CN116731878A - Method for recruiting root endophytic fungi by rhizobium GZHC2-2 strain and application of method in relieving sensitive soybean waterlogging stress - Google Patents
Method for recruiting root endophytic fungi by rhizobium GZHC2-2 strain and application of method in relieving sensitive soybean waterlogging stress Download PDFInfo
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Abstract
The application relates to the technical field of agricultural microorganisms, and particularly discloses a method for recruiting root endophytic fungi by using rhizobium GZHC2-2 strain and application thereof in relieving sensitive soybean waterlogging stress, wherein the rhizobium (Rhizobium multihospitium) GZHC2-2 strain is preserved in the microorganism strain preservation center of Guangdong province at 1 month 4 days 2023, and the preservation number is GDMCC No.63032. The strain has the ability to help soybeans resist waterlogging. Inoculating the rhizobia under waterlogging stress can increase the aboveground and underground biomass of soybeans, the number of rhizomes and the fresh weight of the rhizomes. And simultaneously induce the ability of waterlogging sensitive soybean varieties to enrich beneficial root endophytic fungi (such as Sordariomyceta, moesziomyces, rhynchogastremataceae, eurotiomycetes, ustilaginaceae). The enriched beneficial fungi can also obviously increase the aboveground and underground biomass of the soybeans under the stress of waterlogging caused by purifying and separating the root endophytic fungi, and the number of root nodules and the fresh weight of the root nodules. The application can be used for improving the growth of soybeans under waterlogging stress and increasing the yield, and has wide application prospect.
Description
Technical Field
The application belongs to the technical field of microorganisms, and particularly discloses a method for recruiting root endophytic fungi by rhizobia GZHC2-2 strain and application of the method in relieving sensitive soybean waterlogging stress.
Background
Plants are often exposed to short or long term soil inundations under natural conditions, which has long been recognized as a major abiotic stress. Soil flooding severely limits the growth, development and survival of many plant species, which occurs not only in natural ecosystems but also in agricultural systems. Waterlogging is one of the major natural disasters in many countries and regions of the world at present, the soil suffering from waterlogging in the world accounts for about 10% of the cultivated area of the world, and the waterlogging causes about 20% of crop yield reduction. The impact of some crop production, such as U.S., china, canada, brazil, france, and Japan, is particularly pronounced. The tropical and subtropical areas in China are affected by the weather of the monsoon, the rainfall intensity is high, the season distribution is uneven, waterlogging occurs frequently in the middle and lower reaches of the Yangtze river and in the southern area, and the crop yield is seriously affected. The waterlogging has serious influence on the development of agriculture in China, so that research on the waterlogging tolerance of crops has important significance for improving the yield of crops in waterlogged areas.
Soybean is one of the most important food and oil crops in the world and is also one of the traditional agricultural products in our country. However, long term waterlogging can not only reduce soybean yield and quality, but can also lead to damage to the root system of the soybean, and even death. Thus, it is highly necessary to explore more efficient methods to help soybeans resist waterlogging.
Disclosure of Invention
In order to overcome the defects in the prior art, the application aims to apply a method for inoculating rhizobium to a waterlogging-tolerant root endophytic fungus (Sordariomyceta, moesziomyces, rhynchogastremataceae, eurotiomycetes, ustilaginaceae) on a waterlogging-sensitive variety soybean to resist waterlogging stress.
A method for recruiting rhizobia GZHC2-2 strain to root endophytic fungi, wherein the rhizobia (Rhizobium multihospitium) GZHC2-2 strain is deposited with the cantonese province microorganism strain collection at 2023, 1 and 4, with deposit number GDMCC No.63032;
s1, culturing the rhizobium GZHC2-2 strain in a rhizobium culture medium YMA (Yeast mannitol agar);
s2, culturing the rhizobia GZHC2-2 strain at 28 ℃ and 135rpm/min for 96 hours;
s3, inoculating the rhizobium GZHC2-2 strain to roots of waterlogging sensitive soybean varieties;
s4, collecting roots of the waterlogging sensitive soybeans ten days after inoculation, and carrying out ITS amplicon sequencing on the DNA of the roots in the S3;
s5, separating and culturing the root endophytic fungi in the S4, sequencing,
s6, screening the endophytic fungi with the remarkably increased relative abundance in S5.
Preferably, the rhizobia GZHC2-2 strain is inoculated in an amount of 2X 10 4 -2×10 6 Individuals/strains.
Preferably, the relative abundance in S6 significantly increases the root endophytic fungus including at least any one or any combination of Sordariomyceta, moesziomyces, rhynchogastremataceae, eurotiomycetes, ustilaginaceae.
A method for inoculating rhizobia GZHC2-2 strain to recruit root endophytic fungi is applied to relieving adaptation of sensitive soybean waterlogging stress.
Compared with the prior art, the application has the following advantages:
the rhizobia (Rhizobium multihospitium) has high-efficiency field planting capacity and strong waterlogging tolerance. The waterlogged soybean is well grown under waterlogged stress by inoculating rhizobium, the abundance of the root endophytic fungi (Sordariomyceta, moesziomyces, rhynchogastremataceae, eurotiomycetes, ustilaginaceae) is remarkably increased, and the root length and the aboveground and underground biomass of the soybean under waterlogged stress can be remarkably increased by purifying and separating the root endophytic fungi. The rhizobia can be used as an effective environment-friendly microbial inoculum for relieving the waterlogging tolerance of soybeans, and has remarkable significance for increasing the adaptability of the soybeans to waterlogging stress soil in south China.
Drawings
FIG. 1 shows the growth effects of GZHC2-1, GZHC2-2, GZHC2-3, and GZHC2-4 on waterlogging-sensitive soybeans.
FIG. 2 shows the enriched microbial species in roots screened based on ITS gene sequencing after inoculation with GZHC2-2 strain.
FIG. 3 shows the effect of 5 isolated fungal strains on soybean waterlogging tolerance.
FIG. 4 is the effect of mixed inoculation of 5 strains on soybean root system transcriptome.
Detailed Description
The present application is further illustrated below with reference to specific examples and figures, but the examples are not intended to limit the application in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art. The reagents and materials used in the present application are commercially available unless otherwise specified.
Materials and reagents used in the examples of the present application: prepMan Ultra Kits nucleic acid extractant available from ThermoFisher, inc. of USA; genomic DNA extraction kit: shanghai Biotechnology Co., ltd.
Here, the term recruitment in the present document is interpreted as enrichment of microorganisms of the soybean root system by inoculating rhizobium GZHC2-2 strain.
EXAMPLE 1 isolation and characterization of rhizobia (Rhizobium multihospitium) GZHC2-2 Strain
1. Isolation of strains
Rhizobia (Rhizobium multihospitium) is separated from waterlogging tolerant variety "Huachun No. 2", and 6 rhizobia strains are separated. Rhizobia was placed in rhizobia medium YMA (Yeast mannitol agar) (pH 7.2) and subcultured at 28 ℃ for 96 hours at 135 rpm/min. Then, single colonies were picked up and cultured in YMA medium for 48 hours, then inoculated in YMA medium with 1% of seed solution, cultured for 48 hours, then centrifuged at 3500rpm/min at 4℃to collect the colonies, washed 2 times with PBS buffer having pH=7.2, and then resuspended in 2mLPBS solution for use.
2. Molecular identification:
extracting DNA of GZHC2-2 strain by using nucleic acid extractant, and amplifying the extracted DNA by fungus ITS primer. The forward primer (ITS 1F) has a sequence shown in SEQ ID NO. 2: 5'-CTTGGTCATTTAGAGGAAGTAA-3', the reverse primer (ITS 2R) sequence is shown in SEQ ID NO. 3: 5'-GCTGCGTTCTTCATCGATGC-3' the PCR products were purified and sequenced after amplification and nucleic acid sequencing was performed using a Applied Biosystems 3500 gene analyzer. The sequence is shown as SEQ ID NO. 1.
According to the amplification sequencing of ITS, the sequence similarity of the strain GZHC2-2 and Rhizobium multihospitium is 100% by BLAST in NCBI, so that the strain is finally identified as Rhizobium multihospitium.
EXAMPLE 2 inoculation of rhizobia
Seeds of waterlogging sensitive soybean (Ji bean No. 17) are sterilized by alcohol and sown in a flowerpot. The pot height was 30 cm, the top diameter was 40 cm, the bottom diameter was 30 cm, 5 kg of soil was loaded, and the pot was placed in a greenhouse (20℃and 28℃respectively) of the agricultural university of North China (113℃35'N,23℃15' E). The test used a completely randomized block design with or without inoculation of 4 rhizobia isolated from HC2 in acid soil. The water content of the soil is kept at 80% of the field water capacity. 8 seeds of uniform size were sown per pot and diluted to 4 at day 8 post-sowing. For each treatment there were 6 replicates (pots), 4 seedlings per pot. After sowing the soybeans, carrying out flooding treatment after the soybeans grow to the flowering phase: the flowerpot with the same specification as the flowerpot for planting is taken as the flowerpot for water isolation. Cutting the plastic film into square pieces with proper size, and tightly adhering the inner wall of the flowerpot for water isolation to cover the flowerpot for blocking water, preventing water from losing from the bottom of the flowerpot, and ensuring the flooding treatment effect. After the plastic film is paved, the plastic film needs to be 1-2 cm higher than the flowerpot. After no broken holes and holes are confirmed, the flowerpot for outer water isolation can be directly sleeved outside the flowerpot for planting, and the two flowerpots can be closely attached. And adding water to 2cm above the submerged soil surface, adding water at regular time and quantity for 2 times a day, observing and recording the growth condition, puncturing a plastic film at a low-loophole position of the flowerpot after 3 days after flooding to recover drainage, and observing the effect of 4 rhizobia on the waterlogging tolerance of soybeans (figure 1).
The results show that the effect of the strain 2 is most obvious. Roots of GZHC2-2 strain treated and control were collected for amplicon sequencing. To collect the roots, the roots are shaken to remove loose soil adhering to the roots. The root system and attached soil were then transferred to a beaker filled with phosphate buffered saline. Collecting 10 g of the mixture after root mixing and centrifugation, and preserving at-80 ℃ for extracting microorganism DNA.
EXAMPLE 3 recruitment Effect of rhizobia on root endophytic fungi
Root total DNA was extracted using Fast DNA SPIN Kit for Soil (MP Biomedicals, santa Ana, CA). The forward primer (ITS 1) is used and has a sequence shown in SEQ ID NO. 4: 5'-TCCGTAGGTGAACCTGCGG-3' the sequence of the reverse primer (IST 4) is shown in SEQ ID NO. 5: 5'-TCCTCCGCTTATTGATATGC-3'. Each PCR reaction contained 4. Mu.L of buffer, 2. Mu.L of dNTPs (2 mM), 1. Mu.L of forward/reverse primer (10. Mu.M), 10ng of DNA and 10. Mu.L of ddH2O. PCR was then performed as programmed in the ABI 7900 system. 95 ℃ for 30 seconds; 30 cycles, 95℃for 15 seconds, 55℃for 10 seconds, 72℃for 5 minutes. The PCR products were then purified using the QIAquick gel extraction kit (Qiagen). All samples were pooled at equimolar concentration and sequenced on an Illumina MiSeq platform (Shanghai Meier Biotechnology Co., ltd.). The original FASTQ file of fungal ITS gene sequences was treated with QIIME (v1.9.1) and USEARCH (v 10.0). Primers and low quality sequences (score below 20, length below 200 bp) were trimmed off. The paired ITS readings are then combined into one file. Classification analysis was performed using the Naive Bayes classifier trained on Amplicon Sequence Variants (ASVs). The generalized linear model was performed in R using the "edge" software package, the differences in microorganisms and metabolites between each two treatments were analyzed, then visualized in a volcanic plot, and the significance threshold at P < 0.001.
EXAMPLE 4 isolation of root endophyte and Artificial flora construction
A total of 85 fungi were isolated from the root of soybean. According to the separated microorganisms and sequencing results, 5 strains of fungi (Sordariomyceta, moesziomyces, rhynchogastremataceae, eurotiomycetes, ustilaginaceae) are screened, and the results show that the 5 strains of fungi are singly inoculated or mixed with inoculated fungi to improve the waterlogging pest resistance of soybeans.
Further studies on the growth promoting mechanism of the enriched strain fungi, we performed RNA-seq analysis on soybean root lines with mixed inoculants. The results indicate that the flora treatment significantly increases the abundance of genes controlling the steroid biosynthesis process, the cellular metabolism and the oxidation-reduction process.
The foregoing description of the preferred embodiments of the application is not intended to be limiting, but rather is to cover all modifications, equivalents, alternatives, and improvements that may be made without departing from the spirit and scope of the application.
SEQ ID NO.1(Rhizobium multihospitium GZHC2-2，length：820)
GGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAACTTCATGCACT
CGAGTTGCAGAGTGCAATCCGAACTGAGATGGCTTTTGGAGATTAGCTCACACTCGCGTGCTCGCTGCCCACTGTCA
CCACCATTGTAGCACGTGTGTAGCCCAGCCCGTAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCTCGGCT
TATCACCGGCAGTCCCCTTAGAGTGCCCAACTAAATGCTGGCAACTAAGGGCGAGGGTTGCGCTCGTTGCGGGACTT
AACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCTCTGCGCCACCGAAGTGGACCCCCTATC
TCTAGAGGTAACACAGGATGTCAAGGGCTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCT
TGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGAATGTTTAATGCGTTAGC
TGCGCCACCGAACAGTATACTGCCCGACGGCTAACATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTG
TTTGCTCCCCACGCTTTCGCACCTCAGCGTCAGTAATGGACCAGTGAGCCGCCTTCGCCACTGGTGTTCCTCCGAAT
ATCTACGAATTTCACCTCTACACTCGGAATTCCACTCACCTCTTCCATACTCCAGATCGACAGTATCAAAGGCAGTT
CCAGGGTTGAGCCCTGGGATTTCACCCCTGACTGATCGATCCGCCTACGT
SEQ ID NO.2
CTTGGTCATTTAGAGGAAGTAA
SEQ ID NO.3
GCTGCGTTCTTCATCGATGC
SEQ ID NO.4
TCCGTAGGTGAACCTGCGG
SEQ ID NO.5
TCCTCCGCTTATTGATATGC。
Claims (4)
1. A method for recruiting root endophytic fungi by rhizobia GZHC2-2 strain, characterized in that: the rhizobium (rhizobium) GZHC2-2 strain is preserved in the microorganism strain collection of Guangdong province at 2023, 1 and 4 days, and the preservation number is GDMCCNo.63032;
s1, culturing the rhizobium GZHC2-2 strain in a rhizobium culture medium YMA (Yeastmannitolagar);
s2, culturing the rhizobia GZHC2-2 strain at the culture temperature of 25-35 ℃ and at the speed of 100-150rpm/min for 72-108 hours;
s3, inoculating the rhizobium GZHC2-2 strain to roots of waterlogging sensitive soybean varieties;
s4, collecting roots of the waterlogging sensitive soybean varieties ten days after inoculation, and carrying out ITS amplicon sequencing on the DNA of the roots in the S3;
s5, separating and culturing the root endophytic fungi in the S4, sequencing,
s6, screening the endophytic fungi with the remarkably increased relative abundance in S5.
2. A method of recruiting root endophytic fungi to a rhizobia GZHC2-2 strain according to claim 1, wherein: the inoculation amount of the rhizobium GZHC2-2 strain is 2 multiplied by 10 4 -2×10 6 Individuals/strains.
3. A method of recruiting root endophytic fungi to a rhizobia GZHC2-2 strain according to claim 1, wherein: the relative abundance in S6 significantly increases the root endophytic fungi, including at least any one or any combination of Sordariomyceta, moesziomyces, rhynchogastremataceae, eurotiomycetes, ustilaginaceae.
4. A method of vaccinating rhizobia GZHC2-2 strain against root endophytic fungi according to any of claims 1 or 3 for use in alleviating adaptation to sensitive soybean inula stress.
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| CN116948874A (en) * | 2023-06-08 | 2023-10-27 | 华南农业大学 | Rhizobium GZHC2-2 strain, preparation method thereof, method for recruiting root endophyte by strain and application of strain in relieving sensitive soybean waterlogging stress |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104277994A (en) * | 2014-04-16 | 2015-01-14 | 四川农业大学 | Sinorhizobium SCAUs65 and application thereof |
| CN113215037A (en) * | 2021-05-08 | 2021-08-06 | 华南农业大学 | High-efficiency nitrogen-fixing bradyrhizobium strain and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104277994A (en) * | 2014-04-16 | 2015-01-14 | 四川农业大学 | Sinorhizobium SCAUs65 and application thereof |
| CN113215037A (en) * | 2021-05-08 | 2021-08-06 | 华南农业大学 | High-efficiency nitrogen-fixing bradyrhizobium strain and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116948874A (en) * | 2023-06-08 | 2023-10-27 | 华南农业大学 | Rhizobium GZHC2-2 strain, preparation method thereof, method for recruiting root endophyte by strain and application of strain in relieving sensitive soybean waterlogging stress |
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