CN109370953B - Juemina rhizobium JYN6 and application thereof - Google Patents

Abstract

The invention discloses rhizobium JYN6 and application thereof, and belongs to the technical field of microorganisms. The strain is separated and purified from fresh root nodules of grass of Cassia and PCR detectionnodAgene, identified by 16S rDNA molecular biology as a species of bradyrhizobium: (Bradyrhizobium) The new strain of (1) and is named as Anyu No. 1. And 4, 4 and 10 days in 2018, the culture is preserved in China center for type culture Collection with the preservation number of CCTCC NO: and M2018192. The rhizobium JYN6 disclosed by the invention is proved to have the characteristics of high nodulation efficiency and strong nitrogen fixation capacity through a laboratory pot culture tieback test, and the inoculation of the rhizobium JYN6 can obviously improve the root nodule number, the root nodule nitrogen fixation efficiency, the biomass and the plant nitrogen content of cassia grass, so that the effects of fertilizing the soil and improving the ecological environment are achieved.

Description

Juemina rhizobium JYN6 and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a strain of cassia rhizobium JYN6 and application thereof.

Background

About 5690 thousands hm in China²Acid soil, which accounts for more than 42.2 percent of the total cultivated area. In recent years, soil acidification has become more severe with the application of large amounts of chemical nitrogen fertilizers. Acid soil in China is mainly concentrated in the area south of the Yangtze river, the area is rich in light, heat, water and other resources, but the soil is lean, heavy, low in pH value and low in nutrient availability, so that the rapid development of agriculture in the area is severely restricted. In production, crop yield is often increased by applying large amounts of fertilizers. However, the large amount of fertilizer is added, which not only causes soil hardening and further acidification, but also causes water eutrophication and groundwater pollution, and seriously damages the ecological environment.

The method for improving the soil and improving the soil fertility by planting leguminous plants and combining the rhizobium inoculation technology is an accepted approach at present. The leguminous plants can be combined with rhizobia to form a mutualistic and reciprocal symbiotic relationship. The rhizobia obtains carbohydrates and other nutrients required by growth from host plants, and the host plants obtain nitrogen nutrition from the rhizobia biological nitrogen fixation process, so that the effects of improving the crop yield and reducing the using amount of chemical fertilizers are achieved, and the sustainable development of agriculture is maintained while water and soil pollution is reduced.

Cassia plant (A)Chamaecrista sppGreene.) is an important legume in tropical and subtropical regions, including trees, shrubs, and herbs. Cassia seed, semen Cassiae (Cassia Torae)Chamaecrista rotundifolia(Pers.) Greene native to Australia is a semi-upright perennial legume of the genus CassiaThe plant has the characteristics of high nitrogen fixation efficiency, barren resistance, large biomass, bright color, particular suitability for acid soil and the like. In 1996, the academy of agricultural sciences of Fujian province introduced Cassia tora from the Australian pasture germplasm resource center, and used as leguminous green manure type pasture for improving acid soil, and popularized and planted in acid soil areas such as Fujian, Guangdong and Guangxi. However, the cassia tora has slow and less nodulation and low nitrogen fixation efficiency under natural conditions, and seriously influences the planting, popularization and application of the cassia tora. Therefore, in order to improve the nodulation rate and nitrogen fixation efficiency of the cassia tora rotundifolia, rhizobium strains with high nodulation rate and strong nitrogen fixation capacity need to be screened out urgently so as to improve the nitrogen fixation efficiency of the cassia grass, and further achieve the effects of fertilizing the soil and improving the ecological environment.

Disclosure of Invention

One of the purposes of the invention is to provide a strain of cassia rhizobium JYN6, which is classified and named as rhizobium (A)Bradyrhizobiumsp.) JYN6, is a new strain of bradyrhizobium, is preserved in China center for type culture Collection (CCTCC NO): m2018192, the preservation address is: wuhan university. The strain has the characteristics of high tumor formation rate and strong nitrogen fixation capacity.

The invention also aims to apply the rhizobium JYN6 in symbiotic nitrogen fixation of plants in the genus of Cassia in the family of Leguminosae, promote the growth of Cassia rotundifolia by improving the biological nitrogen fixation capacity of the Cassia rotundifolia, and achieve the purposes of weight reduction and synergism in the production of Cassia tora.

The technical purpose of the invention is realized by the following technical scheme:

1 isolation and purification of Rhizobium strains

1) Taking fresh, mature and large and plump nodules of the Cassia pinnata, washing the nodules with water, and sucking the water on the surfaces of the nodules with filter paper;

2) treating in 95% (w/v) alcohol for 3-5 min, and adding 0.1% (w/v) HgCl₂Sterilizing for 3-5 minutes, taking out, and washing with sterile water for 5-6 times;

3) cut in half on a flame-sterilized slide, grasp half of the tumor with sterile forceps, scribe the incision facing the YMA medium surface, invert and incubate at 28 ℃. The formulation of YMA medium is shown in Table 1:

TABLE 1 YMA (Yeast Mannitol agar) Medium formulation

4) After the bacteria grow out, scraping a small amount of rhizobium colonies from the flat plate by using a gun head, adding 1mL of sterile water for dilution, performing streak culture on the flat plate again, and observing the conditions of the colonies after 3 d until 15 d (the colonies of the slow rhizobium need to appear in 7-15 d) is observed. If no single colony appears, the streaking purification on the plate needs to be repeated until the single colony appears.

5) After the monoclonal thallus grows out, judging whether the thallus is the rhizobium according to the following two aspects:

① colony morphology, the colony of rhizobia is round, milky white, translucent, neat in edge, more or less sticky, fast-growing rhizobia is obtained when the diameter of the colony reaches 2-4 mm after 3-5 days of culture, and slow-growing rhizobia is obtained when the diameter of the colony is 1 mm after 7-10 days of culture.

②nodAPCR detection of gene: selecting the above-mentioned morpholobium to make monoclonal analysisnodAgene PCR detection, primers are respectivelynodA-F andnodA-R, forward primer SEQ ID No. 1:nodA-F5 '-TGCRGTGGARDCTRYGCTGGGAAA-3'; reverse primer SEQ ID NO. 2:nodA-R5 '-GNCCGTCRTCRAASGTCARGTA-3'. The enzyme is selected from 2 XStarmix, and the genome DNA of rhizobium BXYD3 and water are respectively used as templates to be used as a positive control and a negative control.nodAThe gene length is 666 bp, whether a band exists at the position of 666 bp or not is observed by using an electrophoretic imaging technology, and the colony with the band is rhizobium. The PCR reaction system is shown in Table 2:

TABLE 2nodAGene 2 × starMix enzyme reaction system

Reaction conditions are as follows: 5min at 94 ℃; (94 ℃ 30 s, 55 ℃ 30 s, 72 ℃ 1 min) x 35 cycle; 5min at 70 ℃.

Properties of 2 Rhizobium JYN6

1) Morphological characteristics

The strain is slow-growing in colony morphology, small in circle, milky white, translucent and more in mucilage. The diameter of the YMA plate after growing for 5-7 d is 0.8-2.0 mm (figure 1).

2) Characteristics of culture

The optimal growth conditions of the strain are as follows: the pH value is 7.0, the temperature is 28 ℃, the rotating speed is 180 r/min, and mannitol and yeast powder can be respectively used as a carbon source and a nitrogen source.

3) Genetic characterization

Warp beamnodAgene PCR, electrophoresis imaging detection, with a lighter band at 666 bp (FIG. 2).

4) Functional characteristics

The rhizobium JYN6 has the characteristics of high nodulation rate, strong nitrogen fixation capacity and the like. The thalli is released into the natural environment, is harmless to human, animals and plants, does not pollute the environment, enriches the group diversity of rhizobia in soil, and has obvious promotion effect on nodulation and nitrogen fixation of the forage grass of the Mingmu.

16SrDNA molecular biological identification of rhizobium JYN6

To identify the phylogenetic position of the rhizobia strains, the isolated rhizobia was subjected to PCR-specific amplification of 16SrDNA, sequencing the forward primer SEQ ID No. 3:V4V5515-F5 '-GTGCCAGCMGCCGCGGTAA-3'; sequencing reverse primer SEQ ID No. 4:V4V5907-R5'-CCGTCAATTCCTTTGAGTTT-3'; the 16S rDNA sequence of the rhizobium JYN6 obtained by sequencing is shown in SEQ ID No. 5; the PCR reaction system is shown in Table 3:

TABLE 316S rDNA2 × starMix enzyme reaction System

Reaction conditions are as follows: 5min at 95 ℃; (95 ℃ 20 s, 55 ℃ 20 s, 72 ℃ 50 s). times.44 cycle; 5min at 70 ℃.

Obtaining 13 reference strain sequences from NCBI (GenBank) database, analyzing the 16SrDNA partial sequences of the separated strain and the reference strain by using software BioEdit and MEGA6, and constructing a phylogenetic tree of the separated strain and the reference strain. Thereby determining the rhizobium JYN6 as the bradyrhizobium (Bradyrhizobium) The new strain of (FIG. 3) and named as Anyu No. 1.

The invention has the following beneficial effects:

1) the rhizobium JYN6 has the characteristics of high nodulation rate and strong nitrogen fixation capacity. The retrografting test shows that compared with the inoculation without rhizobium, the inoculation of the rhizobium 30 d can respectively increase the fresh weight and the dry weight of the cassia tora by 161.78wt% and 213.49wt%, and can be used for actual production; 2) the cassia tora is inoculated with the rhizobium JYN6 of the invention, so that the total nitrogen content of the plant is remarkably improvedP<0.01), thereby achieving the purposes of promoting the growth of the cassia tora plants and improving the soil fertility; 3) the rhizobium JYN6 is suitable for acid soil areas in south China and can be used as one of important measures for conventional cultivation of the cassia tora.

Drawings

FIG. 1 colony morphology of Rhizobium JYN6 on YMA medium;

FIG. 2 Rhizobium JYN6 warpnodADetecting the image formed by gene PCR and electrophoresis imaging; where, + represents the use of DNA from Rhizobium BXYD3 as a positive control, -the use of water as a negative control.

FIG. 3 is a 16S rDNA partial sequence analysis phylogenetic tree of Rhizobium JYN 6; wherein the numbers on the branches represent confidence levels; the parenthesis indicates the accession number in Gene bank; the scale bar indicates 2 substitutions out of 100 nucleotides.

FIG. 4 is a graph showing the effect of vermiculite tieback by Rhizobium JYN 6; CK: blank control.

FIG. 5 shows the nodulation number and activity of nodule nitrogenase of cassia tora after inoculation of rhizobium JYN 630 d; a: the nodulation number of cassia occidentalis; b: root nodule nitrogenase activity of cassia tora; CK: blank control.

FIG. 6 effect on growth of Cassia tora after inoculation with Rhizobium JYN 630 d; wherein, A: fresh weight of semen Cassiae; b: dry weight of cassia tora;c: the height of the cassia tora; d: cassia tora SPAD value; e: the nitrogen content of the cassia tora plant; CK: blank control; *: 0.01<P<0.05，**：0.001<P<0.01，***：P<0.001。

Detailed Description

Example 1 isolation and purification of Rhizobium JYN6

1 isolation of Rhizobium

Taking fresh, mature and large and plump nodules of Cassia alata, washing with water, and drying surface water by using filter paper. Firstly, the mixture is put into 95 percent (w/v) ethanol for treatment for 3 to 5 minutes, taken out and washed by sterile water for 5 to 6 times, and then 0.1 percent (w/v) HgCl is put into the mixture₂Sterilizing for 3-5 minutes, taking out and washing with sterile water for 5-6 times. Then, the slide glass was cut in half on a flame-sterilized slide glass, and the half tumor was held with a sterile forceps, and the cut was streaked to the surface of YMA (Table 1) medium, which was then inverted and cultured at 28 ℃.

2 purification of

After the bacteria grow out, scraping a small amount of rhizobium colonies from the flat plate by using a gun head, diluting the rhizobium colonies with 1mL of sterile water, performing streak culture on the flat plate again, and observing the conditions of the colonies after 3 d until 15 d (the colonies of the bradyrhizobium need to appear in 7-15 d) is observed.

Whether the rhizobia is rhizobium can be preliminarily judged according to the following two aspects:

① colony morphology, the colony of rhizobia is round, milky white, translucent, with regular edges and more or less mucilaginous, the colony diameter is 2-4 mm when cultured for 3-5 days, and the colony diameter is 1 mm when cultured for 7-10 days, the colony is slow-growing rhizobia.

②nodAgene PCR detection: selecting the above-mentioned morpholobium to make monoclonal analysisnodAgene PCR detection, primers are respectivelynodA-F andnodAand (2) -R, wherein the enzyme is 2 XStarMix, and the genome DNA of rhizobium BXYD3 and water are respectively used as templates to be used as a positive control and a negative control. The length of the gene band is about 666 bp, a brighter band is observed at the position of 666 bp by the strain JYN6 through an electrophoresis imaging technology, and the rhizobium is preliminarily determined. Then adding 50% (w/v) glycerol with the same volume and placing at-80 ℃ for bacteria preservation.

In the embodiment, the rhizobia JYN6 is a slow rhizobia, and is small in circle, milky white, translucent and more in mucilage in colony morphology; the diameter of the YMA plate after growing for 5-7 d is 0.8-2.0 mm (figure 1); the optimal growth conditions are as follows: the pH =7.0, the temperature is 28 ℃, the rotating speed is 180 r/min, and mannitol and yeast powder can be respectively used as a carbon source and a nitrogen source; warp beamnodAgene PCR and electrophoresis imaging detection, wherein the band is consistent with the band taking the genome DNA of the positive control rhizobium BXYD3 as a template (the gene length is 666 bp, as shown in figure 2).

By applying the separation and purification method, after 4 generations of repeated purification, streaking culture is carried out in YMA solid culture medium (table 1) to obtain a plurality of pure bacteria, the pure bacteria are verified to have nodulation and nitrogen fixation capability through a vermiculite tieback test, and the strain JYN6 with high nodulation rate and strong nitrogen fixation capability is separated and purified in the embodiment.

Example 2 molecular biological characterization of 16S rDNA of Rhizobium JYN6

Carrying out 16S rDNA PCR specific amplification on the rhizobium monoclonal bacterial liquid, wherein the forward primer is SEQ ID NO. 3:V4V5515-F5 '-GTGCCAGCMGCCGCGGTAA-3'; the reverse primer is SEQ ID NO. 4:V4V5907-R5'-CCGTCAATTCCTTTGAGTTT-3'. The enzyme is 2 XStar Mix. And detecting the PCR amplification product by an electrophoretic imaging technology, observing whether the PCR amplification product has a band, and using the residual PCR amplification product for sequence determination. The sequencing result is shown in SEQ ID NO. 5. The PCR reaction system is shown in Table 4.

TABLE 416S rDNA2 × starMix enzyme reaction System

Reaction conditions are as follows: 5min at 95 ℃; (95 ℃ 20 s, 55 ℃ 20 s, 72 ℃ 50 s). times.44 cycle; 5min at 70 ℃.

The sequences of 13 reference strains were obtained from the NCBI (GenBank) database, and the 16S rDNA partial sequences of the isolate and reference strains were analyzed using the software BioEdit and MEGA6 to construct phylogenetic trees of the isolate and reference strains (FIG. 3). Thereby identifying Rhizobium JYN6 is a strain of bradyrhizobium (A)Bradyrhizobium) The new strain of (1) and is named as Anyu No. 1.

Example 3 tieback test

The purpose of the test is as follows: screening out rhizobia with high bonding efficiency and strong nitrogen fixation capacity with cassia grass.

Test materials: the test plants: minyun No.1 round-leaf Cassia tora; test strains: isolating the purified rhizobia.

Main test instruments and equipment: a phytotron, a superclean workbench, an autoclave, a constant temperature incubator, 25 pot pots with 15 multiplied by 15 cm, 2 beakers with 1L, tweezers, a culture dish, filter paper, a glass rod, scissors, gauze and the like.

Test drugs and reagents:

(1) test drugs: mannitol, MgSO₄∙ 7H2O, NaCl, yeast powder, K₂HPO₄、KH₂PO₄、CaCO₃、Ca(NO₃)₂∙4H₂O、MgSO₄·7H₂O、CaCl₂·2H₂O、Na₂HPO₄·12H₂O、C₁₀H₁₂N₂O₈FeNa·3H₂O、Na₂MoO₄、MnSO₄、H₃BO₃、CuSO₄·5H₂O and ZnSO₄·7H₂O。

Test reagents:

1) YMA (Yeast Manninitol agar) liquid medium: weighing 10 g of mannitol and MgSO₄∙7H₂O0.2g, NaCl 0.1 g, yeast powder 3 g, K₂HPO₄0.25 g，KH₂PO₄0.25 g，CaCO₃3 g (added during storage) of agar and 15 g of agar were dissolved in 1L of purified water.

2) Plant low-nitrogen nutrient solution: ca (NO)₃)₂∙4H₂O 0.03 g，MgSO₄·7H₂O 0.28 g, CaCl₂·2H₂O0.10 g，KH₂PO₄0.10 g，Na₂HPO₄·12H₂O 0.15 g，C₁₀H₁₂N₂O₈FeNa·3H₂0.0075 g of O, 1mL of trace elements and 1000 mL of distilled water, and the pH value is 5.5. Microelement formula (g ∙ L)^-1): Na₂MoO₄0.03 g，MnSO₄1.81 g，H₃BO₃2.86 g，CuSO₄·5H₂O 0.8 g，ZnSO₄·7H₂O 0.22 g。

Test procedure

(1) Culturing of bacterial strains

Transferring the test strain preserved at-80 ℃ to YMA liquid for culture until logarithmic phase, namely OD value reaches 1-1.2.

(2) Sterilizing treatment

The pot culture pot, beaker, tweezers, culture dish, filter paper, glass rod, gauze and scissors required by the test are wrapped, the vermiculite is packaged by a freshness protection package, and the sterilization is carried out for 30 min at the temperature of 121 ℃. Bottling YMA liquid culture medium and distilled water, and sterilizing at 121 deg.C for 20 min.

Soaking test plant seeds in 80 deg.C hot water for 3 min to soften seed coat, separating out jelly, washing with clear water repeatedly, treating semen Cassiae Torae with 75% (w/v) alcohol for 15 min, washing with sterile water for 5-6 times, and washing with 10% (w/v) H₂O₂Sterilizing the surface for 4 min, washing with sterile water for 7-8 times, and performing germination acceleration for 10 h at 28 deg.C in a culture dish with filter paper until the radicle grows to 0.2 cm.

(3) Cultivation and inoculation of vermiculite

(1) The gauze strips penetrate through the bottom of the sterilized pot culture pot, and then the sterilized vermiculite is contained in the sterilized pot culture pot, so that the plant low-nitrogen nutrient solution is led to the substrate and the roots of the plants through the gauze strips, and the requirements of growth and development of the cassia tora on nutrition and moisture are met. The potted pots were then jacketed in beakers containing nutrient solution. The upper and lower layers of devices are wrapped by tinfoil paper to prevent the nutrient solution from growing moss under the illumination condition.

(2) The treated cassia tora seeds are sown in the vermiculite in a hole mode, the cassia tora seeds are inoculated with rhizobia bacterium liquid, the bacterium content is 10 hundred million per milliliter, and finally the vermiculite with the thickness of 1 cm is covered. The cells were cultured in a growth chamber (photoperiod: day/night =16 h/8 h, temperature: day/night =26 ℃/24 ℃).

And (3) repeating the steps by taking the rhizobium bacteria liquid without inoculation as a blank control, inoculating for 30 d, and then harvesting, wherein the fresh weight, the dry weight, the nodulation number, the plant nitrogen content, the rhizobium azotase activity, the SPAD value and the plant height of the plant are used as judgment indexes for measuring the rhizobium associativity and the nitrogen fixation capacity.

Analysis of results

As shown in fig. 5, the blank Control (CK) showed no nodules, and the tieback treatment yielded 32.08 nodules per 5 round-leaf cassia tora on average. When the interior of the root nodule was found to be red by cutting the root nodule, the test strain JYN6 was judged to be a Rhizobium having nitrogen-fixing efficiency (FIG. 4), and its root nodule nitrogenase activity was measured to find a value of 4.65 umol ∙ (g ∙ h)^-1。

As can be seen from FIG. 6, inoculation of Rhizobium jYN6 in Minyu No.1 of Cassia occidentalis can significantly increase the fresh weight, dry weight, plant height, SPAD value and nitrogen content of the plant (as compared with the blank control without inoculation of Rhizobium) ((P<0.001). The rhizobium JYN6 is high in binding property and nitrogen fixation efficiency.

SEQUENCE LISTING

<110> Fujian agriculture and forestry university

<120> a strain of Juemica rhizobium JYN6 and application thereof

<130>5

<160>5

<170>PatentIn version 3.3

<210>1

<211>24

<212>DNA

<213>nodA-F

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tgcrgtggar dctrygctgg gaaa 24

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<212>DNA

<213>nodA-R

<220>

<221>misc_feature

<222>(2)..(2)

<223>n isa, c, g, or t

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gnccgtcrtc raasgtcarg ta 22

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<213>V4V5515-F

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gtgccagcmg ccgcggtaa 19

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<213>V4V5907-R

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ccgtcaattc ctttgagttt 20

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<213> 16S rDNA of Rhizobium JYN6

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acaaagcagc gtgctcggat cactgggcgt aagggtgcgt aggcgggtct ttaagtcagg 60

ggtgaaatcc tggagctcaa ctccagaact gcctttgata ctgaagatct tgagttcggg 120

agaggtgagt ggaactgcga gtgtagaggt gaaattcgta gatattcgca agaacaccag 180

tggcgaaggc ggctcactgg cccgatactg acgctgaggc acgaaagcgt ggggagcaaa 240

caggattaga taccctggta gtccacgccg taaacgatga atgccagccg ttagtgggtt 300

tactcactag tggcgcagct aacgctttaa gcattccgcc tggggagtac ggtcgcaaga 360

ttaaaactca aaggaaattg acgga 385

Claims (2)

1. A strain of cassia bradyrhizobium JYN6 is characterized in that: (ii) the bradyrhizobium species (Bradyrhizobiumsp.) JYN6, is prepared by separating and purifying fresh root nodule of grass belonging to Cassia, and performing PCR detectionnodAgene, identified by 16S rDNA molecular biology, and preserved in China center for type culture Collection with the preservation number of CCTCC NO: and M2018192.

2. The use of a strain of bradyrhizobium of cassia JYN6 of claim 1 in enhancing biological nitrogen fixation of plants of cassia of leguminosae.

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