Una experiencia de siete años en el laboratorio de Micología del Hospital de Pediatría Juan P. Garrahan / A seven-year experience in the Mycology Laboratory of Hospital de Pediatría Juan P. Garrahan

En noviembre del año 2015 nos incorporamos al Laboratorio de Micología del Servicio de Microbiología del Hospital Garrahan. En este breve resumen queremos compartir los avances logrados a través de nuestra experiencia durante siete años de trabajo profesional. Debido a los diagnósticos realizados y su complejidad, consideramos que el Hospital Garrahan, sus pacientes y la comunidad toda necesitan contar con un laboratorio de Micología que responda a sus necesidades. Creemos haber iniciado un camino que esperamos continúe y culmine con la creación de la Unidad de Micología (AU)

In November 2015 we joined the Mycology Laboratory of the Microbiology Service of the Hospital Garrahan. In this brief summary we want to share the advances achieved through our experience during seven years of professional work. Due to the diagnosis made and their complexity, we believe that the Hospital Garrahan, its patients and the entire community, need to have a Mycology laboratory that responds to their requirements. We believe we have started a path that we hope will continue and culminate with the creation of the Mycology Unit (AU)

Culturing and Mating of Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic human fungal pathogen, capable of causing invasive aspergillosis in patients with compromised immune systems. The fungus was long considered a purely asexual organism. However, a sexual cycle was reported in 2009, with methods described to induce mating under laboratory conditions. The presence of a sexual cycle now offers a valuable tool for classical genetic analysis of the fungus, such as allowing determination of whether traits of interest are mono- or poly-genic in nature. For example, the sexual cycle is currently being exploited to determine the genetic basis of traits of medical importance such as resistance to azole antifungals and virulence, and to characterize the genes involved. The sexual cycle can also be used to assess the possibility of gene flow between isolates. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. This unit describes protocols for culturing of A. fumigatus and for inducing sexual reproduction between compatible MAT1-1 and MAT1-2 isolates of the species. The unit also provides working methods for harvesting sexual structures, isolating single-spore progeny and confirming whether sexual recombination has occurred. © The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Survey of laboratory practices for diagnosis of fungal infection in seven Asian countries: An Asia Fungal Working Group (AFWG) initiative.

An online survey of mycology laboratories in seven Asian countries was conducted to assess the status, competence, and services available. Country representatives from the Asia Fungal Working Group (AFWG) contacted as many laboratories performing mycology diagnosis as possible in their respective countries, requesting that the laboratory heads complete the online survey. In total, 241 laboratories responded, including 71 in China, 104 in India, 11 in Indonesia, 26 in the Philippines, four in Singapore, 18 in Taiwan, and seven in Thailand. Overall, 129/241 (53.5%) surveyed mycology laboratories operate as separate designated mycology laboratories, 75/241 (31.1%) conduct regular formal staff training, 103/241 (42.7%) are accredited, and 88/157 (56.1%) participate in external quality assurance scheme (EQAS) programs. Microscopy and culture methods are available in nearly all laboratories, although few perform DNA sequencing (37/219; 16.9%) or use matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) (27/219; 12.3%) for isolate identification. Antifungal susceptibility testing is performed in 142/241 (58.9%) laboratories, mainly for yeasts. The most commonly performed nonculture diagnostic is cryptococcal antigen testing (66 laboratories), followed by galactomannan testing (55), polymerase chain reaction (PCR) diagnosis (37), and beta-D-glucan testing (24). Therapeutic drug monitoring is conducted in 21 laboratories. There is almost no access to advanced diagnostic tests, like galactomannan, ß-D-glucan, and PCR, in the surveyed laboratories in Indonesia, the Philippines, and Thailand. These results highlight the need for development of quality laboratories, accreditation and training of manpower in existing laboratories, and access to advanced non-culture-based diagnostic tests to facilitate the diagnosis of fungal infections in Asia.

Selection of G1 Phase Yeast Cells for Synchronous Meiosis and Sporulation.

Centrifugal elutriation is a procedure that allows the fractionation of cell populations based upon their size and shape. This allows cells in distinct cell cycle stages can be captured from an asynchronous population. The technique is particularly helpful when performing an experiment to monitor the progression of cells through the cell cycle or meiosis. Yeast sporulation like gametogenesis in other eukaryotes initiates from the G1 phase of the cell cycle. Conveniently, S. cerevisiae arrest in G1 phase when starved for nutrients and so withdrawal of nitrogen and glucose allows cells to abandon vegetative growth in G1 phase before initiating the sporulation program. This simple starvation protocol yields a partial synchronization that has been used extensively in studies of progression through meiosis and sporulation. By using centrifugal elutriation it is possible to isolate a homogeneous population of G1 phase cells and induce them to sporulate synchronously, which is beneficial for investigating progression through meiosis and sporulation. An additionally benefit of this protocol is that cell populations can be isolated based upon size and both large and small cell populations can be tested for progression through meiosis and sporulation. Here we present a protocol for purification of G1 phase diploid cells for examining synchronous progression through meiosis and sporulation.

Cytographic changes on BC-6800 Haematological Analyzer related to the presence of Candida albicans in peripheral blood. A new tool to suspect candidemia?

BACKGROUND: We studied the quantitative and cytographic changes that the presence of Candida albicans (C. albicans) in peripheral blood (PB) samples causes on the Mindray BC-6800 Haematological Analyzer. METHODS: A simulated in vitro candidemia was obtained by adding a different amount of C. albicans to discarded remnants of PB samples. Quantitative data and cytographic features were evaluated immediately as well as after 120 and 240 min of the yeast addition. A microscopic slides review was even performed at the same time. RESULTS: After yeasts addition, an increase of total leucocytes, neutrophils and basophils have been observed, but these increases are not certainly descriptive of C. albicans presence.Instead, extracellular blastospores cause a false increase in nucleated red blood cells (nRBCs), which appear as a new population in the specific counting channel for erytroblasts (NRBC channel). Regardless of the numbers, C. albicans form a pseudo-erythroblastic cluster in the NRBC channel whose resulting shape is so different than the 'normal' nRBC that it demands a microscopic review. Even cytographic changes related with the neutrophilic phagocytic activity have been observed on leucocyte's differential count citogram (DIFF) of the BC-6800. CONCLUSIONS: Our observations suggest that the results of the BC-6800, which are due to C. albicans' presence, might be useful to speculate earlier diagnosis of sepsis.

Comparison of methods for assessing temporal variation of growth of fungi on building materials.

Fungal growth on indoor surfaces can decay building materials and release hazardous substances that affect indoor air quality. Despite the numerous methods available for growth determination, there is no commonly accepted standard. The goal of this study was to compare five different assay methods for the measurement of fungal growth: cultivation, MS-based determination of ergosterol, beta-N-acetylhexosaminidase activity, quantitative PCR and microscopic spore counting. Three fungal species (Aspergillus puulaauensis, Cladosporium montecillanum and Penicillium polonicum) were grown on three different building materials (two types of acoustic board and wood). Fungal load was determined at different time points. Results from all of the methods, except the spore count, showed good correlation between each other (r=0.6-0.8). Results obtained with the cultivation method had the highest variability among replicate samples (65 %), making it the least reproducible in repeated measurements. However, it also displayed the highest variability in incubation times (149 %), indicating its suitability for detecting transient changes in the physiological state of cells. Similar to the cultivation method, quantitative PCR correlated well with the other methods and had high variability in incubation times but had lower variability among replicate samples. Ergosterol and beta-N-acetylhexosaminidase enzyme activity seemed to be the methods least dependent on the physiological state of the cells. Varying growth dynamics were observed for different species over time with the different assay methods. Each one of the tests provides a different perspective on fungal quantification due to its specific responses to the various stages of fungal growth.

[Performance evaluation of Rapid™ Yeast Plus (Remel) system from two different culture media]. / Evaluación del rendimiento del sistema Rapid™ Yeast Plus (Remel) a partir de 2 medios de cultivo diferentes.

Within the genus Candida, Candida albicans is the most commonly isolated species from clinical samples. Due to the emergence of other species which can show a higher index of antifungal resistance, a fast identification of these species is necessary. The aim of this work was to evaluate the performance of the RapID Yeast Plus system from two different subculture media formulations: Sabouraud dextrose agar adjusted by Emmons (the medium is indicated in the equipment insert) and Sabouraud glucose agar, which is the most frequently used in Buenos Aires City laboratories. One hundred and sixty-six clinical sample strains coming from different hospitals belonging to the Mycology Network of Buenos Aires City were studied. From the obtained results, we conclude that the conditions and culture medium indicated by the manufacturer should be followed.

Automated image-based analysis of spatio-temporal fungal dynamics.

Due to their ability to grow in complex environments, fungi play an important role in most ecosystems and have for that reason been the subject of numerous studies. Some of the main obstacles to the study of fungal growth are the heterogeneity of growth environments and the limited scope of laboratory experiments. Given the increasing availability of image capturing techniques, a new approach lies in image analysis. Most previous image analysis studies involve manual labelling of the fungal network, tracking of individual hyphae, or invasive techniques that do not allow for tracking the evolution of the entire fungal network. In response, this work presents a highly versatile tool combining image analysis and graph theory to monitor fungal growth through time and space for different fungal species and image resolutions. In addition, a new experimental set-up is presented that allows for a functional description of fungal growth dynamics and a quantitative mutual comparison of different growth behaviors. The presented method is completely automated and facilitates the extraction of the most studied fungal growth features such as the total length of the mycelium, the area of the mycelium and the fractal dimension. The compactness of the fungal network can also be monitored over time by computing measures such as the number of tips, the node degree and the number of nodes. Finally, the average growth angle and the internodal length can be extracted to study the morphology of the fungi. In summary, the introduced method offers an updated and broader alternative to classical and narrowly focused approaches, thus opening new avenues of investigation in the field of mycology.

Validation of sterility testing of cord blood: challenges and results.

BACKGROUND: Sterility testing for cord blood (CB) products is mandatory to prevent transplantation-transmitted microbial infections. Here, the automated BacT/ALERT (bioMérieux) culture system was validated to detect microbial contamination in CB units processed at the Canadian National Public Cord Blood Bank. STUDY DESIGN AND METHODS: A three-phase validation was developed. CB units were prepared with pentastarch (Phases 1 and 2) or hetastarch (Phase 3). In Phase 1, CB was spiked with approximately 100 colony-forming units/mL of Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Bacteroides fragilis, and Candida albicans. Plasma (8 mL) and buffy coat (BC; 0.5 and 8 mL) were inoculated into culture bottles. In Phases 2 and 3, a mix of red blood cells (RBCs) and plasma (4 mL each) was used as the inoculant. In Phase 3, Aspergillus brasiliensis was added as a test organism and microbial concentrations in the by-product RBCs and plasma were determined. The BC fractions were cryopreserved and tested 3 months later. RESULTS: In Phase 1, bacteria failed to grow in CB units containing antibiotics. Thus, antibiotic-free units were used for the other phases. C. albicans was not always captured in plasma, but using a mix of RBCs and plasma, all organisms were detected. The use of pentastarch or hetastarch did not affect microbial recovery. C. albicans and A. brasiliensis were preferentially recovered in RBCs and BC. Cryopreservation did not affect microbial survival during CB processing. CONCLUSIONS: A mix of plasma and RBCs is appropriate for CB sterility testing. Interestingly, fungi preferentially segregate to cellular fractions. The clinical significance of the bactericidal /or bacteriostatic effect of antibiotics in CB merits further investigation.

[Evaluation of Vitek 2 for the identification of Candida yeasts]. / Evaluación del sistema Vitek 2 para la identificación de las principales especies de levaduras del género Candida.

The aim of this investigation was to evaluate the performance of Vitek 2 YST cards (bioMérieux, Inc., Hazelwood, MO, USA) for the identification of yeasts of the genus Candida. A total of 168 isolates were analyzed and the results were compared to those of the API 20 C AUX (24%) o API ID 32 C (76%) kits (bioMérieux, Marcy L'Etoile, France). Each isolate was grown in chromogenic agar and in corn meal agar (Oxoid, UK) to observe its micromorphology. C. albicans and C. dublininesis were identified by additional biochemical and molecular tests. The agreement observed was 98.3%. Only three isolates were incorrectly identified by Vitek 2: one strain of C .tropicalis and one strain of C. krusei were identified as C. parapsilosis by YST while one strain of C. krusei was identified with low discrimination. The average time for obtaining results was 18.25 h. Vitek 2 is a simple, safe and useful system for the identification of significant Candida species.

Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF).

The heat and mass transfer have been proved to be the important factors in air pressure pulsation for cellulase production. However, as process of enzyme secretion, the cellulase formation has not been studied in the view of microorganism metabolism and metabolic key enzyme activity under air pressure pulsation condition. Two fermentation methods in ATPase activity, cellulase productivity, weight lose rate and membrane permeability were systematically compared. Results indicated that gas double-dynamic solid state fermentation had no obviously effect on cell membrane permeability. However, the relation between ATPase activity and weight loss rate was linearly dependent with r=0.9784. Meanwhile, the results also implied that gas periodic stimulation had apparently strengthened microbial metabolism through increasing ATPase activity during gas double-dynamic solid state fermentation, resulting in motivating the production of cellulase by Trichoderma reesei YG3. Therefore, the increase of ATPase activity would be another crucial factor to strengthen fermentation process for cellulase production under gas double-dynamic solid state fermentation.

Evaluación del sistema Vitek 2 para la identificación de las principales especies de levaduras del género Candida. / [Evaluation of Vitek 2 for the identification of Candida yeasts].

The aim of this investigation was to evaluate the performance of Vitek 2 YST cards (bioMérieux, Inc., Hazelwood, MO, USA) for the identification of yeasts of the genus Candida. A total of 168 isolates were analyzed and the results were compared to those of the API 20 C AUX (24

) o API ID 32 C (76

) kits (bioMérieux, Marcy LEtoile, France). Each isolate was grown in chromogenic agar and in corn meal agar (Oxoid, UK) to observe its micromorphology. C. albicans and C. dublininesis were identified by additional biochemical and molecular tests. The agreement observed was 98.3

. Only three isolates were incorrectly identified by Vitek 2: one strain of C .tropicalis and one strain of C. krusei were identified as C. parapsilosis by YST while one strain of C. krusei was identified with low discrimination. The average time for obtaining results was 18.25 h. Vitek 2 is a simple, safe and useful system for the identification of significant Candida species.

Proyecto épico: formulación de unas recomendaciones educativas con metodología DELPHI para pacientes adultos críticos no neutropénicos y con candidiasis invasiva / Épico project. Development of educational recommendations using the DELPHI technique on invasive candidiasis in non-neutropenic critically ill adult patients

Antecedentes: Aunque en la última década ha mejorado el manejo de la candidiasis invasiva, todavía persisten aspectos controvertidos, en especial por lo que respecta a la estrategia diagnóstica y terapéutica. Objetivos: Identificar los conocimientos clínicos esenciales y formular unas recomendaciones con la obtención de un alto grado de consenso, necesarias en la asistencia de pacientes adultos no neutropénicos en estado crítico con candidiasis invasiva. Métodos: Se preparó una encuesta prospectiva cuyo texto se redactó en español, y se obtuvo un consenso mediante técnica DELPHI (un método de reestructuración de un proceso de comunicación con el que se obtiene un grado de consenso de los especialistas sobre el problema planteado). En primer término, se envió de forma anónima por correo electrónico a 25 especialistas nacionales de diferentes disciplinas médicas, expertos en infecciones fúngicas invasivas, de 5 sociedades científicas nacionales, incluidos intensivistas, anestesistas, microbiólogos, farmacólogos e infectólogos, que respondieron a 47 preguntas preparadas por el grupo de coordinación, tras una revisión exhaustiva de los estudios publicados durante los 5 últimos años. Los objetivos educativos contemplaron 5 categorías: epidemiología, instrumentos diagnósticos, scores, estrategias terapéuticas y de desescalada. Para ser seleccionado, el grado de acuerdo alcanzado entre los expertos del panel en cada uno de los ítems debía superar el 75%. En segundo término, después de extraer las recomendaciones de los ítems seleccionados, se celebró una reunión presencial donde se invitó a participar en una segunda ronda a más de 80 especialistas y se les solicitó la validación de las recomendaciones preseleccionadas. Resultados: En primer término, se realizó una preselección de 20 recomendaciones (epidemiología 4, scores 3, diagnóstico de laboratorio 4, tratamiento 6 y desescalada 3). Después de la segunda ronda, se validaron las 12 recomendaciones siguientes... (AU)

Background. Although there has been an improved management of invasive candidiasis in the last decade, controversial issues still remain, especially in the diagnostic and therapeutic approaches. Aims. We sought to identify the core clinical knowledge and to achieve high level agreement recommendations required to care for critically ill adult patients with invasive candidiasis. Methods. A prospective Spanish survey reaching consensus by the DELPHI technique was made. It was anonymously conducted by electronic mail in a first term to 25 national multidisciplinary experts in invasive fungal infections from five national scientific societies, including intensivists, anesthesiologists, microbiologists, pharmacologists and infectious diseases specialists, who answered to 47 questions prepared by a coordination group after a strict review of the literature in the last five years. The educational objectives spanned five categories, including epidemiology, diagnostic tools, prediction rules, and treatment and de-escalation approaches. The level of agreement achieved among the panel experts in each item should exceed 75% to be selected. In a second term, after extracting recommendations from the selected items, a face to face meeting was performed where more than 80 specialists in a second round were invited to validate the preselected recommendations. Results. In the first term, 20 recommendations were preselected (Epidemiology 4, Scores 3, Diagnostic tools 4, Treatment 6 and De-escalation approaches 3). After the second round, the following 12 were validated: (1) Epidemiology (2 recommendations): think about candidiasis in your Intensive Care Unit (ICU) and do not forget that non-Candida albicans–Candida species also exist. (2) Diagnostic tools (4 recommendations): blood cultures should be performed under suspicion every 2–3 days and, if positive, every 3 days until obtaining the first negative result. Obtain sterile fluid and tissue, if possible (direct examination of the sample is important). Use non-culture based methods as microbiological tools, whenever possible. Determination of antifungal susceptibility is mandatory. (3) Scores (1 recommendation): as screening tool, use the Candida Score and determine multicolonization in high risk patients. (4) Treatment (4 recommendations): start early. Choose echinocandins. Withdraw any central venous catheter. Fundoscopy is needed. (5) De-escalation (1 recommendation): only applied when knowing susceptibility determinations and after 3 days of clinical stability. The higher rate of agreement was achieved in the optimization of microbiological tools and the withdrawal of the catheter, whereas the lower rate corresponded to de-escalation therapy and the use of scores. Conclusions. The management of invasive candidiasis in ICU patients requires the application of a broad range of knowledge and skills that we summarize in our recommendations. These recommendations may help to identify the potential patients... (AU)

Use of image cytometry for quantification of pathogenic fungi in association with host cells.

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

[Simple modified method for fungal slide preparation].

  Slide culture preparations are necessary for accurately identifying dermatophytes. Because the standard slide culture technique is difficult for dermatologists to carry out, there is a need for an economical and easy-to-prepare system without special equipment. We placed a block of agar medium on a square cover glass (24 × 24mm) in the bottom of a case made of polypropylene (Free case, PK-201, HINODEWASHI CO., LTD. Japan). The agar block inoculated with fungi was overlaid with another cover glass on top of it. A plastic petri dish containing 4 cases as described above was sealed with adhesive plastic tape and incubated at 27°C.

[Recommendations for the diagnosis of candidemia in Latin America. Grupo Proyecto Épico]. / Recomendaciones para el diagnóstico de la candidemia en América Latina.

Candidemia is one of the most frequent opportunistic mycoses worldwide. Limited epidemiological studies in Latin America indicate that incidence rates are higher in this region than in the Northern Hemisphere. Diagnosis is often made late in the infection, affecting the initiation of antifungal therapy. A more scientific approach, based on specific parameters, for diagnosis and management of candidemia in Latin America is warranted. 'Recommendations for the diagnosis and management of candidemia' are a series of manuscripts that have been developed by members of the Latin America Invasive Mycosis Network. They aim to provide a set of best-evidence recommendations for the diagnosis and management of candidemia. This publication, 'Recommendations for the diagnosis of candidemia in Latin America', was written to provide guidance to healthcare professionals on the diagnosis of candidemia, as well as on the usefulness and application of susceptibility testing in patients who have a confirmed diagnosis of candidemia. Computerized searches of existing literature were performed by PubMed. The data were extensively reviewed and analyzed by members of the group. The group also met on two occasions to pose questions, discuss conflicting views, and deliberate on a series of management recommendations. 'Recommendations for the diagnosis of candidemia in Latin America' includes diagnostic methods used to detect candidemia, Candida species identification, and susceptibility testing. The availability of methods, their costs and treatment settings are considered. This manuscript is the first of this series that deals with diagnosis and treatment of invasive candidiasis. Other publications in this series include: 'Recommendations for the management of candidemia in adults in Latin America', 'Recommendations for the management of candidemia in children in Latin America', and 'Recommendations for the management of candidemia in neonates in Latin America'.

Pectinolytic enzyme production by Colletotrichum truncatum, causal agent of soybean anthracnose

Antecedentes. Colletotrichum truncatum es el hongo patógeno más comúnmente asociado con la antracnosis de la soja, enfermedad de alta prevalencia en Argentina. Las enzimas pectinolíticas se relacionan con la patogenicidad de un amplio rango de hongos fitopatógenos. Objetivos. Investigar la producción de enzimas pectinolíticas por cepas aisladas de plantas de soja enfermas de diferentes regiones de nuestro país, y con ello contribuir a la caracterización fisiológica de dichos aislamientos como paso preliminar para esclarecer el aún desconocido rol biológico de las enzimas pectinolíticas en la interacción Colletotrichum spp-soja. Métodos. Se investigó la producción in vitro de enzimas pectinolíticas, en un medio sintético con pectina como fuente de carbono, de diez aislamientos de C. truncatum. Resultados. Todas las cepas crecieron en dicho medio, detectándose actividades polimetilgalacturonasa (PMG), poligalacturonasa (PG) y pectin liasa (PL). En general, el pico de galacturonasas precedió al día de máximo crecimiento, en cambio el de PL se registró posteriormente. La cepa BAFC 3097 (originaria de la Provincia de Santa Fe) produjo altos títulos de las tres enzimas tras 7–10 días: 1,08U/ml PG, 1,05U/ml PMG, 156U/ml PL. C. truncatum, cultivado en un medio con glucosa como fuente de carbono, produjo PG y PMG (pero no PL), aunque su síntesis disminuyó marcadamente sugiriendo que estas enzimas son constitutivas. Conclusión. La disparidad registrada en la producción enzimática entre cepas no puede atribuirse al crecimiento fúngico; tampoco se corresponde con su distribución geográfica; pero podría relacionarse con diferencias en su virulencia, que aún no se han investigado(AU)

Background. Colletotrichum truncatum is the most common pathogenic fungus associated with soybean anthracnose, a prevalent disease in Argentina. Pectinolytic enzymes are involved in the pathogenicity of a wide range of plant pathogenic fungi. Objectives. To explore pectinolytic enzyme production in Argentinian Colletotrichum strains isolated from diseased soybean plants from different geographic locations, as a preliminary step to establish the biological role of the pectinolytic enzymes in the Colletotrichum spp.–soybean system, yet unknown. Methods. Ten strains were screened for in vitro pectinolytic enzyme production on a defined medium based on pectin as carbon source. Results. All isolates were able to grow in this medium and polymethylgalacturonase (PMG), polygalacturonase (PG) and pectin lyase (PL) activities were detected. On the whole, the peak of polygalacturonases activities preceded the day of maximum growth, while PL activity reached its highest level afterwards. Strain BAFC 3097 (from Santa Fe province) yielded high titles of the three enzymes (1.08U/ml PG, 1.05U/ml PMG, 156U/ml PL), after a short incubation period (7–10 days). Low synthesis of polygalacturonases in cultures containing glucose as unique carbon source suggests that these enzymes are constitutive in contrast with PL, which was not detected. Conclusions. The disparity observed in enzyme production among strains cannot be related to fungal growth, since no major differences in mycelial yield were found; it was not connected with their geographic origin, but might be associated with differences in virulence among strains not yet evaluated(AU)

Effect of aeration and agitation regimes on lipase production by newly isolated Rhodotorula mucilaginosa-MTCC 8737 in stirred tank reactor using molasses as sole production medium.

The influence of media and process parameters (aeration and agitation) on fermentation broth rheology and biomass formation has been studied in 1.5-l stirred tank reactor for lipase production using Rhodotorula mucilaginosa MTCC 8737. Molasses, as sole production medium, is used for lipase production by varying aeration (1, 2, and 3 vvm) and agitation speeds (100, 200, and 300 rpm). Maximum lipase activity of 72 U/ml was obtained during 96 h of fermentation at 2 vvm, 200 rpm, pH 7, and 25 +/- 2 degrees C temperature. Lipase production kinetics with respect to dry cell weight of biomass showed Y (P/S) of 25.71 U/mg, specific product formation of 10.9 U/mg DC, and Y (X/S) 2.35 mg/mg. Maximum lipase activity (MC 2) of 56 U/ml was observed at 1% molasses, and a further increase in the molasses concentration of (%) 1.5 and 2 inhibited the product formation of lipase with 15 and 8.5 U/ml, respectively. The production kinetics of molasses media showed Y (P/X) was 14 U/mg DC, Y (P/S) 16 U/mg, and Y (X/S) 1.14 mg/mg during 96 h of bioreactor operation. The k(L)a values for all batches (MC 1-MC 4) at 96 h of fermentation were 32, 28, 21, and 19/h, and the |oxygen transfer rate were 54.4, 56, 35.7, and 17.29 mg/l h, respectively. Increase in molasses concentration resulted in decreased lipase activity by increase in viscosity of the fermentation broth.