A Cassia rhizobia txn1 and its application

A technology of Cassia and rhizobia, applied in the field of microorganisms, can solve the problems of low nitrogen fixation efficiency, slow nodulation, affecting planting and popularization and application, etc., and achieves strong nitrogen fixation ability, high nodulation rate, and the effect of promoting fertilization and soil fertility

Inactive Publication Date: 2020-07-31
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, under natural conditions, Cassia rotifera forms nodules slowly and rarely, and its nitrogen fixation efficiency is low, which seriously affects its planting and popularization and application.

Method used

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  • A Cassia rhizobia txn1 and its application
  • A Cassia rhizobia txn1 and its application
  • A Cassia rhizobia txn1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Isolation and purification of Rhizobium TXN1

[0045] 1 Isolation of rhizobia

[0046] Take a fresh, mature and large plump nodule of Cassia pinnatifida, rinse with water, and absorb the surface water with filter paper. Put it in 95% (w / v) ethanol for 3~5 minutes, take it out and rinse with sterile water for 5~6 times, then add 0.1% (w / v) HgCl 2 Sterilize for 3~5 minutes, take out and rinse with sterile water 5~6 times. Then cut into two halves on a flame-sterilized glass slide, clamp half of the tumor with sterile tweezers, scribe the incision facing the surface of the YMA (Table 1) medium, invert it and incubate at 28°C.

[0047] 2 Purification

[0048] After the bacteria grow out, scrape a small amount of rhizobia colonies from the plate with a pipette tip, dilute with 1 mL of sterile water and streak again on the plate. After 3 days, observe the colony situation, and observe it until 15 days (slow). It takes 7-15 days for rhizobia to appear colonies). If no mon...

Embodiment 2

[0054] Example 2 16S rDNA molecular biology identification of Rhizobium TXN1

[0055] PCR specific amplification of 16S rDNA was performed on the Rhizobium monoclonal bacteria liquid, and the forward primer is SEQ IDNO.3: V4V5515 -F 5'-GTGCCAGCMGCCGCGGTAA-3'; the reverse primer is SEQ ID NO. 4: V4V5907 -R 5'-CCGTCAATTCCTTTGAGTTT-3'. Use 2×star Mix as the enzyme. The PCR amplification products are detected by electrophoresis imaging technology to observe whether they have bands, and the remaining PCR amplification products are used for sequence determination. The sequencing result is shown in SEQ ID NO:5. The PCR reaction system is shown in Table 4.

[0056] Table 4 16S rDNA2×starMix enzyme reaction system

[0057]

[0058] Reaction conditions: 95°C 5 min; (95°C 20 s, 55°C 20 s, 72°C 50s) × 44 cycle; 70°C 5 min.

[0059] The 13 reference strain sequences were obtained from the NCBI (GenBank) database, and the 16S rDNA partial sequences of the isolated strains and the reference str...

Embodiment 3

[0060] Example 3 Tie back test

[0061] Test purpose: to screen out rhizobia nitrogen-fixing bacteria with high binding efficiency and strong nitrogen-fixing ability with Cassia forages.

[0062] Test materials: Test plant: Minyu No. 1 Cassia rotundifolia; Test strain: isolated and purified Rhizobium Azotobacter.

[0063] Main test instruments and equipment: artificial climate growth room, ultra-clean workbench, autoclave, constant temperature incubator, 25 15×15 cm pots, 2 1 L beakers, tweezers, petri dishes, filter paper, glass Rods, scissors, gauze, etc.

[0064] Test drugs and reagents

[0065] (1) Test drugs: Mannitol, MgSO 4 ∙7H 2 O, NaCl, yeast powder, K 2 HPO 4 , KH 2 PO 4 , CaCO 3 , Ca(NO 3 ) 2 ∙4H 2 O, MgSO 4 ·7H 2 O, CaCl 2 ·2H 2 O, Na 2 HPO 4 ·12H 2 O, C 10 H 12 N 2 O 8 FeNa·3H 2 O, Na 2 MoO 4 , MnSO 4 , H 3 BO 3 , CuSO 4 ·5H 2 O and ZnSO 4 ·7H 2 O.

[0066] Test reagent:

[0067] 1) YMA (Yeast Mannnitol Agar) liquid medium: weigh 10 g of mannitol, MgSO 4 ∙7H 2 O0.2g, NaCl 0....

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Abstract

The invention discloses a strain of rhizobia TXN1 and its application, belonging to the technical field of microbes. The strain is isolated and purified from fresh root nodules of Cassia forage, and detected by PCR nodA gene, and identified by 16S rDNA molecular biology, identified as Bradyrhizobium ( Bradyrhizobium ), and named it Tianyu No. 1. It was deposited in the China Center for Type Culture Collection on April 10, 2018, and the deposit number is CCTCC NO: M2018191. The nitrogen-fixing bacterium strain TXN1 of the present invention proves that it has the characteristics of high-efficiency nodulation and strong nitrogen fixation ability through laboratory pot plant backgrafting experiments, and the inoculation of the rhizobia can significantly increase the number of nodules, nitrogen fixation efficiency of nodules, biomass and The nitrogen content of the plant can be increased to achieve the effect of fertilizing the soil and improving the ecological environment.

Description

Technical field [0001] The invention belongs to the field of microorganisms, and specifically relates to a Cassia rhizobia TXN1 and its application. Background technique [0002] There are about 56.9 million hm in our country 2 Acidic soil accounts for more than 42.2% of the total cultivated land area. In recent years, with the application of a large amount of chemical nitrogen fertilizers, soil acidification has become increasingly serious. my country’s acidic soils are mainly concentrated in the area south of the Yangtze River. This area is rich in resources such as light, heat, and water, but the soil is lean, heavy, low in pH and low in nutrient availability, which severely restricts the rapid development of agriculture in this area. In production, a lot of chemical fertilizers are often used to increase crop yields. However, the input of a large amount of chemical fertilizer will not only cause soil compaction and further acidification, but also cause eutrophication of wat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A01N63/20A01P21/00C12R1/01
CPCA01N63/00C12N1/205C12R2001/01
Inventor 廖红杨庆李欣欣
Owner FUJIAN AGRI & FORESTRY UNIV
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