Male fertility and cytology of triploid hybrids between tetraploidSolanum commersonii (2n=4x=48, 2EBN) and Phureja-Tuberosum haploid hybrids (2n=2x=24, 2EBN)

Summary Solanum commersonii Dun. is a diploid (2n=2x=24, 1EBN) wild species of potential value for potato breeding. It is a reproductively isolated species and cannot be crossed with Tuberosum haploids (2n=2x=24, 2EBN) or other diploid 2EBNSolanum species. In order to overcome the EBN barriers, triploid hybrids were produced between Phureja-Tuberosum haploid hybrids, which form 2n pollen grains by parallel spindles, and tetraploidS. commersonii. Microsporogenesis analysis of the triploids indicated a trend towards low values of chromosome distribution at Anaphase I; lagging chromosomes were often observed as well. Despite these abnormalities, the percentage of stainable pollen was very high, ranging from 5.0% to 74.3%. A high variation in pollen grain diameter was also evident. Parallel and tripolar orientation of spindles at Metaphase II of microsporogenesis was a common feature of all the triploids analyzed, but dyads and triads were observed at a very low frequency. Therefore, also the frequency of 2n pollen was very low; the different size of stainable pollen appears to represent the ploidy levels which are possible according to the distribution of chromosomes in Anaphase I. The results obtained also suggest thatS. commersonii could have minor genes acting at the end of meiosis in such a way that, despite the presence of parallel/tripolar spindles, dyads/triads are not formed.Contribution no. 124 from the Research Center for Vegetable Breeding.     

Spatial patterns of diversity and genetic erosion of traditional cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz) in the Peruvian Amazon: An evaluation of socio-economic and environmental indicators

This study evaluates quantitatively the suitability of the use of site-specific socio-economic and environmental data as indicators to rapidly assess patterns of diversity and genetic erosion risk in cassava. Socio-economic data as well as farmers’ estimation of genetic erosion were collected in the study area, the Ucayali region of the Peruvian Amazon, through interviews with 285 cassava farmers in 50 communities, while diversity was assessed based on agromorphological characterization of 295 cassava accessions. Using multivariate regression analyses, 50 and 45% of the variation in respectively diversity and genetic erosion estimation could be explained by a selected set of socio-economic and environmental indicators. In both regression models four out of the total of 38 variables proved to contribute significantly (at p < 0.10 level). Additionally, the study revealed that farmers are a good direct source of information on the diversity present at community level, which can contribute to the development of methodologies to assess diversity more rapidly. The results of this study are valuable for the development of models to rapidly assess diversity dynamics in large areas.     

Genetic stability at nuclear and plastid DNA level in regenerated plants of <Emphasis Type="Italic">Solanum</Emphasis> species and hybrids

In this work we detected the extent of variability at nuclear and cytoplasmic DNA level of regenerated plants belonging to Solanum genotypes with a different genetic background and somatic chromosome number. As for the nuclear characterization, a total of 66 (18.5%) polymorphic bands were scored using 13 ISSR primers on 45 randomly selected regenerants. Our results show that the regenerants obtained from clone cmm 1T and, at lower level, those from cph 1C are unstable under in vitro conditions or rather more prone to in vitro-induced stress leading to somaclonal variation than the other genotypes used. Two types of changes were observed: disappearance of parental ISSR fragments, termed “loss”; appearance of novel ISSR fragments, termed “gain”. The most frequent event occurring in the regenerants was the loss of fragments (41 bands). Regenerated plants were analyzed with seven plastid universal primers to determine the cytoplasmic composition at chloroplast level. All cpDNA primer pairs tested produced amplicons of the same size in all genotypes analyzed and no polymorphic fragments were observed with any universal primers used. Our results show that under in vitro culture conditions genotype affects the integrity of the genome. In addition, the absence of polymorphism at plastid level confirms the greater genetic stability of cytoplasmic DNA.     

Culture as an Engine of Local Development Processes: System‐Wide Cultural Districts I: Theory

Building on the early works of Alfred Marshall, analyses of local economies have emphasized the spatial function of clusters and industrial districts in terms of external economies of localization and agglomeration. Recent literature has emphasized the importance of culture and the complementarities between culture and local tangible and intangible assets. This paper aims to provide an analytical foundation for these processes with a view to developing tools for policy design, analysis and evaluation. By “system‐wide cultural districts,” we refer to a new approach to local development where cultural production and participation present significant strategic complementarities with other production chains. In this view, culture drives the accumulation of intangible assets such as human, social, and cultural/symbolic capital, thereby fostering economic and social growth and environmental sustainability.     

Map- vs. homology-based cloning for the recessive gene ol-2 conferring resistance to tomato powdery mildew

The recessive gene ol-2 confers papilla-associated and race-non-specific resistance to tomato powdery mildew caused by Oidium neolycopersici. In order to facilitate marker assisted selection (MAS) in practical breeding programmes, we identified two simple sequence repeat (SSR) markers and one cleaved amplified polymorphic sequence (CAPS) marker which are linked to the resistance locus and co-dominantly inherited. Aiming to provide a base for ol-2 positional cloning, we used a large segregating F₂ population to merge these markers with all the ol-2 linked amplified fragment length polymorphism (AFLP^®) markers previously identified in an integrated genetic map. By screening a tomato bacterial artificial chromosome (BAC) library, we detected two BAC clones containing two expressed sequence tags (ESTs) homologous to the gene mlo, responsible for powdery mildew resistance in barley, as well as an ol-2-linked marker. Chromosomal mapping by Fluorescence in situ Hybridization (FISH) revealed major signals of the two BAC DNAs in the pericentromeric heterochromatin of the short arm of chromosome 4, in the same region where the ol-2 gene was previously mapped. The genetic and cytogenetic co-localisation between ol-2 and tomato mlo-homologue(s), in addition to the similarity of ol-2 and mlo resistances for both genetic and phytopathological characteristics, suggests that ol-2 is likely a mlo-homologue. Thus, a homology-based cloning approach could be more suitable than positional cloning for ol-2 isolation.     

Soil organic C variability and microbial functions in a Mediterranean agro-forest ecosystem

Five soils characterised by different agro-forest managements, typical of Mediterranean environment and with increasing human impact were chosen in Sardinia (Italy): two vineyards with different management systems, a rotation hay crop-pasture and a forest (Quercus suber L.). The study aimed to investigate the relationships between C storage and microbial functionality in soil under different managements. Pools of total organic C and microbial biomass C were determined, as well as the loss of organic C due to microbial respiration (basal and cumulative) and several microbial indices (metabolic, mineralization, and microbial quotient) as indicators of the microbial efficiency in the use of energy and the degree of substrate limitation for soil microbes. Enzymes were chosen on their relevance in the C (β-cellobiohydrolase, N-acetyl-β-glucosaminidase, β-glucosidase, α-glucosidase), N (leucine aminopeptidase), S (arylsulphatase) and P (acid phosphatase) cycling and were used as indicators of functional diversity in soil. Organic C pools and enzyme activities on average increased noticeably in soils with a lower human impact showing the highest values in forest and the lowest in the vineyards, following the trend of organic matter availability. The trend in functional diversity reflected the increase of microbial pool and organic C availability: the vineyards showed a lower Shannon’s diversity index, whilst pasture and forest sites reached the maximum levels of functional diversity. These soils showed an increase of microbial efficiency in the use of available resources and the decrease of substrate limitation for soil microbes.     

Soil chemical and biochemical properties of a salt-marsh alluvial Spanish area after long-term reclamation

Marisma, one of the largest salt-marsh alluvial areas in SW Spain, has been reclaimed since 1970 by artificial drainage and amendment with phosphogypsum (PG) so as to reduce Na⁺ saturation. Within the reclaimed area, two 250- × 20-m plots were treated as follows: (1) amendment with 25 Mg/ha of PG every 2 to 3 years between 1979 and 2003 (plot PY); (2) like PY but PG treatment stopped after 1997 (plot DR). A contiguous virgin Marisma salt-marsh plot (MV), neither drained nor amended, was the control. In MV, soil microbial biomass C, most enzyme activities and total organic C content were much greater than in PY and DR soils, despite the salinity stress. The decrease in soil organic matter content in PY and DR soils was likely due to cotton-cropping practices, which favoured the organic C mineralisation and nutrients removal by crops. Microbial activity of MV soil, probably stimulated by the rhizodepositions of the natural vegetation, did not suffer from the osmotic effect due to the raising of soil solution ionic strength. Microbial quotient could be ranked as MV > PY > DR, whereas the metabolic quotient had an opposite trend. Thus, both quotients suggested that the interruption of PG amendment was not favouring microbial activity. Principal component analysis clearly identified the chemical and biochemical soil properties mostly affected by the reclamation and also indicated the longer PG amendment in PY plot. Stepwise discriminant analysis identified two physiologically different types of soil microflora, one less active present in the MV virgin soil and another more active present in the reclaimed PY and DR soils.     

Ecotoxicological evaluation of Harbour sediments using marine organisms from different trophic levels

Background, Aim and Scope  The toxicity of contaminated sediments should be evaluated considering the direct exposure of laboratory organisms to whole sediments and the indirect exposure to elutriates or extracts (Tay et al. 1992, Byrne and Halloran 1999, Nendza 2002). The alga Dunaliella tertiolecta is indicated for the use in toxicity bioassays because it is highly sensitive to several xenobiotics. Harpacticoid copepods have been already used for toxicity testing and Tigriopus fulvus is a promising Mediterranean target-species in ecotoxicology (Todaro et al. 2001, Faraponova et al. 2003, Pane et al. 2005a). In this study, the toxicity of sediments collected in harbour sites of the Northeastern Adriatic Sea was evaluated by growth inhibition test with free living and alginate-immobilized Dunaliella tertiolecta and acute toxicity test with nauplii and adult Tigriopus fulvus with the aim of pointing out the importance to utilize model organisms from different trophic levels in sediment ecotoxicology. Methodology  Elutriates and whole sediments were tested on free living and immobilized (Pane et al. 1998) algal cells, and on laboratory reared copepods. Free-living D. tertiolecta were exposed to diluted elutriates in a static, multi-well plate system. Naalginate immobilized D. tertiolecta were placed in polystyrene inserts fitted with polyester mesh bottoms and exposed to a thin layer (2 mm) of whole sediments in multi-well plates (EPS 1992, Pane and Bertino 1999). Toxicity tests with copepods were carried out on Tigriopus fulvus nauplii (elutriates) and adults (whole sediments and elutriates). Same-aged nauplii useful for toxicity tests were obtained by egg sac detaching and consequent hatching stimulation (Pane et al. 2006). Newborn nauplii (I–II stage) were exposed to elutriates in multi-well plates provided with polystyrene inserts. Adult T. fulvus maintained in polystyrene inserts fitted with polyester mesh bottoms were placed in contact with a thin layer (2 mm) of whole sediment placed on multi-well plate bottoms. All end-points were evaluated after 96 h. Results  In general, the effects increased with the increasing of elutriate concentration up to 50%; the stimulation or inhibition of algal growth was statistically significant in comparison to the control. The inhibiting elutriates induced EC₅₀ variations of algal growth ranging from 66.9% to 74.3%. The mortality of T. fulvus nauplii was always < 25% after treatment with 100% elutriates and < 10% after treatment with 50% dilution; no effect was shown up with 25% dilution; therefore LC₅₀ was not calculable. The effect of elutriates was negligible on adult copepods and LC₅₀ values were never calculable; percent mortality always resulted in < 10% after treatment with whole sediments. Discussion  Both experimental systems gave substantially similar results after exposition to whole sediments and elutriates. During the experiment with algal cells, the immobilization in Na-alginate and the employment of inserts which allowed the contact of organisms with sediments and their easy counting were particularly useful. Likewise, the employment of inserts of adequate mesh size in the tests with copepods allowed the contact of organisms with the sediment and made organism handling and counting easy, as well as the evaluation of mortality. The methodology here described and the utilization of the proposed test-species could have an importance also considering that the current trend in ecotoxicological research is towards finding the most appropriate organism for specific areas of concern by using indigenous species (Mariani et al. 2006) and towards the major significance of chronic and reproductive end-points. Conclusions  Based on the above results, it can be stated that the bioassay with Dunaliella tertiolecta could be a good estimation tool for the ecotoxicological assessment of marine sediments. The immobilization of algae in Na-alginate was seen to be useful to evaluate the toxicity of whole sediments; the employment of polystyrene inserts allowed an improvement of the procedures. T. fulvus nauplii and adults, as other harpacticoids such as Tigriopus japonicus (Yoon et al. 2006), satisfy the basic criteria for the employment of a standard species in marine bioassays. To date only pelagic Acartia tonsa are utilized in the standardized procedure to evaluate the risk assessment of chemicals or wastewaters (ISO 1999). As, on the contrary, the exposure of copepods to solid-phase contaminants it is not yet standardized, the employment of polystyrene inserts improved the procedures for T. fulvus too. So, the rapidity and the possibility to solve practical problems could be the main attractive features of this technique (Pane et al. 2005a) when applied to whole sediments. Recommendations and Perspectives  The methodology here developed being also applicable to long term and reproduction tests should be recommended because it provides relevant information in comparison with other frequently applied, standardized biotests with crustaceans (ISO 1999). The procedure has been shown to be easily applicable to selected marine organisms. ESS-Submission Editor: Prof. Dr. Henner Hollert (henner.hollert@bio5.rwth-aachen.de)     

Long‐Term Tillage and Cropping System Effects on Chemical and Biochemical Characteristics of Soil Organic Matter in a Mediterranean Semiarid Environment

Several studies have reported how tillage and cropping systems affect quantity, quality, and distribution of soil organic matter (SOM) along the profile. However, the effect of soil management on the chemical structure of SOM and on its hydrophobic and hydrophilic components has been little investigated. In this work, the long‐term (19 years) effects of two cropping systems (wheat monoculture and wheat/faba bean rotation) and three tillage managements (conventional, reduced, and no tillage) on some chemical characteristics of SOM and their relationships with labile carbon (C) pools were evaluated. Soil samples were taken from the topsoil (0–15 cm) of a Chromic Haploxerert (central Sicily, Italy). After 19 years of different tillage and cropping systems management, total organic C significantly differed among treatments with the labile organic C pools showing the greater amount in no till and in wheat/faba bean plots. Hydrophobic and hydrophilic components of SOM, determined by diffuse reflectance infrared Fourier transform spectroscopy, were mainly affected by cropping system, whereas aromatic components of SOM by tillage. Soil organic matter components and characteristics showed significant correlations with the soil biochemical parameters, confirming the expected synergism between chemical and biochemical properties. This study demonstrated that (i) no tillage and crop rotation improve the chemical and biochemical properties of SOM of Vertisols under semiarid environment; and (ii) tillage management and cropping systems have affected, after 19 years, more the chemical and biochemical properties of SOM than its quantity. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of the anthocyanin fraction of sicilian blood orange juice by micro-HPLC-ESI/MS

Anthocyanins of red orange juices were analyzed by micro-HPLC coupled on-line with an MS detector equipped with an ESI source. The use of microcolumn HPLC greatly enhanced detection performance, allowing direct identification of the anthocyanins present in the orange juices. The use of a soft ionization technique allows detection of the molecular ions of the aglycons. Eight components were identified, five of them for the first time in red orange juice. Three additional anthocyanins were detected, of which only the aglycon was identified.