Cultivo de Lentinula edodes e Pleurotus ostreatus em bagaço de cana-de-açúcar

Pós-graduação em Microbiologia - IBILCE

Comparative Genome Analysis of Trichophyton rubrum and Related Dermatophytes Reveals Candidate Genes Involved in Infection

The major cause of athlete's foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response. IMPORTANCE Athlete's foot, jock itch, ringworm, and nail infections are common fungal infections, all caused by fungi known as dermatophytes (fungi that infect skin). This report presents the genome sequences of Trichophyton rubrum, the most frequent cause of athlete's foot, as well as four other common dermatophytes. Dermatophyte genomes are enriched for four gene classes that may contribute to the ability of these fungi to cause disease. These include (i) proteases secreted to degrade skin; (ii) kinases, including pseudokinases, that are involved in signaling necessary for adapting to skin; (iii) secondary metabolites, compounds that act as toxins or signals in the interactions between fungus and host; and (iv) a class of proteins (LysM) that appear to bind and mask cell wall components and carbohydrates, thus avoiding the host's immune response to the fungi. These genome sequences provide a strong foundation for future work in understanding how dermatophytes cause disease.

Mycobiota and mycotoxins in Brazil nut samples from different states of the Brazilian Amazon region

The objective of this study was to evaluate the presence of fungi and mycotoxins (aflatoxins and cyclopiazonic acid) in Brazil nut samples collected in different states of the Brazilian Amazon region: Acre, Amazonas, Amapa, and Para. A total of 200 husk samples and 200 almond samples were inoculated onto Aspergillus flavus-parasiticus agar for the detection of fungi. Mycotoxins were analyzed by high-performance liquid chromatography. The mycobiota comprised the following fungi, in decreasing order of frequency: almonds - Phialemonium spp. (54%), Penicillium spp. (16%), Fusarium spp. (13%), Phaeoacremonium spp. (11%), and Aspergillus spp. (4%), husks - Phialemonium spp. (62%), Phaeoacremonium spp. (11%), Penicillium spp. (10%), Fusarium spp. (9%), and Aspergillus spp. A polyphasic approach was used for identification of Aspergillus species. Aflatoxins were detected in 22 (11%) of the 200 almond samples, with 21 samples presenting aflatoxin B-1 levels above 8 mu g/kg, the limit established by the European Commission for Brazil nuts for further processing. Nineteen (9.5%) of the 200 husk samples contained aflatoxins, but at levels lower than those seen in almonds. Cyclopiazonic acid (CPA) was detected in 44 (22%) almond samples, with levels ranging from 98.65 to 1612 mu g/kg. Aspergillus nomius and A. flavus were the most frequent Aspergillus species. The presence of fungi does not necessarily imply mycotoxin contamination, but almonds of the Brazil nut seem to be a good substrate for fungal growth. (C) 2012 Elsevier B.V. All rights reserved.

Effects of exogenous calcium or oxalic acid on Pinus taeda treatment with the white-rot fungus Ceriporiopsis subvermispora

Pinus taeda wood chips were treated with the biopulping fungus Ceriporiopsis subvermispora in calcium-or oxalic acid-amended cultures. The secretion of hydrolytic and oxidative enzymes was inhibited only in the cultures having the highest concentration of calcium (1400 mg kg(-1) wood). Calcium decreased the availability of free oxalic acid, inhibited fungal growth, and reduced lignin mineralization and transformations. Oxalic acid amendment in the cultures was found not to affect the lignin mineralization and transformations; however, it did inhibit the depolymerization reactions detectable in the residual lignin that was retained in the biotreated wood. C. subvermispora presented catabolic activity for oxalic acid in the cultures amended with 1660 mg acid kg(-1) wood, whereas oxalic acid was synthesized when it was amended at low amounts or initially absent in the cultures. These data suggest one ideal ratio of oxalic acid in C. subvermispora cultures and indicate that its exogenous addition does not necessarily accompany the further degradation of lignin. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

Endophytic fungi from the Amazonian plant Paullinia cupana and from Olea europaea isolated using cassava as an alternative starch media source

Endophytic fungi live inside plants, apparently do not cause any harm to their hosts and may play important roles in defense and growth promotion. Fungal growth is a routine practice at microbiological laboratories, and the Potato Dextrose Agar (PDA) is the most frequently used medium because it is a rich source of starch. However, the production of potatoes in some regions of the world can be costly. Aiming the development of a new medium source to tropical countries, in the present study, we used leaves from the guarana (a tropical plant from the Amazon region) and the olive (which grows in subtropical and temperate regions) to isolate endophytic fungi using PDA and Manihot Dextrose Agar (MDA). Cassava (Manihot esculenta) was evaluated as a substitute starch source. For guarana, the endophytic incidence (EI) was 90% and 98% on PDA and MDA media, respectively, and 65% and 70% for olive, respectively. The fungal isolates were sequenced using the ITS- rDNA region. The fungal identification demonstrated that the isolates varied according to the host plant and media source. In the guarana plant, 13 fungal genera were found using MDA and six were found using PDA. In the olive plant, six genera were obtained using PDA and 4 were obtained using MDA. The multivariate analysis results demonstrated the highest fungal diversity from guarana when using MDA medium. Interestingly, some genera were isolated from one specific host or in one specific media, suggesting the importance of these two factors in fungal isolation specificity. Thus, this study indicated that cassava is a feasible starch source that could serve as a potential alternative medium to potato medium.

Exposure to the Asp f 1 allergen in nonindustrial occupational environments

Sick Building Syndrome is a prevalent problem with patient complaints similar to typical allergy symptoms. Unlike most household allergens, the Asp f 1 allergen is conceivably ubiquitous in the work environment. This project examined levels of the Asp f 1 allergen in office and non-industrial occupational environments, and studied the bioaerosol and dust reservoirs of Aspergillus fumigatus responsible for those levels. ^ Culturable bioaerosols of total mesophilic fungi were sampled with Andersen N6 impactors. Aggressive airborne and bulk dust samples were concurrently collected and assayed for Asp f 1. Bulk dusts were selectively cultured for A. fumigatus. Samples were collected during both wet and dry climatological conditions to examine the possibility of Asp f 1 increases due to fungal growth blooms. ^ Only very low levels of Asp f 1 were detected in relatively few samples. Analysis of wet versus dry period samples showed no differences in Asp f 1 levels, although A. fumigatus counts from dusts did fluctuate significantly with exterior moisture events as did indoor prevalence of total colony forming units. These results indicate that even in the presence of elevated fungal concentrations, levels of Asp f 1 are extremely low. These levels do not correlate with climatological moisture events, despite distinct fungal blooms in the days immediately following those events. Non-industrial office buildings devoid of indoor air quality issues did not demonstrate significant levels or occurrence of Asp f 1 contamination in the geographical region of this study. ^

Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms

Protease inhibitors from plants have been involved in defence mechanisms against pests and pathogens. Phytocystatins and trypsin/α-amylase inhibitors are two of the best characterized protease inhibitor families in plants. In barley, thirteen cystatins (HvCPI-1 to 13) and the BTI-CMe trypsin inhibitor have been previously studied. Their capacity to inhibit pest digestive proteases, and the negative in vivo effect caused by plants expressing these inhibitors on pests support the defence function of these proteins. Barley cystatins are also able to inhibit in vitro fungal growth. However, the antifungal effect of these inhibitors in vivo had not been previously tested. Moreover, their in vitro and in vivo effect on plant pathogenous bacteria is still unknown. In order to obtain new insights on this feature, in vitro assays were made against different bacterial and fungal pathogens of plants using the trypsin inhibitor BTI-CMe and the thirteen barley cystatins. Most barley cystatins and the BTI-CMe inhibitor were able to inhibit mycelial growth but no bacterial growth. Transgenic Arabidopsis plants independently expressing the BTI-CMe inhibitor and the cystatin HvCPI-6 were tested against the same bacterial and fungal pathogens. Neither the HvCPI-6 expressing transgenic plants nor the BTI-CMe ones were more resistant to plant pathogen fungi and bacteria than control Arabidopsis plants. The differences observed between the in vitro and in planta assays against phytopathogenic fungi are discussed

Identification of the causal agent of pistachio dieback in Australia

Symptoms associated with pistachio dieback in Australia include decline (little or no current season growth), xylem staining in shoots two or more years old, trunk mu and limb lesions (often covered by black, superficial fungal growth), excessive exudation of resin, dieback and death of the tree. Bacteria belonging to the genus Xanthomonas have been suggested as the causal agent. To confirm the constant association between these bacteria and the disease syndrome, the absence of other pathogens and the identity of the pathogen, we performed a series of isolations and pathogenicity tests. The only microorganism consistently isolated from diseased tissue was a bacterium that produced yellow, mucoid colonies and displayed morphological and cultural characteristics typical of the genus Xanthomonas. Database comparisons of the fatty acid and whole-cell protein profiles of five representative pistachio isolates indicated that they all belonged to X. translucens, but it was not possible to allocate the isolates to pathovar. Pathogenicity tests on cereals and grasses supported this identification. However, Koch's postulates have been only partially fulfilled because not all symptoms associated with pistachio dieback were reproduced on inoculated two-year-old pistachio trees. While discolouration was observed, dieback, excessive resinous exudate and trunk and limb lesions were not produced; expression of these symptoms may be delayed, and long-term monitoring of a small number of inoculated trees is in progress.

Effects of environmental temperature on members of the mucorales

The following investigation characterises the interaction between temperature and growth in psychrophilic, mesophilic and thermophilic fungi in order to gain further insight into the physiological mechanisms underlying fungal growth at extreme temperatures. In the first part of the investigation, the effect of environmental temperature on the growth of vegetative mycelium and sporangiospore production and germination was considered in order to determine the cardinal temperatures of these activities in different thermal groups. Subsequent investigations of plasma membrane permeability suggested that plasma membrane structure and function may be significant in establishing both the upper and lower growth temperature limits characteristic of psychrophiles, mesophiles and thermophiles. Analysis of the plasma membrane fractions revealed significant differences in membrane phospholipid composition between these thermal groups and it is suggested that the differing cardinal growth temperatures characteristic of psychrophilic, mesophilic and thermophilic fungi reflect the temperature ranges over which these organisms exhibit levels of plasma membrane fluidity sufficient to maintain membrane-associated growth processes. In contrast, the membrane protein components appear uniform in both character and thermostability and are therefore unlikely to contribute to this phenomenon.

The breakdown of plant cell biomass by fungi

Three species of fungi Sporotrichum thermophile, Botrytis cinerea and Trichoderma viride were assessed for their ability to utilize a variety of plant cell substrates (methanol extracted), Catharanthus roseus, Daucus carota, re-autoclaved C. roseus, re-autoclaved D. carota) which preliminary studies had indicated contained the necessary nutrients for fungal growth. Incubated in a suitable manner all three fungal species were able to grow on C. roseus and D. carota plant cell biomass in addition to material which had undergone methanol extraction or a re-autoclaving process to remove soluble components. Fungal biomass yields were markedly influenced by substrate, with each fungal species demonstrating a preference for particular plant cell material. Incubation conditions i.e. static or shaken and temperature also proved important. Release of glucose (i.e. values higher than Day 0) promoted by fungal breakdown of plant cell biomass was only noted with methanol extracted, re-autoclaved C. roseus and re-autoclaved D. carota material. A re-autoclaved substrate was also generally associated with high fungal C1, Cx, B-glucosidase and endo-polygalacturonase activity. In addition for each enzyme highest values were usually obtained from a particular fungal species. Buffering cultures at pH 3 or 5 further influenced enzyme activity, however in a majority of cases when flasks were unbuffered and the pH rose naturally to alkaline values higher enzyme activity was recorded. Likewise Tween 80 addition had only a limited beneficial effect. Finally filtrates containing glucose produced both from the re-autoclaving process and through fungal activity on plant cell biomass were utilized for Fusarium oxysporum, Saccharomyces cerevisiae and C. roseus plant cell culture. Although reasonable fungal biomass was obtained the use of such filtrates proved unsuitable for plant cell growth.

Potencial antifúngico, antioxidante e inibidor da produção de aflatoxina por extratos fenólicos de Chlorella sp. e Spirulina platensis

A contaminação fúngica acarreta alterações na qualidade nutricional e no valor econômico de produtos alimentícios podendo causar danos patológicos em plantas, animais e humanos. A identificação da atividade antioxidante, antifúngica e antimicotoxinas, em extratos de microalgas com propriedade de inibir a multiplicação de fungos e subseqüente produção de micotoxinas abre a perspectiva de empregar substâncias mais eficientes e com maior ação específica contra estes microorganismos. Entre os compostos com propriedades inibidoras de radicais livres, de crescimento fúngico e produção de micotoxinas, destacam-se os compostos fenólicos, que podem inibir a atividade metabólica microbiana, dificultando a atividade de enzimas. Neste estudo foram avaliados o poder de inibição de multiplicação fúngica de Rhizopus oryzae e Aspergillus flavus pelos extratos fenólicos de Chlorella sp. e Spirulina platensis, bem como sua atividade antioxidante, e a atividade antimicotoxinas da última microalga contra Aspergillus flavus. O conteúdo de fenóis totais foi em média 1000 µgfenóis/g Spirulina platensis e 600 µgfenóis/g Chlorella sp., sendo que o acido gálico e o cafeíco foram identificados como compostos majoritários na Spirulina platensis. As determinações de glicosamina (parede celular) e ergosterol (membrana celular) mostraram-se bons indicativos do desenvolvimento microbiano permitindo uma boa estimativa da inibição dele. O extrato fenólico de Spirulina platensis apresentou capacidade de inibir cerca de 50% a formação da parede e da membrana celular para ambos os fungos estudados e de 100% a produção de aflatoxina B1 até o 10º dia de cultivo do Aspergillus flavus. Além disso, o extrato metanólico de Spirulina platensis inativou 53,5% o DPPH reativo, limitou o escurecimento enzimático ocasionado pela peroxidase em 55% e inibiu a peroxidação lipídica em 46% após 14 dias de armazenamento sob luz. Estes resultados mostram que a ação antifúngica, antimicotoxinas e antioxidante está naturalmente presente em alguns tecidos microbianos e que encontrar a forma de extraí-los e aplicá-los como conservantes alimentícios é muito promissor para substituição aos antifúngicos e outros conservantes químicos.

Effect of commercial starter cultures and native yeasts on ochratoxin a production by aspergillus westerdijkiae and penicillium nordicum in meat products

Processed meat products are of worldwide importance and, because of their intrinsic factors as well as the processing methods, they are highly prone to fungal and mycotoxin contamination. Ochratoxin A (OTA) is the most significant mycotoxin in processed meat products. Penicillium nordicum is considered to be responsible for OTA contamination of meat products, as it is highly adapted to salt and protein-rich matrices and is moderately psycrotrophic. However, another OTA-producing fungus, Aspergillus westerdijkiae, adapted to carbon-rich matrices such as cereals and coffee beans, has been recently associated with high levels of OTA in meat products. Several Lactic Acid Bacteria (LAB) and yeasts have been tested as biocontrol agents against P. nordicum growth and OTA production in meat products, with promising results, but none of the studies have considered A. westerdijkiae. The aim of this work was to evaluate in vitro the effect of a commercial starter culture used in sausage fermentation and four yeasts isolated from dry-cured sausage on these two OTA-producing fungi, both in terms of fungal growth and of OTA production, using different meat-based culture media as model systems. The mechanisms underlying the observed effect were also studied. For this purpose, C. krusei, C. zeylanoides, R. mucilaginosa, R. glutinis, a mix of these yeasts and the starter culture were co-inoculated with P. nordicum and A. westerdijkiae in industrial sausage, traditional sausage, and ham-based media, under conditions of water activity, salt concentration and temperature that mimic real conditions at beginning and end of sausage curing process. Fungal growth was determined by measuring colony diameter, and OTA production was quantified by HPLC-FLD after extraction with methanol. Yeasts where found to inhibit significantly the growth of both fungi. P. nordicum was unable to produce detectable OTA in both sausage-based media under any condition. In ham, yeasts reduced OTA production, while the starter culture significantly increased it. Unexpectedly, OTA production by A. westerdijkiae was significantly stimulated in all media tested by all microorganisms. Matrix has a significant effect on OTA production by P. nordicum, but not by A. westerdijkiae, for which only temperature showed to have effect. By testing the mechanisms of action by which starter culture and C. zeylanoides influenced fungal responses, we were able to determine that direct contact and simultaneous growth of test organisms were the mechanisms more significantly involved in the responses. In conclusion, ochratoxigenic fungi do not all respond to antagonistic microorganisms in the same way. The use of biocontrol agents with the intent of reducing fungal growth and mycotoxin production by one fungus can have unexpected effects on others, thus leading to unforeseen safety problems. Further experiments are recommended to properly understand the reasons behind the different effects of microorganisms, to ensure their safe as biocontrol agents.

Metabolism of plant polysaccharides by Leucoagaricus gongylophorus, the symbiotic fungus of the leaf-cutting ant Atta sexdens L.

Atta sexdens L, ante feed on the Fungus they cultivate on cut leaves inside their nests. The fungus, Leucoagaricus gongylophorus, metabolizes plant polysaccharides, such as xylan, starch, pectin, and cellulose, mediating assimilation of these compounds lay the ants, This metabolic integration may be an important part of the ant-fungus symbiosis, and it involves primarily xylan and starch, both of which support rapid fungal growth. Cellulose seems to be less important for symbiont nutrition, since it is poorly degraded and assimilated by the fungus. Pectin is rapidly degraded but slowly assimilated by L. gongylophorus, and its degradation may occur so that the fungus can more easily access other polysaccharides in the leaves.

Synthetic amides toxic to the leaf-cutting ant Atta sexdens rubropilosa L. and its symbiotic fungus

1 Nine synthetic amides similar to natural N-piperidine-3-(4,5-methylenedioxyphenyl)-2-(E)-propenainide and N-pyrrolidine-3-(4,5-methylenedyoxiphenyl)2-(E)-propenamide were synthesized and identified by their spectroscopic data.2 the toxicity of these synthetic amides to the Atta sexdens rubropilosa workers and the antifungal activity against Leticoagaricus gongylophorus, the symbiotic fungus of the leaf-cutting ants, were determined.3 Workers ants that were fed daily on an artificial diet to which these compounds were added had a higher mortality rate than the controls for N-pyrrolidine-3(3',4'-methylenedioxyphenyl)-2-(E)-propenamide and N-benzyl-3-(3',4'-methylenedioxyphenyl)-2-(E)-propenamide at a concentration of 100 mu g/mL.4 the completely inhibition (100%) of the fungal growth was observed with N-piperldine-3-(3',4'-methylenedioxyphenyl)-2-(E)-propenamide and N,N-diethyl-3-(3',4'-methylenedioxyphenyl)-2-(E)-propenamide at concentrations of 50 and 100 mu g/mL and N-pirrolidine-3-(3',4'-methylenedioxyphenyl)-2-(E)-propenamide at a concentration of 100 mu g/mL.5 the possibility of controlling these insects in the future using synthetic piperamides that can simultaneously target both organisms is discussed.