Sugarcane is one of the most important crops for sugar and energy production, and the Guangxi region is the major sugarcane and sugar producing area in China, producing about 60% of China's sugarcane and sugar. Nitrogen fertilizer input for sugarcane production in China is very high. It is essential to reduce nitrogen fertilizer input and thus cultivation cost while achieving a high yield for sustainable and environmentally friendly sugarcane production. Research on biological nitrogen fixation has increased significantly because of its potential importance to the economy and the environment. Sugarcane plants can obtain nitrogen from biological nitrogen fixation via diazotrophs, and ¹⁵N isotope assays have demonstrated that some Brazilian sugarcane varieties are able to obtain considerable nitrogen from biological nitrogen fixation.
The aim of this work was to screen and identify nitrogen-fixing bacteria from the rhizosphere of sugarcane cultivated in the diverse geographical region of Guangxi, investigate their diversity and predominant affiliation, and to demonstrate their potential for associative nitrogen fixation with sugarcane as well as plant growth promotion. One hundred and forty eight bacterial isolates associated with sugarcane were isolated from 20 soil samples collected from 10 major production areas over the Guangxi region using Ashby nitrogen deficient minimal media. Thirty six of these isolates were found to have N₂ fixing ability as demonstrated by the acetylene reduction assay. All 36 isolates also yielded a polymerase chain reaction product with primers targeting the nifH gene, which encodes a key enzyme in the nitrogen fixation process. Further screening and selection indicated that five efficient isolates (GX0703, GX0798, GX07104, GX08125, and GX0797) were obtained which possessed multiple plant growth promoting traits such as higher N₂ fixing, phosphate solubilization and IAA production, and all of the isolates with these attributes may well be valuable for agriculture. To demonstrate their potential for associative nitrogen fixation with sugarcane and growth promotion activities, the five isolates were inoculated into micropropagated sugarcane seedlings, and the seedlings were then incubated in a growth cabinet for 50 d at 28°C. The results indicated that all five isolates significantly promoted the growth of the micropropagated sugarcane seedlings. They also increased the dry weight and nitrogen contents by 29.15 to 57.61% and 29.35 to 45.04%, respectively, compared with the uninoculated controls. Based on phenotypic tests, biolog microplate tests and 16S rRNA gene sequence analysis, the five efficient isolates, GX0703, GX0798, GX07104, GX08125, and GX0797 were identified as Klebsiella sp., Bacillus megaterium, Pseudomonas sp., Pantoea sp. and Burkholderia sp., respectively. These promising associative nitrogen fixation bacteria isolates from the rhizosphere of micropropagated sugarcane showed potential to promote sugarcane production via nitrogen fixation and other plant growth promoting functions. This will improve sugar production efficiency in China and reduce the environmental pollution that results from sugarcane production.
The nifH and 16S rDNA sequences of the N fixing bacterial isolates GX0703, GX0798, GX07104, GX08125 and GX0797 were deposited in the GenBank, and the accession numbers were FJ822998, FJ823001, EU938523, FJ822992 and EU938522 for nifH, and FJ823041, FJ823003, FJ823047, FJ823045 and FJ823002 for 16S rDNA sequences, respectively.