Actinobacteria are Gram-positive bacteria with high G+C DNA content that constitute one of the largest bacterial phyla, and they are ubiquitously distributed in both aquatic and terrestrial ecosystems. Many Actinobacteria have a mycelial lifestyle and undergo complex morphological differentiation. They also have an extensive secondary metabolism and produce about two-thirds of all naturally derived antibiotics in current clinical use, as well as many anticancer, anthelmintic, and antifungal compounds. Consequently, these bacteria are of major importance for biotechnology, medicine, and agriculture. Actinobacteria play diverse roles in their associations with various higher organisms, since their members have adopted different lifestyles, and the phylum includes pathogens (notably, species of Corynebacterium, Mycobacterium, Nocardia, Propionibacterium, and Tropheryma), soil inhabitants (e.g., Micromonospora and Streptomyces species), plant commensals (e.g., Frankia spp.), and gastrointestinal commensals (Bifidobacterium spp.). Actinobacteria also play an important role as symbionts and as pathogens in plant-associated microbial communities. This review presents an update on the biology of this important bacterial phylum.

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Taxonomy, Physiology, and Natural Products of Actinobacteria

The ancient phylum Actinobacteria is composed of phylogenetically and physiologically diverse bacteria that help Earth's ecosystems function. As free-living organisms and symbionts of herbivorous animals, Actinobacteria contribute to the global carbon cycle through the breakdown of plant biomass. In addition, they mediate community dynamics as producers of small molecules with diverse biological activities. Together, the evolution of high cellulolytic ability and diverse chemistry, shaped by their ecological roles in nature, make Actinobacteria a promising group for the bioenergy industry. Specifically, their enzymes can contribute to industrial-scale breakdown of cellulosic plant biomass into simple sugars that can then be converted into biofuels. Furthermore, harnessing their ability to biosynthesize a range of small molecules has potential for the production of specialty biofuels.

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Evolution and Ecology of Actinobacteria and Their Bioenergy Applications.

A Gram-positive, aerobic actinomycete, designated strain NEAU 119T, was isolated from the rhizosphere of a fig tree and was characterized using a polyphasic approach. The isolate formed branching, non-fragmenting vegetative hyphae and produced black pigment on yeast extract/malt extract (ISP medium 2). The G+C content of the DNA was 76.6 mol%. The organism had chemotaxonomic characteristics typical of the genus Actinoalloteichus and was closely related to the type strains of Actinoalloteichus cyanogriseus, Actinoalloteichus spitiensis and Actinoalloteichus hymeniacidonis, currently the only three recognized species of the genus Actinoalloteichus, sharing 16S rRNA gene similarities of 96.4, 96.6 and 98.1 %, respectively. However, the results of DNA–DNA hybridization studies demonstrated that the novel strain showed only 46.8 % relatedness with the type strain of A. hymeniacidonis. In addition, a set of phenotypic characteristics also readily distinguished strain NEAU 119T from the type strains of recognized species of the genus Actinoalloteichus. According to the above data, it is proposed that strain NEAU 119T represents a novel species, Actinoalloteichus nanshanensis sp. nov. The type strain of Actinoalloteichus nanshanensis is NEAU 119T ( = CGMCC 4.5714T = NBRC 106685T).

Actinoalloteichus nanshanensis sp. nov., isolated from the rhizosphere of a fig tree (Ficus religiosa).

Endophytic bacteria associated with medicinal plants possess unique strategies that enhance growth and suvival of host plants, many of which are mediated by distinctive secondary metabolites. These bacteria and their secondary metabolites are important subjects for both basic and applied research aimed at sustainable agriculture. In the present study, 114 endophytic strains isolated from the wild ethnomedicinal plant Glycyrrhiza uralensis (licorice) were screened for their in vitro antimicrobial activities against common fungal pathogens of tomato (Fusarium oxysporum f. sp., Fulvia fulva, Alternaria solani), cotton (Fusarium oxysporum f. sp. Vesinfectum, Verticillium dahliae), pomegranite (Ceratocystis fimbriata), Cymbidinium (Colletotrichum gloeosporioides), and Tsao-ko (Pestalotiopsis microspora and Fusarium graminearum) and the common bacteria Staphylococcus aureus, Bacillus cereus, Salmonella enteritidis, and Escherichia coli. Several Bacillus strains, particularly Bacillus atrophaeus and Bacillus mojavensis, had a broad spectrum of antifungal and antibacterial activity. 16 strains, selected based on broad antimicrobial activity, were shown to contain at least one putative secondary metabolite-encoding gene (i.e., polyketide synthase or non-ribosomal peptide synthetase) and/or one lytic enzyme (i.e., protease, cellulase, lipase, chitinase), which may be important mediators of antagonistic activity against pathogens. Five strains, representing Bacillus atrophaeus and Bacillus mojavensis, were selected for plant growth chamber experiments based on strong in vitro antifungal activities. All five strains significantly reduced disease severity Arabidopsis thaliana plants challenged with V. dahlia infection. GC-MS analysis of cell-free extracts of Bacillus atrophaeus strain XEGI50 showed that at least 13 compounds were produced only during co-cultivation with V. dahlia, including putative compounds known to have antimicrobial activity, such as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester; 9,12-octadecadienoic acid (Z,Z)-, methyl ester; 9-octadecenoic acid, methyl ester, (E)-; and decanedioic acid, bis(2-ethylhexyl) ester. To our knowledge, this study is the ﬁrst to report that bacteria isolated from G. uralensis have biocontrol abilities. Our findings provide new insights into the antimicrobial activities of natural endophytes, particularly B. atrophaeus, and suggest this species may a promising candidate as a biofertilizer to confer resistance to Verticillium wilt disease and other phytopathogens in cotton and other crops.

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Evaluation of the antimicrobial activity of endophytic bacterial populations from Chinese traditional medicinal plant licorice and characterization of the bioactive secondary metabolites produced by Bacillus atrophaeus against Verticillium dahliae.

Grapevine is one of the most important economic crops yielding berries, wine products as well as derivates. However, due to the large array of pathogens inducing diseases on this plant, considerable amounts of pesticides—with possible negative impact on the environment and health—have been used and are currently used in viticulture. To avoid negative impacts of such products and to ensure product quality, a substantial fraction of pesticides needs to be replaced in the near future. One solution can be related to the use of beneficial bacteria inhabiting the rhizo- and/or the endosphere of plants. These biocontrol bacteria and their secondary metabolites can reduce directly or indirectly pathogen diseases by affecting pathogen performance by antibiosis, competition for niches and nutrients, interference with pathogen signaling or by stimulation of host plant defenses. Due to the large demand for biocontrol of grapevine diseases, such biopesticides, their modes of actions and putative consequences of their uses need to be described. Moreover, the current knowledge on new strains from the rhizo- and endosphere and their metabolites that can be used on grapevine plants to counteract pathogen attack needs to be discussed. This is in particular with regard to the control of root rot, grey mould, trunk diseases, powdery and downy mildews, pierce’s disease, grapevine yellows as well as crown gall. Future prospects on specific beneficial microbes and their secondary metabolites that can be used as elicitors of plant defenses and/or as biocontrol agents with potential use in a more sustainable viticulture will be further discussed.

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Use of beneficial bacteria and their secondary metabolites to control grapevine pathogen diseases

One hundred and forty-two different actinomycete strains were isolated from rhizosphere soil of Vitis vinifera L. sampled from four Moroccan areas. To evaluate the antifungal effect of the different collected actinomycete isolates, five fungi known to be phytopathogens (Pythium ultimum, Fusarium oxyysporum f. sp. albedinis, Sclerotium rolfsii,Verticillium dahliae and Botrytis cinerea) were used. Results showed that 24 isolates had an in vitro inhibitory effect toward at least 4 of the indicator fungi, but only 9 inhibited all these phytopathogens. These nine isolates were subsequently evaluated individually using in vitro grapevine plantlets for their ability to protect against plant gray mold. We demonstrate here that pre-inoculation of plantlets with these isolates allow them to withstand Botrytis cinerea. Six of these strains were shown to belong to the genus Streptomyces and three to the genus Micromonospora. These findings indicate the potential of developing effective actinomycetes from Moroccan habitats for the biological control of Botrytis cinerea.

Antagonistic actinomycetes from Moroccan soil to control the grapevine gray mold

In Morocco, there are many sedimentary phosphate mines that produce large quantities of phosphatic limestone wastes (PLW) that contain calcite (46 wt%) and dolomite (16 wt%). These mines are located near contaminated sites, such as the abandoned Kettara pyrrhotite mine. The surface drainage water at the Kettara mine site has a pH of 2.9–4.2 and elevated concentrations of SO4 (from 47 to 5,000 mg/L) and Fe (from 1 to 1,200 mg/L). The efficiency of PLW was assessed in the laboratory as an alternative alkaline material for passive acidic mine drainage (AMD) treatment. A series of experiments were carried out using a synthetic AMD (pH 3) containing Fe (500 mg/L), SO4 (3.4 g/L), Ca (220 mg/L), Al (160 mg/L), Mn (20 mg/L), Zn (15 mg/L), Cu (23 mg/L), and trace amounts of Co, Cr, and Ni. Experiments were done in both anoxic and oxic conditions, in batch and column tests, with hydraulic retention times of 24 and 15 h, respectively. The PLW efficiently increased the alkalinity and pH, inducing precipitation of most metals. The neutralizing capacity of PLW prepared at different particle sizes (0.8 mm–0.5 cm, 0.5–1, 1–2, and 2–3 cm) was found to be similar in batch tests. The initial AMD value increased from 3 to 5–6.5 during the batch tests and 6.5–8 in the columns. In batch tests under anoxic and oxic conditions, there was a significant decrease in concentrations of Fe (500–120 mg/L), Al (160–1.7 mg/L), and Cu (23–0.002 mg/L). In the column tests, Al and Cu decreased (177–2.5 and 26–0.002 mg/L, respectively), while Fe decreased less significantly (618–300 mg/L). The availability and low cost of the PLW make its use in passive AMD treatment potentially feasible.

The Potential Use of Phosphatic Limestone Wastes in the Passive Treatment of AMD: A Laboratory Study

Purification of antibiotics from the biocontrol agent Streptomyces anulatus S37 by centrifugal partition chromatography.

The novel actinomycete strain Ap1T was isolated from rhizosphere soil of the argan tree (Argania spinosa L.) in the south of Morocco. Strain Ap1T has been reported as a novel producer of the pentaene polyene macrolide isochainin, which strongly inhibits the growth of pathogenic yeasts and phytopathogenic fungi. Strain Ap1T shows a greyish-white aerial mycelium with chains of smooth-surfaced spores of the Spiralis type and a cell wall containing ll-diaminopimelic acid. Based on chemotaxonomy and morphological features, strain Ap1T was identified as a member of the genus Streptomyces. 16S rRNA gene sequence similarities based on almost-complete 16S rRNA gene sequences showed that strain Ap1T is closely associated with members of the Streptomyces violaceoruber species group (S. violaceoruber, S. coelescens, S. violaceorubidus, ‘S. caesius’, ‘S. lividans’, S. violaceolatus and S. humiferus) and others (Streptomyces aurantiogriseus, S. lienomycini, S. chattanoogensis, S. rubrogriseus and S. tendae). However, protein profiling, DNA–DNA hybridization and BOX-PCR fingerprinting proved a relationship above the species level. In addition, the phenotype also allowed for the differentiation of strain Ap1T from its closest neighbours. As a result of this polyphasic approach, we conclude that strain Ap1T represents a novel species of the genus Streptomyces, for which the name Streptomyces marokkonensis sp. nov. is proposed. The type strain is Ap1T (=R-22003T =LMG 23016T =DSM 41918T).

Streptomyces marokkonensis sp. nov., isolated from rhizosphere soil of Argania spinosa L.

A novel actinomycete, strain S10T, was isolated from rhizosphere soil of wild Vitis vinifera in Thinghir, Ouarzazate Province, Southern Morocco. The taxonomic status of this strain was established using a polyphasic approach. Strain S10T had white–grey aerial mycelium with long, spiral spore chains bearing smooth surfaced spores and produced a yellow diffusible pigment. Chemotaxonomic analyses showed that the cell wall of strain S10T contained ll-diaminopimelic acid and glycine. Phylogenetic analysis based on the almost complete 16S rRNA gene sequence indicated that strain S10T belonged to the Group I streptomycetes, branching off next to Streptomyces marokkonensis LMG 23016T from the Streptomyces violaceoruber group. DNA–DNA relatedness and phenotypic data distinguished strain S10T from the phylogenetically closest related type strains. It is therefore proposed that strain S10T (=CCMM B35T=DSM 41919T) represents the type strain of a novel species of the genus Streptomyces, for which the name Streptomyces thinghirensis sp. nov. is proposed.

Souad Loqman

Papers

Antagonistic actinomycetes from Moroccan soil to control the grapevine gray mold

The Potential Use of Phosphatic Limestone Wastes in the Passive Treatment of AMD: A Laboratory Study

Purification of antibiotics from the biocontrol agent Streptomyces anulatus S37 by centrifugal partition chromatography.

Streptomyces marokkonensis sp. nov., isolated from rhizosphere soil of Argania spinosa L.

Streptomyces thinghirensis sp. nov., isolated from rhizosphere soil of Vitis vinifera.