Identification of an endophytic nitrogen-fixing bacterium NN08200 from sugarcane and its growth promotion of sugarcane

Abstract:

[Background] Nitrogen fertilizer input for production in China is very high, which is one of the most serious impediments of the sustainable development of agriculture. Research on biological nitrogen fixation (BNF) has increased significantly because of its potential importance to economy and the environment. [Objective] The aim of this work was to screen and identify efficient endophytic nitrogen-fixing bacteria of sugarcane, and to demonstrate their potential for associative nitrogen fixation with sugarcane as well as plant growth promotion. [Methods] An endophytic diazotroph strain NN08200 was isolated from the stem of sugarcane, and its nitrogen fixation was tested further by using the acetylene reduction assay and PCR amplification of the nifH gene encoding the iron protein of nitrogenase. Identification of the strain species according to its culture character and the microscopic observation, automatic bacteria identification system and by analyzing their 16S rRNA gene sequences. To demonstrate its potential for associative nitrogen fixation with sugarcane and growth promotion activities, strain NN08200 was inoculated into micropropagated sugarcane seedlings to investigate their plant growth-promoting and associative BNF activities using the 15N isotope dilution technique. [Results] The acetylene reduction activity of strain NN08200 was 2 445 nmol C2H4/(h·mL), and PCR amplification of nifH amplified approximately 339 bp fragments from the strain. Cloning and sequencing of the amplicon showed that strain NN08200 contained sequences that were the closest match to nifH. Based on phenotypic characteristics, Biolog system test, and 16S rRNA gene sequence analysis, strain NN08200 was identified as Pantoea sp. Compared with the uninoculated controls, strain NN08200 significantly promoted the growth of micropropagated sugarcane seedlings by increasing dry weight and plant height of sugarcane. 15N isotope dilution assays demonstrated that the associative nitrogen fixation rates of NN08200 in the sugarcane roots, stems and leaves were 7.49%, 15.02% and 10.79%, respectively, which were higher than that of Gluconacetobacter diazotrophicus strain PAL5, whose associative nitrogen fixation rates in the sugarcane roots, stems and leaves were 3.53%, 9.44% and 4.87%, respectively. [Conclusion] Strain NN08200 has the potential to efficiently fix N2 in sugarcane and promote sugarcane growth.