CN109337847B

CN109337847B - Cassia rhizobium TXR2 and application thereof

Info

Publication number: CN109337847B
Application number: CN201811480784.7A
Authority: CN
Inventors: 廖红; 杨庆; 李欣欣
Original assignee: Fujian Agriculture and Forestry University
Current assignee: Fujian Agriculture and Forestry University
Priority date: 2018-12-05
Filing date: 2018-12-05
Publication date: 2020-07-31
Anticipated expiration: 2038-12-05
Also published as: CN109337847A

Abstract

The invention discloses rhizobium TXR2 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 strain of the new bacterium of (1) and is named as Tianyuan No. 2. And is preserved in the China center for type culture Collection in 2018, 4 months and 10 days, with the preservation number of CCTCC NO: and M2018193. The rhizobium TXR2 disclosed by the invention has the characteristics of high nodulation efficiency and strong nitrogen fixation capacity as proved by a laboratory pot culture tieback test, and the inoculation of the rhizobium 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 fertility and improving the ecological environment are achieved.

Description

Cassia rhizobium TXR2 and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a strain of cassia rhizobium TXR2 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 rotundifoliaGreene is native to Australia, is a semi-upright perennial bean herb of Cassia, has high nitrogen fixation efficiency, is barren-resistant, has large biomass and bright color, and is particularly suitable for acid soil and the likeAnd (4) sex. 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 purpose of the invention is to provide a cassia grass rhizobium TXR2 strain, a classified name of the strain, namely rhizobium (TXR 2)Bradyrhizobiumsp.) TXR2, a new strain of bradyrhizobium, deposited in China center for type culture Collection in 2018, 4 months and 10 days, with the collection number of CCTCC NO: m2018193, 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 TXR2 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 Cassia rotundifolia, and achieve the purpose 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 tora, washing the nodules with water, and sucking the surface water by using 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 plate by using a gun head, adding 1m L sterile water for dilution, then carrying out streak culture on the plate again, observing the conditions of the colonies after 3 d, and observing for 15d (the slow rhizobium colonies need 7-15 d to appear), if no monoclonal colonies appear, repeatedly carrying out streak purification on the plate until the monoclonal colonies appear.

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' enzyme selects 2 × starMix, and genomic DNA and water of rhizobium BXYD3 are respectively 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 as follows:

TABLE 2nodAGene 2 × starMix enzyme reaction system

The reaction conditions are 94 ℃ for 5min, (94 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 1 min) × 35 cycle and 70 ℃ for 5 min.

2 characteristics of Rhizobium TXR2

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 TXR2 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.

16S rDNA molecular biology identification of 3 rhizobium TXR2

To identify the phylogenetic position of the rhizobia strains, 16S rDNA PCR-specific amplification of the isolated rhizobia strains was performed, 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 as follows:

TABLE 316S rDNA2 × starMix enzyme reaction System

The reaction conditions are 95 ℃ for 5min, (95 ℃ for 20 s, 55 ℃ for 20 s, 72 ℃ for 50 s) × 44 cycles, and 70 ℃ for 5 min.

Obtaining 13 reference strain sequences from NCBI (GenBank) database, and separating the strains and reference bacteria by using software BioEdit and MEGA6And analyzing the 16S rDNA partial sequence of the strain to construct a phylogenetic tree of the isolated strain and the reference strain. Thereby determining the rhizobia TXR2 as a species of bradyrhizobium (II) ((III))Bradyrhizobium) The new strain of (FIG. 3), and named as Tianyuan No. 2.

The invention has the following beneficial effects:

1) the rhizobium TXR2 disclosed by the invention has the characteristics of high nodulation rate and strong nitrogen fixation capacity, and a back grafting test shows that compared with the method without inoculating the rhizobium, the inoculation of the rhizobium for 30 d can respectively increase the fresh weight and dry weight of the cassia tora by 223.16wt% and 293.33wt%, and can be used for actual production; 2) the inoculation of the cassia tora with the rhizobium TXR2 of the invention can remarkably improve the total nitrogen content of plants (P<0.01), thereby achieving the purposes of promoting the growth of the cassia tora plants and improving the soil fertility; 3) the rhizobium TXR2 is suitable for acid soil areas in south China and can be used as one of important measures for conventional cultivation of the cassia occidentalis.

Drawings

FIG. 1 shows the colony morphology of Rhizobium TXR2 on YMA medium.

FIG. 2 shows Rhizobium TXR2nodADetecting the image formed by gene PCR and electrophoresis imaging; wherein, + represents the use of DNA from Rhizobium BXYD3 as a template for a positive control, and-represents the use of water as a negative control

FIG. 3 is a 16S rDNA partial sequence analysis phylogenetic tree of Rhizobium TXR 2; 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 of the effect of vermiculite tieback by Rhizobium TXR 2; CK: blank control.

FIG. 5 shows the nodulation number and activity of nodulation enzyme of cassia tora after inoculation of Rhizobium TXR 230 d; wherein, A: the nodulation number of cassia occidentalis; b: seaear shell nodule nitrogenase activity, CK: blank control.

FIG. 6 effect of inoculation of Rhizobium TXR 230 d on the growth of Cassia rotundifolia; 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 pairIrradiating; *: 0.01<P<0.05，**：0.001<P<0.01，***：P<0.001。

Detailed Description

Example 1 isolation and purification of the Azotobacter nodorum strain TXR2

1 isolation of Rhizobium

Taking fresh, mature and large and plump nodules of the cassia occidentalis, washing the nodules with water, and sucking 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 plate by using a gun head, diluting the rhizobium colonies with 1m L sterile water, and then carrying out streak culture on the plate again, observing the conditions of the colonies after 3 d until 15d (the slow rhizobium colonies need 7-15 d) is observed, if no monoclonal colonies appear, repeatedly carrying out streak purification on the plate until the monoclonal colonies appear.

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 the length of the gene is 666 bp, an electrophoresis imaging technology is used for observing that the strain TXR2 has a brighter band at 666 bp, the strain is preliminarily judged to be rhizobium, and then glycerol with the same volume of 50% w/v) is added for preserving the strain at the temperature of-80 ℃.

The strain TXR2 is a slow rhizobium, 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 TXR2 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 TXR2

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:V4V5907and R5'-CCGTCAATTCCTTTGAGTTT-3', detecting the enzyme by using a2 × star PCR amplification product as an electrophoresis imaging technology, observing whether the enzyme has a band, using the residual PCR amplification product for sequence determination, and obtaining a sequencing result shown as SEQ ID NO.5 and a PCR reaction system shown as a table 4.

TABLE 416S rDNA2 × starMix enzyme reaction System

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 determining the root noduleThe bacterium TXR2 is a bacterium belonging to the genus bradyrhizobium (A), (B), (C), (Bradyrhizobium) The strain of the new bacterium of (1) and is named as Tianyuan No. 2.

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.

The main test instruments and equipment comprise a climatic growth chamber, an ultra-clean workbench, an autoclave, a constant temperature incubator, 25 pot culture pots with 15 × 15 cm, 2 beakers with 1L cm, 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), 15 g of agar was dissolved in 1L pure 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, 1m of trace element L, 1000 m of distilled water L, pH 5.5, trace element 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

The test strains stored at-80 ℃ were transferred to YMA liquid culture and cultured to logarithmic phase. That is, the OD value is 1 to 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 4min, washing with sterile water for 7-8 times, and performing germination acceleration at 28 deg.C for 10 hr until radicle grows out 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 low-nitrogen nutrient solution of the plant is introduced to the matrix and the roots of the plant through the gauze strips, and the requirements of growth and development of the cassia tora pasture 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 vermiculite holes, the cassia tora seeds are inoculated with rhizobia bacterium liquid, the bacterium content is 10 hundred million per milliliter, each pot is 2 m L, and finally, vermiculite with the thickness of 1 cm is covered and placed in a growth chamber (the photoperiod is that day/night =16h/8h, and the temperature is that day/night =26 ℃/24 ℃) for culture.

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 an average of 44.33 nodules per 5 round-leaf cassia tora. The inside of the root nodule was found to be red by cutting the root nodule, and the test strain TXR2 was basically judged to be a root nodule bacterium having nitrogen-fixing efficiency (FIG. 4), and its nitrogen-fixing enzyme activity was measured to be 0.47 umol ∙ (g ∙ h)^-1。

As can be seen from FIG. 6, inoculation of Rhizobium TXR2 in Minyu No.1 of Cassia occidentalis significantly increased the fresh and dry plant weight compared to the control blank without inoculation of Rhizobium (fresh and dry plant weight)P<0.01), the plant height, the SPAD value and the nitrogen content of the plant are greatly improved (P<0.001). The rhizobium TXR2 is high in binding property and nitrogen fixation efficiency.

SEQUENCE LISTING

<110> Fujian agriculture and forestry university

<120> a strain of cassia rhizobium TXR2 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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gnccgtcrtc raasgtcarg ta 22

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<213>V4V5515-F

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gtgccagcmg ccgcggtaa 19

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ccgtcaattc ctttgagttt 20

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<213> 16S rDNA sequence of Rhizobium TXR2

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ctcctaagac gttgcctcgg atcactgggc gtaagggtgc gtaggcgggt cctttaagtc 60

agaggtgaaa tccctggagc tcaactccag aactgccttt gatactgaag atcttgagtt 120

cgggagaggt gagtggaact gcgagtgtag aggtgaaatt cgtagatatt cgcaagaaca 180

ccagtggcga aggcggctca ctggcccgat actgacgctg aggcacgaaa gcgtggggag 240

caaacaggat tagataccct ggtagtccac gccgtaaacg atgaatgcca gccgttagtg 300

ggtttactca ctagtggcgc agctaacgct ttaagcattc cgcctgggga gtacggtcgc 360

aagattaaaa ctcaaaggaa ttgacgga 388

Claims

1. A strain of root nodule bacteria of Cassia genus: (Bradyrhizobiumsp.) TXR2 characterized by: the rhizobia bacterium (A), (B), (C), (Bradyrhizobiumsp.) TXR2 was isolated and purified from fresh root nodules of grass of the genus Cassia and tested by PCRnodAgene, which is obtained by 16S rDNA molecular biology identification, is preserved in China center for type culture Collection in 2018, 4 and 10 months, and the preservation number is CCTCC NO: and M2018193.

2. A strain of Cassia rhizobia (A) as claimed in claim 1Bradyrhizobiumsp.) TXR2 is used for improving biological nitrogen fixation capability of plants in Cassia of Leguminosae.