Esperanza Martínez-Romero

Bacterial endophytes and their interactions with hosts.

Recent molecular studies on endophytic bacterial diversity have revealed a large richness of species. Endophytes promote plant growth and yield, suppress pathogens, may help to remove contaminants, solubilize phosphate, or contribute assimilable nitrogen to plants. Some endophytes are seedborne, but others have mechanisms to colonize the plants that are being studied. Bacterial mutants unable to produce secreted proteins are impaired in the colonization process. Plant genes expressed in the presence of endophytes provide clues as to the effects of endophytes in plants. Molecular analysis showed that plant defense responses limit bacterial populations inside plants. Some human pathogens, such as Salmonella spp., have been found as endophytes, and these bacteria are not removed by disinfection procedures that eliminate superficially occurring bacteria. Delivery of endophytes to the environment or agricultural fields should be carefully evaluated to avoid introducing pathogens.

Rhizobium tropici, a Novel Species Nodulating Phaseolus vulgaris L. Beans and Leucaena sp. Trees

A new Rhizobium species that nodulates Phaseolus vulgaris L. and Leucaena spp. is proposed on the basis of the results of multilocus enzyme electrophoresis, DNA-DNA hybridization, an analysis of ribosomal DNA organization, a sequence analysis of 16S rDNA, and an analysis of phenotypic characteristics. This taxon, Rhizobium tropici sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type II strains) and was recognized by its host range (which includes Leucaena spp.) and nif gene organization. In contrast to R. leguminosarum biovar phaseoli, R. tropici strains tolerate high temperatures and high levels of acidity in culture and are symbiotically more stable. We identified two subgroups within R. tropici and describe them in this paper.

Reclassification of American Rhizobium leguminosarum Biovar Phaseoli Type I Strains as Rhizobium etli sp. nov.

A new Rhizobium species that nodulates Phaseolus vulgaris L. is proposed on the basis of a sequence analysis of 16S ribosomal DNA. This taxon, Rhizobium etli sp. nov., was previously named Rhizobium leguminosarum biovar phaseoli (type I strains) and is characterized by the capacity to establish an effective symbiosis with bean plants, the reiteration of the nitrogenase structural genes, the organization of the common nodulation genes into two separate transcriptional units bearing nodA and nodBC, the presence of the polysaccharide inhibition gene, psi, and the 16S ribosomal DNA sequence. An analysis of the sequence of a fragment of the 16S rRNA gene shows that this gene is quite different from the gene of R. leguminosarum. In addition, all R. etli strains have identical sequences. We describe these analyses and discuss additional evidence supporting our proposal.

Advances in Rhizobium Research

Referee: Prof. Dr. Dietrich Werner, FG Zellbiologie und Angewandte Botanik, Fachbereich Biologie, Philipps-Universität Marburg, Karl-von-Frisch-Strasse, D-35032 Marburg, Germany Rhizobia are well known for their capacity to establish a symbiosis with legumes. They inhabit root nodules, where they reduce atmospheric nitrogen and make it available to the plant. Biological nitrogen fixation is an important component of sustainable agriculture, and rhizobial inoculants have been applied frequently as biofertilizers. In this review we present recently developed technologies and strategies for selecting quality inoculant strains by taking into consideration the complex interaction between the edaphic environment with the genotypes of both the legume and its microsymbiont. Enhanced competitive ability in an inoculant strain is a key requirement for successful colonization of plant roots, nodule formation, and subsequent N2-fixation. We discuss several avenues for the management and manipulation of rhizobial competition as well as genes that influence competition in the rhizosphere. The use of molecular techniques has greatly contributed to our knowledge of nodule-bacterial diversity and phylogeny. Approaches to the study of rhizobial diversity as well as mechanisms for the evolutionary diversification of nodulating bacteria are presented. Rhizobium genomes ranging from 5.5 to 9 Mb have been sequenced recently and deposited in public databases. A comparison of sequence data has led to a better understanding of genes involved in the symbiotic process as well as possible mechanisms responsible for horizontal transfer of genetic elements and symbiosis genes among rhizobia. Furthermore, rhizobia are frequent rhizosphere colonizers of a wide range of plants and may also inhabit nonleguminous plants endophytically. In these rhizospheric and endophytic habitats they may exhibit several plant growth-promoting effects, such as hormone production, phosphate solubilization, and the suppression of pathogens.

Rhizobium Phylogenies and Bacterial Genetic Diversity

Abstract The Leguminosae is one of the largest families of plants. It has a broad geographical distribution. The principal legume species have defined sites of origin and these coincide with the diversification centers for their “specific” symbiotic bacteria. These nitrogen-fixing bacteria, which form nodules in the roots or stems of the plants, belong to different bacterial lineages (Rhizobium, Bradyrhizobium, and Azorhizobium) related to other nonsymbiotic bacteria. A remarkable characteristic of these bacteria is their large genetic diversity. The genetic relationships among the different bacterial groups are being defined based mainly on the analysis of the sequences of the ribosomal genes. Recent results point out the need to have a broader genomic scope. Gene maps, genome sizes, and sequence of metabolic genes would serve to validate the present Rhizobium and Bradyrhizobium phylogenies. More realistic phylogenies should perhaps consider lateral transfer between clusters of bacteria. A compilation of...

Diversity of rhizobia associated with Amorpha fruticosa isolated from Chinese soils and description of Mesorhizobium amorphae sp. nov.

Fifty-five Chinese isolates from nodules of Amorpha fruticosa were characterized and compared with the type strains of the species and genera of bacteria which form nitrogen-fixing symbioses with leguminous host plants. A polyphasic approach, which included RFLP of PCR-amplified 16S rRNA genes, multilocus enzyme electrophoresis (MLEE), DNA-DNA hybridization, 16S rRNA gene sequencing, electrophoretic plasmid profiles, cross-nodulation and a phenotypic study, was used in the comparative analysis. The isolates originated from several different sites in China and they varied in their phenotypic and genetic characteristics. The majority of the isolates had moderate to slow growth rates, produced acid on YMA and harboured a 930 kb symbiotic plasmid (pSym). Five different RFLP patterns were identified among the 16S rRNA genes of all the isolates. Isolates grouped by PCR-RFLP of the 16S rRNA genes were also separated into groups by variation in MLEE profiles and by DNA-DNA hybridization. A representative isolate from each of these DNA homology groups had a separate position in a phylogenetic tree as determined from sequencing analysis of the 16S rRNA genes. A new species, Mesorhizobium amorphae, is proposed for the majority of the isolates, which belonged to a moderately slow- to slow-growing, acid-producing group based upon their distinct phylogenetic position, their unique electrophoretic type, their low DNA homology with reference strains representing the species within the genus Mesorhizobium and their distinct phenotypic features. Strain ACCC 19665 was chosen as the type strain for M. amorphae sp. nov.

Phylogenetic and Genetic Relationships of Mesorhizobium tianshanense and Related Rhizobia

The genetic and phylogenetic relationships for strains of Mesorhizobium tianshanense and its relatives were compared by an analysis of the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins, DNA-DNA hybridization, and full 16S rRNA gene sequencing. The strains of M. tianshanense formed a cluster which was distinct from those of other rhizobium species in the clustering analysis of SDS-PAGE. DNA-DNA relatedness between A-1BS (type strain of M. tianshanense) and the type or reference strains for Mesorhizobium loti, M. huakuii, M. ciceri, M. mediterraneum, and cluster U, an unnamed rhizobial group, ranged from 4.4 to 43.8%. The phylogenetic analysis based on the 16S rRNA gene sequences showed that M. tianshanense was closely related to the Mesorhizobium phylogenetic branch and could be distinguished from the other four species in this branch. These results further confirmed that these bacteria constitute a distinct rhizobial species.

Diversity of Rhizobium-Phaseolus vulgaris symbiosis: overview and perspectives

Common bean (Phaseolus vulgaris) has become a cosmopolitan crop, but was originally domesticated in the Americas and has been grown in Latin America for several thousand years. Consequently an enormous diversity of bean nodulating bacteria have developed and in the centers of origin the predominant species in bean nodules is R. etli. In some areas of Latin America, inoculation, which normally promotes nodulation and nitrogen fixation is hampered by the prevalence of native strains. Many other species in addition to R. etli have been found in bean nodules in regions where bean has been introduced. Some of these species such as R. leguminosarum bv. phaseoli, R. gallicum bv. phaseoli and R. giardinii bv. phaseoli might have arisen by acquiring the phaseoli plasmid from R. etli. Others, like R. tropici, are well adapted to acid soils and high temperatures and are good inoculants for bean under these conditions. The large number of rhizobia species capable of nodulating bean supports that bean is a promiscuous host and a diversity of bean-rhizobia interactions exists. Large ranges of dinitrogen fixing capabilities have been documented among bean cultivars and commercial beans have the lowest values among legume crops. Knowledge on bean symbiosis is still incipient but could help to improve bean biological nitrogen fixation.

Rhizobium huautlense sp. nov., a symbiont of Sesbania herbacea that has a close phylogenetic relationship with Rhizobium galegae

The nitrogen-fixing rhizobial symbionts of Sesbania herbacea growing in the nature reserve at the Sierra de Huautla, Mexico, were isolated and characterized. All 104 isolates together with the type strain for Rhizobium galegae, HAMBI 540T, had similar 16S rRNA genes as revealed by PCR-RFLP analysis. Similarity in the sequences of the 16S rRNA genes placed the isolates on a phylogenetic branch shared with R. galegae. Among 66 randomly selected isolates, three closely related electrophoretic alloenzyme types (ETs) were identified, which were distinct from 10 ETs distinguished among 23 strains of R. galegae. A new species Rhizobium huautlense, represented by the Sesbania isolate SO2T, is proposed based upon low estimates of DNA relatedness between our chosen type strain and the type strains for the other species, the dissimilarity of the nucleotide sequence of the 16S rRNA genes, and their distinct ETs compared with R. galegae. The description of R. huautlense is significant because in the reconstruction of the phylogeny at R. huautlense there was a shift in the node of the branch of Agrobacterium vitis relative to that of R. galegae. The revised phylogenetic tree would tend to indicate common ancestry between R. galegae and Rhizobium leguminosarum.

Polyphasic evidence supporting the reclassification of Bradyrhizobium japonicum group Ia strains as Bradyrhizobium diazoefficiens sp. nov.

Bradyrhizobium japonicum was described from soybean root-nodule bacterial isolates. Since its description, several studies have revealed heterogeneities among rhizobia assigned to this species. Strains assigned to B. japonicum group Ia have been isolated in several countries, and many of them are outstanding soybean symbionts used in inoculants worldwide, but they have also been isolated from other legume hosts. Here, we summarize published studies that indicate that group Ia strains are different from the B. japonicum type strain USDA 6(T) and closely related strains, and present new morphophysiological, genotypic and genomic evidence to support their reclassification into a novel species, for which the name Bradyrhizobium diazoefficiens sp. nov. is proposed. The type strain of the novel species is the well-studied strain USDA 110(T) ( =IAM 13628(T)  =CCRC 13528(T)  =NRRL B-4361(T)  =NRRL B-4450(T)  =TAL 102(T)  =BCRC 13528(T)  =JCM 10833(T)  =TISTR 339(T)  =SEMIA 5032(T)  =3I1B110(T)  =ACCC 15034(T)  =CCT 4249(T)  = NBRC 14792(T)  = R-12974(T)  = CNPSo 46(T)).