The current nitrogen fertilization for sugarcane production in Guangxi, the major sugarcane-producing area in China, is very high. We aim to reduce nitrogen fertilization and improve sugarcane production in Guangxi with the help of indigenous sugarcane-associated nitrogen-fixing bacteria. We initially obtained 196 fast-growing bacterial isolates associated with the main sugarcane cultivar ROC22 plants in fields using a nitrogen-deficient minimal medium and screened out 43 nitrogen-fixing isolates. Analysis of 16S rRNA gene sequences revealed that 42 of the 43 nitrogen-fixing isolates were affiliated with the genera Enterobacter and Klebsiella. Most of the nitrogen-fixing enterobacteria possessed two other plant growth-promoting activities of IAA production, siderophore production and phosphate solubilization. Two Enterobacter spp. strains of NN145S and NN143E isolated from rhizosphere soil and surface-sterilized roots, respectively, of the same ROC22 plant were used to inoculate micropropagated sugarcane plantlets. Both strains increased the biomass and nitrogen content of the sugarcane seedlings grown with nitrogen fertilization equivalent to 180 kg urea ha−1, the recommended nitrogen fertilization for ROC22 cane crops at the seedling stage. 15N isotope dilution assays demonstrated that biological nitrogen fixation contributed to plant growth promotion. These results suggested that indigenous nitrogen-fixing enterobacteria have the potential to fix N2 associated with sugarcane plants grown in fields in Guangxi and to improve sugarcane production.

No abstract

Members of the genus Microbacterium lineage of Gram-positive actinobacteria are increasingly being reported to display significant traits associated with environmental biotechnology and bioengineering. 16SH is a nitrogen-fixing bacterial strain isolated from a surface-sterilized stem of sugarcane grown in Guangxi, China. Analysis of 16S rRNA gene sequences revealed that 16SH belonged to the genus Microbacterium. pPROBE-pTet(r) plasmids were constructed by cloning the promoter region of the Tet(r) gene into the promoterless pPROBE-AT, -OT, and -TT vectors derived from the pBBR1 plasmid that has a broad host range of Gram-negative bacteria and sequence similarities to plasmids from Gram-positive bacteria. The pPROBE-pTet(r) plasmids expressed the gfp reporter gene and were stably maintained in 16SH cells without antibiotic selection in free-living state and in planta. Confocal microscopy on intact roots of micropropagated sugarcane plantlets showed that gfp-tagged 16SH cells formed biofilms on root maturation and elongation zones but not on root meristem zones and root caps, and colonized in intercellular spaces of root cortices. Inoculation of 16SH significantly increased biomass and nitrogen content of micropropagated sugarcane seedlings grown with a nitrogen fertilization of 6.3 mg N/kg soil. ¹⁵N isotope dilution assays demonstrated that biological nitrogen fixation contributed to this plant growth promotion. This study for the first time demonstrated that the pBBR1-based pPROBE plasmids provided an efficient genetic transfer system for a Gram-positive Microbacterium strain, and that a nitrogen-fixing Microbacterium endophyte colonized in intact host plants and fixed N₂ associated with the host plants.

Five nitrogen-fixing bacterial strains (SP1 T , NN143, NN144, NN208 and HX148) were isolated from stem, root or rhizosphere soil of sugar cane (Saccharum officinarum L.) plants. Cells were Gram-negative, motile, rods with peritrichous flagella. DNA G+C content was 55.0±0.5 mol%. Sequence determinations and phylogenetic analysis of 16S rRNA gene and rpoB indicated that the strains were affiliated with the genus Enterobacter and most closely related to E. radicincitans DSM 16656 T and E. oryzae LMG 24251 T . Fluorimetric determination of thermal denaturation temperatures after DNA-DNA hybridization, enterobacterial repetitive intergenic consensus PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry differentiated the whole-genome, genotype and protein profiles from those of E. radicincitans and E. oryzae. The strains' cell fatty acid composition differentiated them from E. radicincitans and E. oryzae by containing a higher level of summed feature 2 (C 16 : 1 v7c and/or C 16 : 1 v6c) and a lower level of C 17 : 0 cyclo. Their physiological and biochemical profiles differentiated them from E. radicincitans by being positive for methyl red test, ornithine decarboxylase and utilization of putrescine, D-arabitol, L-fucose and methyl a-D-glucoside and being negative for arginine dihydrolase, and differentiated them from E. oryzae by being positive for aesculin hydrolysis and utilization of putrescine, D-arabitol and L-rhamnose and being negative for arginine dihydrolase, lysine decarboxylase and utilization of mucate. The five strains therefore represent a novel species, for which the name Enterobacter sacchari sp. nov. is proposed, with the type strain SP1 T (5CGMCC 1.12102 T 5LMG 26783 T ).Both pathogenic and beneficial bacteria to plants have been affiliated with several species of the genus Enterobacter in the family Enterobacteriaceae (Chung et al., 1993; Kämpfer et al., 2005;Madhaiyan et al., 2010;Peng et al., 2009;Zhu et al., 2011). Beneficial nitrogen-fixing bacteria belonging to several genera of the family Enterobacteriaceae have been found and have been shown to fix N 2 in association with sugar cane (Saccharum officinarum L.) worldwide and to promote sugar cane growth (Govindarajan et al., 2007;Lin et al., 2012;Loiret et al., 2004;Magnani et al., 2010;Mehnaz et al., 2010;Mirza et al., 2001;Taulé et al., 2012). Strain SP1 T was isolated from a stem of sugar cane cultivar GT11 grown in Nanning, Guangxi, China in 1994. Sugar cane internodes were surface-sterilized by immersing intoThe GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains SP1 T , HX148, NN143, NN144 and NN208 are JQ001784, HQ204281, HQ204313, HQ204314 and HQ204315, for rpoB sequences are JQ001786, JQ001787, JQ001788, JQ001789 and JQ001790 and for nifH sequences are JQ001785, HQ204226, HQ204258, HQ204259 and HQ204260, respectively. 70 % ethanol for 2 min and 5 % (w/v) chloramine T solution for 5 min; the stem rind was removed and the inner tissues were cut into small pieces with a steril...