The actinobacterium Microbacterium sp. 16SH accepts pBBR1-based pPROBE vectors, forms biofilms, invades roots, and fixes N2 associated with micropropagated sugarcane plants

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.

A nonpathogenic Fusarium oxysporum strain enhances phytoextraction of heavy metals by the hyperaccumulator Sedum alfredii Hance

“…PCR amplifications of the internal transcribed spacer region (ITS) and translation elongation factor (TEF) were performed with the primers ITS1/ITS4 [24] and ef1/ef2 [25]. The respective amplification products of 550 bp and 650 bp were purified, cloned and sequenced as previously described [26]. The obtained putative ITS and TEF sequences of the fungal isolate SaCS12 were BLASTed [27] and deposited in the GenBank database under JQ039192 and JQ039193, respectively.…”

Plant Growth-Promoting Nitrogen-Fixing Enterobacteria Are in Association with Sugarcane Plants Growing in Guangxi, China

“…At present, little is known about the diversity and predominant population of nitrogen-fixing bacteria associated with the sugarcane plants growing in Guangxi. Recently, some nitrogen-fixing bacteria have been isolated from sugarcane plants grown in Guangxi (26, 28, 44, 52, 53) using NFb, JNFb and LGI-P media that were respectively used to isolate Azospirillum (10), Herbaspirillum (6) and Gluconacetobacter diazotrophicus (8); however, nitrogen-fixing bacteria belonging to the genera Azospirillum , Herbaspirillum , and Gluconacetobacter , which are predominantly associated with sugarcane plants in Brazil, have not been isolated. The diversity of nitrogen-fixing bacteria associated with sugarcane plants grown with high N-fertilization in Guangxi may be different from in Brazil.…”

“…The recommended dose of urea fertilization for plant cane crops at the seedling stage is 180 kg ha −1 , 30% of urea fertilization for a season (46). Recent studies have shown that nitrogen-fixing bacterial strains isolated from other sugarcane cultivars are able to provide nitrogen to micropropagated ROC22 sugarcane seedlings via BNF and promote sugarcane growth (26, 29); however, neither indigenous nitrogen-fixing bacteria associated with ROC22 sugarcane plants nor their associative BNF under recommended N-fertilization have been investigated.…”

Microbial community structure and chemical composition from dark earth in a native archaeological site of the lower Amazon

“…Many Microbacterium spp. play a significant role in oil, lactone and xylan degradation, production of biosurfactants, and as a growth promoter in plants (Lin et al, 2012).…”