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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.

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.

Aims: To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains., Methods and Results: The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal., Conclusions: The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations., Significance and Impact of the Study: A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.

Aims: Our aim was to evaluate the effect of acid and alkaline pH on chickpea rhizobia, and on chickpea-rhizobia symbiosis., Methods and Results: Forty-seven rhizobia isolates obtained from 12 Portuguese soils were grown at pH 5, 7 and 9. Among these, 26 grew more at pH 5 than at 7, suggesting the existence of acidophiles. All isolates were identified as mesorhizobia by 16S rDNA partial sequence analysis. Molecular phylogeny of isolates based on partial 16S rDNA sequences suggests that pH tolerance might be species related. Further studies were conducted with six isolates, which were able to grow at acid pH. Isolates PT-35 and 64b grow optimally at pH 6-5-7, with a minimal pH range from 5 to 3, and may thus be considered as moderately acidophilic. Both isolates belong to a previously identified putative new Mesorhizobium species, based on 16S rDNA sequence., Conclusions: Two moderately acidophilic mesorhizobia isolated from chickpea were identified (PT-35 and 64b). A positive correlation was found between the symbiotic effectiveness at low pH and the acid tolerance of rhizobia isolates., Significance and Impact of the Study: This is the first report on moderately acidophilic mesorhizobia, and is an important contribution for the development of highly effective inoculants for chickpea in acid soils.

The development of rhizobial inoculants requires the selection of isolates that are symbiotically efficient as well as adapted to the local environmental conditions. Our aim was to find indigenous chickpea rhizobia tolerant to adverse environmental conditions, such as temperature and pH. Thirteen isolates of chickpea mesorhizobia from southern Portugal were examined. Tolerance to stress temperatures and pH was evaluated by quantification of bacterial growth at 20-37 degrees C and pH 5-9, respectively. Tolerance to heat shocks was studied by submitting isolates to 46 degrees C and 60 degrees C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis revealed qualitative and quantitative differences when isolates were submitted to temperature stress. A 60-kDa protein was overproduced by all isolates under heat stress. Almost all isolates revealed to be more tolerant to 20 degrees C than to 37 degrees C. A positive correlation was found between the maximum growth pH and the isolate origin soil pH. Generally, isolates more tolerant to temperature stress showed a lower symbiotic efficiency.

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.

Chickpea rhizobia isolated from Portuguese soils were assigned to the genus Mesorhizobium by 16S-rDNA sequencing. High species diversity was found within populations of an agricultural region in the south of Portugal. Besides the expected Mesorhizobium ciceri and M. mediterraneum, some isolates were close to M. loti or M. tianshanense and some formed a clade that may represent a new species. A new PCR-based approach, named direct amplified polymorphic DNA (DAPD) analysis, supported the 16S-based phylogeny. This suggests that this method could be used as a molecular tool to assess genetic relationships. Evaluation of genetic diversity by 16S-rDNA sequence, DAPD and protein profiles showed different levels of heterogeneity in natural populations.

Aims: To test the hypothesis that differences in chickpea yields obtained in four distinct Portuguese regions (Beja, Elvas-Casas Velhas, Elvas-Estação Nacional de Melhoramento de Plantas (ENMP) and Evora) could be due to variation between the natural rhizobia populations., Methods and Results: Estimation of the size of the different rhizobial populations showed that Elvas-ENMP population was the largest one. Elvas-ENMP population also revealed a higher proportion of isolates carrying more than one plasmid. Assessment of genetic diversity of the native rhizobia populations by a DNA fingerprinting PCR method, here designated as DAPD (Direct Amplified Polymorphic DNA), showed a higher degree of variation in Elvas-ENMP and Beja populations. The symbiotic effectiveness (SE) of 39 isolates was determined and ranged 13-34%. Statistical analysis showed that SE was negatively correlated with plasmid number of the isolate., Conclusions: The largest indigenous rhizobia population was found in Elvas-ENMP. DAPD pattern and plasmid profile analysis both suggested a higher genetic diversity among the populations of Elvas-ENMP and Beja. No relationship was found between SE of the isolates and their origin site., Significance and Impact of Study: The large native population, rather than the symbiotic performance of individual rhizobia, could contribute to the higher chickpea yields obtained in Elvas-ENMP.