Response of intercropped barley and fenugreek to mono- and co-inoculation with Sinorhizobium meliloti F42 and Variovorax paradoxus F310 under contrasting agroclimatic regions.

In the present research, we aimed to select efficient rhizobia and plant growth-promoting rhizobacteria (PGPR) from fenugreek nodules and assess their performance as bio-inoculum for intercropped fenugreek and barley. Inoculation effects with selected bacteria were investigated firstly on fenugreek plants under greenhouse experiment and secondly on intercropped fenugreek and barley under three different agro-environmental conditions for two consecutive years. Sinorhizobium meliloti F42 was selected due to its ability to nodulate fenugreek and effectively improve plant growth. Among non-nodulating endophytic bacteria, Variovorax paradoxus F310 strain was selected regarding its plant growth-promoting traits showed in vitro and confirmed in vivo under greenhouse experiment. Field inoculation trials revealed a significant improvement in fenugreek nodulation (up to + 97%) as well as in soil enzymes activities (up to + 209%), shoot N content (up to + 18%), shoot dry weight (up to + 40%), photosynthetic assimilation (up to + 34%) and chlorophyll content of both intercropped plants in response to the mono-inoculation with Sinorhizobium meliloti F42, compared to the un-inoculated treatment at the SBR and JBS sites. Variovorax paradoxus F310 inoculation significantly increased shoot P content of both intercropped plants at the three experimental sites compared to the un-inoculated treatment (up to + 48%). It was shown that bacterial inoculation was more efficient at the low-rainfall region than the high-rainfall region. The co-inoculation with Sinorhizobium meliloti F42 and Variovorax paradoxus F310 resulted in a significant reduction in fenugreek nodulation and shoot N content. This survey showed the benefits of rhizobial and PGPR inoculation as efficient bio-inoculums to promote the cereal-legume intercropping system and highlights the influence of site-specific environmental factors on Rhizobium-PGPR-plant interactions.

Inoculation of Lupinus albus with the nodule-endophyte Paenibacillus glycanilyticus LJ121 improves grain nutritional quality.

Metabolic changes occurring in white lupine grain were investigated in response to Plant Growth Promoting Rhizobacteria (PGPR) root inoculation under field condition. We precisely targeted lipids and phenolics changes occurring in white lupine grain in response to Pseudomonas brenneri LJ215 and/or Paenibacillus glycanilyticus LJ121 inoculation. Lipids and phenolic composition were analyzed using an Ultra High-Performance Liquid Chromatography/Tandem Mass Spectrometry Methods. As compared to grain of un-inoculated control plant, Paenibacillus glycaniliticus inoculation highly increased the total lipids content (from 232.55 in seeds of un-inoculated control plant to 944.95 mg/kg) and the relative percentage of several fatty acid such as oleic acid (+20.95%) and linoleic acid (+14.28%) and decreased the relative percentage of glycerophospholipids (- 13.11%), sterol (- 0.2% and - 0.34% for stigmasterol and campesterol, respectively) and prenol (- 17.45%) class. Paenibacillus glycaniliticus inoculation did not affect total phenolic content, while it modulated content of individual phenolic compounds and induced the accumulation of "new" phenolics compounds such as kaempferol. Paenibacillus glycanilyticus LJ121 can be a useful bio-fertilizer to enhance nutritional quality of white lupine grain.

Isolation, identification and plant growth promotion ability of endophytic bacteria associated with lupine root nodule grown in Tunisian soil.

The present study aims to characterize nodule endophytic bacteria of spontaneous lupine plants regarding their diversity and their plant growth promoting (PGP) traits. The potential of PGPR inoculation was investigated to improve white lupine growth across controlled, semi-natural and field conditions. Lupinus luteus and Lupinus angustifolius nodules were shown inhabited by a large diversity of endophytes. Several endophytes harbor numerous plant growth promotion traits such as phosphates solubilization, siderophores production and 1-aminocyclopropane-1-carboxylate deaminase activity. In vivo analysis confirmed the plant growth promotion ability of two strains (Paenibacillus glycanilyticus LJ121 and Pseudomonas brenneri LJ215) in both sterilized and semi-natural conditions. Under field conditions, the co-inoculation of lupine by these strains increased shoot N content and grain yield by 25% and 36%, respectively. These two strains Paenibacillus glycanilyticus LJ121 and Pseudomonas brenneri LJ215 are effective plant growth-promoting bacteria and they may be used to develop an eco-friendly biofertilizer to boost white lupine productivity.

Minimal standards for the description of new genera and species of rhizobia and agrobacteria.

Herein the members of the Subcommittee on Taxonomy of Rhizobia and Agrobacteria of the International Committee on Systematics of Prokaryotes review recent developments in rhizobial and agrobacterial taxonomy and propose updated minimal standards for the description of new species (and genera) in these groups. The essential requirements (minimal standards) for description of a new species are (1) a genome sequence of at least the proposed type strain and (2) evidence for differentiation from other species based on genome sequence comparisons. It is also recommended that (3) genetic variation within the species is documented with sequence data from several clearly different strains and (4) phenotypic features are described, and their variation documented with data from a relevant set of representative strains. Furthermore, it is encouraged that information is provided on (5) nodulation or pathogenicity phenotypes, as appropriate, with relevant gene sequences. These guidelines supplement the current rules of general bacterial taxonomy, which require (6) a name that conforms to the International Code of Nomenclature of Prokaryotes, (7) validation of the name by publication either directly in the International Journal of Systematic and Evolutionary Microbiology or in a validation list when published elsewhere, and (8) deposition of the type strain in two international culture collections in separate countries.

Inoculation of Phaseolus vulgaris with the nodule-endophyte Agrobacterium sp. 10C2 affects richness and structure of rhizosphere bacterial communities and enhances nodulation and growth.

Agrobacterium sp. 10C2 is a nonpathogenic and non-symbiotic nodule-endophyte strain isolated from root nodules of Phaseolus vulgaris. The effect of this strain on nodulation, plant growth and rhizosphere bacterial communities of P. vulgaris is investigated under seminatural conditions. Inoculation with strain 10C2 induced an increase in nodule number (+54 %) and plant biomass (+16 %). Grains also showed a significant increase in phosphorus (+53 %), polyphenols (+217 %), flavonoids (+62 %) and total antioxidant capacity (+82 %). The effect of strain 10C2 on bacterial communities was monitored using terminal restriction fragment length polymorphism of PCR-amplified 16S rRNA genes. When the initial soil was inoculated with strain 10C2 and left 15 days, the Agrobacterium strain did not affect TRF richness but changed structure. When common bean was sown in these soils and cultivated during 75 days, both TRF richness and structure were affected by strain 10C2. TRF richness increased in the rhizosphere soil, while it decreased in the bulk soil (root free). The taxonomic assignation of TRFs induced by strain 10C2 in the bean rhizosphere revealed the presence of four phyla (Firmicutes, Actinobacteria, Bacteroidetes and Proteobacteria) with a relative preponderance of Firmicutes, represented mainly by Bacillus species. Some of these taxa (i.e., Bacillus licheniformis, Bacillus pumilus, Bacillus senegalensis, Bacillus subtilis, Bacillus firmus and Paenibacillus koreensis) are particularly known for their plant growth-promoting potentialities. These results suggest that the beneficial effects of strain 10C2 observed on plant growth and grain quality are explained at least in part by the indirect effect through the promotion of beneficial microorganisms.

Growth capacity and biochemical mechanisms involved in rhizobia tolerance to salinity and water deficit.

The aim of the present study was to evaluate abiotic stress tolerance of rhizobial strains belonging to Mesorhizobium, Sinorhizobium, and Rhizobium genera, as well as to investigate specie specific stress response mechanisms. Effect of NaCl and PEG on growth capacity, protein, lipid peroxydation (MDA), membrane fatty acid composition and antioxidant enzymes were investigated. Growth capacity and viability of overall rhizobia strains decreased proportionally to the increase of NaCl and PEG levels in the medium. Sinorhizobium strains appeared the most tolerant, where 4H41strain was able to grow at 800 mM NaCl and 40% PEG. On the other hand, growth of R. gallicum and M. mediterraneum was inhibited by 200 mM NaCl. The content of MDA was unchanged in Sinorhizobium strains under both stresses. For Mesorhizobium, only PEG treatment increased the content of MDA. Amount of the C19:0 cyclo fatty-acid was increased in both Sinorhizobium and Mesorhizobium tolerant strains. NaCl stress increased Superoxide dismutase (SOD) activity of overall species; especially the most tolerant strain 4H41. Both treatments increased catalase (CAT) activity in 4H41, TII7, and 835 strains. Obtained results suggest that major response of tolerant Sinorhizobium and Mesorhizobium strains to NaCl and PEG stresses is a preferential accumulation of the C19:0 cyclo fatty acid within bacterial membrane as mechanism to reduce fluidity and maintain integrity. Cell integrity and functioning is also assured by maintaining and/or increasing activity of SOD and CAT antioxidant enzymes for tolerant strains to omit structural and functional damages related to reactive oxygen species overproduced under stressful conditions.

Rhizobium laguerreae sp. nov. nodulates Vicia faba on several continents.

Several fast-growing strains nodulating Vicia faba in Peru, Spain and Tunisia formed a cluster related to Rhizobium leguminosarum. The 16S rRNA gene sequences were identical to that of R. leguminosarum USDA 2370(T), whereas rpoB, recA and atpD gene sequences were phylogenetically distant, with sequence similarities of less than 96 %, 97 % and 94 %, respectively. DNA-DNA hybridization analysis showed a mean relatedness value of 43 % between strain FB206(T) and R. leguminosarum USDA 2370(T). Phenotypic characteristics of the novel strains also differed from those of the closest related species of the genus Rhizobium. Therefore, based on genotypic and phenotypic data obtained in this study, we propose to classify this group of strains nodulating Vicia faba as a novel species of the genus Rhizobium named Rhizobium laguerreae sp. nov. The type strain is FB206(T) ( = LMG 27434(T) = CECT 8280(T)).

Rhizobium azibense sp. nov., a nitrogen fixing bacterium isolated from root-nodules of Phaseolus vulgaris.

Three microbial strains isolated from common beans, 23C2T (Tunisia), Gr42 (Spain) and IE4868 (Mexico), which have been identified previously as representing a genomic group closely related to Rhizobium gallicum, are further studied here. Their 16S rRNA genes showed 98.5-99% similarity with Rhizobium loessense CCBAU 7190BT, R. gallicum R602spT, Rhizobium mongolense USDA 1844T and Rhizobium yanglingense CCBAU 71623T. Phylogenetic analysis based on recA, atpD, dnaK and thrC sequences showed that the novel strains were closely related and could be distinguished from the four type strains of the closely related species. Strains 23C2T, Gr42 and IE4868 could be also differentiated from their closest phylogenetic neighbours by their phenotypic and physiological properties and their fatty acid contents. All three strains harboured symbiotic genes specific to biovar gallicum. Levels of DNA-DNA relatedness between strain 23C2T and the type strains of R. loessense, R. mongolense, R. gallicum and R. yanglingense ranged from 58.1 to 61.5%. The DNA G+C content of the genomic DNA of strain 23C2T was 59.52%. On the basis of these data, strains 23C2T, Gr42 and IE4868 were considered to represent a novel species of the genus Rhizobium for which the name Rhizobium azibense is proposed. Strain 23C2T (=CCBAU 101087T=HAMBI3541T) was designated as the type strain.

Characterization of root-nodule bacteria isolated from Vicia faba and selection of plant growth promoting isolates.

A collection of 104 isolates from root-nodules of Vicia faba was submitted to 16S rRNA PCR-RFLP typing. A representative sample was further submitted to sequence analysis of 16S rRNA. Isolates were assigned to 12 genera. All the nodulating isolates (45 %) were closely related to Rhizobium leguminosarum USDA2370(T) (99.34 %). The remaining isolates, including potential human pathogens, failed to nodulate their original host. They were checked for presence of symbiotic genes, P-solubilization, phytohormone and siderophore production, and then tested for their growth promoting abilities. Results indicated that 9 strains could induce significant increase (41-71 %) in shoot dry yield of faba bean. A Pseudomonas strain was further assessed in on-farm trial in combination with a selected rhizobial strain. This work indicated that nodule-associated bacteria could be a valuable pool for selection of effective plant growth promoting isolates. Nevertheless, the possible involvement of nodules in increasing risks related to pathogenic bacteria should not be neglected and needs to be investigated further.

Physicochemical and Biological Characterization of Encapsulated Olive Leaf Extracts for Food Preservation.

Phenolic compounds in olive leaves have an excellent antioxidant activity and good antimicrobial properties. These bioactive molecules have beneficial properties for health, arousing great scientific and commercial interest. This study reports lyophilized olive leaf extracts (OLE) encapsulated by spray-drying using maltodextrins, maltodextrins-pectin and maltodextrins-gum Arabic as encapsulating agents. Lyophilized OLE were collected from two varieties cultivated in a harsh pedo-climatic conditions of the arid region of Tunisia. The effects of the genetic factor and the different encapsulating agents on the physicochemical properties of microcapsules and their behavior during storage, as well as their antimicrobial activities, were studied. Microcapsules successfully passed heat treatment and storage conditions and their antimicrobial activities were preserved. The encapsulating agent combination improved the encapsulation efficiency and the product yield in Zarrazi variety compared to Dhokar one. In addition, Dhokar variety microparticles showed the best heat stability at 4 and 25 °C after 90 days of storage and the higher inhibition percent against bacteria. The results of the present study evidenced that the best conditions for OLE encapsulation were obtained when the maltodextrins-pectin and maltodextrins-gum Arabic were combined to form a hybrid coating material.

Synergistic antileishmanial activity of erythrodiol, uvaol, and oleanolic acid isolated from olive leaves of cv. Chemlali.

This study aimed to assess the antileishmanial activity of biomolecules obtained from Olea europaea L. leaves and twigs recovered from eight Tunisian cultivars. The extraction was first carried out with 80% methanol, and then the obtained extract was fractionated using three solvents of increasing polarity: cyclohexane (CHX), dichloromethane (DCM) and ethyl acetate (EtOAc). The antileishmanial activity was determined against leishmanial strains responsible for cutaneous, visceral, and mucocutaneous leishmaniasis. The cyclohexane fraction of the leaves of cv. Chemlali from the region of Sidi-Bouzid exhibited the strongest leishmanicidal activity against all the tested leishmanial strains. The inhibition concentrations (IC50) were 16.5, 14.5, and 7.4 µg mL-1 for Leishmania mexicana (cutaneous), Leishmania braziliensis (mucocutaneous), and Leishmania donovani (visceral), respectively. Interestingly, low cytotoxicity was observed on THP-1 cells with selective indexes (SI) ranging from 22.8 to 50.5. HPLC-HRMS and full-house NMR analysis allowed the identification of three triterpenic compounds, oleanolic acid (IC50 = 64.1 µg mL-1), erythrodiol (IC50 = 52.0 µg mL-1), and uvaol (IC50 = 53.8 µg mL-1). Antileishmanial activity of uvaol and oleanolic acid has been previously reported. However, this work constitutes the first report of the antileishmanial activity of erythrodiol which showed combinatorial interaction with uvaol (IC50 = 26.1 µg mL-1) against Leishmania tropica. The mixture of the three compounds, as major ones, exhibited an enhanced activity against Leishmania tropica (IC50 = 16.3 µg mL-1) compared to erythrodiol alone or the combination of uvaol and erythrodiol. This finding is of great importance and needs further investigation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03825-3.

The antibiosis of nodule-endophytic agrobacteria and its potential effect on nodule functioning of Phaseolus vulgaris.

The effect of the nodule-endophytic Agrobacterium strain 10C2 on nodulation, plant growth and nodule functioning of Phaseolus vulgaris was investigated using two rhizobial strains differing in their sensitivity to the in vitro antibiosis of the Agrobacterium strain. In the case of the sensitive strain, Agrobacterium sp. 10C2 induced a significant decrease in the proportion of pink nodules, probably by an antibiosis effect leading to the reduction in the number of bacteroids and thereby a decrease in total soluble proteins, leghaemoglobin content, photosynthesis and nitrogen fixation. In this case, the Agrobacterium strain behaved like a plant pathogen and the nodule reacted by increasing guaiacol peroxidase (POX) activity, which assures some physiological processes linked to pathogen control. By contrast, in the case of the resistant strain, the proportion of pink nodules increased, and thereby total soluble proteins, leghaemoglobin content, biomass production and nitrogen fixation were enhanced. The Agrobacterium strain is regarded in this case as a plant growth-promoting rhizobacterium and the POX-pathogen reaction was not observed. There was even a decrease in superoxide dismutase activity. The results suggested also that the Agrobacterium strain may be also involved in retarding nodule senescence in the case of the resistant strain.

Genotypic and symbiotic diversity of native rhizobia nodulating red pea (Lathyrus cicera L.) in Tunisia.

Nodulation and genetic diversity of native rhizobia nodulating Lathyrus cicera plants grown in 24 cultivated and marginal soils collected from northern and central Tunisia were studied. L. cicera plants were nodulated and showed the presence of native rhizobia in 21 soils. A total of 196 bacterial strains were selected and three different ribotypes were revealed after PCR-RFLP analysis. The sequence analysis of the rrs and two housekeeping genes (recA and thrC) from 36 representative isolates identified Rhizobium laguerreae as the dominant (53%) rhizobia nodulating L. cicera. To the best of our knowledge, this is the first time that this species has been reported among wild populations of the rhizobia-nodulating Lathyrus genus. Twenty-five percent of the isolates were identified as R. leguminosarum and isolates LS11.5, LS11.7 and LS8.8 clustered with Ensifer meliloti. Interestingly, five isolates (LS20.3, LS18.3, LS19.10, LS1.2 and LS21.20) were segregated from R. laguerreae and clustered as a separate clade. These isolates possibly belong to new species. According to nodC and nodA phylogeny, strains of R. laguerreae and R. leguminosarum harbored the symbiotic genes of symbiovar viciae and clustered in three different clades showing heterogeneity within the symbiovar. Strains of E. meliloti harbored symbiotic genes of Clade V and induced inefficient nodules.

Potential Hepatoprotective Activity of Super Critical Carbon Dioxide Olive Leaf Extracts against CCl4-Induced Liver Damage.

Virgin olive oil has demonstrated its effective activity against oxidative stress. However, data on the bioactive effect of olive leaves or their major constituents on the liver are scarce. The present research work was conducted to evaluate the hepatoprotective effects of supercritical carbon dioxide (SC-CO2) extracts from fresh and dried olive leaves on hepatotoxicity caused by carbon tetrachloride (CCl4) in rat models. For this purpose, healthy albino rats of 180-250 g weight were used. The assessment of biochemical markers was carried out on blood and liver tissue. Then, a histopathological study was carried out on liver tissue. The obtained results showed that fresh and dried olive leaf extracts ameliorate the perturbed biochemical parameters caused by CCl4 treatment. Furthermore, the results registered for the histopathological study are in accordance with the biochemical parameters and the protective capacity of SC-CO2 extracts against DNA damage, indicating that olive leaf extracts helped to improve liver fibrosis caused by CCl4 treatment.

Agrobacterium strains isolated from root nodules of common bean specifically reduce nodulation by Rhizobium gallicum.

In a previous work, we showed that non-nodulating agrobacteria strains were able to colonize root nodules of common bean. Both rhizobia and agrobacteria co-existed in the infected nodules. No impact on symbiosis was found in laboratory conditions when using sterile gravel as a support for growth. In this study, soil samples originating from different geographic and agronomic regions in Tunisia were inoculated with a mixture of agrobacteria strains isolated previously from root nodules of common bean. A significant effect on nodulation and vegetal growth of common bean was observed. Characterization of nodulating rhizobia and comparison with non-inoculated controls showed a biased genetic structure. It seemed that Rhizobium gallicum was highly inhibited, whereas nodulation by Sinorhizobium medicae was favored. Co-inoculation of non-sterile soils with R. gallicum and agrobacteria confirmed these findings. In vitro antibiosis assays indicated that agrobacteria exercised a significant antagonism against R. gallicum.

High-quality permanent draft genome sequence of Ensifer meliloti strain 4H41, an effective salt- and drought-tolerant microsymbiont of Phaseolus vulgaris.

Ensifer meliloti 4H41 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of common bean (Phaseolus vulgaris). Strain 4H41 was isolated in 2002 from root nodules of P. vulgaris grown in South Tunisia from the oasis of Rjim-Maatoug. Strain 4H41 is salt- and drought-tolerant and highly effective at fixing nitrogen with P. vulgaris. Here we describe the features of E. meliloti 4H41, together with genome sequence information and its annotation. The 6,795,637 bp high-quality permanent draft genome is arranged into 47 scaffolds of 47 contigs containing 6,350 protein-coding genes and 72 RNA-only encoding genes, and is one of the rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project proposal.

Microbial inoculants and their impact on soil microbial communities: a review.

The knowledge of the survival of inoculated fungal and bacterial strains in field and the effects of their release on the indigenous microbial communities has been of great interest since the practical use of selected natural or genetically modified microorganisms has been developed. Soil inoculation or seed bacterization may lead to changes in the structure of the indigenous microbial communities, which is important with regard to the safety of introduction of microbes into the environment. Many reports indicate that application of microbial inoculants can influence, at least temporarily, the resident microbial communities. However, the major concern remains regarding how the impact on taxonomic groups can be related to effects on functional capabilities of the soil microbial communities. These changes could be the result of direct effects resulting from trophic competitions and antagonistic/synergic interactions with the resident microbial populations, or indirect effects mediated by enhanced root growth and exudation. Combination of inoculants will not necessarily produce an additive or synergic effect, but rather a competitive process. The extent of the inoculation impact on the subsequent crops in relation to the buffering capacity of the plant-soil-biota is still not well documented and should be the focus of future research.

Sinorhizobium americanum symbiovar mediterranense is a predominant symbiont that nodulates and fixes nitrogen with common bean (Phaseolus vulgaris L.) in a Northern Tunisian field.

A total of 40 symbiotic bacterial strains isolated from root nodules of common bean grown in a soil located in the north of Tunisia were characterized by PCR-RFLP of the 16S rRNA genes. Six different ribotypes were revealed. Nine representative isolates were submitted to phylogenetic analyses of rrs, recA, atpD, dnaK, nifH and nodA genes. The strains 23C40 and 23C95 representing the most abundant ribotype were closely related to Sinorhizobium americanum CFNEI 156(T). S. americanum was isolated from Acacia spp. in Mexico, but this is the first time that this species is reported among natural populations of rhizobia nodulating common bean. These isolates nodulated and fixed nitrogen with this crop and harbored the symbiotic genes of the symbiovar mediterranense. The strains 23C2 and 23C55 were close to Rhizobium gallicum R602sp(T) but formed a well separated clade and may probably constitute a new species. The sequence similarities with R. gallicum type strain were 98.7% (rrs), 96.6% (recA), 95.8% (atpD) and 93.4% (dnaK). The remaining isolates were, respectively, affiliated to R. gallicum, E. meliloti, Rhizobium giardinii and Rhizobium radiobacter. However, some of them failed to re-nodulate their original host but promoted root growth.

Diversity of nodule-endophytic agrobacteria-like strains associated with different grain legumes in Tunisia.

This study represents the first report describing the genetic diversity of nodule-endophytic agrobacteria isolated from diverse legumes and their phylogenetic relationships with the valid species of agrobacteria, as well as the non-recognized genomospecies of the former Agrobacterium tumefaciens (Rhizobium radiobacter). The genetic diversity of a collection of 18 non-nodulating agrobacteria-like strains, previously isolated from root nodules of Vicia faba, Cicer arietinum and Phaseolus vulgaris from different geographical regions of Tunisia, was studied by REP-PCR and PCR-RFLP of the 16S-23S rDNA IGS, as well as by sequence analysis of the 16S rDNA and the housekeeping genes recA and atpD. The aim of the work was to study the genetic diversity of the different isolates and to check for any host-specificity. The results from the different techniques were congruent and suggested a specific interaction for P. vulgaris, whereas no specific endophytic interaction was observed for V. faba and C. arietinum. The phylogenetic analysis clearly indicated that some isolates were affiliated to R. radiobacter or to its non-recognized genomic species (genomovars G2, G4 and G9). However, the other isolates probably constitute new species within Rhizobium (Agrobacterium) and Shinella.

Effect of on-field inoculation of Phaseolus vulgaris with rhizobia on soil bacterial communities.

The aim of this study was to assess the impact of inoculation of Phaseolus vulgaris with two indigenous rhizobia strains on plant growth promotion, nitrogen turnover processes, richness and structure of the Rhizobiaceae and total bacterial communities in the bulk soil. Both strains used induced a significant increase in nodulation and grain yield. Analysis of bulk soil fertility showed positive, negative and strain-dependent effects of inoculation on nitrate, phosphorus and ammonium, respectively. Terminal-restriction fragment length polymorphism profiling demonstrated that inoculation significantly increased the phylotype richness of the bacterial communities. No significant difference in richness between the strains used and no additive effect of co-inoculation were observed. However, differences between both inoculants and a clear additive effect of co-inoculation on heterogeneity were found. This work gives original insights into the effect of rhizobial inoculation outside the restricted rhizospheric area. Effects on bacterial structure and diversity are clearly sensed in the neighbourhood of 25 cm and in a limited time course. Both Alpha- and Gammaproteobacteria, together with Firmicutes and Actinobacteria, were enhanced by inoculation, No evidence of terminal-restriction fragment inhibition was found. However, it remains to be answered how the impact on taxonomic groups can be related to effects on functional capabilities of soil microbial communities.