Development of a Simple Extraction Method for Tetracycline Analogues from Bovine Milk

Tetracycline and analogues are among the most used antibiotics in the dairy industry. Besides the therapeutic uses, tetracyclines are often incorporated into livestock feed as growth promoters. A considerable amount of antibiotics is released unaltered through milk from dairy animals. The presence of antibiotic residues in milk and their subsequent consumption can lead to potential health impacts, including cancer, hypersensitivity reactions, and the development of antibiotic resistance. Thus, it is important to monitor residual levels of tetracyclines in milk. The purpose of this study is to develop a quick and simple method for simultaneously extracting five tetracycline analogues from bovine milk. Specifically, five tetracycline analogues: Chlortetracycline (CTC), demeclocycline (DEM), doxycycline (DC), minocycline (MC), and tetracycline (TC) were simultaneously extracted from milk using trifluoroacetic acid. Subsequently, the extracted analogues were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and detected at 355 nm using UV/Vis. Calibration curves for all five tetracycline analogues show excellent linearity (r2 value > 0.99). Percent recovery for MC, TC, DEM, CTC, and DC were: 31.88%, 96.91%, 151.29, 99.20%, and 85.58% respectively. The developed extraction method has good precision (RSD < 9.9% for 4 of the 5 analogues). The developed method with minimal sample preparation and pretreatment has the potential to serve as an initial screening test.

The contribution of fungi to the global economy

Fungi provide ecological and environmental services to humans, as well as health and nutritional benefits, and are vital to numerousindustries. Fermented food and beverage products from fungi are circulating in the market, generating billions of USD.However, the highest potential monetary value of fungi is their role in blue carbon trading because of their ability to sequesterlarge amounts of carbon in the soil. There are no conclusive estimates available on the global monetary value of fungi, primarilybecause there are limited data for extrapolation. This study outlines the contribution of fungi to the global economy and providesa first attempt at quantifying the global monetary value of fungi. Our estimate of USD 54.57 trillion provides a starting point thatcan be analysed and improved, highlighting the significance of fungi and providing an appreciation of their value. This paperidentifies the different economically valuable products and services provided by fungi. By giving a monetary value to all importantfungal products, services, and industrial applications underscores their significance in biodiversity and conservation. Furthermore,if the value of fungi is well established, they will be considered in future policies for effective ecosystem management.

Phylogenetic and chemotaxonomic resolution of the genus Annulohypoxylon(Xylariaceae)including four new species

This study deals with an extensive evaluation focusing on phylogenetic and chemotaxonomic infrageneric relationships of the genus Annulohypoxylon(Xylariaceae,Ascomycota),whose species are ubiquitously associated with seed plants as endophytes and saprotrophs in all forested areas of the world.Using evidence from phylogenetic,chemotaxonomic and morphological data,various varieties within the genus are raised to species level,leading to the new combinations for A.areolatum(:A.bovei var.microsporum),A.macrosporum(:A.leptascum var.macrosporum),and A.microdiscum(:A.moriforme var.microdiscum).Annulohypoxylon substygium nom.nov.is applied for A.stygium var.annulatumand the four new tropical and subtropical species A.massivum,A.violaceopigmentum,A.viridistratum and A.yungensis are introduced.Furthermore,A.leucadendri is excluded from the genus as its morphological characters disagree with the generic concept,the recently erected A.palmicola is synonymized with A.leptascum and A.austrobahiense has been reassigned to the genus Hypoxylon.In addition,the key taxa A.annulatum and A.truncatum have been reinvestigated and epitypes have been defined.For the first time,a dichotomous key to the genus is provided.A phylogenetic b-tubulin tree in conjunction with stromatal HPLC profiles clearly shows that Annulohypoxylon comprises two distinct lineages.The A.cohaerens/multiforme group might eventually warrant segregation into a new genus as further molecular data become available.

Elucidation of the life cycle of the endophytic genus Muscodor and its transfer to Induratia in Induratiaceae fam. nov., based on a polyphasic taxonomic approach

Molecular phylogenetic studies of cultures derived from some specimens of plant-inhabiting Sordariomycetes using ITS,LSU,rpb2 and tub2 DNA sequence data revealed close affinities to strains of Muscodor.The taxonomy of this biotechnologi-cally important genus,which exclusively consists of endophytes with sterile mycelia that produce antibiotic volatile secondary metabolites,was based on a rather tentative taxonomic concept.Even though it was accommodated in Xylariaceae,its phylo-genetic position had so far remained obscure.Our phylogeny shows that Muscodor species have affinities to the xylarialean genera Emarcea and Induratia,which is corroborated by the fact that their sexual states produce characteristic apiospores.These data allow for the integration of Muscodor in Induratia,i.e.the genus that was historically described first.The multi-locus phylogenetic tree clearly revealed that a clade comprising Emarcea and Induratia forms a monophylum separate from representatives of Xylariaceae,for which we propose the new family Induratiaceae.Divergence time estimations revealed that Induratiaceae has been diverged from the Xylariaceae+Clypeosphaeriaceae clade at 93(69-119)million years ago(Mya)with the crown age of 61(39-85)Mya during the Cretaceous period.The ascospore-derived cultures were studied for the production of volatile metabolites,using both,dual cultures for assessment of antimicrobial effects and extensive analyses using gas chromatography coupled with mass spectrometry(GC-MS).The antimicrobial effects observed were significant,but not as strong as in the case of the previous reports on Muscodor species.The GC-MS results give rise to some doubt on the validity of the previous identification of certain volatiles.Many peaks in the GC-MS chromatograms could not be safely identified by database searches and may represent new natural products.The isolation of these compounds by preparative chromatography and their subsequent characterisation by nuclear magnetic resonance(NMR)spectroscopy or total synthesis will allow for a more concise identification of these volatiles,and they should also be checked for their individual contri-bution to the observed antibiotic effects.This will be an important prerequisite for the development of biocontrol strains.

Lenormandins A—G,new azaphilones from Hypoxylon lenormandii and Hypoxylon jaklitschii sp.nov.,recognised by chemotaxonomic data

Seven new azaphilone pigments named lenormandins A–G were isolated from stromata(fruiting bodies)of the xylariaceous fungus,Hypoxylon lenormandii using preparative High Performance Liquid Chromatography(HPLC)and their structures were elucidated by NMR spectroscopy,high resolution mass spectrometry and other spectral methods.Their occurrence in herbarium specimens(including various type materials collected in the 19th and early 20th century)and several fresh collections was studied by analytical HPLC with diode array and mass spectrometric detection(HPLC-DAD/MS),revealing that this group of pigments is specific for Hypoxylon lenormandii from various geographic regions and otherwise only occurs in closely related taxa,i.e.H.sublenormandii and an undescribed fungus from Sri Lanka,for which we propose the name H.jaklitschii.The status of the new species was established by using molecular phylogenetic data based on ITS and partial beta-tubulin sequences,and by detailed morphological studies.

High quality genome sequences of thirteen Hypoxylaceae(Ascomycota)strengthen the phylogenetic family backbone and enable the discovery of new taxa

The Hypoxylaceae(Xylariales,Ascomycota)is a diverse family of mainly saprotrophic fungi,which commonly occur in angiosperm-dominated forests around the world.Despite their importance in forest and plant ecology as well as a prolific source of secondary metabolites and enzymes,genome sequences of related taxa are scarce and usually derived from envi-ronmental isolates.To address this lack of knowledge thirteen taxonomically well-defined representatives of the family and one member of the closely related Xylariaceae were genome sequenced using combinations of Illumina and Oxford nanopore technologies or PacBio sequencing.The workflow leads to high quality draft genome sequences with an average N50 of 3.0 Mbp.A backbone phylogenomic tree was calculated based on the amino acid sequences of 4912 core genes reflecting the current accepted taxonomic concept of the Hypoxylaceae.A Percentage of Conserved Proteins(POCP)analysis revealed that 70%of the proteins are conserved within the family,a value with potential application for the definition of family boundaries within the order Xylariales.Also,Hypomontagnella spongiphila is proposed as a new marine derived lineage of Hypom.monticulosa based on in-depth genomic comparison and morphological differences of the cultures.The results showed that both species share 95%of their genes corresponding to more than 700 strain-specific proteins.This difference is not reflected by standard taxonomic assessments(morphology of sexual and asexual morph,chemotaxonomy,phylogeny),preventing species delimitation based on traditional concepts.Genetic changes are likely to be the result of environmental adaptations and selective pressure,the driving force of speciation.These data provide an important starting point for the establishment of a stable phylogeny of the Xylariales;they enable studies on evolution,ecological behavior and biosynthesis of natural products;and they significantly advance the taxonomy of fungi.