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.

Meta-analysis of Arabidopsis thaliana microarray data in relation to heat stress response.

Introduction: Increasing global warming has made heat stress a serious threat to crop productivity and global food security in recent years. One of the most promising solutions to address this issue is developing heat-stress-tolerant plants. Hence, a thorough understanding of heat stress response mechanisms, particularly molecular ones, is crucial. Methods: Although numerous studies have used microarray expression profiling technology to explore this area, these experiments often face limitations, leading to inconsistent results. To overcome these limitations, a random effects meta-analysis was employed using advanced statistical methods. A meta-analysis of 16 microarray datasets related to heat stress response in Arabidopsis thaliana was conducted. Results: The analysis revealed 1,972 significant differentially expressed genes between control and heat-stressed plants (826 over-expressed and 1,146 down-expressed), including 128 differentially expressed transcription factors from different families. The most significantly enriched biological processes, molecular functions, and KEGG pathways for over-expressed genes included heat response, mRNA splicing via spliceosome pathways, unfolded protein binding, and heat shock protein binding. Conversely, for down-expressed genes, the most significantly enriched categories included cell wall organization or biogenesis, protein phosphorylation, transmembrane transporter activity, ion transmembrane transporter, biosynthesis of secondary metabolites, and metabolic pathways. Discussion: Through our comprehensive meta-analysis of heat stress transcriptomics, we have identified pivotal genes integral to the heat stress response, offering profound insights into the molecular mechanisms by which plants counteract such stressors. Our findings elucidate that heat stress influences gene expression both at the transcriptional phase and post-transcriptionally, thereby substantially augmenting our comprehension of plant adaptive strategies to heat stress.

Genotypic and symbiotic diversity studies of rhizobia nodulating Acacia saligna in Tunisia reveal two novel symbiovars within the Rhizobium leguminosarum complex and Bradyrhizobium.

Acacia saligna is an invasive alien species that has the ability to establish symbiotic relationships with rhizobia. In the present study, genotypic and symbiotic diversity of native rhizobia associated with A. saligna in Tunisia were studied. A total of 100 bacterial strains were selected and three different ribotypes were identified based on rrs PCR-RFLP analysis. Sequence analyses of rrs and four housekeeping genes (recA, atpD, gyrB and glnII) assigned 30 isolates to four putative new lineages and a single strain to Sinorhizobium meliloti. Thirteen slow-growing isolates representing the most dominant IGS (intergenic spacer) profile clustered distinctly from known rhizobia species within Bradyrhizobium with the closest related species being Bradyrhizobium shewense and Bradyrhizobium niftali, which had 95.17% and 95.1% sequence identity, respectively. Two slow-growing isolates, 1AS28L and 5AS6L, had B. frederekii as their closest species with a sequence identity of 95.2%, an indication that these strains could constitute a new lineage. Strains 1AS14I, 1AS12I and 6AS6 clustered distinctly from known rhizobia species but within the Rhizobium leguminosarum complex (Rlc) with the most closely related species being Rhizobium indicum with 96.3% sequence identity. Similarly, the remaining 11 strains showed 96.9 % and 97.2% similarity values with R. changzhiense and R. indicum, respectively. Based on nodC and nodA phylogenies and cross inoculation tests, these 14 strains of Rlc species clearly diverged from strains of Sinorhizobium and Rlc symbiovars, and formed a new symbiovar for which the name sv. "salignae" is proposed. Bacterial strains isolated in this study that were taxonomically assigned to Bradyrhizobium harbored different symbiotic genes and the data suggested a new symbiovar, for which sv. "cyanophyllae" is proposed. Isolates formed effective nodules on A. saligna.

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.

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.

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.

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.

Ensifer fredii symbiovar vachelliae nodulates endemic Vachellia gummifera in semiarid Moroccan areas.

The purpose of this work was to study the genetic diversity of the nodule-forming bacteria associated with native populations of Vachellia gummifera growing wild in Morocco. The nearly complete 16S rRNA gene sequences from three selected strains, following ARDRA and REP-PCR results, revealed they were members of the genus Ensifer and the sequencing of the housekeeping genes recA, gyrB, dnaK and rpoB, and their concatenated phylogenetic analysis, showed that the 3 strains belong to the species E. fredii. Based on the nodC and nodA phylogenies, the 3 strains clearly diverged from the type and other reference strains of E. fredii and formed a clearly separated cluster. The strains AGA1, AGA2 and AGB23 did not form nodules on Glycine max, Phaseolus vulgaris and Medicago truncatula, and effectively nodulated V. gummifera, Acacia cyanophylla, Prosopis chilensis and Leucaena leucocephala. Based on similarities of the nodC and nodA symbiotic genes and differences in the host range, the strains isolated from Moroccan endemic V. gummifera may form a different symbiovar within Ensifer species, for which the name "vachelliae" is proposed.

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 CTAW11T 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 CTAW11T, 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 CTAW11T 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.

Diversity of nodular bacteria of Scorpiurus muricatus in western Algeria and their impact on plant growth.

A total of 51 bacterial strains were isolated from root nodules of Scorpiurus muricatus sampled from 6 regions of western Algeria. Strain diversity was assessed by rep-PCR amplification fingerprinting, which grouped the isolates into 28 different clusters. Partial nucleotide sequencing of the 16S rRNA gene and BLAST analysis revealed that root nodules of S. muricatus were colonized by different species close to Rhizobium vignae, Rhizobium radiobacter, Rhizobium leguminosarum, Phyllobacterium ifriqiyense, Phyllobacterium endophyticum, Starkeya sp., and Pseudomonas sp. However, none of these strains was able to form nodules on its host plant; even nodC was present in a single strain (SMT8a). The inoculation test showed a great improvement in the growth of inoculated plants compared with noninoculated control plants. A significant amount of indole acetic acid was produced by some strains, but only 2 strains could solubilize phosphate. In this report we described for the first time the diversity of bacteria isolated from root nodules of S. muricatus growing in different regions in western Algeria and demonstrated their potential use in promoting plant growth.

Ensifer meliloti is the preferred symbiont of Medicago arborea in eastern Morocco soils.

Polyphasic characterization of 61 bacteria isolated from root nodules of Medicago arborea (Medic tree) plants growing in 4 arid soils of the arid eastern area of Morocco was studied. All the isolates characterized were fast growers. The phenotypic, symbiotic, and cultural characteristics analyzed allowed the description of a broad physiological diversity among the isolates. The results obtained suggest that the phenotype of these rhizobia might have evolved to adapt to the local conditions. The genetic characterization consisted of an analysis of the rep-PCR (repetitive extragenic palindromic polymerase chain reaction) fingerprints and a PCR-based RFLP (restriction fragment length polymorphism) of the 16S rDNA patterns. The diversity of the isolates was investigated by rep-PCR, giving a similarity of 62%, delineated into 3 clusters, 4 groups, and 6 subclusters. This wide diversity was also observed by a phenotypic approach, where the carbohydrate assimilation test was the most discriminating. The results show a relationship between rep-PCR fingerprinting and sugar assimilation, which are complementary in diversity investigation. The nearly complete 16S rRNA gene sequence from representative strains of each soil showed they are closely related to members of the genus Ensifer of the family Rhizobiaceae within the Alphaproteobacteria and shows the highest similitude values (99.93%/100%) with Ensifer meliloti LMG 6133(T) (X67222). Sequencing of the symbiotic nodC gene from 7 representative strains revealed they had 94.89% identity with the nodC sequence of the type strain E. meliloti LMG 6133(T) (EF428922). Therefore, the 61 M. arborea isolates from the 4 different soils have the same phylogenetic affiliation, which proves the restricted host specificity among M. arborea species.

Colutea arborescens is nodulated by diverse rhizobia in Eastern Morocco.

Eighteen isolates of rhizobia isolated from root nodules of Colutea arborescens (Bladder senna) grown in different soils of the eastern area of Morocco were characterized by phenotypic and genomic analyses. All the isolates characterized were fast growers. This is may be due to the isolation procedures used. The phenotypic, symbiotic and cultural characteristics analyzed allowed the description of a wide physiological diversity among tested isolates. The results obtained suggest that the phenotype of these rhizobia might have convergent evolved to adapt the local conditions. The genetic characterization consisted in an analysis of the rep-PCR fingerprints and the PCR-based RFLP of the 16S rDNA patterns. The 16S rDNA of six isolates representing the main ribotypes obtained by the PCR-based RFLP was sequenced. A large diversity was observed among these rhizobia, and they were classified as different species of the genera Rhizobium, Sinorhizobium and Mesorhizobium. The nodC gene was also sequenced, and the results confirmed the three lineages corresponding to the three genera. The results of the sequencing of nodC and 16S rDNA genes suggest that the nodulation genes and chromosome might have co-evolved among these bacteria.