Chamaecrista bradyrhizobiumsp. WYS3R1 and application thereof

A Cassia and Rhizobium technology, applied in the field of microorganisms, can solve the problems affecting planting and popularization, slow nodulation, low nitrogen fixation efficiency, etc., and achieves the effects of strong nitrogen fixation ability, high nodulation rate, and improvement of total nitrogen content.

Inactive Publication Date: 2019-03-08
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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  • Chamaecrista bradyrhizobiumsp. WYS3R1 and application thereof
  • Chamaecrista bradyrhizobiumsp. WYS3R1 and application thereof
  • Chamaecrista bradyrhizobiumsp. WYS3R1 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Isolation and Purification of Rhizobium WYS3R1

[0045] 1 Isolation of rhizobia

[0046] Take fresh, mature and large nodules of Cassia rotifera, rinse them with water, and absorb the surface moisture with filter paper. Put it in 95% (w / v) ethanol for 3-5 minutes, take it out and rinse it with sterile water for 5-6 times, then put it in 0.1% (w / v) HgCl 2 Sterilize for 3-5 minutes, take it out and rinse with sterile water 5-6 times. Then cut in half on a flame-sterilized glass slide, clamp half of the tumor with sterile tweezers, make the incision face the surface of YMA (Table 1) culture medium, and incubate at 28°C after being upside down.

[0047] 2 purification

[0048] After the growth of bacteria, a small amount of rhizobia colonies were scraped from the plate with the tip of a pipette, diluted with 1 mL of sterile water, and then cultured by streaking on the plate again. After 3 days, the colonies were observed until 15 days (slow Rhizobia need 7-15...

Embodiment 2

[0054] Example 2 Identification of 16S rDNA Molecular Biology of Rhizobium WYS3R1

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

[0056] Reaction conditions: 95°C for 5 min; (95°C for 20 s, 55°C for 20 s, 72°C for 50 s) × 44 cycle; 70°C for 5 min.

[0057] Obtain 13 reference strain sequences from the NCBI (GenBank) database, use the software BioEdit and MEGA6 to analyze the 16S rDNA partial sequences of the isolated strains and the reference strains, and construct the p...

Embodiment 3

[0058] Example 3 Tieback test

[0059] The purpose of the experiment: to screen out the rhizobia with high binding efficiency and strong nitrogen fixation ability with Cassia forage.

[0060] Test materials: Test plant: Minyu No. 1 Cassia rotifera; Test strain: isolated and purified Rhizobium.

[0061] Main test instruments and equipment: artificial climate growth chamber, ultra-clean workbench, high-pressure sterilization pot, constant temperature incubator, 25 potted pots of 15×15 cm, 2 beakers of 1 L, tweezers, Petri dishes, filter paper, glass Sticks, scissors and gauze etc.

[0062] Test Drugs and Reagents

[0063] (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.

[0064] Test reagent:

[0065] 1) YMA (Yeast Mannitol Agar) liqu...

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Abstract

The invention discloses chamaecrista bradyrhizobiumsp. WYS3R1 and application thereof and belongs to the technical field of microorganisms. A strain line is separated and purified from fresh chamaecrista grass nodule; nodA gene is detected by PCR; a novel strain line of Bradyrhizobium is determined by biological assay of 16S rDNA molecule and is named as Wuyuan 1. The chamaecrista bradyrhizobiumsp. WYS3R1 was preserved in China Center for Type CultureCollection on April 10th, 2018, with a preservation number of CCTCC NO:M2018190. The bonsai tieback test in a laboratory proves that the bradyrhizobiumsp. WYS3R1 disclosed by the invention has the characteristics of efficient nodulation and nitrogen fixation capabilities; by inoculating the bradyrhizobiumsp., nodule number, nodule nitrogen fixation efficiency and the biomass of chamaecrista grass and nitrogen content of plants can be obviously improved, and further the effects of culturing soil fertility and improving ecological environment are realized.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a Cassia Rhizobium WYS3R1 strain and its application. Background technique [0002] my country has about 56.9 million hm 2 Acidic soil accounts for more than 42.2% of the total cultivated land area. In recent years, with the application of large amounts of chemical nitrogen fertilizers, soil acidification has become increasingly serious. The acidic soil in my country is mainly concentrated in the area south of the Yangtze River. This area is rich in light, heat, water and other resources, but the soil is thin, sticky, low in pH and nutrient availability, which seriously restricts the rapid development of agriculture in this area. In terms of production, crop yields are often increased by applying large amounts of chemical fertilizers. However, the input of a large amount of chemical fertilizers will not only lead to soil compaction and further acidification, but also ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C05F11/08A01N63/00A01P21/00C12R1/01
CPCA01N63/00C05F11/08C12N1/205C12R2001/01
Inventor 廖红杨庆李欣欣
Owner FUJIAN AGRI & FORESTRY UNIV
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