ABSTRACT
The phylogeny of symbiotic genes of Robinia pseudoacacia (black locust) rhizobia derived from Poland and Japan was studied by comparative sequence analysis of nodA, nodC, nodH, and nifH loci. In phylogenetic trees, black locust symbionts formed a branch of their own suggesting that the spread and maintenance of symbiotic genes within Robinia pseudoacacia rhizobia occurred through vertical transmission. There was 99-100% sequence similarity for nodA genes of Robinia pseudoacacia nodulators, 97-98% for nodC, and 97-100% for nodH and nifH loci. A considerable sequence conservation of sym genes shows that the symbiotic apparatus of Robinia pseudoacacia rhizobia might have evolved under strong host plant constraints. In the nodA and nodC gene phylograms, Robinia pseudoacacia rhizobia grouped with Phaseolus sp. symbionts, although they were not closely related to our isolates based on 16S rRNA genes, and with Mesorhizobium amorphae. nifH gene phylogeny of our isolates followed the evolutionary history of 16S rDNA and Robinia pseudoacacia rhizobia grouped with Mesorhizobium genus species. Nodulation assays revealed that Robinia pseudoacacia rhizobia effectively nodulated their native host and also Amorpha fruticosa and Amorpha californica resulting in a significant enhancement of plant growth. The black locust root nodules are shown to be of indeterminate type.
Subject(s)
Bacterial Proteins/genetics , Evolution, Molecular , Rhizobiaceae/genetics , Robinia/microbiology , Symbiosis , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Japan , Molecular Sequence Data , Phylogeny , Plant Roots/microbiology , Poland , Polymorphism, Genetic , RNA, Ribosomal, 16S/genetics , Rhizobiaceae/isolation & purification , Rhizobiaceae/physiology , Sequence Analysis, DNA , Sequence Homology , Soil MicrobiologyABSTRACT
Robinia pseudoacacia microsymbionts from plants growing in Poland and Japan were evaluated for phylogeny and taxonomic position by genomic approach. Based on the comparative analyses of atpD (368 bp) and dnaK (573 bp) gene sequences as well as 16S rDNA restriction analysis (RFLP-16S rDNA), R. pseudoacacia microsymbionts were identified as Mesorhizobium strains. In dnaK and atpD gene phylograms R. pseudoacacia nodulators formed robust, monophyletic clusters with Mesorhizobium species with the nucleotide sequence similarity of 91-98% and 90-98%, respectively. The classification of R. pseudoacacia rhizobia to the genus Mesorhizobium was also supported by amplified 16S rDNA restriction analysis. The studied bacteria formed common clusters with Mesorhizobium species, and their DNA patterns were identical or nearly identical to Mesorhizobium genus strains. When DNA-DNA hybridization was performed, the total DNA of the representative R. pseudoacacia rhizobia exhibited 51-75% relatedness to DNA of Mesorhizobium amorphae ICMP15022 strain and below 41% to DNA of other Mesorhizobium species. These results showed that R. pseudoacacia and M. amorphae belong to the same genomospecies. The G+C content of DNA of R. pseudoacacia two microsymbionts was 59.7 and 60.6 mol% compared to 61-64 mol% across M. amorphae strains.
Subject(s)
Alphaproteobacteria/isolation & purification , Alphaproteobacteria/physiology , Plant Root Nodulation , Robinia/microbiology , Robinia/physiology , Symbiosis , Alphaproteobacteria/genetics , Bacterial Proteins/genetics , Base Composition , Cluster Analysis , DNA Fingerprinting , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Japan , Molecular Sequence Data , Nucleic Acid Hybridization , Phylogeny , Poland , Polymorphism, Restriction Fragment Length , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sequence Homology, Nucleic AcidABSTRACT
Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98-99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771-961 kb megaplasmids.
Subject(s)
Bradyrhizobium/classification , Bradyrhizobium/isolation & purification , Rhizobiaceae/classification , Rhizobiaceae/isolation & purification , Robinia/microbiology , Alphaproteobacteria , Amplified Fragment Length Polymorphism Analysis , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Typing Techniques , Bradyrhizobium/genetics , Bradyrhizobium/physiology , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Genomics , Japan , Molecular Sequence Data , Nitrogenase/genetics , Phylogeny , Plant Roots/microbiology , Poland , RNA, Ribosomal, 16S/genetics , Rhizobiaceae/genetics , Rhizobiaceae/physiology , Sequence Analysis, DNA , Sodium Chloride/pharmacologyABSTRACT
Three lytic phages (PhiRP1, PhiRP2, and PhiRP3) specific for Robinia pseudoacacia rhizobia were isolated from the soil under black locust. They were characterized by their morphology, host range, and some other properties including DNA molecular weights. Studied phages have been found to belong to Siphoviridae family that comprises viruses with long, and noncontractile tails. They had broad host ranges and effectively lysed not only Robinia pseudoacacia microsymbionts but also different Mesorhizobium species. The phages were homogenous in latent periods (300 min) but heterogeneous in burst sizes (100-200 phage particles per one infected cell) and rise periods (90-120 min). They showed a distinct adsorption rate to Robinia pseudoacacia rhizobia (70.4-93.94%). The molecular weights of phage DNAs estimated from restriction enzyme digests were in the range from ca. 82 kb to ca. 105 kb.