Effects of Culture Conditions on Antimicrobial Activity of Ganoderma resinaceum (Agaricomycetes) Extracts.

Among many sources of natural bioactive substances, mushrooms constitute a huge and mostly unexplored group. Biologically active secondary metabolites of Ganoderma, a group of wood-degrading mushrooms, have recently been reviewed. Our previous study revealed the antimicrobial activity of extracts from G. resinaceum grown in submerged culture against phytopathogens. Different factors can influence the production of secondary metabolites, including nutritional factors. In this study we evaluated the influence of different culture conditions on the antimicrobial activity of extracts from liquid cultures of G. resinaceum, through use of a factorial design. Minimum inhibitory concentrations for extracts produced under different culture conditions were determined against Staphylococcus aureus and Xanthomonas vesicatoria. Based on the results of these assays, larger-scale cultures in malt extract broth supplemented with 20 g/L glucose and a 15-day incubation time should be performed in order to isolate from G. resinaceum antibiotic compound(s) that are potentially useful against S. aureus. In addition, pH 5 should be considered for the production of antimicrobial metabolites against X. vesicatoria from supernatant broths or extracts from G. resinaceum.

Anti-MRSA Activity of Fruiting Body Extracts of Spectacular Rustgill Mushroom, Gymnopilus junonius (Agaricomycetes).

Despite the great advances in chemotherapeutics, infectious diseases are still one of the leading causes of death worldwide. Among some of the clinically relevant pathogens, methicillin-resistant Staphylococcus aureus (MRSA) ranks as one of the most difficult bacteria to treat. It is a common cause of skin, soft-tissue, and endovascular infections, as well as pneumonia, septic arthritis, endocarditis, osteomyelitis, and sepsis. The research on Basidiomycota is extensive; many species show a broad spectrum of pharmacological activities, including antimicrobial activity. The vast majority of the literature to date generally focuses on screening the antibacterial properties of mushroom extracts. A gap still exists in the identification of the individual compounds responsible for these properties, and few low molecular weight compounds have been described. Gymnopilus junonius, the big laughter mushroom, grows wild in Uruguay, especially on Eucalyptus spp. plantations; it is known as the "eucalyptus fungus." In this work, we report the bioguided isolation, structural elucidation, and antistaphylococcal activity of the main antimicrobial components of fresh basidiocarps of G. junonius.

Functional characterization of two novel purine transporters from the Basidiomycota Phanerochaete chrysosporium.

Purine transporters as substrate entry points in organisms, are involved in a number of cellular processes such as nitrogen source uptake, energy metabolism and synthesis of nucleic acids. In this study, two nucleobase transporter genes (phZ, phU) from Phanerochaete chrysosporium were cloned, identified, and functionally characterized. Our results show that PhZ is a transporter of adenine and hypoxanthine, and a protein belonging to the AzgA-like family, whilst PhU belongs to the NAT/NCS2 family, transporting xanthine and uric acid. No other sequences belonging to these families were detected in P. chrysosporium's genome. Phylogenetic analyses show that AzgA-like sequences form monophyletic groups for each major lineage (Ascomycota, Basidiomycota and Zygomycota). In contrast, Ascomycota and Basidiomycota NAT/NCS2 sequences do not form monophyletic groups and several copies of this protein are distributed across the tree. Expression of phU was significantly downregulated in the presence of a primary source like ammonium, and enhanced if purines were present or if the mycelium was nitrogen starved. phZ was clearly induced by its substrates (hypoxanthine, adenine), very lightly induced by xanthine, suppressed by urea and amino acids and expressed at a basal level when uric acid or ammonium was the nitrogen source or when the mycelium was starved for nitrogen. In order to perform substrate analyses, both P. chrysosporium proteins (PhZ, PhU) were expressed in Aspergillus nidulans. Epifluorescent microscopy showed that under inducing conditions, PhZ-GFP and PhU-GFP were present at the plasma membrane of A. nidulans transformed strains, and were internalized in repressed conditions. Our results suggest that in the white-rot fungus P. chrysosporium, phU has a catabolic role and phZ, (less dependent of the nitrogen source), plays a key role in purine acquisition to provide biosynthetic components. These are the first purine transporters characterized in Basidiomycota.

Screening for Antimicrobial Activity of Wood Rotting Higher Basidiomycetes Mushrooms from Uruguay against Phytopathogens.

In this work, the antimicrobial activity of extracts of wood rotting higher Basidiomycetes mushrooms isolated from Eucalyptus plantations in Uruguay was studied using bacterial and fungal phytopathogens as targets. Fifty-one extracts from mycelia and growth broth were prepared from higher Basidiomycetes mushrooms, from which eight extracts (from Ganoderma resinaceum, Laetiporus sulphureus, Dictyopanus pusillus, and Bjerkandera adusta) showed antimicrobial activity against Xanthomonas vesicatoria, Aspergillus oryzae, Penicillium expansum, Botrytis cinerea, and Rhizopus stolonifer as assayed in the qualitative test. The minimum inhibitory concentration (MIC) for those fungal extracts was determined and the results showed that L. sulphureus deserved further study, with low MIC values against X. vesicatoria. The antimicrobial activity of L. sulphureus culture broth extracts grown under different culture conditions was evaluated against X. vesicatoria. From the results of these assays, larger-scale cultures for the production of the compound(s) with antimicrobial activity should be performed using malt extract broth, at pH 5, at 20°C and static culture conditions.

A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification).

Purification and applications of a lectin from the mushroom Gymnopilus spectabilis.

A lectin was isolated from fruiting bodies of the mushroom Gymnopilus spectabilis (GSL) by ionic exchange chromatography. The lectin agglutinates mouse red cells exhibiting broad specificity towards several monosaccharides including the N-acetylneuraminic acid. Agglutination was also inhibited by the glycoproteins: fetuin, lactoferrin, and recombinant erythropoietin. GSL is a glycoprotein possessing 16 % of carbohydrates; the SDS-PAGE showed two bands with molecular mass of 52.1 and 64.4 kDa. Isoelectric focusing displayed microheterogeneity, with two bands at pIs 5.1 and 5.3. The lectin was stable between pH 2 and pH 8 while at pH 10, the agglutination decayed to 50 % of initial activity. Incubation at 40 and 80 °C led to 50 and 100 % loss in activity of the lectin, respectively. Synthesized GSL-Sepharose interacts with serum pregnant mare gonadotropin, and at least two subpopulations of this glycoprotein were separated. There was no interaction between transferrin and soluble GSL while a partial recognition was achieved with GSL-Sepharose. The terminal sialic acid seems to play an active role in modifying the interaction with GSL, depending if the lectin is in a soluble or immobilized form. The purified lectin inhibited in vitro the growth of Staphylococcus aureus and Aspergillus niger.

Screening for lectins from basidiomycetes and isolation of Punctularia atropurpurascens lectin.

Aqueous extracts of basidiomycete fungi were screened for the presence of lectins by hemagglutination (HA) assays with mouse, rabbit, and sheep red blood cells. From mycelia and/or fruiting bodies, 23 extracts were prepared; 15 extracts exhibited HA activity towards mouse erythrocytes, with specific activities ranging from 12 to 440 lectin units (LU) mg(-1) protein. In HA inhibition assays, 43 carbohydrates including mono-, di-, tri-, tetrasaccharides, glycoproteins, and polysaccharides were tested as haptens, to determine the saccharide-binding specificities of the lectins. A novel lectin with specificity towards N-acetyl-glucosamine was purified from mycelia of Punctularia atropurpurascens using affinity chromatography on chitosan-Sepharose. The lectin has a subunit molecular mass of 67 kDa determined by SDS-PAGE and a pI of 5.0.

Prospección química y microbiológica del bosque de galería del Río Uruguay / Chemical and microbiological prospecting woodland along the Uruguay River / Prospecção química e microbiológica do bosque de galeria do Rio Uruguai

La bioprospección es la búsqueda de información a partir de especies biológicas para su uso posterior en procesos de producción en diversos sectores. Ejemplos de esa información es la contenida en el material genético de todos los seres vivos, en los compuestos químicos que producen, en sus interacciones o en el conocimiento de las personas que de una manera u otra han estudiado a esos seres vivos. En este trabajo se presentan los resultados de la prospección botánica, química y microbiológica del bosque de galería del río Uruguay en la zona del rio Queguay, ecosistema con gran biodiversidad (comprende unas 200 especies) y de características poco comunes en el país dado su carácter subtropical. Se estudiaron 75 extractos provenientes de 31 especies pertenecientes a quince familias cubriendo de esa forma una gran biodiversidad.

Bioprospecting is the search for information from biological species for later use in production processes in various sectors. Examples of this information is contained in the genetic material of all living beings in the chemicals they produce, in their interactions or the knowledge of people who in one way or another, have studied these living beings. This paper presents the results of botanical, chemical and microbiological study of the gallery forest of the Uruguay River in the area of the Queguay river. This ecosystem presents great biodiversity (comprising about 200 species) and unusual features in the country given its subtropical character. We studied 75 extracts from 31 species belonging to fifteen families covering therefore a large biodiversity.

A bioprospecção é a bisca de informações a partir de espécies biológicas para seu uso posterior em processos de produção em diversos setores. Exemplos dessa informação está contida no material genético de todos os seres vivos, nas substâncias químicas que produzem, nas suas interações ou n o conhecimento das pessoas que, de uma maneira ou de outra, estudam os seres vivos. Neste trabalho é apresentado os resultados da prospecção botânica, química e microbiológica do bosque de galeria do Rio Uruguai na zona do Rio Queguay, ecosistema com grande biodiversidade, com cerca de duzentas espécies, e de características pouco comum no país dado seu caráter subtropical. Foram estudados 75 extratos provenientes de 31 espécies pertencentes a quinze famílias, cobrindo, assim, uma grande biodiversidade.

Initial antimicrobial activity studies of plants of the riverside forests of the southern Uruguay River / Estudos antimicrobianos preliminares de plantas da floresta ribeirinha do sul do rio Uruguai

Development of new antimicrobial compounds against different microorganisms is becoming critically important, as infectious diseases are still one of the leading causes of death in the world. Plants can be a useful source of these lead compounds. In this study, 66 extracts of 25 plants of the riverside forest of southern Uruguay River were studied for antimicrobial activity against Staphylococcus aureus, Listeria inocua, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Aspergillus niger and Candida albicans. Fifty-three of these extracts showed some kind of antimicrobial activity. Six of these (Eugenia mansoni, Eugenia repanda, Myrcianthes cisplatensis, Paullinia ellegans, Petunia sp and Ruprechtia laxiflora) presented activity against Mycobacterium tuberculosis with MIC values as low as 50 μg/mL.

As doenças infecciosas ainda são uma das principais causas de morte no mundo, sendo de significativa importância o desenvolvimento de novos compostos antimicrobianos contra diferentes microrganismos. As plantas podem ser uma boa fonte para direcionar a busca destes compostos. Neste estudo, 66 extratos de 25 plantas da floresta ribeirinha do sul do Rio Uruguai foram estudados para a atividade antimicrobiana contra o Staphylococcus aureus, Listeria inocua, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Aspergillus niger e Candida albicans. Cinqüenta e três destes extratos apresentaram algum tipo de atividade antimicrobiana. Seis (Eugenia mansoni, Eugenia repanda, Myrcianthes cisplatensis, Paullinia ellegans, Petunia sp e Ruprechtia laxiflora) apresentaram atividade contra o Mycobacterium tuberculosis com CIM de 50 μg/mL.

Biodegradation of agroindustrial wastes by Pleurotus spp for its use as ruminant feed

The increasing expansion of agro-industrial activity has led to the accumulation of a large quantity of lignocellulosic residues all over the world. In particular, large quantities of rice straw (300.000 t) and citric bagasse (50.000 t) are annually produced in Uruguay. In this work we present the study of the bioconversion of these substrates with the edible mushroom Pleurotus spp so as to increase nutritional values and digestibility for its use as animal feed. The SSF process was optimized and the products after different periods of mushroom growth were evaluated. The microbial counts (cfu/g) for the inoculated substrates 44 days after incubation were 15 x 10(4), < 10 and < 10 for aerobic microorganisms, coliforms and E. coli, respectively. After 14 days of SSF the percentage of dry matter, ADF and NDF decreased, and the content of protein increased. These results show that vegetal cell-wall components were degraded during the period of mushroom incubation. PCR - RFLP analysis of the ITS region was used to characterize the Pleurotus species produced in Uruguay and discriminate between DNAs of Pleurotus ostreatus 814 and other fungi from the different substrates.