Culturable and unculturable potential heterotrophic microbiological threats to the oldest pyramids of the Memphis necropolis, Egypt.

A large percentage of the world's tangible cultural heritage is made from stone; thus, it deteriorates due to physical, chemical, and/or biological factors. The current study explored the microbial community inhabiting two prehistoric sites with high cultural value in the Memphis necropolis of Egypt (Djoser and Lahun Pyramids) using amplicon-based metabarcoding and culture-dependent isolation methods. Samples were examined by epifluorescent microscopy for biological signs before environmental DNA extraction and in vitro cultivation. The metabarcoding analysis identified 644 bacterial species (452 genera) using the 16S rRNA and 204 fungal species (146 genera) using ITS. In comparison with the isolation approach, an additional 28 bacterial species (13 genera) and 34 fungal species (20 genera) were identified. A total of 19 bacterial and 16 fungal species were exclusively culture-dependent, while 92 bacterial and 122 fungal species were culture-independent. The most abundant stone-inhabiting bacteria in the current study were Blastococcus aggregatus, Blastococcus saxobsidens, and Blastococcus sp., among others. The most abundant rock-inhabiting fungi were Knufia karalitana and Pseudotaeniolina globosa, besides abundant unknown Sporormiaceae species. Based on previous reports, microorganisms associated with biodeterioration were detected on color-altered sites at both pyramids. These microorganisms are potentially dangerous as physical and chemical deterioration factors and require proper conservation plans from a microbiological perspective.

aroF and cm2: potential molecular markers for the detection of stone-inhabiting Actinobacteria on cultural heritage sites.

Tangible archeological sites and stone monuments are naturally decayed and deteriorated over time, providing substances that can sustain life, although they provide a complicated ecosystem characterized by low nutrition and desiccation. Stone-inhabiting bacteria (SIB) and especially members of the phylum Actinobacteria dominate such environments, particularly the members of the family Geodermatophilaceae. We used the published data of two confirmed SIB species to mine their genomes for specific molecular markers to rapidly survey the presence of SIB in cultural heritage material prior to further analysis. The search focused on the mycosporine-like amino acids (MAAs) synthesis pathway. MAAs are intracellular compounds biosynthesized by the shikimic acid pathway to synthesize aromatic amino acids and were found related to abiotic resistance features in microorganisms. Based on genome mining, the DAHP II (aroF) and a homolog of the Chorismate mutase gene (cm2) were found mostly in Actinobacteria and few other species. After calibration on five stone-inhabiting Actinobacteria (SIAb) species using conventional PCR, newly designed primers were successfully applied to environmental DNA extracted from two Egyptian pyramidal sites using a qPCR approach. This is the first report of aroF and cm2 as qPCR markers to detect SIAb from cultural heritage material prior to proceeding with further analysis (e.g., metagenomics and meta-barcoding analyses).

An indigenous inland genotype of the black yeast Hortaea werneckii inhabiting the great pyramid of Giza, Egypt.

Within the context of cultural heritage conservation, the biological study of tangible archeological sites is an important task to extend their existence and strengthen the transmission of their cultural value to future generations. In Egypt, a hyper-arid region, a microcolonial fungus with inky black growth was observed on a stone surface in the royal corridor of the Great Pyramid of Giza (King Khufu's pyramid). The isolate was studied and characterized by microscopic morphometric measurements, evaluation of enzymatic activities, and genotyping techniques. The isolate was identified as Hortaea werneckii, a pleomorphic black yeast that naturally inhabits hypersaline environments and infects human skin. It has been reported from humid temperate, subtropical, and tropical zones, mainly from marine habitats and adjacent areas, and is associated with marine life. Since it was observed in an unusual habitat, it raises the question of its type and origin, whether environmental or clinical. The Egyptian Hortaea werneckii GPS5 isolate was profiled and characterized by adaptive extremophilic tolerance to arid salt stress, low portability to infect human skin, and the capability of solubilizing calcite; besides it was phylogenetically clustered with previous recorded environmental accessions. A profile that matches the biodeterioration fungal agents known as rock-inhabiting fungi, a potential threat to cultural heritage sites that requires attention and prevention plans.

Microbial Profiling of Potato-Associated Rhizosphere Bacteria under Bacteriophage Therapy.

Potato soft rot and wilt are economically problematic diseases due to the lack of effective bactericides. Bacteriophages have been studied as a novel and environment-friendly alternative to control plant diseases. However, few experiments have been conducted to study the changes in plants and soil microbiomes after bacteriophage therapy. In this study, rhizosphere microbiomes were examined after potatoes were separately infected with three bacteria (Ralstonia solanacearum, Pectobacterium carotovorum, Pectobacterium atrosepticum) and subsequently treated with a single phage or a phage cocktail consisting of three phages each. Results showed that using the phage cocktails had better efficacy in reducing the disease incidence and disease symptoms' levels when compared to the application of a single phage under greenhouse conditions. At the same time, the rhizosphere microbiota in the soil was affected by the changes in micro-organisms' richness and counts. In conclusion, the explicit phage mixers have the potential to control plant pathogenic bacteria and cause changes in the rhizosphere bacteria, but not affect the beneficial rhizosphere microbes.

A New Extremotolerant Ecotype of the Fungus Pseudotaeniolina globosa Isolated from Djoser Pyramid, Memphis Necropolis, Egypt.

Most of the rock-inhabiting fungi are meristematic and melanized microorganisms often associated with monument biodeterioration. In previous microbial profiling of the Egyptian Djoser pyramid, a Pseudotaeniolina globosa isolate was found. The current study aimed to characterize the P. globosa isolated from the Djoser pyramid compared with an Italian isolate at morphological, physiological, and molecular levels. Experiments were carried out to test temperature, salinity, and pH preferences, as well as stress tolerance to UV radiation and high temperature, in addition to a multi-locus genotyping using ITS, nrSSU or 18S, nrLSU or 28S, BT2, and RPB2 markers. Morphological and molecular data confirmed the con-specificity of the two isolates. However, the Egyptian isolate showed a wider range of growth at different environmental conditions being much more tolerant to a wider range of temperature (4-37 °C) and pH values (3.0-9.0 pH) than the Italian (10-30 °C, 4.0-6.0 pH), and more tolerant to extreme salinity levels (5 M NaCl), compared to the lowest in the Italian isolate (0.2 M NaCl). Besides, the Egyptian isolate was more tolerant to high temperature than the Italian isolate since it was able to survive after exposure to up to 85 °C for 5 min, and was not affected for up to 9 h of UV exposure, while the Italian one could not regrow after the same treatments. The Pseudotaeniolina globosa species was attributed to the family Teratosphaeriaceae of the order Capnodiales, class Dothideomycetes. Our results demonstrated that the Egyptian isolate could be considered an ecotype well adapted to harsh and extreme environments. Its potential bio-deteriorating effect on such an important cultural heritage requires special attention to design and conservation plans and solutions to limit its presence and extension in the studied pyramid and surrounding archaeological sites.

Peripatric speciation associated with genome expansion and female-biased sex ratios in the moss genus Ceratodon.

PREMISE OF THE STUDY: A period of allopatry is widely believed to be essential for the evolution of reproductive isolation. However, strict allopatry may be difficult to achieve in some cosmopolitan, spore-dispersed groups, like mosses. We examined the genetic and genome size diversity in Mediterranean populations of the moss Ceratodon purpureus s.l. to evaluate the role of allopatry and ploidy change in population divergence. METHODS: We sampled populations of the genus Ceratodon from mountainous areas and lowlands of the Mediterranean region, and from Western and Central Europe. We performed phylogenetic and coalescent analyses on sequences from five nuclear introns and a chloroplast locus to reconstruct their evolutionary history. We also estimated genome size using flow cytometry (employing propidium iodide) and determined the sex of samples using a sex-linked PCR marker. KEY RESULTS: Two well-differentiated clades were resolved, discriminating two homogeneous groups: the widespread C. purpureus and a local group mostly restricted to the mountains in Southern Spain. The latter also possessed a genome size 25% larger than the widespread C. purpureus, and the samples of this group consist entirely of females. We also found hybrids, and some of them had a genome size equivalent to the sum of the C. purpureus and Spanish genome, suggesting that they arose by allopolyploidy. CONCLUSIONS: These data suggest that a new species of Ceratodon arose via peripatric speciation, potentially involving a genome size change and a strong female-biased sex ratio. The new species has hybridized in the past with C. purpureus.