Evaluation of cover crops based on characteristics of nitrogen use, and aerial and root growth

La caracterización de los cultivos cubierta (cover crops) puede permitir comparar la idoneidad de diferentes especies para proporcionar servicios ecológicos como el control de la erosión, el reciclado de nutrientes o la producción de forrajes. En este trabajo se estudiaron bajo condiciones de campo diferentes técnicas para caracterizar el dosel vegetal con objeto de establecer una metodología para medir y comparar las arquitecturas de los cultivos cubierta más comunes. Se estableció un ensayo de campo en Madrid (España central) para determinar la relación entre el índice de área foliar (LAI) y la cobertura del suelo (GC) para un cultivo de gramínea, uno de leguminosa y uno de crucífera. Para ello se sembraron doce parcelas con cebada (Hordeum vulgare L.), veza (Vicia sativa L.), y colza (Brassica napus L.). En 10 fechas de muestreo se midieron el LAI (con estimaciones directas y del LAI-2000), la fracción interceptada de la radiación fotosintéticamente activa (FIPAR) y la GC. Un experimento de campo de dos años (Octubre-Abril) se estableció en la misma localización para evaluar diferentes especies (Hordeum vulgare L., Secale cereale L., x Triticosecale Whim, Sinapis alba L., Vicia sativa L.) y cultivares (20) en relación con su idoneidad para ser usadas como cultivos cubierta. La GC se monitorizó mediante análisis de imágenes digitales con 21 y 22 muestreos, y la biomasa se midió 8 y 10 veces, respectivamente para cada año. Un modelo de Gompertz caracterizó la cobertura del suelo hasta el decaimiento observado tras las heladas, mientras que la biomasa se ajustó a ecuaciones de Gompertz, logísticas y lineales-exponenciales. Al final del experimento se determinaron el C, el N y el contenido en fibra (neutrodetergente, ácidodetergente y lignina), así como el N fijado por las leguminosas. Se aplicó el análisis de decisión multicriterio (MCDA) con objeto de obtener un ranking de especies y cultivares de acuerdo con su idoneidad para actuar como cultivos cubierta en cuatro modalidades diferentes: cultivo de cobertura, cultivo captura, abono verde y forraje. Las asociaciones de cultivos leguminosas con no leguminosas pueden afectar al crecimiento radicular y a la absorción de N de ambos componentes de la mezcla. El conocimiento de cómo los sistemas radiculares específicos afectan al crecimiento individual de las especies es útil para entender las interacciones en las asociaciones, así como para planificar estrategias de cultivos cubierta. En un tercer ensayo se combinaron estudios en rhizotrones con extracción de raíces e identificación de especies por microscopía, así como con estudios de crecimiento, absorción de N y 15N en capas profundas del suelo. Las interacciones entre raíces en su crecimiento y en el aprovisionamiento de N se estudiaron para dos de los cultivares mejor valorados en el estudio previo: uno de cebada (Hordeum vulgare L. cv. Hispanic) y otro de veza (Vicia sativa L. cv. Aitana). Se añadió N en dosis de 0 (N0), 50 (N1) y 150 (N2) kg N ha-1. Como resultados del primer estudio, se ajustaron correctamente modelos lineales y cuadráticos a la relación entre la GC y el LAI para todos los cultivos, pero en la gramínea alcanzaron una meseta para un LAI>4. Antes de alcanzar la cobertura total, la pendiente de la relación lineal entre ambas variables se situó en un rango entre 0.025 y 0.030. Las lecturas del LAI-2000 estuvieron correlacionadas linealmente con el LAI, aunque con tendencia a la sobreestimación. Las correcciones basadas en el efecto de aglutinación redujeron el error cuadrático medio del LAI estimado por el LAI-2000 desde 1.2 hasta 0.5 para la crucífera y la leguminosa, no siendo efectivas para la cebada. Esto determinó que para los siguientes estudios se midieran únicamente la GC y la biomasa. En el segundo experimento, las gramíneas alcanzaron la mayor cobertura del suelo (83-99%) y la mayor biomasa (1226-1928 g m-2) al final del mismo. Con la mayor relación C/N (27-39) y contenido en fibra digestible (53-60%) y la menor calidad de residuo (~68%). La mostaza presentó elevadas GC, biomasa y absorción de N en el año más templado en similitud con las gramíneas, aunque escasa calidad como forraje en ambos años. La veza presentó la menor absorción de N (2.4-0.7 g N m-2) debido a la fijación de N (9.8-1.6 g N m-2) y escasa acumulación de N. El tiempo térmico hasta alcanzar el 30% de GC constituyó un buen indicador de especies de rápida cubrición. La cuantificación de las variables permitió hallar variabilidad entre las especies y proporcionó información para posteriores decisiones sobre la selección y manejo de los cultivos cubierta. La agregación de dichas variables a través de funciones de utilidad permitió confeccionar rankings de especies y cultivares para cada uso. Las gramíneas fueron las más indicadas para los usos de cultivo de cobertura, cultivo captura y forraje, mientras que las vezas fueron las mejor como abono verde. La mostaza alcanzó altos valores como cultivo de cobertura y captura en el primer año, pero el segundo decayó debido a su pobre actuación en los inviernos fríos. Hispanic fue el mejor cultivar de cebada como cultivo de cobertura y captura, mientras que Albacete como forraje. El triticale Titania alcanzó la posición más alta como cultiva de cobertura, captura y forraje. Las vezas Aitana y BGE014897 mostraron buenas aptitudes como abono verde y cultivo captura. El MCDA permitió la comparación entre especies y cultivares proporcionando información relevante para la selección y manejo de cultivos cubierta. En el estudio en rhizotrones tanto la mezcla de especies como la cebada alcanzaron mayor intensidad de raíces (RI) y profundidad (RD) que la veza, con valores alrededor de 150 cruces m-1 y 1.4 m respectivamente, comparados con 50 cruces m-1 y 0.9 m para la veza. En las capas más profundas del suelo, la asociación de cultivos mostró valores de RI ligeramente mayores que la cebada en monocultivo. La cebada y la asociación obtuvieron mayores valores de densidad de raíces (RLD) (200-600 m m-3) que la veza (25-130) entre 0.8 y 1.2 m de profundidad. Los niveles de N no mostraron efectos claros en RI, RD ó RLD, sin embargo, el incremento de N favoreció la proliferación de raíces de veza en la asociación en capas profundas del suelo, con un ratio cebada/veza situado entre 25 a N0 y 5 a N2. La absorción de N de la cebada se incrementó en la asociación a expensas de la veza (de ~100 a 200 mg planta-1). Las raíces de cebada en la asociación absorbieron también más nitrógeno marcado de las capas profundas del suelo (0.6 mg 15N planta-1) que en el monocultivo (0.3 mg 15N planta-1). ABSTRACT Cover crop characterization may allow comparing the suitability of different species to provide ecological services such as erosion control, nutrient recycling or fodder production. Different techniques to characterize plant canopy were studied under field conditions in order to establish a methodology for measuring and comparing cover crops canopies. A field trial was established in Madrid (central Spain) to determine the relationship between leaf area index (LAI) and ground cover (GC) in a grass, a legume and a crucifer crop. Twelve plots were sown with either barley (Hordeum vulgare L.), vetch (Vicia sativa L.), or rape (Brassica napus L.). On 10 sampling dates the LAI (both direct and LAI-2000 estimations), fraction intercepted of photosynthetically active radiation (FIPAR) and GC were measured. A two-year field experiment (October-April) was established in the same location to evaluate different species (Hordeum vulgare L., Secale cereale L., x Triticosecale Whim, Sinapis alba L., Vicia sativa L.) and cultivars (20) according to their suitability to be used as cover crops. GC was monitored through digital image analysis with 21 and 22 samples, and biomass measured 8 and 10 times, respectively for each season. A Gompertz model characterized ground cover until the decay observed after frosts, while biomass was fitted to Gompertz, logistic and linear-exponential equations. At the end of the experiment C, N, and fiber (neutral detergent, acid and lignin) contents, and the N fixed by the legumes were determined. Multicriteria decision analysis (MCDA) was applied in order to rank the species and cultivars according to their suitability to perform as cover crops in four different modalities: cover crop, catch crop, green manure and fodder. Intercropping legumes and non-legumes may affect the root growth and N uptake of both components in the mixture. The knowledge of how specific root systems affect the growth of the individual species is useful for understanding the interactions in intercrops as well as for planning cover cropping strategies. In a third trial rhizotron studies were combined with root extraction and species identification by microscopy and with studies of growth, N uptake and 15N uptake from deeper soil layers. The root interactions of root growth and N foraging were studied for two of the best ranked cultivars in the previous study: a barley (Hordeum vulgare L. cv. Hispanic) and a vetch (Vicia sativa L. cv. Aitana). N was added at 0 (N0), 50 (N1) and 150 (N2) kg N ha-1. As a result, linear and quadratic models fitted to the relationship between the GC and LAI for all of the crops, but they reached a plateau in the grass when the LAI > 4. Before reaching full cover, the slope of the linear relationship between both variables was within the range of 0.025 to 0.030. The LAI-2000 readings were linearly correlated with the LAI but they tended to overestimation. Corrections based on the clumping effect reduced the root mean square error of the estimated LAI from the LAI-2000 readings from 1.2 to less than 0.50 for the crucifer and the legume, but were not effective for barley. This determined that in the following studies only the GC and biomass were measured. In the second experiment, the grasses reached the highest ground cover (83- 99%) and biomass (1226-1928 g/m2) at the end of the experiment. The grasses had the highest C/N ratio (27-39) and dietary fiber (53-60%) and the lowest residue quality (~68%). The mustard presented high GC, biomass and N uptake in the warmer year with similarity to grasses, but low fodder capability in both years. The vetch presented the lowest N uptake (2.4-0.7 g N/m2) due to N fixation (9.8-1.6 g N/m2) and low biomass accumulation. The thermal time until reaching 30% ground cover was a good indicator of early coverage species. Variable quantification allowed finding variability among the species and provided information for further decisions involving cover crops selection and management. Aggregation of these variables through utility functions allowed ranking species and cultivars for each usage. Grasses were the most suitable for the cover crop, catch crop and fodder uses, while the vetches were the best as green manures. The mustard attained high ranks as cover and catch crop the first season, but the second decayed due to low performance in cold winters. Hispanic was the most suitable barley cultivar as cover and catch crop, and Albacete as fodder. The triticale Titania attained the highest rank as cover and catch crop and fodder. Vetches Aitana and BGE014897 showed good aptitudes as green manures and catch crops. MCDA allowed comparison among species and cultivars and might provide relevant information for cover crops selection and management. In the rhizotron study the intercrop and the barley attained slightly higher root intensity (RI) and root depth (RD) than the vetch, with values around 150 crosses m-1 and 1.4 m respectively, compared to 50 crosses m-1 and 0.9 m for the vetch. At deep soil layers, intercropping showed slightly larger RI values compared to the sole cropped barley. The barley and the intercropping had larger root length density (RLD) values (200-600 m m-3) than the vetch (25-130) at 0.8-1.2 m depth. The topsoil N supply did not show a clear effect on the RI, RD or RLD; however increasing topsoil N favored the proliferation of vetch roots in the intercropping at deep soil layers, with the barley/vetch root ratio ranging from 25 at N0 to 5 at N2. The N uptake of the barley was enhanced in the intercropping at the expense of the vetch (from ~100 mg plant-1 to 200). The intercropped barley roots took up more labeled nitrogen (0.6 mg 15N plant-1) than the sole-cropped barley roots (0.3 mg 15N plant-1) from deep layers.

Distinct and conserved transcriptomic changes during nematode-induced giant cell development in tomato compared with Arabidopsis: a functional role for gene repression

Root-knot nematodes (RKNs) induce giant cells (GCs) from root vascular cells inside the galls. Accompanying molecular changes as a function of infection time and across different species, and their functional impact, are still poorly understood. Thus, the transcriptomes of tomato galls and laser capture microdissected (LCM) GCs over the course of parasitism were compared with those of Arabidopsis, and functional analysis of a repressed gene was performed. Microarray hybridization with RNA from galls and LCM GCs, infection-reproduction tests and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) transcriptional profiles in susceptible and resistant (Mi-1) lines were performed in tomato. Tomato GC-induced genes include some possibly contributing to the epigenetic control of GC identity. GC-repressed genes are conserved between tomato and Arabidopsis, notably those involved in lignin deposition. However, genes related to the regulation of gene expression diverge, suggesting that diverse transcriptional regulators mediate common responses leading to GC formation in different plant species. TPX1, a cell wall peroxidase specifically involved in lignification, was strongly repressed in GCs/galls, but induced in a nearly isogenic Mi-1 resistant line on nematode infection. TPX1 overexpression in susceptible plants hindered nematode reproduction and GC expansion. Time-course and cross-species comparisons of gall and GC transcriptomes provide novel insights pointing to the relevance of gene repression during RKN establishment.

The role of the secondary cell wall in plant resistance to pathogens

Plant resistance to pathogens relies on a complex network of constitutive and inducible defensive barriers. The plant cell wall is one of the barriers that pathogens need to overcome to successfully colonize plant tissues. The traditional view of the plant cell wall as a passive barrier has evolved to a concept that considers the wall as a dynamic structure that regulates both constitutive and inducible defense mechanisms, and as a source of signaling molecules that trigger immune responses. The secondary cell walls of plants also represent a carbon-neutral feedstock (lignocellulosic biomass) for the production of biofuels and biomaterials. Therefore, engineering plants with improved secondary cell wall characteristics is an interesting strategy to ease the processing of lignocellulosic biomass in the biorefinery. However, modification of the integrity of the cell wall by impairment of proteins required for its biosynthesis or remodeling may impact the plants resistance to pathogens. This review summarizes our understanding of the role of the plant cell wall in pathogen resistance with a focus on the contribution of lignin to this biological process.

Role of fibre in broiler diets - Firend or foe?

The fibrous fraction of the feeds encompasses a group of heterogeneous compounds differing in chemical composition and physical properties (Graham and Aman, 1991, Bach Knudsen, 2001). Dietary fiber is the most used term to define the fiber fraction of ingredients and feeds, and includes cell walls, stored non-starch polysaccharides (NSP), and lignin (Bach Knudsen, 2001). Based on their physico-chemical properties, DF can be divided into soluble and insoluble fractions with distinct effects on digestive physiology and animal metabolism. Consequently, the benefits of fiber inclusion in poultry diets will vary depending on factors such as characteristics of the fiber source, type of bird, and digestive health status.

Ammonia and methane gas emissions from pig slurry: variability induced through feeding strategies

Esta Tesis doctoral fue desarrollada para estudiar las emisiones de amoniaco (NH3) y metano (CH4) en purines de cerdos, y los efectos ocasionados por cambios en la formulación de la dieta. Con este propósito, fueron llevados a cabo tres estudios. El experimento 1 fue realizado con el objetivo de analizar los factores de variación de la composición de purines y establecer ecuaciones de predicción para emisiones potenciales de NH3 y CH4. Fueron recogidas setenta y nueve muestras de piensos y purines durante dos estaciones del año (verano y invierno) de granjas comerciales situadas en dos regiones de España (Centro y Mediterráneo). Se muestrearon granjas de gestación, maternidad, lactación y cebo. Se determinó la composición de piensos y purines, y la emisión potencial de NH3 y CH4. El contenido de nutrientes de los piensos fue usado como covariable en el análisis. La espectroscopia de reflectancia del infrarrojo cercano (NIRS) se evaluó como herramienta de predicción de la composición y potencial emisión de gases del purín. Se encontró una amplia variabilidad en la composición de piensos y purines. Las granjas del Mediterráneo tenían mayor pH (P<0,001) y concentración de cenizas (P =0,02) en el purín que las del Centro. El tipo de granja también afectó al contenido de extracto etéreo (EE) del purín (P =0,02), observando los valores más elevados en las instalaciones de animales jóvenes. Los resultados sugieren un efecto tampón de la fibra de la dieta en el pH del purín y una relación directa (P<0,05) con el contenido de fibra fecal. El contenido de proteína del pienso no afectó al contenido de nitrógeno del purín, pero disminuyó (P=0,003) la concentración de sólidos totales (ST) y de sólidos volátiles (SV). Se obtuvieron modelos de predicción de la emisión potencial de NH3 (R2=0,89) y CH4 (R2=0,61) partir de la composición del purín. Los espectros NIRS mostraron una buena precisión para la estimación de la mayor parte de los constituyentes, con coeficientes de determinación de validación cruzada (R2cv) superiores a 0,90, así como para la predicción del potencial de emisiones de NH3 y CH4 (R2cv=0,84 y 0,68, respectivamente). El experimento 2 fue realizado para investigar los efectos del nivel de inclusión de dos fuentes de sub-productos fibrosos: pulpa de naranja (PN) y pulpa de algarroba (PA), en dietas iso-fibrosas de cerdos de cebo, sobre la composición del purín y las emisiones potenciales de NH3 y CH4. Treinta cerdos (85,4±12,3 kg) fueron alimentados con cinco dietas iso-nutritivas: control comercial trigo/cebada (C) y cuatro dietas experimentales incluyendo las dos fuentes de sub-productos a dos niveles (75 y 150 g/kg) en una estructura 2 × 2 factorial. Después de 14 días de periodo de adaptación, heces y orina fueron recogidas separadamente durante 7 días para medir la digestibilidad de los nutrientes y el nitrógeno (N) excretado (6 réplicas por dieta) en cerdos alojados individualmente en jaulas metabólicas. Las emisiones de NH3 y CH4 fueron medidas después de la recogida de los purínes durante 11 y 100 días respectivamente. La fuente y el nivel de subproductos fibrosos afectó a la eficiencia digestiva de diferentes formas, ya que los coeficientes de digestibilidad total aparente (CDTA) para la materia seca (MS), materia orgánica (MO), fracciones fibrosas y energía bruta (EB) aumentaron con la PN pero disminuyeron con la inclusión de PA (P<0,05). El CDTA de proteína bruta (PB) disminuyó con la inclusión de las dos fuentes de fibra, siendo más bajo al mayor nivel de inclusión. La concentración fecal de fracciones fibrosas aumentó (P<0,05) con el nivel de inclusión de PA pero disminuyó con el de PN (P<0,01). El nivel más alto de las dos fuentes de fibra en el pienso aumentó (P<0,02) el contenido de PB fecal pero disminuyó el contenido de N de la orina (de 205 para 168 g/kg MS, P<0,05) en todas las dietas suplementadas comparadas con la dieta C. Adicionalmente, las proporciones de nitrógeno indigerido, nitrógeno soluble en agua, nitrógeno bacteriano y endógeno excretado en heces no fueron afectados por los tratamientos. Las características iniciales del purín no difirieron entre las diferentes fuentes y niveles de fibra, excepto para el pH que disminuyó con la inclusión de altos niveles de sub-productos. La emisión de NH3 por kg de purín fue más baja en todas las dietas suplementadas con fibras que en la dieta C (2,44 vs.1,81g de promedio, P<0,05). Además, purines de dietas suplementadas con alto nivel de sub-productos tendieron (P<0,06) a emitir menos NH3 por kg de nitrógeno total y mostraron un potencial más bajo para emitir CH4, independientemente de la fuente de fibra. El experimento 3 investigó los efectos de la fuente de proteína en dietas prácticas. Tres piensos experimentales fueron diseñados para sustituir una mescla de harina y cascarilla de soja (SOJ) por harina de girasol (GIR) o por DDGS del trigo (DDGST). La proporción de otros ingredientes fue modificada para mantener los contenidos de nutrientes similares a través de las dietas. El cambio en la fuente de proteína dio lugar a diferencias en el contenido de fibra neutro detergente ligada a proteína bruta (FNDPB), fibra soluble (FS) y lignina ácido detergente (LAD) en la dieta. Veinticuatro cerdos (ocho por dieta), con 52,3 o 60,8 kg en la primera y segunda tanda respectivamente, fueron alojados individualmente en jaulas metabólicas. Durante un periodo de 7 días fue determinado el balance de MS, el CDTA de los nutrientes y la composición de heces y orina. Se realizó el mismo procedimiento del experimento 2 para medir las emisiones de NH3 y CH4 de los purines de cada animal. Ni la ingestión de MS ni el CDTA de la MS o de la energía fueron diferentes entre las dietas experimentales, pero el tipo de pienso afectó (P<0.001) la digestibilidad de la PB, que fue mayor para GIR (0,846) que para SOJ (0,775), mientras que la dieta DDGST mostró un valor intermedio (0,794). La concentración fecal de PB fue por tanto influenciada (P<0,001) por el tratamiento, observándose la menor concentración de PB en la dieta GIR y la mayor en la dieta SOJ. La proporción de N excretado en orina o heces disminuyó de 1,63 en la dieta GIR hasta 0,650 en la dieta SOJ, como consecuencia de perdidas más bajas en orina y más altas en heces, con todas las fracciones de nitrógeno fecales creciendo en paralelo a la excreción total. Este resultado fue paralelo a una disminución de la emisión potencial de NH3 (g/kg purín) en la dieta SOJ con respecto a la dieta GIR (desde 1,82 a 1,12, P<0,05), dando valores intermedios (1,58) para los purines de la dieta DDGST. Por otro lado, el CDTA de la FS y de la fibra neutro detergente (FND) fueron afectados (P<0,001 y 0,002, respectivamente) por el tipo de dieta, siendo más bajas en la dieta GIR que en la dieta SOJ; además, se observó un contenido más alto de FND (491 vs. 361g/kg) en la MS fecal para la dieta GIR que en la dieta SOJ, presentando la dieta DDGST valores intermedios. El grado de lignificación de la FND (FAD/FND x 100) de las heces disminuyó en el orden GIR>DDGST>SOJ (desde 0,171 hasta 0,109 y 0,086, respectivamente) en paralelo a la disminución del potencial de emisión de CH4 por g de SV del purín (desde 301 a 269 y 256 mL, respectivamente). Todos los purines obtenidos en estos tres experimentos y Antezana et al. (2015) fueron usados para desarrollar nuevas calibraciones con la tecnología NIRS, para predecir la composición del purín y el potencial de las emisiones de gases. Se observó una buena precisión (R2cv superior a 0,92) de las calibraciones cuando muestras de los ensayos controlados (2, 3 y Antezana et al., 2015) fueron añadidas, aumentando el rango de variación. Una menor exactitud fue observada para TAN y emisiones de NH3 y CH4, lo que podría explicarse por una menor homogeneidad en la distribución de las muestras cuando se amplía el rango de variación del estudio. ABSTRACT This PhD thesis was developed to study the emissions of ammonia (NH3) and methane (CH4) from pig slurry and the effects caused by changes on diet formulation. For these proposes three studies were conducted. Experiment 1 aimed to analyse several factors of variation of slurry composition and to establish prediction equations for potential CH4 and NH3 emissions. Seventy-nine feed and slurry samples were collected at two seasons (summer and winter) from commercial pig farms sited at two Spanish regions (Centre and Mediterranean). Nursery, growing-fattening, gestating and lactating facilities were sampled. Feed and slurry composition were determined, and potential CH4 and NH3 emissions measured. Feed nutrient contents were used as covariates in the analysis. Near infrared reflectance spectroscopy (NIRS) was evaluated as a predicting tool for slurry composition and potential gaseous emissions. A wide variability was found both in feed and slurry composition. Mediterranean farms had a higher pH (P<0.001) and ash (P=0.02) concentration than those located at the centre of Spain. Also, type of farm affected ether extract (EE) content of the slurry (P=0.02), with highest values obtained for the youngest animal facilities. Results suggested a buffer effect of dietary fibre on slurry pH and a direct relationship (P<0.05) with fibre constituents of manure. Dietary protein content did not affect slurry nitrogen content (N) but decreased (P=0.003) in total solid (TS) and volatile solids (VS) concentration. Prediction models of potential NH3 emissions (R2=0.89) and biochemical CH4 potential (B0) (R2=0.61) were obtained from slurry composition. Predictions from NIRS showed a high accuracy for most slurry constituents with coefficient of determination of cross validation (R2cv) above 0.90 and a similar accuracy of prediction of potential NH3 and CH4 emissions (R2cv=0.84 and 0.68, respectively) thus models based on slurry composition from commercial farms. Experiment 2 was conducted to investigate the effects of increasing the level of two sources of fibrous by-products, orange pulp (OP) and carob meal (CM), in iso-fibrous diets for growing-finishing pig, slurry composition and potential NH3 and CH4 emissions. Thirty pigs (85.4±12.3 kg) were fed five iso-nutritive diets: a commercial control wheat/barley (C) and four experimental diets including two sources of fibrous by-products OP and CM and two dietary levels (75 and 150 g/kg) in a 2 × 2 factorial arrangement. After a 14-day adaptation period, faeces and urine were collected separately for 7 days to measure nutrient digestibility and the excretory patterns of N from pigs (6 replicates per diet) housed individually in metabolic pens. For each animal, the derived NH3 and CH4 emissions were measured in samples of slurry over an 11 and 100-day storage periods, respectively. Source and level of the fibrous by-products affected digestion efficiency in a different way as the coefficients of total tract apparent digestibility (CTTAD) for dry matter (DM), organic matter (OM), fibre fractions and gross energy (GE) increased with OP but decreased with CM (P<0.05). Crude protein CTTAD decreased with the inclusion of both sources of fibre, being lower at the highest dietary level. Faecal concentration of fibre fractions increased (P<0.05) with the level of inclusion of CM but decreased with that of OP (P<0.01). High dietary level for both sources of fibre increased (P<0.02) CP faecal content but urine N content decreased (from 205 to 168 g/kg DM, P<0.05) in all the fibre-supplemented compared to C diet. Additionally, the proportions of undigested dietary, water soluble, and bacterial and endogenous debris of faecal N excretion were not affected by treatments. The initial slurry characteristics did not differ among different fibre sources and dietary levels, except pH, which decreased at the highest by-product inclusion levels. Ammonia emission per kg of slurry was lower in all the fibre-supplemented diets than in C diet (2.44 vs. 1.81g as average, P<0.05). Additionally, slurries from the highest dietary level of by-products tended (P<0.06) to emit less NH3 per kg of initial total Kjeldahl nitrogen (TKN) and showed a lower biochemical CH4 potential , independently of the fibre source. Experiment 3 investigated the effects of protein source in practical diets. Three experimental feeds were designed to substitute a mixture of soybean meal and soybean hulls (SB diet) with sunflower meal (SFM) or wheat DDGS (WDDGS). The proportion of other ingredients was also modified in order to maintain similar nutrient contents across diets. Changes in protein source led to differences in dietary content of neutral detergent insoluble crude protein (NDICP), soluble fibre (SF) and acid detergent lignin (ADL). Twenty-four pigs (eight per diet), weighing 52.3 or 60.8 kg at the first and second batch respectively, were housed individually in metabolic pens to determine during a 7-day period DM balance, CTTAD of nutrients, and faecal and urine composition. Representative slurry samples from each animal were used to measure NH3 and CH4 emissions over an 11 and or 100-day storage period, respectively. Neither DM intake, nor DM or energy CTTAD differed among experimental diets, but type of feed affected (P<0.001) CP digestibility, which was highest for SFM (0.846) than for SB (0.775) diet, with WDDGS-based diet giving an intermediate value (0.794). Faecal DM composition was influenced (P<0.001) accordingly, with the lowest CP concentration found for diet SFM and the highest for SB. The ratio of N excreted in urine or faeces decreased from SFM (1.63) to SB diet (0.650), as a consequence of both lower urine and higher faecal losses, with all the faecal N fractions increasing in parallel to total excretion. This result was parallel to a decrease of potential NH3 emission (g/kg slurry) in diet SB with respect to diet SFM (from 1.82 to 1.12, P<0.05), giving slurry from WDDGS-based diet an intermediate value (1.58). Otherwise, SF and insoluble neutral detergent fibre (NDF) CTTAD were affected (P<0.001 and P=0.002, respectively) by type of diet, being lower for SFM than in SB-diet; besides, a higher content of NDF (491 vs. 361 g/kg) in faecal DM was observed for SFM with respect to SB based diet, with WDDGS diet being intermediate. Degree of lignification of NDF (ADL/NDF x 100) of faeces decreased in the order SFM>WDDGS>SB (from 0.171 to 0.109 and 0.086, respectively) in parallel to a decrease of biochemical CH4 potential per g of VS of slurry (from 301 to 269 and 256 ml, respectively). All slurry samples obtained from these three experiments and Antezana et al. (2015) were used to develop new calibrations with NIRS technology, to predict the slurry composition and potential gaseous emissions of samples with greater variability in comparison to experiment 1. Better accuracy (R2cv above 0.92) was observed for calibrations when samples from controlled trials experiments (2, 3 and Antezana et al., 2015) were included, increasing the range of variation. A lower accuracy was observed for TAN, NH3 and CH4 gaseous emissions, which might be explained by the less homogeneous distribution with a wider range of data.

Quantitative characterization of five cover crop species

The introduction of cover crops in the intercrop period may provide a broad range of ecosystem services derived from the multiple functions they can perform, such as erosion control, recycling of nutrients or forage source. However, the achievement of these services in a particular agrosystem is not always required at the same time or to the same degree. Thus, species selection and definition of targeted objectives is critical when growing cover crops. The goal of the current work was to describe the traits that determine the suitability of five species (barley, rye, triticale, mustard and vetch) for cover cropping. A field trial was established during two seasons (October to April) in Madrid (central Spain). Ground cover and biomass were monitored at regular intervals during each growing season. A Gompertz model characterized ground cover until the decay observed after frosts, while biomass was fitted to Gompertz, logistic and linear-exponential equations. At the end of the experiment, carbon (C), nitrogen (N), and fibre (neutral detergent, acid and lignin) contents, and the N fixed by the legume were determined. The grasses reached the highest ground cover (83–99%) and biomass (1226–1928 g/m2) at the end of the experiment. With the highest C:N ratio (27–39) and dietary fibre (527–600 mg/g) and the lowest residue quality (~680 mg/g), grasses were suitable for erosion control, catch crop and fodder. The vetch presented the lowest N uptake (2·4 and 0·7 g N/m2) due to N fixation (9·8 and 1·6 g N/m2) and low biomass accumulation. The mustard presented high N uptake in the warm year and could act as a catch crop, but low fodder capability in both years. The thermal time before reaching 30% ground cover was a good indicator of early coverage species. Variable quantification allowed finding variability among the species and provided information for further decisions involving cover crop selection and management.

NMR characterization of lignins in Arabidopsis altered in the activity of ferulate 5-hydroxylase

Nuclear magnetic resonance (NMR) of isolated lignins from an Arabidopsis mutant deficient in ferulate 5-hydroxylase (F5H) and transgenic plants derived from the mutant by overexpressing the F5H gene has provided detailed insight into the compositional and structural differences between these lignins. Wild-type Arabidopsis has a guaiacyl-rich, syringyl-guaiacyl lignin typical of other dicots, with prominent β-aryl ether (β–O–4), phenylcoumaran (β–5), resinol (β–β), biphenyl/dibenzodioxocin (5–5), and cinnamyl alcohol end-group structures. The lignin isolated from the F5H-deficient fah1–2 mutant contained only traces of syringyl units and consequently enhanced phenylcoumaran and dibenzodioxocin levels. In fah1–2 transgenics in which the F5H gene was overexpressed under the control of the cauliflower mosaic virus 35S promoter, a guaiacyl-rich, syringyl/guaiacyl lignin similar to the wild type was produced. In contrast, the isolated lignin from the fah1–2 transgenics in which F5H expression was driven by the cinnamate 4-hydroxylase promoter was almost entirely syringyl in nature. This simple lignin contained predominantly β-aryl ether units, mainly with erythro-stereochemistry, with some resinol structures. No phenylcoumaran or dibenzodioxocin structures (which require guaiacyl units) were detectable. The overexpression of syringyl units in this transgenic resulted in a lignin with a higher syringyl content than that in any other plant we have seen reported.

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Gene discovery in the wood-forming tissues of poplar: Analysis of 5,692 expressed sequence tags

A rapidly growing area of genome research is the generation of expressed sequence tags (ESTs) in which large numbers of randomly selected cDNA clones are partially sequenced. The collection of ESTs reflects the level and complexity of gene expression in the sampled tissue. To date, the majority of plant ESTs are from nonwoody plants such as Arabidopsis, Brassica, maize, and rice. Here, we present a large-scale production of ESTs from the wood-forming tissues of two poplars, Populus tremula L. × tremuloides Michx. and Populus trichocarpa ‘Trichobel.’ The 5,692 ESTs analyzed represented a total of 3,719 unique transcripts for the two cDNA libraries. Putative functions could be assigned to 2,245 of these transcripts that corresponded to 820 protein functions. Of specific interest to forest biotechnology are the 4% of ESTs involved in various processes of cell wall formation, such as lignin and cellulose synthesis, 5% similar to developmental regulators and members of known signal transduction pathways, and 2% involved in hormone biosynthesis. An additional 12% of the ESTs showed no significant similarity to any other DNA or protein sequences in existing databases. The absence of these sequences from public databases may indicate a specific role for these proteins in wood formation. The cDNA libraries and the accompanying database are valuable resources for forest research directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

Biochemical Analysis of Plant Protection Afforded by a Nonpathogenic Endophytic Mutant of Colletotrichum magna1

A nonpathogenic mutant of Colletotrichum magna (path-1) was previously shown to protect watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) seedlings from anthracnose disease elicited by wild-type C. magna. Disease protection was observed in stems of path-1-colonized cucurbits but not in cotyledons, indicating that path-1 conferred tissue-specific and/or localized protection. Plant biochemical indicators of a localized and systemic (peroxidase, phenylalanine ammonia-lyase, lignin, and salicylic acid) “plant-defense” response were investigated in anthracnose-resistant and -susceptible cultivars of cucurbit seedlings exposed to four treatments: (1) water (control), (2) path-1 conidia, (3) wild-type conidia, and (4) challenge conditions (inoculation into path-1 conidia for 48 h and then exposure to wild-type conidia). Collectively, these analyses indicated that disease protection in path-1-colonized plants was correlated with the ability of these plants to mount a defense response more rapidly and to equal or greater levels than plants exposed to wild-type C. magna alone. Watermelon plants colonized with path-1 were also protected against disease caused by Colletotrichum orbiculare and Fusarium oxysporum. A model based on the kinetics of plant-defense activation is presented to explain the mechanism of path-1-conferred disease protection.

Regulation of Ferulate-5-Hydroxylase Expression in Arabidopsis in the Context of Sinapate Ester Biosynthesis1

Sinapic acid is an intermediate in syringyl lignin biosynthesis in angiosperms, and in some taxa serves as a precursor for soluble secondary metabolites. The biosynthesis and accumulation of the sinapate esters sinapoylglucose, sinapoylmalate, and sinapoylcholine are developmentally regulated in Arabidopsis and other members of the Brassicaceae. The FAH1 locus of Arabidopsis encodes the enzyme ferulate-5-hydroxylase (F5H), which catalyzes the rate-limiting step in syringyl lignin biosynthesis and is required for the production of sinapate esters. Here we show that F5H expression parallels sinapate ester accumulation in developing siliques and seedlings, but is not rate limiting for their biosynthesis. RNA gel-blot analysis indicated that the tissue-specific and developmentally regulated expression of F5H mRNA is distinct from that of other phenylpropanoid genes. Efforts to identify constructs capable of complementing the sinapate ester-deficient phenotype of fah1 mutants demonstrated that F5H expression in leaves is dependent on sequences 3′ of the F5H coding region. In contrast, the positive regulatory function of the downstream region is not required for F5H transcript or sinapoylcholine accumulation in embryos.

Structural Alterations of Lignins in Transgenic Poplars with Depressed Cinnamyl Alcohol Dehydrogenase or Caffeic Acid O-Methyltransferase Activity Have an Opposite Impact on the Efficiency of Industrial Kraft Pulping1

We evaluated lignin profiles and pulping performances of 2-year-old transgenic poplar (Populus tremula × Populus alba) lines severely altered in the expression of caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) or cinnamyl alcohol dehydrogenase (CAD). Transgenic poplars with CAD or COMT antisense constructs showed growth similar to control trees. CAD down-regulated poplars displayed a red coloration mainly in the outer xylem. A 90% lower COMT activity did not change lignin content but dramatically increased the frequency of guaiacyl units and resistant biphenyl linkages in lignin. This alteration severely lowered the efficiency of kraft pulping. The Klason lignin level of CAD-transformed poplars was slightly lower than that of the control. Whereas CAD down-regulation did not change the frequency of labile ether bonds or guaiacyl units in lignin, it increased the proportion of syringaldehyde and diarylpropane structures and, more importantly with regard to kraft pulping, of free phenolic groups in lignin. In the most depressed line, ASCAD21, a substantially higher content in free phenolic units facilitated lignin solubilization and fragmentation during kraft pulping. These results point the way to genetic modification of lignin structure to improve wood quality for the pulp industry.