RESUMEN
Solid-state fermentation can be used to produce feeds for ruminants, which can provide an enriched population of yeasts to improve ruminal fermentation. Fermentation of apple bagasse was performed to obtain a yeast-rich product, with the objective of isolating, identifying, and characterizing yeast strains and testing their capability to enhance in vitro ruminal fermentation of fibrous feeds. Yeasts were isolated from apple bagasse fermented under in vitro conditions, using rumen liquor obtained from cannulated cows and alfalfa as a fibrous substrate. A total of 16 new yeast strains were isolated and identified by biochemical and molecular methods. The strains were designated Levazot, followed by the isolate number. Their fermentative capacity was assessed using an in vitro gas production method. Strain Levazot 15 (Candida norvegensis) showed the greatest increase in gas production (p 0.05) compared with the yeast-free control and positively affected in vitro ruminal fermentation parameters of alfalfa and oat straw. Based on these results, it was concluded that the Levazot 15 yeast strain could be potentially used as an additive for ruminants consuming high-fiber diets. However, further studies of effects of these additives on rumen digestion, metabolism, and productive performance of ruminants are required.(AU)
Asunto(s)
Malus/citología , Malus/metabolismo , Fermentación , Rumiantes/metabolismo , Levaduras/clasificación , Levaduras/aislamiento & purificaciónRESUMEN
The cell wall of fungi is generally composed of an inner skeletal layer consisting of various polysaccharides surrounded by a layer of glycoproteins. These usually contain both N- and O-linked oligosaccharides, coupled to the proteins by stepwise addition of mannose residues by mannosyltransferases in the endoplasmic reticulum and the Golgi apparatus. In yeast, an essential luminal cofactor for these mannosyltransferases is Mn(2+) provided by the Ca(2+)/Mn(2+)-ATPase known as Pmr1. In this study, we have identified and characterized the Botrytis cinerea pmr1 gene, the closest homolog of yeast PMR1. We hypothesized that bcpmr1 also encodes a Ca(2+)/Mn(2+)-ATPase that plays an important role in the protein glycosylation pathway. Phenotypic analysis showed that bcpmr1 null mutants displayed a significant reduction in conidial production, radial growth and diameter of sclerotia. Significant alterations in hyphal cell wall composition were observed including a 83% decrease of mannan levels and an increase in the amount of chitin and glucan. These changes were accompanied by a hypersensitivity to cell wall-perturbing agents such as Calcofluor white, Congo red and zymolyase. Importantly, the Δbcpmr1 mutant showed reduced virulence in tomato (leafs and fruits) and apple (fruits) and reduced biofilm formation. Together, our results highlight the importance of bcpmr1 for protein glycosylation, cell wall structure and virulence of B. cinerea.
Asunto(s)
Botrytis/fisiología , Pared Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Malus/microbiología , Solanum lycopersicum/microbiología , Botrytis/crecimiento & desarrollo , Botrytis/patogenicidad , Frutas/microbiología , Solanum lycopersicum/citología , Malus/citología , Mutación , Hojas de la Planta/microbiología , Esporas Fúngicas/crecimiento & desarrollo , VirulenciaRESUMEN
Agar is a complex mixture of gel-forming polysaccharides. Gelling agents are very often used to provide proper support for plants grown in semisolid culture media. And agar is the most frequently used gelling agent in plant tissue culture media. Galactomannans, another group of gel-forming polysaccharides, consists of a (1 â 4)-linked ß-D: -mannopyranosyl backbone partially substituted at O-6 with D: -galactopyranosyl side groups. In this work, we demonstrate that a statistically significant 2.7-fold increase on the multiplication rate (MR) for in vitro-grown Marubakaido (Malus prunifolia) shoots was associated with a 12.5% replacement of agar in the semi-solid culture media for a galactomannan obtained from seeds of Schizolobium paraybae. This increase on MR was due mainly to a 1.9-fold increase in the number of main branches and an 8.6-fold increase in the number of primary lateral branches. Gas liquid chromatography and thin layer chromatography analyzes demonstrated that the galactomannan-driven enhancement of the in vitro multiplication rate of the Marubakaido apple rootstock was not related to the galactomannan degradation. To the best of our knowledge, this is the first report on the successful use of partial replacement of high quality agar by a galactomannan from S. paraybae in a micropropagation system for a tree species.
Asunto(s)
Fabaceae/química , Mananos/química , Polisacáridos/química , Agar/química , Galactosa/análogos & derivados , Malus/citología , Malus/crecimiento & desarrollo , Mananos/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/crecimiento & desarrollo , Brotes de la Planta/citología , Brotes de la Planta/crecimiento & desarrollo , Polisacáridos/metabolismo , Semillas/química , Técnicas de Cultivo de TejidosRESUMEN
Images (for example, photomicrographs) are routinely used as qualitative evidence of the microstructure of foods. In quantitative image analysis it is important to estimate the area (or volume) to be sampled, the field of view, and the resolution. The bootstrap method is proposed to estimate the size of the sampling area as a function of the coefficient of variation (CV(Bn)) and standard error (SE(Bn)) of the bootstrap taking sub-areas of different sizes. The bootstrap method was applied to simulated and real structures (apple tissue). For simulated structures, 10 computer-generated images were constructed containing 225 black circles (elements) and different coefficient of variation (CV(image)). For apple tissue, 8 images of apple tissue containing cellular cavities with different CV(image) were analyzed. Results confirmed that for simulated and real structures, increasing the size of the sampling area decreased the CV(Bn) and SE(Bn). Furthermore, there was a linear relationship between the CV(image) and CV(Bn) (.) For example, to obtain a CV(Bn) = 0.10 in an image with CV(image) = 0.60, a sampling area of 400 x 400 pixels (11% of whole image) was required, whereas if CV(image) = 1.46, a sampling area of 1000 x 100 pixels (69% of whole image) became necessary. This suggests that a large-size dispersion of element sizes in an image requires increasingly larger sampling areas or a larger number of images.
Asunto(s)
Análisis de los Alimentos/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Simulación por Computador , Frutas/química , Frutas/citología , Malus/química , Malus/citología , Modelos Estadísticos , Estadística como AsuntoRESUMEN
Botrytis cinerea is a plant-pathogenic fungus that produces the disease known as grey mould in a wide variety of agriculturally important hosts in many countries. This paper describes the development of an immunosensor coupled to carbon-based screen-printed electrodes (SPCE) modified with multi-walled carbon nanotubes (CNTs), which show a rapid and sensitive determination of B. cinerea in apple tissues (Red-delicious) using a competitive immunoassay method. Both the infected plant tissue sample and the B. cinerea-specific monoclonal antibody are allowed to react immunologically with the B. cinerea purified antigens immobilized on a rotating disk. Then, the bound antibodies are quantified by a horseradish peroxidise (HRP) enzyme labeled second antibodies specific to mouse IgG, using 4-tertbutylcatechol (4-TBC) as enzymatic mediators. The HRP, in the presence of hydrogen peroxide, catalyses the oxidation of 4-TBC to 4-tertbutyl o-benzoquinone. The electrochemical reduction back to 4-TBC is detected on SPCE-CNT at -0.15 V. The response current is inversely proportional to the amount of the B. cinerea antigens present in the fruit sample. The time consumed per assay was 30 min and the calculated detection limits for electrochemical method and the ELISA procedure are 0.02 and 10 microg mL(-1), respectively. Moreover the intra- and inter-assay coefficients of variation were below 7%. This electrochemical immunosensor promises to be usefully suited to the detection and quantification of B. cinerea in apparently healthy plant prior to the development of the symptoms.