Discovery of antimalarial drugs from secondary metabolites in actinomycetes culture library.

BACKGROUND: Natural products play a key role as potential sources of biologically active substances for the discovery of new drugs. This study aimed to identify secondary metabolites from actinomycete library extracts that are potent against the asexual stages of Plasmodium falciparum (P. falciparum). METHODS: Secondary metabolites from actinomycete library extracts were isolated from culture supernatants by ethyl acetate extraction. Comprehensive screening was performed to identify novel antimalarial compounds from the actinomycete library extracts (n = 28). The antimalarial activity was initially evaluated in vitro against chloroquine/mefloquine-sensitive (3D7) and-resistant (Dd2) lines of P. falciparum. The cytotoxicity was then evaluated in primary adult mouse brain (AMB) cells. RESULTS: Out of the 28 actinomycete extracts, 17 showed parasite growth inhibition > 50% at a concentration of 50 µg/mL, nine were identified with an IC50 value < 10 µg/mL, and seven suppressed the parasite significantly with an IC50 value < 5 µg/mL. The extracts from Streptomyces aureus strains HUT6003 (Extract ID number: 2), S. antibioticus HUT6035 (8), and Streptomyces sp. strains GK3 (26) and GK7 (27), were found to have the most potent antimalarial activity with IC50 values of 0.39, 0.09, 0.97, and 0.36 µg/mL (against 3D7), and 0.26, 0.22, 0.72, and 0.21 µg/mL (against Dd2), respectively. Among them, Streptomyces antibioticus strain HUT6035 (8) showed the highest antimalarial activity with an IC50 value of 0.09 µg/mL against 3D7 and 0.22 µg/mL against Dd2, and a selective index (SI) of 188 and 73.7, respectively. CONCLUSION: Secondary metabolites obtained from the actinomycete extracts showed promising antimalarial activity in vitro against 3D7 and Dd2 cell lines of P. falciparum with minimal toxicity. Therefore, secondary metabolites obtained from actinomycete extracts represent an excellent starting point for the development of antimalarial drug leads.

Diversity and phylogeny of the bacterial strains isolated from nodules of fenugreek (Trigonella foenum-graecum L.) in Iran.

The diversity of fenugreek (Trigonella foenum-graecum L.) microsymbionts has been barely studied even though it is of great interest for being a spice and a medicinal plant. Here, we analyzed 59 bacterial strains isolated from fenugreek nodules originating from different geographic and climatic areas of Iran. Most of these strains exhibit phenotypic characteristics compatible with rhizobia and they nodulate fenugreek. Analysis of the recA and atpD genes shows that representative strains of ERIC-BOX-PCR groups cluster with the type strains of Ensifer meliloti and E. kummerowiae as well as with strains capable of nodulating different Trigonella species found in other countries. The closeness of E. meliloti and E. kummerowiae suggests there is a need to revise the taxonomic status of the latter species. The nodC gene analysis shows that most Trigonella-nodulating strains belong to the symbiovar meliloti except those nodulating Trigonella arcuata in China, which belong to the symbiovar rigiduloides. This analysis shows that the type strains of E. kummerowiae, E. meliloti, and E. medicae belonged to three well-defined groups within the symbiovar meliloti, with the Iranian strains belonging to the E. kummerowiae subgroup. The small group of strains unable to nodulate fenugreek isolated in this study belong to Enterobacter cloacae, reported for the first time as being a possible endophyte of fenugreek nodules.

Characterization of potential plant growth-promoting rhizobacteria isolated from sunflower (Helianthus annuus L.) in Iran.

PURPOSE: This study aims to characterize plant growth-promoting rhizobacteria (PGPR) in sunflowers growing in different locations at North West of Iran. MATERIALS AND METHODS: Sunflower plants were collected from different regions of West Azarbaijan, and rhizospheric bacterial strains were isolated and screened for PGP traits. Identification and characterization of the PGPR were conducted based on 16s rDNA sequences and phenotypic analysis, the strains clustered for genetic diversity by rep-PCR method. RESULTS: Among the 80 bacterial isolates, 20 showed PGP traits and were selected for other potentials. All the selected isolates produced indole-3-acetic acid at the rate of 9.2-33.7 mg/ml. In addition, 13, 15, 12, and 16 were positive for phosphate solubilization, siderephore, hydrogen cyanide, and ammonia production, respectively. The results from a subsequent pot experiment indicated that PGPRs distinctly increased sun flower shoot and root length, shoot and root fresh weight, as well as shoot and root dry weight. Based on 16S rDNA sequences and biochemical and physiological characteristics, 20 PGPRs were identified as Pseudomonas fluorescens (five isolates), Pseudomonas aeruginosa (four isolates), Pseudomonas geniculata (one isolate), Bacillus subtilis (four isolates), Bacillus pumilus (two isolates), Stenotrophomonas maltophilia (two isolates), and Brevibacterium frigoritolerans (two isolates). In rep-PCR, PGPR isolates were differentiated into seven clusters (A-G) at 65% similarity level. These results demonstrated the existence of a considerable species richness and genetic diversity among PGPRs isolated from different regions of North West of Iran. CONCLUSIONS: To the best of our knowledge, this is first report for the identification and characterization of B. frigoritolerans as PGPR in sunflower plants.

Diversity of Endophytic and Epiphytic Bacteria From Sugarcane in Khuzestan, Iran

Abstract Diverse microorganisms are living as endophytes in plant tissues and as epiphytes on plant surfaces in nature. Commercial formulations of bacteria antagonist to plant pathogenic microbes and ice nucleation active bacteria have been utilized as an environmentally safe method to manage plant disease and to prevent frost damage respectively. Bacteria were isolated from the leaf and sheath of sugarcane (CP69-1026 CP57-614, CP48-103, CP73-21, and CP70-1143 cultivars) verities grown in the field in Khuzestan province, Iran. Bacteria were found in both sheaths and leaves of sugarcane plants which they were significantly higher in density in leaves and which most were endophytic. The bacterial strains were 10 groups on the basis of the biochemical characteristic, which their 16S rRNA encoding gene from representatives were amplified and subjected to sequencing. Results of sequences analyze using blast software from the NCBI website and phylogenetic analysis showed that the representative strains belonged to a wide variety of phylogenetic groups. These results indicated that they were closely related to Burkholderia and Ralstonia from β-Proteobacteria, Mesorhizobium, Ochrobactrum, Sphingomonas from α-Proteobacteria, Microbacterium, Curtobacterium and Leifsonia from Actinobacteria and Xanthomonas from γ-Proteobacteria. This is the first report of the presence of endophytic and epiphytic bacteria from sugarcane in Khuzestan, Iran.

Rhizosphere Bacterial Composition of the Sugar Beet Using SDS-PAGE Methodology

ABSTRACT The rhizosphere zone has been defined as the volume of soil directly influenced by the presence of living plant roots or soil compartment influenced by the root. During the growing season of 2014, the rhizobacteria of 23 sugar beet plants sampled from 12 sites in the west and north west of Iran were inventoried. Using a cultivation-dependent approach, a total of 217 bacteria were isolated from the rhizosphere. The bacterial isolates were tentatively grouped and documented based on polyacrylamide gel electrophoresis of whole-cell proteins and were found to represent 43 different protein electrotypes. The majority of the fingerprint types were found only on a single occasion. Fifty-nine percent of the strains belonged to the five bacterial species and identified as Stenotrophomonas maltophilia, Pseudomonas fluorescens, Pseudomonas aeruginosa, Stenotrophomonas rhizophila and Serratia marcescens. Minor occurring fingerprint types were identified as Flavobacterium spp, Erwinia spp, Acetobacter spp, Agrobacterium spp, Enterobacter spp, Aeromonas spp and Bacillus spp.

Phylogenetic diversity of rhizobial species and symbiovars nodulating Phaseolus vulgaris in Iran.

The phylogenetic diversity of 29 rhizobial strains nodulating Phaseolus vulgaris in Iran was analysed on the basis of their core and symbiotic genes. These strains displayed five 16S rRNA-RFLP patterns and belong to eight ERIC-PCR clusters. The phylogenetic analyses of 16S rRNA, recA and atpD core genes allowed the identification of several strains as Rhizobium sophoriradicis, R. leguminosarum, R. tropici and Pararhizobium giardinii, whereas other strains represented a new phylogenetic lineage related to R. vallis. These strains and those identified as R. sophoriradicis and R. leguminosarum belong to the symbiovar phaseoli carrying the γ nodC allele distributed in P. vulgaris endosymbionts in America, Europe, Africa and Asia. The strain identified as R. tropici belongs to the symbiovar tropici carried by strains of R. tropici, R. leucaenae, R. lusitanum and R. freirei nodulating P. vulgaris in America, Africa and Asia. The strain identified as P. giardinii belongs to the symbiovar giardinii together with the type strain of this species nodulating P. vulgaris in France. It is remarkable that the recently described species R. sophoriradicis is worldwide distributed in P. vulgaris nodules carrying the γ nodC allele of symbiovar phaseoli harboured by rhizobia isolated in the American distribution centers of this legume.

Isolation of borrelidin as a phytotoxic compound from a potato pathogenic streptomyces strain.

Streptomyces species strain GK18, isolated in Iran, induced deep-pitted lesions on potato tubers, lesions different from the raised lesions induced by the usual scab-causing phytotoxin, thaxtomin. In addition, neither thaxtomin production nor hybridization to its biosynthetic probe was detected for strain GK18, suggesting the production of a different phytotoxin. The active component was extracted with ethyl acetate from culture filtrate of strain GK18, purified by gel filtration and silica gel chromatography, and identified as an 18-membered macrolide, borrelidin, by spectroscopic analysis. The purified borrelidin induced necrosis on potato tuber slices and inhibited the growth of shoots and roots of radish seedlings. This is the first report on the phytotoxicity of borrelidin as a possible causative compound of potato scab disease.

Phytoremediation of petroleum-polluted soils: application of Polygonum aviculare and its root-associated (penetrated) fungal strains for bioremediation of petroleum-polluted soils.

Petroleum-polluted soils are a common disaster in many countries. Bioremediation of oil contamination in soils is based on the stimulation of petroleum-hydrocarbon-degrading fungal and microbial communities. A field study was conducted in a petroleum-contaminated site to find petroleum-resistant plants and their root-associated fungal strains for use in bioremediation of petroleum-polluted soils. Results and observations showed that the amounts of petroleum pollution in nonvegetated soils were several times higher than in vegetated soils. Plants collected from petroleum-polluted areas were identified using morphological characters. Results indicated that seven plant species were growing on the contaminated sites: Alhaji cameleron L. (Fabaceae), Amaranthus retroflexus L. var. retroflexus (Amaranthaceae), Convolvulus arvensis L. (Convolvulaceae), Chrozophora hierosolymitana Spreg. (Euphorbiaceae), Noea mucronata L. (Boraginaceae), Poa sp. (Poaceae), and Polygonum aviculare L. (Polygonaceae). The root-associated fungi of each plant were determined and results showed the presence of 11 species that associated with and also penetrated the roots of plants growing in the polluted areas. Altenaria sp. was common to all of the plants and the others had species-specific distribution within the plants. The largest numbers of fungal species (six) were determined for P. aviculare and Poa sp. in polluted areas. However, the variation of fungal strains in the plants collected from petroleum-polluted areas was greater than for nonpolluted ones. Culture of fungi in oil-contaminated media showed that all the studied fungi were resistant to low petroleum pollution (1% v/v) and a few species, especially Fusarium species, showed resistance to higher petroleum pollution (10% v/v) and may be suitable for bioremediation in highly polluted areas. Bioremediation tests with P. aviculare, with and without fungal strains, showed that application of both the plant and its root-associated fungal strains was more effective than of the plant and fungi separately, and Fusarium species were the most effective. Results indicated that fungal strains had the main role in bioremediation of petroleum-polluted soils, but plant roots enhanced the process.

Expression of Cre recombinase during transient phage infection permits efficient marker removal in Streptomyces.

We report a system for the efficient removal of a marker flanked by two loxP sites in Streptomyces coelicolor, using a derivative of the temperate phage phiC31 that expresses Cre recombinase during a transient infection. As the test case for this recombinant phage (called Cre-phage), we present the construction of an in-frame deletion of a gene, pglW, required for phage growth limitation or Pgl in S.coelicolor. Cre-phage was also used for marker deletion in other strains of S.coelicolor.