Soda saline-alkali soil arbuscular mycorrhizal fungi and application thereof

Abstract

The invention discloses a soda saline-alkali soil arbuscular mycorrhizal fungus and application thereof, relates to the field of microorganisms, and aims to provide the soda saline-alkali soil arbuscular mycorrhizal fungus which plays a positive role in growth of saline-alkali soil farmland crops. The arbuscular mycorrhizal fungus is Paraglomus sp. JD5, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is June 24, 2024, and the preservation number is CGMCC No.41381. The arbuscular mycorrhizal fungus has the advantages that the arbuscular mycorrhizal fungus can be used for preparing the arbuscular mycorrhizal fungus; the arbuscular mycorrhizal fungi can improve the saline-alkaline stress tolerance of rice and can promote the growth of the rice.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a soda saline-alkali soil arbuscular mycorrhizal fungus and application thereof. Background Art

[0002] Arbuscular mycorrhizal fungi are a type of beneficial fungi that form a symbiosis with 80% of plants. They can promote the growth of crop root systems and increase crop absorption of nutrients such as water and mineral elements in the soil. At the same time, they play an important role in soil remediation and enhancing crop resistance to salinity, drought, and heavy metal abiotic stresses. It is well known that microbial agents are more environmentally friendly than physical and chemical agents and are a hot topic for application in soil. Therefore, it is crucial to isolate an arbuscular mycorrhizal fungus that can fully utilize beneficial bacteria in the soil and promote crop growth.

[0003] Soda saline-alkali land is currently covering an increasingly large area in Northeast China, and is also the main target of saline-alkali land improvement advocated by the state. Therefore, fully developing the beneficial symbiotic fungi in soda saline-alkali soil can not only promote crop growth, but also make full use of the beneficial resources of the soil. Summary of the invention

[0004] The invention aims to provide a soda saline-alkali land arbuscular mycorrhizal fungus, which plays a positive role in the growth of crops in saline-alkali land farmland.

[0005] The soda saline-alkali arbuscular mycorrhizal fungus of the present invention is Paraglomus sp. JD5, which is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is June 24, 2024, and the deposit number is CGMCC No.41381.

[0006] The spherical fungus JD5 of the present invention can coexist with the roots of alfalfa and rice plants and can be observed in the soil. The spores of the spherical fungus JD5 of the present invention are light yellow in water, have attachments on the surface, have a diameter of 120 μm, and are stained orange in Melzer's reagent.

[0007] The spherical arbuscular mycorrhizal fungus JD5 of the present invention is compared with the gene sequence in the MarrjAM database through Blast analysis, and the homology with the spherical arbuscular mycorrhizal fungus (Paraglomus) is the highest, reaching 96.7%. By combining the identification result of spore morphology characteristics, it is determined that the soda saline-alkali soil arbuscular mycorrhizal fungus JD5 of the present invention is a spherical arbuscular mycorrhizal fungus (Paraglomus).

[0008] The invention also provides application of arbuscular mycorrhizal fungi in soda saline-alkali soil in improving the salt-alkali stress resistance of plants.

[0009] Furthermore, the plant is rice.

[0010] The soda saline-alkali soil arbuscular mycorrhizal fungi of the present invention is used for promoting plant growth.

[0011] Furthermore, the plant is rice.

[0012] Beneficial effects of the present invention:

[0013] The spherical mold JD5 of the present invention can form symbiosis with the roots of alfalfa plants, and the presence of spores is observed in the soil after two months of symbiosis. The JD5 spores are made to form symbiosis with the roots of rice in the same way, wherein the infection rate of JD5 spores in the rice seedling tray is observed to be as high as 45%.

[0014] The Glomus-like fungus JD5 of the present invention has a significant effect on improving the tolerance of rice to moderate salt-alkali stress, and also promotes the growth of rice, and has a positive effect on the growth of crops in saline-alkali land. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the spore staining image of strain JD5 observed under a microscope using Melzer's reagent;

[0016] Figure 2 Phylogenetic tree for molecular identification of strain JD5;

[0017] Figure 3 This is a bar graph of rice root length of strain JD5 under moderate saline-alkali stress and black soil conditions;

[0018] Figure 4 This is a bar graph of rice plant height of strain JD5 under moderate saline-alkali stress and black soil conditions;

[0019] Figure 5 This is a bar graph of rice leaf proline, a physiological and biochemical index of rice under moderate saline-alkali stress and black soil conditions of strain JD5;

[0020] Figure 6 This is a bar graph of soluble sugars of rice physiological and biochemical indicators of strain JD5 under moderate saline-alkali stress and black soil conditions. DETAILED DESCRIPTION

[0021] The embodiments of the present invention are described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0022] Embodiment 1:

[0023] The soda saline-alkali soil arbuscular mycorrhizal fungus in this embodiment is Paraglomus sp. JD5, which is deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date being June 24, 2024, and the deposit number being CGMCC No.41381.

[0024] The method for obtaining the spherical mold JD5 in this embodiment is:

[0025] Initial screening: Wet sieving and decantation method was used to select saline-alkali farmland soil from Da'an City, Baicheng City, Jilin Province, suspend the soil in the aqueous solution, shake it well, and sieve it through 35-mesh and 300-mesh sieves. The soil in the 300-mesh sieve was retained and poured into a disposable plate, a small amount of water was poured, and then the size and shape of the spores were observed and recorded under an inverted microscope. The spores were then stained with Melzer's reagent and the color was observed.

[0026] Example 2: Identification of arbuscular mycorrhizal fungi in soda saline-alkali soil

[0027] Morphological observation:

[0028] The strain JD5 of the present invention exists in soil that coexists with plants. The spore color of the strain JD5 is light yellow, and after being stained with Melzer's reagent, it turns orange-yellow. The spore size of the strain JD5 is 120 μm and is round. The spore morphology of the strain JD5 in Melzer's reagent staining is as follows: Figure 1 shown.

[0029] Molecular identification of strain JD5:

[0030] The single spores were washed with purified water several times, and then placed in a 2 mL centrifuge tube to extract DNA. The DNA extraction method was as follows: add ddH2O to 600 μL in a centrifuge tube, add 30 μL 10% SDS and mix well, heat in a 65°C water bath for 30 min; add 100 μL 5 mol / L NaCl and 80 μL After preheating CTAB / NaCl, mix well and place in a 65°C water bath for 20 minutes, inverting several times during the process; add an equal volume of mixed solution (phenol: chloroform: isoamyl alcohol = 25:24:1) and mix well, centrifuge at 12000rpm for 10 minutes; add an equal volume of chloroform and mix well, centrifuge at 12000rpm for 10 minutes; take the supernatant and add 0.6 times the volume of isoamyl alcohol (stored at 4°C), mix well, store at -20°C for 30 minutes, centrifuge at 12000rpm for 5 minutes, remove the supernatant, add an appropriate amount of 75% ethanol and centrifuge at 12000rpm for 2 minutes, repeat 3 times, finally remove the supernatant, and invert on filter paper until there is no solution, and add 50μL ddH2O to dissolve, and store at -20°C for a long time.

[0031] Take another 2μL DNA solution for PCR amplification. The PCR system is as follows: 2μL template, 2μL primer AM1, 2μL primer NS31, 25μL 2×Hieff PCR Master Mix, 19μL sterilized ddH2O. The PCR amplification system is 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 56℃ for 30s, 72℃ extension for 50s, a total of 35 cycles, 72℃ extension for 10min, and -20℃ storage. 1% agarose gel electrophoresis was used to detect the PCR product and observe the electrophoresis bands. The results showed that there were obvious characteristic bands at 500-750bp.

[0032] The PCR amplification products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the obtained sequences were uploaded to the MarrjAM (https: / / maarjam.botany.ut.ee / ) database for comparison. The homology was compared with the sequences in the gene library by Blast analysis, and the phylogenetic tree was constructed by Neihbor-Joinhing in MEGA11 software. The phylogenetic tree is shown in Figure 2 The strain JD5 has the highest homology with Paraglomus, reaching 96.7%. The strain JD5 is identified as Paraglomus by combining the spore morphological characteristics. The ITS sequence of strain JD5 is shown in SEQ ID NO: 1 in the sequence table.

[0033] Example 3: Propagation of Glomus jd5

[0034] An indoor propagation experiment was conducted on Paraglomus JD5. The Paraglomus JD5 spores observed in the soil were pipetted into a 2mL centrifuge tube, and about 10 spores were pipetted. Sterilized black soil was placed inside the flowerpot, and the spores in the centrifuge tube were rinsed to the middle of the flowerpot. After multiple rinses, alfalfa seeds disinfected with alcohol were placed in the center of the flowerpot, and finally covered with a layer of black soil. It was cultured in a greenhouse for 2 months, and then about 70 spores / g were observed in the soil under a microscope. This shows that Paraglomus JD5 can coexist with alfalfa.

[0035] The JD5 spores were propagated and then symbiotically grown with the roots of rice in the same manner. The infection rate of JD5 spores in the rice seedling tray was observed to be as high as 45%.

[0036] Example 4: Effect of Glomus-like fungus JD5 on the tolerance of rice to moderate saline-alkali stress

[0037] Set up four treatment groups:

[0038] Four groups were set up: the black soil control group (CK) was rice grown in the seedling stage transplanted into a pot with ordinary black soil, without JD5 spores; the black soil treatment group was rice grown in the seedling stage transplanted into a pot with ordinary black soil and inoculated with JD5 spores; the saline-alkali soil control group (CK) was rice grown in the seedling stage transplanted into a pot with saline-alkali soil with a pH of 8.5, without JD5 spores; the saline-alkali soil treatment group was rice grown in the seedling stage transplanted into a pot with saline-alkali soil with a pH of 8.5 and inoculated with JD5 spores. Each group was replicated 6 times and cultured in a greenhouse for 2 months to observe the salt-alkali tolerance of rice. The physiological and biochemical indicators of rice were calculated.

[0039] Glomus-like fungus JD5 grows like a rice root under moderate saline-alkali stress and black soil conditions Figure 3 As shown in Figure 2, the plant height of rice grown under moderate saline-alkali stress and black soil conditions was significantly increased by 2.3 % (P < 0.05). Figure 4 The measurement results show that under saline-alkali soil conditions, the root length and plant height of rice in the treatment group increased by an average of 59.73% and 14.27% compared with the control group. Under black soil conditions, the root length and plant height of rice in the treatment group increased by an average of 17.21% and 6% compared with the control group.

[0040] Physiological and biochemical indicators of rice grown under moderate saline-alkali stress and black soil conditions such as Figure 5 and Figure 6 As shown, Figure 5 is the proline content, Figure 6 The measurement results showed that under saline-alkali soil conditions, the proline and soluble sugar contents of rice leaves in the treatment group increased by 64.9% and 7.6% on average compared with the control. Under black soil conditions, the proline and soluble sugar contents of rice leaves in the treatment group increased by 50.93% and 3.6% on average compared with the control.

[0041] Conclusion: Glomus-like fungi JD5 can significantly improve the growth of rice under moderate saline-alkali stress, and Glomus-like fungi JD5 can promote the growth of rice in black soil.

Claims

1. A soda saline-alkali soil arbuscular mycorrhizal fungus, characterized in that: The arbuscular mycorrhizal fungus is Paraglomus sp. JD5, which is preserved in the General Microbiology Center of China Culture Collection Administration. The preservation address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The preservation date is June 24, 2024, and the preservation number is CGMCC No. 41381.

2. Use of the soda saline-alkali soil arbuscular mycorrhizal fungi as claimed in claim 1 in improving plant tolerance to salt-alkali stress.

3. The use according to claim 2, characterized in that: The plant is rice.

4. The soda saline-alkali soil arbuscular mycorrhizal fungi as claimed in claim 1 are used to promote plant growth.

5. The use according to claim 4, characterized in that: The plant is rice.

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