rhizosphere of Tamarix ramosissima in phosphorus solubilization under saline-alkali stress.

[0010] Furthermore, the wild Priestia aryabhattai strain TC214 from the rhizosphere of Tamarix ramosissima can survive in a saline-alkali environment with a pH of 9 and 10% NaCl.

[0011] Furthermore, the application of the wild Priestia aryabhattai strain TC214 from the rhizosphere of Tamarix ramosissima in promoting seed germination.

[0012] Furthermore, a saline-alkali-tolerant microbial inoculant, including the wild Priestia aryabhattai strain TC214 from the rhizosphere of Tamarix ramosissima.

[0013] Through the above technical solutions, compared with the prior art, the present invention discloses a wild Priestia aryabhattai strain TC214 from the rhizosphere of Tamarix ramosissima and its application in phosphorus solubilization under saline-alkali stress. The Priestia aryabhattai strain TC214 can tolerate saline-alkali conditions with a pH of 9 and 10% NaCl, and at the same time has a high phosphorus solubilization ability. After culturing for 84 h, the phosphorus solubilization amount reaches 106.94 mg / L; and after inoculating this strain under saline-alkali conditions, the germination rate of Suaeda salsa seeds is greatly improved, having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0015] Figure 1 This shows the phosphorus solubilization effect of the Priestia aryabhattai strain TC214 of the present invention in inorganic phosphorus and organic phosphorus solid media; among them, A: inorganic phosphorus solid medium; B: organic phosphorus solid medium;

[0016] Figure 2 This is the inorganic phosphorus solubilization kinetic curve of Priestia aryabhattai TC214 of the present invention; AP: available phosphorus; Bacterial absorbance: bacterial absorbance.

[0017] Figure 3 This is the effect of different NaCl concentrations on the inorganic phosphorus solubilization ability of Priestia aryabhattai TC214 of the present invention; AP: available phosphorus; Terminal pH: end point pH value.

[0018] Figure 4 This is the effect of different pH values on the inorganic phosphorus solubilization ability of Priestia aryabhattai TC214 of the present invention; AP: available phosphorus; Terminal pH: end point pH value. Detailed implementation manners

[0019] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0020] Example 1

[0021] (1) Strain source

[0022] The strain screening sample was collected from the rhizosphere soil of wild Tamarix chinensis Lour. in the Yellow River Delta tidal flat (37°33′17″N, 118°56′2″E). The medium used for strain cultivation was LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L).

[0023] The inorganic phosphorus solubilization ability was determined using an inorganic phosphorus medium (without agar) (HaiBo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park); the organic phosphorus solubilization ability was determined using an organic phosphorus medium (without agar) (HaiBo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park).

[0024] (2) Strain identification

[0025] The strain was screened by the dilution plate coating method. The screened strain was subjected to PCR amplification using the 16S rDNA universal primer sequences F27 and R1492, and after completion, it was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results are shown in SEQ ID NO.1. The gene sequence was logged in to the website of the National Center for Biotechnology Information, USA (http: / / www.ncbi.nlm.nih.gov) for nucleotide sequence Blast alignment, and several nucleotide sequences homologous to the gene sequences of related strains were obtained. The results showed that the homology of the strain with the gene sequence of Priestia aryabhattai was 100%. The isolated strain was identified as Priestia aryabhattai and named Priestia aryabhattai TC214.

[0026]

[0027] Priestia aryabhattai strain TC214, with the preservation number CGMCC No. 31822, has been deposited in the China General Microbiological Culture Collection Center, abbreviated as CGMCC, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was September 2, 2024, and the taxonomic name is Priestia aryabhattai.

[0028] Example 2: Determination of the growth and phosphorus-solubilizing ability of strain TC214

[0029] (1) Using the spot inoculation method, inoculate the test strains on the inorganic phosphorus solid medium plate and the organic phosphorus solid medium plate respectively, with 3 replicates for each. Incubate in an incubator at 28 °C for 5 days. Judge the phosphorus-solubilizing ability of the strains according to whether there is a phosphorus-solubilizing circle around the colonies and its size, and use Digimizer software to calculate the phosphorus-solubilizing coefficient of the strains. The calculation formula for the phosphorus-solubilizing coefficient is as follows:

[0030]

[0031] The phosphorus-solubilizing effects of Priestia aryabhattai TC214 in the inorganic phosphorus and organic phosphorus solid media are shown in Figure 1 , the inorganic phosphorus-solubilizing coefficient is 1.402, and the organic phosphorus-solubilizing coefficient is 1.567.

[0032] (2) After activating strain TC214 with LB liquid medium, take the strain seed liquid (OD 600 is about 1), transfer it to the inorganic phosphorus liquid medium for cultivation according to an inoculation amount of 1%, mix evenly, place it in a shaker at 28 °C and 180 r / min for cultivation. Continuously draw the fermentation broth every 12 h for 7 days, set 3 replicates, and take the average value. Zero the instrument with the fermentation broth as the blank control. Calculate the net release amount of available phosphorus in the strain fermentation broth according to the calculation formula (as follows) to judge the phosphorus-solubilizing ability of the strain, and measure the absorbance value of the cell mass at 600 nm to judge the growth of the cell mass, and draw the phosphorus-solubilizing kinetic curve of the phosphorus-solubilizing bacteria.

[0033] The calculation formula is as follows:

[0034] P = K × (V1 ÷ V2)

[0035] Where: P is the available phosphorus content; K is the phosphorus content (mg / L) obtained from the standard curve; V1 is the volume (mL) of the solution for constant volume during color development; V2 is the volume (mL) of the supernatant taken during color development.

[0036] Using the non-inoculated medium as a blank control, the soluble phosphorus content in the phosphate-solubilizing bacteria fermentation broth was determined by the molybdenum blue colorimetric method, with 4 replicates.

[0037] The growth of strain TC214 gradually increased with time, growing rapidly in the first 12 h and then slowing down; the amount of phosphorus solubilized from liquid inorganic phosphorus by the strain showed an increasing trend in the first 60 h, then fluctuated, and gradually decreased after 120 h, reaching a peak of 106.94 mg / L at 84 h( Figure 2 ).

[0038] Example 3 Determination of the salt and alkali tolerance ability of strain TC214

[0039] Based on the inorganic phosphorus liquid medium as the basal medium, after activating the preserved strain, take the seed liquid of strain TC214 (OD 600 about 1), transfer it to the liquid medium according to an inoculation amount of 1%, determine the culture time according to the growth curve, and the basis for selecting the culture time is the peak of phosphorus solubilization in the phosphorus solubilization kinetic curve of the strain (i.e., 84 h). Media with different salt concentrations (0%, 1%, 2.5%, 4%, 5.5%, 7%, 8%, 9%, 10%, 20% and 30%) and pH values (7, 8, 9, 10, 11 and 12) were prepared (3 replicates for each group), and cultured in a shaker at 28 °C and 180 r / min. After the culture was completed, 1 mL of the fermentation broth was taken and centrifuged at 6000 r / min for 12 min to determine the available phosphorus content and the final pH of the fermentation broth.

[0040] The ability of strain TC214 to solubilize inorganic phosphorus gradually increased within the range of 7% salt concentration, reaching 98.96 mg / L, and then gradually decreased. The amount of phosphorus solubilized within the salt concentration range of 1% - 10% was above 50 mg / L; the amount of phosphorus solubilized was inversely proportional to the final pH value, indicating that acidic substances were produced during the phosphorus solubilization process( Figure 3 ).

[0041] Under alkaline conditions, with the increase of pH, the inorganic phosphorus solubilization ability of strain TC214 first increased and then decreased, and it could withstand the stress of pH = 9 at most (the amount of phosphorus solubilized was above 60 mg / L). When pH = 8, the phosphorus solubilization ability of the strain was the strongest, reaching 114.964 mg / L( Figure 4 ).

[0042] Example 4 Determination of the effect of strain TC214 on the germination rate of Suaeda salsa

[0043] Suaeda salsa seeds were collected from wild plants growing on the Yellow River Delta tidal flat (37°33′17″N, 118°56′2″E).

[0044] Select seeds of Suaeda salsa that are plump, uniform, disinfect them with 75% ethanol for 1 min, wash them 3 times with sterile water, then disinfect them with 10% sodium hypochlorite for 10 min, and wash them 5 times with sterile water before air-drying. Immerse the disinfected Suaeda salsa seeds in the seed solution of strain TC214 (OD 600 is about 1) for 3 h, soak them in sterile water as a control for 3 h, then rinse them 5 times with sterile water, and blot the surface moisture dry with absorbent paper for standby.

[0045] Add 10 mL of treatment solutions with different concentrations into sterilized petri dishes respectively, lay a single layer of sterilized filter paper flat on the bottom of the petri dishes, place 30 seeds of Suaeda salsa pretreated differently evenly in each petri dish, and seal them with sealing film to keep the concentration of each treatment unchanged.

[0046] This experiment adopted a two-factor experimental design. One factor was the treatment of salt, alkali, and saline-alkali mixed stress, and the control group 1 (CK1) was treated with sterile water. The other factor was the strain inoculation treatment, and the control group 2 (CK2) was treated with an uninoculated LB medium instead of the strain suspension. The specific experimental design scheme is shown in Table 1, and each treatment was set with 3 replicates. The experiment was carried out in an artificial climate chamber, and the conditions set for the artificial climate chamber were: during the day: light time 12 h / d, relative humidity 70%, daytime light intensity 80%, constant temperature of 25 °C, and constant temperature of 20 °C at night.

[0047] Table 1 Two-factor experimental design

[0048]

[0049] The experiment used the breakthrough of the seed radicle through the seed coat by 1 mm as the germination standard, observed it regularly every day, and recorded the number of germinated seeds every day. The germination potential was calculated on the 4th day, and the observation lasted for 5 days in total. Calculate the germination rate, vigor index, growth promotion rate of strain inoculation, germination potential, germination index, etc. of the seeds according to the following formulas.

[0050] Seed germination rate (G) = actual number of germinated seeds / total number of seeds × 100%;

[0051] Growth promotion rate = (germination rate of treatment group - germination rate of control group) / germination rate of control group × 100%;

[0052] Germination potential = number of germinated seeds of the tested seeds within 4 days / number of tested seeds × 100%;

[0053] Germination index GI = ∑Gt / Dt, where Gt is the number of germinations on day t and Dt is the corresponding germination days;

[0054] Vigor index = GI × fresh weight (g).

[0055] When the phosphate-solubilizing bacterium TC214 was not inoculated, the germination rate of Suaeda salsa seeds gradually decreased under saline-alkali stress conditions. Through the treatment of soaking seeds with the phosphate-solubilizing bacterium for 3 h, under different saline-alkali stress treatments, compared with the control group, the vigor index, germination potential, germination rate, and germination index of the seeds all increased. Under the treatment condition of a salt concentration of 450 mM, the growth promotion rate of strain TC214 on Suaeda salsa seeds reached 30.34%; under the treatment condition of an alkali concentration of 50 mM, the growth promotion rate of strain TC214 on Suaeda salsa seeds reached 20.83%, and then the growth promotion effect decreased with the increase of the alkali stress concentration; while under the condition of a salt-alkali concentration of 450 mM - 150 mM, the growth promotion rate of strain TC214 on Suaeda salsa seeds was 37.66%. Generally, under high-salt and saline-alkali mixed conditions, the growth promotion effect of Priestia aryabhattai TC214 on the germination rate of Suaeda salsa was more obvious (Table 2).

[0056] Table 2 Growth conditions of Suaeda salsa under different treatments and different saline-alkali conditions

[0057]

[0058] Note: Y1, Y2, and Y3 represent the treatments with salt concentrations of 150 mM, 300 mM, and 450 mM, J1, J2, and J3 represent the treatments with alkali concentrations of 50 mM, 100 mM, and 150 mM, and Y1-J1, Y2-J2, and Y3-J3 represent the treatments with salt-alkali concentrations of 150 mM - 50 mM, 300 mM - 100 mM, and 450 mM - 150 mM.

[0059] By inoculating Priestia aryabhattai TC214 in saline-alkali areas, the available phosphorus content in the soil increased, the plant breeding rate was enhanced, and the ecological environment was improved.

[0060] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A wild Priestia aryabhattai TC214 in the rhizosphere of Tamarix chinensis, characterized in that, Its preservation number is CGMCC No. 31822, and it has been preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation date is September 2, 2024.

2. Application of the strain of Priestia aryabhattai TC214 from the rhizosphere of wild Tamarix chinensis described in claim 1 in phosphorus solubilization.

3. Application of the strain of Priestia aryabhattai TC214 from the rhizosphere of wild Tamarix chinensis described in claim 1 in phosphorus solubilization under saline-alkali stress.

4. The application according to claim 3, characterized in that, The strain of Priestia aryabhattai TC214 from the rhizosphere of wild Tamarix chinensis can survive in a saline-alkali environment with a pH of 9 and 10% NaCl.

5. Application of the strain of Priestia aryabhattai TC214 from the rhizosphere of wild Tamarix chinensis described in claim 1 in promoting seed germination.

6. A salt-tolerant and alkali-tolerant microbial inoculant, characterized in that, It includes the strain of Priestia aryabhattai TC214 from the rhizosphere of wild Tamarix chinensis described in claim 1.

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