Study Of The Relationship Between Rhizobium And Agrobacterium Isolated From Nodules Of Clover

Hbh54.2 and hbh3.1, two strains isolated from the same nodule of white clover in China's northwest region , are used in this thesis for the preliminary study of the relationship of Rhizobium and Agrobacterium. A phylogenetic tree is constructed on the basis of the 16S rDNA sequences of the two strains, which locates hbh54.2 to the genus of Agrobaterium and hbh3.1 to the genus of Rhizobium. As we know, in the study of rhizobia diversity, it is very common to isolate Agrobacterium-related strains from root nodules. However, up to now, there is no precise explanation to account for this phenomenon. In our study, a series of experiments are designed under the aim of revealing the relationship of Rhizobium and Agrobacterium that can exist in the same nodules of the host plant, which can provide theoretical basis for the further research.In our study, through the PCR amplification of the functional genes of the two tested strains, we can verify whether the tested strain is symbiotic or pathogenic. As is shown from the results, tested strain hbh54.2 habours no nodulation gene(nodA) and nitrogen-fixation gene(nifH), which is consistent with the results of the plant nodulation pre-experiment as is demonstrated that it cannot induce root nodules in the white clover. In addition, virulence gene(virC) can not be amplified when using the template of the two tested strains, indicating that both of the tested strains do not carry Ti plasmid, which is representative of pathogenecity.Through pot plant-nodulation experiments, it can be seen from the consequence that there is difference on the mass growth of the host plant among different kinds of disposal condition, one is designed to inoculate with only one kind of strain, another is co-inoculating with two tested strains simutaneously or unsimutaneously. Additionally, it also shows that co-inoculation can promote the nodulation process. In a word, the results indicate that the significance of the interaction of hbh54.2 with hbh3.1 can be reflected in aspects of promoting the growth and development of the original host plant.After a series of experiments testing the physiological characteristics of plant-growth promoting bacteria, we find an interesting phenomenon. Compared with hbh3.1, hbh54.2 has a strong ability of producing siderophores and secreting plant-growth hormone (indole acetic acid, IAA). Therefore, it may be inferred that hbh54.2 can make up some of the defects and shortcomings of hbh3.1, when the two strains co-exist in the same nodules, the former can provide exogenous growth hormones to host plants through the secretion of IAA, controling the concentration of IAA of the plants in vivo and regulating plant growth condition, what's more, through the secretion of siderophore, it can also chelate Fe element which is limited around the cells, not only for its own requirements, but also better for the latter in enhancing the efficiency of nitrogen fixation.This study also used the EGFP(enhanced green fluorescent protein) gene as the marker gene of hbh54.2, expecting to find out how hbh54.2 could locate in the nodule. As the results demonstrate, only under the circumstance of inoculating with hbh54.2(marked with egfp) and hbh3.1 together, can hbh54.2 show its existence in the nodules, while there is no fluorescence detected in the nodules in other situation, which indicates that hbh54.2 do not have the ability to invade the white clover alone, only with the help of the appropriate rhizobia(hbh3.1) can it be carried into the host plants and colonize in the root nodule tissues.This paper integrated the results of experiments testing the physiological characteristics of plant-growth promoting bacteria and plant nodulation tests with egfp marked strain for the first time. After analyzing the whole results, we conclude that Agrobaterium can enter the root nodules under the carrying of the appropriate Rhizobium; in the nodules, in order to make host-plant provide sufficient carbon source for Rhizobium, Agrobaterium can regulate plant-growth condition through the secretion of IAA, and also it can secrete siderophores to help Rhizobium absorb Fe for the maintenance of the normal physiological activity of nitrogenase (MoFe protein). The inferred conclusion revealed the significance of the coexistence of Agrobacterium with Rhizobium in the nodules, opening a new direction for further study of the relationship of Agrobacterium and Rhizobium in the future.