Rare Halophilic 
Hypersaline ecosystems represent an extreme environment in which a relatively low diversity of microbial species can be found and host a particular native microflora adapted to these habitats such as halophilic bacteria . The occurrence of actinobacteria in high salinity environments and tolerance of these organisms to high concentrations of salt have been previously studied by many authors, and several novel genera and species of halophilic and halotolerant have been described such as Streptomonospora litoralis , Phytoactinopolyspora halophila , P. mesophila , Glycomyces salinus , Nesterenkonia pannonica , N. natronophila , Prauserella isguenensis , P. oleivorans , Mzabimyces algeriensis , Actinopolyspora salinaria , and Brachybacterium halotolerans . Halophiles and halotolerant actinobacteria have attracted a great attention owing to produce various bioactive natural compounds, such as antibiotics actinopolysporins A-C , persiamycin A , anticancer salternamide A , cytotoxic compounds nocarbenzoxazoles A-G , salternamides A-D , antiviral xiamycins C-E , and enzymes . The Sahara is the third largest (after the cold deserts of the Antarctic and the Arctic) and hot desert in the world. The Sahara is one of the harshest environments on Earth, covering more than 9 million km 2 , spanning nearly a third of the African continent . Microbial species found in the Algerian Sahara are adapted to the extreme environment characterized by high temperature and often by high salinity encountered in this area. They develop particular metabolic pathways to survive, and special phenetic properties are acquired, which can lead to a production of new substances . Numerous studies have demonstrated the abundance of actinobacterial biodiversity in Algerian Saharan soil . This biodiversity can lead to the discovery of novel species and secondary metabolites [ – ]. As part of this program, we focused our study on halophilic Nocardiopsis isolates, which were characterized using a polyphasic approach, and their antagonistic properties were evaluated against several pathogenic and nonpathogenic microorganisms. Halophilic actinobacteria are a less explored source for the discovery of novel bioactive secondary metabolites . At present, seven of the thirteen validly published genera in the family Nocardiopsaceae , including Haloactinospora , Lipingzhangella , Nocardiopsis , Salinactinospora , Spinactinospora , Streptomonospora , and Thermobifida , contain one or several halophilic species. The genus Nocardiopsis currently contains 45 validly published species names among which 23 species are halophilic. The strains of Nocardiopsis are also known for their potential to produce bioactive metabolites [ – ]. The goal of the present study is to investigate the biodiversity of halophilic actinobacteria isolated from Algerian Saharan soils by using a polyphasic approach and to evaluate their potential to produce bioactive molecules. Actinobacteria are remarkable sources of novel antibiotics and compounds which possess medical and industrial importance. 

2.1. Strain Isolation Twenty-three halophilic actinobacteria were isolated by the dilution agar plating method , from extremely saline soil samples obtained from different regions of the Algerian Sahara. The soils in these regions are characterized by a sandy loam texture, slightly basic pH (7.5 to 8.5), and high salinity (electric conductivity at 1/5 comprised between 8 and 15 mS cm −1 ) . Each dry soil sample was suspended in sterile distilled water and diluted (10 g soil in 90 mL of water). Aliquots (0.2 mL) of each dilution were spread onto a humic-vitamin agar medium  supplemented with NaCl (10%) and with antifungal cycloheximide (50 μ g/mL) to inhibit the growth of fungi. The culture plates were incubated at 30°C for two weeks, and all the colonies were directly examined by light microscopy to detect Nocardiopsis -like isolates. After isolation, the purified strains were stored at 4°C on agar slants of Bacto tryptic soy agar (TSA) medium supplemented with NaCl (10%) for further use. 2.2. Morphological Study The cultural and morphological characteristics of selected actinobacteria were studied on yeast extract-malt extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salt-starch agar (ISP 4) , TSA medium, and complex medium agar (CMA) . All culture media were supplemented with 10% NaCl. The colors of aerial and substrate mycelia were determined with the ISCC-NBS centroid color charts . The morphology of the strains grown on various media at 30°C for 12 days was examined by light microscopy for mycelia organization and sporulation. 2.3. Chemotaxonomic Study For the chemotaxonomic analysis, biomass was obtained from a culture grown on a TSB medium modified with the addition of 10% NaCl and incubated at 30°C for 5 days. Analysis of diaminopimelic acid isomers and whole-cell sugar pattern was carried out using the method of Becker et al.  and M. Lechevalier and H. Lechevalier . Phospholipids were analyzed according to the procedures developed by Minnikin et al. . 2.4. Physiological Study and Numerical Taxonomy Fifty physiological tests were performed, including the utilization of 16 carbohydrate compounds ; assimilation of alanine, proline, and serine as nitrogen source; degradation of adenine, guanine, xanthine, hypoxanthine, milk casein, testosterone, Tween 80, starch, gelatin, esculin, and arbutin; decarboxylation of sodium acetate and sodium butyrate; production of nitrate reductase; growth in the presence of 0, 7, 10, 15, 20, and 25% NaCl; sensitivity to lysozyme at 0.005% and to five antibiotics; and growth at pH 5 and 9 (pH was adjusted accordingly using HCl/NaOH solutions) and at temperatures 20, 30, 37, and 42°C. All the results of the physiological study were analyzed by numerical taxonomy. The data of phenetic characters were coded in a binary system (1/0). The degree of similarity between the studied halophilic strains was calculated by simple matching (SM), and clustering was performed by the unweighted-pair group method using average linkages (UPGMA) in the SPSS package (v.16.0.1). 2.5. 16S rRNA Gene Sequencing and Phylogenetic Analysis DNA preparation was performed according to the method of Liu et al. . The actinobacterial isolates were grown in a shaker (250 rpm) at 30°C for 7 days. PCR amplification of the 16S rRNA gene of the isolates was performed using two primers: 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-GTTACCTTGTTACGAC TT-3′). The sequencing reaction was performed by MilleGen Company (Toulouse, France). The same primers as before and an automated sequencer were used for this purpose. The similarities of the 16S rRNA gene sequences between strains were calculated on the basis of pairwise alignment using the EzTaxon-e server . Phylogenetic and molecular evolutionary analyses were carried out using MEGA version 7.0 . The 16S rRNA gene sequences of the strains were aligned using the CLUSTAL W program  against corresponding nucleotide sequences of Nocardiopsis retrieved from GenBank and EzTaxon-e. Phylogenetic trees were reconstructed by using neighbor-joining  with the model of Jukes and Cantor , maximum likelihood  with Kimura's two-parameter model , and maximum parsimony . Bootstrap analysis  was performed to evaluate the reliability of the tree topology. 2.6. Antagonistic Properties The antimicrobial spectrum was determined by the conventional agar method (agar cylinder method) as described by Patel and Brown  subsequently against the pathogenic and nonpathogenic fungi ( Aspergillus niger , A . flavus , Botrytis cinerea , Rhizopus nigricans , Saccharomyces cerevisiae ATCC 4226, and Kluyveromyces lactis ), Gram-positive bacteria ( Bacillus subtilis ATCC 6633 and Staphylococcus aureus CIP 7625), and Gram-negative bacteria ( Agrobacterium tumefaciens No. 2410, Klebsiella pneumoniae CIP 82·91, Pseudomonas syringae , Salmonella enterica , and Serratia marcescens ). The actinobacterial isolates were grown on TSA plates supplemented with 10% NaCl for 7 days at 30°C, and then, agar cylinders (5 mm in diameter) were cut out and placed onto the agar surface (nutrient agar or Sabouraud agar, covered by 0.2 mL of culture containing 5 × 10 5 cfu/mL for bacteria or 5 × 10 4 cfu/mL for fungi, respectively). A sterile cylinder of TSA, supplemented with 10% NaCl, was used as control, and the plates were incubated at 30°C for 24-48 h after a diffusion process for 4 h at 4°C. The diameters of inhibition zones formed around the cylinder were then measured. 

Twenty-three halophilic actinobacteria were isolated by the dilution agar plating method , from extremely saline soil samples obtained from different regions of the Algerian Sahara. The soils in these regions are characterized by a sandy loam texture, slightly basic pH (7.5 to 8.5), and high salinity (electric conductivity at 1/5 comprised between 8 and 15 mS cm −1 ) . Each dry soil sample was suspended in sterile distilled water and diluted (10 g soil in 90 mL of water). Aliquots (0.2 mL) of each dilution were spread onto a humic-vitamin agar medium  supplemented with NaCl (10%) and with antifungal cycloheximide (50 μ g/mL) to inhibit the growth of fungi. The culture plates were incubated at 30°C for two weeks, and all the colonies were directly examined by light microscopy to detect Nocardiopsis -like isolates. After isolation, the purified strains were stored at 4°C on agar slants of Bacto tryptic soy agar (TSA) medium supplemented with NaCl (10%) for further use. 

The cultural and morphological characteristics of selected actinobacteria were studied on yeast extract-malt extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salt-starch agar (ISP 4) , TSA medium, and complex medium agar (CMA) . All culture media were supplemented with 10% NaCl. The colors of aerial and substrate mycelia were determined with the ISCC-NBS centroid color charts . The morphology of the strains grown on various media at 30°C for 12 days was examined by light microscopy for mycelia organization and sporulation. 

For the chemotaxonomic analysis, biomass was obtained from a culture grown on a TSB medium modified with the addition of 10% NaCl and incubated at 30°C for 5 days. Analysis of diaminopimelic acid isomers and whole-cell sugar pattern was carried out using the method of Becker et al.  and M. Lechevalier and H. Lechevalier . Phospholipids were analyzed according to the procedures developed by Minnikin et al. . 

Fifty physiological tests were performed, including the utilization of 16 carbohydrate compounds ; assimilation of alanine, proline, and serine as nitrogen source; degradation of adenine, guanine, xanthine, hypoxanthine, milk casein, testosterone, Tween 80, starch, gelatin, esculin, and arbutin; decarboxylation of sodium acetate and sodium butyrate; production of nitrate reductase; growth in the presence of 0, 7, 10, 15, 20, and 25% NaCl; sensitivity to lysozyme at 0.005% and to five antibiotics; and growth at pH 5 and 9 (pH was adjusted accordingly using HCl/NaOH solutions) and at temperatures 20, 30, 37, and 42°C. All the results of the physiological study were analyzed by numerical taxonomy. The data of phenetic characters were coded in a binary system (1/0). The degree of similarity between the studied halophilic strains was calculated by simple matching (SM), and clustering was performed by the unweighted-pair group method using average linkages (UPGMA) in the SPSS package (v.16.0.1). 

DNA preparation was performed according to the method of Liu et al. . The actinobacterial isolates were grown in a shaker (250 rpm) at 30°C for 7 days. PCR amplification of the 16S rRNA gene of the isolates was performed using two primers: 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-GTTACCTTGTTACGAC TT-3′). The sequencing reaction was performed by MilleGen Company (Toulouse, France). The same primers as before and an automated sequencer were used for this purpose. The similarities of the 16S rRNA gene sequences between strains were calculated on the basis of pairwise alignment using the EzTaxon-e server . Phylogenetic and molecular evolutionary analyses were carried out using MEGA version 7.0 . The 16S rRNA gene sequences of the strains were aligned using the CLUSTAL W program  against corresponding nucleotide sequences of Nocardiopsis retrieved from GenBank and EzTaxon-e. Phylogenetic trees were reconstructed by using neighbor-joining  with the model of Jukes and Cantor , maximum likelihood  with Kimura's two-parameter model , and maximum parsimony . Bootstrap analysis  was performed to evaluate the reliability of the tree topology. 

The antimicrobial spectrum was determined by the conventional agar method (agar cylinder method) as described by Patel and Brown  subsequently against the pathogenic and nonpathogenic fungi ( Aspergillus niger , A . flavus , Botrytis cinerea , Rhizopus nigricans , Saccharomyces cerevisiae ATCC 4226, and Kluyveromyces lactis ), Gram-positive bacteria ( Bacillus subtilis ATCC 6633 and Staphylococcus aureus CIP 7625), and Gram-negative bacteria ( Agrobacterium tumefaciens No. 2410, Klebsiella pneumoniae CIP 82·91, Pseudomonas syringae , Salmonella enterica , and Serratia marcescens ). The actinobacterial isolates were grown on TSA plates supplemented with 10% NaCl for 7 days at 30°C, and then, agar cylinders (5 mm in diameter) were cut out and placed onto the agar surface (nutrient agar or Sabouraud agar, covered by 0.2 mL of culture containing 5 × 10 5 cfu/mL for bacteria or 5 × 10 4 cfu/mL for fungi, respectively). A sterile cylinder of TSA, supplemented with 10% NaCl, was used as control, and the plates were incubated at 30°C for 24-48 h after a diffusion process for 4 h at 4°C. The diameters of inhibition zones formed around the cylinder were then measured. 

3.1. Cultural Characteristics and Morphology All isolates have the same morphology except AH25 and AH26. The growth after 12 days at 30°C was abundant on CMA and TSA media, moderate on ISP 2 medium, and poor on ISP 4 medium. All isolates, except AH25 and AH26, produced abundant white to pale yellow aerial mycelium, a pale yellow substrate mycelium, and no diffusible pigment. Isolate AH25 produced an abundant pale pink aerial mycelium, a pink to light reddish brown substrate mycelium, and a moderate reddish brown diffusible pigment on ISP 2, CMA, and TSA media. Isolate AH26 showed only traces (microscopic) of aerial mycelium, orange substrate mycelium, and no diffusible pigment on all media tested ( and ). All isolates produced long sporulated aerial hyphae, branched, straight to irregularly curved, often with a zigzag shape (except AH26). At the maturation state, these hyphae fragmented irregularly into long chains of elongated spores. 3.2. Chemical Analysis of Cellular Constituents Cell wall hydrolysate of the 23 actinobacterial isolates contained meso-diaminopimelic acid, but glycine was not detected. Glucose and ribose were detected in whole-cell hydrolysates. Diagnostic sugars such as arabinose, madurose, xylose, or rhamnose were absent. Thus, isolates had cell walls of type III and sugar pattern type C . The diagnostic phospholipids detected were phosphatidylcholine. This pattern corresponds to phospholipids type PIII . Based on the morphological and chemical characteristics, isolates are attached to the genus Nocardiopsis described by Li et al. . 3.3. Physiological Characteristics and Numerical Taxonomy The physiological characteristics based on 50 assays were subjected to numerical analysis (based on the similarity SM coefficient and UPGMA clustering). This physiological classification gave five cluster groups, designated I to V, and nine single isolates at 93.5% ( ). Cluster I contains six strains, and clusters II, III, IV, and V contain two strains each. The numerical taxonomy used in this study enabled a rational distinction between isolates. The 23 Nocardiopsis isolates have many similarities between them, but they also have many differences in physiological characteristics, as shown in . All isolates can grow on culture media with a NaCl concentration ranging from 7 to 15% (except AH19 which grows at 10 to 15% NaCl), and almost no isolates grow at 0% (except AH12). Some of them can tolerate up to 20% of NaCl, and optimal growth was observed at 10% for all isolates. Thus, they can be considered as moderate halophilic microorganisms, except AH12 which is halotolerant. A number of physiological characteristic differences were observed between clusters: 4 tests (clusters I-II), 5 tests (clusters I-III and III-V), 6 tests (clusters I-IV), 7 tests (clusters III-IV), and 9 tests (clusters I-V, II-III, II-IV, II-V, and IV-V). Physiological characteristic differences, sometimes more significant, were observed in the case of some single isolates such as AH26, which differs from the 22 other isolates of Nocardiopsis by its inability to degrade adenine, starch, D-glucose, xanthine, hypoxanthine, and Tween 80, and also several other characteristics. 3.4. Phylogenetic Studies The 16S rRNA gene sequences (1529-1544 bp) of the 23 isolates were determined and deposited in the GenBank data library under the accession numbers JF777508-JF777530. The sequences were aligned with those of Nocardiopsis reference species available in the GenBank database, which confirmed that these 23 strains belong to the genus Nocardiopsis . It is interesting to note that the phylogenetic study corresponded in most cases with phenotypic taxonomy. The dendrogram constructed by the neighbor-joining method is shown in . The isolates of cluster I (AH15, AH17, AH33, AH47, AH57, and AH65), cluster II (AH36 and AH44), and cluster III (AH25 and AH62), as well as the single isolate AH4, were related to Nocardiopsis xinjiangensis , with 99.4 to 99.8% 16S rRNA gene sequence similarity. The isolates of cluster IV (AH0 and AH67) were closely related to Nocardiopsis litoralis , with 99.7% similarity, followed by N . kunsanensis , with 99.5% similarity. The isolates of cluster V (AH1 and AH52) were also shown to be closely related to N . litoralis , but with lower percentages of similarity (99.3 to 99.6%), and more distant from N . kunsanensis (99.0 to 99.4%). Concerning the single isolates AH38, AH19, and AH37, they exhibited relatively lower percentages of similarity, respectively, to N . litoralis (99.2%), N . halotolerans  (99.3%), and N . terrae  (99.4%). These percentages of similarity are even lower for the single isolates AH63 (98.4% with N . litoralis ), AH46 (98.4% with N . xinjiangensis ), AH12 (98.7% with N . litoralis ), AH26 (98.8%) with N . salina , and AH24 (98.9% with N . xinjiangensis ). High 16S rRNA similarity values were found between representatives of validly described Nocardiopsis species, such as the type strains of N . valliformis and N . exhalans (99.9%) , N . sinuspersici and N . arvandica (99.9%) , N . halophila and N . baichengensis (99.9%) , N . litoralis and N . kunsanensis (99.6%), N. metallicus and N. prasina (99.3%), and N . metallicus  and N . exhalans (99.4%) . It is known that strains with 16S rRNA similarity range between 98.65 and 99.99% could be a potential novel species; however, the isolates need to be the subject of more detailed molecular systematic studies based on DNA-DNA reassociation or whole-genome sequencing to determine their taxonomic status. The results were also supported by the physiological differences observed between these isolates and the nearest relatives in the genus Nocardiopsis . The physiological differences are given in Tables – . 3.5. Antimicrobial Activities The antimicrobial activities of Nocardiopsis isolates against several bacteria and fungi (listed in ) were determined by the cylinder plate method on TSA medium supplemented with 10% NaCl. All Nocardiopsis isolates (except isolate AH37) showed activities against Pseudomonas syringae and Salmonella enterica (with moderate to high activity). Some isolates showed activities against Agrobacterium tumefaciens (16 isolates), Serratia marcescens (15 isolates), and Klebsiella pneumoniae (16 isolates). In contrast, the activity against Gram-positive bacteria was observed only by five isolates of Nocardiopsis against Staphylococcus aureus . However, no isolates were active against Bacillus subtilis , Aspergillus flavus , or Aspergillus niger . From these results, we can deduce that the halophilic actinobacteria produce molecules mainly active against Gram-negative bacteria, which appears to be interesting. There are many works reported that Gram-positive bacteria are potential biocontrol agents against Gram-negative pathogens [ – ]. High activity is obtained against Salmonella enterica and Pseudomonas syringae , which is interesting, given the toxicity and pathogenicity of S . enterica and the phytopathogenicity of P . syringae . The antifungal activity is only limited to Kluyveromyces lactis (16 isolates) and Rhizopus nigricans (12 isolates). Up to now, Nocardiopsis strains and species have been shown to be the source of many secondary metabolites, such as 3-trehalosamine , griseusin D , macrolide NWA52-A , thiopeptide , nocapyrones , diketopiperazines , 4-oxo-1,4-dihydroquinoline-3-carboxamide and N -acetyl-anthranilic acid , and more recently angucyclinones  and kenalactams . Moreover, Nocardiopsis strains are known to produce many novel extracellular enzymes such as amylases, inulinases, chitinases, proteases, xylanases, glucanases, and cellulases . It is interesting to mention that some products derived from Nocardiopsis species are commercially available such as the protein kinase and NGF inhibitors K-252a . The K-252a, a kinase inhibitor isolated from the culture broth of Nocardiopsis sp., selectively inhibits the actions of nerve growth factor (NGF) on PC12 cells (neuroendocrine tumor) . An understanding on the gene clusters involved in the biosynthesis of bioactive secondary metabolites produced by many species and strains of Nocardiopsis would allow to increase their production by following genetic manipulation techniques and their diversity by using mutasynthetic approaches . With further understanding on the biosynthetic capabilities of the several new strains and/or species, the range of molecules derived from the members of this genus and the field of “ Nocardiopsis Biotechnology” is projected to grow in the upcoming years . 

All isolates have the same morphology except AH25 and AH26. The growth after 12 days at 30°C was abundant on CMA and TSA media, moderate on ISP 2 medium, and poor on ISP 4 medium. All isolates, except AH25 and AH26, produced abundant white to pale yellow aerial mycelium, a pale yellow substrate mycelium, and no diffusible pigment. Isolate AH25 produced an abundant pale pink aerial mycelium, a pink to light reddish brown substrate mycelium, and a moderate reddish brown diffusible pigment on ISP 2, CMA, and TSA media. Isolate AH26 showed only traces (microscopic) of aerial mycelium, orange substrate mycelium, and no diffusible pigment on all media tested ( and ). All isolates produced long sporulated aerial hyphae, branched, straight to irregularly curved, often with a zigzag shape (except AH26). At the maturation state, these hyphae fragmented irregularly into long chains of elongated spores. 

Cell wall hydrolysate of the 23 actinobacterial isolates contained meso-diaminopimelic acid, but glycine was not detected. Glucose and ribose were detected in whole-cell hydrolysates. Diagnostic sugars such as arabinose, madurose, xylose, or rhamnose were absent. Thus, isolates had cell walls of type III and sugar pattern type C . The diagnostic phospholipids detected were phosphatidylcholine. This pattern corresponds to phospholipids type PIII . Based on the morphological and chemical characteristics, isolates are attached to the genus Nocardiopsis described by Li et al. . 

The physiological characteristics based on 50 assays were subjected to numerical analysis (based on the similarity SM coefficient and UPGMA clustering). This physiological classification gave five cluster groups, designated I to V, and nine single isolates at 93.5% ( ). Cluster I contains six strains, and clusters II, III, IV, and V contain two strains each. The numerical taxonomy used in this study enabled a rational distinction between isolates. The 23 Nocardiopsis isolates have many similarities between them, but they also have many differences in physiological characteristics, as shown in . All isolates can grow on culture media with a NaCl concentration ranging from 7 to 15% (except AH19 which grows at 10 to 15% NaCl), and almost no isolates grow at 0% (except AH12). Some of them can tolerate up to 20% of NaCl, and optimal growth was observed at 10% for all isolates. Thus, they can be considered as moderate halophilic microorganisms, except AH12 which is halotolerant. A number of physiological characteristic differences were observed between clusters: 4 tests (clusters I-II), 5 tests (clusters I-III and III-V), 6 tests (clusters I-IV), 7 tests (clusters III-IV), and 9 tests (clusters I-V, II-III, II-IV, II-V, and IV-V). Physiological characteristic differences, sometimes more significant, were observed in the case of some single isolates such as AH26, which differs from the 22 other isolates of Nocardiopsis by its inability to degrade adenine, starch, D-glucose, xanthine, hypoxanthine, and Tween 80, and also several other characteristics. 

The 16S rRNA gene sequences (1529-1544 bp) of the 23 isolates were determined and deposited in the GenBank data library under the accession numbers JF777508-JF777530. The sequences were aligned with those of Nocardiopsis reference species available in the GenBank database, which confirmed that these 23 strains belong to the genus Nocardiopsis . It is interesting to note that the phylogenetic study corresponded in most cases with phenotypic taxonomy. The dendrogram constructed by the neighbor-joining method is shown in . The isolates of cluster I (AH15, AH17, AH33, AH47, AH57, and AH65), cluster II (AH36 and AH44), and cluster III (AH25 and AH62), as well as the single isolate AH4, were related to Nocardiopsis xinjiangensis , with 99.4 to 99.8% 16S rRNA gene sequence similarity. The isolates of cluster IV (AH0 and AH67) were closely related to Nocardiopsis litoralis , with 99.7% similarity, followed by N . kunsanensis , with 99.5% similarity. The isolates of cluster V (AH1 and AH52) were also shown to be closely related to N . litoralis , but with lower percentages of similarity (99.3 to 99.6%), and more distant from N . kunsanensis (99.0 to 99.4%). Concerning the single isolates AH38, AH19, and AH37, they exhibited relatively lower percentages of similarity, respectively, to N . litoralis (99.2%), N . halotolerans  (99.3%), and N . terrae  (99.4%). These percentages of similarity are even lower for the single isolates AH63 (98.4% with N . litoralis ), AH46 (98.4% with N . xinjiangensis ), AH12 (98.7% with N . litoralis ), AH26 (98.8%) with N . salina , and AH24 (98.9% with N . xinjiangensis ). High 16S rRNA similarity values were found between representatives of validly described Nocardiopsis species, such as the type strains of N . valliformis and N . exhalans (99.9%) , N . sinuspersici and N . arvandica (99.9%) , N . halophila and N . baichengensis (99.9%) , N . litoralis and N . kunsanensis (99.6%), N. metallicus and N. prasina (99.3%), and N . metallicus  and N . exhalans (99.4%) . It is known that strains with 16S rRNA similarity range between 98.65 and 99.99% could be a potential novel species; however, the isolates need to be the subject of more detailed molecular systematic studies based on DNA-DNA reassociation or whole-genome sequencing to determine their taxonomic status. The results were also supported by the physiological differences observed between these isolates and the nearest relatives in the genus Nocardiopsis . The physiological differences are given in Tables – . 

The antimicrobial activities of Nocardiopsis isolates against several bacteria and fungi (listed in ) were determined by the cylinder plate method on TSA medium supplemented with 10% NaCl. All Nocardiopsis isolates (except isolate AH37) showed activities against Pseudomonas syringae and Salmonella enterica (with moderate to high activity). Some isolates showed activities against Agrobacterium tumefaciens (16 isolates), Serratia marcescens (15 isolates), and Klebsiella pneumoniae (16 isolates). In contrast, the activity against Gram-positive bacteria was observed only by five isolates of Nocardiopsis against Staphylococcus aureus . However, no isolates were active against Bacillus subtilis , Aspergillus flavus , or Aspergillus niger . From these results, we can deduce that the halophilic actinobacteria produce molecules mainly active against Gram-negative bacteria, which appears to be interesting. There are many works reported that Gram-positive bacteria are potential biocontrol agents against Gram-negative pathogens [ – ]. High activity is obtained against Salmonella enterica and Pseudomonas syringae , which is interesting, given the toxicity and pathogenicity of S . enterica and the phytopathogenicity of P . syringae . The antifungal activity is only limited to Kluyveromyces lactis (16 isolates) and Rhizopus nigricans (12 isolates). Up to now, Nocardiopsis strains and species have been shown to be the source of many secondary metabolites, such as 3-trehalosamine , griseusin D , macrolide NWA52-A , thiopeptide , nocapyrones , diketopiperazines , 4-oxo-1,4-dihydroquinoline-3-carboxamide and N -acetyl-anthranilic acid , and more recently angucyclinones  and kenalactams . Moreover, Nocardiopsis strains are known to produce many novel extracellular enzymes such as amylases, inulinases, chitinases, proteases, xylanases, glucanases, and cellulases . It is interesting to mention that some products derived from Nocardiopsis species are commercially available such as the protein kinase and NGF inhibitors K-252a . The K-252a, a kinase inhibitor isolated from the culture broth of Nocardiopsis sp., selectively inhibits the actions of nerve growth factor (NGF) on PC12 cells (neuroendocrine tumor) . An understanding on the gene clusters involved in the biosynthesis of bioactive secondary metabolites produced by many species and strains of Nocardiopsis would allow to increase their production by following genetic manipulation techniques and their diversity by using mutasynthetic approaches . With further understanding on the biosynthetic capabilities of the several new strains and/or species, the range of molecules derived from the members of this genus and the field of “ Nocardiopsis Biotechnology” is projected to grow in the upcoming years . 

The 23 isolates were characterized by morphological characteristics and had chemotaxonomic properties consistent with their assignment to the genus Nocardiopsis. These isolates were compared phylogenetically and physiologically with the nearest species. The comparison showed that they are clearly different from the known species of Nocardiopsis and suggested the presence of a potential novel species. The antagonistic properties of the isolates showed antibacterial activity directed mainly against Gram-negative bacteria for the majority of isolates, which is interesting given the known resistance of this group of bacteria to many antibiotics. The strong activities are obtained against Salmonella enterica , which is interesting, given the pathogenicity and toxicity of this germ for humans. The results imply that unexplored extreme ecosystems such as the Sahara Desert potentially contain new species of actinobacteria as the source of novel bioactive secondary metabolites that may serve as a structural foundation for the development of novel drugs to be used in medicine and/or other industries. 

