CN116694496A - Qigou rhizobium for promoting chickpea nodulation and application thereof - Google Patents

Qigou rhizobium for promoting chickpea nodulation and application thereof Download PDF

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CN116694496A
CN116694496A CN202211704961.1A CN202211704961A CN116694496A CN 116694496 A CN116694496 A CN 116694496A CN 202211704961 A CN202211704961 A CN 202211704961A CN 116694496 A CN116694496 A CN 116694496A
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rhizobium
chickpea
nodulation
rhizobia
nitrogen
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沈红玲
万传星
梁红丽
丁培治
马亮亮
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Tarim University
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Abstract

The invention discloses a rhizobium of a new species of rhizobium capable of promoting nodulation and nitrogen fixation of chickpeas, which is preserved in the microorganism strain collection of Guangdong province in the period of 2022 and 7 months and has the preservation number of GDMCC No.62650. The application of the strain in nodulation and nitrogen fixation is also disclosed, and the nitrogen fixation microbial preparation containing the rhizobium strain and/or bacterial suspension thereof is provided, after the strain is inoculated to chickpea plants, the plant height, root weight, overground part weight, root nodule weight and the like of chickpea are obviously improved, and the strain is suitable for cultivation and production of chickpea in saline-alkali areas, and is beneficial to promoting the nodulation and nitrogen fixation of chickpea, increasing nitrogen nutrition and promoting the high quality and high yield of chickpea.

Description

Qigou rhizobium for promoting chickpea nodulation and application thereof
Technical Field
The invention relates to the field of microorganisms, in particular to rhizobium mirabilis capable of promoting chickpea nodulation and application thereof.
Background
Chickpea, academic name:Cicer arietinumthe other names of the pears, the chickpeas and the chickpeas belong to the field pea family and are rare varieties in leguminous plants. Chickpea is a leguminous herb, originating in the western asia and in the near east. The leguminous plants with larger cultivation area in the world are about one hundred million mu, wherein the cultivation area of two countries of India and Pakistan accounts for more than 80% of the world, the leguminous plants are distributed only sporadically in China and are mainly distributed in northwest provinces of Xinjiang, qinghai, gansu and the like, and the Xinjiang Mubase county is the main production area of chickpeas in China. The chickpea has the characteristics of drought resistance, barren resistance, nitrogen fixation, good management and the like, and is suitable for planting in the mountain dry land in the Mubang county. Accelerating the development of chickpea industry, and has important significance for promoting regional economic development and driving peasants in mountain areas to increase income and become rich. The starch of chickpea has chestnut flavor. The chickpea powder and milk powder are added to prepare soybean milk powder, which is easy to absorb and digest, and is a nutritional food for infants and the elderly. Chickpea can also be made into various snack and fried bean.
Rhizobium speciesRhizobium) Is a soil bacterium which can establish symbiotic relation with leguminous plants and comprises azotembotrious genusAzorhizobium) Genus OncomelaniaAllorhizobium) Rhizobium slow-growing genusBradyrhizobium) Rhizobium mesochrous genusMesorhizobium) Rhizobium genusRhizobium) And sinorhizobium spSinorhizobium). Symbiotic nitrogen fixation systems between leguminous plants and rhizobia have been a research hotspot. At present, the chickpea nodulation phenomenon is only found in Xinjiang ChangJi, gansu Huning and Ningxia Guyuan in China, chickpea rhizobium resources are obtained by separation, and other newly introduced areas such as Yunnan thalonil, jilin Baicheng, shandong Qingdao, henan Daqing, henan Ru and the like are not found, so that the chickpea has higher nodulation specificity, only two chickpea rhizobium symbiotic populations are found in China so far, namely, slow rhizobium in the Mubase and slow rhizobium in the Wenzhen, the chickpea rhizobium populations found abroad are not distributed in China, and the special biological and geographic distribution characteristics and regional characteristics of the chickpea rhizobium are shown.
In order to meet the requirement of chickpea inoculation rhizobia in the large-scale inoculation process of chickpea, the microbial strain resource library of nitrogen fixation rhizobia is expanded, more new rhizobia strains with high-efficiency nodulation nitrogen fixation capacity are necessary to be screened, and the microbial strain resource library is enriched.
Disclosure of Invention
The invention aims to overcome the defects and the shortcomings of the problems and provide a brand new rhizobium mirabilis suitable for saline-alkali soil to promote chickpea nodulation and nitrogen fixation.
The invention separates and screens 2 rhizobium strains TRM95796 and TRM96647 from chickpea rhizomes. The invention carries out sequencing analysis on the 16S rDNA gene sequence of the strain, which shows that the 16S rDNA gene sequence of the rhizobium strain and rhizobium genus provided by the inventionRhizobiumsp.) strainsRhizobium alvei TNR-22T sequence similarity was 96.3%. TRM96647 and RhizobiumRhizobium azooxidifex Po 20/26 T ) The sequence similarity of the 16S rRNA gene of (2) was 97.5%.
Two rhizobia strains were deposited: rhizobium mirabilis (L.) SpRhizobium qitaienseTRM95796Rhizobium muleienseTRM96647 was deposited with microorganisms from Guangdong province at 2022, 7, 27 and 3, 8, and 2022The collection of species (GDMCC) has the collection numbers of GDMCC No.62650 and GDMCC No. 62640 respectively, and the collection address is the building 5 of the 100 th Mitsui Hirsu, guangzhou, guangdong.
The invention provides a nitrogen fixation microbial preparation, which contains the rhizobiaRhizobium qitaienseTRM95796 strain and/or bacterial suspension thereof. OD value of the bacterial suspension is 0.8-1.2, fermentation condition of the bacterial suspension is 25 ℃, pH value is 7.0, and YMA culture medium. The two bacterial suspension are respectively inoculated to chickpea plants, and the plant height, root weight, overground part weight, root nodule weight and the like of the chickpea are obviously improved, so that the bacterial suspension is suitable for cultivating and producing chickpea, is particularly suitable for cultivating and producing chickpea in saline-alkali areas, is beneficial to promoting plant nodulation and nitrogen fixation, increasing nitrogen nutrition of plants and promoting high-quality and high-yield chickpea.
The invention has the following beneficial effects:
the rhizobium strain is separated and screened from chickpea rhizomes, and has the characteristics of high rhizobium rate and strong nitrogen fixation capacity; the tieback test shows that the strain suspension of the field inoculated TRM95796 strain has obvious improvement on the plant height, root weight, overground part weight, root nodule weight and the like of chickpeas. The strain provides an important rhizobium resource for producing chickpea microbial fertilizer.
Drawings
FIG. 1 Rhizobium mirabilis TRM95796 T Rhizobium lignans TRM96647 T Electron microscope photographs of (2);
FIG. 2A strain TRM95796 T And TRM96647 T Phylogenetic tree based on 16S rRNA gene orthography.
FIG. 2B strain TRM95796 T And TRM96647 T The system evolved tree based on the 16S rRNA gene maximum conciseness method.
FIG. 2C strain TRM95796 T And TRM96647 T Phylogenetic tree based on the 16S rRNA gene maximum likelihood method.
FIG. 3 strain TRM95796 T And TRM96647 T Whole genome (TYGS) phylogenetic tree
FIG. 4 strain TRM95796 T And TRM96647 T Housekeeper(s)recA-atpD) Gene orthography phylogenetic tree
FIG. 5 strain TRM95796 T And TRM96647 T Polar lipid type (PE: phosphatidylethanolamine; PIM: phosphatidylinositol mannoside; DPG: biphospholipid glycerol; PI: phosphatidylinositol; L1, L2: unknown compound)
FIG. 6 inoculation of strain TRM95796 T And TRM96647 T Results of potted chickpea experiments.
FIG. 7 inoculation of strain TRM95796 T And TRM96647 T And (5) a field test chickpea growth map.
Detailed Description
The invention is further illustrated by the following detailed description of specific embodiments, which is not intended to be limiting, but is made merely by way of example.
Example one, source and isolation culture of Rhizobium mirabilis TRM95796 and Rhizobium lignans TRM96647
Fresh and full rhizomes are collected from chickpea planting areas in Qigong county and Mubase county in Xinjiang, surfaces are cleaned by running water after the rhizomes are collected, surface disinfection is respectively carried out by 75% ethanol and 2% sodium hypochlorite solution, sterile water is used for washing repeatedly, after a single rhizomes are cut by a sterile knife, the rhizomes are clamped by forceps to be streaked on a solid YMA (containing Congo red with the final concentration of 25 ppm) culture medium, the solid YMA culture medium is inversely cultured for 5-7 d in a constant temperature incubator at the temperature of 28 ℃ until clear colonies grow out, streaking and purifying are carried out on the isolated rhizobium monoclonals for 3-4 times according to the characteristics of the shape, size, transparency, viscosity, color, gloss and the like of the thalli until clear monoclonals grow out, a single colony transfer freeze-drying tube is selected, the single colony transfer freeze-drying tube is preserved at the temperature of 4 ℃, and the obtained strain is named as Qigobium TRM95796 and Mubase rhizobium TRM96647.
Example two identification of Rhizobium mirabilis TRM95796 and Rhizobium lignan TRM96647
In order to further determine the taxonomic status of rhizobia TRM95796 and TRM96647, the present invention uses a heterogeneous classification method to classify and identify the strain.
Culture medium for morphological observation
LB medium: tryptone 10g/L, naCl10g/L, yeast extract 5g/L, pH 7.0-7.2, and culturing at 30deg.C for 3 days.
Primer(s)
The bacterial universal primer is synthesized by Shanghai division of Beijing qingke biotechnology Co. The sequences are respectively as follows: 27F (5'-AGAGTTTGATCCTGGCTC-3') and 1492R (5'-CGGCTACCTTGTTACGACTT-3').
Test method
Morphological observation method for rhizobia TRM95796 and TRM96647
Scanning electron microscope and transmission electron microscope observation: rhizobium TRM95796 and TRM96647 single colonies are selected and cultured in LB liquid medium at 30 ℃, bacterial cells are centrifugally collected, and 2.5% glutaraldehyde is added for fixation. The strain morphology, cell shape and size, presence or absence of flagellum, etc. were recorded by scanning and transmission electron microscopy.
Extraction method of rhizobia TRM95796 and TRM96647 genome DNA
TRM95796 and TRM96647 cells on the collection plates were placed in a 1.5mL sterile centrifuge tube, and 480. Mu.L of 1 XTE buffer was added. Add 20. Mu.L lysozyme (50 mg. M L) -1 ) Placing in a shaking table at 37 deg.C, and shaking overnight at 200 rpm. mu.L of 20% SDS was added to each tube, and 5. Mu.L of 20mg. m L was added -1 Is put into a shaking table at 60 ℃ and is oscillated for 1h at 200 r/min. 550. Mu.L of phenol/chloroform/isoamyl alcohol (25:24:1) was added, centrifuged at 12000rpm for 10min, the supernatant was transferred to another centrifuge tube, and repeated extraction was performed 2-3 times. Taking the supernatant, adding the same volume of absolute ethyl alcohol, adding 0.1 times volume of sodium acetate (3 mol.L) -1 ) The DNA was precipitated in a refrigerator at 4℃for about 0.5h. Centrifuge at 12000rpm for 10min, discard supernatant. The centrifuged product was washed 2 times with 200. Mu.L of 70% ethanol, centrifuged at 12000rpm for 5min, and the supernatant was discarded to evaporate the ethanol completely. The DNA at the bottom was sufficiently dissolved with 50. Mu.L of sterile ultra-pure water, and the quality of DNA extraction was checked by agarose gel electrophoresis at 1%, and the extracted DNA was kept in a refrigerator at-20℃for further use.
Amplification method of rhizobia TRM95796 and TRM96647 16S r RNA genes
The 16S R RNA gene fragment in bacterial genomic DNA was amplified using bacterial 16S R RNA gene universal primers 27F (5'-AGAGTTTGATCCTGGCTC-3') and 1492R (5'-CGGCTACCTTGTTACGACTT-3').
The 25. Mu.L PCR reaction system was: dd H 2 O20.4. Mu.L, 10 XBuffer (Buffer containing Mg 2+ ) 2.5. Mu.L, dNTPs 0.5. Mu.L, primer 27F (10. Mu. Mol. L) -1 ) 0.5. Mu.L, primer 1492R (10. Mu. Mol. L) -1 ）
0.5. Mu.L of Taq DNA polymerase 0.1. Mu.L, template DNA 0.5. Mu.L.
The PCR reaction conditions were: pre-denaturation at 94℃for 4min; denaturation at 94℃for 1min, annealing at 56℃for 1min, extension at 72℃for 2min,30 cycles; total extension was carried out at 72℃for 8min. After completion of the reaction, the reaction mixture was subjected to 1% agarose gel electrophoresis. Sequencing the PCR products meeting the conditions.
Comparison of sequencing results.
The sequencing results are spliced by SeqMan software, sequences are compared through the effectively published strain sequences in an EzBioCloud database, the 16S r RNA gene sequences of the effectively published strains with higher similarity are downloaded, and the MEGAX software is used for constructing a phylogenetic tree of the sequences to determine the taxonomic position of actinomycetes.
The genome-wide splice results were compared using the GenBank database in NCBI to further determine the taxonomic status of the strain.
Test results
Morphological observations of rhizobia TRM95796 and TRM96647
Rhizobium TRM95796 and TRM96647 are negative in gram staining, the optimal growth temperature is 25 ℃, the optimal growth p H is 7, the optimal NaCl (W/V) is 0%, and the rhizobium is subjected to plate culture on LB culture medium at 25 ℃ for 3d, so that the bacterial colony is round, light yellow, and the edge of the bacterial colony is neat and convex. The observation by a scanning electron microscope shows that: rhizobium TRM95796 is in a long rod shape, the width of the thallus is 0.3-0.4 mu m, and the length is 1.5-1.7 mu m; TRM96647 is in the shape of rod, and has thallus width of 0.4-0.5 μm and length of 1.1-1.5 μm; the observation by a transmission electron microscope shows that: the thallus has flagella, as shown in figure 1. Determination of TR based on rhizobia TRM95796 and TRM96647 colonies, cell morphology and physiological characterizationM95796 and TRM96647 belong to the genus RhizobiumRhizobium）。
Molecular biological identification results of rhizobia TRM95796 and TRM96647
16S r RNA gene sequence determination results of rhizobium TRM95796 and TRM96647
The sequencing results were spliced using SeqMan software, and it was determined that TRM95796 and TRM96647 consisted of 1501 and 1383 bases, respectively, to give the 16S rRNA gene sequences of Rhizobium TRM95796 and TRM96647, respectively, as follows:
SEQ ID NO:1
>TRM95796
CCTTGTTACGACTTCACCCCAGTCGCTGACCCTACCGTGGTCGCCTGCTTCCCTTGCGGGTTGGCGCAGCGCCTTCGGGTAAAACCAACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCGTTCTGATCCACGATTACTAGCGATTCCAACTTCATGCACCCGAGTTGCAGAGTGCAATCCGAACTGAGACGGCTTTTGGAGATTAGCTCACCCTCGCGGGTTCGCTGCCCATTGTCACCGCCATTGTAGCACGTGTGTAGCCCAGCCCGTAAGGGCCATGAGGACTTGACGTCATCCCCACCTTCCTCTCGGCTTATCACCGGCAGTCCCTCTAGAGTGCCCAACTGAATGATGGCAACTAGAGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTCTCCGGTCTAGCCGAACTGAAGGACAATGTCTCCACTGTCCGGAACCGGGATGTCAAGGGCTTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCCTTGTGCGGGCCCCCGTCAATTTCCCTTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGAATGGTTTAATGGCGTTAGCTGCGCCACCGACATGCATGCATGCCGACGGCTAACATTCATCGTTTTACGGCGTGGACTACCAGGGTATCTAAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGCGGTCAGTAATGGGACCAGTAAGCCGCTTCCGGCCACTGGTGTTCCTCCGAATATCTACGAATTTCACCTCTACACTCGGAATTCCACTTACCTCTTCCATACTCGAGACACCCAGTATCAAAGGCAGTTCCAGAGTTGAGCTCTGGGATTTCACCCCTGACTTAAATGTCCGCCTACGTGCGCTTTACGCCCAGTAATTCCGAACAACGCTAGCCCCCTTCGTATTACCGCGGCTGCTGGCACGAAGTTAGCCGGGGCTTCTTCTCCGGATACCGTCATTATCTTCTCCGGTGAAAGAGCTTTACAACCCTAGGGCCTTCATCACTCACGCGGCATGGCTGGATCAGGCTTGCGCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCTGATCATCCTCTCAGACCAGCTATGGATCGTCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATCCAACGCGGGCTCATCCATCCCCGATAAATCTTTCCCCCCCATAAAATGCCAAGGGCATTCTATGATTCTTGGTATTTTAAAGGGGGGCGTATACGGTATTAGCACACGTTTCCATGCGTTATTCCGTAGGGTTGGGTAGATTCCCACGCGTTACTCACCCGTCTGCCGCTGACATATTGCTATGCCCGCTCGACTTGCATGTGTTAAGCCTGCCGCCAGCGTTCGTTCTGAGCCATGATCAAACTCT
SEQ ID NO:2
>TRM96647
CGGCGTTGATCTGAACTCAGGATCACTCGTGTTACACTGCAATCGCGTGTCGCCCTTAGAGTTTGATCCTGGCTCAGAACGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGCCCCGCAAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAACGTACCCTTTGCTACGGAATAACTCCGGGAAACTGGAACTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATCGGCAAAGGATCGGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCCTAGGGTTGTAAAGCTCTTTCACCGATGAAGATAATGACGGTAGTCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGGTATTTAAGTCAGGGGTGAAATCCCGGAGCTCAACTCCGGAACTGCCTTTGATACTGGGTACCTAGAGTATGGAAGAGGTAAGTGGAATTCCGAGTGGAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTTTACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATCCCGATCGCGGTTCGTGGAGACACGTTCCTTCAGTTCGGCTGGATCGGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTCAGTTGGGGACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGACCGCGAGGTCGAGCTAATCTCCAAAAGCCATCCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCCACCATGGGGAGTTGGTTTTACCGAAAGGTAGTGTGCTAACCGCAAGGAGGCAGCTAACCACGGTAGGGTCAGCGACTGGGGGTGAAGTCGTAACAAGGTAAGCCGTAAAGGGCGACACGCGATTGCAGTCTTGAGTCCACCTGAAGGATGTCAAACTTGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTTCCATAAAAACAACATACGAGCCGGAACAAGAAAGTCAAAAGCCTCCGACCGGAGGCTTTTGAC
rhizobia TRM95796 and TRM96647 homologous evolutionary tree construction result
By comparing in EzBioCloud database, sequences with similarity to the 16S rRNA gene sequence of rhizobium to be detected are compared by MEGA softwareAnd (5) carrying out multiple sequence comparison on actinomycetes 16S r RNA gene sequences, and constructing a phylogenetic tree. The evolutionary tree of rhizobia TRM95796 and TRM96647 is shown in figures 2A, 2B and 2C, and rhizobia TRM95796 and rhizobia are shown in the specificationRhizobium alvei TNR-22T)The 16S r RNA gene sequence (the consistency is 96.31%) is gathered on the same systematic evolution branch, TRM96647 and rhizobia are @Rhizobium azooxidifex Po 20/26 T ) The 16S r RNA gene sequence (identity 97.5%) but with low similarity, so the novel species of rhizobia was named rhizobia mirabilis according to the sampling placeRhizobium qitaienseTRM95796 and Rhizobium lignans Rhizobium muleiense TRM96647。
Rhizobia TRM95796 and TRM96647 whole genome and housekeeping thereofrecA-atpD) Gene evolutionary tree construction results
Uploading the genome of strains TRM95796, TRM96647, TRM96650 and kindred rhizobium strain to TYGS (http:// types. Dsmz. De) to construct a whole genome system developmental evolutionary tree, as can be seen from FIG. 3, TRM95796 and strain @Rhizobium alvei TNR-22T) is clustered on one branch, TRM96647T and%Rhizobium azooxidifexPo 20/26 T ) Clustered together, the whole genome cluster results were consistent with the 16S rRNA results.
The housekeeping genes of rhizobia TRM95796 and TRM96647 are prepared by adopting a Neighbor-Joining (NJ) methodrecA、atpD) And constructing a phylogenetic tree of housekeeping genes with the housekeeping gene sequences of the near-edge rhizobia, wherein the housekeeping gene sequences of the near-edge rhizobia can be downloaded from an NCBI database. Rhizobium TRM95796 and rhizobium @Rhizobium alvei TNR-22T) are gathered on the same systematic evolution branch, TRM96647 and rhizobiaRhizobium azooxidifex Po 20/26 T ) The housekeeping gene sequences of (a) are gathered on the same systematic evolution branch (as shown in figure 4), and consistent results are presented through the 16S rRNA cluster analysis, the whole genome cluster analysis and the housekeeping gene cluster analysis, so that TRM95796 and TRM96647 are both novel rhizobia species.
Strains TRM95796T and TRM96647T polar lipids
Analyzing the type of the bacterial strain polar lipid by adopting a biphasic thin layer chromatography, and analyzing by using an ninhydrin reagent, an anisaldehyde color developing agent and a molybdenum phosphoric acid color developing agent to obtain the bacterial strain TRM95796, wherein the type of the bacterial membrane phospholipid lipid is Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), unknown type of Amino Glycolipid (AGL), double Phosphatidylglycerol (DPG), unknown type of phospholipid (PL 1) and glucosamine-containing unknown structure phosphate lipid (NPG), and compared with similar bacterial strains, the similar bacterial strains contain L1-L4 and PL2; whereas TRM95796 was absent, indicating a difference between the two, the color development results are shown in fig. 5. The TRM96647 cell membrane phospholipid lipid type is Phosphatidylcholine (PC), phosphatidylglycerol (PG), biphospholipid glycerol (DPG), and glucosamine-containing unknown structure phosphate lipid (NPG), unknown type phosphoramidate APL1 and APL2 and unknown type phosphoramidate (AL 1), and unknown type phospholipids PL1 and PL2; the strains were compared with the nearest strains, and the similar strains lack Phosphatidylglycerol (PG), biphospholipid glycerol (DPG) and glucosamine-containing phosphate lipid (NPG) with unknown structures, which indicate that the strains of two new species are different from the similar strains, and are potential new species, and the color development results are shown in FIG. 5.
TABLE 1 heterogeneous classification index comparison of strains TRM95796 and TRM96647 with similar strains
Note that: strains: 1,Rhizobium qitaienseTRM95796 T ; 2, R.alveiTNR-22 T ( Sheu SY et al.); 3, R. tarimense PL-41 T (Turdahon M et al.); 4, R. muleiense TRM96647 T ; 5, R. azooxidifex Po 20/26 T (BehrendtUet al.); 6,R. subbaraonis JC85 T (Ramana CVet al) +, positive; -, negative;
TABLE 2 Strain TRM95796 T And TRM96647 T dDDH and ANI values for strains of rhizobium kindred species
Example three, tiebacteria TRM95796 and TRM96647 to chickpea potting test
Sterilizing chickpea seeds: selecting uniform chickpea seeds with full size, treating with 75% ethanol for 30s, sterilizing with sodium hypochlorite for 2min, washing with sterile water for 8-10 times, spreading on 1% water agar plate, and germinating at 28deg.C for 2-3d or soaking in water directly after washing, and placing in a 28 deg.C incubator for 1-2h.
Culturing the bacterial cells: preparing 1L YMA liquid culture medium, subpackaging in 5 triangular bottles, and sterilizing at 121deg.C for 20min; and (3) sucking 2mL of rhizobia bacterial liquid which is purified and reaches the logarithmic growth phase into a triangular flask filled with YMA culture medium, oscillating overnight at a rotating speed of 180r/min on a shaking table at 28 ℃ until the OD 600 value of the bacterial liquid reaches 0.6+/-0.05, subpackaging into a sterilized 50mL centrifuge tube, centrifuging at 5000rpm in the centrifuge for 10min, taking out, pouring out supernatant, and re-suspending with nitrogen-free nutrient solution to obtain bacterial suspension for later use.
Potted chickpea and inoculation: sowing the germinated or soaked seeds in sterilized vermiculite, 6 seeds in each pot until the first cotyledon grows out, leaving three strong seedlings in the pot, inoculating 5mL rhizobium suspension in the step, culturing in an illumination room for 30-45 days, and watering the plants with the Fahraeus nitrogen-free nutrient solution during the growth period. The temperature of the illumination chamber is 20 ℃, the illumination is 16 hours, and the darkness is 8 hours.
Taking non-inoculation as a control, repeating each step for 6 times, harvesting chickpea plants after 60 days of inoculation, and taking the fresh weight, nodulation number and nitrogen fixation enzyme activity value of the plants as judgment indexes for measuring the binding property and nitrogen fixation capacity of rhizobia.
In the blank (-N) no nodulation was observed, but inoculation with rhizobia TRM95796 and TRM96647 was able to promote nodulation of chickpea, i.e. tieback treatment averaged 30 and 89 per chickpea cocoa nodulation, the nodulation profile showed a dark red color (see FIG. 6), and the rhizobia nitrogen fixation enzyme activities were measured at 26.21. Mu. Mol/h/g and 68.08. Mu. Mol/h/g, giving a preliminary determination that the test TRM95796 and TRM96647 strains were rhizobia with nitrogen fixation efficiency.
Example four, application of rhizobia TRM95796 and TRM96647 bacteria suspension chick pea field test
The total test area was 27m2. Each cell 3m2. The 3 ridge areas of the cells are 6 m long, each cell is separated by a plastic film, and the depth is 50cm. A total 9 cell arrangement is as follows:
granule 1 Granule group 2 Granule 3
CK 1 Inoculation of TRM95796 treatment 1 Inoculation of TRM96647 treatment 3
Inoculation of TRM96647 treatment 1 CK 2 Inoculation of TRM95796 treatment 2
Inoculation of TRM95796 treatment 3 Inoculation of TRM96647 treatment 2 CK 3
Randomly arranged, 3 different treatments. Each treatment was repeated 3 times.
Culturing the bacterial cells: preparing 10L YMA liquid culture medium, subpackaging in 500mL triangular bottles with 200mL of each bottle, and sterilizing at 121deg.C for 20min; and (3) sucking 2mL of rhizobia bacterial liquid which is purified and reaches the logarithmic growth phase into a triangular flask filled with YMA culture medium, oscillating overnight at a rotating speed of 180r/min on a shaking table at 28 ℃ until the OD 600 value of the bacterial liquid reaches 0.6+/-0.05, subpackaging into a sterilized 200mL centrifuge tube, centrifuging at 5000rpm in the centrifuge for 10min, taking out, pouring out supernatant, and re-suspending with nitrogen-free nutrient solution to obtain bacterial suspension for later use.
Sowing and inoculating: sowing the germinated or soaked seeds in a field, inoculating rhizobia suspension in the steps until the first cotyledon grows out of the plants, performing daily management in the field, performing growth period investigation of chickpeas in the test process, and recording investigation results. Plant height, aerial part biomass (fresh weight and dry weight), root nodule number, root nodule weight and other characters of chickpea. Investigation is carried out from the emergence of chickpea in the seedling emergence stage, the flowering stage and the pod bearing stage of chickpea respectively; 3 individuals were randomly selected and measured for each treatment cell.
The chickpea leaf plants of the control group (not treated with the bacteria) were short and thin in stem, while the chickpea leaves of the experimental group inoculated with rhizobium TRM95796 and TRM96647 were greener, the plants were higher and thicker in stem, and the aerial part biomass (fresh weight and dry weight) and the number of nodules (as in fig. 7), the weight of the nodules were superior to those of the control group.

Claims (10)

1. A rhizobium capable of promoting chickpea nodulation and fixing nitrogen is characterized by being rhizobium of the singular stageRhizobium qitaiense) The deposit number is GDMCC No.62650.
2. The method according to claim 1, wherein the culture medium used for the rhizobia is YMA medium: mannitol 10g/L, yeast extract 0.4g/L, K 2 HPO 4 0.5g/L，MgSO 4 0.2g/L, trace elements 4mL; the microelement nutrition liquid comprises 0.22g ZnSO 4 ，1.81g MnSO 4 ，2 .86g H 3 BO 3 ，0 .8g ，CuSO 4 ·5H 2 O and 0.02. 0.02 g H 2 MoO 4 18g/L agar, 1L water, and adjusting pH to 7.0-7.2.
3. The method of claim 2, wherein the fermentation conditions are 25 ℃.
4. Use of rhizobia as claimed in claim 1 in nodulation and nitrogen fixation of plants of the leguminous family.
5. The use according to claim 4, wherein the leguminous plant is chickpea.
6. The use according to claim 4, wherein the rhizobia is inoculated onto the plant separately or in combination to promote nodulation and nitrogen fixation of the plant and to increase nitrogen nutrition of the plant.
7. Use according to any one of claims 4 to 6, characterized in that it is for the cultivation of chickpeas, in particular in saline-alkaline areas.
8. A nitrogen fixing microbial preparation comprising a culture of rhizobia as claimed in claim 1.
9. The nitrogen-fixing microbial formulation of claim 8, wherein said culture is a bacterial suspension.
10. The nitrogen-fixing microbial formulation of claim 9, wherein the OD of the bacterial suspension 600 The value is 0.8-1.2.
CN202211704961.1A 2022-12-07 2022-12-07 Qigou rhizobium for promoting chickpea nodulation and application thereof Pending CN116694496A (en)

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Publication number Priority date Publication date Assignee Title
CN116083299A (en) * 2022-12-07 2023-05-09 塔里木大学 Mubase rhizobium for promoting chickpea nodulation and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116083299A (en) * 2022-12-07 2023-05-09 塔里木大学 Mubase rhizobium for promoting chickpea nodulation and application thereof
CN116083299B (en) * 2022-12-07 2024-06-11 塔里木大学 Mubase rhizobium for promoting chickpea nodulation and application thereof

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