A protozoan strain of Renilla and its application in promoting salt-alkali tolerance growth of corn

Abstract

A protozoan strain of Renilla spp. and its application in promoting salt-alkali tolerance in corn, the protozoan Renilla spp. ISS-TY1, has been deposited in a designated depository institution designated by the State Intellectual Property Office on April 22, 2025. The depository institution is the China Center for Type Culture Collection, and the deposit number is CCTCC NO: C202521. The study found that treatment with Renilla spp. ISS-TY1 increased the aboveground biomass of corn by 183% to 197%, significantly reduced the abscisic acid content in the roots by 30.3% to 43.1%, and reduced the K content in the leaves by 1.5%. + The content increased by 20.7%~29.2%, while Na + The content was reduced by 18.5% to 26.8%. The present invention reveals that ISS-TY1 has great application potential and prospects in improving the salt-alkali tolerance of crops and promoting crop growth.

Description

Technical Field

[0001] The invention belongs to the field of microbial protists, and particularly relates to a protozoan Renilla and an application thereof in promoting the salt-alkali resistant growth of corn. Background Art

[0002] Corn is a globally important food crop, widely grown in a variety of soil types. However, the presence of saline-alkali land severely limits corn cultivation and yield. High salinity in saline-alkali land can deteriorate soil structure, reduce nutrient availability, and significantly inhibit corn growth and development, resulting in poor root development, stunted plants, and impaired photosynthesis, ultimately leading to a significant decrease in corn yield.

[0003] Salt-alkali stress can also trigger a series of secondary problems. In high-salt environments, sodium ions in the soil displace beneficial ions such as calcium and magnesium in soil colloids, causing soil compaction and reduced aeration and water permeability, further exacerbating corn's growth difficulties. Furthermore, the high pH in saline-alkali land reduces the availability of nutrients such as phosphorus, zinc, and iron in the soil, making it difficult for corn to absorb sufficient nutrients to meet its growth needs.

[0004] In this unfavorable soil environment, the activity and diversity of soil microorganisms will also be seriously affected. Many beneficial microorganisms find it difficult to survive under high saline-alkali conditions, resulting in impaired functions of the soil micro-ecosystem. Microorganisms play a key role in soil nutrient cycling, organic matter decomposition, and plant rhizosphere growth promotion. A reduction in their number and activity will weaken the fertility and ecological functions of the soil, further restricting the growth of corn. However, recent studies have found that protozoa play a key role in saline-alkali soil ecosystems. They are not only widely present in extreme environments, but also abundant in number, with about 10 protozoa per gram of soil. 4 ~10 8 Furthermore, studies have shown that protists play an important role in promoting plant growth and health. A deeper understanding of how protists help saline-alkali-tolerant crops grow and mitigate barriers can help us improve saline-alkali soils more efficiently and environmentally friendly.

[0005] Currently, there are no systematic reports on protists significantly improving both salinity and alkaline tolerance in maize and promoting its growth. Therefore, in-depth research on the potential application of protists in maize cultivation on saline-alkali soils is of great scientific and practical significance for overcoming the limitations of saline-alkali soils and increasing maize yields. Summary of the Invention

[0006] Technical Problem to be Solved: The present invention provides a protozoan Renilla and its application in promoting saline-alkali tolerant growth of corn. The protozoan Renilla is derived from soda saline-alkali soil, is easy to culture, and reproduces quickly. The protozoan Renilla plays a significant role in promoting corn growth. At the same time, the protozoan Renilla can significantly reduce the Na content in corn leaves. + Content and Na + / K + The ratio has a positive effect on improving the salt-alkali tolerance of corn.

[0007] Technical solution: A protozoan strain of Renilla spp. ISS-TY1 ( Colpoda inflata ISS-TY1), the expanded reniformis ISS-TY1 was deposited in the China Center for Type Culture Collection (CCTCC), address: Wuhan University, Wuhan, China, deposit date: April 22, 2025, deposit number: CCTCC NO: C202521.

[0008] The above-mentioned protozoan Renilla spp. ISS-TY1 ( Colpoda inflata Application of ISS-TY1) in promoting corn growth and improving corn salt-alkali tolerance.

[0009] The culture method of the above-mentioned ISS-TY1 of the pneumocystis pulex comprises the following steps: inoculating the pneumocystis pulex ISS-TY1 into a PAS buffer; adding inactivated Escherichia coli as a food source; and culturing at a constant temperature of 25° C. for 72 hours.

[0010] The preparation method of the above-mentioned PAS buffer is as follows: prepare buffer A: dissolve 0.142 g Na2HPO4 and 0.136 g KH2PO4 in 500 mL of sterile water; prepare buffer B: dissolve 4 mg MgSO4·7H2O, 4 mg CaCl2·2H2O and 0.12 g NaCl in 500 mL of sterile water; buffer A and buffer B are sterilized at 121°C for 15 minutes respectively and then mixed to make 1000 mL of PAS buffer.

[0011] The above-mentioned ISS-TY1 protozoa suspension after enrichment culture is applied at 10% per corn plant. 4 ~10 6 The concentration of 10 cells was applied to the rhizosphere soil of corn.

[0012] The application comprises applying the enlarged kidneyworm ISS-TY1 and corn seeds simultaneously when planting corn in saline-alkali soil.

[0013] The above-mentioned corn varieties include Pengcheng No. 10 and Ketai 881.

[0014] The amount of the inactivated E. coli added was 10 6 ~10 7 A bacterium.

[0015] A soil conditioner for promoting corn growth and improving corn's salt-alkali tolerance, wherein the active ingredient includes the above-mentioned protozoan Renilla sphaeroides ISS-TY1.

[0016] Beneficial effects: The present invention isolates and screens the protozoan ISS-TY1 from soda saline-alkali soil, which improves the salt-alkali tolerance of corn and promotes the growth of corn. Pot experiments show that ISS-TY1 can significantly increase the biomass of corn. In addition, ISS-TY1 can also improve the resistance of corn to salt-alkali stress. Therefore, the ISS-TY1 of the present invention plays an important role in improving the salt-alkali tolerance of corn. The study found that the aboveground biomass of corn treated with ISS-TY1 increased by 183% to 197%, the abscisic acid content in the roots was significantly reduced by 30.3% to 43.1%, and the K content in the leaves was significantly reduced by 30.3% to 43.1%. + The content increased by 20.7% to 29.2%, while Na + The content was reduced by 18.5% to 26.8%. The present invention reveals that ISS-TY1 has great application potential and prospects in improving the salt-alkali tolerance of crops and promoting crop growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The protozoan Renilla inflata ISS-TY1 of the present invention ( Colpoda inflata ISS-TY1) Photographs of the morphological characteristics of vegetative bodies A and dormant cysts B.

[0018] Figure 2 The potted plant test in Example 3 of the present invention was inoculated with ISS-TY1 ( Colpoda inflata Comparison of corn growth between the treatments of ISS-TY1) and blank control (CK), where S1 represents Pengcheng 10 and S2 represents Ketai 881.

[0019] Figure 3 The potted plant test in Example 3 of the present invention was inoculated with ISS-TY1 ( Colpoda inflata Comparison of salt-alkali tolerance growth effects of corn treated with ISS-TY1) and blank control (CK), including A biomass comparison, B root base abscisic acid content comparison, C leaf potassium content comparison, and D leaf sodium content comparison. S1 represents Pengcheng No. 10, and S2 represents Ketai 881. DETAILED DESCRIPTION

[0020] The following examples are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent transformations or modifications made according to the spirit of the present invention should be included within the scope of protection of the present invention. Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent companies.

[0021] Page's saline (PAS) buffer solution: Dissolve 0.142 g Na₂HPO₄ and 0.136 g KH₂PO₄ in 500 mL of sterile water to make buffer A. Separately, dissolve 4 mg MgSO₄·7H₂O, 4 mg CaCl₂·2H₂O, and 0.12 g NaCl in 500 mL of sterile water to make buffer B. Buffers A and B are sterilized separately in an autoclave at 121°C for 15 minutes. After cooling, mix and shake in a clean hood to make 1000 mL of PAS buffer. Water agar plates (1.5% wateragar plates): Add 1.5 g agar to 100 mL of sterile water and dissolve thoroughly. Autoclave at 121°C for 15 minutes. Pour a small amount of the medium onto the plates to a thickness of approximately 1.5 mm. Cool and set aside. Nutrient Broth (NB) medium: Dissolve 10.0 g peptone, 3.0 g beef extract powder, and 5.0 g NaCl in 1000 mL of distilled water and autoclave at 121°C for 15 min. Yeast Extract Powder (1.0% YPD) medium: Dissolve 2.0 g peptone, 2.0 g glucose, and 1.0 g yeast extract powder in 1000 mL of distilled water and autoclave at 121°C for 15 min.

[0022] Preparation of a humid chamber: According to the method for making a humid chamber in Protozoology Experimental Techniques (Pang Yanbin, 1991), a thin layer of absorbent cotton was spread on the bottom of the foam box. An appropriate amount of distilled water was added to allow the cotton to absorb enough water. Two glass rods were then inserted close to the cotton layer to fix the 96-well plate and culture dish.

[0023] Example 1 Isolation and purification of protists

[0024] Because saline-alkali soil has low soil moisture content and low protozoan activity, it is necessary to pre-cultivate the soil sample. Weigh 10.0 g of soil, add appropriate amount of water to maintain the soil moisture at 20%, add appropriate amount of food source along with the water, and incubate at 25°C for 7 to 14 days. Then collect the soil sample and separate the protozoa. The separation steps are as follows: weigh 1.0 g of rhizosphere soil of salt-alkali tolerant corn varieties into a 50 mL centrifuge tube, add 30 mL of PAS buffer solution, place the centrifuge tube in a shaker at 200 rpm and 25°C for 30 minutes to fully mix the soil particles and the buffer solution, and then use a vortex shaker to shake vigorously for 20 seconds to separate the protozoa from the soil particles as much as possible. Let the centrifuge tube stand for 30 minutes. When the soil suspension is slightly clarified, add 95 μL of PAS buffer solution to each well of a 96-well plate in a clean bench, add 5 μL of soil suspension, and then add 10 μL of PAS buffer solution to each well of the 96-well plate. -1 , 10 -2 , 10 -3 and 10 -4 Perform serial dilutions and add 10 μL of bacterial food source to each well. Completely seal the 96-well plate with sealing film and incubate in a humidified room at 15°C incubator in the dark. Observe the growth of the protists under an inverted microscope (×200 magnification) on days 3 and 7 of culture. Separate and dilute the protists stepwise under an inverted microscope to obtain single protists.

[0025] Example 2 Identification of protists

[0026] Total DNA from protists was extracted using the Neasy Blood & Tissue Kit (QIAGEN NV, Maryland, USA) and the kit protocol was followed. PCR amplification of the protist 18S rRNA gene was performed using primers RibA (5'-ACCTGGTTGATCCTGCCAGT-3') as shown in SEQ ID NO:1 and RibB (5'-TGATCCATCTGCAGGTTCACCTAC-3') as shown in SEQ ID NO:2. The PCR amplification system was prepared in a 50 μL volume consisting of 25 μL of 2× Taq PCR Master Mix, 15 μL of DNA template, 1.25 μL each of primers RibA and RibB, and ddH2O added to bring the volume up to 50 μL. The PCR amplification program was as follows: preheat denaturation at 98°C for 3 min, followed by 30 cycles of 98°C for 45 s, 55°C for 45 s, and 72°C for 45 s, and a final step of 72°C for 10 min. The amplified products were sent to a testing company for sequencing. The obtained 18S rDNA gene sequences were compared with the NT (Nucleotide Sequence Database) database on the NCBI website using BLAST software. The phylogenetic analysis of the isolated protozoa was performed using MEGA software. Colpoda inflata The similarity was the highest, so the isolated protozoan ISS-TY1 was identified as Colpoda inflata The strain was deposited with the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on April 22, 2025, with the accession number CCTCC NO: C202521. Protist names were compared with those at the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov).

[0027] Example 3 Protists Improve Corn Growth and Salt-Alkali Tolerance in Saline-Alkali Soils

[0028] Experimental setup: (1) blank control group CK; (2) treatment group inoculated with ISS-TY1. We provided two corn varieties: Pengcheng No. 10 and Ketai 881. Each treatment was repeated three times, for a total of 2×2×3 = 12 samples (inoculated and non-inoculated treatments, two corn varieties, and three replicates). The protozoa tested were ISS-TY1 isolated in Example 1. After the purified and enriched protozoa samples were evenly mixed, 10 μL of the enrichment solution was evenly added to a Neubauer counting plate for counting, and the abundance was observed using an inverted microscope. Each sample was counted at least 3-5 times, and the average value was taken as the final concentration. The enriched protozoa were added near the root zone, and the corn growth and salt-alkali tolerance indicators were tested after harvest. The study found that the aboveground biomass of corn treated with the protozoan ISS-TY1 increased by 183% to 197% ( Figure 3 :A), the ABA content in the roots decreased significantly by 30.3% to 43.1% ( Figure 3 :B), K in leaves + The content increased by 20.7% ~ 29.2% ( Figure 3 :C), and Na + The content decreased by 18.5% to 26.8% ( Figure 3 :D). This indicates that the salt-alkali resistance, biomass, and growth of corn plants have been significantly improved.

Claims

1. A strain of protozoan Nephroderma inflatum ISS-TY1 ( Colpoda inflata ISS-TY1), characterized by The ISS-TY1 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C202521 and a deposit date of April 22, 2025.

2. The protozoan Renilla spp. ISS-TY1 according to claim 1 ( Colpoda inflata Application of ISS-TY1) in promoting corn growth and improving corn salt-alkali tolerance.

3. The use according to claim 2, characterized in that The culture method of the ISS-TY1 reniformis comprises the following steps: inoculating the ISS-TY1 reniformis into a PAS buffer; adding inactivated Escherichia coli as a food source; and culturing at a constant temperature of 25°C.

4. The use according to claim 3, characterized in that The preparation method of the PAS buffer is as follows: preparing buffer A: dissolving 0.142 g Na2HPO4 and 0.136 g KH2PO4 in 500 mL of sterile water; preparing buffer B: dissolving 4 mg MgSO4·7H2O, 4 mg CaCl2·2H2O and 0.12 g NaCl in 500 mL of sterile water; buffer A and buffer B are sterilized at 121°C for 15 minutes respectively and then mixed to prepare 1000 mL of PAS buffer.

5. The use according to claim 2, characterized in that The application method of ISS-TY1 is as follows: the protozoan suspension after enrichment culture is applied at 10 4 ~10 6 The concentration of 10 cells was applied to the rhizosphere soil of corn.

6. The use according to claim 2, characterized in that The application comprises applying the enlarged kidneyworm ISS-TY1 and corn seeds simultaneously when planting corn in saline-alkali soil.

7. The use according to claim 2, characterized in that The corn varieties include Pengcheng No. 10 and Ketai 881.

8. The use according to claim 3, characterized in that The amount of inactivated Escherichia coli added is 10 6 ~10 7 A bacterium.

9. A soil conditioner for promoting corn growth and improving corn salt and alkaline tolerance, characterized in that: The active ingredient comprises the protozoan Renilla magna ISS-TY1 according to claim 1.

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