Use of confocal scanning laser microscopy to measure the concentrations of aerial and penetrative hyphae during growth of Rhizopus oligosporus on a solid surface

In order to develop a method for use in investigations of spatial biomass distribution in solid-state fermentation systems, confocal scanning laser microscopy was used to determine the concentrations of aerial and penetrative biomass against height and depth above and below the substrate surface, during growth of Rhizopus oligosporus on potato dextrose agar. Penetrative hyphae had penetrated to a depth of 0.445 cm by 64 h and showed rhizoid morphology, in which the maximum biomass concentration, of 4.45 mg dry wt cm(-3), occurred at a depth of 0.075 cm. For aerial biomass the maximum density of 39.54 mg dry wt(-3) occurred at the substrate surface. For both aerial and penetrative biomass, there were two distinct regions in which the biomass concentration decayed exponentially with distance from the surface. For aerial biomass, the first exponential decay region was up to 0.1 cm height. The second region above the height of 0.1 cm corresponded to that in which sporangiophores dominated. This work lays the foundation for deeper studies into what controls the growth of fungal hyphae above and below the surfaces of solid substrates. (C) Wiley Periodicals, Inc.

Use of confocal microscopy to follow the development of penetrative hyphae during growth of Rhizopus oligosporus in an artificial solid-state fermentation system

Two methods were compared for determining the concentration of penetrative biomass during growth of Rhizopus oligosporus on an artificial solid substrate consisting of an inert gel and starch as the sole source of carbon and energy. The first method was based on the use of a hand microtome to make sections of approximately 0.2- to 0.4-mm thickness parallel to the substrate surface and the determination of the glucosamine content in each slice. Use of glucosamine measurements to estimate biomass concentrations was shown to be problematic due to the large variations in glucosamine content with mycelial age. The second method was a novel method based on the use of confocal scanning laser microscopy to estimate the fractional volume occupied by the biomass. Although it is not simple to translate fractional volumes into dry weights of hyphae due to the lack of experimentally determined conversion factors, measurement of the fractional volumes in themselves is useful for characterizing fungal penetration into the substrate. Growth of penetrative biomass in the artificial model substrate showed two forms of growth with an indistinct mass in the region close to the substrate surface and a few hyphae penetrating perpendicularly to the surface in regions further away from the substrate surface. The biomass profiles against depth obtained from the confocal microscopy showed two linear regions on log-linear plots, which are possibly related to different oxygen availability at different depths within the substrate. Confocal microscopy has the potential to be a powerful tool in the investigation of fungal growth mechanisms in solid-state fermentation. (C) 2003 Wiley Periodicals, Inc.

Isolation and characterization of a cone snail protease with homology to CRISP proteins of the pathogenesis-related protein superfamily

The pathogenesis-related (PR) protein superfamily is widely distributed in the animal, plant, and fungal kingdoms and is implicated in human brain tumor growth and plant pathogenesis. The precise biological activity of PR proteins, however, has remained elusive. Here we report the characterization, cloning and structural homology modeling of Tex31 from the venom duct of Conus textile. Tex31 was isolated to >95% purity by activity-guided fractionation using a para-nitroanilide substrate based on the putative cleavage site residues found in the propeptide precursor of conotoxin TxVIA. Tex31 requires four residues including a leucine N-terminal of the cleavage site for efficient substrate processing. The sequence of Tex31 was determined using two degenerate PCR primers designed from N-terminal and tryptic digest Edman sequences. A BLAST search revealed that Tex31 was a member of the PR protein superfamily and most closely related to the CRISP family of mammalian proteins that have a cysteine-rich C-terminal tail. A homology model constructed from two PR proteins revealed that the likely catalytic residues in Tex31 fall within a structurally conserved domain found in PR proteins. Thus, it is possible that other PR proteins may also be substrate-specific proteases.

Revestimentos comestíveis na conservação pós-colheita de morangos cultivar Camarosa produzidos em sistema orgânico e convencional

As perdas pós-colheita de frutas promovem a elevação do custo dos produtos e diminuem a oferta ao consumidor. Suas principais causas estão na colheita, transporte e armazenamento inadequados. A aplicação de revestimentos comestíveis juntamente com a redução da temperatura de armazenamento constitui um dos métodos empregados para a conservação pós-colheita de produtos com vida útil curta, como frutas e hortaliças. O morango é um fruto consumido preferencialmente in natura. Desta forma, torna-se promissora a utilização de revestimento comestível para aumentar seu período de armazenamento e comercialização, sem alteração do sabor, da cor e do aroma dos frutos. A produção orgânica frente a convencional de frutos podem apresentar diferenças, sendo interessante o estudo envolvendo as formas de cultivo. Este estudo teve como objetivo avaliar a conservação pós-colheita de morangos cv. Camarosa, oriundos de cultivo orgânico e convencional revestidos com coberturas comestíveis. Os morangos foram revestidos com fécula de mandioca, gelatina e cera de carnaúba, armazenados durante 10 dias a 10 ºC. A cada 2 dias de armazenamento foram determinados perda de massa, sólidos solúveis, pH, acidez titulável, firmeza, antocianinas totais e podridão fúngica. Análise sensorial foi realizada para verificar a aceitação dos morangos e para avaliar a influência da informação nesta aceitação. A perda de massa foi maior no cultivo orgânico a partir do 8º dia de armazenamento, chegando a 11,47% contra 8,88% do cultivo convencional no final do 10º dia de armazenamento. O revestimento que possibilitou menor perda de massa foi o de cera de carnaúba em relação ao controle. A contaminação fúngica iniciou-se no 4º dia de armazenamento em ambos os tipos de cultivo. No 8º dia de armazenamento observou-se diferenças na podridão entre os tipos de cultivo, sendo o orgânico visualmente mais contaminado. O revestimento de cera de carnaúba apresentou menor podridão fúngica em relação aos outros revestimentos, porém não diferiu da amostra controle. Das variáveis físico-químicas avaliadas, apenas o teor de sólidos solúveis apresentou diferenças entre os tipos de cultivo, sendo o morango convencional o que obteve maiores valores. O teor de antocianinas dos morangos revestidos com fécula de mandioca diferiu do controle, porém o revestimento com fécula não diferiu dos revestidos com gelatina e cera de carnaúba. Foram verificadas diferenças na firmeza dos frutos em relação aos revestimentos. Ao longo do tempo foi observado diferenças no pH, teor de antocianinas e firmeza. O revestimento com cera de carnaúba se mostrou mais adequado em relação aos demais revestimentos, porém sua aparência mostrou-se com pouco brilho e esbranquiçado. Os frutos avaliados apresentaram vida útil pós-colheita de aproximadamente 6 dias. Os morangos, do ponto de vista microbiológico, se mostraram aptos para consumo. A aceitação dos morangos foi boa, não tendo ix diferenças significativas entre morango orgânico e convencional. O fornecimento da informação de “morango orgânico” e a apresentação de um texto adicional informativo não influenciaram na aceitação dos morangos. Entre os revestimentos testados o de cera de carnaúba se mostrou mais aplicável, os demais nas condições testadas não mostraram bons resultados.

Atividade fungicida de triazóis obtidos a partir de glicerol sobre Colletotrichum gloeosporioides

A antracnose é responsável por significativos danos no pós-colheita em frutos de mamão, ocasionando expressivas perdas aos produtores. Desta forma, objetivou-se avaliar a eficiência de novas moléculas fungicidas do grupo dos triazóis obtidas a partir de glicerol no manejo in vitro de Colletotrichum gloeosporioides. O experimento foi conduzido em delineamento inteiramente casualizado, em esquema fatorial 18x5+1 (17 moléculas inéditas de triazóis (T1 a T17), 1 fungicida comercial (tebuconazol), cinco concentrações e uma testemunha adicional, com cinco repetições por tratamento. Para avaliar o efeito das moléculas no crescimento micelial e esporulação do fungo foi empregado o método de incorporação de cada molécula ao meio de cultura batata-dextrose-ágar nas concentrações de 1, 10, 100, 500 e 1000 ppm. Com base nos resultados obtidos foi observada diferença significativa entre os tratamentos e concentrações tanto para o crescimento micelial quanto esporulação, sendo que quanto maior a concentração testada maior o efeito fungistático e/ou fungicida dos compostos triazólicos. O efeito inibitório dos fungicidas foi maior sobre a esporulação do que no crescimento micelial, sendo os triazóis T12 e T14 os mais eficazes, havendo inibição completa do patógeno a partir das concentrações de 414,17 e 393,79 ppm, respectivamente. É válido ressaltar que além destes os triazóis T11, T16 e T17 também apresentaram completa inibição do patógeno em concentrações acima de 500 ppm, merecendo ser também estudados em experimentos futuros in vivo. Os valores médios de ED50 e ED100 foram menores para esporulação em relação ao crescimento micelial, demonstrando a eficiência antiesporulante dos novos triazóis sintetizados a partir do glicerol. Assim, conclui-se que compostos com anel 1,2,3-triazol sintetizados a partir do glicerol possuem atividade fungicida sobre C. gloeosporioides, sendo promissores para o manejo da antracnose em mamoeiro.

Uniformity of liquid distribution in the canopy of the bean plant, using the spectrophotometric analysis

Fungal diseases are important factors limiting common bean yield. White mold is one of the main diseases caused by soil pathogens. The objective of this study was to quantify the distribution of a fungicide solution sprayed into the canopy of bean plants by spectrophotometry, using a boom sprayer with and without air assistance. The experiment was arranged in a 2 x 2 x 2 factorial (two types of nozzles, two application rates, and air assistance on and off) randomized block design with four replications. Air assistance influenced the deposition of solution on the bean plant and yield increased significantly with the increased rate of application and air assistance in the boom sprayer.

Prevalence of fungi in indoor air with reference to gymnasiums with swimming pools

Fungal contamination of air in 10 gymnasiums with swimming pools was monitored. Fifty air samples of 200 L each were collected, using a Millipore air tester, from the area surrounding the pool, in training studios, in showers and changing rooms for both sexes, and also, outside premises, since these are the places regarded as reference. Simultaneously, environmental parameters – temperature and humidity – were also monitored. Some 25 different species of fungi were identified. The six most commonly isolated genera were the following: Cladosporium sp. (36.6%), Penicillium sp. (19.0%), Aspergillus sp. (10.2%), Mucor sp. (7%), Phoma sp. and Chrysonilia sp. (3.3%). For yeasts, three different genera were identified, namely, Rhodotorula sp. (70%), Trichosporon mucoides and Cryptococcus uniguttulattus (10%).

Possíveis implicações da contaminação fúngica num aviário

Introdução – Apesar de em Portugal se verificar o aumento da indústria da produção de aves para consumo humano, apenas alguns estudos incidem sobre a qualidade do ar interior e as implicações da sua degradação. Objectivos – Descrever a contaminação fúngica num aviário, analisar possíveis associações com a temperatura ambiente e a humidade relativa e o possível impacto na saúde dos consumidores e trabalhadores desta unidade. Métodos – Foi desenvolvido um estudo descritivo para avaliar a contaminação fúngica num aviário. Colheram‑se 5 amostras de ar de 100 litros através do método de compactação e 4 amostras de superfícies, utilizando a técnica da zaragatoa e um quadrado de 10 cm de lado de metal. Simultaneamente, os parâmetros ambientais – temperatura ambiente e humidade relativa – também foram medidos. Resultados – Foram identificadas vinte espécies de fungos no ar, sendo os seguintes os quatro géneros mais comummente isolados: Cladosporium (40,5%), Alternaria (10,8%), Chrysosporium e Aspergillus (6,8%). Nas superfícies, 21 espécies de fungos foram identificadas, sendo os 4 géneros mais identificados Penicillium (51,8%), Cladosporium (25,4%), Alternaria (6,1%) e Aspergillus (4,2%). Importa referir o facto de Aspergillus flavus, também isolado no ar, ser reconhecido como produtor de micotoxinas (aflatoxina) e Aspergillus fumigatus, uma das espécies isoladas no ar e superfícies, ser capaz de causar aspergilose grave ou fatal. Não se verificou relação significativa (p> 0,05) entre a contaminação fúngica e as variáveis ambientais. Conclusão – Caracterizou‑se a distribuição fúngica no ar e superfícies do aviário e analisou‑se a possível influência das variáveis ambientais. Foi reconhecido um potencial problema de Saúde Pública devido à contaminação fúngica e à possível produção de micotoxinas com a eventual contaminação dos produtos alimentares. A contaminação fúngica, particularmente causada pelo Aspergillus fumigatus, e a possível presença de micotoxinas no ar, devem ser encaradas também como fatores de risco neste contexto ocupacional. ABSTRACT - Background – Although there is an increasingly industry that produce whole chickens for domestic consumption in Portugal, only few investigations have reported on the indoor air of these plants and the consequences of their degradation. Objectives – Describe one poultry environmental fungal contamination analyse possible associations between temperature and relative humidity and its possible impact on the health of consumers and of the poultry workers. Methods – A descriptive study was developed to monitor one poultry fungal contamination. Five air samples of 100 litres through impaction method were collected and 4 swab samples from surfaces were also collected using a 10 cm square of metal. Simultaneously, environmental parameters – temperature and relative humidity – were also measured. Results – Twenty species of fungi in air were identified, being the 4 most commonly isolated the following genera: Cladosporium (40.5%), Alternaria (10.8%), Chrysosporium and Aspergillus (6.8%). In surfaces, 21 species of fungi were identified, being the 4 genera more identified Penicillium (51.8%), Cladosporium (25.4%), Alternaria (6.1%) and Aspergillus (4.2%). In addition, Aspergillus flavus also isolated in the poultry air is a well‑known producer of potent mycotoxins (aflatoxin), and Aspergillus fumigatus, one of the species isolated in air and surfaces, is capable of causing severe or fatal aspergillosis. There was no significant relationship (p>0,05) between fungal contamination and environmental variables. Conclusions – Was characterized fungal distribution in poultry air and surfaces and analyzed the association of environmental variables. It was recognized the Public Health problem because of fungal contamination and also due to probable mycotoxins production with the possible contamination of food products. Fungal contamination, particularly due to the presence of Aspergillus fumigatus and also the possible presence of mycotoxins in the air, should be seen as risk factor in this occupational setting.

Risk assessment of exposure to multiple mycotoxins in food

Moulds may produce a diversity of toxins such as aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins and others. Although toxicological, environmental and epidemiological studies have addressed the problem of these toxins one by one, more than one mycotoxin are found usually in the same contaminated food. Risk assessment for humans potentially exposed to multimycotoxins suffers very much from the lack of adequate food consumption data. Furthermore, for a given mycotoxin, synergism and antagonism with other mycotoxins, found in the same food commodities, are not taken into account. Aflatoxin B1 and ochratoxin A belong to the most frequently occurring mycotoxins. This has repeatedly been demonstrated, however, normally, the risk resulting from their simultaneous occurrence is not considered. A descriptive study was developed to monitor air fungal contamination in one hospital food unit.

Are air-borne mycotoxins a public health concern in Portugal?

Introduction - Microscopic filamentous fungi, under suitable environmental conditions, can lead to the production of highly toxic chemical substances, commonly known as mycotoxins. The most widespread and studied mycotoxins are metabolites of some genera of moulds such as Aspergillus, Penicillium and Fusarium. Quite peculiar conditions may influence mycotoxin biosynthesis, such as climate, geographical location, cultivation practices, storage and type of substrate. Toxicity has been extensively investigated for the most important mycotoxins, such as aflatoxins, ochratoxin A and Fusarium toxins, and much information derived from toxicokinetics in animal models has also been obtained. The adverse effects are mainly related to genotoxicity, carcinogenicity, mutagenicity, teratogenicity and immunotoxicity. Aim of the study - To identify fungal species able to produce important mycotoxins in different Portuguese settings.

Ventilation influence in occupational exposure to fungi and volatile organic compounds: poultry case

Introduction - In poultry houses, large-scale production has led to increased bird densities within buildings. Such high densities of animals kept within confined spaces are a source of human health problems related to occupational organic dust exposure. This organic dust is composed of both non-viable particles and viable particulate matter (also called bioaerosols). Bioaerosols are comprised by airborne bacteria, fungi, viruses and their by-products, endotoxins and mycotoxins. Exposure to fungi in broiler houses may vary depending upon the applied ventilation system. Ventilation can be an important resource in order to reduce air contamination in these type of settings. Nevertheless, some concerns regarding costs, sensitivity of the animal species to temperature differences, and also the type of building used define which type of ventilation is used. Aim of the study - A descriptive study was developed in one poultry unit aiming to assess occupational fungal and volatile organic compounds (VOCs) exposure.

Air contaminants in animal production: the poultry case

A descriptive study was developed in order to assess air contamination caused by fungi and particles in seven poultry units. Twenty seven air samples of 25 litters were collected through impaction method. Air sampling and particle concentration measurement were performed in the pavilions’ interior and also outside premises, since this was the place regarded as reference. Simultaneously, temperature and relative humidity were also registered. Regarding fungal load in the air from the seven poultry farms, the highest value obtained was 24040 CFU/m3 and the lowest was 320 CFU/m3. Twenty eight species/genera of fungi were identified, being Scopulariopsis brevicaulis (39.0%) the most commonly isolated species and Rhizopus sp. (30.0%) the most commonly isolated genus. From the Aspergillus genus, Aspergillus flavus (74.5%) was the most frequently detected species. There was a significant correlation (r=0.487; p=0.014) between temperature and the level of fungal contamination (CFU/m3). Considering contamination caused by particles, in this study, particles with larger dimensions (PM5.0 and PM10) have higher concentrations. There was also a significant correlation between relative humidity and concentration of smaller particles namely, PM0.5 (r=0.438; p=0.025) and PM1.0 (r=0.537; p=0.005). Characterizing typical exposure levels to these contaminants in this specific occupational setting is required to allow a more detailed risk assessment analysis and to set exposure limits to protect workers’ health.

Comparison of indoor and outdoor fungi and particles in poultry units

A descriptive study was developed in order to compare indoor and outdoor air contamination caused by fungi and particles in seven poultry units. Twenty eight air samples of 25 litters were collected through the impaction method on malt extract agar. Air sampling and particles concentration measurement were done in the interior and also outside premises of the poultries’ pavilions. Regarding the fungal load in the air, indoor concentration of mold was higher than outside air in six poultry units. Twenty eight species / genera of fungi were identified indoor, being Scopulariopsis brevicaulis (40.5%) the most commonly isolated species and Rhizopus sp. (30.0%) the most commonly isolated genus. Concerning outdoor, eighteen species/genera of fungi were isolated, being Scopulariopsis brevicaulis (62.6%) also the most isolated. All the poultry farms analyzed presented indoor fungi different from the ones identified outdoors. Regarding particles’ contamination, PM2.5, PM5.0 and PM10 had a statistically significant difference (Mann-Whitney U test) between the inside and outside of the pavilions, with the inside more contaminated (p=.006; p=.005; p=.005, respectively). The analyzed poultry units are potential reservoirs of substantial amounts of fungi and particles and could therefore free them in the atmospheric air. The developed study showed that indoor air was more contaminated than outdoors, and this can result in emission of potentially pathogenic fungi and particles via aerosols from poultry units to the environment, which may post a considerable risk to public health and contribute to environmental pollution.

Indoor air quality in Portuguese archives: a snapshot on exposure levels

Indoor air quality recently entered legislation in Portugal. Several parameters must be evaluated and kept within limits in order to obtain a certification for air quality and energy consumption. Certification parameters were analyzed in two Portuguese archives in order to assess indoor air quality both for people attending or working on these premises and for maintenance of a written heritage that must be retained for future generations. Carbon monoxide (CO) and carbon dioxide (CO2), formaldehyde, and fungal counts were kept within stipulated limits. Relative humidity (RH), volatile organic compounds (VOC), particulate matter (PM10), and ozone (O3) showed values above legislated levels and justified the implementation of corrective measures. In terms of conservation, studies on the limit values are still needed, but according to the available international guidelines, some of the analyzed parameters such as PM10, O3, and RH were also above desirable values. Corrective measures were proposed to these institutions. Although this study was only of a short duration, it proved valuable in assessing potential eventual problems and constitutes the first Portuguese indoor air quality assessment taking into consideration both aspects of an archive such as human health and heritage safekeeping.

Occupational exposure to aflatoxin (AFB1) in poultry production

Aflatoxin B1 (AFB1) has been recognized to produce cancer in human liver. In addition, epidemiological and laboratory studies demonstrated that the respiratory system was a target for AFB1. Exposure occurs predominantly through the food chain, but inhalation represents an additional route of exposure. The present study aimed to examine AFB1 exposure among poultry workers in Portugal. Blood samples were collected from a total of 31 poultry workers from six poultry farms. In addition, a control group (n = 30) was included comprised of workers who undertook administrative tasks. Measurement of AFB1 in serum was performed by enzyme-linked immunosorbent assay (ELISA). For examining fungi contamination, air samples were collected through an impaction method. Air sampling was obtained in pavilion interior and outside the premises, since this was the place regarded as the reference location. Using molecular methods, toxicogenic strains (aflatoxin-producing) were investigated within the group of species belonging to Aspergillus flavus complex. Eighteen poultry workers (59%) had detectable levels of AFB1 with values ranging from <1 ng/ml to4.23 ng/ml and with a mean value of 2 ± 0.98ng/ml. AFB1 was not detected in the serum sampled from any of the controls. Aspergillus flavus was the fungal species third most frequently found in the indoor air samples analyzed (7.2%) and was the most frequently isolated species in air samples containing only Aspergillus genus (74.5%). The presence of aflatoxigenic strains was only confirmed in outdoor air samples from one of the units, indicating the presence of a source inside the building in at least one case. Data indicate that AFB1 inhalation represents an additional risk in this occupational setting that needs to be recognized, assessed, and prevented.