Isolation, Identification And Partial Genomics Of Phages Of Rhizobium Leguminosae

Rhizobiophages infection resulted in the decrease of effective rhizobia quantity, decrease of nodulation rate and decrease of nitrogen fixation, which resulted in a significant decrease of crop yield. However, few reports on rhizobiophages have been reported. In this study, three model rhizobium strains with different genotypes, Sinorhizobium meliloti USDA1002 T, Sinorhizobium sojae CCBAU05684 and Bradyrhizobium diazoefficiens USDA110, were served as host rhizobium to isolate their rhizobiophages from soil environment using the double-layer plate method. Then the morphological characteristics of rhizobiophages were observed through transmission electron microscope, and the biological characteristics of the phages were investigated. In addition, the morphology and genetic characteristics of rhizobiophages were determined by PCR-clone, high-throughout sequencing, genomic structural analysis and comparative whole genome analysis to reveal the similarities and differences of the phages infecting different rhizobium hosts, and to analyze the interactions of rhizobiophages, their hosts and legumes, to provide basic data and theoretical basis for the study of biological agents for reducing or even eliminating Rhizobium phage.Rhizobiophages were isolated from soil environment three model strains with different genotypes, viz., Sinorhizobium meliloti USDA1002 T, Sinorhizobium sojae CCBAU05684 and Bradyrhizobium diazoefficiens USDA110, by using the double-layer plate methodThe morphological characteristic of rhizobiophage was performed by transmission electron microscope. The biological characteristics of the phage were determined. The genome DNA was extracted and PCR-clone sequenced, high-throughout sequenced, genomic strutural analysis and comparative whole genome analysis. Therefore, the cultural methods and molecular biological technique will be used to study morphology and genetic characteristics of rhizobiophages and to reveal the similarities and differences of the phages infecting different Rhizobium hosts, and to analyze the relationship between Rhizobium phages and its hosts and legumes.The main findings are as follows:1. Three rhizobiophages were isolated from different rhizobium with different genotypes according to their plaque sizesindividually. Transmission electron microscope revealed that the phages of Sinorhizobium meliloti USDA1002 T and Bradyrhizobium diazoefficiens USDA110 were Myoviridae family and the phages of Sinorhizobium sojae CCBAU05684 were Siphoviridae family.2. The optimal numbers of phage infection among nine phages were differed from each other significantly, but all varied in the range of 0.001-1. The latent period and rise period of phages of Bradyrhizobium diazoefficiens were longest, the second were the phages of Sinorhizobium meliloti and the third were the phages of Sinorhizobium sojae.3. All nine phages of this study can survive stablely at 30-40 ? and pH 6.0–9.0. The phages ofSinorhizobium meliloti and Bradyrhizobium diazoefficiens were more tolerant to Ultra Violet(UV) than the phages of Sinorhizobium sojae. After 3 minutes of UV irradiation, the survival rate of the bacteriophages of Sinorhizobium sojae was reduced by more than 90%, and all phages were inactivated after 12 minutes of UV irradiation. In addition, the host range of all phages were very narrow. The biological characteristics of the phages isolatied from the same host were also different.4. Basic local alignment search tool(BLAST) searches in website of National Center for Biotechnology Information(NCBI) revealed that the g23 amino acid sequences obtained in this study had high similarity to 98%-99% with each other, but had very lower similarity with those from T-evens, PseudoT-evens, SchizoT-evens and ExoT-evens, just is 30%-57%. Phylogenetic analysis showed that the isolated g23 sequences formed a unique clade with those clones from different ecosystem.5. The results of high-throughput whole genome sequencing, genome structural analysis and comparative genomic analysisshowed that BDU-3 genome was a double-stranded closed circular phage, and had low similarity with 13 reported phages ofrhizobium leguminosae the whole genome of Through phylogenetic analysis based on amino acid sequences of the major capsid protein, baseplate assembly protein J and phage tail sheath monomer, BDU-3 was clustered with Bradyrhizobium sp.th.b2 ? Bradyrhizobium elkaniiand Bradyrhizobium diazoefficiens, the similarity of them was 74%-84%, while exhibited a low similarity to other phages in terms of genome organization and structural protein amino acid sequences. These results suggested that BDU-3 appeared to encode structural and functional proteins involved in interaction with the host and may have been acquired by horizontal transfer.