High-Efficiency Electroporation for Genetic Improvement of Fungal Strains.

Electroporation is a method for the introduction of molecules (usually nucleic acids) into a cell, consisting of submitting the cells to high-voltage and short electric pulses in the presence of the exogenous DNA/molecule. It is a versatile method, adaptable to different types of cells, from bacteria to cultured cells to higher eukaryotes, and thus has applications in many diverse fields, such as environmental biology, biotechnology, genetic engineering, and medicine. Electroporation has some advantages over other genetic transformation strategies, including the simplicity of the method, a wide range of adjustable parameters (possibility of optimization), high reproducibility and avoidance of the use of chemicals toxic to cells. Here we describe an optimized electroporation procedure for the industrially important fungus Acremonium chrysogenum, using germinated conidia and fragmented young mycelium. In both cases, the transformation efficiency was higher compared to the conventional polyethylene glycol (PEG)-mediated transformation of protoplasts.

Sensitivity of hyperparasitic fungi to alternative products for use in the control of papaya black spot / Sensibilidade de fungos hiperparasitas a produtos alternativos para uso no controle da pinta preta do mamoeiro

Abstract The use of more than one control technique can maximize the reduction of the damages caused by the fungus Asperisporium caricae causal agent of the black spot in the papaya crop. The objective of this work was to evaluate the sensitivity of the fungi Hansfordia pulvinata and Acremonium spp. to alternative products with potential for use in the control of the black-spotted ptarmigan. Three isolates of Acremonium spp. (A-598, A-602 and A-617) and an isolate of H. pulvinata (H-611) were grown in BDA medium containing Agro-Mos®, Bion®, Ecolife®, Hortifospk®, Matriz G®, Vitaphol® separately. The Amistar 500WG ® fungicide was used as a positive control and pure BDA as a negative control. The toxicity of the tested products was determined based on the values of the biological index, derived from the means of mycelial growth, sporulation and germination of conidia, in each experimental unit. In this way it was possible to select the products classified as compatible for all isolates, and to test them in vivo. In the greenhouse, only the isolates and isolates with selected products, were applied in papaya plants with foliar symptoms of black-spotted. The incidence of leaves with hyperparasites and the percentage of black-painted lesions colonized by the tested isolates were evaluated. Thus, the H-611 isolate proved to be compatible with most of the alternative products tested, except with Hortifos®. Bion® and Matrix® products were compatible with all tested isolates and could be used in conjunction with Acremonium spp. and H. pulvinata to control the papaya black spot, since these products did not present toxicity on the hyperparasitic fungi.

Resumo O uso de mais de uma técnica de controle pode maximizar a redução dos danos causados pelo fungo Asperisporium caricae agente causal da pinta preta na cultura do mamoeiro. O objetivo deste trabalho foi avaliar a sensibilidade dos fungos Hansfordia pulvinata e Acremonium spp. a produtos alternativos com potencial para uso no controle da pinta-preta. Três isolados de Acremonium spp. (A-598, A-602 e A-617) e um isolado de H. pulvinata (H-611) foram cultivados em meio BDA contendo Agro-Mos®, Bion®, Ecolife®, Hortifospk®, Matriz G®, Vitaphol® separadamente. Fungicida Amistar 500WG® foi utilizado como controle positivo e BDA puro, como controle negativo. A toxicidade dos produtos testados foi determinada com base nos valores do índice biológico, provenientes das médias do crescimento micelial, esporulação e germinação de conídios, em cada unidade experimental. Dessa forma foi possível selecionar os produtos classificados como compatíveis para todos os isolados, e testá-los in vivo. Em casa de vegetação, somente os isolados e os isolados com produtos selecionados, foram aplicados em plantas de mamoeiro com sintomas foliares de pinta-preta. Avaliaram-se a incidência de folhas com hiperparasitas e a porcentagem de lesões de pinta-preta colonizadas pelos isolados testados. Assim o isolado H-611 mostrou-se compatível com a maioria dos produtos alternativos testados, exceto com Hortifos®. Os produtos Bion® e Matriz® apresentaram compatibilidade com todos os isolados testados, podendo ser utilizados em conjunto com o Acremonium spp. e H. pulvinata para controlar a pinta-preta do mamoeiro, uma vez que estes produtos não apresentaram toxidade sobre os fungos hiperparasitas.

Sensitivity of hyperparasitic fungi to alternative products for use in the control of papaya black spot / Sensibilidade de fungos hiperparasitas a produtos alternativos para uso no controle da pinta preta do mamoeiro

The use of more than one control technique can maximize the reduction of the damages caused by the fungus Asperisporium caricae causal agent of the black spot in the papaya crop. The objective of this work was to evaluate the sensitivity of the fungi Hansfordia pulvinata and Acremonium spp. to alternative products with potential for use in the control of the black-spotted ptarmigan. Three isolates of Acremonium spp. (A-598, A-602 and A-617) and an isolate of H. pulvinata (H-611) were grown in BDA medium containing Agro-Mos®, Bion®, Ecolife®, Hortifospk®, Matriz G®, Vitaphol® separately. The Amistar 500WG ® fungicide was used as a positive control and pure BDA as a negative control. The toxicity of the tested products was determined based on the values of the biological index, derived from the means of mycelial growth, sporulation and germination of conidia, in each experimental unit. In this way it was possible to select the products classified as compatible for all isolates, and to test them in vivo. In the greenhouse, only the isolates and isolates with selected products, were applied in papaya plants with foliar symptoms of black-spotted. The incidence of leaves with hyperparasites and the percentage of black-painted lesions colonized by the tested isolates were evaluated. Thus, the H-611 isolate proved to be compatible with most of the alternative products tested, except with Hortifos®. Bion® and Matrix® products were compatible with all tested isolates and could be used in conjunction with Acremonium spp. and H. pulvinata to control the papaya black spot, since these products did not present toxicity on the hyperparasitic fungi.(AU)

O uso de mais de uma técnica de controle pode maximizar a redução dos danos causados pelo fungo Asperisporium caricae agente causal da pinta preta na cultura do mamoeiro. O objetivo deste trabalho foi avaliar a sensibilidade dos fungos Hansfordia pulvinata e Acremonium spp. a produtos alternativos com potencial para uso no controle da pinta-preta. Três isolados de Acremonium spp. (A-598, A-602 e A-617) e um isolado de H. pulvinata (H-611) foram cultivados em meio BDA contendo Agro-Mos®, Bion®, Ecolife®, Hortifospk®, Matriz G®, Vitaphol® separadamente. Fungicida Amistar 500WG® foi utilizado como controle positivo e BDA puro, como controle negativo. A toxicidade dos produtos testados foi determinada com base nos valores do índice biológico, provenientes das médias do crescimento micelial, esporulação e germinação de conídios, em cada unidade experimental. Dessa forma foi possível selecionar os produtos classificados como compatíveis para todos os isolados, e testá-los in vivo. Em casa de vegetação, somente os isolados e os isolados com produtos selecionados, foram aplicados em plantas de mamoeiro com sintomas foliares de pinta-preta. Avaliaram-se a incidência de folhas com hiperparasitas e a porcentagem de lesões de pinta-preta colonizadas pelos isolados testados. Assim o isolado H-611 mostrou-se compatível com a maioria dos produtos alternativos testados, exceto com Hortifos®. Os produtos Bion® e Matriz® apresentaram compatibilidade com todos os isolados testados, podendo ser utilizados em conjunto com o Acremonium spp. e H. pulvinata para controlar a pinta-preta do mamoeiro, uma vez que estes produtos não apresentaram toxidade sobre os fungos hiperparasitas.(AU)

Penicillin and cephalosporin biosyntheses are also regulated by reactive oxygen species.

In an earlier work on lovastatin production by Aspergillus terreus, we found that reactive oxygen species (ROS) concentration increased to high levels precisely at the start of the production phase (idiophase) and that these levels were sustained during all idiophase. Moreover, it was shown that ROS regulate lovastatin biosynthesis. ROS regulation has also been reported for aflatoxins. It has been suggested that, due to their antioxidant activity, aflatoxins are regulated and synthesized like a second line of defense against oxidative stress. To study the possible ROS regulation of other industrially important secondary metabolites, we analyzed the relationship between ROS and penicillin biosynthesis by Penicillium chrysogenum and cephalosporin biosynthesis by Acremonium chrysogenum. Results revealed a similar ROS accumulation in idiophase in penicillin and cephalosporin fermentations. Moreover, when intracellular ROS concentrations were decreased by the addition of antioxidants to the cultures, penicillin and cephalosporin production were drastically reduced. When intracellular ROS were increased by the addition of exogenous ROS (H2O2) to the cultures, proportional increments in penicillin and cephalosporin biosyntheses were obtained. It was also shown that lovastatin, penicillin, and cephalosporin are not antioxidants. Taken together, our results provide evidence that ROS regulation is a general mechanism controlling secondary metabolism in fungi.

Sensitivity of hyperparasitic fungi to alternative products for use in the control of papaya black spot

Abstract The use of more than one control technique can maximize the reduction of the damages caused by the fungus Asperisporium caricae causal agent of the black spot in the papaya crop. The objective of this work was to evaluate the sensitivity of the fungi Hansfordia pulvinata and Acremonium spp. to alternative products with potential for use in the control of the black-spotted ptarmigan. Three isolates of Acremonium spp. (A-598, A-602 and A-617) and an isolate of H. pulvinata (H-611) were grown in BDA medium containing Agro-Mos®, Bion®, Ecolife®, Hortifospk®, Matriz G®, Vitaphol® separately. The Amistar 500WG ® fungicide was used as a positive control and pure BDA as a negative control. The toxicity of the tested products was determined based on the values of the biological index, derived from the means of mycelial growth, sporulation and germination of conidia, in each experimental unit. In this way it was possible to select the products classified as compatible for all isolates, and to test them in vivo. In the greenhouse, only the isolates and isolates with selected products, were applied in papaya plants with foliar symptoms of black-spotted. The incidence of leaves with hyperparasites and the percentage of black-painted lesions colonized by the tested isolates were evaluated. Thus, the H-611 isolate proved to be compatible with most of the alternative products tested, except with Hortifos®. Bion® and Matrix® products were compatible with all tested isolates and could be used in conjunction with Acremonium spp. and H. pulvinata to control the papaya black spot, since these products did not present toxicity on the hyperparasitic fungi.

Resumo O uso de mais de uma técnica de controle pode maximizar a redução dos danos causados pelo fungo Asperisporium caricae agente causal da pinta preta na cultura do mamoeiro. O objetivo deste trabalho foi avaliar a sensibilidade dos fungos Hansfordia pulvinata e Acremonium spp. a produtos alternativos com potencial para uso no controle da pinta-preta. Três isolados de Acremonium spp. (A-598, A-602 e A-617) e um isolado de H. pulvinata (H-611) foram cultivados em meio BDA contendo Agro-Mos®, Bion®, Ecolife®, Hortifospk®, Matriz G®, Vitaphol® separadamente. Fungicida Amistar 500WG® foi utilizado como controle positivo e BDA puro, como controle negativo. A toxicidade dos produtos testados foi determinada com base nos valores do índice biológico, provenientes das médias do crescimento micelial, esporulação e germinação de conídios, em cada unidade experimental. Dessa forma foi possível selecionar os produtos classificados como compatíveis para todos os isolados, e testá-los in vivo. Em casa de vegetação, somente os isolados e os isolados com produtos selecionados, foram aplicados em plantas de mamoeiro com sintomas foliares de pinta-preta. Avaliaram-se a incidência de folhas com hiperparasitas e a porcentagem de lesões de pinta-preta colonizadas pelos isolados testados. Assim o isolado H-611 mostrou-se compatível com a maioria dos produtos alternativos testados, exceto com Hortifos®. Os produtos Bion® e Matriz® apresentaram compatibilidade com todos os isolados testados, podendo ser utilizados em conjunto com o Acremonium spp. e H. pulvinata para controlar a pinta-preta do mamoeiro, uma vez que estes produtos não apresentaram toxidade sobre os fungos hiperparasitas.

Identification of Insect-Deterrent Metabolites from Acremonium masseei strain CICY026, a Saprophytic Fungus from a Sinkhole in Yucatán.

Micromycetes from unexplored sources represent an opportunity to discover novel natural products to control insect pests. With this aim, a strain of Acremonium masseei CICY026 isolated from a tropical sinkhole was identified, cultured on fermented rice, and its ethyl acetate extract (EAE) was evaluated against three serious phytophagous insects (Bemisia tabaci, Myzus persicae, and Rhopalosiphum padi). DNA from A. masseei CICY026 was used to confirm its identity. EAE caused settling inhibition (SI) of M. persicae and R. padi (67.5% and 75.3%, respectively). Bioassay-guided fractionation of the active EAE led to the isolation of a novel metabolite, named hexahydroacremonintriol (1), and of acremonin A glucoside (2). The structures of 1 and 2 were determined using IR, one- and two-dimensional NMR, HRMS, and confirmed by theoretical data. The aphid M. persicae was noticeably sensitive to 1 and 2 (SI: 55.6% and 67.2%, respectively), whereas R. padi was only slightly affected by 1 (SI: 59%). This new knowledge about mycobiota from these special sinkhole ecosystems will inform the development of new biorational pesticides.

Ceratite micótica por Acremonium spp. ­ Relato de caso / Mycotic keratitis by Acremonium spp.: case report

Ceratite micótica é uma infecção corneana fúngica de aspecto supurativo e normalmente ulcerativo. Representa uma condição potencialmente devastadora e de difícil tratamento. Mais de sessenta espécies de fungos foram reportadas como agentes de ceratites, dependendo da região geográfica. O gênero Acremonium spp. é um agente incomum de infecções de córnea. Paciente masculino, agricultor, portador de transplante de córnea, procurou atendimento devido à dor e vermelhidão na região ocular. Apresentava lesão ulcerada, a qual teve progressão mesmo com o tratamento inicial proposto, sendo necessária a realização de retransplante de córnea a quente. A cultura do material coletado mostrou-se positiva para o fungo Acremonium spp. Existem na literatura poucos relatos de ceratite por Acremonium spp., sendo estes, em sua maioria, ocorridos após procedimentos, como Laser in situ keratomileusis (LASIK). A ceratite fúngica pode ser causada por fungos filamentosos ou leveduras e requer um tratamento específico orientado pela análise microbiológica. O prognóstico destas lesões e o grau de acometimento visual dependem do uso de antifúngicos efetivos contra o agente em questão.

Brote de fungemias por Sarocladium kiliense en ocho hospitales públicos por contaminación intrínseca de ondansetrón intravenoso / Outbreak of fungemias by Sarocladium kiliense in eight public hospitals per intrinsic contamination of ondansetron intravenous

Resumen Introducción: Sarocladium kiliense es un hongo saprófito que puede generar infecciones oportunistas asociadas a procedimientos invasores. Se informa un brote multicéntrico nosocomial de fungemias de fuente común por este agente. Luego del reporte de cinco casos en pacientes en tres hospitales al Programa de Control de Infecciones del Ministerio de Salud de Chile en julio de 2013, se estudiaron a nivel nacional todos los pacientes con hemocultivo positivo para este agente. Se trató de cuadros clínicos leves a moderados, sin muertes atribuibles. El estudio identificó 65 casos en 8 hospitales, en su mayoría pacientes pediátricos en quimioterapia. Estudios iniciales de 94 muestras de cuatro fármacos y dispositivos usados en todos los casos resultaron negativas hasta que, en un segundo análisis de lotes seleccionados por criterios epidemiológicos y su matriz farmacéutica, se identificó la contaminación intrínseca de ampollas de ondansetrón de un productor específico, que se usó en todos los casos. Se realizó un retiro nacional de las ampollas de los tres lotes contaminados del fármaco, después de lo cual se contuvo el brote. La vigilancia de infecciones en los hospitales y el programa nacional coordinado con los laboratorios de microbiología fueron claves para identificar un brote multicéntrico de fuente común por contaminación de un fármaco por un hongo inusual.

Sarocladium kiliense is a saprophyte fungus that can cause opportunistic infections associated to invasive procedures. We report a multi-hospital nosocomial outbreak of fungemias due to this agent. Patients with positive blood culture to this agent were studied after six bloodstream infections identified in three Chilean hospitals in July 2013 were reported to Ministry of Health National Infection and Prevention Control Program. In general, there were mild clinical manifestations, without deaths attributable to the infection. Epidemiological and micro-biological study identified 65 cases in 8 hospitals, mostly pediatric patients in chemotherapy. Initial studies of 94 different drugs and medical devices had negative results, until a second analysis of specific blisters and their pharmaceutical matrix selected by epidemiological criteria identified an intrinsic contamination of ondansetron blisters from a specific producer used in all the patients. A recall of contaminated ondansetron blisters was performed in all the country, after which the outbreak was contained. Surveillance and response of local and national infection prevention and control programs and laboratory support were key to control of a national multi-hospital common source outbreak due to contamination of a drug by an unusual fungus.

Contact lens-related bilateral and simultaneous Acremonium keratitis / Ceratite bilateral e simultânea por Acremonium relacionada ao uso de lentes de contato

Abstract This is a case of bilateral and simultaneous Acremonium keratitis related to intermittent and alternating eye soft contact lens use, which has a delayed diagnose, presented amphotericin B resistance with persistent hypopyon and had a positive response to topical natamycin. Besides the unusual presentation, there was no history of trauma or contact with vegetable matter, usually associated to the majority of cases of keratomicosys by filamentous fungi.

Resumo Trata-se de um caso de ceratite bilateral e simultânea por Acremonium relacionada ao uso intermitente e sem respeitar a lateralidade do uso de lentes de contato gelatinosas. Houve diagnóstico tardio, apresentando resistência a anfotericina B e hipópio persistente, com resposta positiva a natamicina tópica. Além da forma incomum de apresentação, não houve histórico de trauma ou contato com material vegetal, associado à maioria dos casos de ceratite por fungos filamentosos.

Sensitivity of hyperparasitic fungi to alternative products for use in the control of papaya black spot

Abstract The use of more than one control technique can maximize the reduction of the damages caused by the fungus Asperisporium caricae causal agent of the black spot in the papaya crop. The objective of this work was to evaluate the sensitivity of the fungi Hansfordia pulvinata and Acremonium spp. to alternative products with potential for use in the control of the black-spotted ptarmigan. Three isolates of Acremonium spp. (A-598, A-602 and A-617) and an isolate of H. pulvinata (H-611) were grown in BDA medium containing Agro-Mos®, Bion®, Ecolife®, Hortifospk®, Matriz G®, Vitaphol® separately. The Amistar 500WG ® fungicide was used as a positive control and pure BDA as a negative control. The toxicity of the tested products was determined based on the values of the biological index, derived from the means of mycelial growth, sporulation and germination of conidia, in each experimental unit. In this way it was possible to select the products classified as compatible for all isolates, and to test them in vivo. In the greenhouse, only the isolates and isolates with selected products, were applied in papaya plants with foliar symptoms of black-spotted. The incidence of leaves with hyperparasites and the percentage of black-painted lesions colonized by the tested isolates were evaluated. Thus, the H-611 isolate proved to be compatible with most of the alternative products tested, except with Hortifos®. Bion® and Matrix® products were compatible with all tested isolates and could be used in conjunction with Acremonium spp. and H. pulvinata to control the papaya black spot, since these products did not present toxicity on the hyperparasitic fungi.

Resumo O uso de mais de uma técnica de controle pode maximizar a redução dos danos causados pelo fungo Asperisporium caricae agente causal da pinta preta na cultura do mamoeiro. O objetivo deste trabalho foi avaliar a sensibilidade dos fungos Hansfordia pulvinata e Acremonium spp. a produtos alternativos com potencial para uso no controle da pinta-preta. Três isolados de Acremonium spp. (A-598, A-602 e A-617) e um isolado de H. pulvinata (H-611) foram cultivados em meio BDA contendo Agro-Mos®, Bion®, Ecolife®, Hortifospk®, Matriz G®, Vitaphol® separadamente. Fungicida Amistar 500WG® foi utilizado como controle positivo e BDA puro, como controle negativo. A toxicidade dos produtos testados foi determinada com base nos valores do índice biológico, provenientes das médias do crescimento micelial, esporulação e germinação de conídios, em cada unidade experimental. Dessa forma foi possível selecionar os produtos classificados como compatíveis para todos os isolados, e testá-los in vivo. Em casa de vegetação, somente os isolados e os isolados com produtos selecionados, foram aplicados em plantas de mamoeiro com sintomas foliares de pinta-preta. Avaliaram-se a incidência de folhas com hiperparasitas e a porcentagem de lesões de pinta-preta colonizadas pelos isolados testados. Assim o isolado H-611 mostrou-se compatível com a maioria dos produtos alternativos testados, exceto com Hortifos®. Os produtos Bion® e Matriz® apresentaram compatibilidade com todos os isolados testados, podendo ser utilizados em conjunto com o Acremonium spp. e H. pulvinata para controlar a pinta-preta do mamoeiro, uma vez que estes produtos não apresentaram toxidade sobre os fungos hiperparasitas.

Polyethyleneimine coating of magnetic particles increased the stability of an immobilized diglycosidase.

The diglycosidase, α-rhamnosyl-ß-glucosidase, from Acremonium sp. DSM24697 was immobilized by adsorption and cross-linking onto polyaniline-iron (PI) particles. The immobilization yield and the immobilization efficiency were relatively high, 31.2% and 8.9%, respectively. However, the heterogeneous preparation showed lower stability in comparison with the soluble form of the enzyme in operational conditions at 60 °C. One parameter involved in the reduced stability of the heterogeneous preparation was the protein metal-catalyzed oxidation achieved by iron traces supplied from the support. To overcome the harmful effect, iron particles were coated with polyethyleneimine (PEI; 0.84 mg/g) previously for the immobilization of the catalyst. The increased stability of the catalyst was correlated with the amount of iron released from the support. Under operational conditions, the uncoated particles lost between 76% and 52% activity after two cycles of reuse, whereas the PEI-coated preparation reduced 45-28% activity after five cycles of reuse in the range of pH 5.0-10, respectively. Hence, polymer coating of magnetic materials used as enzyme supports might be an interesting approach to improve the performance of biotransformation processes.

Dermoscopy assisting the diagnosis of mycetoma: case report and literature review

Mycetomas are a chronic skin infection characterized by perilesional edema, formation of sinus tracts, and discharge of purulent or seropurulent exudate containing grains. This report aims to demonstrate the clinical diagnosis (by dermoscopy) of a skin lesion that initially bared no clinical features of a mycetoma.

Direct ethanol production from glucose, xylose and sugarcane bagasse by the corn endophytic fungi Fusarium verticillioides and Acremonium zeae.

Production of ethanol with two corn endophytic fungi, Fusarium verticillioides and Acremonium zeae, was studied. The yield of ethanol from glucose, xylose and a mixture of both sugars were 0.47, 0.46 and 0.50g/g ethanol/sugar for F. verticillioides and 0.37, 0.39 and 0.48g/g ethanol/sugar for A. zeae. Both fungi were able to co-ferment glucose and xylose. Ethanol production from 40g/L of pre-treated sugarcane bagasse was 4.6 and 3.9g/L for F. verticillioides and A. zeae, respectively, yielding 0.31g/g of ethanol per consumed sugar. Both fungi studied were capable of co-fermenting glucose and xylose at high yields. Moreover, they were able to produce ethanol directly from lignocellulosic biomass, demonstrating to be suitable microorganisms for consolidated bioprocessing.

Purification and characterization of AsES protein: a subtilisin secreted by Acremonium strictum is a novel plant defense elicitor.

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2(˙)) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

Caracterización morfológica de Acremonium sp. asociado a Neonectria fuckeliana en Pinus radiata en Chile / Morphological characterization of Acremonium-like asociated with Neonectria fuckeliana in Pinus radiata in Chile

En este estudio se realizaron los primeros aislamientos del estado asexual del patógeno Neonectria fuckeliana asociado a cancros o ®revirado del pino¼ en plantaciones de Pinus radiata. Se caracterizaron morfológicamente las cepas del sinanamorfo semejante a Verticillium (Acremonium), obtenidas en cultivo in vitro a partir de peritecios. El material para los aislamientos consistió en trozos de corteza de P. radiata con presencia de peritecios, colectados en Toltén, región de La Araucanía, lugar donde se realizó el primer reporte de N. fuckeliana en Chile. Se utilizaron diez cepas del semejante a Acremonium para la caracterización morfológica, mediciones de estructuras fúngicas, ritmo de crecimiento in vitro y morfología de las colonias. Las colonias presentaron un micelio flocoso y ralo de bordes blanquecinos e irregulares, destacándose tres tipos de colonias, blancas, naranjo oscura y naranjo claro. Taxonómicamente, las cepas coinciden con las estructuras mencionadas en la literatura, caracterizándose por la presencia de glioconidios. Las fialides midieron entre 7 – 78,4 x 1,4 - 4,9 ìm. Los conidios, de formas ovoides y algunas bicelulares, midieron entre 4,2 - 8,4 x 2,6 - 3,5 ìm. El ritmo de crecimiento in vitro fue lento, completando su desarrollo a los 19 días con un promedio de 71 +/- 0,3 mm de diámetro, a una tasa de crecimiento diario de 3,8 mm. Los resultados obtenidos hacen necesario futuros estudios de carácter molecular para analizar la variabilidad genética poblacional que puede presentarse en Chile.

First Neonectria fuckeliana isolates in Chile. Strains of Acremonium obtained from in vitro peritecia culture were morphologically characterized. The samples were collected in Toltén, La Araucanía region, were Neonectria fuckeliana was first reported in Chile. The material used for the isolation came from pieces of Pinus radiata bark with peritecia. Ten Acremonium-like strains were used for characterization of fungal structures, in vitro growth and strains morphology. The colonies were a floccose mycelium and thin edges whitish and irregular, varying color highlighting three types of strains, white, dark orange and pale orange. Taxonomically, the strains match the structures referred in the literature, characterized by the presence of gliospores. The phialides dimensions ranged from 7 to 7.8 ìm long and 1.4 to 4.9 ìm wide. The conidio of ovoid shapes and some bicelular measured between 4.2 to 8.4 ìm in length and width 2.6 and 3.5 ìm. In vitro growth rates were slow, the complete development take19 days with a daily growth average of 71 +/-3 mm in diameter, at a rate of 3.8 mm. It is necessary future molecular studies to analyze the population genetic variation that may occur in Chile.

Recurrent Acremonium infection in a kidney transplant patient treated with voriconazole: a case report / Infecção recidivante por Acremonium em paciente transplantado renal tratado com voriconazol: relato de caso

Acremonium infection is rare and associated with immunosuppression. A case of recurrent cutaneous Acremonium infection after short term voriconazole use is described. Surgical resection was the definitive therapy. Oral voriconazole was used in the treatment of Acremonium infection, but recurrence was associated with short therapy. Prolonged antifungal therapy and surgical resection are discussed for the treatment of localized lesions.

Infecção por Acremonium é rara e pode estar associada com imunossupressão. Descrevemos um caso de infecção recorrente de pele por Acremonium após tratamento breve com voriconazol. Ressecção cirúrgica foi o tratamento definitivo. Terapia prolongada com antifúngicos e ressecção cirúrgica são discutidas para o tratamento de doenças fúngicas localizadas.

Viabilidade, confirmação taxonômica e detecção enzimática de espécies de Acremonium preservadas sob óleo mineral na Coleção de Culturas University Recife Mycology / Viability, taxonomic confirmation and enzymatic detection of Acremonium species preserved under mineral oil in the URM Culture Collection

Enzimas hidrolíticas secretadas por fungos têm um papel importante na patogenicidade das infecções. Objetivando avaliar a atividade enzimática foram testados 31 isolados de Acremonium mantidos na Coleção de Culturas University Recife Mycology. Fragmentos das culturas foram transferidos para caldo glicosado para reativação e posterior crescimento em meio ágar batata dextrose, para verificar viabilidade, pureza e confirmação taxonômica pela observação das características macroscópicas e microscópicas. Para detecção enzimática foram utilizados substratos de caseína do leite e gelatina para protease, amido para amilase e lecitina de soja para fosfolipase. Das 31 culturas, 26 (83,9 por cento) mantiveram-se viáveis e 24 (92,3 por cento) foram confirmadas taxonomicamente. Das 24 culturas, 12 (50 por cento) apresentaram atividade proteásica, duas (16,7 por cento) em caseína do leite, uma (8,3 por cento) em gelatina e nove (75 por cento) em ambos os substratos; 16 (66,7 por cento) degradaram amido. Nenhuma cultura apresentou atividade fosfolipásica. Conclui-se que espécies de Acremonium são capazes de produzir enzimas envolvidas na patogenicidade das infecções fúngicas.

Hydrolytic enzymes secreted by fungi play an important role in the pathogenesis of infection. With the aim of evaluating the enzymatic activity, 31 isolates of Acremonium stored in the University of Recife Mycology (URM) Culture Collection were tested. Culture fragments were transferred to glycoside broth for reactivation and further growth in potato dextrose agar medium in order to investigate viability and purity and to confirm the taxonomy through observing the macroscopic and microscopic characteristics. To detect enzymes, milk casein and gelatin were used as substrates for proteinase, starch for amylase and soy lecithin for phospholipase. Among the 31 cultures, 26 (83.9 percent) remained viable and 24 (92.3 percent) were confirmed taxonomically. Out of these 24 cultures, 12 (50 percent) presented proteinase activity, of which two (16.7 percent) were on milk casein, one (8.3 percent) on gelatin and nine (75 percent) on both substrates; 16 (66.7 percent) degraded starch. None of the cultures presented phospholipase activity. It was concluded that Acremonium species are able to produce enzymes that are involved in the pathogenicity of fungal infections.

Casos clínicos: Microsporum praecox y Acremonium strictum nuevos agentes de micosis cutáneas oportunistas en la zona central de Chile / Clinical reports: Microsporum praecox and Acremonium strictum new agents of opportunistic cutaneous mycoses in the central zone of Chile

Se presentan 2 casos clínicos de agentes de micosis cutáneas no descritos en el país. El primer caso corresponde a un paciente femenino de 18 años de edad, sin antecedentes mórbidos de importancia, ni viajes al exterior, que presenta una lesión pruriginosa en cara externa del brazo izquierdo de apariencia erito – pápulodescamativa de 2 semanas de evolución. Al examen directo, presentó micelios hialinos y en los cultivos micológicos un abundante desarrollo de Microsporum praecox. La lesión remitió completamente al tratamiento con Terbinafina tópica 2 veces al día por 21 días. Se comenta la rara presencia en Chile de M.praecox, especie presente en equinos y su hábitat, pero de dispersión restringida a Francia y Bélgica principalmente. Se discute el posible origen de este agente y las interrogantes en cuanto a su presencia en el país. El segundo caso corresponde a un paciente femenino de 24 años de edad, sin antecedentes mórbidos de importancia, que presenta alteraciones de la uña del primer ortejo derecho durante 2 años, caracterizadas por: engrosamiento subungueal, destrucción distal y cambios de coloración. Consulta con un dermatólogo, quien por su aspecto clínico típico, diagnostica onicomicosis e indica Terbinafina 250 mg diarios por 2 meses, sin embargo, el cuadro clínico se hace refractario en el tiempo frente a diversos antimicóticos usados.

Two clinical cases of agents of cutaneous mycoses not described in the country are reported in this paper. The first case deals with an 18-aged female patient, free from any significant morbid history, who has never traveled abroad, which bears a erytho-papule-descamative pruriginous lesion on the outside of her left arm and 2-week evolution. A direct exam revealed the presence of hyaline mycelia while an abundant development of Microsporum praecox was detected in the mycological cultures. The lesion had a favorable reaction to the treatment with topic Terbinafine twice a day for 21 days. The rare occurrence of M.praecox in Chile, a species commonly found in horses and their habitat yet with a restricted distribution mainly in France and Belgium and the possible origin of this agent in the country is commented. The second case is focused on a 24-age female patient, free from any significant morbid history, who exhibits variations in her first right toe since two years and which are characterized by the following: subungueal thickening, distal destruction and change in colour. She consults a dermatologist, who after considering a typical clinical aspect, determined an onimycosis and recommends 250mg Terbinafine for two months; however the clinical pattern becomes refractory in time due to the use of several antimycotic.

Fungemia asintomática causada por Acremonium sp asociada a colonización de catéter venoso central (CVC) / Asymptomatic fungemia caused by Acremonium sp associated with colonization of a central venous catheter

Introducción: En años recientes se ha reportado que los pacientes inmunocomprometidos presentan infecciones por organismos considerados habitualmente como saprófitos. Material y métodos: Detectamos un caso de fungemia por Acremonium sp. en un paciente asintomático, 5 semanas después apareció un segundo caso. Ambos pacientes portaban catéter venoso central (CVC) de larga estancia y acudían a la sesión semanal de cuidados que lleva a cabo el Equipo de Terapia Intravenosa. Los dos pacientes recibieron nutrición parenteral total (NPT) durante 5 meses antes del diagnóstico de fungemia. Se estudiaron en forma retrospectiva todos los pacientes que habían recibido NPT durante el mismo periodo de tiempo. Resultados: Los dos casos de fungemia por Acremonium sp. en pacientes con adenocarcinoma gástrico habían recibido NPT por un promedio de 19 días. Se retiró el CVC y recibieron tratamiento antifúngico con resolución de la infección. Otros 8 pacientes que habían recibido NPT preparado por la misma casa comercial durante este periodo por un promedio de 9.5 días (rango 6 a 20). No se encontró ningún otro hemocultivo ni punta de catéter con Acremonium sp. Conclusiones: Se sugiere mantener una estrecha vigilancia en los pacientes con CVC que reciben NPT para identificar la colonización por gérmenes de baja patogenicidad, aún en pacientes asintomáticos.

BACKGROUND: Microorganisms considered saprophytes have emerged as invasive or indolent pathogens among immuno-compromised patients. MATERIAL AND METHODS: We detected an initial case of catheter-related Acremonium sp fungemia on a previously asymptomatic patient. We diagnosed a second case five weeks later. Both patients had a non-tunneled central venous catheter (CVC) that had been cared for following routine protocol by nurses in the Intravenous Therapy Team on a weekly basis. The sole risk factor that both patients shared was that they had received total parenteral nutrition (TPN) by a CVC 5 months prior to the date the catheter-related fungemia was detected. We retrospectively studied all patients who had received TPN during this period. RESULTS: We found two cases ofAcremonium fungemia, patients had gastric adenocarcinoma and received TPN for an average of 19 days. Infection was resolved with catheter removal and antifungic therapy. Another eight patients received TPN from the same commercial firm during this period; average administration was 9.5 days (range, 6-20). Neither blood cultures nor tip-catheters culture reported Acremonium sp. CONCLUSIONS: Patients with CVC for TPN should be closely monitored to identify colonization with a low pathogenic microorganism that could be mistakenly diagnosed as asymptomatic.

Influence of medium constituents on the biosynthesis of cephalosporin-C

Acremonium chrysogenum NCIM 1069 was used for the biosynthesis of cephalosporin-C (CPC) in batch mode of cultivation. The effect of different medium constituents for better yield of CPC was thoroughly investigated. From the results of the fermentation, it was found that ammonium sulphate as inorganic nitrogen source and methionine at the concentration of 0.4 percent are most suitable for higher yield of antibiotic. The variation in the C/N ratio on the biosynthesis of CPC showed that a C/N ratio of 8.0 is most suitable for maximum production of CPC