Plant growth promoting activities of Pseudomonas sp. and Enterobacter sp. isolated from the rhizosphere of Vachellia gummifera in Morocco.

The Moroccan endemic Vachellia gummifera grows wild under extreme desert conditions. This plant could be used as an alternative fodder for goats, and camels, in order to protect the Argan forests against overgrazing in Central and Southwestern Moroccan semiarid areas. With the aim to improve the V. gummifera population's density in semiarid areas, we proposed its inoculation with performing plant growth-promoting bacteria. Hence, 500 bacteria were isolated from the plant rhizosphere. From these, 291 isolates were retained for plant growth-promoting (PGP) activities assessment. A total of 44 isolates showed the best phosphates solubilization potential, as well as siderophore and auxin production. The combination of REP-PCR (repetitive extragenic palindromic-polymerase chain reaction) fingerprinting, PGP activities, and phenotypic properties, allowed the selection of three strains for the inoculation experiments. The three selected strains' 16S rRNA sequencing showed that they are members of the Enterobacter and Pseudomonas genera. The inoculation with three strains had diverse effects on V. gummifera growth parameters. All single and combined inoculations improved the plant shoot weight by more than 200%, and the root length by up to 139%, while some combinations further improved protein and chlorophyll content, thereby improving the plant's forage value. The three selected strains constitute an effective inoculum for use in the arid and semiarid zones of southern Morocco.

Different species of Bradyrhizobium from symbiovars genistearum and retamae nodulate the endemic Retama dasycarpa in the High Atlas Mountains.

Retama dasycarpa is an endemic Retama species native to the cold semi-arid bioclimates of the High Atlas Mountains in Morocco. In this work, we analyzed the diversity of the microsymbionts nodulating this plant and their different phenotypic and symbiotic characteristics. Phylogenetic analysis of the 16S rRNA gene revealed that the tested isolates clustered in the Bradyrhizobium genus. Multilocus sequence analyses of four housekeeping genes (recA, gyrB, glnII and atpD) for 12 selected strains grouped them into four clusters close to B. lupini USDA 3051T, B. frederickii CNPSo 3446T, B. valentinum LmjM3T and B. retamae Ro19T. The individual phylogenies of these core genes and the symbiotic genes nodC, nodA and nifH were congruent. These isolates showed a broad host range, being able to nodulate different legume hosts, such as R. sphaerocarpa, R. monosperma, Lupinus luteus, Cytisus grandiflorus andChamaecytisus albidus, but not Phaseolus vulgaris or Glycine max. They all had a similar metabolic capacity, using the majority of the carbohydrates and amino acids tested as sole sources of carbon and nitrogen. Furthermore, out of the 12 selected strains, some displayed plant growth-promoting features, with six of them solubilizing phosphate and three of them producing siderophores. The present work provides, for the first time, a detailed description about the microsymbionts associated with the endemic legume R. dasycarpa.

Characterization of plant growth-promoting bacteria isolated from the rhizosphere of Robinia pseudoacacia growing in metal-contaminated mine tailings in eastern Morocco.

BACKGROUND: Mining activity in the Touissit district of Eastern Morocco has led to an unprecedented accumulation of heavy metals, mainly lead and zinc, in the tailing ponds of the open-air mines. This poses a real danger to both the environment and local population. OBJECTIVES: The goal of this work was to characterize the Plant Growth Promoting Rhizobacteria (PGPR) isolated from the rhizosphere soil of R. pseudoacacia plants grown wild in the abandoned Pb- and Zn-contaminated tailing ponds in the mining district of Touissit, in Eastern Morocco. MAIN RESULTS: One hundred bacterial strains were isolated from the rhizosphere of black locust (Robinia pseudoacacia L.) plants growing naturally in the Touissit mine tailings. Quantitative determination of indole-acetic and siderophores production, inorganic phosphate solubilization, hydrolysis of 1-aminocyclopropane-1-carboxylic acid (ACC deaminase activity), and ability to act as a biocontrol agent allowed selection of the 3 strains, 7MBT, 17MBT and 84MBT with improved PGP properties. The three strains grew well in the presence of high concentration of Pb-acetate and ZnCl2; and the addition of Pb or Zn to the culture medium differently affected the PGP properties analyzed. NOVELTY STATEMENT: Inoculation of black locust grown with the 3 selected strains, in the presence 1000 µg ml-1 of Pb-acetate, produced varying effects on the plant dry weight. The strain 84MBT alone or in combination with strains 7MBT and 17MBT increased significantly the dry weight of the plants by 91, 62, and 85% respectively. The 16S rRNA gene sequence analysis of each strain showed that the strains 7MBT 17MBT and 84MBT had 99.34, 100, and had 99.72% similarity with Priestia endophytica (formerly B. endophyticus), B. pumilus NBRC 12092T, and B. halotolerans NBRC 15718T, respectively.

The Fodder Legume Chamaecytisus albidus Establishes Functional Symbiosis with Different Bradyrhizobial Symbiovars in Morocco.

In this work, we analyzed the symbiotic performance and diversity of rhizobial strains isolated from the endemic shrubby legume Chamaecytisus albidus grown in soils of three different agroforestry ecosystems representing arid and semi-arid forest areas in Morocco. The analysis of the rrs gene sequences from twenty-four representative strains selected after REP-PCR fingerprinting showed that all the strains belong to the genus Bradyrhizobium. Following multi-locus sequence analysis (MLSA) using the rrs, gyrB, recA, glnII, and rpoB housekeeping genes, five representative strains, CA20, CA61, CJ2, CB10, and CB61 were selected for further molecular studies. Phylogenetic analysis of the concatenated glnII, gyrB, recA, and rpoB genes showed that the strain CJ2 isolated from Sahel Doukkala soil is close to Bradyrhizobium canariense BTA-1 T (96.95%); that strains CA20 and CA61 isolated from the Amhach site are more related to Bradyrhizobium valentinum LmjM3T, with 96.40 and 94.57% similarity values; and that the strains CB10 and CB60 isolated from soil in the Bounaga site are more related to Bradyrhizobium murdochi CNPSo 4020 T and Bradyrhizobium. retamae Ro19T, with which they showed 95.45 and 97.34% similarity values, respectively. The phylogenetic analysis of the symbiotic genes showed that the strains belong to symbiovars lupini, genistearum, and retamae. All the five strains are able to nodulate Lupinus luteus, Retama monosperma, and Cytisus monspessilanus, but they do not nodulate Glycine max and Phaseolus vulgaris. The inoculation tests showed that the strains isolated from the 3 regions improve significantly the plant yield as compared to uninoculated plants. However, the strains of Bradyrhizobium sp. sv. retamae isolated from the site of Amhach were the most performing. The phenotypic analysis showed that the strains are able to use a wide range of carbohydrates and amino acids as sole carbon and nitrogen source. The strains isolated from the arid areas of Bounaga and Amhach were more tolerant to salinity and drought stress than strains isolated in the semi-arid area of Sahel Doukkala.

Characterization of Retama sphaerocarpa microsymbionts in Zaida lead mine tailings in the Moroccan middle Atlas.

In the Moroccan Middle Atlas, the tailings rich in lead and other metal residues, in the abandoned Zaida mining district, represent a real threat to environment and the neighboring villages' inhabitants' health. In this semi-arid to arid area, phytostabilisation would be the best choice to limit the transfer of heavy metals to populations and groundwater. The aim of this work was to characterize the bacteria that nodulate Retama sphaerocarpa, spontaneous nitrogen fixing shrubby legume, native to the Zaida mining area, with great potential to develop for phytostabilisation. Forty-three bacteria isolated from root nodules of the plant were characterized. Based on REP-PCR and ARDRA, four strains were selected for further molecular analyzes. The 16S rRNA gene sequences analysis revealed that the isolated strains are members of the genus Bradyrhizobium, and the phylogenetic analysis of the housekeeping genes glnII, atpD, gyrB, rpoB, recA and dnaK individual sequences and their concatenation showed that the strains are close to B. algeriense RST89T and B. valentinum LmjM3T with similarity percentages of 89.07% to 95.66% which suggest that the newly isolated strains from this mining site may belong to a potential novel species. The phylogeny of the nodA and nodC genes showed that the strains belong to the symbiovar retamae of the genus Bradyrhizobium. These strains nodulate also R. monosperma, R. dasycarpa and Lupinus luteus.

The endemic Chamaecytisus albidus is nodulated by symbiovar genistearum of Bradyrhizobium in the Moroccan Maamora Forest.

Out of 54 isolates from root nodules of the Moroccan-endemic Chamaecytisus albidus plants growing in soils from the Maamora cork oak forest, 44 isolates formed nodules when used to infect their original host plant. A phenotypic analysis showed the metabolic diversity of the strains that used different carbohydrates and amino acids as sole carbon and nitrogen sources. The isolates grew on media with pH values ranging from 6 to 8. However, they did not tolerate high temperatures or drought and they did not grow on media with salt concentrations higher than 85 mM. REP-PCR fingerprinting grouped the strains into 12 clusters, of which representative strains were selected for ARDRA and rrs analyses. The rrs gene sequence analysis indicated that all 12 strains were members of the genus Bradyrhizobium and their phylogeny showed that they were grouped into two different clusters. Two strains from each group were selected for multilocus sequence analysis (MLSA) using atpD, recA, gyrB and glnII housekeeping genes. The inferred phylogenetic trees confirmed that the strains clustered into two divergent clusters. Strains CM55 and CM57 were affiliated to the B. canariense/B. lupini group, whereas strains CM61 and CM64 were regrouped within the B. cytisi/B. rifense lineage. The analysis of the nodC symbiotic gene affiliated the strains to the symbiovar genistearum. The strains were also able to nodulate Retama monosperma, Lupinus luteus and Cytisus monspessulanus, but not Phaseolus vulgaris or Glycine max. Inoculation tests with C. albidus showed that some strains could be exploited as efficient inocula that could be used to improve plant growth in the Maamora forest.

Bradyrhizobium sp. sv. retamae nodulates Retama monosperma grown in a lead and zinc mine tailings in Eastern Morocco.

The aim of this work was to characterize and identify some bacteria isolated from the root nodules of Retama monosperma grown in Sidi Boubker lead and zinc mine tailings. Very few root nodules were obtained on the root nodules of R. monosperma grown in these soils. The three bacteria isolated from the root nodules were tolerant in vitro to different concentrations of heavy metals, including lead and zinc. The rep-PCR experiments showed that the three isolates have different molecular fingerprints and were considered as three different strains. The analysis of their 16S rRNA gene sequences proved their affiliation to the genus Bradyrhizobium. The analysis and phylogeny of the housekeeping genes atpD, glnII, gyrB, recA, and rpoB confirmed that the closest species was B. valentinum with similarity percentages of 95.61 to 95.82%. The three isolates recovered from the root nodules were slow-growing rhizobia capable to renodulate their original host plant in the presence of Pb-acetate. They were able to nodulate R. sphaerocarpa and Lupinus luteus also but not Glycine max or Phaseolus vulgaris. The phylogeny of the nodA and nodC nodulation genes as well as the nifH gene of the three strains showed that they belong to the symbiovar retamae of the genus Bradyrhizobium. The three strains isolated could be considered for use as inoculum for Retama plants before use in phytoremediation experiments.

Characterization of Pisum sativum and Vicia faba microsymbionts in Morocco and definition of symbiovar viciae in Rhizobium acidisoli.

In this work, we analyzed the diversity of seventy-six bacteria isolated from Pea and faba bean nodules in two regions of Morocco. The molecular diversity was realized using the analysis of the sequences of 16S rRNA and six housekeeping genes (recA, glnII, atpD, dnaK, rpoB and gyrB) and two symbiotic genes (nodA and nodC). The phylogeny of the 16S rRNA gene sequences revealed that all strains belong to the genus Rhizobium, being related to the type strains of R. leguminosarum, R. laguerreae, R. indigoferae, R. anhuiense and R. acidisoli. The housekeeping genes phylogenies showed that some strains formed a subclade distinct from the rhizobial species that usually nodulate Vicia faba and Pisum sativum which are closely related to R. acidisoli FH23 with sequence similarity of 98.3%. Analysis of the PGPR activities of the different isolates showed that the strains related to R. laguerreae were able to solubilize phosphates and to produce siderophores and auxin phytohormone. However, R. acidisoli strain F40D2 was unable to solubilize phosphates although they produce siderophores and IAA. The phylogenetic analysis of the nodA and nodC sequences showed that all isolated strains were closely related with the strains of symbiovar viciae. The nodulation tests confirmed the ability to nodulate V. faba and P. sativum but not Cicer arietinum or Phaseolus vulgaris. Hence, in Morocco P. sativum is nodulated by R. laguerreae; whereas V. faba is nodulated by R. laguerreae and the symbiovar viciae of R. acidisoli which has been not previously described in this species.