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In order to investigate the presence of thermo-tolerant rock phosphate (RP) solubilizing anaerobic microbes during the fermentation process, we used grassland as sole organic substrate to evaluate the RP solubilization process under anaerobic thermophilic conditions. The result shows a significant decrease of pH from 6.5 to 4.8, and solubilizing from 7 to 15.8% of the phosphorus from the RP in the reactors after 90 days of incubation at 45°C. In these conditions, the organic acids produced were qualitatively and quantitatively identified as: acetic, butyric and propionic acids. This biological RP solubilization is due to the presence of a single thermo-tolerant bacterium isolated and identified as Bacillus subtilis from the anaerobic reactors. This B. subtilis strain was shown to be able to solubilize RP in liquid cultures containing insoluble RP as sole phosphate source. The mechanisms involved in these weathering processes confirmed the production of organic acids which were identified and quantified. This study is expected to lead to the development of novel, non-polluting farming practices by entering in the formulation of novel multi-functional biofertilizer by inoculating this thermo-tolerant phosphate-solubilizing bacterium into agricultural wastes as a practical and environmental strategy. Key words: Grassland, phosphate, solubilization, Bacillus subtilis , thermo-anaerobic conditions.

A novel actinomycete, strain X4T, was isolated from a phosphate mine in Youssoufia, 100 km north of Marrakesh, Morocco. The taxonomic status of this strain was evaluated by a polyphasic approach. Strain X4Thad white aerial mycelium withRectiflexibilesspore chains bearing smooth-surfaced spores and did not produce diffusible pigments. Chemotaxonomic analysis showed that the cell wall of strain X4Tcontainedll-diaminopimelic acid and glycine. Phylogenetic analysis based on the almost complete 16S rRNA gene sequence indicated that strain X4Tbelongs to the Group I streptomycetes, branching off next toStreptomyces ramulosusNRRL B-2714TandStreptomyces kasugaensisM338-M1T. DNA–DNA relatedness and phenotypic data enabled strain X4Tto be distinguished from the phylogenetically most closely related type strains. It is therefore proposed that strain X4Trepresents a novel species of the genusStreptomyces, for which the nameStreptomyces youssoufiensissp. nov. is proposed; the type strain is X4T( = CCMM B709T = DSM 41920T).

Actinomycete strains originating from Moroccan phosphate mines (MPM) were selected for their ability to use the insoluble ground hydroxyapatite called rock phosphate (RP), present in their biotope, as sole phosphate (P) source. Physiological studies carried out with these strains and with the reference strains, Streptomyces lividans and Streptomyces griseus, demonstrated that all strains were able to grown in a synthetic minimal medium (SMM) containing either soluble (SP) or insoluble (RP) phosphate as sole P source. The MPM strains and S. griseus took up glucose much more actively and exhaustively than S. lividans, constituting more abundant glycogen reserves than the latter. All strains took up soluble P at comparable rates, storing it as polyphosphates. In SMM + RP, a sharp increase in the concentration of soluble P was detected in the culture broths of all MPM strains and S. griseus, at stationary phase, but not in that of S. lividans. The P peak detected in the supernatant of these strains correlated with the successive appearance of two compounds absorbing at 320 nm and 430 nm, respectively. These compounds are thought to be strong ion chelators involved in the destruction of the hydroxyapatite structure leading to soluble P release. The good growth of S. lividans in SMM + RP indicated that this strain was also able to release P from RP but consummed it as soon as it was released, unlike the other strains. Our study is expected to lead to the development of a novel type of slow release bio-phosphate fertilizer constituted by the association of the MPM strains and ground RP. This novel product would precisely supply plant needs and thus limit the pollution of the environment.

In order to select Actinobacteria that could improve plant growth and thus agricultural yield, we assessed different plant growth promoting (PGP) abilities of eight rock phosphate (RP)-solubilizing Actinomycete isolates originating from Moroccan phosphate mines. Six of these strains were able to grow on root exudates of the wheat plant as sole nutrient sources and were efficiently releasing soluble phosphate from RP. These strains also inhibited the growth of potentially phytopathogenic fungi, bacteria (Gram +/-) or yeasts. Five of these strains pro- duced indoleacetic acid and four showed endophytic properties. When these strains were grown, in the presence of the wheat plant, in a synthetic minimum medium (SMM) containing insoluble RP as unique phosphate source or in soil experiment, the most active RP-solubi- lizing strains had the highest stimulatory effect on the production of plant biomass. The most efficient strain Streptomyces griseus-related strain (BH7), stimulated aerial growth of the plant more than 70% in test tubes and more than 30% in RP soil compared to the non-inoculated con- trol treatment. This study demonstrated that our selected Actinomycete strains could be used for the development of novel, non-polluting; farming practices by entering in the formulation of novel biofertilizer and biocontrol products constituted by spores and/or mycelium of the ad hoc Ac- tinobacteria in association with pulverized RP.

A phosphate mine from Togo, an original biotope rich in insoluble rock phosphate (RP), was explored for the presence of RP solubilizing actinobacteria. One hundred and fifty actinobacteria isolates originating from this mine was tested for their ability to grow on a synthetic minimum medium (SMM) containing insoluble RP as unique phosphate source. Only 29 isolates (19%) were able to weather RP in SMM medium. Five isolates showed the most active growth and solubilization capability. These isolates were shown to be able to solubilize RP in liquid cultures. The study of mechanisms involved in these weathering processes indicated that the isolates produce siderophores but not organic acids. Four of these strains were shown to belong to the genus Micromonospora and one, to the genus Streptomyces. Key words: Actinobacteria, isolation, characterization, Togolese phosphate mine, rock phosphate solubilization.

Micromonospora aurantiaca- and Streptomyces griseus-related strains isolated from Moroccan phosphate mines (MAMPM and SGMPM) were previously selected for their rock phosphate (RP) solubilizing abilities and their multiple plant growth promoting properties demonstrated in laboratory conditions. In order to assess whether these interesting properties could have a direct effect on plant growth and fitness, seeds of the wheat plant (Triticum durum L. cv. Vitron) coated or not with mycelium of these strains and of the reference strain S. griseus M1323, were grown in a sterile soil deficient in soluble phosphate supplemented or not with soluble phosphate or with the insoluble RP, under greenhouse conditions. These studies revealed that the presence of the actinomycete strains in the soil supplemented with RP significantly promoted the growth of the wheat plants. MAMPM and SGMPM had the greatest stimulatory effect on plant growth with 50–47% and 80–78% weight increase of shoots and roots, respectively, in comparison with the sterile control. This increase correlated with a significant increase in the N and P content of plant tissues. The MAMPM- and SGMPM-dependent growth promotion in the RP supplemented soil was on average 10–13% lower than that achieved by the soluble phosphate supplement. Furthermore, in a soil infested with Pythium ultimum, the mediator of damping-off disease, the coating of wheat seeds with the mycelium of MAMPM strain resulted in a clear protection of the plant. The level of protection achieved by MAMPM was 14% lower than that conferred by the commercial bio-fungicide agent (Mycostop®). This study demonstrated that MAMPM in association with pulverized RP could constitute a novel and non-polluting bio-fertilizer/biocontrol product useful for the development of sustainable agriculture.

Three Moroccan phosphate mines, an original biotope rich in insoluble rock phosphate (RP), were explored for the presence of RP solubilizing Actinobacteria. Three hundred actinomycete isolates originating from these mines were tested for their ability to grow on a synthetic minimum medium (SMM) containing insoluble RP as unique phosphate source. Only 55 isolates (18.3%) were able to weather RP in SMM medium. Eight isolates showed the most active growth and solubilization capability. These isolates were shown to be able to solubilize RP in liquid cultures. The study of mechanisms involved in these weathering processes indicated that the isolates produce siderophores but not organic acids. Seven of these strains were shown to belong to the genus Streptomyces and one, to the genus Micromonospora. This study is expected to lead to the formulation of novel bio-phosphate fertilizers constituted by the association of pulverized RP and spores of the ad hoc actinomycete strains.