Polyporoid fungi of Tanzania : Taxonomy, transcriptomics and biochemical analyses of Kusaghiporia usambarensis and Piptoporellus baudonii

Polyporoid fungi refers to basidiomycetes with fruiting bodies with the hymenium located to the inner surfaces of pores or narrow tubes. The majority of polyporoids belongs to Polyporales. Most Polyporales are saprobes, but some are plant pathogens. The overall aim of this thesis was to study the taxonomy, systematics and chemistry of the two species Kusaghiporia usambarensis (saprobic) and Piptoporellus baudonii (a plant pathogen) collected from Tanzania, using morphological and molecular approaches, combined with transcriptomics and pharmacognostic investigations. The main contribution of this thesis includes the description a new genus with the new species K. usambarensis from the Usambara Mountains, Tanzania; investigation of the chemical composition of volatile compounds from this medicinal mushroom; isolation and structure determination of a novel and most abundant peptide in K. usambarensis, and further to elucidate the phylogenetic position of Piptoporellus baudonii (formerly known as Laetiporus baudonii) by using a four molecular markers dataset. Paper I was conducted applying a classical taxonomic approach, including both morphological and phylogenetic analyses, to describe a new genus and species K. usambarensis. Paper II, investigated volatiles and volatile derivatives in dichloromethane extracts of K. usambarensis analysed by GC-MS and NMR spectroscopy. The main elements were phenols, and esters, compounds that may explain the formerly reported antioxidant activity and traditional medicinal use of the mushroom. In paper III, screening of peptides in K. usambarensis revealed a novel cysteine-rich peptide, highly expressed at gene level and the most abundant compound in the fruiting body. Combined LC-MS and transcriptome analyses were used to determine the peptide sequence, and subsequently NMR spectroscopy to determine the 3D structure of the novel peptide, kusaghitide. In paper IV molecular techniques were used to elucidate the phylogenetic position of the parasitic Laetiporus baudonii. Phylogenetic analyses of combined 5.8S, nrLSU, nrSSU, and TEF1 gene sequences placed L. baudonii in the genus Piptoporellus, hence the new combination Piptoporellus baudonii was proposed. This thesis has contributed to build capacity in the fields of mycology, systematics and pharmacognosy in order to reinforce ecological knowledge and ethnopharmaceutical research for future drug discovery in Tanzania and Africa at large

Polyporoid fungi of Tanzania : Taxonomy, transcriptomics and biochemical analyses of Kusaghiporia usambarensis and Piptoporellus baudonii

Polyporoid fungi refers to basidiomycetes with fruiting bodies with the hymenium located to the inner surfaces of pores or narrow tubes. The majority of polyporoids belongs to Polyporales. Most Polyporales are saprobes, but some are plant pathogens. The overall aim of this thesis was to study the taxonomy, systematics and chemistry of the two species Kusaghiporia usambarensis (saprobic) and Piptoporellus baudonii (a plant pathogen) collected from Tanzania, using morphological and molecular approaches, combined with transcriptomics and pharmacognostic investigations. The main contribution of this thesis includes the description a new genus with the new species K. usambarensis from the Usambara Mountains, Tanzania; investigation of the chemical composition of volatile compounds from this medicinal mushroom; isolation and structure determination of a novel and most abundant peptide in K. usambarensis, and further to elucidate the phylogenetic position of Piptoporellus baudonii (formerly known as Laetiporus baudonii) by using a four molecular markers dataset. Paper I was conducted applying a classical taxonomic approach, including both morphological and phylogenetic analyses, to describe a new genus and species K. usambarensis. Paper II, investigated volatiles and volatile derivatives in dichloromethane extracts of K. usambarensis analysed by GC-MS and NMR spectroscopy. The main elements were phenols, and esters, compounds that may explain the formerly reported antioxidant activity and traditional medicinal use of the mushroom. In paper III, screening of peptides in K. usambarensis revealed a novel cysteine-rich peptide, highly expressed at gene level and the most abundant compound in the fruiting body. Combined LC-MS and transcriptome analyses were used to determine the peptide sequence, and subsequently NMR spectroscopy to determine the 3D structure of the novel peptide, kusaghitide. In paper IV molecular techniques were used to elucidate the phylogenetic position of the parasitic Laetiporus baudonii. Phylogenetic analyses of combined 5.8S, nrLSU, nrSSU, and TEF1 gene sequences placed L. baudonii in the genus Piptoporellus, hence the new combination Piptoporellus baudonii was proposed. This thesis has contributed to build capacity in the fields of mycology, systematics and pharmacognosy in order to reinforce ecological knowledge and ethnopharmaceutical research for future drug discovery in Tanzania and Africa at large

The effects of bee species and vegetation on the antioxidant properties of honeys produced by Afrotropical stingless bees (Hymenoptera, Apidae, Meliponini)

Stingless bee honey is widely utilized as a traditional medicine in tropical and subtropical regions. The rich diversity of stingless bees and floral resources in the African tropics presents an opportunity for the production of honeys with diverse therapeutic properties. This study investigated the influence of bee species and vegetation on the antioxidant properties of stingless bee honey. Honey samples were collected from six species of Afrotropical stingless bees in six different vegetation zones of Tanzania, and tested for phytochemical composition and antioxidant activity. Our findings demonstrate that the antioxidant properties of stingless bee honey were influenced by both bee species and vegetation, with the former exhibiting a stronger effect. Honeys produced by different stingless bee species varied significantly in all of the assayed parameters, whereas honey samples from different vegetation zones differed in 80% of the parameters. Further, there was a clear separation of honey samples based on bee species on the non-metric multidimensional scaling (NMDS) and principal component analysis (PCA) plots which suggests that the antioxidant properties can be used as markers for discriminating honeys produced by different bee species. The studied stingless bee species across different vegetation zones produced honeys with remarkable phenolic content (46.80–365.17 mg GAE/100 g), flavonoid content (27.49–210.83 mg QE/100 g), vitamin C (7.42–60.50 mg/100 g), radical scavenging activity (32.75–92.50%) and ferric reducing antioxidant power (68.11–221.23 μmol Fe (II)/100 g). Stingless bee honey presents a promising niche for pharmaceutical research and innovation, particularly for Dactylurina schmidti honey which demonstrated exceptional levels of phytochemicals and antioxidant activity. We encourage future studies to investigate the biologically active components in honeys produced by Afrotropical stingless bees, paving the way for potential applications of stingless bee honey in clinical therapy

Discrimination of honeys produced by Tanzanian stingless bees (Hymenoptera, Apidae, Meliponini) based on physicochemical properties and sugar profiles

Honey composition vary according to floral origin and bee species from which it is produced. Understanding the composition of honey is essential for guaranteeing its quality and authenticity, and revealing its potential benefits. Studies on honeys produced by Afrotropical stingless bees are scarce resulting into limited understanding and underutilization. The current study investigated the physicochemical properties and sugar profiles of honeys produced by six stingless bee species (Dactylurina schmidti, Hypotrigona gribodoi, Meliponula beccarii, Meliponula ferruginea, Meliponula togoensis and Plebeina armata) from different vegetation zones of Tanzania. Permutation analysis of variance (PERMANOVA) and Kruskal-Wallis tests were conducted to assess how bee species and vegetation influenced the studied parameters. Furthermore, we employed principal component analysis (PCA) and linear discriminant analysis (LDA) to discriminate honey samples according to bee species and vegetation zones. Our findings show that honey composition was greatly influenced by the identity of bee species which affected 84.6 % of the studied parameters, compared to vegetation zones which affected only 23.1 % of the studied parameters. Stingless bee honey showed significant variation from the international quality standards established for Apis mellifera honey. Also, the levels of free acidity (32.05–99.95 meq/Kg), ash content (0.09–0.81 % w/w) and reducing sugars (43.79–50.82 g/100 g) in some honey samples deviated from the East African Community standards for stingless bee honey. PCA and LDA revealed that the physicochemical properties and sugar profiles can be used to classify stingless bee honeys according to their bee species origin. We recommend further studies to elucidate essential markers that can be used in identifying the entomological source of stingless bee honey

Phylogenetic position and taxonomy of <i>Kusaghiporia usambarensis</i> gen. et sp. nov. (Polyporales)

<p>A large polyporoid mushroom from the West Usambara Mountains in North-eastern Tanzania produces dark brown, up to 60-cm large fruiting bodies that at maturity may weigh more than 10 kg. It has a high rate of mycelial growth and regeneration and was found growing on both dry and green leaves of shrubs; attached to the base of living trees, and it was also observed to degrade dead snakes and insects accidentally coming into contact with it. Phylogenetic analyses based on individual and concatenated data sets of nrLSU, nrSSU and the RPB2 and TEF1 genes showed it, together with <i>Laetiporus, Phaeolus, Pycnoporellus</i> and <i>Wolfiporia</i>, to form a monophyletic group in <i>Polyporales</i>. Based on morphological features and molecular data, it is described as <i>Kusaghiporia usambarensis</i>.</p