Chickpea (Cicer arietinum) is nodulated by unexpected wide diversity of Mesorhizobium species in Eastern Algeria

Using a local Algerian cultivar of chickpea, from 9 sites in Eastern Algeria, along a sub-humid to arid gradient, we isolated 60 nodulating isolates, among which 19 were assigned to Mesorhizobium species and 41 to Ensifer meliloti. Trapping revealed great differences among sites for their ability to induce nodules on plant, but no correlation with chemical and physical characterizations of soil samples could be found. Based on 16S rRNA gene sequences, 16S-23S IGS, recA, nodC and nifH sequences, Mesorhizobium isolates were assigned to at least 5 different species, among which one had never been described as nodulating chickpea before. The symbiotic diversity is conversely low, and shared by several species, reflecting horizontal gene transfer. The ability of Mesorhizobium genus to recombine and exchange symbiotic clusters among different species must play a great role in this ability to be associated with chickpea. The question to what extent the symbiotic species would end in this genus is discussed

Phylogenetic diversity of Bradyrhizobium strains isolated from root nodules of Lupinus angustifolius grown wild in the North East of Algeria

From a total of 80 bacterial strains isolated from root nodules of Lupinus angustifolius grown wild in the North-Eastern Algerian region of El Tarf, 64 plant host-nodulating strains clustered into 17 random amplified polymorphic DNA (RAPD) fingerprinting groups. The nearly complete 16S rRNA gene sequence from the representative strain of each group revealed they were closely related to members of the genus Bradyrhizobium of the Alphaproteobacteria, but their affiliation at the species level was not clear. Sequencing of the housekeeping genes glnII and recA, and their concatenated phylogenetic analysis, showed that 12 strains belong to B. lupini, other 2 strains affiliated with B. diazoefficiens and that 1 strain was closely related to B. japonicum. The remaining two strains showed similarity values ≤95% with B. cytisi and could represent new lineages within the genus Bradyrhizobium. Sequencing of the symbiotic nodC gene from 4 selected bradyrhizobial strains showed they were all similar to those of the species included in symbiovar genistearum.This study was supported by the ERDF-cofinanced grants PEAGR2012-1968 from Consejería de Economía, Innovación y Ciencia (Junta de Andalucía, Spain), AGL2015-64582-C3-3-R from MINECO. ACH is recipient of a grant of MECD (FPU 2014/01633).Peer Reviewe

Phylogenetic diversity of Bradyrhizobium strains isolated from root nodules of Lupinus angustifolius grown wild in the North East of Algeria

From a total of 80 bacterial strains isolated from root nodules of Lupinus angustifolius grown wild in the North-Eastern Algerian region of El Tarf, 64 plant host-nodulating strains clustered into 17 random amplified polymorphic DNA (RAPD) fingerprinting groups. The nearly complete 16S rRNA gene sequence from the representative strain of each group revealed they were closely related to members of the genus Bradyrhizobium of the Alphaproteobacteria, but their affiliation at the species level was not clear. Sequencing of the housekeeping genes glnII and recA, and their concatenated phylogenetic analysis, showed that 12 strains belong to B. lupini, other 2 strains affiliated with B. diazoefficiens and that 1 strain was closely related to B. japonicum. The remaining two strains showed similarity values ≤95% with B. cytisi and could represent new lineages within the genus Bradyrhizobium. Sequencing of the symbiotic nodC gene from 4 selected bradyrhizobial strains showed they were all similar to those of the species included in symbiovar genistearum.This study was supported by the ERDF-cofinanced grants PEAGR2012-1968 from Consejería de Economía, Innovación y Ciencia (Junta de Andalucía, Spain), AGL2015-64582-C3-3-R from MINECO. ACH is recipient of a grant of MECD (FPU 2014/01633).Peer Reviewe

Non-culturable Rhizobia and diverse endophytic bacteria co-inhabiting wild legume nodules.

Analyses of the bacterial content of legume nodules does generally yield cultures of the symbiotic partner responsible of nodule induction, invasion and nitrogen fixation. Species involved in such interaction include examples among the alpha and beta-rhizobia. In the present survey, exploring the nodular content of several species of wild legumes in the Mediterranean basin, we witnessed a consistent occurrence of the following situation: the bacteria which could be isolated from surface- sterilized nodules do not represent the actual nodule-inducing microorganism but rather a variety of endophytic invaders. Their presence in the nodules appeared related to their more general and systemic distribution throughout the whole plant, in close analogy with the situation observed in many other herbaceous and woody species which host an array of on-pathogenic endophytic taxa. At the same time the presence and quantitative abundance of the real nodule symbiont in the wild legumes covered by our study could be demonstrated by 16S rRNA gene PCR and nucleotide sequencing, which revealed that, despite their failure to grow on plates, alpha-proteobacterial rhizobia do represent the bona-fide symbiont responsible of nodule organogenesis

Coexistence of predominantly nonculturable rhizobia with diverse,endophytic bacterial taxa within nodules of wild legumes

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed