Nemr, R. A., M. Khalil, M. S. Sarhan, M. Abbas, H. ElSawey, H. H. Youssef, M. A. Hamza, A. T. Morsi, M. El-Tahan, M. Fayez, et al., "“In situ similis” Culturing of Plant Microbiota: A Novel Simulated Environmental Method Based on Plant Leaf Blades as Nutritional Pads", Frontiers in Microbiology, vol. 11, pp. 454, 2020. AbstractWebsite

High-throughput cultivation methods have recently been developed to accelerate the recovery of microorganisms reluctant to cultivation. They simulate in situ environmental conditions for the isolation of environmental microbiota through the exchange of growth substrates during cultivation. Here, we introduce leaf-based culture media adopting the concept of the plant being the master architect of the composition of its microbial community. Pre-physical treatments of sunflower plant leaves, namely punching, freezing, and/or autoclavation, allowed the diffusion of electrolytes and other nutrients to configure the leaf surface as a natural pad, i.e., creating an “in situ similis” environment suitable for the growth of rarely isolated microbiota. We used surface inoculation and membrane-filtration methods to assess the culturability of endophytic bacteria from the sunflower phyllosphere and rhizosphere. Both methods supported excellent colony-forming unit (CFU) development when compared to standard R2A medium, with a special affinity to support better growth of epiphytic and endophytic populations of the phyllosphere compared with the rhizosphere. A 16S rRNA gene analysis of >122 representative isolates indicated the cultivation of a diverse set of microorganisms by application of the new methods. It indicated the predominance of 13 genera of >30 potential species, belonging to Firmicutes, Proteobacteria, and Actinobacteria, and especially genera not commonly reported for sunflower, e.g., Rhizobium, Aureimonas, Sphingomonas, Paracoccus, Stenotrophomonas, Pantoea, Kosakonia, and Erwinia. The strategy successfully extended diversity and richness in the endophyllosphere compared to the endorhizosphere, while CFUs grown on the standard R2A medium mainly pertain to Firmicutes, especially Bacillus spp. MALDI-TOF MS analysis clustered the isolates according to their niche and potential functions, where the majority of isolates of the endorhizosphere were clustered away from those of the endophyllosphere. Isolates identified as Gammaproteobacteria and Alphaproteobacteria were distinguishably sub-clustered, which was in contrast to the heterogeneous isolates of Firmicutes (Bacillus spp.). In conclusion, leaf in situ similis cultivation is an effective strategy to support the future application of culturomics of plant microbiota. This is an effort to access novel isolates that are more adapted and competitive in their natural environments, especially those subjected to abiotic stresses like those prevailing in arid/semi-arid zones, and, consequently, to support the application of agro-biotechnologies, among other technologies, to improving agriculture in such zones.

ElSawey, H., S. Patz, R. A. Nemr, M. S. Sarhan, M. A. Hamza, H. H. Youssef, M. R. Abdelfadeel, H. - S. A. Daanaa, M. El-Tahan, M. Abbas, et al., "Plant Broth- (Not Bovine-) Based Culture Media Provide the Most Compatible Vegan Nutrition for In Vitro Culturing and In Situ Probing of Plant Microbiota", Diversity, vol. 12, no. 11, 2020. AbstractWebsite

Plant microbiota support the diversity and productivity of plants. Thus, cultivation-dependent approaches are indispensable for in vitro manipulation of hub taxa. Despite recent advances in high-throughput methods, cultivability is lagging behind other environmental microbiomes, notably the human microbiome. As a plant-based culturing strategy, we developed culture media based on a broth of cooked aqueous mixtures of host plants. This improved the in vitro growth of representative isolates of plant microbiota and extended the in situ recovery of plant microbiota. With clover, 16S rRNA gene sequencing of representative isolates confirmed the predominance of Firmicutes, Alphaproteobacteria and Gammaproteobacteria, and less frequently Bacteroidetes and Actinobacteria. Whereas bovine-based culture media (modified R2A) confined the diversity to Firmicutes, the plant broth-based culture media revealed a wider scope of endophytes beyond rhizobia, i.e., multiple genera such as Chryseobacterium, Cronobacter, Kosakonia, Tsukamurella, and a potentially/presumptive novel species. Matrix-assisted laser desorption/ionization time-of-flight (MADI-TOF) analysis clustered isolates according to their plant niches, the endo-phyllosphere/endo-rhizosphere. We recommend the plant broth for simplicity, reproducibility and perdurable storage, supporting future culturomics applications, good laboratory practice (GLP) and good manufacturing practice (GMP). The strategy creates an “in-situ-similis” vegan nutritional matrix to analyze microbial diversity and reveal novel microbial resources pertinent to biotechnological and environmental applications.

A. Daanaa, H. - S., M. Abdou, H. A. Goda, M. T. Abbas, M. A. Hamza, M. S. Sarhan, H. H. Youssef, R. Hamed, M. El-Tahan, M. Fayez, et al., "Plant Pellets: A Compatible Vegan Feedstock for Preparation of Plant-Based Culture Media and Production of Value-Added Biomass of Rhizobia", Sustainability, vol. 12, no. 20, 2020. AbstractWebsite

Although plant-based culture media enhances in vitro cultivation of rhizobacteria, studies assessing their biomass potential for large-scale applications are lacking. Here, we advance plant pellets (PPs) as a novel technology to unlock the potential of such vegan culture media for biomass production of Rhizobium leguminosarum. PP formulations were based on mixtures of Egyptian clover powder and the agro-byproducts glycerol and molasses. These mixtures were either contained or not contained in teabags during culture media preparation. Metrics of biomass included colony forming units, optical density (OD600nm), and cell dry weight (DW). Biomass comparisons between culture media based on PPs and standard yeast extract mannitol (YEM) revealed that the following PPs composition, contained in teabags, cultivated rhizobia at levels comparable to YEM: 16 g clover powder, 5% molasses, and 0.8% glycerol. This PPs composition enabled shorter generation times of rhizobia (PP: 3.83 h, YEM: 4.28 h). Strikingly, PPs mixtures supplemented with 10% molasses and not contained in teabags promoted rhizobia without apparent lag phases and produced 25% greater DW than YEM. PPs potentiate the use of dehydrated vegan feedstocks for both plant microbiota cultivation and biomass production and appear as cost- and labor-effective tools, easy to handle and store for plant-based culture media preparation.

Mourad, E. F., M. S. Sarhan, H. - S. A. Daanaa, M. Abdou, A. T. Morsi, M. R. Abdelfadeel, H. ElSawey, R. Nemr, M. El-Tahan, M. A. Hamza, et al., Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for in vitro Culturing of the Plant Microbiota, , vol. 33, issue 1, pp. 40 - 49, 2018. Abstract
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Sarhan, M. S., S. Patz, M. A. Hamza, H. H. Youssef, E. F. Mourad, M. Fayez, B. Murphy, S. Ruppel, and N. A. Hegazi, G3 PhyloChip Analysis Confirms the Promise of Plant-Based Culture Media for Unlocking the Composition and Diversity of the Maize Root Microbiome and for Recovering Unculturable Candidate Divisions/Phyla, , vol. 33, issue 3, pp. 317 - 325, 2018. Abstract
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Sarhan, M. S., M. A. Hamza, H. H. Youssef, S. Patz, M. Becker, H. ElSawey, R. Nemr, H. - S. A. Daanaa, E. F. Mourad, A. T. Morsi, et al., "Culturomics of the plant prokaryotic microbiome and the dawn of plant-based culture media – A review", Special Issue on Plant Microbiome, vol. 19, pp. 15 - 27, 2019. AbstractWebsite
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Hegazi, N., A. Hartmann, and S. Ruppel, "The plant microbiome: Exploration of plant-microbe interactions for improving agricultural productivity", Special Issue on Plant Microbiome, vol. 19, pp. 1 - 2, 2019. AbstractWebsite
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, "Editorial Board", Special Issue on Plant Microbiome, vol. 19, pp. iii, 2019. AbstractWebsite
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Hegazi, N., A. Hartmann, and S. Ruppel, "The plant microbiome: Exploration of plant-microbe interactions for improving agricultural productivity", Journal of Advanced Research, vol. 19, pp. 1 - 2, 2019. AbstractWebsite
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Kayed, A. S., A. Kandeil, M. R. Gomaa, R. El-Shesheny, S. Mahmoud, M., N. Hegazi, M. Fayez, B. Sheta, P. P. McKenzie, R. J. Webby, et al., "Surveillance for avian influenza viruses in wild birds at live bird markets, Egypt, 2014-2016", Influenza and Other Respiratory VirusesInfluenza and Other Respiratory Viruses, vol. 13, issue 4: John Wiley & Sons, Ltd, pp. 407 - 414, 2019. AbstractWebsite

Aim Egypt is the habitat for a large number of bird species and serves as a vital stopover for millions of migratory birds during their annual migration between the Palearctic and Afrotropical ecozones. Surveillance for avian influenza viruses (AIVs) is critical to assessing risks for potential spreading of these viruses among domestic poultry. Surveillance for AIV among hunted and captured wild birds in Egypt was conducted in order to understand the characteristics of circulating viruses. Methods Sampling of wild bird species occurred in two locations along the Mediterranean Coast of Egypt in the period from 2014 to 2016. A total of 1316 samples (cloacal and oropharyngeal swabs) were collected from 20 different species of hunted or captured resident and migratory birds sold at live bird markets. Viruses were propagated then sequenced. Phylogenetic analysis and receptor binding affinities were studied. Results Eighteen AIVs (1.37%) were isolated from migratory Anseriformes at live bird markets. Further characterization of the viral isolates identified five hemagglutinin (H3, H5, H7, H9, and H10) and five neuraminidase (N1, N2, N3, N6, and N9) subtypes, which were related to isolates reported in the Eurasian region. Two of the 18 isolates were highly pathogenic H5N1 viruses related to clade 2.2.1, while three isolates were G1-like H9N2 viruses. Conclusions Our data show significant diversity of AIVs in Anserifromes sold at live bird markets in Egypt. This allows for genetic exchanges between imported and enzootic viruses and put the exposed humans at a higher risk of infection.

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