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Sugarcane endogenous nitrogen-fixing Pantoea bacteria and application thereof

A Pantoea and sugarcane technology, applied in the field of sugarcane endogenous Pantoea nitrogen-fixing bacteria, can solve the problems of soil water retention, poor fertilizer retention capacity, and low utilization rate of chemical fertilizers

Inactive Publication Date: 2012-10-17
MICROBIOLOGY RES INST GUANGXI ZHUANG AUTONOMOUS REGION ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, more than 90% of the sugarcane land in my country's main sugarcane production areas is dry slope land, the soil water and fertilizer retention capacity is poor, and the utilization rate of chemical fertilizers is very low.

Method used

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  • Sugarcane endogenous nitrogen-fixing Pantoea bacteria and application thereof
  • Sugarcane endogenous nitrogen-fixing Pantoea bacteria and application thereof
  • Sugarcane endogenous nitrogen-fixing Pantoea bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] The isolation of embodiment 1 bacterial strain of the present invention

[0019] Take the sugarcane stems, soak them in 70% alcohol for 30 seconds, wash them with sterile water three times, then soak them in 0.1% mercury chloride for 1 minute, soak them in 70% alcohol for 30 seconds, and wash them with sterile water five times. Weigh 1g of the surface-sterilized tissue block, soak it in 10mL of phosphate buffer, grind it in a sterile mortar, and let it stand for 5min. Take the supernatant and make a 10-fold serial dilution with sterile water, and take 100μL of the original solution and the diluted solution respectively. Spread on Ashby nitrogen-free medium (ingredients are sucrose 10g, NaCl 0.2g, KH 2 PO 4 0.2g, MgSO 4 ·7H 2 O0.2g, CaSO 4 2H 2 O 0.1g, CaCO 3 5g, distilled water 1000mL, pH 7.0) on a plate, culture at 28°C for 2-3d until a single colony grows. After the colony grows, strains are purified by streaking on the Ashby nitrogen-free medium plate.

Embodiment 2

[0020] The nitrogenase activity of embodiment 2 bacterial strains of the present invention

[0021] Nitrogenase activity was determined by acetylene reduction assay (ARA). After NN08200 was activated, it was grown in Ashby liquid medium for 48 hours, and 1 mL of bacterial liquid was inoculated into a sterilized 60 mL ground-mouth bottle containing 10 mL of Ashby liquid medium. After 24 hours of cultivation, 5 mL of gas was extracted from the bottle, and then 5 mL was injected. Acetylene gas, continue to cultivate for 24h, use gas chromatography to detect the amount of ethylene production, in nmolC 2 h 4 / h.mL represents nitrogenase activity. The Brazilian sugarcane endogenous nitrogen-fixing bacterium G.diazotrophicusPAL5 was used as the positive control, and the treatment of inoculating 1mL of inactivated bacterial solution was used as the blank control.

[0022] Experimental result shows, the acetylene reducing ethylene activity of the nitrogenase of bacterial strain of t...

Embodiment 3

[0023] Cloning analysis of the nifH gene and 16S rDNA of the strain of the present invention in embodiment 3

[0024]Preparation of PCR amplification template: activate the strain NN08200 and inoculate it into LB liquid medium (composition: tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1000mL, pH 7.0), culture for 20h and centrifuge to collect the bacteria for later use . Use a bacterial genomic DNA extraction kit to extract the genomic DNA of the strain of the present invention from fresh bacterial LB cultures according to the product instructions, store at -20°C, and the DNA content is about 50-100ng / μL.

[0025] nifH gene analysis: the amplification primers are PolyF (TGCGAYCCSAARGCBGACTC) and PolyR (ATSGCCATCATYTCRCCGGA), the PCR reaction program is: 94°C 3min pre-denaturation, then 94°C 1min, 55°C 1min, 72°C 1min, 30 cycles, and finally 72°C 10min. PCR amplification products were detected by 1.5% agarose gel electrophoresis. Gluconoacetic acid ba...

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Abstract

The invention relates to a sugarcane endogenous nitrogen-fixing bacteria strain and an application thereof. The classification of the strain is named as Pantoea sp.NN08200 with a preservation number of CGMCC NO.5438. The strain disclosed herein can significantly promote the growth of plants and revisiting various plant pathogenic fungi. According to the invention, after inoculating the strain to sugarcane to conduct tissue culture plantlet for 50 days, dry weight of the plants and the total nitrogen content are respectively increased by 46.74% and 41.5% over blank control group, the nitrogen fixation efficiency is 12.85%; the dry weight of the overground part of corn seedling of the inoculated strain NN08200 is increased by 74.5% over control group, the dry weight of the underground part is increased by 84.6%, the plant height is increased by 16.3%, the chlorophyll content is increased by 29.2%, and the nitrogen content is increased by 27.0%. The strain has the effects of association nitrogen fixation, production of hormone, and the like for promoting the growth of plants, and can be applied for producing nitrogen-fixing microbial agents and bio-organic fertilizers with the effects of promoting growth and inhibiting plant diseases.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to a sugarcane endophytic Azopantoea bacterium with nitrogen fixation and resistance to pathogenic fungi. Background technique [0002] Agricultural microbial resources are an important part of the treasure house of life science resources. They are the source of microbial preservation strains and the source of microbial development and utilization. Give full play to the role of the agricultural microbial resource treasure house and actively explore new biocontrol microbial resources. Agricultural microbial resources Important content of development research. Endophytic nitrogen-fixing bacteria refer to those microorganisms that colonize inside plants and jointly fix nitrogen with host plants, mainly bacteria. Part or the entire life cycle of them resides in plant tissues, especially vegetative reproduction tissues, to survive, reproduce, and spread without Causes obvious damage...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01N63/00A01P21/00A01P3/00C05F11/08C12R1/01
CPCY02P60/21
Inventor 胡春锦李杨瑞林丽史国英魏源文
Owner MICROBIOLOGY RES INST GUANGXI ZHUANG AUTONOMOUS REGION ACADEMY OF AGRI SCI
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