Mokhtar Rejili

Microbiological and chemical characterisations of organic and conventional date pastes (Phoenix dactylifera L.) from Tunisia

Chemical and microbiological properties of organic and conventional date pastes produced from the Deglet Nour cultivar were investigated. Significant differences were observed in water content, sugar profiles, and pH among the date pastes. The low moisture and the high acidity of organic date paste were two important positive attributes for its storage and potential manufacturing uses. Before pasteurisation, moulds CFU’s of the organic paste was significantly (P <0.01) lower than those of the conventional one but no significant differences were observed for yeasts, coliforms and the total aerobic flora among the two date pastes. Considerable interest has developed on the preservation of conventional date paste by the use of natural additives such asJuniperus phoenicea and olive oil (conditioned paste). At a given mixture ofJuniperus and olive oil and after pasteurisation, mould CFU’s were reduced by 95% (P <0.01) whereas the reduction was only 84% for the yeast populations, after 45 days at 25 °C.

Legume-nodulating bacteria (LNB) from three pasture legumes ( Vicia sativa , Trigonella maritima and Hedysarum spinosissimum ) in Tunisia

Sixty-one bacterial isolates were recovered from surface-sterilized root nodules of Vicia sativa, Trigonella maritima and Hedysarum spinosissimum plants growing in two arid Tunisian soils. The natural nodulation resource of these legumes, prospected from the two sites, was investigated. The occurrence of nodulation and the morphology of the nodules were observed. The isolates were examined by phenotypic characterization and 16S rDNA analysis. Among the 61 isolates that were screened, the majority (92%) were fast-growing rhizobia. Twenty-eight strains tolerated high concentration of salt (3% NaCl) and grew at temperatures up to 40°C. PCR restriction fragment length polymorphism (PCR-RFLP) and 16S rRNA gene sequencing revealed that the majority of the isolates belonged to the genera Rhizobium (54%) and Sinorhizobium (42%). Five H. spinosissimum isolates failed to nodulate their host plant, and were affiliated to Pseudomonas and Kocuria genera. This study is the first report that describes bacteria of genus Kocuria occupying root nodules of legumes to the best of our knowledge.

Members of Microvirga and Bradyrhizobium genera are native endosymbiotic bacteria nodulating Lupinus luteus in Northern Tunisian soils

ABSTRACT The genetic diversity of bacterial populations nodulating Lupinus luteus (yellow lupine) in Northern Tunisia was examined. Phylogenetic analyses of 43 isolates based on recA and gyrB partial sequences grouped them in three clusters, two of which belong to genus Bradyrhizobium (41 isolates) and one, remarkably, to Microvirga (2 isolates), a genus never previously described as microsymbiont of this lupine species. Representatives of the three clusters were analysed in‐depth by multilocus sequence analysis of five housekeeping genes (rrs, recA, glnII, gyrB and dnaK). Surprisingly, the Bradyrhizobium cluster with the two isolates LluI4 and LluTb2 may constitute a new species defined by a separate position between Bradyrhizobium manausense and B. denitrificans. A nodC‐based phylogeny identified only two groups: one formed by Bradyrhizobium strains included in the symbiovar genistearum and the other by the Microvirga strains. Symbiotic behaviour of representative isolates was tested, and among the seven legumes inoculated only a difference was observed i.e. the Bradyrhizobium strains nodulated Ornithopus compressus unlike the two strains of Microvirga. On the basis of these data, we conclude that L. luteus root nodule symbionts in Northern Tunisia are mostly strains within the B. canariense/B. lupini lineages, and the remaining strains belong to two groups not previously identified as L. luteus endosymbionts: one corresponding to a new clade of Bradyrhizobium and the other to the genus Microvirga. &NA; Graphical Abstract Figure. Our results show the diversity of Lupinus luteus root nodule symbionts in Northern Tunisia, where most of the strains are within the Bradyrhizobium canariense/B. lupini lineages, widely described as microsymbionts of Old World Lupinus; and the remaining strains belong to two minority groups never identified as L. luteus endosymbionts, one corresponding to a new clade of Bradyrhizobium and the other to the genus Microvirga.

Diverse Bacteria Affiliated with the Genera Microvirga, Phyllobacterium, and Bradyrhizobium Nodulate Lupinus micranthus Growing in Soils of Northern Tunisia.

ABSTRACT The genetic diversity of bacterial populations nodulating Lupinus micranthus in five geographical sites from northern Tunisia was examined. Phylogenetic analyses of 50 isolates based on partial sequences of recA and gyrB grouped strains into seven clusters, five of which belong to the genus Bradyrhizobium (28 isolates), one to Phyllobacterium (2 isolates), and one, remarkably, to Microvirga (20 isolates). The largest Bradyrhizobium cluster (17 isolates) grouped with the B. lupini species, and the other five clusters were close to different recently defined Bradyrhizobium species. Isolates close to Microvirga were obtained from nodules of plants from four of the five sites sampled. We carried out an in-depth phylogenetic study with representatives of the seven clusters using sequences from housekeeping genes (rrs, recA, glnII, gyrB, and dnaK) and obtained consistent results. A phylogeny based on the sequence of the symbiotic gene nodC identified four groups, three formed by Bradyrhizobium isolates and one by the Microvirga and Phyllobacterium isolates. Symbiotic behaviors of the representative strains were tested, and some congruence between symbiovars and symbiotic performance was observed. These data indicate a remarkable diversity of L. micranthus root nodule symbionts in northern Tunisia, including strains from the Bradyrhizobiaceae, Methylobacteriaceae, and Phyllobacteriaceae families, in contrast with those of the rhizobial populations nodulating lupines in the Old World, including L. micranthus from other Mediterranean areas, which are nodulated mostly by Bradyrhizobium strains. IMPORTANCE Lupinus micranthus is a legume broadly distributed in the Mediterranean region and plays an important role in soil fertility and vegetation coverage by fixing nitrogen and solubilizing phosphate in semiarid areas. Direct sowing to extend the distribution of this indigenous legume can contribute to the prevention of soil erosion in pre-Saharan lands of Tunisia. However, rhizobial populations associated with L. micranthus are poorly understood. In this context, the diversity of endosymbionts of this legume was investigated. Most Lupinus species are nodulated by Bradyrhizobium strains. This work showed that about half of the isolates from northern Tunisian soils were in fact Bradyrhizobium symbionts, but the other half were found unexpectedly to be bacteria within the genera Microvirga and Phyllobacterium. These unusual endosymbionts may have a great ecological relevance. Inoculation with the appropriate selected symbiotic bacterial partners will increase L. micranthus survival with consequent advantages for the environment in semiarid areas of Tunisia.

Definition of two new symbiovars, sv. lupini and sv. mediterranense , within the genera Bradyrhizobium and Phyllobacterium efficiently nodulating Lupinus micranthus in Tunisia

In this study, a polyphasic approach was used to analyze three representative strains (LmiH4, LmiM2 and LmiT21) from a collection of six previously described strains isolated in Tunisia from root nodules of Lupinus micranthus. The phylogenetic analysis of the concatenated rrs, recA and glnII genes showed that strain LmiH4 had 100% concatenated gene sequence identity with the type strain Bradyrhizobium retamae Ro19T. Similarly, strain LmiM2 shared 100% concatenated gene sequence identity with the species Bradyrhizobium valentinum LmjM3T. However, strain LmiT21 showed an identical concatenated gene sequence with reference strain Phyllobacterium sophorae CCBAU03422T. The recA-glnII concatenated protein-coding genes used produced incongruent phylogenies compared with 16S rDNA phylogeny. The nodC gene analysis showed that the strains were phylogenetically divergent to the Bradyrhizobium symbiovars defined to date, and represented two new symbiovars. Plant infection analysis revealed that the three strains showed moderate host range and symbiotic specificities. Based on their symbiotic characteristics, we propose that the three strains isolated from Lupinus micranthus nodules belong to two new symbiovars, with the first denominated lupini within the two species Bradyrhizobium valentinum (type strain LmiM2) and B. retamae (type strain LmiH4), and the second denominated mediterranense within the species P. sophorae (type strain LmiT21).

Characterization of Rhizobial Bacteria Nodulating Astragalus corrugatus and Hippocrepis areolata in Tunisian Arid Soils

Fifty seven bacterial isolates from root nodules of two spontaneous legumes (Astragalus corrugatus and Hippocrepis areolata) growing in the arid areas of Tunisia were characterized by phenotypic features, 16S rDNA PCR-RFLP and 16S rRNA gene sequencing. Phenotypically, our results indicate that A. corrugatus and H. areolata isolates showed heterogenic responses to the different phenotypic features. All isolates were acid producers, fast growers and all of them used different compounds as sole carbon and nitrogen source. The majority of isolate grew at pHs between 6 and 9, at temperatures up to 40°C and tolerated 3% NaCl concentrations. Phylogenetically, the new isolates were affiliated to four genera Sinorhizobium, Rhizobium, Mesorhizobium and Agrobacterium. About 73% of the isolates were species within the genera Sinorhizobium and Rhizobium. The isolates which failed to nodulate their host plants of origin were associated to Agrobacterium genus (three isolates).