Nitrogen-fixing Klebsiella sp. with high anti-glyphosate activity and its application

A Klebsiella, glyphosate technology, applied in bacteria, microorganism-based methods, chemicals for biological control, etc., can solve problems such as limiting the scope of use and use time, achieve good application prospects, promote The effect of sustainable agricultural development and seedling growth promotion

Inactive Publication Date: 2013-03-13
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

However, glyphosate can also kill crops while killi

Method used

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  • Nitrogen-fixing Klebsiella sp. with high anti-glyphosate activity and its application
  • Nitrogen-fixing Klebsiella sp. with high anti-glyphosate activity and its application
  • Nitrogen-fixing Klebsiella sp. with high anti-glyphosate activity and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1 Isolation of bacterial strain of the present invention

[0017] Weigh 5g of the surface soil attached to the sugarcane root and put it into 45mL of sterilized water, shake on a shaker at 180r / min, shake at a constant temperature of 28°C for 30min to disperse the microbial cells, and let it stand for 10min to obtain 10 -1 Dilution, 10-fold serial dilution with sterilized water, each 100 μL coated on Ashby medium (ingredients are sucrose 10g, NaCl 0.2g, KH 2 PO 4 0.2g, MgSO 4 ·7H 2 O 0.2g, 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. Different single colonies were picked with an inoculation needle, streaked and purified three times on an Ashby plate to obtain a single colony. Inoculate the purified single colony on LB medium (composition: tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1000mL, pH 7.0) plate, store at 4°C for recent use; purified s...

Embodiment 2

[0018] Example 2 Nitrogenase activity of bacterial strains of the present invention

[0019] Nitrogenase activity was determined by acetylene reduction assay (ARA). GG08160 was activated and grown in Ashby liquid medium for 48 hours. Take 1 mL of bacterial liquid and inoculate it into a sterilized 60 mL ground-mouth bottle containing 10 mL of Ashby liquid medium. After 24 hours of cultivation, extract 5 mL of gas from the bottle, and then inject 5 mL 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.diazotrophicus PAL5 was used as the positive control, and the treatment of inoculating 1 mL of inactivated bacterial solution was used as the blank control.

[0020] Experimental result shows, the acetylene reducing ethylene activity of the nitrogenase of bacterial strain of the present invention is 1...

Embodiment 3

[0021] Example 3 Cloning analysis of the nifH gene and 16S rDNA of the bacterial strain of the present invention

[0022] Preparation of PCR amplification template: activate the strain GG08160 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.

[0023] 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 ac...

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Abstract

The invention relates to a Klebsiella sp. strain with a nitrogen-fixing effect and high anti-glyphosate capability and its application. The strain is obtained from sugarcane rhizosphere separation. After nitrogen-free culture medium screening, nitrogenase activity determination, nitrogenase gene nif H amplification and sequence analysis, the strain is determined as an associative nitrogen-fixing bacterium. And by strain morphology and of 16S rDNA sequence analysis, the strain is identified as a Klebsiella sp. bacterium. Named as GG08160, the strain provided in the invention has an associative nitrogen-fixing capability and an inorganic phosphorus degradation function, can significantly promote sugarcane seedling growth, and can still grow normally in a culture medium with a glyphosate content up to 250mM. With high anti-glyphosate activity and good application prospects, the strain can serve as a plant growth-promoting bacterium, and also provides a new microorganism resource or gene resource for study of glyphosate biodegradation, as well as provides resources and support for promoting agricultural sustainable development.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Klebsiella bacterium with nitrogen fixation and high glyphosate resistance and application thereof. Background technique [0002] Giving full play to the role of the treasure house of agricultural microbial resources and actively exploring new biocontrol microbial resources is an important content of the development and research of agricultural microbial resources. In recent years, the waste of resources and the pollution of soil and groundwater caused by the extensive use of chemical fertilizers and pesticides have attracted widespread attention from all over the world. The use of biological nitrogen fixation is the most effective way to reduce the amount of nitrogen fertilizer. The research, development and utilization of biological nitrogen fixation resources play an important role in the sustainable development of agriculture in my country. Although the efficiency of combined ...

Claims

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Application Information

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IPC IPC(8): C12N1/20A01P21/00C12R1/22
Inventor 胡春锦李杨瑞史国英林丽魏源文邓智年李楠
Owner MICROBIOLOGY RES INST GUANGXI ZHUANG AUTONOMOUS REGION ACADEMY OF AGRI SCI
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