Electron microscopy of the uredinial and telial stages of the rust fungus, Puccinia imperatae.

The purpose of this work was to assess the morphology and ontogeny of Puccinia imperatae urediniospores and teliospores growing on its host, Imperata cylindrica, for the first time using scanning (SEM) and transmission (TEM) electron microscopy. The first evidence of uredinial development is the aggregation of hyphae in host intercellular spaces under the epidermis to form an uredinial initial. Uredinial primordia become evident as compact masses of fungal hyphae. The mycelia are dikaryotic and originated from dikaryotic urediniospores, which infect a new host. Dikaryotic sporogenous cells begin to form from dikaryotic mycelium as the uredinium develops further. The fundamental cell in the urediniospore and teliospore formation is the sporogenous cell. The teliospores with their persistent pedicels arise from dense dikaryotic sporogenous tissue, which later becomes highly vacuolated. The sporogenous cell forms a spore bud, which later divides into a pedicel and teliospore mother cell with two nuclei that produce the mature teliospore. SEM examination revealed that the urediniospores are echinulate bearing conical spines on their surfaces, sometimes curving near their tips, and the echinulate ornaments are distributed over the entire urediniospore surface. SEM examination also indicated that the teliospore surface shows rugose ornamentations. This investigation proved that the morphology of the uredinia of different Puccinia species is not related to the host species, but to the rust species. The possibility of using this fungus as a biological control agent for the noxious weed, Imperata cylindrica, was also discussed.

A Coculture Based, 3D Bioprinted Ovarian Tumor Model Combining Cancer Cells and Cancer Associated Fibroblasts.

Ovarian cancer remains a major public health issue due to its poor prognosis. To develop more effective therapies, it is crucial to set-up reliable models that closely mimic the complexity of the ovarian tumor's microenvironment. 3D bioprinting is currently a promising approach to build heterogenous and reproducible cancer models with controlled shape and architecture. However, this technology is still poorly investigated to model ovarian tumors. In this study, a 3D bioprinted ovarian tumor model combining cancer cells (SKOV-3) and cancer associated fibroblasts (CAFs) are described. The resulting tumor models show their ability to maintain cell viability and proliferation. Cells are observed to self-assemble in heterotypic aggregates. Moreover, CAFs are observed to be recruited and to circle cancer cells reproducing an in vivo process taking place in the tumor microenvironment. Interestingly, this approach also shows its ability to rapidly generate a high number of reproducible tumor models that can be subjected to usual characterizations (cell viability and metabolic activity; histology and immunological studies; and real-time imaging). Therefore, these ovarian tumor models can be an interesting tool for high throughput drug screening applications.

Biological and molecular characterization of fEg-Eco19, a lytic bacteriophage active against an antibiotic-resistant clinical Escherichia coli isolate.

Characterization of bacteriophages facilitates better understanding of their biology, host specificity, genomic diversity, and adaptation to their bacterial hosts. This, in turn, is important for the exploitation of phages for therapeutic purposes, as the use of uncharacterized phages may lead to treatment failure. The present study describes the isolation and characterization of a bacteriophage effective against the important clinical pathogen Escherichia coli, which shows increasing accumulation of antibiotic resistance. Phage fEg-Eco19, which is specific for a clinical E. coli strain, was isolated from an Egyptian sewage sample. Phage fEg-Eco19 formed clear, sharp-edged, round plaques. Electron microscopy showed that the isolated phage is tailed and therefore belongs to the order Caudovirales, and morphologically, it resembles siphoviruses. The diameter of the icosahedral head of fEg-Eco19 is 68 ± 2 nm, and the non-contractile tail length and diameter are 118 ± 0.2 and 13 ± 0.6 nm, respectively. The host range of the phage was found to be narrow, as it infected only two out of 137 clinical E. coli strains tested. The phage genome is 45,805 bp in length with a GC content of 50.3% and contains 76 predicted genes. Comparison of predicted and experimental restriction digestion patterns allowed rough mapping of the physical ends of the phage genome, which was confirmed using the PhageTerm tool. Annotation of the predicted genes revealed gene products belonging to several functional groups, including regulatory proteins, DNA packaging and phage structural proteins, host lysis proteins, and proteins involved in DNA/RNA metabolism and replication.

Antifungal activity of silver/silicon dioxide nanocomposite on the response of faba bean plants (Vicia faba L.) infected by Botrytis cinerea.

Silicon (Si) and its nanomaterials could help plants cope with different negative effects of abiotic and/or biotic stresses. In this study, the antifungal role of silver/silicon dioxide nanocomposite (Ag/SiO2NC) biosynthesized using a free-cell supernatant of Escherichia coli D8 was investigated for controlling the growth parameters and yield of faba bean (Vicia faba L.) infected by Botrytis cinerea. This nanocomposite was characterized using UV-Vis spectroscopy, Fourier transform-infrared (FTIR), transmission electron microscopy (TEM), zeta analysis, and X-ray diffraction pattern (XRD). Positively charged Ag/SiO2NC (+ 31.0 mV) with spherical-shaped silver nanoparticles (AgNPs) showed strong in vitro antifungal activity with minimal inhibition concentration (MIC) value equal to 40 ppm. In vivo experiments revealed the good resistance of Ag/SiO2NC-treated plants against the B. cinerea infection due to the increase of total phenolic content, peroxidase, and polyphenol oxidase activity. The ultrastructure of Ag/SiO2NC-treated plants showed normal morphology of cells including cell membranes and ellipsoidal-shaped chloroplasts with big starch grains. The concentration of silver content in Ag/SiO2NC-treated plants was similar to the untreated control plant indicating the low realizability of AgNPs. All of these results are promising outcomes for the application of the biosynthesized Ag/SiO2NC as a safe and effective antifungal agent against B. cinerea.

Green Synthesis of Silver Nanoparticles Using Pomegranate and Orange Peel Extracts and Their Antifungal Activity against Alternaria solani, the Causal Agent of Early Blight Disease of Tomato.

This study aimed to synthesize silver nanoparticles (AgNPs) by pomegranate and orange peel extracts using a low concentration of AgNO3 solution to controlearly blight of tomato caused by Alternaria solani. The pathogen was isolated from infected tomato plants growing in different areas of Saudi Arabia. The isolates of this pathogen were morphologically and molecularly identified. Extracts from peels of pomegranate and orange fruits effectively developed a simple, quick, eco-friendly and economical method through a synthesis of AgNPs as antifungal agents against A. solani. Phenolic content in the pomegranate peel extract was greater than orange peel extract. Phenolic compounds showed a variation of both peel extracts as identified and quantified by High-Performance Liquid Chromatography. The phenolic composition displayed variability as the pomegranate peel extract exhibited an exorbitant amount of Quercitrin (23.62 mg/g DW), while orange peel extract recorded a high amount of Chlorogenic acid (5.92 mg/g DW). Biosynthesized AgNPs were characterized using UV- visible spectroscopy which recorded an average wavelength of 437 nm and 450 nm for pomegranate and orange peels, respectively. Fourier-transform infrared spectroscopy exhibited 32x73.24, 2223.71, 2047.29 and 1972.46 cm-1, and 3260.70, 1634.62, 1376.62 and 1243.76 cm-1 for pomegranate and orange peels, respectively. Transmission electron microscopy showed spherical shape of nanoparticles. Zetasizer analysis presented negative charge values; -16.9 and -19.5 mV with average particle sizes 8 and 14 nm fin case of pomegranate and orange peels, respectively. In vitro, antifungal assay was done to estimate the possibility of biosynthesized AgNPs and crude extracts of fruit peels to reduce the mycelial growth of A. solani. AgNPs displayed more fungal mycelial inhibition than crude extracts of two peels and AgNO3. We recommend the use of AgNPs synthesized from fruit peels for controlling fungal plant pathogens and may be applied broadly and safely in place by using the chemical fungicides, which display high toxicity for humans.

In vivo toxicity and antitumor activity of newly green synthesized reduced graphene oxide/silver nanocomposites.

A novel biosynthesis of dual reduced graphene oxide/silver nanocomposites (rGO/AgNC) using the crude metabolite of Escherichia coli D8 (MF06257) strain and sunlight is introduced in this work. Physicochemical analysis of these rGO/AgNC revealed that they are sheet-like structures having spherically shaped silver nanoparticles (AgNPs) with an average particle size of 8 to 17 nm, and their absorption peak ranged from 350 to 450 nm. The biosynthesized rGO/AgNC were characterized by UV-vis and FT-IR spectra, X-ray diffraction, Zeta potential and transmission electron microscopy. After the injection of these nanocomposites to mice, their uptake by the kidney and liver has been proven by the ultrastructural observation and estimation of the hepatic and renal silver content. These nanocomposites caused a moderate toxicity for both organs. Changes in the liver and kidney functions and histopathological effects had been observed. The rGO/AgNC revealed a remarkable antitumor effect. They showed a dose-dependent cytotoxic effect on Ehrlich ascites carcinoma (EAC) cells in vitro. Treatment of mice bearing EAC tumors intraperitoneally with 10 mg/kg rGO/AgNC showed an antiproliferative effect on EAC cells, reduced ascites volume, and maintained mice survival. The results indicate that this green synergy of silver nanoparticles with reduced graphene oxide may have a promising potential in cancer therapy.

Identification and Functional Analysis of Temperate Siphoviridae Bacteriophages of Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen that presents a serious clinical challenge due to its increasing resistance to all available antibiotics. Phage therapy has been introduced recently to treat antibiotic-incurable A. baumannii infections. In search for new A. baumannii specific bacteriophages, 20 clinical A. baumannii strains were used in two pools in an attempt to enrich phages from sewage. The enrichment resulted in induction of resident prophage(s) and three temperate bacteriophages, named vB_AbaS_fEg-Aba01, vB_AbaS_fLi-Aba02 and vB_AbaS_fLi-Aba03, all able to infect only one strain (#6597) of the 20 clinical strains, were isolated. Morphological characteristics obtained by transmission electron microscopy together with the genomic information revealed that the phages belong to the family Siphoviridae. The ca. 35 kb genomic sequences of the phages were >99% identical to each other. The linear ds DNA genomes of the phages contained 10 nt cohesive end termini, 52-54 predicted genes, an attP site and one tRNA gene each. A database search revealed an >99% identical prophage in the genome of A.baumannii strain AbPK1 (acc. no. CP024576.1). Over 99% identical prophages were also identified from two of the original 20 clinical strains (#5707 and #5920) and both were shown to be spontaneously inducible, thus very likely being the origins of the isolated phages. The phage vB_AbaS_fEg-Aba01 was also able to lysogenize the susceptible strain #6597 demonstrating that it was fully functional. The phages showed a very narrow host range infecting only two A.baumannii strains. In conclusion, we have isolated and characterized three novel temperate Siphoviridae phages that infect A.baumannii.

Occurrence and distribution of tomato seed-borne mycoflora in Saudi Arabia and its correlation with the climatic variables.

One hundred samples of tomato seeds were collected in 2011 and 2012 from tomato-cultivated fields in Saudi Arabia and screened for their seed-borne mycoflora. A total of 30 genera and 57 species of fungi were recovered from the collected seed samples using agar plate and deep-freezing blotter methods. The two methods differed as regards the frequency of recovered seed-borne fungi. Seven fungi among those recovered from tomato seeds, which are known as plant pathogens, were tested for their pathogenicity and transmission on tomato seedlings. The recovery rate of these pathogens gradually decreased from root up to the upper stem, and did not reach to the stem apex. The distribution of tomato seed-borne fungi was also investigated throughout Saudi Arabia. In this concern, Al-Madena governorate recorded the highest incidence of fungal flora associated with tomato seeds. The impact of meteorological variables on the distribution of tomato seed-borne mycoflora was explored using the ordination technique (canonical correspondence analysis). Among all climatic factors, relative humidity was the most influential variable in this regard. Our findings may provide a valuable contribution to our understanding of future global disease change and may be used also to predict disease occurrence and fungal transfer to new uninfected areas.

Occurrence of Puccinia isiacae on Phragmites australis in Saudi Arabia and its possibility as a biological control agent.

During the summer of 2002, symptoms of rust disease were observed for the first time on Phragmites australis in Saudi Arabia. Light brown lesions of regular shape indicating uredinia of Puccinia isiacae appeared on the leaves. The morphology and characteristics of the fungus were described in detail with both light and scanning electron microscopy. The possibility of using this fungus as a biological control agent was also discussed.

Ultrastructure of intercellular hypha and haustorium of the rust fungus, Uromyces euphorbiae.

The ultrastructure of intercellular hyphae and dikaryotic haustoria of Uromyces euphorbiae, and the host response to haustorial invasion was investigated. The intercellular hyphae share common characteristics with those of other uredinial stages of rust fungi. Three types of septa were recognized inside the intercellular hypha. This study showed that the extrahaustorial membrane was possibly formed before the development of the haustorium. The periodic acid-thiocharbohydrazide-silver proteinate technique showed that the haustorial mother cell wall at the penetration site, and the haustorial wall contained more carbohydrates than other fungal structures. In addition, the neckband, present around the haustorial neck, contains different material from those of the rest of the haustorial neck wall. The close associations of host organelles, such as the nucleus, chloroplasts, mitochondria, endoplasmic reticulum and microtubules, with the haustorium, is described.