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Nitrogen-fixing Paenibacillus sp. 1-49 and application thereof

A bacillus and nitrogen-fixing technology, applied in application, bacteria, fungicides, etc., can solve the problems of late start of research on autogenous nitrogen-fixing bacteria, limited application of Paenibacillus strains, etc. The effect of promotion

Inactive Publication Date: 2012-02-15
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the late start of research on autogenous nitrogen-fixing bacteria at home and abroad, especially in China, the application of Paenibacillus species as a potential microbial agent in agriculture has been greatly limited.

Method used

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  • Nitrogen-fixing Paenibacillus sp. 1-49 and application thereof
  • Nitrogen-fixing Paenibacillus sp. 1-49 and application thereof
  • Nitrogen-fixing Paenibacillus sp. 1-49 and application thereof

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Experimental program
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Embodiment 1

[0024] The cultivation and identification of embodiment 1 Paenibacillus nitrogen-fixing 1-49 bacterial strain

[0025] (1) Cultivation of strains

[0026] Production of medium: liquid medium (1L): 20g sucrose; 12.06g K 2 HPO 4 ;3.4gKH 2 PO 4 ;0.2g MgSO 4 ·7H 2 O; 0.01g NaCl; 0.01g FeCl 3 ;0.002gNaMoO 4 2H 2 O; H 2 O 1L. Sterilize at 120°C for 25 minutes. The production of solid medium is to add 14g agar per liter of liquid medium. Inoculate Paenibacillus nitrogen-fixing bacteria into solid or liquid medium, and cultivate them in a 30°C incubator for 3 days.

[0027] (2) Extraction of bacterial total DNA The extraction method refers to the method in the literature [Ausubel et al. Current Protocols in Molecular Biology. New York: Wiley.1987].

[0028] (3) Identification of strains and amplification of nitrogenase structural gene nifH and 16S rDNA

[0029] Extract genomic DNA from the pure culture of bacterial strain Paenibacillus sp.1-49 of the present invention, a...

Embodiment 2

[0042] The determination of embodiment 2 nitrogenase activity

[0043] 1. Inoculate strains 1-49 in 5mL medium for measuring enzyme activity, culture overnight at 30°C, transfer to a 50mL Erlenmeyer flask at 1% inoculum size, culture overnight again at 30°C, and then collect the bacteria. Suspend the specific medium with an appropriate amount of medium for measuring enzyme activity, and adjust the OD 600 to 1. Inoculate and culture 1.5mL of bacterial liquid for 2h, take 100μL of gas and measure the ethylene content by gas chromatography.

[0044] The enzyme activity medium is: 26.3g Na 2 HPO 4 12H 2 O; 3.4 g KH 2 PO 4 ; 10 μg biotin; 26 mg CaCl 2 2H 2 O; 30mg MgSO 4 ; 0.33mg MnSO 4 ·H 2 O; 36 mg ferric citrate; 7.6 mg Na 2 MoO 4 2H 2 O; 10 μg p-aminobenzoic acid; 4 g glucose. Add glucose separately after sterilizing at 115°C for 30 minutes, and other reagents can be sterilized at 121°C for 30 minutes after preparation.

[0045] 2. Determination of protein conte...

Embodiment 3

[0061] Antibacterial activity experiment of embodiment 3 Paenibacillus nitrogen-fixing 1-49 bacterial strains

[0062] PDA medium (potato 200g, glucose 20g, agar powder 16g, add distilled water to 1L). Plant pathogenic bacteria and Paenibacillus 1-49 provided by the present invention were inoculated respectively at two points at 2 cm from the center of the PDA plate, and then the plate was placed in a 30° C. Inhibition, the results are shown in Figure 3. The plant pathogens used in this example are: Fusarium oxysporum, Rhizoctonia cerealis, Sclerotinia sclerotiorum, and Botrytis cinerea.

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Abstract

The invention provides a Paenibacillus sp. strain 1-49 with nitrogen-fixing capability, the collection number of which is CGMCC No.4966. The Paenibacillus sp. 1-49 has a nitrogenase-structured gene nifH, and the acetylene reduction method determines that the nitrogenase activity of the Paenibacillus sp. 1-49 is 969. 49nmolC2H4 / (mg protein h) negative 1. The nitrogen-fixing Paenibacillus sp. strain 1-49 provided by the invention has a remarkable inhibition effect on a variety of plant pathogenic fungi, and nitrogen-fixing microorganism fungicide or bioorganic fertilizer prepared with the strain 1-49 can remarkably promote the germination and growth of plant seeds, and can be popularized to use in agricultural production to effectively reduced the usage of urea.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nitrogen-fixing Paenibacillus 1-49 with nitrogen-fixing ability and application thereof. Background technique [0002] Grass crops such as wheat, maize and rice are the main food for about 6.5 billion people in the world. With the rise of the chemical fertilizer industry, increasing the yield of crops mainly depends on increasing the application of chemical nitrogen fertilizers. my country is the world's largest producer and consumer of chemical fertilizers. The annual amount of chemical fertilizers used is as high as 48 million tons, including 28 million tons of nitrogen fertilizers and 12 million tons of phosphate fertilizers (P 2 o 5 ), and when the area of ​​arable land decreases, the total annual demand for fertilizers will rise instead of falling. However, less than 30% of the applied fertilizers can be used (Van Kessel et al., 2009), and the impact of nitrogen oxides prod...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/00C12N15/52A01N63/02A01P3/00A01P21/00C05F11/08C12R1/01
Inventor 陈三凤王莉瑛陈曦
Owner CHINA AGRI UNIV
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