【作者】 韩继刚；

【导师】 宋未；

【作者基本信息】 首都师范大学 ， 植物学， 2004， 博士

【摘要】 本文鉴定了从水稻根面分离的一个植物内生固氮新菌株Delftia tsuruhatensis HR4，并对其在系统发育上的地位和生物学特性进行了的研究。 对HR4进行了形态特征和生理生化特性鉴定，PCR扩增了16S rDNA全序列共1498 bp并进行了系统发育学分析。结果表明HR4与Delftia tsuruhatensis(ATCC BAA-554~T)的相似性达到99％，是Delftia tsuruhatensis的一个新菌株。 筛选了HR4利福平突变株HR4A和HR4B，并对其进行了gfp基因标记。标记菌株HR4A∷gfp同野生型菌株相比，其生长只受到较小的影响，并且有较高的标记稳定性。激光共聚焦显微镜观察了HR4A∷gfp在水稻根部的定殖，发现幼根伸长区和侧根发生处是其侵染和定殖的重要部位，从而证明HR4是一株兼性植物内生细菌。 HR4具有较广的拮抗谱，对实验的14株植物病原菌，尤其是水稻三大病害病原菌(稻瘟病菌、白叶枯病菌和纹枯病菌)都表现出了较强的平板拮抗活性。但对黄瓜黑星病原菌、黄瓜枯萎病原菌、辣椒疫霉病原菌和辣椒立枯病原菌的温室生防功能比较低，还有待于进一步的研究。 用地高辛标记的nifHDK探针进行的斑点杂交实验将HR4的固氮酶基因定位于染色体DNA上。PCR扩增了全长3715bp的部分固氮酶结构基因，得到nifD基因的完整序列、nifH和nifK基因的部分序列以及nifH-nifD的IGS和nifD-nifK的IGS序列。氨基酸序列系统发育学分析表明固氮酶具有很强的保守性，与HR4亲缘关系最近的是Klebsiella pneumoniae。 本文还首次将非培养方法(变性梯度凝胶电泳，DGGE)应用于水稻植物内生细菌研究，进行了初步的探索。 本文是第一次报道Delftia属菌株具有固氮活性并可以拮抗多种植物病原首都师范大学博l一学位论文菌等生物学功能，同时本文也第一次报道从水稻植株中分离到可以固氮的Delftia)禹菌株。更多还原

【Abstract】 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.更多还原