Methods of recruiting rhizospheric fungi by rhizobium GZHC2-2 strain and its application in alleviating sensitive soybean waterlogging stress

Abstract

The present invention relates to the field of agricultural microbial technology, and specifically discloses a method for recruiting rhizosphere fungi by rhizobium GZHC2-2 strain and its application in alleviating sensitive soybean waterlogging stress. The rhizobium (Rhizobium multihospitium) GZHC2-2 strain was deposited in Guangdong Microbial Culture Collection Center on January 4, 2023, with a deposit number of GDMCC No: 63032. This strain has the ability to help soybeans resist waterlogging. Inoculation of the rhizobium under waterlogging stress can increase the aboveground and underground biomass, the number of nodules and the fresh weight of nodules of soybeans. At the same time, the ability of waterlogging-sensitive soybean varieties to enrich beneficial rhizosphere fungi (such as Dothideomyceta, Corticiales, Kondoaceae, Cryptococcaceae, Polyporales, Cladosporium) is induced. After purifying and isolating rhizosphere fungi, the enriched beneficial fungi can also significantly increase the aboveground and underground biomass, the number of nodules and the fresh weight of nodules of soybeans under waterlogging stress. The invention can be used to improve the growth and increase the yield of soybeans under waterlogging stress, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically discloses a method for recruiting rhizosphere fungi by a rhizobium GZHC2-2 strain and an application thereof in alleviating waterlogging stress of sensitive soybeans. Background Art

[0002] Under natural conditions, plants are often exposed to short-term or long-term soil waterlogging, which has long been considered a major abiotic stress. Soil waterlogging severely limits the growth, development and survival of many plant species, not only in natural ecosystems but also in agricultural systems. Therefore, studying the waterlogging tolerance of crops is of great significance for improving the yield of crops in waterlogged areas.

[0003] Soybean is one of the most important food and oil crops in the world and one of my country's traditional agricultural products. However, long-term waterlogging will not only reduce soybean yield and quality, but also damage the root system of soybeans and even cause death. Therefore, it is very necessary to explore more effective methods to help soybeans resist waterlogging. Summary of the invention

[0004] In order to make up for the shortcomings of the prior art, the purpose of the present invention is to use a method in which rhizobia (Rhizobiummultihospitium) are inoculated on waterlogging-sensitive soybean varieties to recruit waterlogging-resistant rhizospheric fungi (Dothideomyceta, Corticiales, Kondoaceae, Cryptococcaceae, Polyporales, Cladosporium) to resist waterlogging stress.

[0005] A method for recruiting rhizospheric fungi by a rhizobium GZHC2-2 strain, wherein the rhizobium (Rhizobium multihospitium) GZHC2-2 strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on January 4, 2023, with a deposit number of GDMCC No. 63032, and the address of the depository unit is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] S1. The rhizobium GZHC2-2 strain is cultured in rhizobium medium YMA (Yeast mannitol agar);

[0007] S2. The culture temperature of the rhizobium GZHC2-2 strain is 25-35°C, 100-150rpm / min, and the culture time is 72-108h;

[0008] S3. The rhizobium GZHC2-2 strain is inoculated into the roots of waterlogging-sensitive soybean varieties;

[0009] S4. The roots of the waterlogging-sensitive soybean varieties were collected ten days after inoculation, and the rhizosphere soil DNA in S3 was sequenced by ITS amplicon;

[0010] S5. Isolate, culture and sequence the fungi in the rhizosphere soil in S4;

[0011] S6. Screen the fungi in the rhizosphere soil whose relative abundance increased significantly in S5.

[0012] Preferably, the inoculation amount of the rhizobium GZHC2-2 strain is 2×10 4 -2×10 6 Piece / strain.

[0013] Preferably, the rhizospheric fungi with significantly increased relative abundance in S6 include at least any one or any combination of Dothideomyceta, Corticiales, Kondoaceae, Cryptococcaceae, Polyporales, and Cladosporium.

[0014] A method for recruiting rhizospheric fungi by inoculating the rhizobium GZHC2-2 strain, and its application in alleviating the adaptability of sensitive soybean to waterlogging stress.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0016] The rhizobium (Rhizobium multihospitium) in the present invention has efficient colonization ability and strong waterlogging resistance ability. Waterlogging-sensitive soybeans grow well when inoculated with rhizobium (Rhizobium multihospitium) under waterlogging stress, and the abundance of rhizosphere fungi (Dothideomyceta, Corticiales, Kondoaceae, Cryptococcaceae, Polyporales, Cladosporium) is significantly increased. After purification and separation of the above rhizosphere fungi, they can significantly increase the root length and above-ground and underground biomass under waterlogging stress. The rhizobium can be used as an effective environmentally friendly bacterial agent to alleviate soybean waterlogging resistance, and has significant significance for increasing the adaptability of soybeans in waterlogging-stressed soils in South China. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 To study the effects of inoculation with GZHC2-1, GZHC2-2, GZHC2-3, and GZHC2-4 on the growth of soybeans that are sensitive to waterlogging.

[0018] Figure 2 The rhizosphere-enriched microbial species were screened based on ITS gene sequencing after inoculation with GZHC2-2 strain.

[0019] Figure 3 This is the effect of 6 isolated fungal strains on soybean waterlogging resistance.

[0020] Figure 4 Effects of mixed inoculation of six strains on soybean root transcriptome. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with specific examples and accompanying drawings, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0022] Materials and reagents used in the examples of the present invention: PrepMan Ultra Kits nucleic acid extraction reagent, purchased from ThermoFisher, USA; genomic DNA extraction kit: Shanghai Sangon Biotechnology Co., Ltd.

[0023] It should be noted here that the term "recruitment" in this application document is interpreted as enriching the microorganisms in the soybean root system after inoculation with the rhizobium GZHC2-2 strain.

[0024] Example 1 Isolation and identification of Rhizobium multihospitium GZHC2-2 strain

[0025] 1. Strain Isolation

[0026] Rhizobium multihospitium was isolated from the waterlogging-resistant variety "Huachun No. 2", and 6 strains of rhizobia were isolated. The rhizobia were placed in the rhizobium culture medium YMA (Yeast mannitol agar) (pH 7.2) and subcultured for 96 hours at 28°C and 135rpm / min. Then, a single colony was picked and cultured in the YMA culture medium for 48 hours, and then inoculated in the YMA culture medium with 1% inoculation amount of seed liquid, cultured for 48 hours, and then centrifuged at 3500rpm / min and 4°C to collect the cells, washed twice with PBS buffer at pH=7.2, and then resuspended in 2mL PBS solution for use.

[0027] 2. Molecular identification:

[0028] The DNA of the GZHC2-2 strain was extracted using a nucleic acid extractant, and the extracted DNA was amplified using fungal ITS primers. The sequence of the forward primer (ITS1F) is shown in SEQ ID NO.2: 5'-CTTGGTCATTTAGAGGAAGTAA-3', and the sequence of the reverse primer (ITS2R) is shown in SEQ ID NO.3: 5'-GCTGCGTTCTTCATCGATGC-3'. After amplification, the PCR product was purified and sequenced, and nucleic acid sequencing was performed using an Applied Biosystems 3500 genetic analyzer. The sequence is shown in SEQ ID NO.1.

[0029] According to the amplified sequencing of ITS, homology analysis and comparison were performed by BLAST in NCBI, and the sequence similarity between strain GZHC2-2 and Rhizobium multihospitium was 100%. Therefore, the strain was finally identified as Rhizobium multihospitium.

[0030] Example 2 Inoculation of Rhizobium

[0031] Soybean seeds sensitive to waterlogging (Jidou No. 17) were sterilized with alcohol and sown in pots. The pots were 30 cm high, 40 cm in top diameter, 30 cm in bottom diameter, and filled with 5 kg of soil. They were placed in a greenhouse (113°35′N, 23°15′E) at South China Agricultural University (with a day and night temperature difference of 20°C and 28°C, respectively). The experiment adopted a completely randomized block design, inoculating or not inoculating 4 strains of rhizobia isolated from HC2 in acidic soil. The soil moisture content was maintained at 80% of the field water capacity. Eight seeds of uniform size were sown in each pot, and the seeds were diluted to 4 seeds on the 8th day after sowing. For each treatment, there were 6 replicates (pots), and each pot had 4 seedlings. After sowing soybeans, they were allowed to grow to the flowering stage and then flooded: pots of the same specifications as the planting pots were taken as water-proof pots. Cut the plastic film into square pieces of suitable size, and cover the flowerpot with the inner wall of the water-proof flowerpot tightly to block moisture and prevent moisture from escaping from the bottom of the flowerpot to ensure the flooding treatment effect. After the plastic film is laid, it needs to be 1 to 2 cm higher than the flowerpot. After confirming that there are no holes or leaks, the outer water-proof flowerpot can be directly inserted into the planting flowerpot, and the two flowerpots can fit tightly. Add water to submerge 2 cm above the soil surface, add water at regular intervals and in fixed quantities twice a day, observe and record the growth situation, and puncture the plastic film at the low holes of the flowerpot 3 days after flooding to restore drainage, and observe the effect of 4 strains of rhizobia on soybean waterlogging resistance ( Figure 1), and the results showed that strain 2 had the most obvious effect. The rhizosphere soil of the GZHC2-2 strain treatment and the control was collected for amplicon sequencing. To collect the rhizosphere soil, the roots were shaken to remove the loose soil attached to the roots. The roots and attached soil were then transferred to a beaker filled with phosphate-buffered saline. After collecting the rhizosphere soil, 10 grams of the mixed centrifugation were stored at -80°C for microbial DNA extraction.

[0032] Example 3 Effect of rhizobia on recruitment of rhizosphere fungi

[0033] Total soil DNA was extracted using Fast DNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA). The forward primer (ITS1) sequence was shown in SEQ ID NO.4: 5′-TCCGTAGGTGAACCTGCGG-3′, and the reverse primer (IST4) sequence was shown in SEQ ID NO.5: 5′-TCCTCCGCTTATTGATATGC-3′. Each PCR reaction contained 4 μL buffer, 2 μL dNTPs (2 mM), 1 μL forward / reverse primers (10 μM), 10 ng DNA, and 10 μL ddH2O. PCR was then performed in an ABI 7900 system according to the program. 95°C for 30 seconds; 30 cycles of 95°C for 15 seconds, 55°C for 10 seconds, and 72°C for 5 minutes. The PCR products were then purified using the QIAquick Gel Extraction Kit (Qiagen). All samples were pooled at equimolar concentrations and sequenced on the Illumina MiSeq platform (Shanghai Mindray Biopharmaceutical Technology Co., Ltd.). The original FASTQ files of fungal ITS gene sequences were processed with QIIME (v1.9.1) and USEARCH (v10.0). Primers and sequences with low quality (scores below 20 and lengths less than 200 bp) were trimmed. Paired ITS reads were then merged into a single file. Classification analysis was performed using a Naive Bayes classifier trained on amplicon sequence variants (ASVs). Generalized linear models were performed in R using the "edgeR" package to analyze the differences in microorganisms and metabolites between each two treatments, which were then visualized in volcano plots and at a significance threshold of P < 0.001.

[0034] Example 4 Isolation of rhizosphere microorganisms and construction of artificial bacterial colonies

[0035] A total of 85 fungi were isolated from the rhizosphere of soybean. Based on the isolated microorganisms and sequencing results, we screened out 6 fungi (Dothideomyceta, Corticiales, Kondoaceae, Cryptococcaceae, Polyporales, Cladosporium). The results showed that the 6 fungi inoculated alone or mixed with other fungi could improve the ability of soybean to resist waterlogging.

[0036] To further investigate the growth-promoting mechanism of the enriched fungi, we performed RNA-seq analysis on soybean roots inoculated with the mixed inoculum. The results showed that the bacterial consortium treatment significantly increased the abundance of genes controlling steroid biosynthesis, cellular metabolism, and oxidation-reduction processes.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for recruiting rhizosphere fungi by rhizobium GZHC2-2 strain, characterized in that: The Rhizobium multihospitium GZHC2-2 strain was deposited in Guangdong Microbiological Culture Collection Center on January 4, 2023, with the deposit number GDMCC No.63032; S1. The rhizobium GZHC2-2 strain is cultured in rhizobium medium YMA (Yeast mannitol agar); S2. The culture temperature of the rhizobium GZHC2-2 strain is 25-35°C, 100-150rpm / min, and the culture time is 72-108h; S3. The rhizobium GZHC2-2 strain is inoculated into the roots of soybean varieties that are sensitive to waterlogging; S4. The roots of the waterlogging-sensitive soybean varieties were collected ten days after inoculation, and the rhizosphere soil DNA in S3 was sequenced by ITS amplicon; S5. Isolate, culture and sequence the fungi in the rhizosphere soil in S4; S6. Screening fungi in rhizosphere soil whose relative abundance significantly increases in S5, wherein the rhizosphere fungi whose relative abundance significantly increases include at least any one or any combination of Dothideomyceta, Corticiales, Kondoaceae, Cryptococcaceae, Polyporales, and Cladosporium.

2. The method for recruiting rhizosphere fungi by the rhizobium GZHC2-2 strain according to claim 1, characterized in that: The inoculation amount of the rhizobium GZHC2-2 strain was 2×10 4 -2×10 6 Pieces / plant.

3. Use of the method for recruiting rhizospheric fungi by the rhizobium GZHC2-2 strain according to claim 1 or 2 in alleviating the adaptability of sensitive soybeans to waterlogging stress.

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