Numerical Taxonomy And Identification Of Rhizobia Isolated From Mimosa Spp.

This thesis reviewed recently developed rhizobial systematics, summarized the main methods applied in rhizobia identification and studied on the heavy metal and phenol tolerance.The phylogenetic positions of representative strains symbiotic with mimosa were revealed by 16S rRNA gene sequences. It indicated that all strains separated into Rhizobium,Burkholderia and cupriavidus.The representative strains HBU66117,HBU66074,HBU66003,HBU66305,HBU66304,HBU66247,HBU66049,HBU66044,HBU66029,HBU66286 were located in the subbranch of Burkholderia.The strains HBU66294,HBU66322,HBU66194,HBU66166,HBU66232,HBU66133,HBU66131,HBU65028,HBU66045 distributed in the cupriavidus branch, and HBU66289 in the Rhizobium genus.Sixty-six rhizobial strains were studied by numerical analysis of 89 phenotypic characteristics.The results showed that 45 of all tested strains were clustered into 5 groups at a similarity level of 80%. The strains in this study with a high level resistance to saline,pH,antibiotics were found. Some strains can resist to 5% NaCl, and grow at YMA medium with pH12.0, some strains even resisted to 300μg/mL of antibiotic. This result showed that there is a great phenotypic diversity among the isolates.In order to indentify the strain furtherly, whole cell protein SDS-PAGE and BOX-PCR were applied to central rhizobial strains. The patterns of SDS-PAGE and BOX-PCR showed that it can get more message from the strains. Based on the results of above methods, all strains were classified 55 different styles of all strains .Central strain HBU66286 which were related to Burkholderia were chosen for determination of DNA base composition and DNA-DNA hybridization in comparison with the type strains of related described rhizobial species. The DNA relatedness between HBU66286 and B.phymatum was 83%, which demonstrated that HBU66286 belonged to B.phymatum. And the DNA homology of the representative strains HBU66322,HBU66194,HBU66166,HBU66232 with reference strain C.taiwanensis LMG19424T ranged from 0～60%.the DNA homology of the representative strains HBU66049,HBU66029,HBU66044,HBU66305 with reference strain B.phymatum LMG21445T ranged from 0-60%.the G+C mol% of them were all in the G+C mol% range of the genu Burkholderia or cupriavidus.It showed that they may be new distinct new species.To determine that the strains HBU66322,HBU66194,HBU66166,HBU66232,HBU66049,HBU66029,HBU66044,HBU66305 that isolated from Mimosa spp. related to Burkholderia,cupriavidus species, the ability to renodulate the 10 species of leguminous plants were checked, Over a four-week period, all of test strains only can formed nodules with the Leucaena glaucaplant on the roots of plant.Nine rizobium strains from mimosa were used to analyse resistance and the maximum resistance leve(lMRL)to heavy metals and phenol.The result showed that most of strains had strong resistance for heavy metals and phenol.Some strains even were tolerant to Zn2+of 5 mmol/L,Hg2+ of 0.5 mmol/L,Cu2+ of 0.6 mmol/L,phenol of 1700 mg/L. Meantime the strains of Burkholderia have stronger resistance than the strains from cupriavidus.From above analysis, we discovered that strains isolated from nodules of Mimosa have great diversity. The strains isolated from mimosa were mainly distributed in Rhizobium,Burkholderia and cupriavidus branches respectively.Meantime 8 strains may be new speises. These finding also shows that the strains of Burkholderia have stronger resistance than the strains of cupriavidus. This will be especially meaningful if we can apply these strains with stronger rolerance of heavy metals and phenol to the contaminated region.