Phenotypic, molecular, and symbiotic characterization of the rhizobial symbionts isolated from Acacia saligna grown in different regions in Morocco: a multivariate approach.

The introduced species Acacia saligna is a very promiscuous host as it can be efficiently nodulated with a wide range diversity of rhizobia taxa, including both fast and slow-growing strains. Fourteen nitrogen (N)-fixing bacteria were isolated from root nodules of wild Acacia saligna growing in distinct geographic locations in Morocco and were examined for their symbiotic efficiency and phenotypic properties. Multivariate tools, such as principal component analysis (PCA) and hierarchical clustering analysis (HCA), were used to study the correlation between phenotypic and symbiotic variables and discriminate and describe the similarities between different isolated bacteria with respect to all the phenotypic and symbiotic variables. Phenotypic characterization showed a variable response to extreme temperature, salinity and soil pH. At the plant level, the nodulation, nitrogen fixation, and the shoot and root dry weights were considered. The obtained results show that some of the tested isolates exhibit remarkable tolerances to the studied abiotic stresses while showing significant N2 fixation, indicating their usefulness as effective candidates for the inoculation of acacia trees. The PCA also allowed showing the isolates groups that present a similarity with evaluated phenotypic and symbiotic parameters. The genotypic identification of N2-fixing bacteria, carried out by the 16S rDNA approach, showed a variable genetic diversity among the 14 identified isolates, and their belonging to three different genera, namely Agrobacterium, Phyllobacterium and Rhizobium.

Simplex-centroid design as innovative approach in the optimization of antimicrobial effect of Thymus satureioides, Myrtus communis and Artemisia herba alba essential oils against Escherichia coli, Staphylococcus aureus and Candidatropicalis.

Essential Oils (EOs) known since Antiquity, and initially obtained by maceration of aromatic plants, are used as diseases' remedies because they contain valuable therapeutic components. Antimicrobial effect's evaluation of formulations established by an augmented centered mixing plan for three Moroccan medicinal and aromatic plants' essential oils (EOs): Thymus satureioides (T. satureioides), Myrtus communis (M. communis) and Artemisia herba alba (A. herba alba) against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Candida tropicalis (C. tropicalis). The identification of E.Os chemical compounds was made using Gaz chromatography (GC) and Gaz chromatography coupled to Mass spectrometry (GC/MS).Whereas, the optimization of their antimicrobial effect was performed by experimental design methodology (mixture design) coupled to microdilution method based on Minimal Inhibitory Concentrations (MICs) determination. Results showed that the main components are borneol (34.26%) and α-pinene (31.21%); borneol (27.15%) and 1,8-cineole (21.33%); camphor (14.67%), chrysanthenone (14.10%), and ß-thujone (12.60%) for T. satureioides, M. communis and A. herba alba respectively. Optimal mixtures composed of 60% and 40% of T. satureioides and M. communis E.Os; 72% and 28% of T. satureioides and A. Herba alba E.Os; 75% of thyme E.O and 25% of mugwort E.O; predicted the highest antimicrobial effect against E. coli, S. aureus and C. tropicalis, respectively. This study highlights the antimicrobial effect of EOs tertiary mixtures through using mixture design methodology, which may contribute to a successful application in pharmaceutical, food, or cosmetic industries.

Screening and optimization of indole-3-acetic acid production and phosphate solubilization by rhizobacterial strains isolated from Acacia cyanophylla root nodules and their effects on its plant growth.

BACKGROUND: Plant growth-promoting rhizobacteria (PGPR) are known to improve plant growth and are used as biofertilizers, thanks to their numerous benefits to agriculture such as phosphorus solubilization and phytohormone production. In this paper, four rhizospheric bacteria (Phyllobacterium sp., Bacillus sp., Agrobacterium sp., and Rhizobium sp.) isolated from surface-sterilized root nodules of Acacia cyanophylla were tested for their ability to solubilize inorganic phosphate and to produce indole-3-acetic acid (IAA) under laboratory conditions. Then, the best IAA producer (Rhizobium sp.) was selected to test optimized conditions for IAA production. Finally, the effect of the four strains on plant growth for A. cyanophylla was evaluated in vivo. RESULTS: The results showed that the totality of the tested isolates had solubilized inorganic phosphate (P) in both NBRIP (National Botanical Research Institute Phosphate) and PVK (Pikovskaya) media. Bacillus sp. was a high P-solubilizer and showed maximum solubilization in PVK (519 µg ml-1) and NBRIP (782 µg ml-1). The optimization of maximum phosphate solubilization was done using different sources of carbon (1%) and nitrogen (0.1%). Glucose and ammonium sulfate were selected to be the best carbon and nitrogen source for phosphate solubilization by all tested strains, except for Phyllobacterium sp., which recorded the highest phosphate solubilization with ammonium nitrate. The IAA production by the tested strains indicated that Rhizobium sp. produced the highest amount of IAA (90.21 µg ml-1) in culture media supplemented with L-tryptophan. The best production was observed with L-Trp concentration of 0.2% (116.42 µg ml-1) and at an initial pH of 9 (116.07 µg ml-1). The effect of NaCl on IAA production was tested at concentrations of 0 to 5% and the maximum production of 89.43 µg ml-1 was found at 2% NaCl. The extraction of crude IAA from this strain was done and purity was confirmed with Thin Layer Chromatography (TLC) analysis. A specific spot from the extracted IAA production was found to correspond with a standard spot of IAA with the same Rf value. Finally, the tested PGPR demonstrated growth stimulatory effects on Acacia cyanophylla seedlings in vivo, with a great increase of shoots' and roots' dry weights, and shoot length compared to control. The rhizobacterial isolates were identified by 16S rDNA sequence analysis as Agrobacterium sp. NA11001, Phyllobacterium sp. C65, Bacillus sp. CS14, and Rhizobium sp. V3E1. CONCLUSION: This study highlights the importance of the use of phosphate solubilizing and IAA producer microorganisms as biofertilizers to increase crop yields. The studied strains showed a significant phosphate solubilization potential and IAA production. The use of selected strains as inoculants would be interesting, in particular with a view of promoting sustainable agriculture. However, further studies to verify the efficacy of the best isolates in situ is certainly required.

Screening and optimization of indole-3-acetic acid production by Rhizobium sp. strain using response surface methodology.

BACKGROUND: The production of indole-3-acetic acid (IAA) is an essential tool for rhizobacteria to stimulate and facilitate plant growth. For this, eighty rhizobial bacteria isolated from root nodules of Acacia cyanophylla grown in different regions of Morocco were firstly screened for their ability to produce IAA. Then, IAA production by a combination of isolates and the inoculation effect on the germination of Acacia cyanophylla seeds was studied using the best performing isolates in terms of IAA production. The best IAA producer bacterial isolate (I69) was selected to optimize IAA production using response surface methodology based on the central composite design. RESULTS: Results showed that the majority of tested isolates were able to produce IAA with a relatively higher concentration of 135 µg/ml for the isolate I69, followed by isolates I22 and I75 with respective concentrations of 116 µg/ml and 105 µg/ml IAA. The IAA production and the seed germination rate were relatively increased by the synergistic effect of I69 and I22. Later, response surface methodology was used to determine optimal operating conditions leading to IAA production optimization. Thus, an incubation temperature of 36 °C, a pH of 6.5, an incubation time of 1 day, and respective tryptophan and NaCl concentrations of 1 g/l and 0.1 g/l were optimal parameters leading to 166 µg/ml IAA which was the maximal produced concentration. CONCLUSION: The present study highlighted that IAA-producing rhizobacteria could be harnessed to improve plant growth. Furthermore, their production can be easily controlled using response surface methodology, which represents a very useful tool for optimization.

Combined treatment of Thymus vulgaris L., Rosmarinus officinalis L. and Myrtus communis L. essential oils against Salmonella typhimurium: Optimization of antibacterial activity by mixture design methodology.

To increase the sensibility of Salmonella typhimurium strain, a mixture of Thymus vulgaris L. (T. vulgaris L.), Rosmarinus officinalis L. (R. officinalis L.) and Myrtus communis L. (M. communis L.) essential oils (EOs) was used in combined treatment by experimental design methodology (mixture design). The chemical composition of EOs was firstly identified by GC and GC/MS and their antibacterial activity was evaluated. The results of this first step have shown that thymol and borneol were the major compounds in T. vulgaris and M. communis L. EOs, respectively, while 1,8-cineole and α-pinene were found as major compounds in R. officinalis L. The same results have shown a strong antibacterial activity of T. vulgaris L. EO followed by an important power of M. communis L. EO against a moderate activity of R. officinalis L. EO. Besides, 1/20 (v/v) was the concentration giving a strain response classified as sensitive. From this concentration, the mixture design was performed and analyzed. The optimization of mixtures antibacterial activities has highlighted the synergistic effect between T. vulgaris L. and M. communis L. essential oils. A formulation comprising 55% of T. vulgaris L. and 45% of M. communis L. essential oils, respectively, can be considered for the increase of Salmonella typhimurium sensibility.

Chemometric investigation of light-shade effects on essential oil yield and morphology of Moroccan Myrtus communis L.

BACKGROUND: To investigate the effect of environmental factors such as light and shade on essential oil yield and morphological traits of Moroccan Myrtus communis, a chemometric study was conducted on 20 individuals growing under two contrasting light environments. RESULTS: The study of individual's parameters by principal component analysis has shown that essential oil yield, altitude, and leaves thickness were positively correlated between them and negatively correlated with plants height, leaves length and leaves width. Principal component analysis and hierarchical cluster analysis have also shown that the individuals of each sampling site were grouped separately. The one-way ANOVA test has confirmed the effect of light and shade on essential oil yield and morphological parameters by showing a statistically significant difference between them from the shaded side to the sunny one. Finally, the multiple linear model containing main, interaction and quadratic terms was chosen for the modeling of essential oil yield in terms of morphological parameters. CONCLUSIONS: Sun plants have a small height, small leaves length and width, but they are thicker and richer in essential oil than shade plants which have shown almost the opposite. The highlighted multiple linear model can be used to predict essential oil yield in the studied area.