Evaluation of the API 50CH and API ZYM systems for rapid characterization of Clavibacter michiganensis subsp. sepedonicus, causal agent of potato ring rot

API 50CH and API ZYM systems were used to characterize fifty-three strains of Clavibacter michiganensis subsp. sepedonicus from different geographic locations and several reference strains of the same and different species, including other potato pathogens. Clavibacter michiganensis subsp. sepedonicus strains displayed a high level of homogeneity, both in carbohydrate utilization and in enzymatic activity. Using API 50CH and API ZYM it was possible to differentiate C. michiganensis subsp. sepedonicus strains from the remaining taxa analysed in this study, which included representative strains of the other subspecies of C. michiganensis as well as other bacterial pathogens affecting potatoes. Therefore, these systems could be used as an effective method to characterize C. michiganensis subsp. sepedonicus. Such a procedure would constitute an alternative system to the conventional nutritional and physiological identification tests currently included in the official methods employed in the European Union to detect and identify this bacterium. The results obtained with the API systems agreed with the current taxonomic classification of C. michiganensis, clearly separating sepedonicus from the other subspecies belonging to this species.     

Phenotypic variability in different strains of Pseudomonas syringae subsp. savastanoi isolated from different hosts

One hundred and sixty strains of Pseudomonas syringae subsp. savastanoi from Olea europaea, Olea europaea var. sylvestris, Nerium oleander, Fraxinus angustifolia and Retama sphaerocarpa, and four type strains of other pathovars were studied, investigating 102 phenotypic traits, among which we include biochemical characteristics, assimilation of different carbon sources, sensitivity or resistance to antibiotics and indoleacetic acid (IAA) production. Results were analysed with an affinity dendrogram via the Jaccard coefficient. They indicate an influence of environmental factors on the formation of the 15 phenons obtained, since isolated (knot) strains from the same species but different geographical areas are segregated. Segregation, also detected in strains from different hosts within the same area, added to the pathogenicity test helps to characterise these strains as different pathovars.     

Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.     

The influence of the ethanol pumping of wheat straw and of the beating of pulp on the resulting paper sheets

This paper reports on the influence of independent variables in the pulping of wheat straw (viz. cooking temperature and time, and ethanol concentration), and of the number of PFI beating revolutions to which the pulp is subjected, on the yield and Shopper-Riegler index of the pulp, and on the breaking length, stretch, burst index and tear index of the resulting paper sheets. By using a central composite factor design and the BMDP software suite, equations that relate each dependent variable to the different independent variables were obtained that reproduce the experimental results for the dependent variables with errors less than 25% at temperatures, times, ethanol concentrations and numbers of PFI beating revolutions in the ranges 140–180°C, 60–120 min, 40–80% and 0–1750, respectively. Ensuring the optimal Shopper-Riegler index for the pulp (73.18°SR), and also the optimal breaking length (6276 m), stretch (2.57%) and burst index (3.72 kN/g) for the papers sheets, entails using a high temperature (180°C), a large number of PFI beating revolutions (1750) and medium-to-high ethanol concentration and time values; these conditions, however, decrease the tear index for the paper sheets below the optimum level (4.60 mNm²/g). A high temperature and a large number of PFI beating revolutions, in conjunction with a low ethanol concentration (60%) and a short time (60 min), can be used to save solvent and increase the production; the values thus obtained for the dependent variables differ by less than 30% from their optimum levels in the worst case.     

<Emphasis Type="Italic">Pinus halepensis</Emphasis> somatic embryogenesis is affected by the physical and chemical conditions at the initial stages of the process

Pinus halepensis has been described as a drought-tolerant species with high plasticity to growth in different environments. Its eco-physiological characteristics could facilitate the use of this species in large afforestations in the future scenery of climate change. Somatic embryogenesis is a biotechnological tool with potential for large-scale clonal propagation. In order to establish an improved regeneration protocol for Pinus halepensis, the effects of different temperatures (18, 23, and 28 °C) and water availability conditions (2, 3, and 4 g L^?1 Gelrite^®), during initiation of embryonal masses on the rate of initiation, proliferation, maturation, and the number of embryos developed, were evaluated. It was found that environmental conditions during the initiation stage of Pinus halepensis somatic embryogenesis influence the success of initiation and proliferation. In contrast, there was no effect of these conditions on the maturation rates and the number of somatic embryos. Somatic embryos were obtained in all treatments tested, indicating that plants can be produced from extreme conditions of induction, such as high temperatures (28 °C) and low water availability conditions (4 g L^?1).     

Effect of drought on diameter increment of Quercus ilex, Phillyrea latifolia, and Arbutus unedo in a holm oak forest of NE Spain

The present study was carried out to elucidate the drought growth responses of Quercus ilex L., Phillyrea latifolia L., Arbutus unedo L., and other accompanying woody species of the Mediterranean holm oak forest. We submitted holm oak forest stands in Prades mountains (NE Spain) to a 2-year experimental drought. We reduced soil water availability about 15% by plastic strips and funnels that partially excluded rain throughfall and by ditch interception of water runoff. Mean stem diameter increment showed a great variation depending on the species. A. unedo had larger growth rates than Q. ilex and P. latifolia, but it was also the species that experimented the highest growth reduction in the drought plots (77%), suggesting a higher drought sensitivity than Q. ilex (55%) and P. latifolia (no drought effect). The growth reduction was specially marked in the larger trees. Aboveground stand biomass increment, estimated from stem diameter by allometric relationships, was 1.9 Mg ha⁻¹ per year in the control plots. The 15% reduction in the upper soil moisture produced 42% reduction in this biomass increment. In the drier conditions predicted in this Mediterranean area in the frame of climate change, an important reduction of growth rates can be hence expected, accompanied by a gain of dominance of drought-tolerant species such as P. latifolia in detriment of more mesic species such as Q. ilex.     

Vegetation dynamics in burnt heather-gorse shrublands under different grazing management with sheep and goats

Vegetation dynamics in previously burnt shrublands dominated by gorse (Ulex gallii) in north-west Spain were studied during 4 years under different grazing managements with sheep (Gallega breed) and goats (Cashmere or local breed). Initially (Period 1), treatments consisted of sheep or goat grazing on four plots (two replicates). After two grazing seasons, the plots were split in two and the treatments were reversed in four of the subplots; sheep were placed in subplots previously grazed by goats and vice versa (Period 2). The results from Period 1 showed that goats control the regrowth of gorse (from 19.6 to 14.2% of cover) more effectively than sheep (17.8 to 27.0% cover; P < 0.05), favouring the increase in herbaceous plants (from 21.5 to 42.1% of cover under goat grazing and from 22.1 to 26.6% under sheep grazing; P < 0.01). In Period 2, after changing treatments, there was a clear effect of the previous management (P < 0.01), conditioning the dynamics of the different plant components. Where the same animal species were maintained for 4 years, the differences between sheep or goat grazing on gorse and herbaceous plant dynamics increased (24.4% of gorse and 35.2% of herbaceous plant cover under goat grazing vs 43.8% of gorse cover and 19.1% of herbaceous plant cover under sheep grazing). Animal species change buffered the differences between sheep and goats during the second Period.     

Ecological interactions,management lessons and design tools in tropical agroforestry systems

During the 1980s, land- and labor-intensive simultaneous agroforestry systems (SAFS) were promoted in the tropics, based on the optimism on tree-crop niche differentiation and its potential for designing tree-crop mixtures using high tree-densities. In the 1990s it became clearer that although trees would yield crucial products and facilitate simultaneous growing of crops, they would also exert strong competitive effects on crops. In the meanwhile, a number of instruments for measuring the use of growth resources, exploratory and predictive models, and production assessment tools were developed to aid in understanding the opportunities and biophysical limits of SAFS. Following a review of the basic concepts of interspecific competition and facilitation between plants in general, this chapter synthesizes positive and negative effects of trees on crops, and discusses how these effects interact under different environmental resource conditions and how this imposes tradeoffs, biophysical limitations and management requirements in SAFS. The scope and limits of some of the research methods and tools, such as analytical and simulation models, that are available for assessing and predicting to a certain extent the productive outcome of SAFS are also discussed. The review brings out clearly the need for looking beyond yield performance in order to secure long-term management of farms and landscapes, by considering the environmental impacts and functions of SAFS.

     

Effects of various densities of Ophiostoma ips inoculations on Pinus sylvestris in north‐western Spain

The aim was to determine the inoculation density above which Scots pine (Pinus sylvestris) is overcome by the blue‐stain fungus Ophiostoma ips that is associated with the bark beetle Ips sexdentatus. In north‐western Spain, stems of 16 Scots pines were inoculated at various densities (0, 400, 800 or 1600 inoculi/m²) along circumferential 100 or 150 cm wide inoculation belts. Each inoculum consisted of a 5 mm diameter cylinder of malt extract agar colonized by the fungus. Three months later, all trees were harvested and trunk resinosis and foliage colour were visually assessed. The percentage of healthy, desiccated, resin soaked, and blue‐stained sapwood, as well as growth productivity indices, were calculated from stem disks cut from within the inoculated zone of each tree. Sapwood‐specific hydraulic conductivity (Ks) of each tree was measured in the middle of the inoculated zone. All parameters of tree vigour changed dramatically to the worse when inoculation densities were above 400 inoculi/m², and foliage changed from green to yellow‐green or yellow when an inoculation density of 800 instead of 400 was used. The percentage loss of sapwood‐specific conductivity (PLC) increased from 30 to 90% and the percentage of healthy, conductive sapwood dropped from 85 to 35% at 800 inoculi/m². No effect of the width of the inoculation belt was observed, and there was no relationship between tree productivity indices and the level of resistance. A non‐linear negative relationship was found between PLC and the percentage of healthy sapwood. It is concluded that tree resistance was overcome and that trees were going to die when the inoculation density was ≥800 inoculi/m².