Ana Alexandre

Legume growth-promoting rhizobia: An overview on the Mesorhizobium genus?

The need for sustainable agricultural practices is revitalizing the interest in biological nitrogen fixation and rhizobia-legumes symbioses, particularly those involving economically important legume crops in terms of food and forage. The genus Mesorhizobium includes species with high geographical dispersion and able to nodulate a wide variety of legumes, including important crop species, like chickpea or biserrula. Some cases of legume-mesorhizobia inoculant introduction represent exceptional opportunities to study the rhizobia genomes evolution and the evolutionary relationships among species. Complete genome sequences revealed that mesorhizobia typically harbour chromosomal symbiosis islands. The phylogenies of symbiosis genes, such as nodC, are not congruent with the phylogenies based on core genes, reflecting rhizobial host range, rather than species affiliation. This agrees with studies showing that Mesorhizobium species are able to exchange symbiosis genes through lateral transfer of chromosomal symbiosis islands, thus acquiring the ability to nodulate new hosts. Phylogenetic analyses of the Mesorhizobium genus based on core and accessory genes reveal complex evolutionary relationships and a high genomic plasticity, rendering the Mesorhizobium genus as a good model to investigate rhizobia genome evolution and adaptation to different host plants. Further investigation of symbiosis genes as well as stress response genes will certainly contribute to understand mesorhizobia-legume symbiosis and to develop more effective mesorhizobia inoculants.

Chickpea rhizobia symbiosis genes are highly conserved across multiple Mesorhizobium species

Chickpea has been considered as a restrictive host for nodulation by rhizobia. However, recent studies have reported that several Mesorhizobium species may effectively nodulate chickpea. With the purpose of investigating the evolutionary relationships between these different species with the ability of nodulating the same host, we analysed 21 Portuguese chickpea rhizobial isolates. Symbiosis genes nifH and nodC were sequenced and used for phylogenetic studies. Symbiotic effectiveness was determined to evaluate its relationship with symbiosis genes. The comparison of 16S rRNA gene-based phylogeny with the phylogenies based on symbiosis genes revealed evidence of lateral transfer of symbiosis genes across different species. Chickpea is confirmed as a nonpromiscuous host. Although chickpea is nodulated by many different species, they share common symbiosis genes, suggesting recognition of only a few Nod factors by chickpea. Our results suggest that sequencing of nifH or nodC genes can be used for rapid detection of chickpea mesorhizobia.

Survey of Chickpea Rhizobia Diversity in Portugal Reveals the Predominance of Species Distinct from Mesorhizobium ciceri and Mesorhizobium mediterraneum

Several Mesorhizobium species are able to induce effective nodules in chickpea, one of the most important legumes worldwide. Our aims were to examine the biogeography of chickpea rhizobia, to search for a predominant species, and to identify the most efficient microsymbiont, considering Portugal as a case study. One hundred and ten isolates were obtained from continental Portugal and Madeira Island. The 16S ribosomal RNA gene phylogeny revealed that isolates are highly diverse, grouping with most Mesorhizobium type strains, in four main clusters (A–D). Interestingly, only 33% of the isolates grouped with Mesorhizobium ciceri (cluster B) or Mesorhizobium mediterraneum (cluster D), the formerly described specific chickpea microsymbionts. Most isolates belong to cluster A, showing higher sequence similarity with Mesorhizobium huakuii and Mesorhizobium amorphae. The association found between the province of origin and species cluster of the isolates suggests biogeography patterns: most isolates from the north, center, and south belong to clusters B, A, and D, respectively. Most of the highly efficient isolates (symbiotic effectiveness >75%) belong to cluster B. A correlation was found between species cluster and origin soil pH of the isolates, suggesting that pH is a key environmental factor, which influences the species geographic distribution. To our knowledge, this is one of the few surveys on chickpea rhizobia and the first systematic assessment of indigenous rhizobia in Portugal.

Response to temperature stress in rhizobia

It is well established that soil is a challenging environment for bacteria, where conditions may change rapidly and bacteria have to acclimate and adapt in order to survive. Rhizobia are an important group of soil bacteria due to their ability to establish atmospheric nitrogen-fixing symbioses with many legume species. Some of these legumes are used to feed either humans or cattle and therefore the use of rhizobia can reduce the need for synthetic N-fertilizers. Several environmental factors shape the composition and the activity of rhizobia populations in the rhizosphere. Soil pH and temperature are often considered to be the major abiotic factors in determining the bacterial community diversity. The present review focuses on the current knowledge on the molecular bases of temperature stress response in rhizobia. The effects of temperature stress in the legume-rhizobia symbioses are also addressed.

Natural Populations of Chickpea Rhizobia Evaluated by Antibiotic Resistance Profiles and Molecular Methods

The aims of this study were to investigate the hypothesis that intrinsic antibiotic resistance (IAR) profiles of chickpea rhizobia are correlated with the isolates site of origin, and to compare the discriminating power of IAR profiles with molecular approaches in rhizobial strain identification and differentiation. Rhizobial diversity from five Portuguese soils was assessed by IAR profiles and molecular methods [16S rDNA restriction fragment length polymorphism (RFLP) analysis, direct amplified polymorphic DNA (DAPD) fingerprinting, and SDS–PAGE analysis of protein profiles]. For each analysis, a dendrogram was generated using the software BioNumerics. All three molecular methods generated analogous clustering of the isolates, supporting previous results on 16S rDNA sequence-based phylogeny. Clusters obtained with IAR profile are similar to the species groups generated with the molecular methods used. IAR groups do not correlate significantly with the geographic origin of the isolates. These results may indicate a chromosomal location of antibiotic resistance genes, and suggest that IAR is species related. DAPD and IAR profiles proved to be the most discriminating approaches in strain differentiation and can be used as fast methods to screen diversity in new isolates.

Moderately acidophilic mesorhizobia isolated from chickpea

Aims:  Our aim was to evaluate the effect of acid and alkaline pH on chickpea rhizobia, and on chickpea–rhizobia symbiosis.

Transcriptional analysis of major chaperone genes in salt-tolerant and salt-sensitive mesorhizobia

Salinity is an important abiotic stress that limits rhizobia-legume symbiosis, affecting plant growth, thus reducing crop productivity. Our aims were to evaluate the tolerance to salinity of native chickpea rhizobia as well as to investigate the expression of chaperone genes groEL, dnaKJ and clpB in both tolerant and sensitive isolates. One hundred and six native chickpea mesorhizobia were screened for salinity tolerance by measuring their growth with 1.5% and 3% NaCl. Most isolates were salt-sensitive, showing a growth below 20% compared to control. An association between salt tolerance and province of origin of the isolates was found. The transcriptional analysis by northern hybridization of chaperone genes was performed using tolerant and sensitive isolates belonging to different Mesorhizobium species. Upon salt shock, most isolates revealed a slight increase in the expression of the dnaK gene, whereas the groESL and clpB expression was unchanged or slightly repressed. No clear relationship was found between the chaperone genes induction and the level of salt tolerance of the isolates. This is the first report on transcriptional analysis of the major chaperones genes in chickpea mesorhizobia under salinity, which may contribute to a better understanding of the mechanisms that influence rhizobia salt tolerance.

Global Transcriptional Response to Heat Shock of the Legume Symbiont Mesorhizobium loti MAFF303099 Comprises Extensive Gene Downregulation

Rhizobia, the bacterial legume symbionts able to fix atmospheric nitrogen inside root nodules, have to survive in varied environmental conditions. The aim of this study was to analyse the transcriptional response to heat shock of Mesorhizobium loti MAFF303099, a rhizobium with a large multipartite genome of 7.6 Mb that nodulates the model legume Lotus japonicus. Using microarray analysis, extensive transcriptomic changes were detected in response to heat shock: 30% of the protein-coding genes were differentially expressed (2067 genes in the chromosome, 62 in pMLa and 57 in pMLb). The highest-induced genes are in the same operon and code for two sHSP. Only one of the five groEL genes in MAFF303099 genome was induced by heat shock. Unlike other prokaryotes, the transcriptional response of this Mesorhizobium included the underexpression of an unusually large number of genes (72% of the differentially expressed genes). This extensive downregulation of gene expression may be an important part of the heat shock response, as a way of reducing energetic costs under stress. To our knowledge, this study reports the heat shock response of the largest prokaryote genome analysed so far, representing an important contribution to understand the response of plant-interacting bacteria to challenging environmental conditions.

The Symbiotic Performance of Chickpea Rhizobia Can Be Improved by Additional Copies of the clpB Chaperone Gene

The ClpB chaperone is known to be involved in bacterial stress response. Moreover, recent studies suggest that this protein has also a role in the chickpea-rhizobia symbiosis. In order to improve both stress tolerance and symbiotic performance of a chickpea microsymbiont, the Mesorhizobium mediterraneum UPM-Ca36T strain was genetically transformed with pPHU231 containing an extra-copy of the clpB gene. To investigate if the clpB-transformed strain displays an improved stress tolerance, bacterial growth was evaluated under heat and acid stress conditions. In addition, the effect of the extra-copies of the clpB gene in the symbiotic performance was evaluated using plant growth assays (hydroponic and pot trials). The clpB-transformed strain is more tolerant to heat shock than the strain transformed with pPHU231, supporting the involvement of ClpB in rhizobia heat shock tolerance. Both plant growth assays showed that ClpB has an important role in chickpea-rhizobia symbiosis. The nodulation kinetics analysis showed a higher rate of nodule appearance with the clpB-transformed strain. This strain also induced a greater number of nodules and, more notably, its symbiotic effectiveness increased ~60% at pH5 and 83% at pH7, compared to the wild-type strain. Furthermore, a higher frequency of root hair curling was also observed in plants inoculated with the clpB-transformed strain, compared to the wild-type strain. The superior root hair curling induction, nodulation ability and symbiotic effectiveness of the clpB-transformed strain may be explained by an increased expression of symbiosis genes. Indeed, higher transcript levels of the nodulation genes nodA and nodC (~3 folds) were detected in the clpB-transformed strain. The improvement of rhizobia by addition of extra-copies of the clpB gene may be a promising strategy to obtain strains with enhanced stress tolerance and symbiotic effectiveness, thus contributing to their success as crop inoculants, particularly under environmental stresses. This is the first report on the successful improvement of a rhizobium with a chaperone gene.

Can stress response genes be used to improve the symbiotic performance of rhizobia

Rhizobia are soil bacteria able to form symbioses with legumes and fix atmospheric nitrogen, converting it into a form that can be assimilated by the plant. The biological nitrogen fixation is a possible strategy to reduce the environmental pollution caused by the use of chemical N-fertilizers in agricultural fields. Successful colonization of the host root by free-living rhizobia requires that these bacteria are able to deal with adverse conditions in the soil, in addition to stresses that may occur in their endosymbiotic life inside the root nodules. Stress response genes, such as otsAB, groEL, clpB, rpoH play an important role in tolerance of free-living rhizobia to different environmental conditions and some of these genes have been shown to be involved in the symbiosis. This review will focus on stress response genes that have been reported to improve the symbiotic performance of rhizobia with their host plants. For example, chickpea plants inoculated with a Mesorhizobium strain modified with extra copies of the groEL gene showed a symbiotic effectiveness approximately 1.5 fold higher than plants inoculated with the wild-type strain. Despite these promising results, more studies are required to obtain highly efficient and tolerant rhizobia strains, suitable for different edaphoclimatic conditions, to be used as field inoculants.