Aims: To determine the biodiversity of rhizobial strains nodulating Cicer arietinum L. in representative soils from various areas of Morocco. Methods and Results: Symbiotic traits, utilization of 49 carbohydrate sources, resistance to antibiotics and heavy metals, tolerance to salinity, to extreme temperatures and pH were studied as phenotypic markers. In addition, restriction fragment length polymorphism (RFLP) of PCR-amplified 16S rDNAs were compared with those of reference strains. Numerical analysis of the phenotypic characteristics showed that the 48 strains studied fell into three distinct groups. RFLP analysis of 16S rRNA genes revealed an additional heterogeneity and four ribotypes were identified.

Phenotypic characteristics of fifty-six rhizobia strains isolated from root nodules of two chickpea (Cicer arietinum L.) cultivars, growing in soils collected from different areas of Morocco, were studied. Tolerance to salinity, high temperatures, acid and alkaline pHs, heavy metals and to antibiotics as well as symbiotic and cultural characteristics allowed the description of a wide physiological diversity among tested isolates. Numerical analysis of the phenotypic characteristics showed that, below the boundary level of 82% average similarity, isolates fell into at least five distinct groups. A number of potential isolates have been identified for inoculation trials. They were effective and able to grow at pH ranging from 5 to 8, tolerated salt concentration (1-1.5%) and grew at a maximum temperature between 30 and 35 o C. biodiversity / Cicer arietinum / phenotypic characterization / Rhizobium Résumé-Caractérisation phénotypique des rhizobia isolés du pois chiche (Cicer arietinum) cultivé sur les sols marocains. Cinquante six souches de rhizobium nodulant deux cultivars de pois chiche (Cicer arietinum L.) et isolées de différentes régions du Maroc, ont été caractérisées sur le plan phénotypique. L'analyse de leur tolérance à la salinité, aux températures élevées, aux pH acides et alcalins, aux antibiotiques, aux métaux lourds ainsi que leurs caractéristiques symbiotiques et culturales ont permis de mettre en évidence une large diversité physiologique au sein de ces populations de rhizobium nodulant le pois chiche. L'analyse numérique de ces caractéristiques phénotypiques a montré qu'à un niveau de 82 % de similarité, ces souches bactériennes se sont réparties en cinq groupes distincts. Des souches intéressantes pour les essais d'inoculation ont été identifiées. Elles sont efficientes et capables de tolérer des pHs allant de 5 à 8, des concentrations en NaCl de 1 à 1,5 % et des températures maximales comprises entre 30 à 35 o C. biodiversité / caractérisation phénotypique / pois chiche / Rhizobium Communicated by Jean-Jacques Drevon (Montpellier, France)

Cicer arietinum (chickpea) is a legume very sensitive to salinity, and so are most of its rhizobial symbionts belonging to the species Mesorhizobium ciceri. We observed that exogenous trehalose (i.e., added to the growth medium) can significantly improve growth of M. ciceri strain Rch125 under moderate salinity. In order to test if endogenous trehalose (i.e., synthesized by the cell) could also enhance salt tolerance, strain Rch125 was genetically modified with various trehalose biosynthesis genes from Sinorhizobium meliloti 1021 (otsA, treS, treY) and Mesorhizobium loti MAFF 303099 (otsAB). We found that overexpression of otsA or otsAB, but not treS or treY, significantly improved M. ciceri Rch125 growth in saline media. This growth improvement correlated with enhanced trehalose accumulation in otsA-and otsABmodified cells, suggesting that increased trehalose synthesis via trehalose-6-phosphate can enhance bacterial salt tolerance. Chickpea plants inoculated with M. ciceri Rch125 derivatives carrying extra otsAB or otsA genes formed more nodules and accumulated more shoot biomass than wild type inoculated plants when grown in the presence of NaCl. These results support the notion that improved salt tolerance of the bacterial symbiont can alleviate the negative effects of salinity on chickpeas, and that such improvement in M. ciceri can be achieved by manipulating trehalose metabolism.

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SUMMARY. Diversity analysis of moroccan carob (Ceratonia siliqua L.) accessions using phenotypic traits and RAPD markers. The carob (Ceratonia siliqua L.) is a perennial leguminous (Caesalpinioideae) that grows as an evergreen shrub or tree. It’s an important component of the Mediterranean vegetation and its adaptation in marginal soils of the Mediterranean regions is important environmentally and economically. Phenotypic and genetic diversity among 10 Ceratonia siliqua accessions coming from different areas of Morocco were studied with morphometric and RAPD approaches. The analysis of discriminative fruit characters showed highly significant differences among these accessions, the closely related ones have a similarity level less than 65%. No molecular studies have been carried out so far on Ceratonia siliqua. To provide markers useful for molecular diversity study of the carob tree, genomic DNA extraction and amplification conditions were finalized and 67 arbitrary primers were screened. 52 of them showed clearly reproducible banding patterns. The analysis of RAPD profiles revealed a high degree of genetic diversity within these carob accessions and enabled each of them to be uniquely fingerprinted. Overall, in our study we note that morphological relationship between carob accessions is deeply different to their molecular relationship. Concerning the distribution of the accessions according to their geographical origin, clustering based on RAPD data revealed a rough distribution of theses accessions. Indeed, Sidi Bou Ottman, Demnate and Essaouira accessions coming from the south of Morocco seem to be linked in the PCA plot. However, two geographically distant accessions, Aïn Sfa and Ouazzane coming from the north (about 700 Km) were clustered with Essaouira and Demanate - Sidi Bou Ottman, respectively.Key words. Carob, Ceratonia siliqua L., genetic variability, morphometry, RAPD.RÉSUMÉ. Analyse de la diversite des accessions du caroubier marocain (Ceratonia siliqua L.) utilisant des traits phenotypiques et des marqueurs RAPD. Le caroubier (Ceratonia siliqua L.) est une légumineuse pérenne (Caesalpinioideae), à feuillage persistant et pouvant croître entant qu’arbrisseau ou arbre. C’est un composant important de la végétation méditerranéenne et son adaptation aux sols marginaux des régions méditerranéennes est d’une grande importance écologique et économique. La diversité phénotypique et génétique au sein de 10 accessions de caroubier provenant de différentes régions du Maroc a été étudiée par des approches morphométriques et moléculaires (RAPD). L’analyse des caractères morphologiques discriminants du fruit a montré des différences hautement significatives au sein des accessions, puisque les apparentées d’entre elles n’ont approximativement que 65% de similarité. Jusqu’à nos jours, aucune étude moléculaire n’a été entreprise sur Ceratonia siliqua. Pour mettre à disposition des marqueurs utiles pour l’étude de la diversité moléculaire du caroubier, l’extraction de l’ADN génomique et les conditions de l’amplification ont été mises au point et 67 amorces arbitraires ont été criblées. 52 d’entre elles ont montré clairement des profils de bandes reproductibles. L’analyse des profils RAPD a révélé une diversité génétique de haut niveau entre les accessions de caroubier ce qui a permis à chacune d’elles d’être distinguée génétiquement de façon singulière. De manière générale, nous avons noté que la relation morphologique entre les accessions du caroubier est profondément différente de leur relation moléculaire. Concernant la distribution des accessions selon leur origine géographique, le regroupement basé sur les données de la RAPD a révélé une distribution grossière. En effet, les provenances de Sidi Bou Ottman, Demnate et Essaouira, originaires du sud de Maroc, semblent être liés par l’analyse des composantes principales (PCA). Cependant, deux accessions géographiquement distantes, Aïn Sfa et Ouazzane, originaires du Nord (approximativement 700 Kms) ont été liées avec Essaouira et Demanate - Sidi Bou Ottman, respectivement.Mots clés. Caroubier, Ceratonia siliqua L., diversité génétique, morphométrie, RAPD.