Renan do Nascimento Barbosa

Endophytic fungi from medicinal plant Bauhinia forficata: Diversity and biotechnological potential

Bauhinia forficata is native to South America and used with relative success in the folk medicine in Brazil. The diversity, antibacterial activity, and extracellular hydrolytic enzymes of endophytic fungi associated with this plant were studied. Plant samples, which included leaves, sepals, stems, and seeds, were used. Ninety-five endophytic fungal were isolated (18 from leaves, 22 from sepals, 46 from stems, and nine from seeds), comprising 28 species. The most frequently isolated species were Acremonium curvulum (9.5%), Aspergillus ochraceus (7.37%), Gibberella fujikuroi (10.53%), Myrothecium verrucaria (10.53%) and Trichoderma piluliferum (7.37%). Diversity and species richness were higher in stem tissues, and Sorensen’s index of similarity between the tissues was low. Eleven fungi showed antibacterial activity. Aspergillus ochraceus , Gibberella baccata , Penicillium commune , and P. glabrum were those with the greatest antibacterial activity against Staphylococcus aureus and/or Streptococcus pyogenes . Thirteen species showed proteolytic activity, particularly Phoma putaminum . Fourteen species were cellulase positive, particularly the Penicillium species and Myrmecridium schulzeri . All isolates tested were xylanase positive and 10 showed lipolytic activity, especially Penicillium glabrum . It is clear that the endophytic fungi from B. forficata have potential for the production of bioactive compounds and may be a source of new therapeutic agents for the effective treatment of diseases in humans, other animals, and plants. To our knowledge, this is the first study of endophytic fungi from different tissues of B. forficata and their biotechnological potential.

Phylogenetic analysis of Monascus and new species from honey, pollen and nests of stingless bees

The genus Monascus was described by van Tieghem (1884) to accommodate M. ruber and M. mucoroides, two species with non-ostiolate ascomata. Species delimitation in the genus is still mainly based on phenotypic characters, and taxonomic studies that include sequence data are limited. The genus is of economic importance. Species are used in fermented Asian foods as food colourants (e.g. ‘red rice’ (ang-kak, angka)) and found as spoilage organisms, and recently Monascus was found to be essential in the lifecycle of stingless bees. In this study, a polyphasic approach was applied combining morphological characters, ITS, LSU, β-tubulin, calmodulin and RNA polymerase II second largest subunit sequences and extrolite data, to delimit species and to study phylogenetic relationships in Monascus. Furthermore, 30 Monascus isolates from honey, pollen and nests of stingless bees in Brazil were included. Based on this polyphasic approach, the genus Monascus is resolved in nine species, including three new species associated with stingless bees (M. flavipigmentosus sp. nov., M. mellicola sp. nov., M. recifensis sp. nov., M. argentinensis, M. floridanus, M. lunisporas, M. pallens, M. purpureus, M. ruber), and split in two new sections (section Floridani sect. nov., section Rubri sect. nov.). Phylogenetic analysis showed that the xerophile Monascus eremophilus does not belong in Monascus and monophyly in Monascus is restored with the transfer of M. eremophilus to Penicillium (P. eremophilum comb. nov.). A list of accepted and excluded Monascus and Basipetospora species is given, together with information on (ex-)types cultures and barcode sequence data.

Experimental white piedra: a robust approach to ultrastructural analysis, scanning electron microscopy, and etiological discoveries

White piedra is a fungal infection characterized by nodules comprised of Trichosporon species and restricted to the extrafollicular portion of the hair shaft. The diagnosis is based on clinical and mycological characteristics, and must be confirmed with a precise identification of the etiological agent. This research aimed to develop an in vitro infection model of white piedra and analyze its morphological and ultra‐structural aspects. In the process, hair infection was induced using eight isolates of the genus Trichosporon maintained in the Culture Collection Micoteca URM. The ITS and IGS1 regions were sequenced for taxonomic confirmation. Scanning Electron Microscope (SEM) was performed at the Strategic Center for Northeast Technologies (CETENE). The scanning electron microscope was equipped with an Energy Dispersion Spectrometer (EDS). The Trichosporon isolates were identified as Trichosporon asahii (6) and Trichosporon montevideense (2) by internal transcript spacer (ITS) region and intergenic spacer 1 region (IGS1) sequencing. All eight strains were used to induce the in vitro hair infection, and nodules formed after the incubation period. Temperature variations and high humidity were not observed to be related to the development of this hair disease. The main chemical constituents detected in the nodules were carbon, nitrogen and oxygen, as well as a low level of sulfur. The absence of calcium, combined with the low level of sulfur, might explain the soft nature of the white piedra nodules. This study demonstrated that several Trichosporon species may be responsible for causing white piedra.

New Penicillium and Talaromyces species from honey, pollen and nests of stingless bees

Penicillium and Talaromyces species have a worldwide distribution and are isolated from various materials and hosts, including insects and their substrates. The aim of this study was to characterize the Penicillium and Talaromyces species obtained during a survey of honey, pollen and the inside of nests of Melipona scutellaris. A total of 100 isolates were obtained during the survey and 82% of those strains belonged to Penicillium and 18% to Talaromyces. Identification of these isolates was performed based on phenotypic characters and β-tubulin and ITS sequencing. Twenty-one species were identified in Penicillium and six in Talaromyces, including seven new species. These new species were studied in detail using a polyphasic approach combining phenotypic, molecular and extrolite data. The four new Penicillium species belong to sections Sclerotiora (Penicillium fernandesiae sp. nov., Penicillium mellis sp. nov., Penicillium meliponae sp. nov.) and Gracilenta (Penicillium apimei sp. nov.) and the three new Talaromyces species to sections Helici (Talaromyces pigmentosus sp. nov.), Talaromyces (Talaromyces mycothecae sp. nov.) and Trachyspermi (Talaromyces brasiliensis sp. nov.). The invalidly described species Penicillium echinulonalgiovense sp. nov. was also isolated during the survey and this species is validated here.

Requalification of a Brazilian Trichoderma Collection and Screening of Its Capability to Decolourise Real Textile Effluent

Water contamination with large amounts of industrial textile coloured effluents is an environmental concern. For the treatment of textile effluents, white-rot fungi have received extensive attention due to their powerful capability to produce oxidative (e.g., ligninolytic) enzymes. In addition, other groups of fungi, such as species of Aspergillus and Trichoderma, have also been used for textile effluents treatment. The main aim of the present study was to requalify a Brazilian Trichoderma culture collection of 51 Trichoderma strains, isolated from different sources in Brazil and preserved in the oldest Latin-American Fungal Service Culture Collection, The Micoteca URM WDCM 804 (Recife, Brazil). Fungal isolates were re-identified through a polyphasic approach including macro- and micro-morphology and molecular biology, and screened for their capability to decolourise real effluents collected directly from storage tanks of a textile manufacture. Trichoderma atroviride URM 4950 presented the best performance on the dye decolourisation in real textile effluent and can be considered in a scale-up process at industrial level. Overall, the potential of Trichoderma strains in decolourising real textile dye present in textile effluent and the production of the oxidative enzymes Lac, LiP and MnP was demonstrated. Fungal strains are available in the collection e-catalogue to be further explored from the biotechnological point of view.

INIBIÇÃO DE PATÓGENOS POR BACTEIAS ESPORULANTES ISOLADAS DE MEL DE Melipona sp. (APIDAE: APINAE: MELIPONINI)

The aim of this study was to isolate sporogenic bacteria from the honey of stingless bees Melipona sp., in dry forest, and to evaluate their antagonistic potential for medicinal employment purposes and animal production. The honey samples were collected in Serra Talhada-PE, where honey was taken from four different hives (in triplicate), totaling 12 samples. The samples were diluted and subjected to 80 oC for 20 minutes to eliminate vegetative cells. The dilutions were plated onto nutrient agar and incubated at 30 oC for 72 hours. Then the colony forming units (CFU) were quantified. The samples were also plated onto malt agar and Sabouraud agar, and incubated at 30 oC for 14 days for the growth of yeast and molds. Total and fecal coliforms were quantified by the most probable number method (MPN). Seven isolates (I) of sporogenic bacteria (Bacillus) were obtained, however only four showed probiotic potential. Isolate I-5 showed the greatest probiotic potential and inhibited the growth of Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Salmonella sp., and Staphylococcus aureus. The growth of the Sarcina sp. was not inhibited by any isolate. No yeast, molds or coliforms were found. The Melipona sp. honey is a source of spore-forming bacteria and is antagonistic to microorganisms that contaminate honey. It has good microbiological quality.