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.The authors want to thank all the persons who contributed to the achievement of this work. This work was funded by the Hassan II Academy of Science and Technology (AH2ST), in Morocco. B.M. received a grant from the AH2ST. She received also the excellency fellowship Khadija Baddouri in life sciences, from the Horizons foundation, Franc

Germination, growth and nodulation of Trigonella foenum graecum (Fenu Greek) under salt stress

In this work, we analyzed the effects of salinity on seed germination, growth and nodulation of fenugreek plants. The germination of fenugreek seeds was not affected by salt concentrations lower than 140 mM. Two saline tolerant indigenous rhizobia strains were isolated from the root nodules of fenugreek grown in two different soils. The two rhizobial strains were tested for their ability to grow under high salt stress and then assessed for their ability to nodulate fenugreek grown under different salt stress levels. We have found that the two strains have differential sensitivity to high salt levels. The inoculation of fenugreek with these strains results in better plant growth under salt stress thanuninoculated plants. Twenty percent of plants inoculated with strains S9D or S3G survived in 175 mM NaCl, whereas all controls supplemented or not with nitrogen did not grow. Although plants survived in these salt concentrations, many phenotypic alterations were observed, such as stems short length, small number of leaves and reduced fresh weight. No fenugreek plant grew at salt concentrationshigher than 175 mM NaCl. Stem and root proteins content was also affected by salinity. However, plants inoculated with the two rhizobial strains were more tolerant to salt than controls. The strains infectivityas estimated by the plants nodules number was also reduced by salinity

Nodulation of Retama monosperma by Ensifer aridi in an Abandonned Lead Mine Soils in Eastern Morocco

Millions tons of lead and zinc wastes from the abandoned Touissit mine are stored in the open air as dikes in the vicinity of the villages in Eastern Morocco and pose a real danger to both the environment and local populations. To prevent the movement of minerals to the nearby villages and limit the damages to the environment and health, we proposed the nitrogen-fixing leguminous shrub Retama monosperma, as a model plant to use for phytostabilization experimentations. This plant species is known by its ability to grow in hard climatic conditions and in heavy metals contaminated soils. The isolation of bacterial strains nodulating R. monosperma in the abandoned mine soils will permit the selection of rhizobia to inoculate young plant seedlings before their use for the phytostabilization of the mine tailings. In this work, 44 bacteria were isolated from the root nodules of R. Monosperma grown in the Touissit abandoned mine. Twenty-four isolates were considered as true rhizobia as they possess a copy of the nodC symbiotic gene and were able to renodulate their original host. The phenotypic characterization showed that all the strains are tolerant in vitro to different concentrations of heavy metals. The analysis of the 16S rRNA sequences of two selected representative strains showed they were related to different strains of Ensifer aridi isolated from different legumes in three continents deserts. The glnII, recA, and gyrB housekeeping genes analysis confirmed the affiliation of the strains to E. aridi. Moreover, the phylogenic analysis of nodA, nodC, and nifH symbiotic genes showed that the strains are more related to E. aridi JNVUTP6 species isolated from Tephrosia purpurea root nodules in the Thar Desert in India. To our knowledge, this is the first report about the isolation of E. aridi from R. monosperma root nodules.HL was granted a fellowship from the Moroccan Ministry of National Education, Vocational Training, Higher Education, and Scientific Research within the frame of the “Priority projects for scientific research and technological development, PPR2”. The Hassan II Academy of Science and Technology participated also to the funding of this work.Peer Reviewe

Identification of the endosymbionts from Sulla spinosissima growing in a lead mine tailings in Eastern Morocco as Mesorhizobium camelthorni sv. aridi

Aims: To identify the bacteria nodulating Sulla spinosissima growing profuselyin a lead and zinc mine tailings in Eastern Morocco. Methods and Results: In all, 32 rhizobial cultures, isolated from root nodules of S. spinosissima growing in soils of the mining site, were tolerant to different heavy metals. The ERIC-polymerase chain reaction (PCR) finger printing analysis clustered the isolates into seven different groups, and the analysis of the 16SrRNA sequences of four selected representative strains, showed they were related to different species of the genus Mesorhizobium.TheatpD, glnII and rec Ahousekeeping genes analysis confirmed the affiliation of the four representativestrains to Mesorhizobium camelthorni CCNWXJ40-4T, with similarity percentages varying from 96.30 to 98.30%. The sequences of the nifH gene had 97.33–97.78% similarities with that of M. camelthorni CCNWXJ40-4T; however, the nodC phylogeny of the four strains diverged from the type and other reference strains of M. camelthorni and formed a separated cluster.The four strains nodulate also Astragalus gombiformis and A. armatus but did not nodulate A. boeticus, Vachellia gummifera, Prosopis chilensis, Cicer arietinum, Lens culinaris, Medicago truncatula, Lupinus luteus or Phaseolus vulgaris. Conclusions: Based on similarities of the nodC symbiotic gene and differences in the host range, the strains isolated from S. spinosissima growing in soils ofthe Sidi Boubker mining site may form a different symbiovar within Mesorhizobium for which the name aridi is proposed. Significance and Impact of the Study: In this work, we show that strains ofM. camelthorni species nodulating S. spinosissima in the arid area of Eastern Morocco constitute a distinct phylogenetic clade of nodulation genes; wenamed symbiovar aridi, which encompasses also mesorhizobia from other Mediterranean desert legumes.The authors thank the Academy Hassan II of Science andTechnology and the Moroccan Ministry of National Education, Vocational Training, Higher Education and Scientific Research for financial suppor

Characterization of Bradyrhizobium spp. nodulating Lupinus cosentinii and L. luteus microsymbionts in Morocco

In this work, we analyzed the diversity of the nodule-forming bacteria associated with Lupinus luteus and Lupinus cosentinii grown in the Maamora Cork oak forest acidic soils in Morocco. The phenotypic analysis showed the high diversity of the strains nodulating the two lupine's species. The strains were not tolerant to acidity or high alkalinity. They do not tolerate salinity or high temperatures either. The strains isolated from L. luteus were more tolerant to antibiotics and salinity than those isolated from L. cosentinii. The plant growth promoting (PGP) activities of our strains are modest, as among the 28 tested isolates, only six produced auxins, six produced siderophores, whereas three solubilized phosphates. Only two strains possess the three activities. The rrs gene sequences from eight representative strains selected following ARDRA and REP-PCR results revealed that they were members of the genus Bradyrhizobium. Six strains were then retained for further molecular analysis. The glnII, recA, gyrB, dnaK, and rpoB housekeeping gene sequence phylogeny showed that some strains were close to B. lupini LMG28514 whereas others may constitute new genospecies in the genus Bradyrhizobium. The strains were unable to nodulate Glycine max and Phaseolus vulgaris and effectively nodulated L. luteus, L. cosentinii, L. angustifolius, Chamaecytisus albidus, and Retama monosperma. The nodC and nodA symbiotic gene phylogenies showed that the strains are members of the genistearum symbiovar

Astragalus algarbiensis is nodulated by the genistearum symbiovar of Bradyrhizobium spp. in Morocco

Astragalus algarbiensis is a wild herbaceous legume growing in Maamora, the most important cork oak forest in northern Africa. It is a plant of great importance as fodder in silvopastoral systems, and in the restoration of poor and degraded soils. The purpose of this study was to describe the biodiversity of rhizobia nodulating this plant and determine their identity. Out of 80 bacterial isolates, 56 strains isolated from root nodules of A. algarbiensis were characterized. ERIC-PCR fingerprinting grouped the strains in two main clusters containing 29 and 27 isolates, respectively, and the amplified ribosomal DNA restriction analysis (ARDRA) generated two different ribotypes. Based on both the ERIC-PCR and ARDRA results, representative strains As21 and As36 were selected for further genetic studies. The nearly complete 16S rRNA gene sequences of As21 and As36 showed that they were closely related to Bradyrhizobium cytisi CTAW11 with similarity values of 99.84% and 99.77%, respectively. Concatenation of atpD, recA, gyrB and dnaK housekeeping gene sequences indicated that strains As21 and As36 had a 95.22% similarity but they showed values of 95.80% and 94.97% with B. cytisi CTAW11, respectively. The sequencing of the symbiotic nodC gene of the two strains revealed 97.20% and 97.76% identities, respectively, with that of B. cytisi CTAW11 isolated from Cytisus villosus growing in the Moroccan Rif Mountains. Furthermore, the phylogenic analysis showed that the strains isolated from A. algarbiensis clustered with B. cytisi and B. rifense within the bradyrhizobia genistearum symbiovar and may constitute two novel genospecies.The authors thank the Hassan II Academy of Sciences, as well the Moroccan Ministry of National Education, Vocational Training, Higher Education and Scientific Research for the financial support of this work. This study was also supported by the ERDF-cofinanced grant PEAGR2012-1968 from the Consejería de Economía, Innovación y Ciencia (Junta de Andalucía, Spain)

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.This work was supported by the Academy Hassan II of Sciences and Techniques in Morocco as well as the Ministry of Higher Education, Scientific Research and Vocational Training of Morocco

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 RST89 and B. valentinum LmjM3 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 authors want to thank the Hassan II Academy of Sciences for funding this research in the frame of the BIOMICS project, as well as the ministry of National Education, Vocational training, Higher Education and Scientific Research in the frame of PPR2-BIOMIVER project. Financial support was also obtained from the ERDF-cofinanced project AGL2017–85676R from Ministerio de Economía, Industria y Competitividad (Spain)

Microvirga sp. symbiovar mediterranense nodulates Lupinus cosentinii grown wild in Morocco

Aim: To analyse the diversity of nodule-forming bacteria isolated from Lupinus cosentinii naturally grown in the Maamora cork oak forest (Rabat, Morocco). Methods and Results: Of the 31 bacterial strains, four were selected based on their REP-PCR fingerprinting that were studied by sequencing and phylogenetic analysis of their 16S rRNA, gyrB, dnaK, recA and rpoB housekeeping genes as well as the nodC symbiotic gene. The nearly complete 16S rRNA gene sequence of the four representative strains showed that they are related to Tunisian strains of genus Microvirga isolated from L. micranthus with nucleotide identity values ranging from 98·67 to 97·13%. The single and concatenated sequences of the 16S rRNA, gyrB, dnaK, recA and rpoB housekeeping genes indicated that the L. cosentinii-isolated strains had 99·2–99·9% similarities with the Tunisian L. micranthus microsymbionts. The nodC gene phylogeny revealed that the Moroccan strains clustered in the newly described mediterranense symbiovar, and nodulation tests showed that they nodulated not only L. cosentinii but also L. angustifolius, L. luteus and L. albus. Conclusions: To the best of our knowledge, this is the first report concerning the isolation, molecular identification and phylogenetic diversity of L. cosentinii nodule-forming endosymbionts and of their description as members of the Microvirga genus. Significance and Impact of the Study: In this work, we show that Microvirga sp. can be isolated from root nodules of wild-grown L. cosentinii in Northeast Africa, that selected strains also nodulate L. angustifolius, L. luteus and L. albus, and that they belong to symbiovar mediterranense. In addition, our data support that the ability of Microvirga to nodulate lupines could be related to the soil pH, its geographical distribution being more widespread than expected.This project was supported by the Hassan II Academy of Sciences and Techniques (Morocco). Support was also obtained from the Moroccan Ministry of National Education, Vocational Training, Higher Education and Scientific Research within the frame of the ‘Priority projects for scientific research and technological development, PPR2’. Financial support was also obtained from the ERDF-cofinanced project PEAGR2012-1968 granted by Consejería de Economía, Innovacion y Ciencia (Junta de Andalucía, Spain)