Protozoa reniform worm and application thereof in promoting saline-alkaline tolerant growth of corn

Abstract

The invention relates to protozoa reniform worm and application thereof in promoting saline-alkaline tolerant growth of corn, protozoa swollen reniform worm ISS-TY1 has been preserved in a preservation unit specified by the State Intellectual Property Office on April 22, 2025, the preservation unit name is China Center for Type Culture Collection, and the preservation number is CCTCC NO: C202521. Research finds that the biomass of the upper part of the corn field treated by applying the reniform enlargement worm ISS-TY1 is increased by 183%-197%, the abscisic acid content of the root is remarkably reduced by 30.3%-43.1%, the K < + > content of the leaf is increased by 20.7%-29.2%, and the Na < + > content is reduced by 18.5%-26.8%. The invention reveals that the reniform swollen worm ISS-TY1 has huge application potential and prospect in the aspects of improving the saline-alkaline resistance of crops and promoting the growth of the crops.

Description

Technical Field

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

[0002] Corn is one of the most important food crops in the world and is widely grown in a variety of soil types. However, the presence of saline-alkali land has severely limited the planting range and yield of corn. The high salt and alkali content of saline-alkali land will lead to the deterioration of soil structure, reduced nutrient effectiveness, and have a significant inhibitory effect on the growth and development of corn, such as poor root development, short plants, and hindered photosynthesis, which ultimately leads to a significant decrease in corn yield.

[0003] Salt-alkali stress can also cause a series of secondary problems. In a high-salt environment, sodium ions in the soil will replace beneficial ions such as calcium and magnesium in the soil colloid, causing soil compaction, poor air permeability and water permeability, and further exacerbating the growth difficulties of corn. In addition, the high pH value in saline-alkali land will reduce the effectiveness of nutrients such as phosphorus, zinc, and iron in the soil, making it difficult for corn to absorb enough 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 damage to the function 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 function of the soil, further restricting the growth of corn. However, recent studies have found that protists 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 protists per gram of soil. 4 ~10 8 In addition, studies have shown that protists play an important role in promoting plant growth and plant health. A deeper understanding of how protists help salt-tolerant crops grow and reduce barriers can help us improve saline-alkali soils more efficiently and greenly.

[0005] At present, there is no systematic report on protists significantly improving corn salt tolerance and promoting corn growth at the same time. Therefore, in-depth research on the application potential of protists in corn cultivation in saline-alkali land has important scientific and practical significance for breaking through the limitations of saline-alkali land and increasing corn yields. Summary of the invention

[0006] Technical problem to be solved: The present invention provides a protozoan Colpoda inflata and its application in promoting the growth of salt-tolerant maize. This protozoan Colpoda inflata is from soda saline-alkali soil, easy to culture and has a fast reproduction rate. This strain of protozoan Colpoda inflata has a significant effect in promoting the growth of maize. At the same time, this strain of protozoan Colpoda inflata can significantly reduce the Na + content and the Na + / K + ratio in maize leaves, and has a positive effect on improving the salt tolerance of maize.

[0007] Technical solution: A strain of protozoan Colpoda inflata ISS-TY1 ( Colpoda inflata ISS-TY1), the Colpoda inflata ISS-TY1 is 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 application of the above-mentioned protozoan Colpoda inflata ISS-TY1 ( Colpoda inflata ISS-TY1) in promoting the growth of maize and improving the salt tolerance of maize.

[0009] The culture method of the above-mentioned Colpoda inflata ISS-TY1 includes the following steps: inoculating the Colpoda inflata ISS-TY1 into PAS buffer; adding inactivated Escherichia coli as a food source; 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 of Na2HPO4 and 0.136 g of KH2PO4 in 500 mL of sterile water; Prepare buffer B: Dissolve 4 mg of MgSO4·7H2O, 4 mg of CaCl2·2H2O and 0.12 g of NaCl in 500 mL of sterile water; Sterilize buffer A and buffer B at 121°C for 15 minutes respectively and then mix them to make 1000 mL of PAS buffer.

[0011] The application method of the above-mentioned Colpoda inflata ISS-TY1 is: applying the enriched protozoan suspension to the rhizosphere soil of maize at a concentration of 10 4 ~10 6 cells per maize plant.

[0012] The above application includes applying the Colpoda inflata ISS-TY1 synchronously with maize seeds when planting maize in saline-alkali soil.

[0013] The above maize varieties include Pengcheng 10 and Kete 881.

[0014] The addition amount of the above-inactivated Escherichia coli is 10 6 ~10 7 bacteria per milliliter of PAS buffer.

[0015] A soil conditioner for promoting maize growth and improving maize tolerance to saline-alkali soil, the active ingredient of which includes the above-mentioned protozoan Inflata nephridia ISS-TY1.

[0016] Beneficial effects: The present invention isolates and screens the protozoan Inflata nephridia ISS-TY1 from soda saline-alkali soil, which can improve the saline-alkali tolerance of maize and promote maize growth. Pot experiments show that Inflata nephridia ISS-TY1 can significantly increase the biomass of maize. In addition, Inflata nephridia ISS-TY1 can also improve maize's resistance to saline-alkali stress. Therefore, the Inflata nephridia ISS-TY1 of the present invention plays an important role in improving the saline-alkali tolerance and growth of maize. Research finds that the aboveground biomass of maize treated with Inflata nephridia ISS-TY1 increases by 183% - 197%, the abscisic acid content in the roots decreases significantly by 30.3% - 43.1%, the K + content in the leaves increases by 20.7% - 29.2%, while the Na + content decreases by 18.5% - 26.8%. The present invention reveals that Inflata nephridia ISS-TY1 has great application potential and prospects in improving the saline-alkali tolerance of crops and promoting crop growth. Description of the Drawings

[0017] Figure 1 It is a photograph of the morphological characteristics of the trophozoite A and dormant cyst B of the protozoan Inflata nephridia ISS-TY1 ( Colpoda inflata ISS-TY1) of the present invention.

[0018] Figure 2 It is a comparison diagram of the growth of maize in the pot experiment of Example 3 of the present invention, inoculating Inflata nephridia ISS-TY1 ( Colpoda inflata ISS-TY1) treatment and blank control (CK) treatment, where S1 represents Pengcheng No. 10 and S2 represents Kete 881.

[0019] Figure 3 It is a comparison diagram of the saline-alkali tolerance growth effect of maize in the pot experiment of Example 3 of the present invention, inoculating Inflata nephridia ISS-TY1 ( Colpoda inflata ISS-TY1) treatment and blank control (CK) treatment, where A is the comparison of biomass, B is the comparison of abscisic acid content in the root base, C is the comparison of potassium content in the leaves, D is the comparison of sodium content in the leaves, and S1 represents Pengcheng No. 10 and S2 represents Kete 881. Detailed Embodiments

[0020] The following examples are only for illustrating the technical concept and characteristics of the present invention. The purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and it should not be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention. The test materials used in the following examples are all purchased from regular biochemical reagent companies unless otherwise specified.

[0021] Page’s saline (PAS) buffer solution: Dissolve 0.142 g of Na2HPO4 and 0.136 g of KH2PO4 into 500 mL of sterile water to make buffer solution A; separately dissolve 4 mg of MgSO4·7H2O, 4 mg of CaCl2·2H2O and 0.12 g of NaCl into 500 mL of sterile water to make buffer solution B. After buffer solutions A and B are sterilized at 121 °C for 15 min in a high-pressure steam sterilizer respectively, and cooled, they are mixed and shaken well in a laminar flow hood to make 1000 mL of PAS buffer solution. Water agar medium (1.5 % water agar plates): Add 1.5 g of agar to 100 mL of sterile water, fully dissolve it, sterilize it at 121 °C for 15 min, pour a small amount of the medium into a petri dish, with a thickness of about 1.5 mm, and set aside after cooling. Nutrient Broth (NB) medium: Dissolve 10.0 g of peptone, 3.0 g of beef extract powder and 5.0 g of NaCl into 1000 mL of distilled water, and sterilize it at 121 °C for 15 min. Yeast extract powder (1.0 % YPD) medium: Heat and fully dissolve 2.0 g of peptone, 2.0 g of glucose and 1.0 g of yeast extract powder into 1000 mL of distilled water, and sterilize it at 121 °C for 15 min.

[0022] Preparation of the humid chamber: According to the method of making a humid chamber in "Experimental Techniques of Protozoology" (Pang Yanbin, 1991), spread a thin layer of absorbent cotton at the bottom of a foam box, add an appropriate amount of distilled water to make the cotton fully absorb the water, and then insert two glass rods closely on the cotton layer to fix the 96-well plate and the petri dish.

[0023] Example 1 Isolation and purification of protists Due to the low soil moisture content and low native biological activity in saline-alkali soil, it is necessary to pre-cultivate the soil sample first. Weigh 10.0 g of soil, add an appropriate amount of water to maintain the soil moisture at 20%, and add an appropriate amount of food source with the water. Incubate at 25°C for 7 to 14 days, then collect the soil sample and isolate the native organisms. The isolation steps are as follows: Weigh 1.0 g of rhizosphere soil of salt-tolerant corn varieties and put it 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 and shake for 30 min to fully mix the soil particles with the buffer solution. Then use a vortex oscillator to shake vigorously for 20 s to strip the native organisms from the soil particles as much as possible. Let the centrifuge tube stand for 30 min. Wait until the soil suspension is slightly clarified. In a laminar flow hood, add 95 μL of PAS buffer solution to each well of a 96-well plate, add 5 μL of the soil suspension, and dilute it according to the gradients of 10 -1 、10 -2 、10 -3 and 10 -4 . Then add 10 μL of bacterial food source to each well. Seal the 96-well plate completely with a sealing film. After placing it in a humid chamber, incubate it in the dark in an incubator at 15°C. Observe the growth of native organisms under an inverted microscope (×200 magnification) on the 3rd and 7th days of incubation respectively. Gradually isolate and dilute under the inverted microscope to obtain single native organisms.

[0024] Example 2 Identification of Native Organisms Total DNA of protists was extracted using the Neasy Blood&Tissue Kit (QIAGEN N.V, Maryland, USA) according to the kit instructions. The primers for PCR amplification were RibA (5’-ACCTGGTTGATCCTGCCAGT-3’) shown in SEQ ID NO:1 and RibB (5’-TGATCCATCTGCAGGTTCACCTAC-3’) shown in SEQ ID NO:2 to perform PCR amplification on the 18S rRNA gene of protists. The total volume of the PCR amplification system was 50 μL: 25 μL of 2×Taq PCR MasterMix, 15 μL of DNA template, 1.25 μL each of primers RibA and RibB, and ddH2O was added to make up to 50 μL. The PCR amplification program was: pre-denaturation at 98 °C for 3 min, then 98 °C for 45 s, 55 °C for 45 s, 72 °C for 45 s, for a total of 30 cycles, and finally 72 °C for 10 min. The amplified product was sent to a testing company for sequencing. Using the BLAST software, the obtained 18S rDNA gene sequence was aligned with the NT (Nucleotide Sequence Database) database on the NCBI website, and the MEGA software was used to perform phylogenetic analysis on the isolated protozoa. The sequencing result of Colpoda inflata ISS-TY1 had the highest similarity with the protozoan Colpoda Colpoda inflata , so the isolated protozoan ISS-TY1 was identified as Colpoda inflata . This strain was deposited at the China Center for Type Culture Collection on April 22, 2025, at Wuhan University, Wuhan, China, with the deposit number: CCTCC NO:C202521. The name of the protist was compared on the National Center for Biotechnology Information NCBI in the United States (http: / / www.ncbi.nlm.nih.gov).

[0025] Example 3 Growth promotion and salt tolerance improvement of maize in saline-alkali soil by protists Experimental setup: (1) Blank control group CK; (2) Treatment group inoculated with Colpoda inflata ISS-TY1. We provided two maize varieties: Pengcheng 10 and Ketai 881. Each treatment had 3 replicates, for a total of 2×2×3 = 12 samples (inoculation and non-inoculation treatments, two maize varieties, 3 replicates). The test protist was Colpoda inflata ISS-TY1 isolated in Example 1. After uniformly mixing the purified and enriched protist samples, 10 μL of the enrichment solution was pipetted and evenly added to a Neubauer counting chamber for counting. An inverted microscope was used to observe its abundance. Each sample was counted at least 3 - 5 times, and the average value was taken as its final concentration. The enriched protists were added near the root zone, and after harvesting, maize growth and its salt and alkali tolerance indicators were detected. It was found that the aboveground biomass of maize treated with the protist Colpoda inflata ISS-TY1 increased by 183% - 197% ( Figure 3 :A), the abscisic acid content in the roots decreased significantly by 30.3% - 43.1% ( Figure 3 :B), the K + content in the leaves increased by 20.7% - 29.2% ( Figure 3 :C), while the Na + content decreased by 18.5% - 26.8% ( Figure 3 :D). This indicates that the salt and alkali resistance characteristics, biomass, and growth of maize plants have been significantly improved.

Claims

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

2. The protozoan Renilla sphaeroides 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 ISS-TY1 comprises the following steps: inoculating ISS-TY1 into 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 sterile water; preparing buffer B: dissolving 4 mgMgSO4·7H2O, 4 mg CaCl2·2H2O and 0.12 g NaCl in 500 mL sterile water; sterilizing buffer A and buffer B at 121° C. for 15 minutes respectively, and then mixing 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 to each corn plant at 10 4 ~10 6 The concentration of 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 bacteria.

9. A soil conditioner for promoting corn growth and improving corn salt-alkali tolerance, characterized in that: The active ingredient comprises the protozoan Renilla sphaeroides ISS-TY1 as described in claim 1.

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