The Studies On The Identification And Biological Characteristics Of A Novel Rice Endophytic Bacteria Strain Delftia Tsuruhatensis HR4 With N₂-fixing Activity

A novel endophytic strain Delftia tsuruhatensis HR4 with N2-fixing activity isolated from rice rhizoplane were systematically studied on classification and identification, phylogenetic systematic status as well as its biological characteristics.Based on the phenotypic, physiological, biochemical and phylogenetic studies, strain HR4 could be classified as a member of Delftia tsuruhatensis. The complete 16S rDNA of strain HR4 in length 1498 bp were sequenced and a phylogenetic tree was constructed based on a consensus of length 1368 bp of 16S sequences homology. It is shown HR4 was most closed to Delftia tsuruhatensis (ATCC BAA-554T) with 16S rDNA similarity 99%. However, comparisons of characteristics with other known species of the genus Delftia suggested that strain HR4 was a novel dizotrophic strain.The rifampicin resistant mutant strains HR4A and HR4B were screened and used to mark with green fluorescent protein gene. Compared with the wild type strain, the growth of HR4A::gfp was little influenced and the marking stability of the labeled strain was high. Microscopic observation of colonization of Delftia tsuruhatensis HR4A::gfp on rice roots using laser scanning confocal microscope showed that the elongation region of young roots and the lateral root junctions were important infection and colonization sites. Moreover, it was demonstrated that strain HR4 was a facultative endophytic bacteria.The in vitro antagonism assays showed that strain HR4 has a widerantagonistic spectrum to all 14 plant pathogens tested in this work, especially exhibited strong anti-fungal activity against to the three main rice pathogens: Pyricularia grisea, Xanthomonas oryzae pv. oryzae and Rhizoctonia Solani. But after treatment of Cucumis sativus L. cv. Jinyan 2 and Capsicum frutescens L. cv. Qiemen with strain HR4 culture, strong biocontrol activity to Cladosporium cucumerinum Ellis et Arthur, Fusarium oxysporium (Schl.) f. sp. cucumerinum Qwen, Phytophthora capsici Leonian and Rhizoctonia solani in the greenhouse was not showed, and further studies are needed.HR4 genomic DNA and nifHDK-plasmid DNA spot blotting with labeled probe showed that the nifHDK gene located in chromosome of HR4. The partial nz/HDK gene of strain HR4 in length 3 715 bp was sequenced, which included partial nifH gene and nifK. gene, complete nifD gene, two IGS sequences. Phylogenetic trees were constructed based on amino acid sequence homology of Fe protein, Fe-Mo protein a subunit and B subunit. It was shown HR4 was most closed to Klebsiella pneumoniae.A preliminary study on rice endophytic bacteria using culture-independent method (DGGE) was done in this paper.This is the first report about genus Delftia with strong antagonistic activity in vitro to a variety of fungal plant pathogens and high nitrogen-fixing activity. Moreover, this paper is also the first report for a new diazotroph genus isolated from rice plant.