Effect of fungicide on Fusarium verticillioides mycelial morphology and fumonisin B1 production

The effect of fludioxonil + metalaxyl-M on the mycelial morphology, sporulation and fumonisin B1 production by Fusarium verticillioides 103 F was evaluated. Scanning electron microscopy analysis showed that the fungicide caused inhibition of hyphal growth and defects on hyphae morphology such as cell wall disruption, withered hyphae, and excessive septation. In addition, extracellular material around the hyphae was rarely observed in the presence of fludioxonil + metalaxyl-M. While promoting the reduction of mycelial growth, the fungicide increased sporulation of F. verticillioides compared to the control, and the highest production occurred on the 14th day in the treatments and on the 10th day in the control cultures. Fumonisin B1 production in the culture media containing the fungicide (treatment) was detected from the 7th day incubation, whereas in cultures without fungicide (control) it was detected on the 10th day. The highest fumonisin B1 production occurred on the 14th day, both for the control and for the treatment. Fludioxonil + metalaxyl - M can interfere in F. verticillioides mycelial morphology and sporulation and increase fumonisin B1 levels. These data indicate the importance of understanding the effects of fungicide to minimize the occurrence of toxigenic fungi and fumonisins.

Candida albicans morphologies revealed by scanning electron microscopy analysis

Scanning electron microscope (SEM) observations were used to analyze particular morphologies of Candida albicans clinical isolate (strain 82) and mutants defective in hyphae-promoting genes EFG1 (strain HLC52) and/ or CPH1 (strains HLC54 and Can16). Transcription factors Efg1 and Cph1 play role in regulating filamentation and adhesion of C. albicans' morphologies. Comparative analysis of such mutants and clinical isolate showed that Efg1 is required for human serum-induced cell growth and morphological switching. In the study, distinct differences between ultrastructural patterns of clinical strain's and null mutants' morphologies were observed (spherical vs tube-like blastoconidia, or solid and fragile constricted septa vs only the latter observed in strains with EFG1 deleted). In addition, wild type strain displayed smooth colonies of cells in comparison to mutants which exhibited wrinkled phenotype. It was observed that blastoconidia of clinical strain exhibited either polarly or randomly located budding. Contrariwise, morphotypes of mutants showed either multiple polar budding or a centrally located single bud scar (mother-daughter cell junction) distinguishing tube-like yeast/ pseudohyphal growth (the length-to-width ratios larger than 1.5). In their planktonic form of growth, blastoconidia of clinical bloodstream isolate formed constitutively true hyphae under undiluted human serum inducing conditions. It was found that true hyphae are essential elements for developing structural integrity of conglomerate, as mutants displaying defects in their flocculation and conglomerate-forming abilities in serum. While filamentation is an important virulence trait in C. albicans the true hyphae are the morphologies which may be expected to play a role in bloodstream infections.

Optimization of biomass production of a mutant of Yarrowia lipolytica with an increased lipase activity using raw glycerol

The yeast Yarrowia lipolytica accumulates oils and is able to produce extracellular lipases when growing in different carbon sources including glycerol, the principal by-product of the biodiesel industry. In this study, biomass production of a novel mutant strain of Y. lipolytica was statistically optimized by Response Surface Methodology in media containing biodiesel-derived glycerol as main carbon source. This strain exhibited distinctive morphological and fatty acid profile characteristics, and showed an increased extracellular lipase activity. An organic source of nitrogen and the addition of 1.0 g/l olive oil were necessary for significant lipase production. Plackett-Burman and Central Composite Statistical Designs were employed for screening and optimization of fermentation in shaken flasks cultures, and the maximum values obtained were 16.1 g/l for biomass and 12.2 Units/ml for lipase, respectively. Optimized batch bioprocess was thereafter scaled in aerated bioreactors and the values reached for lipase specific activity after 95 % of the glycerol had been consumed, were three-fold higher than those obtained in shaken flasks cultures. A sustainable bioprocess to obtain biomass and extracellular lipase activity was attained by maximizing the use of the by-products of biodiesel industry.

Optimización de la producción de biomasa usando glicerol crudo, de una cepa mutante de Yarrowia lipolytica con actividad incrementada de lipasa. La levadura Yarrowia lipolytica acumula aceites y produce una lipasa extracelular al crecer en diferentes fuentes de carbono, entre ellas el glicerol, principal subproducto de la creciente industria del biodiésel. En el presente trabajo, se optimizó mediante la metodología de superficies de respuesta la producción de biomasa de una nueva cepa mutante de Y. lipolytica, empleando medios con glicerol derivado de la industria del biodiésel como principal fuente de carbono. Esta cepa presentó características morfológicas y perfil de ácidos grasos distintivos, y una mayor actividad de lipasa extracelular. Para obtener una producción significativa de lipasa extracelular, fue necesario el agregado de una fuente orgánica de nitrógeno y de 1 g/l de aceite de oliva. Se utilizaron los diseños estadísticos de Plackett-Burman y central compuesto para la selección y la optimización de las fermentaciones en frascos agitados; los máximos valores de biomasa y de lipasa obtenidos fueron de 16,1 g/l y 12,2 unidades/ml, respectivamente. Luego, el bioproceso en lote optimizado se escaló a biorreactores aireados, y los valores de actividad específica de lipasa alcanzados después de haberse consumido el 95 % del glicerol fueron tres veces más altos que los obtenidos en los cultivos en frascos agitados. En suma, se desarrolló un bioproceso sostenible para la obtención de biomasa y de una actividad de lipasa extracelular, que a la vez maximiza el uso de subproductos de la industria del biodiésel.

In vitro synthesis of Frankia and mycorrhiza with Casuarina equisetifolia and ultrastructure of root system.

Casuarina equisetifolia is one of the ecologically and economically important tropical coastal trees nodulated by nitrogen-fixing actinomycete Frankia and forming symbiotic associations with both ecto- and endomycorrhizal fungi. The present study aims at the ultrastructural study of interactions between C. equisetifolia, Frankia, and mycorrhiza. C. equisetifolia seeds were sterilised and germinated under in vitro condition. The seedlings were transferred to conical flasks containing vermiculite and saw dust with Hoagland's solution. After 30 days, the inoculum of AM fungus--Glomusfasciculatum (A), ectomycorrhizal fungus-Pisolithus tinctorius (E) and actinorhizal Frankia (F) were inoculated individually and in various combinations, (A+E), (A+F), E+F) and (A+E+F). After 90 days, the experimental plant roots and nodules were harvested for assessment of growth characters of mycorrhizal and actinorhizal association by light and scanning electron microscope methods. C. equisetifolia roots were infected with arbuscles and vesicles of G. fasciculatum; P. tinctorius formed fungal sheath but no Hartig net. Large number of cortical cells were seen infected with Frankia, hyphae of Frankia were frequently seen penetrating from cell to cell directly through cell walls and Frankia occupied majority of the cell volume.

Alterations in growth and branching of Neurospora crassa caused by sub-inhibitory concentrations of antifungal agents / Alteraciones de crecimiento y ramificación en Neurospora crassa provocadas por concentraciones subinhibitorias de agentes antimicóticos

Six antifungal agents at subinhibitory concentrations were used for investigating their ability to affect the growth and branching in Neurospora crassa. Among the antifungals herein used, the azole agent ketoconazole at 0.5 μg/ml inhibited radial growth more than fluconazole at 5.0 μg/ml while amphotericin B at 0.05 μg/ml was more effective than nystatin at 0.05 μg/ml. Morphological alterations in hyphae were observed in the presence of griseofulvin, ketoconazole and terbinafine at the established concentrations. The antifungal agents were more effective on vegetative growth than on conidial germination. Terbinafine markedly reduced growth unit length (GU) by 54.89%, and caused mycelia to become hyperbranched. In all cases, there was a high correlation between hyphal length and number of tips (r > 0.9). All our results showed highly significant differences by ANOVA, (p < 0.001, α = 0.05). Considering that the hyphal tip is the main interface between the fungus and its environment /through which enzymes and toxins are secreted and nutrients absorbed, it would not be desirable to obtain a hyperbranched mycelia with inefficient doses of antifungal drugs.

Se investigó el efecto de seis agentes antimicóticos en concentraciones subinhibitorias sobre el crecimiento y la ramificación en Neurospora crassa. El agente azólico ketoconazol a la concentración de 0,5 μg/ml inhibió el crecimiento radial más que el fluconazol a 5,0 μg/ml, y la anfotericina B a 0,05 μg/ ml fue más eficiente que 0,05 μg/ml de nistatina, entre los agentes poliénicos usados. En presencia de griseofulvina, ketoconazol y terbinafina a las concentraciones establecidas se observaron alteraciones morfológicas en las hifas. Los agentes antimicóticos fueron más eficientes sobre el crecimiento vegetativo que sobre la germinación conidial. La terbinafina redujo marcadamente (54,89%) la longitud de la unidad de crecimiento y provocó la hiperramificación del micelio. En todos los casos, existió gran correlación entre la longitud y el número de ápices de las hifas (r > 0,9). Todos los resultados mostraron diferencias altamente significativas de acuerdo con ANOVA (p < 0,001, α = 0,05). Considerando que el ápice de la hifa es la principal interfase entre el hongo y su ambiente, a través de la cual las enzimas y las toxinas son secretadas y los nutrientes son absorbidos, un micelio hiperramificado resultante de dosis ineficientes de agentes antimicóticos sería perjudicial.