Molecular biodiversity and population structure in common ash (Fraxinus excelsior L.) in Britain: implications for conservation

Current forestry policy promotes the use of local seed for new plantings, on the assumption that local material may be better adapted to local conditions. However, landscape‐scale genetic studies which are necessary to underpin conservation and breeding strategies are often lacking. We investigated molecular diversity in common ash (Fraxinus excelsior L.) sampled from 42 British and six French sites with microsatellites. Chloroplast haplotype H04 was the most common and widespread in Britain, although rare and localized individuals with H02 and H09 were also detected. In addition, three new chloroplast haplotypes were identified, and these were rare and highly localized. In terms of nuclear microsatellite markers, allelic richness differed between sites and decreased in an east to west direction. Differentiation between sites was often very low (mean F_ST 0.025), indicating few differences between the majority of sites. There was a clear excess of homozygotes (mean H_O 0.669, mean H_E 0.818) and a relatively high F_IS (mean 0.182), suggests a consistent level of inbreeding or a widespread Wahlund effect in many F. excelsior sites. Gene pool ancestry analysis suggested that the majority of British F. excelsior belongs to a single meta‐population which covers mainland western and central Europe. Three northern and western sites diverged markedly from the dominant population, and may represent remnants of two late potential Ice Age refugia in northern Britain. The data provide new information which will aid development of appropriate conservation policies for ash and other wind pollinated tree species.     

Underwater germination potential of common ash seed (Fraxinus excelsior L.) originating from flooded and non-flooded sites

Oxygen availability is one of the main factors affecting seed germination capacity. On floodplains, oxygen supply is seasonally restricted because of flooding events. Seed ecotypes are able to cope with total submergence to different extents, depending on the species and its position in a given environmental gradient. This investigation tested three provenances of common ash (Fraxinus excelsior L.), one from a lowland site and two from upland sites, for germination capacity and germination velocity under water. The final germination capacity of all provenances was unaffected, but the germination velocity was decreased for all provenances. The adaptational significance of these results is discussed.     

Micropropagation of hornbeam (Carpinus betulus L.) and ash (Fraxinus excelsior L.)

Shoot multiplication of hornbeam was stimulated onWPM, QL andDKW medium supplemented with a low concentration of BAP or BPA (0.1-0.2 mg I -1) andIBA (0.1 mg I -1). Low concentration of thidiazuron promoted axillary bud formation, higher concentration inhibited shoot elongation. Microshoots were rooted onWPM supplemented with a low auxin concentration (IBA or NAA 0.2-0.5 mg I -1). High rooting percentages were obtained. Shoot proliferation of ash was stimulated on MS andDKW medium supplemented withBAP orBPA (2.0-5.0 mg I -1) andIBA (0.1 mg I -1). Root formation was promoted onWPM containing a low auxin concentration. Rooted plantlets were transplanted into soil and after hardening off the micropropagated trees were planted in the field. The planted trees grew normally without showing signs of abnormality.     

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.     

Utility of the polymerase chain reaction-restriction fragment length polymorphisms of the intergenic spacer region of the rDNA for characterizing Gibberella fujikuroi isolates

In the present report, a total of thirty-one isolates of Gibberella fujikuroi (Sawada) Wollenw. species complex of Fusarium (section Liseola) morphologically classified as F. moniliforme according to the taxonomy of Nelson, Toussoun and Marasas (1983) were analyzed for their ability to produce fumonisin B₁ and fumonisin B₂ by an optimized liquid chromatographic method. They were isolated from three hosts (Zea mays, Musa sapientum and Pinus pinea). The results indicate that M. sapientum is a preferential host for G. fujikuroi isolates with low or null capacity for producing fumonisins, while isolates from Z. mays and P. pinea are generally high fumonisin producers.

The molecular characterization of isolates was carried out in parallel using an optimized, simple and low-cost method for isolating DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the rDNA intergenic spacer (IGS) region. The haplotypes obtained with Hha I enzyme and combinations of Hha I, EcoR I, Alu I, Pst I and Xho I enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin B₁ and B₂ producing capacity. IGS region restriction patterns showed no relationship to isolate geographical origin. This is the first report on this method's capacity to detect polymorphism permitting discrimination between G. fujikuroi isolates from different hosts and with different toxigenic profiles.     

Nitrate reduction and nitrate content in ash trees (Fraxinus excelsior L.): distribution between compartments,site comparison and seasonal variation

Summary Nitrate reductase activity (NRA), nitrate content and biomass components of leaflets, leaf stalks, old stem, current-year stem and roots of ash trees (Fraxinus excelsior L.) growing in their natural habitats were investigated. In addition, NRA, total nitrogen and nitrate concentration were analyzed in the leaves and roots of ash trees from four different field sites. The highest NRA per gram biomass and also per total compartment biomass was found in the leaflets, even though root biomass was much higher than total leaflet biomass. The highest nitrate concentrations were found in the leaf stalks. Correlations between nitrate availability in the soil and NRA in leaves were not significant due to high variability of the actual soil nitrate concentrations. The seasonal variation in foliar NRA, nitrate concentration and total nitrogen concentration is much smaller in F. excelsior than reported for herbaceous species and is mainly caused by changes in the actual soil nitrate availability and by senescence of the leaves.     

Cumulative temperatures for prediction of the beginning of ash (Fraxinus excelsior L.) pollen season

The aim of this study is the analysis of the meteorological conditions leading to the release of ash pollen in order to predict the beginning of the pollen season in Zurich, Switzerland. For 12 years over a period of 14 consecutive years, it appears that ash pollen release begins when the cumulated mean daily temperatures starting on January 1 reach 220.7°C, admitting a variation of ±30°C that more or less corresponds to 3 consecutive days at 10°C.     

Changes in ecosystem carbon stocks in a grassland ash (Fraxinus excelsior) afforestation chronosequence in Ireland

Aims Government policy in Ireland is to increase the national forest cover from the current 10% to 18% of the total land area by 2020. This represents a major land use change that is expected to impact on the national carbon (C) stocks. While the C stocks of ecosystem biomass and soils of Irish grasslands and coniferous forests have been quantified, little work has been done to assess the impact of broadleaf afforestation on C stocks.Methods In this study, we sampled a chronosequence of ash (Fraxinus excelsior) forests aged 12, 20, 27, 40 and 47 years on brown earth soils. A grassland site, representative of the pre-afforestation land use, was sampled as a control.Important findings Our results show that there was a significant decline (P < 0.05) in the carbon density of the soil (0–30cm) following afforestation from the grassland (90.2 Mg C ha^-1) to the 27-year-old forest (66.7 Mg C ha^-1). Subsequently, the forest soils switched from being a C source to a C sink and began to sequester C to 71.3 Mg C ha^-1 at the 47-year-old forest. We found the amount of C stored in the above- and belowground biomass increased with age of the forest stands and offset the amount of C lost from the soil. The amount of C stored in the above- and belowground biomass increased on average by 1.83 Mg C ha^-1 year^-1. The increased storage of C in the biomass led to an increase in the total ecosystem C, from 90.2 Mg C ha^-1 at the grassland site to 162.6 Mg C ha^-1 at the 47-year-old forest. On a national scale, projected rates of ash afforestation to the year 2020 may cause a loss of 290 752 Mg C from the soil compared to 2 525 936 Mg C sequestered into the tree biomass. The effects of harvesting and reforestation may further modify the development of ecosystem C stocks over an entire ash rotation.     

Frost periods and beginning of the ash (Fraxinus excelsior L.) pollen season in Basel (Switzerland)

In order to be able to forecast the beginning of the ash pollen season, several meteorological factors have been considered. During these research activities it appeared that the ash pollen release is closely linked to frost periods happening during the months of February and March. For 24 years over a period of 29 consecutive years, a daily concentration of 30 ash pollen grains/m³/day has been reached when the cumulated mean daily temperatures starting on the last day when the absolute minimum temperature is lower or equal to −2 °C amounts on average to 214 °C – admitting a variation of plus or minus 42 °C. As for the five remaining years, rain is three times responsible for the delay of the pollen release. This revised version was published online in June 2006 with corrections to the Cover Date.     

Patterns of ash (Fraxinus excelsior L.) colonization in mountain grasslands: the importance of management practices

Woody colonization of grasslands is often associated with changes in abiotic or biotic conditions or a combination of both. Widely used as fodder and litter in the past traditional agro-pastoral system, ash (Fraxinus excelsior L.) has now become a colonizing species of mountain grasslands in the French Pyrenees. Its present distribution is dependent on past human activities and it is locally controlled by propagule pressure and abiotic conditions. However, even when all favourable conditions are met, all the potentially colonizable grasslands are not invaded. We hypothesize that management practices should play a crucial role in the control of ash colonization. From empirical field surveys we have compared the botanical composition of a set of grasslands (present and former) differing in management practices and level of ash colonization. We have displayed a kind of successional gradient positively linked to both ash cover and height but not to the age of trees. We have tested the relationships between ash presence in grassland and management types i.e. cutting and/or grazing, management intensity and some grassland communities’ features i.e. total and local specific richness and species heterogeneity. Mixed use (cutting and grazing) is negatively linked to ash presence in grassland whereas grazing alone positively. Mixed use and high grazing intensity are directly preventing ash seedlings establishment, when low grazing intensity is allowing ash seedlings establishment indirectly through herbaceous vegetation neglected by livestock. Our results show the existence of a limit between grasslands with and without established ashes corresponding to a threshold in the intensity of use. Under this threshold, when ash is established, the colonization process seems to become irreversible. Ash possesses the ability of compensatory growth and therefore under a high grazing intensity develops a subterranean vegetative reproduction. However the question remains at which stage of seedling development and grazing intensity these strategies could occur.     

Sequence of the plastid rDNA spacer region of the brown alga Pylaiella littoralis (L.) Kjellm. Evolutionary significance

The DNA segment situated between the 16S and 23S rRNA genes belonging to the plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm. has been sequenced. This small region (322 bp) contains two unsplit tRNA genes separated by 3 bp. A comparison with similar regions from different plants shows that this region has evolved in two different ways according to the place of plants in evolution. In the primitive group, this region is reduced in size when compared to prokaryotes. In the other groups, it is considerably enlarged by insertion of repetitive sequences, open reading frames and introns.     

PCR amplification of species specific sequences of 16S rDNA and 16S-23S rDNA intergenic spacer region for identification of Streptococcus phocae

Streptococcus phocae, a bacterial pathogen of seals, could reliably be identified by PCR amplification using oligonucleotide primers designed according to species specific segments of the previously sequenced 16S rRNA gene and the 16S-23S rDNA intergenic spacer region of this species. The PCR mediated assay allowed an identification of S. phocae isolated from harbor and gray seals and from Atlantic salmons. No cross-reaction could be observed with 13 different other streptococcal species and subspecies and with Lactococcus garvieae strains investigated for control purposes.