Phenotypic characteristics of root-nodulating bacteria isolated from Acacia spp. grown in Libya

Thirty isolates of root-nodulating bacteria obtained from Acacia cyanophylla, A. karroo, A. cyclops, A. tortilis (subsp.raddiana), Faidherbia albida and Acacia sp., grown in different regions of Libya, were studied by performing numerical analysis of 104 characteristics. Three fast- and one slow-growing reference strains from herbaceous and woody legumes were included. Five distinct clusters were formed. The fast-growing reference strains were separated from the isolates whereas the slow-growing was included in cluster 4. With the exception of one cluster, the majority of clusters were formed regardless of the host plant or site of origin. Based on plant tests, generation times, acid production and carbon utilization the isolates were diverse (fast and slow-growing isolates). Like slow-growing isolates, most of the fast-growing isolates appeared to be non-specific, nodulated many species from the same genus notably F. albida, known to nodulate only with slow-growing strains. Most clusters grew at temperatures 35 °C and 37 °C; some grew at temperatures above 40 °C. The majority of isolates grew at acid and alkaline pH and only one isolate grew below pH 4. Most isolates were able to utilize many amino acids as nitrogen sources and to reduce nitrate. Urea was hydrolysed by all clusters. Monosaccharides and polyols were used by slow and fast-growing isolates as the only carbon sources whereas assimilation of disaccharides varied: Some isolates, like slow-growing isolates, failed to utilize these carbon sources. Most isolates were unable to utilize polysaccharides. Regarding tolerance to NaCl on agar medium, the majority of isolates were unable to grow at a concentration of 2% NaCl, but some were highly resistant and there was one isolate which grew at 8% NaCl. Most isolates were resistant to heavy metals and to antibiotics.     

Diversity and spatial genetic structure of natural Moroccan <Emphasis Type="Italic">Quercus susber</Emphasis> L. assessed by ISSR markers for conservation

Morocco is one of the most important regions of the world in terms of Quercus suber L. number and variation. This species is in decline due to several factors, which can lead to permanent loss of this resource. It would be essential to evaluate the genetic diversity in order to conserve maximum genetic variability of this species. The genetic diversity and differentiation of 16 sites from five regions representing the entire range of Moroccan Cork Oak were assessed. Twenty-three ISSR primers used generated 985 polymorphic fragments with an average of 42.8 bands per primer and showed 100% of polymorphism. The 173 individuals revealed a moderate level of genetic diversity at species level (I?=?0.27, He?=?0.161). The AMOVA showed that the highest level of diversity occurred within populations (64%), this was also confirmed by the coefficient of differentiation (Gst?=?0.47). The estimated gene flow (Nm?=?0.56) and the Mantel test revealed a significant correlation between geographic and genetic diversity (r?=?0.266; p?=?0.001). This genetic structure was further shown by the topology of the UPGMA, sPCA and STRUCTURE analysis. In addition, a core collection of 34 genotypes was established representing the essential diversity detected. This research advocates populations and individuals to preserve in order to improve and conserve this resource in the future.     

Osmoadaptative responses in the rhizobia nodulating Acacia isolated from south-eastern Moroccan Sahara

Four strains of rhizobia nodulating Acacia were isolated from the Moroccan desert soil by trapping with seedlings of Acacia gummifera and Acacia raddiana, and were studied for their ability to tolerate high salinity and dryness conditions. The strains MDSMC 2, MDSMC 18 and MDSMC 50 were halotolerant (they tolerated up to 1 M NaCl) and they accumulated glutamate and mannosucrose. The synthesis of the latter solute, which is the major endogenous osmolyte, is partially repressed in the presence of glycine betaine. The strain MDSMC 34 was less halotolerant (growth inhibited by a concentration greater than 0.5 M NaCl), and accumulated trehalose (as the main endogenous osmolyte) and glutamate. Rhizobia from the Moroccan desert soil were highly resistant to desiccation and their tolerance to dryness was stimulated by osmotic pretreatment. Thus, the accumulation of mannosucrose or trehalose by desert rhizobia represents both an osmoadaptative response and a part of a desiccation tolerance mechanism.     

Isolation of a bacteriocin-producing Lactococcus lactis subsp. lactis and application to control Listeria monocytogenes in Moroccan jben

AIM: Use of a bacteriocin-producing lactococcal strain to control Listeria monocytogenes in jben. METHODS AND RESULTS: A Lactococcus lactis strain isolated from lben was shown, by the spot technique, to produce a bacteriocin different from nisin. Inhibitory activity of the bacteriocin-producing strain against Listeria monocytogenes was investigated in jben, made from cow's milk fermented with the producer organism and contaminated with 104 or 107 cfu ml-1. Listeria counts were monitored during manufacture, and during conservation at room and at refrigeration temperatures. Results showed that the pathogen was reduced by 2.7 logarithmic units after 30 h of jben processing when the initial inoculum of 107 cfu ml(-1) was used. For the initial inoculum of 104 cfu ml(-1), the bacterium was completely eliminated at 24 h. Furthermore, the use of the bacteriocin-producing starter culture extended the shelf-life of jben by 5 days. CONCLUSIONS: In situ production of the lactococcal bacteriocin is an efficient biological means of controlling L. monocytogenes in jben and of allowing shelf-life extension. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed technology will essentially benefit minimally processed dairy products and those made with raw milk.     

Hydrous and photosynthetic adaptations of common and durum wheat to saline stress

Seven varieties of bred wheat and seven varieties of durum wheat were cultivated in three different sites from the area of Errachidia (southeastern Morocco). These sites differ by the degree of salinity in the irrigation water. Results obtained showed that the reduction in leaf area is the principal strategy that makes it possible to attenuate the effects of the reduction in the availability of water under saline stress. Bread wheat, which limited the reduction in the leaf area, with the risk to undergo some hydrous problems, seems to better preserve its photosynthetic potentialities and grain productivity.     

Seaweed polysaccharides as bio-elicitors of natural defenses in olive trees against verticillium wilt of olive

Ulvan, carrageenan, alginate and laminarin were tested in olive trees’ twigs to elicit phenolic metabolism and control verticillium wilt of olive (VWO) caused by Verticillium dahliae. The elicitation effect was determined through phenylalanine ammonia-lyase activity, total polyphenol content and lignin content. VWO was assessed in twigs previously elicited (24?h) and maintained in a solution containing bio-elicitors (2?g/L) and conidial suspension (10⁶?conidia/mL). Our results showed stimulation of the phenolic metabolism and the decline of wilt symptoms. Ulvan reduced significantly the area under the disease progress curve for severity to 39.9% and the final incidence to 28.9%. Ulvan and alginate produced significant inhibitory rates on mycelial growth of the fungus in vitro. Seaweed polysaccharides might help to overcome VWO by strengthening the host defense metabolism and restricting the pathogen’s growth.     

Alcaligenes aquatilis GTE53: Phosphate solubilising and bioremediation bacterium isolated from new biotope “phosphate sludge enriched-compost”

The isolation and identification of beneficial bacteria from the active phase of composting is considered to be a key bio-quality parameter for the assessment of the process. The aim of this work was the selection and identification of beneficial bacteria from a co-composting experiment of vegetable waste (VW), olive oil mill waste (O₂MW), and phosphate sludge (PS). Phosphate-solubilizing strains were isolated from the thermophilic phase using Pikovskaya (PVK) solid medium supplemented with tricalcium phosphate Ca3(PO4) (TCP) as the sole source of phosphorus (P). Therefore, the selected isolate Alcaligenes aquatilis GTE53 was tested to tolerate abiotic stresses (different levels of temperature, variable pH, high salinity and water stress). The isolate was also assessed for indole acetic acid (IAA) and siderophores synthesis, nitrogen fixation, phenol degradation and pathogens inactivation. The quality of the co-composting process was also investigated by monitoring the physico-chemical parameters. The obtained results showed that A. aquatilis GTE53 displayed a higher solubilization index of 2.4 and was efficiently dissolved, up to 162.8 and 247.4 mg·mL⁻¹ of inorganic phosphate from PS and phosphate rock (PR), respectively. A. aquatilis GTE53 exhibited siderophores and IAA release, along with atmospheric nitrogen fixation. In addition to that, A. aquatilis GTE53 showed a high resistance to heat and tolerance to acidic and alkaline pH, high salinity and water stress. Moreover, A. aquatilis GTE53 could degrade 99.2% of phenol from a high-concentrated medium (1100 mg·L⁻¹ of phenol) and can inactivate the most abundant pathogens in industrial wastes: Escherichia coli, Streptococcus sp., Salmonella sp., and Fusarium oxysporum albedinis. Analysis of temperature, pH, electrical conductivity, carbon/nitrogen (C/N) ratio, indicated successful co-composting. An efficient transformation of P to the available form and a great abatement of polyphenols, were also recorded during the process. The findings of this study will help to advance the understanding of A. aquatilis GTE53 functions and will facilitate its application to promote beneficial microbial organisms during composting, thus obtaining a high-quality product.     

Characterization of a secreted cholesterol oxidase from Rhodococcus sp.GKI (CIP 105335)

Extracellular cholesterol oxidase (COX) (EC 1.1.3.6) was produced by Rhodococcus sp. GK1 cells grown in a defined mineral salt medium containing a mixture of phytosterols (sitosterol, campesterol, stigmasterol) as the sole source of carbon and energy. In the same time, the sterols acted as enzyme inducers. The medium was enriched with yeast extract in order to stimulate enzyme secretion. COX was purified from the culture supernatants by affinity-like chromatography on a column packed with kieselguhr and cholesterol. Enzyme bound onto the column was eluted with 0.05 M phosphate buffer pH 7.0 containing Triton X-100 at 0.1% (w/v). Some properties of the purified COX were determined. Its specific activity at pH 7.0 and 30 °C, was around 5.5 units mg^–1. The molecular mass of the enzyme, as estimated by SDS-PAGE, was 59 kDa. Its isoelectrofocusing point was around pH 8.9. The C-5 double bond and the alkyl chain moiety in sterol molecules were necessary for an adequate oxidation of the sterol 3-ol. Enzyme inhibition by the ions (0.1 mM): AsO₂ ^–, Ba²⁺, Co²⁺, Cd²⁺, Cu²⁺, N₃ ^–, Ni²⁺, and Pb²⁺ was negligible (around 10%). However, COX inhibition by 0.1 mM of either Zn²⁺, 2-[(ethylmercurio)-thio]benzoic acid, or Hg²⁺ was 18%, 22% and 93% respectively. Inhibition of activity by Hg²⁺ was significant, even at 1 M. The purified COX (0.1–0.15 mg ml^–1 in 0.05 M phosphate pH 7.0) was relatively heat-stable at temperatures up to 50 °C. At this temperature, the half-life of its activity was around 70 min. However, 90% of the enzyme initial activity was lost by 20 min incubation at 60 °C. The aminoacid sequence of the COX N-terminal segment was: H2N–Ala–Pro–Pro–Val–Ala–Ser–X–Arg–Tyr–X–(Phe)– (X might be 2 Cys residues).     

Molecular Typing of Mycobacterium Tuberculosis Complex by 24-Locus Based MIRU-VNTR Typing in Conjunction with Spoligotyping to Assess Genetic Diversity of Strains Circulating in Morocco

Background

Standard 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing allows to get an improved resolution power for tracing TB transmission and predicting different strain (sub) lineages in a community.

Methodology

During 2010–2012, a total of 168 Mycobacterium tuberculosis Complex (MTBC) isolates were collected by cluster sampling from 10 different Moroccan cities, and centralized by the National Reference Laboratory of Tuberculosis over the study period. All isolates were genotyped using spoligotyping, and a subset of 75 was genotyped using 24-locus based MIRU-VNTR typing, followed by first line drug susceptibility testing. Corresponding strain lineages were predicted using MIRU-VNTRplus database.

Principal Findings

Spoligotyping resulted in 137 isolates in 18 clusters (2–50 isolates per cluster: clustering rate of 81.54%) corresponding to a SIT number in the SITVIT database, while 31(18.45%) patterns were unique of which 10 were labelled as “unknown” according to the same database. The most prevalent spoligotype family was LAM; (n = 81 or 48.24% of isolates, dominated by SIT42, n = 49), followed by Haarlem (23.80%), T superfamily (15.47%), >Beijing (2.97%), > U clade (2.38%) and S clade (1.19%). Subsequent 24-Locus MIRU-VNTR typing identified 64 unique types and 11 isolates in 5 clusters (2 to 3isolates per cluster), substantially reducing clusters defined by spoligotyping only. The single cluster of three isolates corresponded to two previously treated MDR-TB cases and one new MDR-TB case known to be contact a same index case and belonging to a same family, albeit residing in 3 different administrative regions. MIRU-VNTR loci 4052, 802, 2996, 2163b, 3690, 1955, 424, 2531, 2401 and 960 were highly discriminative in our setting (HGDI >0.6).

Conclusions

24-locus MIRU-VNTR typing can substantially improve the resolution of large clusters initially defined by spoligotyping alone and predominating in Morocco, and could therefore be used to better study tuberculosis transmission in a population-based, multi-year sample context.     

Effect of high salts concentrations on the growth of rhizobia and responses to added osmotica

Twenty-eight reference strains, 79 rhizobia isolated from Trigonella foenum graecum (fenugreek), 26 strains isolated from root nodules of Ceratonia siliqua (carob tree), 30 strains isolated from nodules of Adenocarpus decorticans and five isolated from Cytisus arboreus root nodules, were screened for their tolerance to increased concentrations of NaCl. Nine isolates of fenugreek were able to grow on medium containing 10% NaCl and one strain still grew at 14%. The effect of a range of salts at 2·5 and 5% (w/v) on the growth of rhizobia was assessed and it was shown that this effect depended on the ion form and the strains used. In general, NaCl appeared to be a good indicator of the tolerance of the strains to salts. The concentrations of the salts used were high and even at 5%, growth was not completely arrested in the less tolerant strains. Some substances, such as glutamate, proline, glycine betaine and CaCl₂, were tested as osmotica. The effect of the two amino acids and betaine was confirmed with all the strains used whereas the alleviating effect of CaCl₂ was not observed in all strains. This salt had different effects on two isolates of fenugreek. These results revealed a great diversity in salt tolerance, correlated with different responses to other stress conditions, which may be due to diversity in microbial ecology.