Coincidence of small-scale spatial discontinuities in leaf morphology and nuclear microsatellite variation of Quercus petraea and Q. robur in a mixed forest

BACKGROUND AND AIMS: The taxon complex comprising Quercus petraea and Q. robur shows distinct morphologies and ecological preferences, but mostly low differentiation in various types of molecular markers at a broad spatial range. Local, spatially explicit analyses may reveal patterns induced by microevolutionary processes operating mainly over short distances. However, no attempts have been made to date to explore the potential of spatial analyses combining morphological and genetic data of these oaks. METHODS: A mixed oak stand was studied to elucidate the small-scale population genetic structure. All adult individuals were classified and putative hybrids were identified using multivariate discrimination analysis of leaf morphological characters. Likewise, all trees were genotyped with five nuclear microsatellites, and a Bayesian assignment method was applied based on maximum likelihood of multilocus genotypes for taxon and putative hybrid classification. KEY RESULTS: Multivariate analyses of leaf morphological data recognized two groups with few individuals as putative hybrids. These groups were significantly differentiated at the five microsatellites, and genetic taxon assignment coincided well with morphological classification. Furthermore, most putative hybrids were assigned to the taxon found in their spatial neighbourhood. When grouping trees into clusters according to their spatial positions, these clusters were clearly dominated by one taxon. Discontinuities in morphological and genetic distance matrices among these clusters showed high congruence. CONCLUSIONS: The spatial-genetic analyses and the available literature led to the assumption that reproductive barriers, assortative mating, limited seed dispersal and microsite-induced selection in favour of the locally adapted taxon at the juvenile stage may reinforce taxon-specific spatial aggregation that fosters species separation. Thus, the results tend to support the hypothesis that Q. petraea and Q. robur are distinct taxa which share a recent common ancestry. Occasional hybrids are rarely found in adults owing to selection during establishment of juveniles.     

Analysis of fungal diversity of grapes by application of temporal temperature gradient gel electrophoresis - potentialities and limits of the method

AIMS: Fungi could be responsible for several problems in wines but the fungal ecosystem of grapes remains little known. The use of traditional methods does not allow to describe quickly this ecosystem. Therefore, we need to improve the knowledge about these fungi to prevent defects in wine. This study aims at evaluating the potentialities of the temporal temperature gradient gel electrophoresis (TTGE) method to describe the fungal ecosystem of grapes. METHODS AND RESULTS: The internal transcribed spacer (ITS) region was amplified and analysed using TTGE. A reference database of 56 fungal species was set up to evaluate the discrimination power of the method. The database was used for the direct identification of the fungal species present in complex samples. The sensitivity of the method is below 10(4) spores per ml. CONCLUSIONS: This method allows to describe the fungal diversity of grapes, but does not always allow to directly identify all fungal species, because of the taxonomic resolution of the ITS sequences. However, this identification strategy is less time consuming than traditional analysis by cloning and sequencing the bands. SIGNIFICANCE AND IMPACT OF THE STUDY: With this method, it will be possible to compare the fungal species present in different vineyards and to connect the presence of some fungi with particular defects in wine.     

Biological and physico-chemical assessment of hydroxyapatite (HA) with different porosity

HA with specific internal porosities was loaded with different antibiotics (ATBs) and then tested on its microbiological effectiveness. The HA purity was controlled with X-ray diffraction, IR and Raman spectrometry. Varying the sintering temperature and/or adding graphite and PMMA as porogenous agents lead to obtained micro- and meso-porosities. The biological tests concerned cell viability, proliferation and morphology (SEM), and the cytochemical staining of actin and vinculin. The micro- and meso-porous HA samples had an internal pore size of 1-10 microm and 10-50 microm, respectively. X-ray diffraction and FTIR confirmed the high purity of the HA. The cell viability tests with L132 cells confirmed the excellent cytocompatibility of HA, the graphite powder and the ATB vancomycine. Proliferation rate was assessed with MC3T3-E1 osteoblasts. All HA samples produced a higher proliferation than the controls; the micro-porous HA inducing the highest cell growth. The ATB impregnated HA also stimulated cell proliferation but in lower extend. Cytochemical staining of osteoblasts revealed a well-developed cytoskeleton with strong stress fibres. Labelling of the focal adhesion contacts with anti-vinculin showed a less developed adhesion process in the cells on the different HA substrates. It was possible to realize a highly pure hydroxyapatite with different but controlled porosities by varying the sintering temperature and/or addition of a porogenous agents. This purity and the micro-porosity stimulate significantly cell growth.     

Antibacterial activation of hydroxyapatite (HA) with controlled porosity by different antibiotics

In order to prevent the increasing frequency of per-operative infections, bioceramics can be loaded with anti-bacterial agents, which will release with respect to their chemical characteristics. A novel hydroxyapatite (HA) was elaborated with specific internal porosities for using as a bone-bioactive antibiotic (ATB) carrier material. UV spectrophotometry and bacteria inhibition tests were performed for testing the ATB adsorption and the microbiological effectiveness after loading with different antibiotics. The impregnation time, ATB impregnating concentration, impregnation condition and other factors, which might influence the ATB loading effect, were studied by exposure to different releasing solvents and different pathogenic bacteria: Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli. It clearly showed that the facility of ATB loading on this porous HA is even possible just under simple non-vacuum impregnation conditions in a not-so-long impregnating interval. The results also showed that, for all three types of ATB (vancomycin, ciprofloxacin and gentamicin), adsorbed amount on the micro-porous HA were hugely higher than that on dense HA. The micro-porosity of test HA had also significantly prolonged the release time of antibiotics even under mimic physiological conditions. Furthermore, it also has primarily proved by a pilot test that the antibacterial efficiency of crude micro-porous HA could be further significantly improved by other methods of functionalization such as cold plasma technique.